Search results for: tissue specific genes

Authors: Ryeong-Hyeon Kim, Ah-Reum Lee, Seong-Soo Roh, Gyo-Nam Kim

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Appropriate regulation of melanogenesis is important for the management of skin pigmentation-related disease. Although several beneficial effects of fisetin (3,7,3’,4’-tetrahydroxyflavone) have been reported, the precise role and molecular mechanisms of fisetin in skin health both remain unclear. Here, we induced melanogenesis of HRM2 mice (n=7/group) by UVB irradiation for 20 days. UVB-induced HRM2 mice showed that the significantly increased melanin accumulation, however, fisetin treatment (25mg and 50mg/kg of body weight) dose-dependently and significantly inhibits UVB-induced melanogenesis. In line with this, fisetin treatment effectively down-regulated m RNA and expression levels of tyrosinase, TRP2, and MITF. In addition, our inhibitor assay revealed the down-regulated melanogenic marker genes by fisetin treatment were mediated with connective tissue growth factor (CCN2)/TGF-β signaling pathway. Useful information is provided for development of functional foods using fisetin for skin health.

Keywords: connective tissue growth factor, fisetin, melanogenesis, skin, TGF-beta

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Authors: Charalabos Antonatos, Mariza Panoutsopoulou, Georgios K. Georgakilas, Evangelos Evangelou, Yiannis Vasilopoulos

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The extended tissue damage and severe clinical outcomes of autoimmune diseases, accompanied by the high annual costs to the overall health care system, highlight the need for an efficient therapy. Increasing knowledge over the pathophysiology of specific chronic inflammatory diseases, namely Psoriasis (PsO), Inflammatory Bowel Diseases (IBD) consisting of Crohn’s disease (CD) and Ulcerative colitis (UC), and Rheumatoid Arthritis (RA), has provided insights into the underlying mechanisms that lead to the maintenance of the inflammation, such as Tumor Necrosis Factor alpha (TNF-α). Hence, the anti-TNFα biological agents pose as an ideal therapeutic approach. Despite the efficacy of anti-TNFα agents, several clinical trials have shown that 20-40% of patients do not respond to treatment. Nowadays, high-throughput technologies have been recruited in order to elucidate the complex interactions in multifactorial phenotypes, with the most ubiquitous ones referring to transcriptome quantification analyses. In this context, a random effects meta-analysis of available gene expression cDNA microarray datasets was performed between responders and non-responders to anti-TNFα therapy in patients with IBD, PsO, and RA. Publicly available datasets were systematically searched from inception to 10th of November 2020 and selected for further analysis if they assessed the response to anti-TNFα therapy with clinical score indexes from inflamed biopsies. Specifically, 4 IBD (79 responders/72 non-responders), 3 PsO (40 responders/11 non-responders) and 2 RA (16 responders/6 non-responders) datasetswere selected. After the separate pre-processing of each dataset, 4 separate meta-analyses were conducted; three disease-specific and a single combined meta-analysis on the disease-specific results. The MetaVolcano R package (v.1.8.0) was utilized for a random-effects meta-analysis through theRestricted Maximum Likelihood (RELM) method. The top 1% of the most consistently perturbed genes in the included datasets was highlighted through the TopConfects approach while maintaining a 5% False Discovery Rate (FDR). Genes were considered as Differentialy Expressed (DEGs) as those with P ≤ 0.05, |log2(FC)| ≥ log2(1.25) and perturbed in at least 75% of the included datasets. Over-representation analysis was performed using Gene Ontology and Reactome Pathways for both up- and down-regulated genes in all 4 performed meta-analyses. Protein-Protein interaction networks were also incorporated in the subsequentanalyses with STRING v11.5 and Cytoscape v3.9. Disease-specific meta-analyses detected multiple distinct pro-inflammatory and immune-related down-regulated genes for each disease, such asNFKBIA, IL36, and IRAK1, respectively. Pathway analyses revealed unique and shared pathways between each disease, such as Neutrophil Degranulation and Signaling by Interleukins. The combined meta-analysis unveiled 436 DEGs, 86 out of which were up- and 350 down-regulated, confirming the aforementioned shared pathways and genes, as well as uncovering genes that participate in anti-inflammatory pathways, namely IL-10 signaling. The identification of key biological pathways and regulatory elements is imperative for the accurate prediction of the patient’s response to biological drugs. Meta-analysis of such gene expression data could aid the challenging approach to unravel the complex interactions implicated in the response to anti-TNFα therapy in patients with PsO, IBD, and RA, as well as distinguish gene clusters and pathways that are altered through this heterogeneous phenotype.

