Molecular identification of neuraminidase gene mutations in influenza A/H1N1 and A/H3N2 isolates of Mazandaran province, north of Iran.

OBJECTIVES: The neuraminidase (NA) mutations causing resistance to NA inhibitors (NAIs) mostly compromise the fitness of influenza viruses. Considering the importance of these mutations, constant monitoring of the effectiveness of available drugs is critical. This study aimed to identify NA mutations in the influenza A/H1N1 and A/H3N2 subtypes in the samples of Mazandaran, Iran from 2016 to 2020. METHODS: In this cross-sectional study, 20 influenza A/H1N1 and 20 influenza A/H3N2 samples were included in the study. After design of appropriate primers for NA gene, all samples subjected to RT-PCR and electrophoresis. Then the PCR product was sequenced to determine the mutations. RESULTS: In the present study, no oseltamivir resistance-related mutations were detected. Still, NA gene showed variations compared to the vaccine strains. In A/H1N1, a total of 43 mutations were detected. Similarly, in A/H3N2, a total of 66 mutations were observed. In all isolates of H1N1, N200S, N248D and I321V mutations were detected in the antigenic site of NA protein, which can affect vaccine incompatibility and virus escape from the host's immune system. Also, H150R mutation was observed in the NA active site of H3N2, which is the cause of agglutination by NA protein. Also, S245N mutation was identified as a new N-Glycosylation site of H3N2 subtype. CONCLUSIONS: The study of NA gene sequences revealed no oseltamivir resistance mutations. In H1N1 isolates, ca. 97% identities and in the H3N2 subtype, 96% identities were observed compared to reference isolate of 2009, which indicates the importance of constant monitoring of the emergence of the drug resistance mutations.

Opium use and risk of colorectal cancer: a multi-center case-referent study in Iran.

BACKGROUND: Opium use has been associated with an increased risk of cancers of the lung, oesophagus, and pancreas, and it was recently classified by the International Agency for Cancer Research as carcinogenic to humans. It is not clear whether opium also increases the risk of colorectal cancer (CRC). The aim of our study was to assess the association between various metrics of opium use and the risk of CRC. METHODS: This case-referent study from seven provinces in Iran comprised 848 CRC cases and 3215 referents. Data on opium use (duration, amount, frequency) and potential confounders were collected by trained interviewers. Multivariable unconditional logistic regression models were used to measure odds ratios (OR) adjusted for age, gender, province, marital status, family history of CRC-linked cancers, consumption of red meat, fruits and vegetables, body shape, occupational physical activity, and socioeconomic status. RESULTS: Regular opium consumption was not associated with the risk of CRC (OR 0.9, 95% confidence interval, CI: 0.7, 1.2) compared to subjects who never used opium. However, frequent opium use more than twice a day was associated with an increased risk of CRC compared to non-users of opium (OR: 2.0, 95% CI: 1.1, 3.8; p for quadratic trend 0.008). CONCLUSION: There seems to be no overall association between opium use and CRC, but the risk of CRC might be increased among persons who use opium many times a day.

Implementation of an In-House Platform for Rapid Screening of SARS-CoV-2 Genome Variations.

BACKGROUND: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings. METHODS: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021. RESULTS: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants. CONCLUSION: Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.

NSP4 as adjuvant for immunogenicity and design of effective therapeutic HPV16 E6/E7/L1 DNA vaccine in tumor-bearing and healthy C57BL/6 mice.

