Autophagy induction plays time-dependent role in viral load of HCV infected Huh7.5 cell line.

Autophagy provides an initial membranous platform for incoming hepatitis C virus (HCV) RNA translation and immune evasion. Once HCV replication is established, this infrastructure will be unnecessary for translation of HCV RNA progeny. So, the autophagy plays key role in the replication and immune pathogenesis of HCV virus. The aim of this study was to study the effect of autophagy induction in Huh7.5 cell on virus titer. The Huh7.5 cell was transfected with recombinant pcDNA-Beclin1. The autophagy induction was evaluated via microtubule associated protein 1 light chain 3 staining as autophagy formation marker using flow cytometry. The HCV (JFH1) was inoculated 12-h post-transfection. Next, to evaluate the viral load, viral RNA was extracted after 24 and 48 h and virus titer was calculated using real-time PCR. The result of the current study shows that the induction of autophagy before virus infection was able to enhance virus yield from 4 × 103 copies/mL to 1 × 104 copies/mL at 24-h post-infection, but reduced viral load after 48 h up to 6 × 103 copies/mL. The study of cross-talk between autophagy and HCV may bring new hope for human intervention and treatment of HCV. Also, it opens new avenue to improve virus cultivation in cell culture and understanding HCV and host cell responses. © 2018 IUBMB Life, 71(1):41-44, 2019.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production.

OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Intranasal administration of cold-adapted live-attenuated SARS-CoV-2 candidate vaccine confers protection against SARS-CoV-2.

With the COVID-19 pandemic globally, the ongoing threat of new challenges of mucosal infections was once again reminded human beings. Hence, access to the next-generation vaccine to elicit mucosal immunity is required to reduce virus shedding. SARS-CoV-2 retains a unique polybasic cleavage motif in its spike protein, recognized by the host furin protease. The proteolytic furin cleavage site at the junction of S1/S2 glycoprotein plays a key role in the pathogenesis of SARS-CoV-2. Here, we examined the protective immunity of a double-deleted PRRA/GTNGTKR motifs cold-adapted live-attenuated candidate vaccines as a called "KaraVac." using a hamster animal model of infected attenuated SARS-CoV-2. The KaraVac vaccinated hamsters were challenged against the wild-type (WT) SARS-CoV-2. No apparent bodyweight loss and histopathological lesions were observed in the hamsters. The establishment of sterilizing immunity was induced via stimulating a robust neutralizing antibody (NAb) response in a hamster model. Consequently, deletions in the spike sequence and inoculation into hamsters provide resistance to the subsequent challenge with WT SARS-CoV-2. We have suggested that deletion of the furin cleavage site and GTNGTKR motifs in the spike sequence attenuates the virus from the parental strain and can be used as a potent immunogen.

Autophagy Modulation and Cancer Combination Therapy: A Smart Approach in Cancer Therapy.

The autophagy pathway is the process whereby cells keep cellular homeostasis and respond to stress via recycling their damaged cellular proteins, organelles, and other cellular components. In the context of cancer, autophagy is a dual-edge sword pro- and anti-tumorigenic role depending on the oncogenic context and stage of tumorigenesis. Cancer cells have a higher dependency on autophagy compared with normal cells because of cellular damages and high demands for energy. The carbon, nitrogen, and molecular oxygen are building blocks for highly proliferative cancer cells which extremely depend on glutaminolysis and aerobic glycolysis; when a cancer cell is restricted to glucose and glutamine, it initiates to activate a stress response pathway using autophagy. Oncogenic tyrosine kinases (OncTKs) and receptor tyrosine kinases (RTKs) activation result in autophagy modulation through activation of the PI3K/AKT/mTORC1 and RAS/MAPK signaling pathways. Targeted inhibition of tyrosine kinases (TKs) and RTKs have recently been considered as cancer therapy but drug resistance and cancer relapse continue to be a major limitation of tyrosine kinase inhibitors (TKIs). Manipulation of autophagy pathway along with TKIs may be a promising strategy to circumvent unknown existing drug-resistance mechanisms that may emerge in a treated patient. In this way, clinical trials are ongoing to modulate autophagy to treat cancer. This review aims to summarize the combination therapy of autophagy affecting compounds with anticancer drugs which target cell signaling pathways, metabolism mechanisms, and epigenetics modification to improve therapeutic efficacy against cancers.