Keywords: anti-TNFα, autoimmune, meta-analysis, microarrays

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Authors: S. S. Salehi, A. Shamloo

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Poor ability of cartilage tissue when experiencing a damage leads scientists to use tissue engineering as a reliable and effective method for regenerating or replacing damaged tissues. An artificial tissue should have some features such as biocompatibility, biodegradation and, enough mechanical properties like the original tissue. In this work, a composite hydrogel is prepared by using natural and synthetic materials that has high porosity. Mechanical properties of different combinations of polymers such as modulus of elasticity were tested, and a hydrogel with good mechanical properties was selected. Bone marrow derived mesenchymal stem cells were also seeded into the pores of the sponge, and the results showed the adhesion and proliferation of cells within the hydrogel after one month. In comparison with previous works, this study offers a new and efficient procedure for the fabrication of cartilage like tissue and further cartilage repair.

Keywords: cartilage tissue engineering, hydrogel, mechanical strength, mesenchymal stem cell

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Authors: Jianhua Zhang, Weiping Chen, Guanjie Chen, Jason Flannick, Emma Fikse, Glenda Smerin, Yanqin Yang, Yulong Li, John A. Hanover, William F. Simonds

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Ethnic disparities in many diseases are well recognized and reflect the consequences of genetic, behavior, and environmental factors. However, direct scientific evidence connecting the ethnic genetic variations and the disease disparities has been elusive, which may have led to the ethnic inequalities in large scale genetic studies. Through the genome-wide analysis of data representing 185,934 subjects, including 14,955 from our own studies of the African America Diabetes Mellitus, we discovered sets of genetic variants either unique to or conserved in all ethnicities. We further developed a quantitative gene function-based high-risk variant index (hrVI) of 20,428 genes to establish profiles that strongly correlate with the subjects' self-identified ethnicities. With respect to the ability to detect human essential and pathogenic genes, the hrVI analysis method is both comparable with and complementary to the well-known genetic analysis methods, pLI and VIRlof. Application of the ethnicity-specific hrVI analysis to the type 2 diabetes mellitus (T2DM) national repository, containing 20,791 cases and 24,440 controls, identified 114 candidate T2DM-associated genes, 8.8-fold greater than that of ethnicity-blind analysis. All the genes identified are defined as either pathogenic or likely-pathogenic in ClinVar database, with 33.3% diabetes-associated and 54.4% obesity-associated genes. These results demonstrate the utility of hrVI analysis and provide the first genetic evidence by clustering patterns of how genetic variations among ethnicities may impede the discovery of diabetes and foreseeably other disease-associated genes.

Keywords: diabetes-associated genes, ethnic health disparities, high-risk variant index, hrVI, T2DM

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Authors: Ghulam Muhammad Ali

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Unveiling of genes involved in drought and root architecture using transcriptomic analyses remained fragmented for further improvement of wheat through genome editing. The purpose of this research endeavor was to unveil the variations in different genes implicated in drought tolerance and root architecture in wheat through RNA-seq data analysis. In this study seedlings of 8 days old, 6 cultivars of wheat namely, Batis, Blue Silver, Local White, UZ888, Chakwal 50 and Synthetic wheat S22 were subjected to transcriptomic analysis for root and shoot genes. Total of 12 RNA samples was sequenced by Illumina. Using updated wheat transcripts from Ensembl and IWGC references with 54,175 gene models, we found that 49,621 out of 54,175 (91.5%) genes are expressed at an RPKM of 0.1 or more (in at least 1 sample). The number of genes expressed was higher in Local White than Batis. Differentially expressed genes (DEG) were higher in Chakwal 50. Expression-based clustering indicated conserved function of DRO1and RPK1 between Arabidopsis and wheat. Dendrogram showed that Local White is sister to Chakwal 50 while Batis is closely related to Blue Silver. This study flaunts transcriptomic sequence variations in different cultivars that showed mutations in genes associated with drought that may directly contribute to drought tolerance. DRO1 and RPK1 genes were fetched/isolated for genome editing. These genes are being edited in wheat through CRISPR-Cas9 for yield enhancement.

