Investigating neural differentiation of mouse P19 embryonic stem cells in a time-dependent manner by bioinformatic, microscopic and transcriptional analyses.

BACKGROUND: As an available cell line, mouse pluripotent P19 has been widely employed for neuronal differentiation studies. In this research, by applying the in vitro differentiation of this cell line into neuron-like cells through retinoic acid (RA) treatment, the roles of some genes including DNMT3B, ICAM1, IRX3, JAK2, LHX1, SOX9, TBX3 and THY1 in neural differentiation was investigated. METHODS AND RESULTS: Bioinformatics, microscopic, and transcriptional studies were conducted in a time-dependent manner after RA-induced neural differentiation. According to bioinformatics studies, we determined the engagement of the metabolic and developmental super-pathways and pathways in neural cell differentiation, particularly focusing on the considered genes. According to our qRT-PCR analyses, JAK2, SOX9, TBX3, LHX1 and IRX3 genes were found to be significantly overexpressed in a time-dependent manner (p < 0.05). In addition, the significant downregulation of THY1, DNMT3B and ICAM1 genes was observed during the experiment (p < 0.05). The optical microscopic investigation showed that the specialized extensions of the neuron-like cells were revealed on day 8 after RA treatment. CONCLUSION: Accordingly, the neural differentiation of P19 cell line and the role of the considered genes during the differentiation were proved. However, our results warrant further in vivo studies.

Association of xenobiotic-metabolizing genes polymorphisms with cervical cancer risk in the Tunisian population.

BACKGROUND: Host genetic characteristics and environmental factors interactions may play a crucial role in cervical carcinogenesis. We investigated the impact of functional genetic variants of four xenobiotic-metabolizing genes (AhR, CYP1A1, GSTM1, and GSTT1) on cervical cancer development in Tunisian women. METHODS: The AhR gene polymorphism was analyzed using the tetra-primer ARMS-PCR, whereas the CYP1A1 polymorphism genotypes were identified by PCR-RFLP. A multiplex ligation-dependent polymerase chain reaction approach was applied for the analysis of GSTM1 and GSTT1 polymorphisms. RESULTS: The homozygous A/A genotype of the AhR gene (rs2066853) and the heterozygous T/C genotype of the CYP1A1 SNP (CYP1A1-MspI) appeared to be associated with an increased risk of cervical tumorigenesis (ORa = 2.81; ORa = 5.52, respectively). Furthermore, a significantly increased risk of cervical cancer was associated with the GSTT1 null genotype (ORa = 2.65). However, the null GSTM1 genotype showed any significant association with the risk of cervical cancer compared to the wild genotype (ORa = 1.18; p = 0.784). Considering the combined effect, we noted a significantly higher association with cancer risk for individuals with at least two high-risk genotypes of CYP1A1/GSTT1 (ORa = 4.2), individuals with at least two high-risk genotypes of CYP1A1/GSTT1/AhR (ORa = 11.3) and individuals with at least two high-risk genotypes of CYP1A1/GSTM1/GSTT1/AhR exploitation low-risk genotype as a reference. CONCLUSION: This study indicated that the single-gene contribution and the combined effect of xenobiotic-metabolizing gene polymorphisms (AhR, CYP1A1-MspI, GSTM1, and GSTT1) may have a considerable association with increased cervical cancer risk.

Comparative analysis of protein-protein interaction networks in neural differentiation mechanisms.

Neural differentiation as a major process during neural cell therapy is one of the main issues that is not fully characterized. This study focuses on the major deconstruction of the transcriptional networks that regulate cell fate determination during neural differentiation under the influence of RA signalling. In our studies, we used four different microarray datasets containing a total of 15,660 genes to determine which genes were differentially expressed during neural differentiation from pluripotent stem cells (P19), among the 17 samples from four different datasets that were integrated via meta-analysis approaches. Of the 15,660 gene expression in our data integration, 443 DEGs are induced during neural differentiation. Upstream dissection of these 443 DEGs revealed a network of protein-protein interactions (PPIs) from TFs and kinases, as well as intermediate proteins between them, which are indicated by three (POU51, NANOG, and FOXO1) down-expression genes and one PAX6 up-expression gene playing roles in up-stream of these 443 induced DEGs during neural differentiation. The constructed network from the PPIs database revealed that four novel sub-networks play major roles in neuron differentiation in cluster 3, retinol metabolism in cluster 4, Rap1 signalling pathways in cluster 2, and axonogenesis in cluster 6. These four clusters have revealed very useful information about how neural characterization will be created from pluripotent stem cells. This research reveals a plethora of information on the neural differentiation process, including cell commitment and neural differentiation, and lays the groundwork for future research into particular pathways involving protein-protein interactions in neurogenesis.