INTRODUCTION: In humans, approximately 5% of all cancers are attributable to HPV infection. Prophylactic vaccines can inhibit viral migration and persistence. However, further studies are still required to develop such treatments. To achieve this goal, we designed a therapeutic HPV DNA vaccine encoding a construct of E6/E7/L1 and used NSP4 antigen as an adjuvant to assess the efficiency of this construct in generating antigen-specific antitumor immune responses. MATERIALS AND METHODS: Sixty female C57BL/6 mice (6-8 weeks old) were purchased from the Institute Pasteur of Iran. Through a subcutaneous (s.c) injection of a suspension of 100 µl PBS containing 106 TC-1 cells/mouse in the back side, 30 of them became cancerous, while 30 of them were healthy control mice. To amplify E6/E7/L1-pcDNA3 and NSP4-pcDNA3, the competent cells of DH5α and to generate a tumor, TC-1 cell line was used. Mice were then immunized with the HPV DNA vaccine. Cell proliferation was assessed by MTT assay. Finally, cytokine responses (IL-4, IL-12, IFN- γ) were measured in the supernatant of mice spleen cells. RESULT: Mice receiving the NSP4/E6-E7-L1 vaccine had the highest stimulatory index compared to other groups, although it was not statistically significant. Interleukin 4/12 and IFN-γ production were significantly higher in E6-E7-L1 / NSP4 group and E6-E7-L1 group compared to other groups (P < 0.05). Among different groups, E6/E7/L1 + NSP4 group was able to slow down the tumor growth process, but it was not significant (p > 0.05). Among the aforementioned cytokines, IFN-γ and IL-12 are among the cytokines that stimulate the Th1 pathway and IL-4 cytokine stimulates the Th2 pathway and B lymphocytes. CONCLUSION: Our data revealed that the present vaccine can reduce tumor size, and cytokine measurement showed that it stimulates innate and acquired immune responses, thus it can be a therapeutic vaccine in the tumor-bearing mice model.

Mutations in the S gene of hepatitis B virus in three generations of patients with chronic hepatitis B.

Failure to neutralize HBsAg and subsequent escape from the host immune system may be caused by HBsAg mutations, particularly in the "a" determinant, which alters the antigenicity of the protein. The purpose of this study was to examine the frequency of S gene mutations in three generations of HBV cases in northeastern Iran. In this study, 90 patients with chronic HBV were assigned to three groups according to the inclusion criteria. The plasma were utilized to extract viral DNA, and the PCR was applied. Direct sequencing and alignment were performed on the S gene, using reference sequence. The results indicated that all HBV genomes were categorized as the genotype D/ayw2. Among 79 point mutations detected, 36.8% were silent, and 56.2% were missense. In the S region, mutations were observed in 88.9% of CHB subjects studied. In the three-generation group, 21.5% of mutations were in the "a" determinant, and 2.6%, 19.5%, and 87.0% of these mutations were observed in antigenic epitopes of CTLs, CD4+, and B cells, respectively. In addition, 56.7% of mutations occurred at Major Hydrophilic Region. S143L and G145R mutations which the most prevalent in the three-generation (36.7%, 20%), and two-generation (42.5%, 20%) groups, related to the failure of HBsAg detection, vaccine, and immunotherapy escape. The findings showed that most of the mutations were concentrated in the B cell epitope. Most CHB cases from the three-generation, especially grandmothers, had HBV S gene mutations and subsequent amino acid mutations, suggesting that these mutations may be critical for pathogenesis and vaccine evasion.

EBNA1 Upregulates P53-Inhibiting Genes in Burkitt's Lymphoma Cell Line.

Background: Suppression of p53 is an important mechanism in Epstein-Barr virus associate-tumors and described as EBNA1-USP7 which is a key axis in p53 suppression. Thus, in this study, we aimed to evaluate the function of EBNA1 on the expression of p53-inhibiting genes including HDAC-1, MDM2, MDM4, Sirt-3, and PSMD10 and the influence of USP7 inhibition using GNE-6776 on p53 at protein/mRNA level. Methods: The electroporation method was used to transfect the BL28 cell line with EBNA1. Cells with stable EBNA1 expression were selected by Hygromycin B treatment. The expression of seven genes, including PSMD10, HDAC-1, USP7, MDM2, P53, Sirt-3, and MDM4, was evaluated using a real-time PCR assay. For evaluating the effects of USP7 inhibition, the cells were treated with GNE-6776; after 24 hours and 4 days, the cells were collected and again expression of interest genes was evaluated. Results: MDM2 (P=0.028), MDM4 (P=0.028), USP7 (P=0.028), and HDAC1 (P=0.015) all showed significantly higher expression in EBNA1-harboring cells compared to control plasmid transfected cells, while p53 mRNA expression was only marginally downregulated in EBNA1 harboring cells (P=0.685). Four-day after treatment, none of the studied genes was significantly changed. Also, in the first 24-hour after treatment, mRNA expression of p53 was downregulated (P=0.685), but after 4 days it was upregulated (P=0.7) insignificantly. Conclusion: It seems that EBNA1 could strongly upregulate p53-inhibiting genes including HDAC1, MDM2, MDM4, and USP7. Moreover, it appears that the effects of USP7 suppression on p53 at protein/mRNA level depend on the cell nature; however, further research is needed.

