CRISPR/Cas9 Ablated BCL11A Unveils the Genes with Possible Role of Globin Switching.

Purpose: Fetal hemoglobin (HbF) upregulation is a mitigating factor in ß-hemoglobinopathies therapy like ß-thalassemia and sickle cell diseases. Finding molecular mechanisms and the key regulators responsible for globin switching could be helpful to develop effective ways to HbF upregulation. In our prior in silico report, we identified a few factors that are likely to be responsible for globin switching. The goal of this study is to experimentally validate the factors. Methods: We established K562 cell line with BCL11A knock down leading to increase in HBG1/2 using CRISPR/Cas9 system. Then, using quantitative polymerase chain reaction (qPCR), we determined the expression level of the factors which were previously identified in our prior in silico study. Results: our analysis showed that BCL11A was substantially knocked down, resulting in the upregulation of HBG1/2 in the BCL11A-ablated K562 cells using CRISPR/Cas9 system. Additionally, the experimental data acquired in this study validated our prior bioinformatics findings about three potentially responsible genes for globin switching, namely HIST1H2Bl, TRIM58, and Al133243.2. Conclusion: BCL11A is a promising candidate for the treatment of ß-hemoglobinopathies, with high HbF reactivation. In addition, HIST1H2BL, TRIM58 and Al133243.2 are likely to be involved in the mechanism of hemoglobin switching. To further validate the selected genes, more experimental in vivo and in vitro studies are required.

Promoter Methylation of Two HOXA9 and NISCH Genes in Opium Users.

Background: Opiate abuse has been critically increased in the world, especially in Iran. Owing to the association of opiate use with multiple human cancers and neurological disorders, seeking for genetic and epigenetic effects of opium can pave the way for early diagnosis of major health defects in addicted users. Accordingly, the present study aimed to determine the methylation status of the promoter of two genes, which are actively involved in neurodevelopment and cancer evolution. Methods: DNA was isolated from peripheral blood of 28 opium abusers and 19 healthy controls and then subjected to sonication. Sonicated DNAs undergone methylated DNA immunoprecipitation-real time polymerase chain reaction (MeDIP-Real Time PCR) using specific primer pairs designed for HOXA9 and NISCH genes. Obtained data were analyzed using SPSS software. Findings: HOXA9 and NISCH genes were found to be significantly methylated in addicted users compared to controls (P<0.001) which was significantly associated with the mean of the age regarding HOXA9 gene (P=0.002). Neither opium amount nor duration or route of using was associated with the methylation status of HOXA9 or NISCH genes. Conclusion: Hypermethylation of HOXA9 and NISCH genes as tumor suppressor in opium-addicted individuals can be considered as confirmatory evidence for carcinogenesis of opium. Further studies are required to figure out the role of epigenetic alterations in cancer evolution among opium users.

Identification of Potential Key Genes Linked to Gender Differences in Bladder Cancer Based on Gene Expression Omnibus (GEO) Database.

Background: Growing evidence strongly indicates pivotal roles of gender differences in the occurrence and survival rate of patients with bladder cancer, with a higher incidence in males and poorer prognosis in females. Nevertheless, the molecular basis underlying gender-specific differences in bladder cancer remains unknown. The current study has tried to detect key genes contributing to gender differences in bladder cancer patients. Materials and Methods: The gene expression profile of GSE13507 was firstly obtained from the Gene Expression Omnibus (GEO) database. Further, differentially expressed genes (DEGs) were screened between males and females using R software. Protein-protein interactive (PPI) network analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Kaplan-Meier survival analyses were also performed. Results: We detected six hub genes contributing to gender differences in bladder cancer patients, containing IGF2, CCL5, ASPM, CDC20, BUB1B, and CCNB1. Our analyses demonstrated that CCNB1 and BUB1B were upregulated in tumor tissues of female subjects with bladder cancer. Other genes, such as IGF2 and CCL5, were associated with a poor outcome in male patients with bladder cancer. Additionally, three signaling pathways (focal adhesion, rheumatoid arthritis, and human T-cell leukemia virus infection) were identified to be differentially downregulated in bladder cancer versus normal samples in both genders. Conclusion: Our findings suggested that gender differences may modulate the expression of key genes that contributed to bladder cancer occurrence and prognosis.

Evaluation of IGF2, KRT14, and KRT20 as Urinary Biomarkers in Patients with Bladder Cancer.

