Search results for: genes expression

Authors: Othman E. Othman, Amira M. Nowier, Medhat El-Denary

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Camels play an important socio-economic role within the pastoral and agricultural system in the dry and semidry zones of Asia and Africa. Camels are economically important animals in Egypt where they are dual purpose animals (meat and milk). The analysis of chemical composition of camel milk showed that the total protein contents ranged from 2.4% to 5.3% and it is divided into casein and whey proteins. The casein fraction constitutes 52% to 89% of total camel milk protein and it divided into 4 fractions namely αs1, αs2, β and κ-caseins which are encoded by four tightly genes. In spite of the important role of casein genes and the effects of their genetic polymorphisms on quantitative traits and technological properties of milk, the studies for the detection of genetic polymorphism of camel milk genes are still limited. Due to this fact, this work focused - using PCR-RFP and sequencing analysis - on the identification of genetic polymorphisms and SNPs of two casein genes in Maghrabi camel breed which is a dual purpose camel breed in Egypt. The amplified fragments at 488-bp of the camel κ-CN gene were digested with AluI endonuclease. The results showed the appearance of three different genotypes in the tested animals; CC with three digested fragments at 203-, 127- and 120-bp, TT with three digested fragments at 203-, 158- and 127-bp and CT with four digested fragments at 203-, 158-, 127- and 120-bp. The frequencies of three detected genotypes were 11.0% for CC, 48.0% for TT and 41.0% for CT genotypes. The sequencing analysis of the two different alleles declared the presence of a single nucleotide polymorphism (C→T) at position 121 in the amplified fragments which is responsible for the destruction of a restriction site (AG/CT) in allele T and resulted in the presence of two different alleles C and T in tested animals. The nucleotide sequences of κ-CN alleles C and T were submitted to GenBank with the accession numbers; KU055605 and KU055606, respectively. The primers used in this study amplified 942-bp fragments spanning from exon 4 to exon 6 of camel αS1-Casein gene. The amplified fragments were digested with two different restriction enzymes; SmlI and AluI. The results of SmlI digestion did not show any restriction site whereas the digestion with AluI endonuclease revealed the presence of two restriction sites AG^CT at positions 68^69 and 631^632 yielding the presence of three digested fragments with sizes 68-, 563- and 293-bp.The nucleotide sequences of this fragment from camel αS1-Casein gene were submitted to GenBank with the accession number KU145820. In conclusion, the genetic characterization of quantitative traits genes which are associated with the production traits like milk yield and composition is considered an important step towards the genetic improvement of livestock species through the selection of superior animals depending on the favorable alleles and genotypes; marker assisted selection (MAS).

Keywords: genetic polymorphism, SNP polymorphism, Maghrabi camels, κ-Casein gene, αS1-Casein gene

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Authors: Agumah N. B. Orji, M. U., Oru C. M., Ugbo, E. N., Onwuliri E. A Nwakaeze, E. A.,

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ERG11 gene has been reported to be one of the genes whose expression is responsible for resistance of Candida to various triazole drugs, which are first line treatment for candidiasis. This study was carried out to determine the prevalence of Triazole (Fluconazole and voriconazole) resistant Candida albicans expressing ERG11 gene from adult females in Abakaliki. Urine and vaginal swab samples were randomly collected from volunteers after obtaining their consent to participate in the study. A total of 565 adult females participated in the study. A total of 340 urine specimens and 288 vaginal swab specimens were collected. Direct wet mount technique, as well as culture in Trichomonas broth, were used to examine the urine and vaginal swab specimens for the presence of motile Trichomonads. The Trichomonas broth used was selective for both T. vaginalis and C. albicans. Broths that yielded budding yeast cells after microscopy were subcultured on to Sabouraud dextrose agar, after which Germ tube test was carried out to confirm the presence of C. albicans. Biochemical tests, including carbohydrate fermentation and urease utilization, were also performed. Antibiogram of C. albicans isolates obtained from this study was carried out using commercially available azole drugs. Fluconazole and voriconazole were selected as Triazole drugs used for this study. Nystatin was used as a tangential control. An MIC test was carried out with E-strips on some of the resistant C. albicans isolates A total of 6 isolates that resisted all the azole drugs were selected and screened for the presence of ERG11 gene using Reverse transcriptase polymerase chain reaction technique. The total prevalence recorded for C. albicans was 13.0%. Frequency was statistically higher in Pregnant (7.96%) than non pregnant (5.09%) volunteers (X2=0.94 at P=0.05). With respect to clinical samples, frequency was higher in vaginal swabs samples (7.96%) than Urine samples (5.09%) (X2=9.05 at P=0.05). Volunteers within the age group 26-30 years recorded the highest prevalence (4.46%), while those within the age group 36-40 years recorded the lowest at 1.27%(X2=4.34 at P=0.05). In pregnant female participants, the highest frequency was recorded with those in their 3rd trimester (4.14%), while lowest incidence was recorded for those in their first trimester (0.80%). Antibiogram results from this study showed that C. albicans isolates obtained from this study resisted Fluconazole (72%) more than Voriconazole (57%). Only one out of the six selected isolates yielded resistance in the MIC test. Results obtained from the RT-PCR showed that there was no expression of ERG11 gene among the fluconazole resistant isolates of C. albicans. Observed resistance may be due to other factors other than expression of ERG11 gene.

Keywords: candida, ERG11, triazole, nigeria

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Authors: Chengcao Sun, Shujun Li, Cuili Yang, Yongyong Xi, Liang Wang, Feng Zhang, Dejia Li

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Recently, the long non-coding RNA (lncRNA) NEAT1 has been identified as an oncogenic gene in multiple cancer types and elevated expression of NEAT1 was tightly linked to tumorigenesis and cancer progression. However, the molecular basis for this observation has not been characterized in progression of non-small cell lung cancer (NSCLC). In our studies, we identified NEAT1 was highly expressed in NSCLC patients and was a novel regulator of NSCLC progression. Patients whose tumors had high NEAT1 expression had a shorter overall survival than patients whose tumors had low NEAT1 expression. Further, NEAT1 significantly accelerates NSCLC cell growth and metastasis in vitro and tumor growth in vivo. Additionally, by using bioinformatics study and RNA pull down combined with luciferase reporter assays, we demonstrated that NEAT1 functioned as a competing endogenous RNA (ceRNA) for has-miR-377-3p, antagonized its functions and led to the de-repression of its endogenous targets E2F3, which was a core oncogene in promoting NSCLC progression. Taken together, these observations imply that the NEAT1 modulated the expression of E2F3 gene by acting as a competing endogenous RNA, which may build up the missing link between the regulatory miRNA network and NSCLC progression.