The impact of training chemotherapy safety standards with a smartphone application on the knowledge, attitude, and performance of nurses.

BACKGROUND: Nurses who play the leading role in caring for patients, especially nurses in the chemotherapy department who are constantly exposed to high-risk drugs and their side effects, should pay more attention to occupational safety. This study was performed to determine the effect of training chemotherapy safety standards using a smartphone application on nurses' knowledge, attitude, and performance. METHODS: The whole enumeration of fifty oncology nurses was recruited who they worked in 3 hospitals affiliated with Lorestan University of Medical Sciences (west of Iran). The study was conducted from June to November 2021. The training was done for four weeks with a smartphone application, including six main courses of familiarity with hazardous drugs, Personal Protective Equipment, preparation, storage and transfer, spilling, and wastes disposal of hazardous drudges . The nurses' knowledge, attitude, and performance questionnaire were completed before, immediately after, and one month after the intervention. Data analysis was performed using SPSS version 26, descriptive and inferential statistical tests of independent t-test, one-way analysis of variance, Spearman's rank correlation coefficient, repeated measures analysis of variance, and the Generalized Estimating Equation (GEE) model. RESULTS: Mean knowledge score of participants before, immediately after, and one month after the intervention was (47.18 ± 8.19), (60.08 ± 3.82), and (61.88 ± 3.45), respectively. The mean attitude score of participants before, immediately after, and one month after the intervention was (30.34 ± 3.94), (34.32 ± 3.25), and (34.98 ± 2.88), in order, and the mean performance score of participants before, immediately after, and one month after the intervention was (43.60 ± 5.11), (51.78 ± 3.15) and (52.88 ± 3.06), respectively. The mean nurses' knowledge, attitude, and performance score increased significantly over time (P < 0.001). CONCLUSIONS: Teaching chemotherapy safety standards using the application improved oncology nurses' knowledge, attitude, and performance. Appropriate educational programs, especially by new methods such as E-learning, are recommended for providing safety for nurses.

A precise review on NAATs-based diagnostic assays for COVID-19: A motion in fast POC molecular tests.

BACKGROUND: Diagnosis is one of the main strategies to deal with infectious and deadly diseases such as coronavirus disease 2019 (COVID-19). The global pandemic of COVID-19 has led to an immediate need to expand rapid diagnostic techniques. New isothermal-based methods are being developed for COVID-19 detection aiming to resolve the limitations related to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method through immediate samples processing and minimizing false-negative or ambiguous results. Advances in nucleic acid amplification techniques (NAATs) can provide affordable and easy-to-use diagnostic platforms with high sensitivity and specificity in order to be available to the public as approved commercial kits. AIMS: The development of point-of-care (POC) testing can assist in rapid clinical decision-making and mitigate burdens on health care facilities. Finally, we discussed the different diagnostic methods based on NAATs for COVID-19 in detail. Comparative parameters are addressed for all assays and Emergency Use Authorizations (EUA)-approved commercial tests are cited. CONCLUSIONS: Isothermal-coupled methods and LAMP-based molecular methods have been suggested as suitable portable tests with high diagnostic speed for use in POC testing.

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells.

BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

Computational study for suppression of CD25/IL-2 interaction.

Cancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

The Effects of Self-cleavage Intein-ELK16 Tag in the Transcript Steric Hindrance of IFN.

Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNß) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of Mycobacterium tuberculosis RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNß was also cloned into pBudCE4.1 vector. In the next step, the rhIFNß was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF - 261.10 kcal/mol. Our results indicate that IFNß was upregulated (37.8-fold, p < 0.0001) in cells which transfected by rhIFNß-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNß had no interference in transcription of rhIFNß in the HEK293T cell line.

DMLR: A toolkit for investigation of deoxyribozyme-mediated ligation based on real time PCR.