A New Insight Into p53-Inhibiting Genes in Epstein­Barr Virus-Associated Gastric Adenocarcinoma

Background: The p53 mutation is uncommon in Epstein­Barr virus-linked gastric carcinoma, but its suppression occurs through mechanisms such as ubiquitin specific peptidase 7 (USP7) inhibitions via Epstein­Barr virus nuclear antigen-1 (EBNA1) activity. This study aimed to evaluate the effect of EBNA1 on p53-inhibiting gene expression and the impact of USP7 inhibition on p53 suppression. Methods: MKN-45 cells were transfected with the EBNA1 plasmid. A stable EBNA1 expression cell line was developed through selection based on hygromycin B resistance. Murine double minute (MDM)4, MDM2, sirtuin (SIRT)3, histone deacetylase (HDAC)1, proteasome 26S subunit, Non-ATPase (PSMD)10, USP7, and p53 expression were checked using real-time PCR. Also, cells containing EBNA1 or control plasmid were treated with GNE-6776, and the expression of the interested genes and cell survival were assessed. Results: MDM4, MDM2, and PSMD10 were significantly upregulated in the MKN-45 cell line following EBNA1 transfection. Morphological changes were observed in the cells harboring EBNA1 after 20 days. In the control cells, USP7 inhibition significantly upregulated the HDAC1, PSMD10, MDM4, and MDM2 genes after 24 h, but downregulated these genes after four days. In the EBNA1-harboring cells, MDM2, MDM4, and PSMD10 genes were significantly upregulated after 24 h, and this effect was sustained for all genes except for MDM4, even after four days. Furthermore, USP7 inhibition induced apoptosis in both cell groups. Conclusion: EBNA1 enhances the expression of p53-inhibiting genes. Two events­p53 protein overexpression and apoptosis activation­followed the suppression of the USP7 protein and provided evidence for its possible function. The significance of the EBNA1-USP7 interaction in p53 suppression warrants additional investigation and possibly reconsideration.

Association of HLADQ-B1 polymorphisms in three generations of chronic hepatitis B patients.

The presence of polymorphisms in the human leukocyte antigen (HLA)-DQB1 gene, along with its expression, has been demonstrated to be correlated with spontaneous clearance and susceptibility to HBV infection. The present study aimed to evaluate the possible role of genetic polymorphisms in HLA-DQB1 in three generations of patients with chronic hepatitis B (CHB). Based on the inclusion criteria, 90 CHB patients, 18 individuals recovered from HBV infection, and 40 healthy subjects were chosen. The DNA contents of the whole blood samples were extracted in order to perform HLA-DQB1 typing by the PCR technique. Besides whole blood samples, sera were applied to measure liver function tests (LFTs), as well as the titers of anti-HDV and anti-HCV. Also, in all CHB patients were measured liver stiffness (LSM) by Fibro Scan. The results of HLA-DQB1 polymorphisms (rs2856718 and rs7453920) demonstrated that the majority of polymorphisms in CHB patients were HLA-DQB1*03, HLA-DQB1*05, HLA-DQB1*04:01 and HLA-DQB1*03:01 that associated with HBV persistence and chronicity. Among the patients who showed these polymorphisms, the mean±SD, LSM was 4±1.57 KPa and most of them, F grade was reported as F2, which was a sign of disease progression towards chronicity. HLA polymorphisms imputation revealed that HLA-DQB1*06:04 (3.4%, P-Value= 0.2) was detected only in healthy subjects as protective polymorphism, while the allele HLA-DQB1*03:03 was reported in both healthy subjects (P-Value= 0.06) and recovered patients (P-Value= 0.1) as suppressor of CHB formation. The allele HLA-DQB1*05:02 was found in both healthy subjects (3.4%) and CHB patients (4.5%) which was associated with risk to liver cirrhosis (P-Value= 0, OR: 0.002 0.95CI: 0.000-0.15). HLA polymorphism analysis indicated that 17.39% of patients who were seropositive for anti-HCV carried the HLA-DQB1*03:01. HBV resistance or infection risk could be assessed by DBQ1 typing. The existence of polymorphisms in HLA gene could influence the clearance (HLA-DQB1*03:03) or susceptibility and persistence of infection (HLA-DQB1*03, HLA-DQB1*05, HLA-DQB1*04:01 and HLA-DQB1*03:01). These results have the potential to improve personalized therapy and prognosis for HBV infection.

