ABSTRACT
Iran was among countries which was hard hit at the early stage of the coronavirus disease 2019 (COVID-19) pandemic and dealt with the second wave of the pandemic in May and June 2020; however, there are a very limited number of complete genome sequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Iran. In this study, complete genome sequences of the virus in the samples obtained from three patients in Alborz province in May and June 2020 were generated and analyzed using bioinformatic methods. The sequenced genomes were positioned in a cluster with B.4 lineage along with the sequences from other countries namely, United Arab Emirates and Oman. There were seven single nucleotide variations (SNVs) in common in all samples and only one of the sequenced genomes showed the D614G amino acid substitution. Three SNVs, 1397 G > A, 28688T > C, 29742 G > T, which had already been reported in February, were found with high frequency in all the sequenced genomes in this study, implying that viral diversity reflected in the early stages of viral transmission in Iran were established in the second wave. Considering the importance of molecular epidemiology in response to ongoing pandemic, there is an urgent need for more complete genome sequencing and comprehensive analyses to gain insight into the transmission, adaptation and evolution of the virus in Iran.
ABSTRACT
OBJECTIVE: One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells. METHODS: The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot. RESULTS: Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis. CONCLUSIONS: In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19+ while keeping the normal cells intact.