Deoxyribozymes or DNAzyme are identified as catalytic DNA sequences which catalyze different chemical reactions. Ligating deoxyribozymes catalyze the formation of branched and linear products. Due to the lack of efficient read-out systems, there is no report on in vivo application of ligating deoxyribozymes. To expand the biological application of branched-RNA forming deoxyribozymes, we performed our study in order to suggest a practical toolkit for measurement of in vivo real-time activity of ligating deoxyribozymes. Further in vitro studies were designed to analyze the effects of the location of branch site on reverse transcriptase (RT) interference. With this toolkit even the activity of RT was measured precisely. Our results indicate that the activity of RT enzyme significantly affected by a 17 nt branched adaptor synthesized by 10DM24 ligating deoxyribozyme. The RT stalls at or near the RNA branch point during both initiation and elongation phases. The DNA synthesis is decreased 4.3 and 2.7 fold during initiation and elongation phases respectively. In conclusion, we introduce a general and practical toolkit called "DMLR" which is based on Real-time PCR method. The use of DMLR precisely determines RT behavior when encountered with any backbone modification with the ability of stopping the enzyme activity.

Network-based expression analyses and experimental validations revealed high co-expression between Yap1 and stem cell markers compared to differentiated cells.

The Hippo signaling pathway is identified as a potential regulatory pathway which plays critical roles in differentiation and stem cell self-renewal. Yap1 is a primary transcriptional effector of this pathway. The importance of Yap1 in embryonic stem cells (ESCs) and differentiation procedure remains a challenging question, since two different observations have been reported. To answer this question we used co-expression network and differential co-expression analyses followed by experimental validations. Our results indicate that Yap1 is highly co-expressed with stem cell markers in ESCs but not in differentiated cells (DCs). The significant Yap1 down-regulation and also translocation of Yap1 into the cytoplasm during P19 differentiation was also detected. Moreover, our results suggest the E2f7, Lin28a and Dppa4 genes as possible regulatory nuclear factors of Hippo pathway in stem cells. The present findings are actively consistent with studies that suggested Yap1 as an essential factor for stem cell self-renewal.

Evaluation of Silencing Effect of miR-133a-5p Mimic on TIM-3 Expression in AML (HL-60) Cell Line.

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% (p = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.

Increased expression of microRNAs, miR-20a and miR-326 in PBMCs of patients with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disorder which is characterized by autoimmune attack on ß cells of pancreas and lack of insulin. The involvement of microRNAs (miRNAs) in the development of immune system and their differential expression in various autoimmune diseases including T1D have been well established. In this study, the association between expression levels of miR-20a, miR-326 and T1D were evaluated. The expression levels of miR-20a and miR-326 were measured in the PBMCs of 21 T1D patients and 16 healthy controls using qPCR method. In silico analysis was also performed on targetome of miR-20a and miR-326. Both miR-20a (p value: 0.015) and miR-326 (p value: 0.005) were upregulated in the PBMCs of T1D patients compared to healthy controls. Furthermore, different dysregulated miR326-mRNA and miR20a-mRNA interactions were also suggested using integrative computational analysis. The expression level of miR-20a and miR-326 indicates significant association with T1D which suggests the possible regulatory effects of these non-coding RNAs in T1D.

In vitro and computational studies on the effects of ARE deletion and targeted mutations on the expression of interferon beta-1a in HEK293T cells.

Interferon beta (IFNß) is transiently expressed in response to viral infections and widely used to treat relapsing-remitting multiple sclerosis (MS). We introduced mutations in the IFNß gene (in the 27th and 101st codons and in the Kozak sequence, and also deletion of 3' and 5' unstable, untranslated region, UTR) with the aim of increasing the expression of IFNß. Computational analyses of mutant and wild-type RNAs and proteins of IFNß by RNAfold, ASAView, HOPE and Ramachandran plot, and iStable web servers showed that the mutations could decrease RNA stability, protein solvent accessibility, and protein stability but could not change correct folding. Two recombinant IFNß101 and IFNß101+27 constructs were designed by site-directed mutagenesis. The wild-type IFNß gene also was used as a control. In vitro experiments by quantitative real-time PCR, dot blot, SDS-PAGE, and Western blot assays showed an increased expression level of recombinant IFNßs. 79.9-fold, 99.7-fold, and 190-fold elevations in the mRNA expression of IFNßw, IFNß101, and IFNß101+27 were seen, respectively, in comparison with the endogenous IFNß mRNA in untransfected HEK293T cells. The IFNß mRNA expression was increased 2.38-fold and 1.25-fold for 101+27 and 101 mutated forms, respectively, in comparison with the IFNß wild-type construct. An elevation in IFNß protein production was also clearly detected in the transfected HEK293T cell containing recombinant IFNß101 and IFNß101+27 constructs. Finally, these directed mutations in the IFNß gene successfully elevated protein and mRNA production but in silico analyses of mutant mRNAs showed decreased mRNA stability, unlike previous studies, in comparison with the wild-type mRNA.