Opium use and risk of lung cancer: A multicenter case-control study in Iran.

Opium use was recently classified as a human carcinogen for lung cancer by the International Agency for Research on Cancer. We conducted a large, multicenter case-control study evaluating the association between opium use and the risk of lung cancer. We recruited 627 cases and 3477 controls from May 2017 to July 2020. We used unconditional logistic regression analyses to estimate the odds ratios (OR) and 95% confidence intervals (CI) and measured the association between opium use and the risk of lung cancer. The ORs were adjusted for the residential place, age, gender, socioeconomic status, cigarettes, and water pipe smoking. We found a 3.6-fold risk of lung cancer for regular opium users compared to never users (95% CI: 2.9, 4.6). There was a strong dose-response association between a cumulative count of opium use and lung cancer risk. The OR for regular opium use was higher for small cell carcinoma than in other histology (8.3, 95% CI: 4.8, 14.4). The OR of developing lung cancer among opium users was higher in females (7.4, 95% CI: 3.8, 14.5) than in males (3.3, 95% CI: 2.6, 4.2). The OR for users of both opium and tobacco was 13.4 (95% CI: 10.2, 17.7) compared to nonusers of anything. The risk of developing lung cancer is higher in regular opium users, and these results strengthen the conclusions on the carcinogenicity of opium. The association is stronger for small cell carcinoma cases than in other histology.

EBV and HPV Infections in Colorectal Cancer and Their Effect on P53 and P16 Protein Expression.

Viral infections contribute to 15-20% of newly diagnosed cancers worldwide. There is evidence of a possible etiological role of Epstein-Barr virus (EBV) and high-risk human papillomaviruses (HR-HPVs) in colorectal carcinoma (CRC). Loss of p53 and p16 function has been found in many cancers and this may occur in many different ways, including gene mutation or interaction with viral oncoproteins. This study aimed to evaluate the presence of EBV and HPV in CRC patients in northern Iran and to assess p53 and p16 protein expression related to these viral infections. Real-time PCR was used to amplify the DNA sequences of these viruses in 55 colorectal tumoral tissues, along with their corresponding non-tumoral adjacent tissues. Additionally, immunohistochemistry (IHC) was utilized to determine p53 and p16 protein expression. EBV DNA was detected in 49.1% of CRC tissues. Furthermore, HPV DNA was present in 7.3% of CRC tissues. Notably, the prevalence of EBV infection in tumoral tissues was significantly higher than in non-tumoral tissues (P=0.001). The EBV DNA polymerase catalytic subunit (BALF5) copy number in tumoral tissues was higher than in non-tumoral tissues and this difference was statistically significant (P=0.008). P53 was positive in 21/26 (80.8%) EBV-positive and in 11/25 (44%) EBV-negative samples and this difference was significant (P=0.007). P16 was positive in 13/26 (50%) EBV-positive and in 14/25 (58.3%) EBV-negative samples (P= 0.668). Our findings suggest that EBV infection can increase the risk of CRC. In addition, EBV seems to stabilize p53 in EBV-positive CRC which needs further research. No significant correlation was detected between EBV infection and p16 expression. Also, we could not find a causal relationship between HPV infection and CRC in the study population.

Live-attenuated poliovirus-induced extrinsic apoptosis through Caspase 8 within breast cancer cell lines expressing CD155.