Background: Many researchers have tried to identify bladder cancer biomarkers to reduce the need for cystoscopy. The aim of this study was to identify and measure appropriate transcripts in patient urine to develop a non-invasive screening test. Methods: From February 2020 to May 2022, 49 samples were obtained from Velayat Hospital, Qazvin University of Medical Sciences, Qazvin, Iran. Twenty-two samples were obtained from bladder cancer patients and 27 from bladder cancer-free subjects. RNA was extracted from participant samples, quantitative RT-PCR was performed, and TNP plots were used to assess IGF2 (NCBI Gene ID: 3481), KRT14 (NCBI Gene ID: 3861) and KRT20 (NCBI Gene ID: 54474) expression. For UCSC Xena analysis, Dataset ID: TCGA-BLCA was used to compare transitional cell carcinoma (TCC) and normal samples for survival rates. Results: IGF and KRT14 were more greatly expressed in patient urine samples than in those of the normal group. However, KRT20 expression did not significantly differ between the two groups. IGF2 had 45.45 and 88.89% sensitivity and specificity, respectively, for detecting TCC in urine samples while KRT14 had 59 and 88.89% sensitivity and specificity, respectively. Also, these results infer that overexpression of IGF would be prognosticators of poor TCC outcomes. Conclusion: Our study showed that IGF2 and KRT14 are overexpressed in bladder cancer patient urine, and IGF2 could be a potential biomarker for poor prognoses in TCC.

Roles of non-coding RNAs in cell death pathways involved in the treatment of resistance and recurrence of cancer.

Considering the burden of cancer, a number of methods have been applied to control or stop it. However, because of drug resistance or cancer recurrence, these treatments usually face failure. Combination of modulation of expression of non-coding RNAs (ncRNAs) with other treatments can increase treatment-sensitivity of tumors but these approaches still face some challenges. Gathering information in this field is a prerequisite to find more efficient cures for cancer. Cancer cells use ncRNAs to enhance uncontrolled proliferation originated from inactivation of cell death routs. In this review article, the main routes of cell death and involved ncRNAs in these routes are discussed. Moreover, extant information in the role of different ncRNAs on cell death pathways involved in the treatment resistance and cancer recurrence is summarized.

Using chitosan-stabilized, hyaluronic acid-modified selenium nanoparticles to deliver CD44-targeted PLK1 siRNAs for treating bladder cancer.

Aims: Achieving an effective biocompatible system for siRNAs delivery to the tumor site remains a significant challenge. Materials & methods: Selenium nanoparticles (SeNPs) modified by chitosan (CS) and hyaluronic acid (HA) were fabricated for PLK1 siRNAs (siPLK1) delivery to the bladder cancer cells. The HA-CS-SeNP@siPLK1 efficacy was evaluated using in vitro and in vivo models. Results: HA-CS-SeNP@siPLK1 was selectively internalized into T24 cells through clathrin-mediated endocytosis. Treatment with HA-CS-SeNP@siPLK1 successfully silenced the PLK1 gene, inhibited cell proliferation and induced cell cycle arrest in vitro. HA-CS-SeNP@siPLK1 could also inhibit tumor growth in vivo without causing systemic toxicity. Conclusion: Our results suggest that HA-CS-SeNPs may provide a good vehicle for delivering siPLK1 to the bladder tumor site.

siRNAs are small biomolecules shown as novel insights in cancer gene therapy because of their capability to silence target genes. However, achieving an effective biocompatible system for siRNA delivery to the tumor site remains a significant challenge. This work aimed to develop a nanoparticle-based delivery system consisting of selenium nanoparticles modified by chitosan and hyaluronic acid to sustain the release of siRNAs to bladder cancer cells. The results of this study demonstrated that this nanosystem successfully silenced the PLK1 gene and reduced the proliferation in vitro and in vivo. These findings suggest that hyaluronic acid-chitosan-selenium nanoparticles may open a new insight for targeted gene therapy for bladder cancer.

Identification of TSGA10 and GGNBP2 splicing variants in 5' untranslated region with distinct expression profiles in brain tumor samples.

