Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy.     

Overlapping Genes Produce Proteins with Unusual Sequence Properties and Offer Insight into De Novo Protein Creation

It is widely assumed that new proteins are created by duplication, fusion, or fission of existing coding sequences. Another mechanism of protein birth is provided by overlapping genes. They are created de novo by mutations within a coding sequence that lead to the expression of a novel protein in another reading frame, a process called “overprinting.” To investigate this mechanism, we have analyzed the sequences of the protein products of manually curated overlapping genes from 43 genera of unspliced RNA viruses infecting eukaryotes. Overlapping proteins have a sequence composition globally biased toward disorder-promoting amino acids and are predicted to contain significantly more structural disorder than nonoverlapping proteins. By analyzing the phylogenetic distribution of overlapping proteins, we were able to confirm that 17 of these had been created de novo and to study them individually. Most proteins created de novo are orphans (i.e., restricted to one species or genus). Almost all are accessory proteins that play a role in viral pathogenicity or spread, rather than proteins central to viral replication or structure. Most proteins created de novo are predicted to be fully disordered and have a highly unusual sequence composition. This suggests that some viral overlapping reading frames encoding hypothetical proteins with highly biased composition, often discarded as noncoding, might in fact encode proteins. Some proteins created de novo are predicted to be ordered, however, and whenever a three-dimensional structure of such a protein has been solved, it corresponds to a fold previously unobserved, suggesting that the study of these proteins could enhance our knowledge of protein space.Since their discovery (76), overlapping genes, i.e., DNA sequences simultaneously encoding two or more proteins in different reading frames, have exerted a fascination on evolutionary biologists. Among several mechanisms, they can be created by a process called “overprinting” (43), in which a DNA sequence originally encoding only one protein undergoes a genetic modification leading to the expression of a second reading frame in addition to the first one (Fig. ​(Fig.1).1). The resulting overlap encodes an ancestral, “overprinted” protein region and a protein region created de novo (i.e., not by duplication) called an “overprinting” or “novel” region (Fig. ​(Fig.1).1). At present, it is widely thought that the creation of proteins de novo is very rare, contrary to their emergence by gene duplication, which is thought to be the major factor (for reviews, see references 55 and 94). However, this belief might actually reflect the fact that proteins created de novo are in general very difficult to identify (55). Indeed, a long-standing question is whether a protein that has no detectable homolog in other organisms (called an “orphan” protein or “ORFan” [27] or “taxonomically restricted” [110]) represents a protein created de novo in a particular organism or merely a protein that is a member of a larger family whose other members have diverged beyond recognition or have become extinct (115). Proteins created de novo by overprinting provide a valuable opportunity to address these questions, and this constitutes one of the two strands of our study.Open in a separate windowFIG. 1.Creation of a novel protein region (C-terminal extension) by overprinting. Top, a DNA sequence encodes two proteins in different reading frames. Notice the potential, unused stop codon downstream of protein X. Middle, a mutation abolishes the stop codon of protein X, causing its elongation (“overprinting”) to the preexisting stop codon. This results in a gene overlap. Bottom, the overlap encodes an overprinted (ancestral) protein region (dark gray) and an overprinting (novel) protein region (light gray).Practically all studies of overlapping genes have been focused on evolutionary constraints and informational characteristics at the DNA level (see, e.g., references 46, 71, 75, 84, 85, and 114). However, very little has been done to assess potential effects of the overlap on the corresponding protein products. Two studies reported that overlapping proteins are enriched in amino acids with a high codon degeneracy (arginine, leucine, and serine) (68) and that they often simultaneously encode a cluster of basic amino acids in one frame and a stretch of acidic amino acids in the other frame (66).The other strand of the present study is based on earlier observations of the overlapping gene set of measles virus (41), which suggested that protein regions encoded by overlapping genes might have a propensity toward structural disorder.Structural disorder is an essential state of numerous proteins, in which it is associated mostly with signaling and regulation roles (21, 96, 111). The key feature of intrinsically disordered proteins (also called “unstructured” or “natively unfolded”) is that under physiological conditions, instead of a particular three-dimensional (3D) structure, they adopt ensembles of rapidly interconverting structural forms. Different degrees of disorder exist, from random coils to molten globules (100), and some disordered regions can become ordered under certain conditions (21, 96, 117). A variety of computer programs have been developed to predict these regions (19, 23, 101). Each predictor typically differs in what kind of “disorder” it identifies (23, 78), matching only some of the types of disorder mentioned above. Therefore, in order to choose a proper predictor, it was necessary to define precisely what kind of structural disorder we expected to find in proteins encoded by overlapping genes.At least two nonexclusive hypotheses can explain why overlapping genes might encode disordered proteins: (i) the newly created (overprinting) protein of each overlap might tend to be disordered, and (ii) structural disorder in proteins encoded by overlapping genes might alleviate evolutionary constraints imposed on their sequence by the overlap. These hypotheses are clarified below.Intuitively, the conditions required for a protein to fold into a stable 3D configuration, including sequence composition, periodicity, and complexity, are such that structurally ordered proteins represent a vanishingly small fraction of all possible amino acid sequences. Indeed, proteins artificially created from random nucleotide sequences generally have a low secondary structure content (107, 112). Hence our first hypothesis: novel, overprinting proteins are not expected to have a fixed 3D structure at birth, given the low probability of generating structure from a completely new sequence.Disordered proteins are generally subject to less structural constraint than ordered ones (13). Hence our second hypothesis: the presence of disorder in one or both products of an overlapping gene pair could greatly alleviate evolutionary constraints imposed by the overlap, allowing both protein products to scan a wider sequence space without losing their function.Both hypotheses suppose only the lack of a rigid structure, as opposed to a total lack of structure (e.g., some proteins created de novo from a random nucleotide sequence, though lacking secondary structure, have a certain degree of order [112]). For that reason, in this work, we use the widest possible definition of disorder, i.e., the lack of a rigid 3D structure, and we use a program whose predictions of disorder correspond to this definition, PONDR VSL2 (69) (see Results).In this work, we collected a large number of experimentally proven cases of proteins encoded by overlapping genes in unspliced eukaryotic RNA viruses and analyzed their sequence properties.     