INTRODUCTION: Breast cancer is one of the most common cancers among women in the world. Different therapeutic strategies such as radiotherapy, chemotherapy and surgery have been used either individually or in combination. Oncolytic virotherapy is a rising treatment methodology, which utilizes replicating viruses to eliminate tumor cells. The aim of this study was to investigate the oncolytic activity of live-attenuated poliovirus in breast cancer cell lines. MATERIALS AND METHODS: The CD155 expression level in two human breast cancer cell lines and a normal breast cell line were evaluated using real-time PCR and flow cytometry. Virus titration was assessed by TCID50. The cytotoxicity of poliovirus on cell line and apoptosis response was investigated by MTT and Caspase 8 and Caspase 9 ELISA kits, respectively. RESULTS: This study showed that CD155 gene was expressed significantly (p = 0.001) higher in both human breast cancer cell lines compared to the normal cell line. The protein expression level of CD155 was 98.1%, 96.7%, in MDA_MB231 and MCF_7 cell lines, respectively, whereas the CD155 expression level was 1.3% in MCF_10A. The cytopathic effect of poliovirus in breast cancer cell lines was significantly higher than normal cells (p < 0.05). Extrinsic apoptosis response was more effective than intrinsic apoptosis in both breast cancer cell lines (p < 0.05). CONCLUSION: In summary, administration of live-attenuated poliovirus can be a promising treatment to breast cancer. However, in vitro and in vivo studies will be required to evaluate the safety of this strategy.

Anti-inflammatory effects of curcumin-loaded niosomes on respiratory syncytial virus infection in a mice model.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in paediatrics. While antivirals are apparent candidates to treat RSV-induced diseases, they have not yet met expectations and have remained in infancy. There is growing evidence to suggest that modulating the exacerbated inflammation during RSV infection can improve disease outcome. Curcumin-loaded niosomes have anti-inflammatory effects against RSV-induced respiratory disease by reducing immune cells' infiltration and inflammatory cytokines' production. This study evaluated the effects of curcumin-loaded niosomes on RSV-induced immunopathology in a mice model. Curcumin-loaded niosomes were prepared using the thin-film hydration method and characterized in vitro. Female Balb/c mice were infected by RSV-A2 and treated daily with curcumin-loaded niosomes. The potential anti-inflammatory effects of curcumin-loaded niosomes were evaluated on day 5 after infection. Using curcumin-loaded niosomes decreased immune cell influx and the inflammatory mediators (MIP-1α, TNF-α and IFN-γ) production in the lung, resulting in alleviated lung pathology following RSV infection. These findings indicate that curcumin-loaded niosomes have anti-inflammatory potential and could be a promising candidate to alleviate RSV-associated immunopathology.

Opium use and risk of bladder cancer: a multi-centre case-referent study in Iran.

BACKGROUND: Bladder cancer (BC) is the 10th most common type of cancer worldwide and the fourth most common type of cancer in Iran. Opium use is considered as one of the risk factors for BC. We aim to assess the association between various parameters of opium use, which in Iran is mainly ingested or smoked in various forms, and the risk of BC. METHOD: In this multi-centre case-referent study in Iran, 717 BC cases and 3477 referents were recruited to the study from May 2017 until July 2020. Detailed histories of opium use (duration, amount, frequency) and potential confounders were collected by trained interviewers. Multivariable unconditional logistic regression models were used to measure adjusted odds ratio (OR) and 95% confidence intervals (CI). The ORs were adjusted for age, gender, place of residence and pack-years of cigarette smoking. RESULTS: Regular opium consumption was associated with an increased risk of BC (OR 3.5, 95% CI: 2.8, 4.3) compared with subjects who never used opium. Compared with continuous users, the risk decreased to one-third for those who stopped opium more than 10 years ago. The adjusted OR for those who used both crude opium (teriak) and opium juice was 7.4 (95% CI: 4.1, 13.3). There was a joint effect of opium and tobacco (OR for users of both opium and tobacco 7.7, 95% CI: 6.0, 9.7). CONCLUSIONS: Regular opium use is associated with an approximately 4-fold risk for BC. The OR decreases along with the increasing time since stopping opium use.

Disease Waves of SARS-CoV-2 in Iran Closely Mirror Global Pandemic Trends.