Introduction: Brain tumors (BTs) are perceived as one of the most common malignancies among children. The specific regulation of each gene can play a critical role in cancer progression. The present study aimed to determine the transcripts of the TSGA10 and GGNBP2 genes, considering the alternative 5'UTR region, and investigating the expression of these different transcripts in BTs. Material and methods: Public data on brain tumor microarray datasets in GEO were analyzed with R software to evaluate the expression levels of TSGA10 and GGNBP2 genes (the Pheatmap package in R was also used to plot DEGs in a heat map). In addition, to validate our in-silico data analysis, RT-PCR was performed to determine the splicing variants of TSGA10 and GGNBP2 genes in testis and brain tumor samples. The expression levels of splice variants of these genes were analyzed in 30 brain tumor samples and two testicular tissue samples as a positive control. Results: In silico results show that the differential expression levels of TSGA10 and GGNBP2 were significant in the GEO datasets of BTs compared to normal samples (with adjusted p-value<0.05 and log fold change > 1). This study's experimental results showed that the TSGA10 gene produces four different transcripts with two distinct promoter regions and splicing exon 4. The relative mRNA expression of transcripts without exon 4 was higher than transcripts with exon 4 in BT samples (p-value<001). In GGNBP2, exon 2 in the 5'UTR region and exon 6 in the coding sequence were spliced. The expression analysis results showed that the relative mRNA expression of transcript variants without exon 2 was higher than other transcript variants with exon 2 in BT samples (p-value<001). Conclusion: The decreased expression levels of transcripts with longer 5'UTR in BT samples than in testicular or low-grade brain tumor samples may decrease their translation efficiency. Therefore, decreased amounts of TSGA10 and GGNBP2 as potential tumor suppressor proteins, especially in high-grade brain tumors, may cause cancer development by angiogenesis and metastasis.

Genetic and epigenetic effects on couple adjustment in context of romantic relationship: A scoping systematic review.

Introduction: Couples' relationships defined by a complex interaction between the two partners and their intrapersonal traits. Romantic; relationships and love are associated with marital satisfaction and stability, as well as couples' happiness and health. Personality traits influence romantic relationships and, personality influenced by genetical and non-genetically factors. The roles of non-genetically factors such as socioeconomic position and external appearance have revealed in determining the quality of romantic relationships. Methods: We; performed a scoping systematic review to assess the association between genetics and epigenetic factors and romantic relationship. Relevant articles were identified by PubMed, EMBASE, Web of Science, Scopus, and the APA PsycInfo searching between inception and 4 June 2022. Results: Different studies evaluated the associated polymorphisms in 15 different genes or chromosomal regions. In the first step; we classified them into four groups: (1) Oxytocin-related signaling pathway (OXTR, CD38, and AVPR1A); (2) Serotonin-related signaling pathway (SLC6A4, HTR1A, and HTR2A); (3) Dopamine and catecholamine-related signaling pathway (DRD1, DRD2, DRD4, ANKK1, and COMT); and (4) other genes (HLA, GABRA2, OPRM1, and Y-DNA haplogroup D-M55). Then, we evaluated and extracted significant polymorphisms that affect couple adjustment and romantic relationships. Discussion: Overall, the findings suggest that genetic and epigenetics variants play a key role in marital adjustment and romantic relationships over time.

A comprehensive overview of SMN and NAIP copy numbers in Iranian SMA patients.

Spinal muscular atrophy (SMA) is among the most common autosomal recessive disorders with different incidence rates in different ethnic groups. In the current study, we have determined SMN1, SMN2 and NAIP copy numbers in an Iranian population using MLPA assay. Cases were recruited from Genome-Nilou Laboratory, Tehran, Iran and Pars-Genome Laboratory, Karaj, Iran during 2012-2022. All enrolled cases had a homozygous deletion of exon 7 of SMN1. Moreover, except for 11 cases, all other cases had a homozygous deletion of exon 8 of SMN1. Out of 186 patients, 177 (95.16%) patients showed the same copy numbers of exons 7 and 8 of SMN2 gene. In addition, 53 patients (28.49%) showed 2 copies, 71 (38.17%) showed 3 copies and 53 patients (28.49%) showed 4 copies of SMN2 gene exons 7 and 8. The remaining 9 patients showed different copy numbers of exons 7 and 8 of SMN2 gene. The proportions of SMA patients with different numbers of normal NAIP were 0 copy in 73 patients (39.24%), 1 copy in 59 patients (31.72%), 2 copies in 53 patients (28.49%) and 4 copies in one patient (0.5%). These values are different from values reported in other populations. Integration of the data of the SMN1/2 and NAIP genes showed 17 genotypes. Patients with genotype 0-0-3-3-1 (0 copies of SMN1 (E7,8), 3 copies of SMN2 (E7,8) and 1 copy of NAIP (E5)) were the most common genotype in this study. Patients with 0-0-2-2-0 genotype were more likely to have type I SMA. The results of the current study have practical significance, particularly in the genetic counseling of at-risk families.

Extreme ßHCG levels in first trimester screening are risk factors for adverse maternal and fetal outcomes.