Inhibition of LPS-induced apoptosis in differentiated-PC12 cells by new triazine derivatives through NF-κB-mediated suppression of COX-2

Anti-inflammatory therapy approaches have been in the focus of attention in the treatment of neurodegenerative diseases, such as Alzheimer's disease (AD). In this study, we examined the role of new 1,2,4-triazine derivatives against cytotoxicity exerted by lipopolysaccharide (LPS) in differentiated rat pheochromocytoma (PC12) cell line. Our results indicated that LPS-induced cell death can be inhibited in the presence of some of these compounds, as measured by MTT test, acridine orange/ethidium bromide staining and caspase-3 expression assay. We further showed that these compounds exert their protective effects through the inhibition of LPS-induced generation of nitric oxide and reactive oxygen species. Triazine derivatives inhibited LPS-induced nuclear translocation of nuclear factor- κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. Pretreatment of PC12 cells with triazine derivatives also suppressed LPS-induced cyclooxygenase-2 expression while up-regulated heat shock protein-70 (Hsp-70). Moreover, the treatment of brain diseases is limited by the insufficiency in delivering therapeutic drugs into brain relating to highly limited transport of compounds through blood-brain barrier (BBB). Using a reliable model based on the artificial neural network, we indicated that these compounds are capable of penetrating BBB and may be useful agents for preventing neuroinflammatory diseases like AD.     

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.     

Toward a preliminary assessment of the diversity and origin of Cyprinid fish genus Carassius in Iran

Introduction of non-native fish species has been criticized as one of the major threats to freshwater ecosystems. During recent years, the cyprinid fish genus Carassius has been the subject of various studies in terms of introduction biology and ecology. The taxonomy and identification of described species in the genus Carassius is still ambiguous. In order to increase the knowledge of introduced species of the genus in Iran, molecular approaches were employed to identify species based on the mitochondrial gene, cytochrome b (Cyt b). In total, 417 specimens across Eurasia and some other parts of the world have been analyzed. Afterwards, investigation of the possible origin and pathways of introduction was done using analysis of haplotype networks based on the Cyt b gene. The results revealed that there are three introduced species i.e., Carassius auratus, Carassius gibelio and Carassius langsdorfii, in inland waters of Iran. Carassius langsdorfii was recorded from Iran and western Asia for the first time. Analysis of haplotype network showed various potential sources of introduction for each species in Iran and regions beyond their native distribution ranges.     

Antibacterial effects of Octenicept,and benzalkonium chloride on Acinetobacter baumannii strains isolated from clinical samples and determination of genetic diversity of isolates by RAPD-PCR method

Molecular Biology Reports - Acinetobacter baumannii (A. baumannii) is among the important causes of nosocomial infections. Due to the emergence of antibiotic resistance, many problems have been...     

Tumour suppressor ING1b maintains genomic stability upon replication stress

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.     

Induced pluripotent stem cells (iPSCs) as game-changing tools in the treatment of neurodegenerative disease: Mirage or reality?