Molecular Genetic and Epigenetic Basis of Multiple Sclerosis.

Multiple Sclerosis (MS) is a chronic immune-mediated disease of spinal cord and brain. The initial event in MS occurs when activated CD4+ T cells in periphery exacerbates immune responses by stimulating immune cells such as B cells, CD8+ cells, mast cells, granulocytes and monocytes. These proinflammatory cells pass blood brain barrier by secreting proinflammatory cytokines including TNF-α and INF-γ which activate adhesion factors. APCs (antigen-presenting cells) reactivate CD4+ T cells after infiltrating the CNS and CD4+ T cells produce cytokines and chemokines. These proinflammatory cytokines aggravate inflammation by inducing myelin phagocytosis through microglia and astrocytes activation. MS is believed to have a multifactorial origin that includes a combination of multiple genetic, environmental and stochastic factors. Although the exact component of MS risks that can be explained by these factors is difficult to determine, estimates based on genetic and epidemiological studies suggest that up to 60-70 % of the total risk of MS may be contribute to genetic factors. In continue, firstly we provide an overview of the current understanding of epigenetic mechanisms, and so present evidence of how the epigenetic modifications contribute to increased susceptibility of MS. We also explain how specified epigenetic modifications may influence the pathophysiology and key aspects of disease in MS (demyelination, remyelination, inflammation, and neurodegeneration). Finally, we tend to discuss how environmental factors and epigenetic mechanisms may interact to have an effect on MS risk and clinical outcome and recommend new therapeutic interventions that might modulate patients' epigenetic profiles.

Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities.

Interferon ß (IFNß) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNß by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNß mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNß101 or IFNß101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNß101 and IFNß101+27 constructs increase IFNß protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNß with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNß protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNß101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNß in such a level is promising not only for the patients but also for the pharmaceutical industries.

The Effect of Education and Telephone Follow-up on the Burden of Family Caregivers of Patients With Cancer.

Introduction: Family caregivers of patients with cancer face a care burden due to the responsibilities and problems of providing care to the patients. Applying appropriate strategies to reduce the burden is essential. Objective: The study aimed to determine the effect of education and telephone follow-up on family caregivers' burden on patients with cancer. Methods: In this quasi-experimental study, 69 family caregivers of patients with cancer referred to only one chemotherapy center of a hospital in Lorestan province in Iran were recruited by convenience sampling method. They were randomly assigned to intervention (n = 33) and control (n = 36) groups. For the intervention group, two face-to-face training sessions and six telephone counseling sessions were held related to the care of the patients and self-care. The control group received only routine care. The family caregiver burden was measured by Novak and Gast Caregiver Burden Inventory (1989) completed before, immediately, and 6 weeks after the study. Data were analyzed by SPSS21 using independent t-tests, paired t-tests, and repeated measures. Results: Both groups were homogeneous regarding demographic characteristics and the baseline care burden. The caregiver burden decreased significantly in the intervention group, so its score was 77.33 ± 8.49, 58.93 ± 8.03, and 52.78 ± 6.86 before the study, immediately after and 6 weeks later, respectively (p < .001). In the control group, there were no significant changes. Conclusion: Education and telephone counseling reduced the burden on family caregivers. Therefore, this type of support is beneficial for providing holistic care and preserving the health of family caregivers.

A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing.

Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA-seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA-seq, we delineated a deep intronic TAPT1 mutation (c.1237-52 G>A) that segregated with the disease. Using SI-NET-seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre-mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein-null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease-relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non-coding variants, as well as in illuminating the molecular mechanisms of human diseases.

The stromelysin-1 5A/5A genotype enhances colorectal cancer cell invasion in Iranian population.