Keywords: long non-coding RNA NEAT1, hsa-miRNA-377-3p, E2F3, non-small cell lung cancer, tumorigenesis

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Authors: N. Zarghami, M. Mohammadinejad, A. Akbarzadeh, Y. Pilehvar-Soltanahmadi, F. Zarghami

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Breast cancer is known to be the most common cancer in women. Cyclin D1 is a proto-oncogene and over expression of cyclin D1 is directly associated with tumorgenesis. Cyclin D1 is overexpressed in more than 50% of breast cancer cases. Curcumin is derived from turmeric (curcuma longa) and chrysin is a component that could be extracted from many plants and honey. These two plants derived compounds are believed to assist in inhibition of the cancer cells growth and reducing cyclin D1 expression. In this work, the hypothesis is to combine curcumin and chrysin in order to analyze the potential synergistic effect in inhibition of cell proliferation and down regulation of cyclin D1. In addition, use of PLGA-PEG to improve bioavailability of pure curcumin and chrysin, while reinforcing the potential effect of this combination. PLGA-PEG nanoparticles were synthesized and characterized with FT-IR and 1HNMR methods. Although morphological features were analyzed by SEM. Afterward curcumin and chrysin were encapsulated with synthesized PLGA-PEG and MTT-assay was performed to measure cytotoxicity effect of these plant constitutes. T-47D cells were treated with proper concentration of these constituents and Real-time PCR was carried out to evaluate cyclin D1 expression levels. Curcumin, chrysin and combination of curcumin –chrysin in intact and nano-capsulated form affected T-47D cells in time and dose dependent manner and the combination of these compounds had synergistic effects. Real-time PCR results, revealed that curcumin, chrysin and combination of curcumin-chrysin in pure and encapsulated form inhibited cyclin D1 expression. Compared to pure components, different concentrations of nano-curcumin, nano chrysin and nano-combination caused further decline in cyclin D12 expression by 5-11%, 8-22% and 6-18% respectively. Our results demonstrated that, combination of chrysin-curcumin had synergistic effect and nano capsulated form of this component had grater inhibition on cyclin D1 expression.

Keywords: breast cancer, cyclin D1, curcumin, chrysin, nanoparticles

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Authors: Ouafae Baida, Najma Hamzaoui, Maha Akbib, Abdelfettah Sedqui, Abdelouahid Lyhyaoui

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Numerous scientific works present various methods to analyze the data for several domains, specially the comparison of classifications. In our recent work, we presented a new approach to help the user choose the best classification method from the results obtained by every method, by basing itself on the distances between the trees of classification. The result of our approach was in the form of a dendrogram contains methods as a succession of connections. This approach is much needed in phylogeny analysis. This discipline is intended to analyze the sequences of biological macro molecules for information on the evolutionary history of living beings, including their relationship. The product of phylogeny analysis is a phylogenetic tree. In this paper, we recommend the use of a new method of construction the phylogenetic tree based on comparison of different classifications obtained by different molecular genes.

Keywords: hierarchical classification, classification methods, structure of tree, genes, phylogenetic analysis

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Authors: Sajad Haghshenas

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Emerging evidence from clinical, genetic, and animal model studies suggests that the N-methyl-D-aspartate (NMDA) glutamate receptors (NMDAR) may contribute to the pathophysiology and aetiology of neurological and psychiatric disorders and the patients with impaired NMDR receptors experience psychological symptoms. Therefore, we hypothesised that NMDAR receptors play a key role in the development of attention deficit hyperactivity disorder (ADHD). In this comparative analytical study, we utilized western blotting method to assay the expression levels of NMDA subunits NR1 and NR2 in the blood plasma of 50 male individuals diagnosed with ADHD in comparison to 20 healthy controls. The findings from the western blotting analysis provide support for the hypothesis that individuals with ADHD exhibit significantly lower levels of NR1/2 receptors compared to those without the disorder. Further research is needed to explore the potential causal relationship between reduced NR1/NR2 receptor levels and the development of ADHD.

Keywords: expression, glutamate receptors, NR1, NR2, ADHD

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Authors: Renata Checiches, Thierry Coche, Nicolas F. Delahaye, Albert Linder, Fernando Ulloa Montoya, Olivier Gruselle, Karen Langfeld, An de Creus, Bart Spiessens, Vincent G. Brichard, Jamila Louahed, Frédéric F. Lehmann

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Background: The RNA-expression levels of cancer-testis antigens MAGE A3 and PRAME were determined in resected tissue from patients with primary non-small-cell lung cancer (NSCLC) and related to clinical outcome. EGFR, KRAS and BRAF mutation status was determined in a subset to investigate associations with MAGE A3 and PRAME expression. Methods: We conducted a single-centre, uncontrolled, retrospective study of 1260 tissue-bank samples from stage IA-III resected NSCLC. The prognostic value of antigen expression (qRT-PCR) was determined by hazard-ratio and Kaplan-Meier curves. Results: Thirty-seven percent (314/844) of tumours expressed MAGE-A3, 66% (723/1092) expressed PRAME and 31% (239/839) expressed both. Respective frequencies in squamous-cell tumours and adenocarcinomas were 43%/30% for MAGE A3 and 80%/44% for PRAME. No correlation with stage, tumour size or patient age was found. Overall, no prognostic value was identified for either antigen. A trend to poorer overall survival was associated with MAGE-A3 in stage IIIB and with PRAME in stage IB. EGFR and KRAS mutations were found in 10.1% (28/311) and 33.8% (97/311) of tumours, respectively. EGFR (but not KRAS) mutation status was negatively associated with PRAME expression. Conclusion: No clear prognostic value for either PRAME or MAGE A3 was observed in the overall population, although some observed trends may warrant further investigation.