Multiples of the normal median (MoM) of free ßHCG is a valuable parameter in evaluation of risk of adverse pregnancy outcomes. In the current retrospective study, we assessed the maternal and fetal outcomes in pregnant women having free ßHCG MoM levels < 0.2 or > 5 in their first trimester screening (FTS). Relative risk of trisomy 21 was significantly higher in patients having free ßHCG MoM > 5. On the other hand, relative risk of trisomies 13 and 18 and Turner syndrome were higher in those having free ßHCG MoM < 0.2. Other chromosomal abnormalities were nearly equally detected between those having free ßHCG MoM < 0.2 or > 5. Relative risk of hydrocephaly and hydrops fetalis was higher when free ßHCG MoM was below 0.2. On the other hand, relative risk of low birth weight was higher when free ßHCG MoM was above 5. Moreover, frequency of gestational diabetes mellitus, preeclampsia, preterm delivery and vaginal bleeding increased with levels of free ßHCG MoM. However, polyhydramnios had the opposite trend. Frequencies of premature rupture of membranes and pregnancy induced hypertension were highest among pregnant women having levels of free ßHCG MoM < 0.2. The current study indicates importance of free ßHCG MoM in identification of at-risk pregnancies in terms of both fetal and maternal outcomes. In fact, ßHCG MoM < 0.2 or > 5 can be regarded as risk factors for adverse maternal or fetal outcomes irrespective of the presence of other abnormalities in the FTS results.

Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4 µM and the PCR product to 5 µl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1 h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

Antioxidant and anti-apoptotic effects of selenium nanoparticles and Lactobacillus casei on mice testis after X-ray.

Radiation can lead to various damages in the process of spermatogenesis that lead to a decrease in the number of sperm, an increase in spermatogenesis disorders, and defective sperm function. Radioprotectors are considered a good approach to reducing the damage caused by radiation. The goal of this work was to study how X-ray radiation affects testicular tissue and the process of spermatogenesis, as well as the radioprotective effects of selenium nanoparticles (SeNPs) and Lactobacillus casei (L. casei) as probiotic compounds, given alone or together. This study included 64 adult Syrian male mice weighing approximately 20 ± 5 g and aged 10 ± 1 weeks. Animals were randomly divided into eight groups: control group, SeNPs, probiotic, SeNPs and probiotic, X-ray radiation, SeNPs (X-ray), probiotic (X-ray), and SeNPs and probiotic (X-ray). Histology parameters and levels of oxidative stress biomarkers such as catalase, malondialdehyde, superoxide dismutase, and glutathione peroxidase were examined. In addition, the level of apoptosis was measured in testicular cells that had been treated with SeNPs and L. casei as a probiotic. The results showed that the administration of SeNPs or probiotic diminished the effects of X-ray radiation. These compounds induced a significant decreased in malondialdehyde, caspase 3, and caspase 9 gene levels and a remarkable increased in catalase, superoxide dismutase, and Catsper gene expression. SeNPs and probiotic exhibited a potent antioxidant effect and elevated the mean number of spermatogonia cells, sperm cell count, spermatogenesis percentage, and sperm motility percentage. The prescribed compound exhibited an ideal radioprotective effect with the ability to reduce the side effects of ionizing radiation and to protect normal tissues. SeNPs and probiotic inhibit testicular injury and improve the antioxidant state in male mice.

A novel variant in TLE6 is associated with embryonic developmental arrest (EDA) in familial female infertility.

This study aims to identify genetic causes of familial female infertility characterized by embryonic developmental arrest (EDA) and repeated implantation failure (RIF) with oocyte donation IVF cycle. We used Whole-exome sequencing and Sanger validation to find causative genes in an Iranian consanguineous family that had 3 infertile daughters, 4 fertile daughters, and 2 fertile sons. All patients in this consanguineous family exhibited typical manifestations of unexplained RIF and EDA. Genetic analysis identified a homozygous missense variant (c.G1054C:p.G352R) in exon 13 of the TLE6 gene that cosegregated with the EDA phenotype in an autosomal recessive pattern. Other members of the family, the gene carriers, remain clinically asymptomatic and fertile. Our findings identify a novel nonsynonymous variant, c.G1054C:p.G352R, in the TLE6 gene within a consanguineous Iranian family with autosomal-recessive female infertility and broaden the genetic spectrum of TLE6-associated EDA.

Bcl11a and the Correlated Key Genes Ascribable to Globin Switching: An In-silico Study.