BACKGROUND: Complete SARS-CoV-2 genome sequencing in the early phase of the outbreak in Iran showed two independent viral entries. Subsequently, as part of a genome surveillance project, we aimed to characterize the genetic diversity of SARS-CoV-2 in Iran over one year after emerging. METHODS: We provided 319 SARS-CoV-2 whole-genome sequences used to monitor circulating lineages in March 2020-May 2021 time interval. RESULTS: The temporal dynamics of major SARS-CoV-2 clades/lineages circulating in Iran is comparable to the global perspective and represent the 19A clade (B.4) dominating the first disease wave, followed by 20A (B.1.36), 20B (B.1.1.413), 20I (B.1.1.7), leading the second, third and fourth waves, respectively. We observed a mixture of circulating B.1.36, B.1.1.413, B.1.1.7 lineages in winter 2021, paralleled in a fading manner for B.1.36/B.1.1.413 and a growing rise for B.1.1.7, prompting the fourth outbreak. Entry of the Delta variant, leading to the fifth disease wave in summer 2021, was detected in April 2021. This study highlights three lineages as hallmarks of the SARS-CoV-2 outbreak in Iran; B4, dominating early periods of the epidemic, B.1.1.413 (B.1.1 with the combination of [D138Y-S477N-D614G] spike mutations) as a characterizing lineage in Iran, and the co-occurrence of [I100T-L699I] spike mutations in half of B.1.1.7 sequences mediating the fourth peak. It also designates the renowned combination of G and GR clades' mutations as the top recurrent mutations. CONCLUSION: In brief, we provided a real-time and comprehensive picture of the SARS-CoV-2 genetic diversity in Iran and shed light on the SARS-CoV-2 transmission and circulation on the regional scale.

SARS-CoV-2 outbreak in Iran: The dynamics of the epidemic and evidence on two independent introductions.

The SARS-CoV-2 virus has been rapidly spreading globally since December 2019, triggering a pandemic, soon after its emergence. While Iran was among the first countries confronted with rapid spread of virus in February 2020, no real-time SARS-CoV-2 whole-genome tracking in early phase of outbreak was performed in the country. To address this issue, we provided 50 whole-genome sequences of viral isolates ascertained from different geographical locations in Iran during March-July 2020. The corresponding analysis on origins, transmission dynamics and genetic diversity of SARS-CoV-2 virus, represented at least two introductions of the virus into the country, constructing two major clusters defined as B.4 and B.1*. The first entry of the virus might have occurred around very late 2019/early 2020, as suggested by the time to the most recent common ancestor, followed by a rapid community transmission that led to dominancy of B.4 lineage in early epidemic till the end of June. Gradually, reduction in dominancy of B.4 occurred possibly as a result of other entries of the virus, followed by surge of B.1* lineages, as of mid-May. Remarkably, variation tracking of the virus indicated the increase in frequency of D614G mutation, along with B.1* lineages, which showed continuity till October 2020. The increase in frequency of D614G mutation and B.1* lineages from mid-May onwards predicts a rapid viral transmission that may push the country into a critical health situation followed by a considerable change in composition of viral lineages circulating in the country.

Novel X gene point mutations in chronic hepatitis B and HBV related cirrhotic patients.

INTRODUCTION: HBx is a multifunctional modulator viral protein with key roles in various biological processes such as signal transduction, transcription, proliferation, and cell apoptosis. Also, HBx has an important role in the progression of cirrhosis and hepatocellular carcinoma (HCC). This study aimed to determine mutations in X gene, enhancer II (EnhII), and basal core promoter (BCP) of genotype D of Hepatitis B Virus (HBV) in cirrhotic and chronic HBV patients. MATERIAL AND METHODS: This cross-sectional study was performed on 68 cases with chronic HBV (cHBV) and 50 cases with HBV related cirrhosis. Serum samples were obtained for genomic DNA extraction. Semi-nested PCR was used to amplify the HBx region. Point mutations in the HBx region were detected by sequencing. RESULT: Novel mutations were detected, including C1491G, C1500T, G1613T, and G1658T in the N-terminal of the X gene. The frequency of C1481T/G1479A, T1498C, C1500T, G1512A, A1635T, C1678T, A1727T, and A1762T/ G1764A/ C1773T was significantly higher in cirrhotic patients compared to chronically HBV infected ones. A higher rate of A1635T, C1678T, A1727T, A1762T, G1764A, and C1773T was observed in cirrhotic patients. CONCLUSION: Our findings showed that the frequency of mutations in the basal-core promoter, enhancer II, and regulatory region of the HBx gene was more seen in cirrhotic patients than in chronic HBV cases. Novel mutations were detected in the HBx gene, causing amino acid substitutions; however, the clinical impact of these novel mutations is yet to be cleared.

Molecular detection of HHV-1, HHV-2, HHV-5 and HBV in semen of fertile and infertile men by multiplex PCR method.