Keywords: MAGE A3, PRAME, cancer-testis gene, NSCLC, survival, EGFR

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Authors: H. Sakamoto, S. Kurosawa, M. Suzuki, S. Oguri

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In Arabidopsis, several genes encoding proteins with ankyrin repeats and trans-membrane domains (AtANKTM) have been identified as mediators of biotic and abiotic stress responses. It has been known that the expression of an AtANKTM gene, At2g24600, is induced in response to abiotic stress and that there are four splicing variants derived from this locus. In this study, by RT-PCR and sequencing analysis, an unknown splicing variant of the At2g24600 transcript was identified. Based on differences in the predicted amino acid sequences, the five splicing variants are divided into three groups. The three predicted proteins are highly homologous, yet have different numbers of ankyrin repeats and trans-membrane domains. It is generally considered that ankyrin repeats mediate protein-protein interaction and that the number of trans-membrane domains affects membrane topology of proteins. The protein variants derived from the At2g24600 locus may have different molecular functions each other.

Keywords: alternative splicing, ankyrin repeats, trans-membrane domains, arabidopsis

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Authors: Abdullahi Musa, Yakubu Kukure Enebe Ibrahim, Adeshina Gujumbola

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The emergence of multidrug resistance in clinical Escherichia coli has been associated with plasmid-mediated genes. DNA transfer among bacteria is critical to the dissemination of resistance. Plasmids have proved to be the ideal vehicles for dissemination of resistance genes. Plasmids coding for antibiotic resistance were long being recognized by many researchers globally. The study aimed at determining the antibiotic susceptibility pattern of ESBL E. coli isolates claimed to be multidrug resistance using disc diffusion method. Antibacterial activity of the test isolates was carried out using disk diffusion methods. The results showed that, majority of the multidrug resistance among clinical isolates of ESBL E. coli was as a result of acquisition of plasmid carrying antibiotic-resistance genes. Production of these ESBL enzymes by these organisms which are normally carried by plasmid and transfer from one bacterium to another has greatly contributed to the rapid spread of antibiotic resistance amongst E. coli isolates, which lead to high economic burden, increase morbidity and mortality rate, complication in therapy and limit treatment options. To curtail these problems, it is of significance to checkmate the rate at which over the counter drugs are sold and antibiotic misused in animal feeds. This will play a very important role in minimizing the spread of resistance bacterial strains in our environment.

Keywords: Escherichia coli, plasmid, multidrug resistance, ESBL, pan drug resistance

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Authors: Mohd Adil, Rosina Khan, Asad U. Khan, Vasantha Rupasinghe HP

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The study examined the effects of Syzygium aromaticum extracts on the virulence properties of Streptococcus mutans. The activity of glucosyltransferases in the presence of crude and diethylether fraction was reduced to 80% at concentration 78.12μg/ml and 39.06μg/ml respectively. The glycolytic pH drop by S. mutans cells was also disrupted by these extracts without affecting the bacterial viability. Microscopic analysis revealed morphological changes of the S. mutans biofilms, indicating that these plant extracts at sub-MICs could significantly affect the ability of S. mutans to form biofilm with distorted extracellular matrix. Furthermore, with the help of quantitative RT-PCR, the expression of different genes involved in adherence, quorum sensing, in the presence of these extracts were down regulated. The crude and active fractions were found effective in preventing caries development in rats. The data showed that S. aromaticum holds promise as a naturally occurring source of compounds that may prevent biofilm-related oral diseases.

Keywords: biofilm, quorum sensing, Streptococcus mutans, Syzygium aromaticum extract

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Authors: Nichole Haag, Kimberly Velk, Tyler McCune, Chun Wu

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Methicillin/multiple-resistant Staphylococcus aureus (MRSA) are infectious bacteria that are resistant to common antibiotics. A previous in silico study in our group has identified a hypothetical protein SAV1226 as one of the potential drug targets. In this study, we reported the bioinformatics characterization, as well as cloning, expression, purification and kinetic assays of hypothetical protein SAV1226 from methicillin/vancomycin-resistant Staphylococcus aureus Mu50 strain. MALDI-TOF/MS analysis revealed a low degree of structural similarity with known proteins. Kinetic assays demonstrated that hypothetical protein SAV1226 is neither a domain of an ATP dependent dihydroxyacetone kinase nor of a phosphotransferase system (PTS) dihydroxyacetone kinase, suggesting that the function of hypothetical protein SAV1226 might be misannotated on public databases such as UniProt and InterProScan 5.

Keywords: Methicillin-resistant Staphylococcus aureus, dihydroxyacetone kinase, essential genes, drug target, phosphoryl group donor

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Authors: Parvaneh Mahmoudi, Ahmad Moeni, Seyed Mojtaba Khayam Nekoei, Mohsen Mardi, Mehrshad Zeinolabedini, Ghasem Hosseini Salekdeh

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Saffron (Crocus sativus L.) is one of the most important spice and medicinal plants. The three-branch style of C. sativus flowers are the most important economic part of the plant and known as saffron, which has several medicinal properties. Despite the economic and biological significance of this plant, knowledge about its molecular characteristics is very limited. In the present study, we, for the first time, constructed a comprehensive dataset for C. sativus stigma through de novo transcriptome sequencing. We performed de novo transcriptome sequencing of C. sativus stigma using the Illumina paired-end sequencing technology. A total of 52075128 reads were generated and assembled into 118075 unigenes, with an average length of 629 bp and an N50 of 951 bp. A total of 66171unigenes were identified, among them, 66171 (56%) were annotated in the non-redundant National Center for Biotechnology Information (NCBI) database, 30938 (26%) were annotated in the Swiss-Prot database, 10273 (8.7%) unigenes were mapped to 141 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, while 52560 (44%) and 40756 (34%) unigenes were assigned to Gen Ontology (GO) categories and Eukaryotic Orthologous Groups of proteins (KOG), respectively. In addition, 65 candidate genes involved in three stages of crocin biosynthesis were identified. Finally, transcriptome sequencing of saffron stigma was used to identify 6779 potential microsatellites (SSRs) molecular markers. High-throughput de novo transcriptome sequencing provided a valuable resource of transcript sequences of C. sativus in public databases. In addition, most of candidate genes potentially involved in crocin biosynthesis were identified which could be further utilized in functional genomics studies. Furthermore, numerous obtained SSRs might contribute to address open questions about the origin of this amphiploid spices with probable little genetic diversity.