BACKGROUND: Reactivation of HbF is a potential strategy to ameliorate symptoms of hemoglobinopathies such as sickle cell disease and b-thalassemia. After birth, there is a switch from fetal to adult hemoglobin, for which the molecular mechanisms and key regulators await further understanding in order to develop effective methods for HbF reactivation. Bcl11a, one of the major HbF reactivation regulators, demonstrates no significant changes at transcriptional levels in F erythroblasts compared to the non-HbF expressing cells. Therefore, it is possible that posttranscriptional regulation and epigenetic effects, for which the miRNAs play an important role, are the primary causes of the decreased Bcl11a protein level in adult erythroblasts. OBJECTIVE: This paper aims to determine the differentially expressed mRNAs and miRNAs of erythroblasts in HSCs from the fetal liver and bone marrow. METHODS: Raw high-throughput sequencing data (GSE110936, GSE90878) was downloaded from Gene Expression Omnibus (GEO) database. After RNAseq analysis, several data sets and tools were used to select key genes and examine selection validation. RESULTS: We selected 42 DEmRNAs and nine DEmiRs, including hsa-let-7f-5p, hsa-miR-21-5p, hsamiR- 22-3p, hsa-miR-126-5p, hsa-miR-146b-5p, hsa-miR-181a-5p, hsa-miR-92a-3p, hsa-miR-25-3p and hsa-miR-191-5p. Furthermore, hub genes including hist1h2bl, al133243.2, trim58, abcc13, bpgm, and fam210b were identified in the coexpression network, as well as RPS27A in the PPI network. Functional analysis revealed that these DEmRNAs and DEmiRs might play a role in gene expression regulation at multiple levels. Gene set enrichment analysis, in particular, revealed a possible role for genes in the globin switching process. CONCLUSION: According to our findings, a number of the DEmRNAs and DEmiRs may play significant roles in globin switching regulation and thus have the potential to be applied for HbF reactivation.

Antiproliferative effects of AAV-delivered CRISPR/Cas9-based degradation of the HPV18-E6 gene in HeLa cells.

Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.

Antimicrobial and anti-biofilm potencies of dermcidin-derived peptide DCD-1L against Acinetobacter baumannii: an in vivo wound healing model.

BACKGROUND: The global emergence of Acinetobacter baumannii resistance to most conventional antibiotics presents a major therapeutic challenge and necessitates the discovery of new antibacterial agents. The purpose of this study was to investigate in vitro and in vivo anti-biofilm potency of dermcidin-1L (DCD-1L) against extensively drug-resistant (XDR)-, pandrug-resistant (PDR)-, and ATCC19606-A. baumannii. METHODS: After determination of minimum inhibitory concentration (MIC) of DCD-1L, in vitro anti-adhesive and anti-biofilm activities of DCD-1L were evaluated. Cytotoxicity, hemolytic activity, and the effect of DCD-1L treatment on the expression of various biofilm-associated genes were determined. The inhibitory effect of DCD-1L on biofilm formation in the model of catheter-associated infection, as well as, histopathological examination of the burn wound sites of mice treated with DCD-1L were assessed. RESULTS: The bacterial adhesion and biofilm formation in all A. baumannii isolates were inhibited at 2 × , 4 × , and 8 × MIC of DCD-1L, while only 8 × MIC of DCD-1L was able to destroy the pre-formed biofilm in vitro. Also, reduce the expression of genes involved in biofilm formation was observed following DCD-1L treatment. DCD-1L without cytotoxic and hemolytic activities significantly reduced the biofilm formation in the model of catheter-associated infection. In vivo results showed that the count of A. baumannii in infected wounds was significantly decreased and the promotion in wound healing by the acceleration of skin re-epithelialization in mice was observed following treatment with 8 × MIC of DCD-1L. CONCLUSIONS: Results of this study demonstrated that DCD-1L can inhibit bacterial attachment and biofilm formation and prevent the onset of infection. Taking these properties together, DCD-1L appears as a promising candidate for antimicrobial and anti-biofilm drug development.

Cellular Immunity in Mice Vaccinated with Recombinant Phospholipase D Toxoid of Hemiscorpius lepturus Scorpion.

Background: Hemiscorpius lepturus is one of the most dangerous scorpions in Iran and the world. Numerous studies have been conducted on phospholipases, especially phospholipase D, in this scorpion's venom, and the results have shown this protein to be the main cause of death. Therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. The present study was conducted to assess the non-toxicity of this toxoid and the safety of the vaccine candidate in BALB/c mice. Methods: The production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using ELISpot assay 28 days following immunization with rPLD toxoid. Results: The unpaired t-test results showed a significant increase in the concentration of IFN-γ cytokine in the vaccinated mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant difference was observed in the levels of IL-4 (P= 0.16) despite an increase in this cytokine. The in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the LD100 of the venom. Moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin. Conclusion: As a vaccine, the toxoid has the potential to increase the Th1 cytokine response and, subsequently, increase acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the venom of Hemiscorpius lepturus.