BACKGROUND: Matrix metalloproteinases comprise a family of enzyme degrade components of extra cellular matrix. There are single nucleotide polymorphisms in the promoter regions of several genes with ability to influence cancer susceptibility. The aim of this study was to analyze association between MMP3 promoter polymorphisms and colorectal cancer occurrence and progression. MATERIALS AND METHODS: In this case-control study 120 colorectal cancer patients and 100 controls were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the genomic deoxyribonucleic acid (DNA). The patients group was divided into different subgroups: a subgroup without metastatic activity (M(-)) and a subgroup that had developed metastasis (M(+)). RESULTS: There was a significant difference in frequency of the MMP-3 genotype between cases and controls (χ΂ = 16.17; P = 0.0003). The 5A homozygote in patients and controls was significantly different. The frequency of the 5A allele among affected patients (67.91%) was significantly higher than among the healthy controls (49%; χ(2) = 16.17, P = 0.00005). At the time of diagnosis, individual who was carrying the 5A allele was more represented in the M(+) subgroup than in M(-) subgroup (χ² = 7.49; P = 0.006, OR = 3.86; 95% CI, 1.43-10.33). The difference between M(-) and controls did not observe statistically significant (χ² = 0.009; P = 0.92). CONCLUSIONS: Our results suggest that the presence of 5A polymorphism at the MMP-3 promoter region may favor the growth and the metastasis process in colorectal cancer patients and could be looked at as a risk factor for a worse prognosis.

The isolation and expression analysis of cinnamate 4-hydroxylase and chalcone synthase genes of Scrophularia striata under different abiotic elicitors.

The phenylpropanoid pathway serves as a rich source of metabolites in plants, and it is considered as a starting point for the production of many other important compounds such as the flavonoids, flavonols, coumarins, and lignans. Scrophularia striata is a member of the Lamiaceae family with some biological activities similar to flavonoid compounds such as antioxidant, antibacterial, anti-inflammatory and analgesic activities. Cinnamate 4-hydroxylase (C4H) and Chalcone synthase (CHS) are key enzymes of the phenylpropanoid pathway, leading to the biosynthesis of several secondary metabolites. In this study, two S. striata CHS and C4H were isolated and then analyzed. The investigation of the expression of these genes was performed under the effects of three salicylic acid (SA), jasmonic acid (JA), and gibberellic acid (GA) at concentrations of 100 and 300 ppm with a completely randomized design at the transcript level using Real Time PCR method. These have different expression patterns at developmental stages. Moreover, these genes present different sensitivities to hormonal treatment. Considering the total results, it was found that the amount of expression of these genes during the reproductive phase is higher than that of the vegetative phase. Additionally, the treatment of 300 ppm SA in the reproductive phase is the most effective treatment on increasing the corresponding phenylpropanoid compounds. A correlation analysis was performed between the phenylpropanoid compounds content and both CHS and C4H expression values at different phenological development stages. The results indicate that the expression variations of both CHS and C4H are significantly related to the changes in total phenolic content. We believe that the isolation of CHS and C4H can be helpful in better understanding phenylpropanoid metabolis.

Identification of Circulating hsa-miR-324-3p and hsa-miR-331-3p Exchanges in The Serum of Alzheimer's Patients and Insights into The Pathophysiological Pathways.

OBJECTIVE: Alzheimer's disease (AD) is a type of dementia. Currently, there are not any existing and reliable methods for the prognosis or diagnosis of AD. Hence, finding a diagnostic/prognostic biomarker for AD helps physicians to prescribe the treatments and methods preventing disease progression. Circulating microRNAs (miRNAs) are the most promising biomarkers due to their non-invasive and easily accessible for diagnosis and prognosis of AD. The aim of current study is to evaluate expression levels of two unwell-known circulating miRNAs including hsa-miR-324-3p and hsa-miR-331-3p in serums of AD patients and to understand their roles in AD physiopathogenesis by in silico analysis. MATERIALS AND METHODS: In this case and control study, to get the gene targets related to these two miRNAs, TargetScan, miRTargetLink Human and mirDIP web servers were applied. In addition, gene networks and gene ontology enrichment analysis were performed by STRING 10.5, KEGG and ShinyGO v0.41. Experimentally, expression levels of these two miRNAs in the serum of 21 patients with AD and 23 healthy individuals were compared using the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. RESULTS: The pathophysiological pathways associated with these two miRNAs were nucleotide metabolism and cellular response to stress pathway. Furthermore, the upregulated expression levels of hsa-miR-324-3p and hsa-miR-331-3p in comparison with the healthy control serums were not statistically significant (P>0.05). CONCLUSION: Non-significant results were obtained from the expression levels of AD patients and two significant pathways were obtained by networks and gene enrichment analysis.