Keywords: saffron, transcriptome, NGS, bioinformatic

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Authors: Jae-Hyeon Kim, Michael Lee

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Intrinsic and acquired resistance limits the therapeutic benefits of oncogenic BRAF inhibitors in melanoma. MicroRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation. Thus, we investigated miRNA expression patterns in melanoma cell lines to identify candidate biomarkers for acquired resistance to BRAF inhibitor. Here, we used Affymetrix miRNA V3.0 microarray profiling platform to compare miRNA expression levels in three cell lines containing BRAF inhibitor-sensitive A375P BRAF V600E cells, their BRAF inhibitor-resistant counterparts (A375P/Mdr), and SK-MEL-2 BRAF-WT cells with intrinsic resistance to BRAF inhibitor. The miRNAs with at least a two-fold change in expression between BRAF inhibitor-sensitive and –resistant cell lines, were identified as differentially expressed. Averaged intensity measurements identified 138 and 217 miRNAs that were differentially expressed by 2 fold or more between: 1) A375P and A375P/Mdr; 2) A375P and SK-MEL-2, respectively. The hierarchical clustering revealed differences in miRNA expression profiles between BRAF inhibitor-sensitive and –resistant cell lines for miRNAs involved in intrinsic and acquired resistance to BRAF inhibitor. In particular, 43 miRNAs were identified whose expression was consistently altered in two BRAF inhibitor-resistant cell lines, regardless of intrinsic and acquired resistance. Twenty five miRNAs were consistently upregulated and 18 downregulated more than 2-fold. Although some discrepancies were detected when miRNA microarray data were compared with qPCR-measured expression levels, qRT-PCR for five miRNAs (miR-3617, miR-92a1, miR-1246, miR-1936-3p, and miR-17-3p) results showed excellent agreement with microarray experiments. To further investigate cellular functions of miRNAs, we examined effects on cell proliferation. Synthetic oligonucleotide miRNA mimics were transfected into three cell lines, and proliferation was quantified using a colorimetric assay. Of the 5 miRNAs tested, only miR-1246 altered cell proliferation of A375P/Mdr cells. The transfection of miR-1246 mimic strongly conferred PLX-4720 resistance to A375P/Mdr cells, implying that miR-1246 upregulation confers acquired resistance to BRAF inhibition. We also found that PLX-4720 caused much greater G2/M arrest in A375P/Mdr cells transfected with miR-1246mimic than that seen in scrambled RNA-transfected cells. Additionally, miR-1246 mimic partially caused a resistance to autophagy induction by PLX-4720. These results indicate that autophagy does play an essential death-promoting role inPLX-4720-induced cell death. Taken together, these results suggest that miRNA expression profiling in melanoma cells can provide valuable information for a network of BRAF inhibitor resistance-associated miRNAs.

Keywords: microRNA, BRAF inhibitor, drug resistance, autophagy

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Authors: Sobia Shafqat, Saeed Ahmad Qaisrani

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MicroRNAs play an essential role in the regulation and development of all processes in most eukaryotes because of their prospective part as mediators controlling cell growth and differentiation towards the exact position of RNAs response in plants under biotic and abiotic factors or stressors. In a few cases, Zn is oblivious poisonous for plants due to its heavy metal status. Some other metals are extremely toxic, like Cd, Hg, and Pb, but these elements require in rice for the programming of genes under abiotic stress resembling Zn stress when micro RNAs268 was importantly introduced in rice. The micro RNAs overexpressed in transgenic plants with an accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in the seedlings stage. Let out results for rice pliability under Zn stress micro RNAs act as negative controllers. But the role of micro RNA268 act as a modulator in different ecological condition. It has been explained clearly with a long understanding of the role of micro RNA268 under stress conditions; pliability and practically showed outcome to increase plant sufferance under Zn stress because micro RNAs is an intervention technique for gene regulation in gene expression. The proposed study was experimented with by using genetic factors of Zn stress and toxicity effect on rice plants done at District Vehari, Pakistan. The trial was performed randomly with three replications in a complete block design (RCBD). These blocks were controlled with different concentrations of genetic factors. By overexpression of micro RNA268 rice, seedling growth was not stopped under Zn deficiency due to the accumulation of a large amount of melanin dialdehyde, hydrogen peroxide, and an excessive quantity of Zn in their seedlings. Results showed that micro RNA268 act as a negative controller under Zn stress. In the end, under stress conditions, micro RNA268 showed the necessary function in the tolerance of rice plants. The directorial work sketch gave out high agronomic applications and yield outcomes in rice with a specific amount of Zn application.

Keywords: micro RNA268, zinc, rice, agronomic approach

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Authors: Mengfei Peng, Serajus Salaheen, Debabrata Biswas

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Lactobacillus casei found in the human intestine and mouth is commonly applied for dairy production. Recently, it was found that some byproducts produced by Lactobacillus exhibited antimicrobial activities against multiple bacteria. Meanwhile, introduction of prebiotic-like foods (e.g. cocoa) or probiotics or both of them as food supplements in human diets as well as in farm animal feeds is believed to be an effective ways in control/reduce the colonization of foodborne bacterial pathogens infection in the gut environment. We hypothesized that cocoa may stimulate the production antimicrobial components of Lactobacillus casei and may potentially inhibit/reduce the colonization and infection of foodborne bacterial pathogens in the gut. Mixed culture of L. casei (LC) with enterohemorrhagic E. coli EDL933 (EHEC), Salmonella Typhimurium LT2 (ST), or Listeria monocytogenes LM2 (LM) showed that LC could competitively exclude (100%) them within 72 h. Further, investigation of cell-free culture supernatant (CFCS) revealed that the antimicrobial effects of LC came from CFCS. CFCS of LC eliminated (100%) EHEC, ST, and LM within 72 h, and 2 h CFCS treatment increased the hydrophobicity of EHEC (5.10 folds), ST (8.48 folds), and LM (2.03 folds). In addition, LC cells exhibited more inhibitive effects than CFCS on cell adhesive and invasive activities of EHEC (52.14% & 90.45%), ST (66.89% & 93.83%), and LM (61.10% & 83.40%). Two clusters of poly-peptides in CFCS were identified by SDS-PAGE, the molecular weights of which are ≈5 KD and 40-45 KD. LC CFCS with overnight growth in the presence of 3% strengthened all of the antimicrobial activities (growth inhibition, outer membrane disruption, and cell infective ability reduction). Liquid chromatography/Mass spectrometry analysis detected 5 unique components in class of flavonoids in LC CFCS with overnight 3% cocoa supplement. Furthermore, qPCR results showed that CFCSs up-regulated the expression level of genes responsible for flagellin synthesis and motility, but down-regulated genes for specific binding and invasion-associated proteins synthesis. The stimulatory effects of cocoa in producing bioactive components of probiotics may aid prevention of foodborne illness caused by major foodborne enteric bacterial pathogens.