Keywords: transcriptomic, wheat, genome editing, drought, CRISPR-Cas9, yield enhancement

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Authors: Fulufhelo Amanda Doboro, Kgomotso Sebeko, Stephen Njiro, Moritz Van Vuuren

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Ovine herpesvirus 2 (OvHV-2) genome obtained from the lymphopblastoid cell line of a BJ1035 cow was recently sequenced in the United States of America (USA). Information on the sequences of OvHV-2 genes obtained from South African strains from bovine or other African countries and molecular characterization of OvHV-2 is not documented. Present investigation provides information on the nucleotide and derived amino acid sequences and genetic diversity of Ov 7, Ov 8 ex2, ORF 27 and ORF 73 genes, of these genes from OvHV-2 strains circulating in South Africa. Gene-specific primers were designed and used for PCR of DNA extracted from 42 bovine blood samples that previously tested positive for OvHV-2. The expected PCR products of 495 bp, 253 bp, 890 bp and 1632 bp respectively for Ov 7, Ov 8 ex2, ORF 27 and ORF 73 genes were sequenced and multiple sequence analysis done on the selected regions of the sequenced PCR products. Two genotypes for ORF 27 and ORF 73 gene sequences, and three genotypes for Ov 7 and Ov 8 ex2 gene sequences were identified, and similar groupings for the derived amino acid sequences were obtained for each gene. Nucleotide and amino acid sequence variations that led to the identification of the different genotypes included SNPs, deletions and insertions. Sequence analysis of Ov 7 and ORF 27 genes revealed variations that distinguished between sequences from SA and reference OvHV-2 strains. The implication of geographic origin among SA sequences was difficult to evaluate because of random distribution of genotypes in the different provinces, for each gene. However, socio-economic factors such as migration of people with animals, or transportation of animals for agricultural or business use from one province to another are most likely to be responsible for this observation. The sequence variations observed in this study have no impact on the antibody binding activities of glycoproteins encoded by Ov 7, Ov 8 ex2 and ORF 27 genes, as determined by prediction of the presence of B cell epitopes using BepiPred 1.0. The findings of this study will be used for selection of gene candidates for the development of diagnostic assays and vaccine development as well.

Keywords: amino acid, genetic diversity, genes, nucleotide

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Authors: Masoud Soltanialvar, Ali Bagherpour

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Documented cases of human infection with H9N2 avian influenza viruses, first detected in 1999 in Hong Kong and China, indicate that these viruses can be directly transmitted from birds to humans. In this study, we characterized the mutation in the Hemagglutinin (HA) genes and proteins that correlates with a shift in affinity of the Hemagglutinin (HA) protein from the “avian” type sialic receptors to the “human” type in 10 Iranian isolates. We delineated the genomes and receptor binding profile of HA gene of some field isolates and established their phylogenetic relationship to the other Asian H9N2 sub lineages. A total of 1200 tissue samples collected from 40 farms located in various states of Iran during 2008 – 2010 as part of a program to monitor Avian Influenza Viruses (AIV) infection. To determine the genetic relationship of Iranian viruses, the Hemagglutinin (HA) genes from ten isolates were amplified and sequenced (by RT-PCR method). Nucleotide sequences (orf) of the (HA) genes were used for phylogenetic tree construction. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) in all ten Iranian isolates which indicates a preference to binding of α (2–6) sialic acid receptors, so these Iranian H9N2 viruses have the potential to infect human beings. These isolates showed high degree of homology with 2 human H9N2 isolates A/HK/1073/99, A/HK/1074/99. Phylogenetic analysis of showed that all the HA genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage which had contributed as donor of six internal genes to H5N1 highly pathogenic avian influenza. The results of this study indicated that all Iranian viruses have the potential to emerge as highly pathogenic influenza virus, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.

Keywords: influenza virus, hemagglutinin, neuraminidase, Iran

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Authors: Ki-Yeo Kim

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Trends in genetics are transforming in order to identify differential coexpressions of correlated gene expression rather than the significant individual gene. Moreover, it is known that a combined biomarker pattern improves the discrimination of a specific cancer. The identification of the combined biomarker is also necessary for the early detection of invasive oral squamous cell carcinoma (OSCC). To identify the combined biomarker that could improve the discrimination of OSCC, we explored an appropriate number of genes in a combined gene set in order to attain the highest level of accuracy. After detecting a significant gene set, including the pre-defined number of genes, a combined expression was identified using the weights of genes in a gene set. We used the Principal Component Analysis (PCA) for the weight calculation. In this process, we used three public microarray datasets. One dataset was used for identifying the combined biomarker, and the other two datasets were used for validation. The discrimination accuracy was measured by the out-of-bag (OOB) error. There was no relation between the significance and the discrimination accuracy in each individual gene. The identified gene set included both significant and insignificant genes. One of the most significant gene sets in the classification of normal and OSCC included MMP1, SOCS3 and ACOX1. Furthermore, in the case of oral dysplasia and OSCC discrimination, two combined biomarkers were identified. The combined genomic expression achieved better performance in the discrimination of different conditions than in a single significant gene. Therefore, it could be expected that accurate diagnosis for cancer could be possible with a combined biomarker.