The Prioritization of Lean Techniques in Emergency Departments Using VIKOR and SAW Approaches.

BACKGROUND: Considering various researches were carried out to implement Lean techniques in healthcare centers, this study has tried to investigate how lean principles could be prioritized in the Emergency Department (ED) by comparing physicians and nurses viewpoints. METHODOLOGY: In the first stage, relevant Lean techniques and several criteria to evaluate the ED performance were selected by reviewing the literature. Then, weight factors for each criterion were calculated using the Entropy method, and Lean techniques were compared and ranked via a questionnaire by which the physicians' and nurses' opinions were obtained separately. In the last stage, the final ranking of Lean techniques was done using VIKOR and SAW methods as two powerful means of Multi-Criteria Decision-Making (MCDM). RESULTS: Theory of Constraints (TOC) was selected as the most appropriate principle from the physicians' viewpoints by both decision-making methods. However, according to the nurses' opinions, Jiduka was the best approach by the VIKOR method, while with the SAW method, 5S was chosen as the most practical Lean technique. CONCLUSION: This study has illustrated that although all Lean techniques are useable for ED, these techniques' prioritization has a key role in choosing the more suitable Lean approach. Moreover, it provides a chance for the emergency wards to keep down different costs and improve staff and patient satisfaction and the quality of treatment simultaneously.

Comparing 2 Appointment Scheduling Policies Using Discrete-Event Simulation.

BACKGROUND AND OBJECTIVES: Two of the most important policies for dealing with the negative effects of high rates of no-show patients and appointment cancellations include double-booking and walk-in admission policies. This study aimed to compare these policies to identify their differences and their effects as well as the best circumstances for using each one. METHODS: The main approach used in this study was discrete-event simulation using the Arena software application. Moreover, the average waiting time (considering patients' lateness) and the number of missed patients (considering no-show and cancelled patients) were accounted for in the performance evaluation criteria for both of the selected policies. RESULTS: When the patients' arrival rate was high, the double-booking system resulted in higher productivity, while when it was low, the walk-in admission policy was the best policy for patient admission. The successful appointment rates of the current system, the walk-in admission system, and the double-booking system were 61.18%, 89.45%, and 93.24%, respectively. CONCLUSIONS: Although both double-booking and walk-in policies reduced the negative effects of cancelled and no-show patients, they had significantly different results in different situations. In general, there is no best system for appointment scheduling, and the choice of the superior system depends on the demand rate and its fluctuations.

Reconstruction of H3N2 influenza virus based virosome in-vitro.

BACKGROUND AND OBJECTIVES: Virosomes are Virus Like Particles (VLP) assembled in-vitro. Influenza virosomes maintain the cell binding and membrane fusion activity of the wild type virus but are devoid of viral genetic material or internal proteins. Influenza virosomes mimic the natural antigen presentation route of the influenza virus. METHODS: Virosomes were prepared by membrane solubilization and reconstitution. Briefly, the Madine-Darby Canine kidney (MDCK) cell line was cultivated on microcarrier beads inoculated with influenza virus strain A/X-47 (H3N2). The culture medium was harvested and clarified. Subsequently, virus was concentrated and purified by ultrafiltration and ultracentrifugation. The purified viral membrane was dissolved by adding 375 µl of 200 mM 1, 2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) in HEPES-buffered saline (HBS). Nucleocapsid was removed by ultracentrifugation. The supernatant consisting of phospholipids and glycoproteins of the influenza virus was reconstituted by removal of DCPC using overnight dialysis against Hank's Buffered Saline (HBS) solution at 4°C. After dialysis, crude virosome preparation was layered over a discontinuous sucrose gradient in order to separate non-incorporated material from the reconstituted virus membranes. RESULTS: The virosome harvested from the boundary of the two sucrose layers successfully was identified by the Hemagglutination assay and western blotting. CONCLUSION: Use of a dialyzable short-chain phospholipid (DCPC) is an efficient procedure for solubilization and reconstitution of influenza virus virosomes and has not caused structural changes in a major envelope glycoprotein (hemagglutinin protein) on the surface of virosome.