Keywords: foodborne pathogens, probiotics, prebiotics, pathogen exclusion

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Authors: Tripureshwari Paul

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We are born with nature molded by nurture. Studies have confirmed the productive role of genes and environment on an individual. This study examines the relationship of parental genotype values on the intellectual ability of their children. Keeping in mind that academic achievement-learning capacity of student through normative education, a function of exposure to family environment and pathology with intellectual quotient of the individual. Purposive sampling was used and children between ages 11 and 12 years and their respective parents were involved. Raven’s Standard Progressive Matrices (RSPM), Family Pathology Scale (FPS) and Family Environment Scale (FES) were administered. The results found significant relationship of Offspring IQ to Parental IQ, maternal IQ demonstrating higher values of correlation. Female IQ was significant to maternal IQ and male IQ was significant to paternal IQ. With Academic Achievement not significantly correlated to IQ, it was determined that Competitive framework, freedom to expression and Recreational Orientation in family affect a child’s intellectual performance.

Keywords: academic achievement, environment, family environment, family pathology, genotype, intelligence quotient, maternal IQ, paternal IQ

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Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

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5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN

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Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

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Authors: Anca Dinischiotu, Sorina Nicoleta Voicu

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Silica nanoparticles (SiO2-NPs) are widely used in consumer products such as paints, plastics, insulation materials, tires, concrete production, as well as in gene delivery systems and imaging procedures. Environmental human exposure to them occurs during utilization of these products, in a time-dependent manner, the uptake being by topic and inhalation route especially. SiO2-NPs enter cells and induce membrane damage, oxidative stress and inflammatory reactions in a concentration-dependent manner. In this study, MRC-5 cells (human fetal lung fibroblasts) were exposed to amorphous SiO2-NPs at a dose of 62.5 μg/ml for 24, 48 and 72 hours. The size distribution of NPs was a lognormal function, in the range 3-14 nm. A time-dependent decrease of total reduced glutathione concentration by 36%, 50%, and 78% and an increase of NO level by 62%, 32%, respectively 24% compared to control were noticed. An up-regulation of NF-kB expression by 20%, 50% respectively 10% and of Nrf-2 by 139%, 58%, and 16% compared to control after 24, 48 and 72 hours was noticed also. The expression of IL-1β, IL-6, IL-8, and COX-2 was up-regulated in a time-dependent manner. Also, the expression of MMP-2 and MMP-9 were down-regulated after 48 and 72 hours, whereas their activities raised in a time-dependent manner. Exposure of cells to NPs up-regulated the expression of inducible NO synthase, as previously was shown, and probably this is the reason for the increased level of NO, that can react with the thiol groups of reduced glutathione molecules, diminishing its concentration Nrf2 is a transcription factor translocated in nucleus, under oxidative stress, where downstream gene expression activates in order to modulate the adaptive intracellular response against oxidative stress. The cross-talk between Nrf2 and NF-kB activities regulates the inflammatory processes. The activation of NF-kB could activate up-regulation of IL-1β, IL-6, and IL-8. The increase of COX-2 expression could be correlated with IL-1β one. Also, probably in response to the pro-inflammatory cytokines, MMP-2 and MMP-9 were induced and activated. In conclusion, the exposure of MRC-5 cells to SiO2-NPs generated inflammation in a time-dependent manner.

Keywords: inflammation, MRC-5 cells, oxidative stress, silica nanoparticles

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Authors: Hasanah Md. Amin

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The existence of cyberspace and its growing contents is real and overwhelming. Visual as one of the properties of cyber contents is increasingly becoming more significant and popular among creator and user. The visual and aesthetic of the content is consistent with many similarities. Aesthetic, although universal, has slight differences across the world. Aesthetic power could impress, influence, and cause bias among the users. The content creator who knows how to manipulate this visuals and aesthetic expression can dominate the scenario and the user who is ‘expressive literate’ will gain much from the scenes. User who understands aesthetic will be rewarded with competence, confidence, and certainly, a personality enhanced experience in carrying out a task when participating in this chaotic but promising cyberworld. The aim of this article is to gain knowledge from related literature and research regarding User-Generated Content (UGC), which focuses on aesthetic expression by the Net generation. The objective of this preliminary study is to analyze the aesthetic expression linked to visual from the participatory cultural viewpoint looking for meaning, value, patterns, and characteristics.

Keywords: visual overloaded, user-generated content, net generation, visual arts

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Authors: S. H. Gasa, L. Singh, B. Pillay, A. O. Olaniran