Based on investigations, there exist tight correlations between neurodegenerative diseases' incidence and progression and aberrant protein aggregreferates in nervous tissue. However, the pathology of these diseases is not well known, leading to an inability to find an appropriate therapeutic approach to delay occurrence or slow many neurodegenerative diseases' development. The accessibility of induced pluripotent stem cells (iPSCs) in mimicking the phenotypes of various late-onset neurodegenerative diseases presents a novel strategy for in vitro disease modeling. The iPSCs provide a valuable and well-identified resource to clarify neurodegenerative disease mechanisms, as well as prepare a promising human stem cell platform for drug screening. Undoubtedly, neurodegenerative disease modeling using iPSCs has established innovative opportunities for both mechanistic types of research and recognition of novel disease treatments. Most important, the iPSCs have been considered as a novel autologous cell origin for cell-based therapy of neurodegenerative diseases following differentiation to varied types of neural lineage cells (e.g. GABAergic neurons, dopamine neurons, cortical neurons, and motor neurons). In this review, we summarize iPSC-based disease modeling in neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Moreover, we discuss the efficacy of cell-replacement therapies for neurodegenerative disease.     

Phylogenetic relationships of Old World Brassicaceae from Iran based on nuclear ribosomal DNA sequences

Phylogenetic analysis of 155 nuclear rDNA ITS sequences among them 19 Iranian endemic genera were used to elucidate phylogenetic relationships of Old World Brassicaceae from Iran in the context of the most recent tribal system suggested by Al-Shehbaz et al. [Al-Shehbaz, I.A., Beilstein, M.A, Kellogg, E.A., 2006. Systematics and phylogeny of the Brassicaceae (Cruciferae): an overview. Plant Syst. Evol. 259, 89–120]. Iranian endemic taxa are assigned to 16 clades, 15 of these correspond to recognized tribes. Our data support the recent tribal recognition of Calepina and relatives and further indicate that the Orychophragmus clade (with Conringia planisiliqua and Orychophragmus) may be recognized as a new tribe. Our data also support the inclusion of 13 genera not previously studied, or with unresolved positions in previous phylogenetic analyses in 10 tribes with the tribal assignment given in parentheses: Acanthocardamum (Aethionemeae), Alyssopsis (Camelineae), Anastatica (Malcolmieae), Asperuginoides (Cochlearieae), Camelinopsis (Thlaspideae), Didymophysa (Thlaspideae), Dielsiocharisi (Camelineae), Lachnoloma (Anchonieae), Micrantha (Anchonieae), Noccidium (Camelineae), Octoceras (Euclidieae), Pseudofortuynia (Sisymbrieae) and Streptoloma (Euclidieae). ITS data and morphological characters further indicate that the remaining five genera, i.e., Acanthocardamum, Olimarabidopsis, Brossardia, Noccidium and Zuvanda may be subsumed under Aethionema, Alyssopsis, Noccaea, Capsella and Conringia, respectively. Alyssum, Chorispora, Fibigia and Goldbachia are paraphyletic and Conringia, Malcolmia, Matthiola are polyphyletic taxa.     

Upregulation of Fucosyltransferase 3, 8 and protein O-Fucosyltransferase 1, 2 genes in esophageal cancer stem-like cells (CSLCs)

Recently, studies have shown that Fucosylation plays an important role in the invasion and metastatic process of CSLCs. Understanding the expression pattern of fucosyltransferase (FUT) genes may help to suggest better-targeted therapy strategies for esophageal squamous cell carcinoma (ESCC). The study aimed to address the expression pattern of FUT gene variants in esophageal CSLCs and parental adherent cells. Sphere formation method was used to enrich CSLCs. Expression of FUT genes was examined in tumor sphere and parental adherent cells using the RT-PCR method and then relative expression of detected variants was performed by the Real-Time PCR method in both groups. The detected FUTs, also, were assessed in fresh ESCC tumors and the matched healthy controls. Analysis of The cell surface carbohydrate Lewis x (LeX, CD15) was performed by flow cytometry. Molecular analysis showed that the expression of FUT 3, 8 and POFUT1, 2 genes in tumorsphere were significantly higher than parental adherent cells. Analysis of fresh ESCC tumor tissues and the matched healthy controls showed that FUT8 and POFUT1, 2 genes in contrast to FUT 3 have higher expression in tumor tissues than controls. Flow cytometric analyses revealed that tumorsphere and their parent cells do not differ significantly in Lewis x surface marker. The present study showed that FUT 3, 8 and POFUT1, 2 genes upregulated in esophageal CSLCs in comparison to adherent cells. Understanding the expression pattern of FUT gene variants may help to suggest better-targeted therapy strategies for ESCC.