Background and Objectives: A great diversity of factors including viruses such as human herpes virus 1&2 (HHV-1&2), human herpes virus 5 (HHV-5), and hepatitis B virus (HBV) play key roles in sterility and it is worth noting that male infertility accounts for nearly 50% of barrenness, globally. In this regard, we evaluated the prevalence of the aforementioned viruses in semen specimens of two distinct groups of men referred to Novin Infertility Center in Mashhad, Iran. Materials and Methods: In this cross-sectional study, 300 semen samples were collected from 150 infertile and 150 fertile men. Subsequently, genomic DNA was extracted before performing multiplex polymerase chain reaction (PCR). Eventually, the results were analyzed via SPSS Statistics V.16.0. Results: Out of 300 specimens, 183 (61.1%) were positive at least for one of the forenamed viruses; genome detection of HHV-1&2, HHV-5, and HBV were 27%, 18%, 36.66%, and 4%, respectively. Conclusion: The current study found no correlation between infertility and HBV, HHV-5, and HHV-1&2, which may have to do with factors like sample size, the geographical distribution of the viruses, and the lifestyle (sexual behavior) of the participants. These results emphasize the implementation of such studies on a broader scale to determine the exact factors involved in infertility.

Functional variation (Q63R) in the cannabinoid CB2 receptor may affect the severity of COVID-19: a human study and molecular docking.

Evidence supports a role of host genetic diversity in the clinical course of coronavirus disease 2019 (COVID-19). Variation in the cannabinoid CB2 receptor gene (CNR2) could affect the regulatory action of endocannabinoids on the immune system, resulting in an increased risk of various inflammatory diseases. The present study investigated the relationship between the CNR2-Q63R variant and COVID-19 severity. A total of 200 Iranian COVID-19 patients were enrolled in the study and genotyped using a TaqMan assay. The co-dominant, dominant, recessive, over-dominant, and additive inheritance models were analyzed using SNPStats software. In silico molecular docking was also performed to simulate the effects of the Q63R variation on CB2 binding with a ligand and with the G-protein. A significant difference in the Q63R allele and genotype distribution was found between expired and discharged COVID-19 patients in co-dominant, recessive, and additive inheritance models. The molecular docking results showed that the predicted structure of mutant CB2 (63R type) could not bind to the G-protein in the correct position. The data indicated that the Q63R variation in the CNR2 gene may affect the severity of COVID-19. Identification of genes related to susceptibility and severity of COVID-19 may lead to specific targets for drug repurposing or development.

Correction: Sensitivity of self-reported opioid use in case-control studies: Healthy individuals versus hospitalized patients.

[This corrects the article DOI: 10.1371/journal.pone.0183017.].

Identification of differentially expressed microRNAs in primary esophageal achalasia by next-generation sequencing.

Molecular knowledge regarding the primary esophageal achalasia is essential for the early diagnosis and treatment of this neurodegenerative motility disorder. Therefore, there is a need to find the main microRNAs (miRNAs) contributing to the mechanisms of achalasia. This study was conducted to determine some patterns of deregulated miRNAs in achalasia. This case-control study was performed on 52 patients with achalasia and 50 nonachalasia controls. The miRNA expression profiling was conducted on the esophageal tissue samples using the next-generation sequencing (NGS). Differential expression of miRNAs was analyzed by the edgeR software. The selected dysregulated miRNAs were additionally confirmed using the quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fifteen miRNAs were identified that were significantly altered in the tissues of the patients with achalasia. Among them, three miRNAs including miR-133a-5p, miR-143-3p, and miR-6507-5p were upregulated. Also, six miRNAs including miR-215-5p, miR-216a-5p, miR-216b-5p, miR-217, miR-7641 and miR-194-5p were downregulated significantly. The predicted targets for the dysregulated miRNAs showed significant disease-associated pathways like neuronal cell apoptosis, neuromuscular balance, nerve growth factor signaling, and immune response regulation. Further analysis using qRT-PCR showed significant down-regulation of hsa-miR-217 (p-value = 0.004) in achalasia tissue. Our results may serve as a basis for more future functional studies to investigate the role of candidate miRNAs in the etiology of achalasia and their application in the diagnosis and probably treatment of the disease.