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Wastewater treatment plants (WWTPs) have been implicated as the leading reservoir for antibiotic resistant bacteria (ARB), including Enterococci spp. and antibiotic resistance genes (ARGs), worldwide. Enterococci are a group of clinically significant bacteria that have gained much attention as a result of their antibiotic resistance. They play a significant role as the principal cause of nosocomial infections and dissemination of antimicrobial resistance genes in the environment. The main objective of this study was to ascertain the role of WWTPs in Durban, South Africa as potential reservoirs for antibiotic resistant Enterococci (ARE) and their related ARGs. Furthermore, the antibiogram and resistance gene profile of Enterococci species recovered from treated wastewater effluent and receiving surface water in Durban were also investigated. Using membrane filtration technique, Enterococcus selective agar and selected antibiotics, ARE were enumerated in samples (influent, activated sludge, before chlorination and final effluent) collected from two WWTPs, as well as from upstream and downstream of the receiving surface water. Two hundred Enterococcus isolates recovered from the treated effluent and receiving surface water were identified by biochemical and PCR-based methods, and their antibiotic resistance profiles determined by the Kirby-Bauer disc diffusion assay, while PCR-based assays were used to detect the presence of resistance and virulence genes. High prevalence of ARE was obtained at both WWTPs, with values reaching a maximum of 40%. The influent and activated sludge samples contained the greatest prevalence of ARE with lower values observed in the before and after chlorination samples. Of the 44 vancomycin and high-level gentamicin-resistant isolates, 11 were identified as E. faecium, 18 as E. faecalis, 4 as E. hirae while 11 are classified as “other” Enterococci species. High-level aminoglycoside resistance for gentamicin (39%) and vancomycin (61%) was recorded in species tested. The most commonly detected virulence gene was the gelE (44%), followed by asa1 (40%), while cylA and esp were detected in only 2% of the isolates. The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(3')-IIIa, and ant(6')-Ia detected in 43%, 45% and 41% of the isolates, respectively. Positive correlation was observed between resistant phenotypes to high levels of aminoglycosides and presence of all aminoglycoside resistance genes. Resistance genes for glycopeptide: vanB (37%) and vanC-1 (25%), and macrolide: ermB (11%) and ermC (54%) were detected in the isolates. These results show the need for more efficient wastewater treatment and disposal in order to prevent the release of virulent and antibiotic resistant Enterococci species and safeguard public health.

Keywords: antibiogram, enterococci, gentamicin, vancomycin, virulence signatures

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Authors: Lu Yang, Keng Po Lai

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Metformin, a well-known clinical drug against diabetes, is found with potential anti-diabetic and anti-obese benefits, as reported in increasing evidences. However, the current clinical and experimental investigations are not to reveal the detailed mechanisms of metformin-anti-obesity/hypertension. We have used the bioinformatics strategy, including network pharmacology and molecular docking methodology, to uncover the key targets and pathways of bioactive compounds against clinical disorders, such as cancers, coronavirus disease. Thus, in this report, the in-silico approach was utilized to identify the hug targets, pharmacological function, and mechanism of metformin against obesity and hypertension. The networking analysis identified 154 differentially expressed genes of obesity and hypertension, 21 interaction genes, and 6 hug genes of metformin treating hypertensive obesity. As a result, the molecular docking findings indicated the potent binding capability of metformin with the key proteins, including interleukin 6 (IL-6) and chemokine (C-C motif) Ligand 2 (CCL2), in hypertensive obesity. The metformin-exerted anti-hypertensive obesity action involved in metabolic regulation, inflammatory reaction. And the anti-hypertensive obesity mechanisms of metformin were revealed, including regulation of inflammatory and immunological signaling pathways for metabolic homeostasis in tissue and microenvironmental melioration in blood pressure. In conclusion, our identified findings with bioinformatics analysis have demonstrated the detailed hug and pharmacological targets, biological functions, and signaling pathways of metformin treating hypertensive obesity.

Keywords: metformin, obesity, hypertension, bioinformatics findings

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Authors: Shan-Ying Wu, Sheng-Hui Lan, Xi-Zhang Lin, Ih-Jen Su, Ting-Fen Tsai, Chia-Jui Yen, Tsung-Hsueh Lu, Fu-Wen Liang, Huey-Jen Su, Chun-Li Su, Hsiao-Sheng Liu

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In hepatocelluar carcinoma (HCC), dysregulated expression of cyclin D1 and impaired autophagy has been reported separately. However, the relationship between them has not been explored. In this study, we demonstrated that autophagy was inversely correlated with cyclin D1 expression in 147 paired HCC patient specimens. HCC specimen with highly expression of cyclin D1 shows correlation with poor overall survival rate. Furthermore, induction of autophagy by amiodarone (antiarrhythmic drug) in Hep 3B cells, cyclin D1 was recruited into autophagosomes demonstrated by immune-gold labeling of cyclin D1 after extraction of autophagosomes. We further demonstrated that autophagy suppresses Hep 3B cell proliferation, and further analysis revealed that cell cycle was arrested at G1 phase. The interaction between LC3 (maker of autophagy) and cyclin D1 was increased after autophagy induction. In addition, ubiquitinated-cyclin D1 was also increased after autophagy induction, which is selectively degraded by autophagosome through binding with SQSTM1/p62 (an adaptor protein). In vivo study showed that amiodarone induced autophagy suppresses liver tumor formation in xenograft mouse and orthotopic rat model through decreasing cyclin D1 expression and inhibition of cell proliferation. Altogether, we reveal a novel mechanism that ubiquitinated cyclin D1 degraded by autophagic pathway by p62 and amiodarone is a promising drug for targeting cyclin D1 in liver cancer therapy.

Keywords: autophagy, cyclin D1, hepatocellular carcinoma, amiodarone

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Authors: Abdoulie K. Ceesay

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Within the sequence of the whole genome, it is known that 99.9% of the human genome is similar, whilst our difference lies in just 0.1%. Among these minor dissimilarities, the most common type of genetic variations that occurs in a population is SNP, which arises due to nucleotide substitution in a protein sequence that leads to protein destabilization, alteration in dynamics, and other physio-chemical properties’ distortions. While causing variations, they are equally responsible for our difference in the way we respond to a treatment or a disease, including various cancer types. There are two types of SNPs; synonymous single nucleotide polymorphism (sSNP) and non-synonymous single nucleotide polymorphism (nsSNP). sSNP occur in the gene coding region without causing a change in the encoded amino acid, while nsSNP is deleterious due to its replacement of a nucleotide residue in the gene sequence that results in a change in the encoded amino acid. Predicting the effects of cancer related nsSNPs on protein stability, function, and dynamics is important due to the significance of phenotype-genotype association of cancer. In this thesis, Data of 5 oncogenes (ONGs) (AKT1, ALK, ERBB2, KRAS, BRAF) and 5 tumor suppressor genes (TSGs) (ESR1, CASP8, TET2, PALB2, PTEN) were retrieved from ClinVar. Five common in silico tools; Polyphen, Provean, Mutation Assessor, Suspect, and FATHMM, were used to predict and categorize nsSNPs as deleterious, benign, or neutral. To understand the impact of each variation on the phenotype, Maestro, PremPS, Cupsat, and mCSM-NA in silico structural prediction tools were used. This study comprises of in-depth analysis of 10 cancer gene variants downloaded from Clinvar. Various analysis of the genes was conducted to derive a meaningful conclusion from the data. Research done indicated that pathogenic variants are more common among ONGs. Our research also shows that pathogenic and destabilizing variants are more common among ONGs than TSGs. Moreover, our data indicated that ALK(409) and BRAF(86) has higher benign count among ONGs; whilst among TSGs, PALB2(1308) and PTEN(318) genes have higher benign counts. Looking at the individual cancer genes predisposition or frequencies of causing cancer according to our research data, KRAS(76%), BRAF(55%), and ERBB2(36%) among ONGs; and PTEN(29%) and ESR1(17%) among TSGs have higher tendencies of causing cancer. Obtained results can shed light to the future research in order to pave new frontiers in cancer therapies.

