Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.     

Initiation of Hepatitis C Virus Infection Requires the Dynamic Microtubule Network: ROLE OF THE VIRAL NUCLEOCAPSID PROTEIN*

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified β- and α-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.HCV⁵ infection is a major cause of chronic liver disease, which frequently progresses to cirrhosis and hepatocellular carcinoma. HCV represents a global public health problem, with 130 million people infected worldwide. There is currently no vaccine directed against HCV and the available antiviral treatments eliminate the virus in 40-80% of patients, depending on the virus genotype (for review, see Ref. 1).HCV has a single-stranded, positive-sense RNA genome of ∼9.6 kilobases encoding a large polyprotein that is processed by both host and viral proteases to produce three structural proteins (core protein and the envelope glycoproteins E1 and E2), p7, and six nonstructural proteins, which are involved in polyprotein processing and replication of the virus genome (for review, see Ref. 2).HCV core is a basic protein, synthesized as the most N-terminal component of the polyprotein, and is followed by the signal sequence of the E1 envelope glycoprotein (3). The polypeptide is cleaved by signal peptidase and signal peptide peptidase, resulting in the release of core from the endoplasmic reticulum membrane and its trafficking to lipid droplets (3-5). Mature core protein forms the viral nucleocapsid (6) and consists of two domains, D1 and D2. D1 lies at the protein N terminus, is composed of about 117 amino acids (aa), and is involved in RNA binding (7). D2 is relatively hydrophobic, has a length of about 55 aa, and targets HCV core to lipid droplets (8).Microtubules (MTs) are ubiquitous cytoskeleton components that play a key role in various cellular processes relating to cell shape and division, motility, and intracellular trafficking (9). MTs are dynamic, polarized polymers composed of α/β-tubulin heterodimers that undergo alternate phases of growth and shrinkage, dependent on so-called “dynamic instability” (10). Active transport by MTs is bidirectional and involves both plus and minus end-directed motors: kinesin and dynein (11, 12).Another mechanism of cytosolic transport on MTs, called “treadmilling” (13, 14) involves polymerization at the plus end and depolymerization at the minus end after severing of MTs by cellular katenin (15).MTs have important functions in the life cycle of most viruses (13, 16, 17). Cytoplasmic transport on MTs provides viruses with the means to reach sites of replication or enables progeny virus to leave the infected cell. Some viruses, such as Ebola virus (18) or reovirus (19), are transported on MTs within membranous compartments, whereas other viruses like herpes simplex virus type 1 (20), murine polyoma virus (21), human cytomegalovirus (22), or adenovirus (23) interact with MT motors or MT-associated proteins to allow their transport along microtubules.Previous studies have established that the cell cytoskeleton is involved in HCV replication, since HCV replication complexes are subjected to intracellular transport and their formation is closely linked to the dynamic organization of endoplasmic reticulum, actin filaments, and the microtubule network (24-26). In addition, intact microtubules are essential for viral morphogenesis and the secretion of progeny virus from infected cells (27). The role of microtubules in HCV cell entry and the initiation of productive HCV infection has not yet been addressed.In this study, we provide evidence that the MT network plays a key role in HCV cell entry and postfusion steps of the virus cycle that lead to the establishment of productive HCV infection. The initial steps of the viral cycle are sensitive to MT-affecting drugs that inhibit MT formation or depolymerize or stabilize microtubules. We also show a unique property of the HCV core protein, its capacity to directly bind to tubulin and to enhance MT polymerization in vitro. Our findings suggest that HCV could exploit the MT network by polymerization-related mechanisms to productively infect its target cell. Thus, microtubules may provide a novel target for therapeutic interventions against HCV infection.     

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-α treatment

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG₂) under IFN-α treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG₂ cells as the most stable reference genes under IFN-α treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG₂ group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG₂ cells would greatly improve the normalization of the RT-qPCR data under IFN-α. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway.     

Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues

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COVID-19-related coagulopathy: A review of pathophysiology and pharmaceutical management

December 2019 will never be forgotten in the history of medicine when an outbreak of pneumonia of unknown etiology in Wuhan, China sooner or later prompted the World Health Organization to issue a public health warning emergency. This is not the first nor will it be the last time that a member of β-coronaviruses (CoVs) is waging a full-scale war against human health. Notwithstanding the fact that pneumonia is the primary symptom of the novel coronavirus (2019nCoV; designated as SARS-CoV-2), the emergence of severe disease mainly due to the injury of nonpulmonary organs at the shadow of coagulopathy leaves no choice, in some cases, rather than a dreadful death. Multiple casual factors such as inflammation, endothelial dysfunction, platelet and complement activation, renin-angiotensin-aldosterone system derangement, and hypoxemia play a major role in the pathogenesis of coagulopathy in coronavirus disease 2019 (COVID-19) patients. Due to the undeniable role of coagulation dysfunction in the initiation of several complications, assessment of coagulation parameters and the platelet count would be beneficial in early diagnosis and also timely prediction of disease severity. Although low-molecular-weight heparin is considered as the first-line of treatment in COVID-19-associated coagulopathy, several possible therapeutic options have also been proposed for better management of the disease. In conclusion, this review would help us to gain insight into the pathogenesis, clinical manifestation, and laboratory findings associated with COVID-19 coagulopathy and would summarize management strategies to alleviate coagulopathy-related complications.     

Age-specific reference intervals for routine biochemical parameters in healthy neonates,infants, and young children in Iran

Age and sex need to be considered in the establishment of reference intervals (RIs), especially in early life when there are dynamic physiological changes. Since data for important biomarkers in healthy neonates and infants are limited, particularly in Iranian populations, we have determined age-specific RIs for 7 laboratory biochemical parameters. This cross-sectional study comprised a total of 344 paediatric participants (males: 158, females: 186) between the ages of 3 days and 30 months (mean age: 12.91 ± 7.15 months). Serum levels of creatinine, urea, uric acid, calcium, phosphate, vitamin D and high-sensitivity C-reactive protein (hs-CRP) were measured using an Alpha classic-AT plus auto-analyser. We determined age-specific RIs using CLSI Ep28-A3 and C28-A3 guidelines. No sex partitioning was required for any of the biomarkers. Age partitioning was required for kidney function tests and phosphate. The serum concentration of urea and creatinine increased with age, while phosphate and uric acid decreased with age. Age partitioning was not required for serum calcium, vitamin D, and hs-CRP, which remained relatively constant throughout the age range. Age-specific RIs for 7 routine biochemical markers were determined to address critical gaps in RIs in early life to help improve clinical interpretation of blood test results in young children, including neonates. Established age partitions demonstrate the biochemical changes that take place during child growth and development. These novel data will ultimately better disease management in the Iranian paediatric population and can be of value to clinical and hospital laboratories with similar populations.     

Toll-like receptors (TLRs): An old family of immune receptors with a new face in cancer pathogenesis

In the dark path of tumorigenesis, the more carefully the cancer biology is studied, the more brilliant answers could be given to the countless questions about its orchestrating derivers. The identification of the correlation between Toll-like receptors (TLRs) and different processes involved in carcinogenesis was one of the single points of blinding light highlighting the interconnection between the immune system and cancer. TLRs are a wide family of single-pass membrane-spanning receptors that have developed through the evolution to recognize the structurally conserved molecules derived from microorganisms or damaged cells. But this is not everything about these receptors as they could orchestrate several downstream signalling pathways leading to the formation or suppression of cancer cells. The present review is tempted to provide a concise schematic about the biology and the characters of TLRs and also summarize the major findings of the regulatory role of TLRs and their associated signalling in the pathogenesis of human cancers.     

Genome-scale metabolic model-based engineering of Escherichia coli enhances recombinant single-chain antibody fragment production

Biotechnology Letters - Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a...     

Mathematical models for mobile network member’s coordination through coverage development-based contract

Flexible Services and Manufacturing Journal - This study examines a mobile phone network in three models: (1) mobile network model: one mobile operator, one mobile phone manufacturer, and one...