Keywords: tumor suppressor genes (TSGs), oncogenes (ONGs), non synonymous single nucleotide polymorphism (nsSNP), single nucleotide polymorphism (SNP)

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Authors: Feng Ji Mervin Goh, Edmund Chee Jian Pua, Stuart Alexander Cook

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Dilated cardiomyopathy (DCM) is a common cause of heart failure. Genetic mutations account for 50% of DCM cases with TTN mutations being the most common, accounting for up to 25% of DCM cases. However, the genetic architecture underlying Asian DCM patients is unknown. We evaluated 68 patients (female= 17) with DCM who underwent follow-up at the National Heart Centre, Singapore from 2013 through 2014. Clinical data were obtained and analyzed retrospectively. Genomic DNA was subjected to next-generation targeted sequencing. Nextera Rapid Capture Enrichment was used to capture the exons of a panel of 169 cardiac genes. DNA libraries were sequenced as paired-end 150-bp reads on Illumina MiSeq. Raw sequence reads were processed and analysed using standard bioinformatics techniques. The average age of onset of DCM was 46.1±10.21 years old. The average left ventricular ejection fraction (LVEF), left ventricular diastolic internal diameter (LVIDd), left ventricular systolic internal diameter (LVIDs) were 26.1±11.2%, 6.20±0.83cm, and 5.23±0.92cm respectively. The frequencies of mutations in major DCM-associated genes were as follows TTN (5.88% vs published frequency of 20%), LMNA (4.41% vs 6%), MYH7 (5.88% vs 4%), MYH6 (5.88% vs 4%), and SCN5a (4.41% vs 3%). The average callability at 10 times coverage of each major gene were: TTN (99.7%), LMNA (87.1%), MYH7 (94.8%), MYH6 (95.5%), and SCN5a (94.3%). In conclusion, TTN mutations are not common in Singaporean DCM patients. The frequencies of other major DCM-associated genes are comparable to frequencies published in the current literature.

Keywords: heart failure, dilated cardiomyopathy, genetics, next-generation sequencing

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Authors: Haveesha Buddhdev

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The most important right for a human being in a society is the freedom of expression as stated by Article 18 and 19 of the Universal Declaration of Human rights pledged by member states of United Nations. Will it be fair to expect him/her to be of sound mental health if this right is taken away? Religion as a primary social institution controls many rights, freedoms and duties of people in a society. It does so by imposing certain values and beliefs on people which would either enhance quality of life or curb their freedom adversely thus affecting individual mental health. This paper aims to study the positive and negative role that religion plays in influencing one’s freedom of expression. This paper will focus on reviewing existing studies on the positive and negative impacts of religion on mental health. It will also contain data collected by the researcher about the impacts of religion on freedom of expression which will be obtained by surveying a sample of 30 adolescents and young adults. The researcher will use a Likert scale for these purpose, with response options ranging from strongly disagree to strongly agree and quantify it accordingly. Descriptive statistics would be used to analyse the data. Such research would help to identify possible problems faced by adolescents and young adults when it comes to religio-cultural ethos and also facilitate further researches to study the role that religion plays in mental health.

Keywords: cultural Ethos, freedom of expression, adolescent mental health, social science

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Authors: Shahid Hussain Abro, Siamak Zohari, Lena H. M. Renström, Désirée S. Jansson, Faruk Otman, Karin Ullman, Claudia Baule

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Infectious bronchitis is an acute, highly contagious respiratory, nephropathogenic and reproductive disease of poultry that is caused by infectious bronchitis virus (IBV). The present study used a large data set of structural gene sequences, including newly generated ones and sequences available in the GenBank database to further analyze the diversity and to identify selective pressures and recombination spots. There were some deletions or insertions in the analyzed regions in isolates of the Italy-02 and D274 genotypes. Whereas, there were no insertions or deletions observed in the isolates of the Massachusetts and 4/91 genotype. The hypervariable nucleotide sequence regions spanned positions 152–239, 554–582, 686–737 and 802–912 in the S1 sub-unit of the all analyzed genotypes. The nucleotide sequence data of the E gene showed that this gene was comparatively unstable and subjected to a high frequency of mutations. The M gene showed substitutions consistently distributed except for a region between nucleotide positions 250–680 that remained conserved. The lowest variation in the nucleotide sequences of ORF5a was observed in the isolates of the D274 genotype. While, ORF5b and N gene sequences showed highly conserved regions and were less subjected to variation. Genes ORF3a, ORF3b, M, ORF5a, ORF5b and N presented negative selective pressure among the analyzed isolates. However, some regions of the ORFs showed favorable selective pressure(s). The S1 and E proteins were subjected to a high rate of mutational substitutions and non-synonymous amino acids. Strong signals of recombination breakpoints and ending break point were observed in the S and N genes. Overall, the results of this study revealed that very likely the strong selective pressures in E, M and the high frequency of substitutions in the S gene can probably be considered the main determinants in the evolution of IBV.

Keywords: IBV, avian infectious bronchitis, structural genes, genotypes, genetic diversity

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Authors: Seong Gu Hwang, Young Kyoon Oh, Joseph F. dela Cruz

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Marbling, or intramuscular fat, has been consistently identified as one of the top beef quality problems. Intramuscular adipocytes distribute throughout the perimysial connective tissue of skeletal muscle and are the major site for the deposition of intramuscular fat, which is essential for the eating quality of meat. The stromal vascular fraction of the skeletal muscle contains progenitor cells that can be enhanced to differentiate to adipocytes and increase intramuscular fat. Primary cultures of bovine intramuscular stromal vascular cells were used in this study to elucidate the effects of extracellular calcium and retinoic acid concentration on adipocyte differentiation. Cell viability assay revealed that even at different concentrations of calcium and retinoic acid, there was no significant difference on cell viability. Monitoring of the adipocyte differentiation showed that bovine intramuscular stromal vascular cells cultured in a low concentration of extracellular calcium and retinoic acid had a better degree of fat accumulation. The mRNA and protein expressions of PPARγ, C/EBPα, SREBP-1c and aP2 were analyzed and showed a significant upregulation upon the reduction in the level of extracellular calcium and retinoic acid. The upregulation of these adipogenic related genes means that the decreasing concentration of calcium and retinoic acid is able to stimulate the adipogenic differentiation of bovine intramuscular stromal vascular cells. To further elucidate the effect of calcium, the expression level of calreticulin was measured. Calreticulin which is known to be an inhibitor of PPARγ was down regulated by the decreased level of calcium and retinoic acid in the culture media. The same tendency was observed on retinoic acid receptors RARα and CRABP-II. These receptors are recognized as adipogenic inhibitors, and the downregulation of their expression allowed a better level of differentiation in bovine intramuscular stromal vascular cells. In conclusion, data show that decreasing the level of extracellular calcium and retinoic acid can significantly promote adipogenesis in intramuscular stromal vascular cells of Hanwoo beef cattle. These findings may provide new insights in enhancing intramuscular adipogenesis and marbling in beef cattle.

Keywords: calcium, calreticulin, hanwoo beef, retinoic acid

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Authors: Precious Barnes, Abraham Mensah, Leonard Derkyi-Kwarteng, Benjamin Amoani, George Adjei, Ernest Adankwah, Faustina Pappoe, Kwabena Dankwah, Daniel Amoako-Sakyi, Samuel Victor Nuvor, Dorcas Obiri-Yeboah, Ewura Seidu Yahaya, Patrick Kafui Akakpo, Roland Osei Saahene

Abstract:

Context: Breast cancer is a prevalent and aggressive type of cancer among African women, with high mortality rates in Ghana. Nuclear factor kappa B (NF-kB) is a transcription factor that has been associated with tumor progression in breast cancer. However, there is a lack of published data on NF-kB in breast cancer patients in Ghana or other African countries. Research Aim: The aim of this study was to assess the prognostic significance of NF-kB (p65) expression and its association with various clinicopathological features in breast cancer patients at the Cape Coast Teaching Hospital in Ghana. Methodology: A total of 90 formalin-fixed breast cancer tissues and 15 normal breast tissues were used in this study. The expression level of NF-kB (p65) was examined using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Findings: The study found that NF-kB (p65) was expressed in 86.7% of breast cancer tissues. There was a significant relationship between NF-kB (p65) expression and tumor grade, proliferation index (Ki67), and molecular subtype. High-level expression of NF-kB (p65) was more common in tumor grade 3 compared to grade 1, and Ki67 > 20 had higher expression of NF-kB (p65) compared to Ki67 ≤ 20. Triple-negative breast cancer patients had the highest overexpression of NF-kB (p65) compared to other molecular subtypes. There was no significant association between NF-kB (p65) expression and other clinicopathological parameters. Theoretical Importance: This study provides important insights into the expression of NF-kB (p65) in breast cancer patients in Ghana, particularly in relation to tumor grade and proliferation index. The findings suggest that NF-kB (p65) could serve as a potential biological marker for cancer stage, progression, prognosis and as a therapeutic target. Data Collection and Analysis Procedures: Formalin-fixed breast cancer tissues and normal breast tissues were collected and analyzed using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Question Addressed: This study addressed the question of the prognostic significance of NF-kB (p65) expression and its association with clinicopathological features in breast cancer patients in Ghana. Conclusion: This study, the first of its kind in Ghana, demonstrates that NF-kB (p65) is highly expressed among breast cancer patients at the Cape Coast Teaching Hospital, especially in triple-negative breast cancer patients. The expression of NF-kB (p65) is associated with tumor grade and proliferation index. NF-kB (p65) could potentially serve as a biological marker for cancer stage, progression, prognosis, and as a therapeutic target.

Keywords: breast cancer, Ki67, NF-kB (p65), tumor grade

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Authors: Yen-Ni Teng, Hsing-Yi Chen, Hsien-An Pan, Yung-Ming Lin, Hany A. Omar, Jui-Hsiang Hung

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Leucine-rich repeats and WD repeat domain containing 1 (LRWD1) is highly expressed in the testes of healthy males. On the other hand, LRWD1 is significantly down-regulated in the testicular tissues of patients with severe spermatogenic defects. In our study, the downregulation of LRWD1 expression by shRNA caused a significant reduction of cell growth and mitosis and a noteworthy increase in the cell microtubule atrophy rate. Here, we used EMBOSS CpG plot analysis to explore the promoter region of LRWD1 gene. We found that CpG islands are located between positions -253 to +5 nucleotides upstream from the LRWD1 transcription start site. Luciferase reporter assay revealed that the hypermethylation of the LRWD1 promoter reduced the transcription activity in cells. In addition, quantitative methylation-specific PCR and immunostaining showed that the methylation inhibitor, 5-Aza-2'-deoxycytidine, increased LRWD1 promoter activity, LRWD1 mRNA, protein expression and cell viability. Whereas, the methylation activator, S-adenosylmethionine, caused opposite effects. The overexpression of p53 and Nrf2 in NT2/D1 cells increased LRWD1 promoter activity while 5-fluorodeoxyuridine decreased it. In conclusion, this study highlights evidence that the methylation status of LRWD1 promoter is associated with LRWD1 expression. Since the expression level of LRWD1 plays an important role in spermatogenesis, the methylation status of LRWD1 may serve as a novel molecular diagnostic or therapeutic approach in male's infertility.

Keywords: LRWD1, DNA methylation, p53, Nrf2

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