Keywords: oral squamous cell carcinoma, combined biomarker, microarray dataset, correlated genes

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Authors: Hyuk Sang Yoo, Young Ju Son, Wei Mao, Myung Gu Kang, Sol Lee

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Biomedical applications of electrospun nanofibrous meshes have been received tremendous attentions because of their unique structures and versatilities as biomaterials. Incorporation of growth factors in fibrous meshes can be performed by surface-modification and encapsulation. Those growth factors stimulate differentiation and proliferation of specific types of cells and thus lead tissue regenerations of specific cell types. Topographical cues of electrospun nanofibrous meshes also increase differentiation of specific cell types according to alignments of fibrous structures. Wound healing treatments of diabetic ulcers were performed using nanofibrous meshes encapsulating multiple growth factors. Aligned nanofibrous meshes and those with random configuration were compared for differentiating mesenchymal stem cells into neuronal cells. Thus, nanofibrous meshes can be applied to drug delivery carriers and matrix for promoting cellular proliferation.

Keywords: nanofiber, tissue, mesh, drug

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Authors: Evans Iyamu

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Genes encoding hydrophobins play distinct roles at different stages of the life cycle of fungi, and they foster hyphal attachment to surfaces. The hydrophobin Sc4 is known to provide a hydrophobic membrane lining of the gas channels within Schizophyllum commune fruiting bodies. Here, we cultivated non-fruiting, monokaryotic S. commune 12-43 on glass surfaces that could be verified by micrography. Differential gene expression profiling of nine hydrophobin genes and the hydrophobin-like sc15 gene by quantitative PCR showed significant up-regulation of sc4 when S. commune was attached to glass surfaces, also confirmed with RNA-Seq data analysis. Another silicate, namely quartz sand, was investigated, and induction of sc4 was seen as well. The up-regulation of the hydrophobin gene sc4 may indicate involvement in S. commune hyphal attachment to glass as well as quartz surfaces. We propose that the covering of hyphae by Sc4 allows for direct interaction with the hydrophobic surfaces of silicates and that differential functions of specific hydrophobin genes depend on the surface interface involved. This study could help with the clarification of the biological functions of hydrophobins in natural surroundings, including hydrophobic surface attachment. Therefore, the analysis of growth on glass serves as a basis for understanding S. commune interaction with glass surfaces while providing the possibility to visualize the interaction microscopically.

Keywords: hydrophobin, structured glass surfaces, differential gene expression, quartz sand

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Authors: Ariadna Jalife Gómez, Claudia Rangel Escareño

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The Acropora coral genus has experienced impactful consequences of climate change, especially in terms of population reduction related to limited thermal tolerance, however, comprehensive resources for genetic responses of these corals to phenomena are lacking. Thus, this study aims to identify key genes expressed across different developmental stages and conditions of Acropora spp. highlighted in published studies given the shared tissue and polyp-level characteristics among the species comprising the genus, as it is hypothesized that common reproductive, developmental, and stress response mechanisms are conserved. The presented resources, aiming to streamline the genus’ biology, elucidate several signaling pathways of development and stress response that contribute to the understanding of researchers of overall biological responses, while providing a genetic framework for potential further studies that might contribute to reef preservation strategies.

Keywords: acropora, development, genes, transcriptomics

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Authors: Taru Singh, Shukla Das, V. G. Ramachandran

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Objectives: To develop SYBR green based real-time PCR assay to detect carbapenemases (NDM, IMP) genes in E. coli. Methods: A total of 40 E. coli from stool samples were tested. Six were previously characterized as resistant to carbapenems and documented by PCR. The remaining 34 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial RNA was extracted using manual method. The real-time PCR was performed using the Light Cycler III 480 instrument (Roche) and specific primers for each carbapenemase target were used. Results: Each one of the two carbapenemase gene tested presented a different melting curve after PCR amplification. The melting temperature (Tm) analysis of the amplicons identified was as follows: blaIMP type (Tm 82.18°C), blaNDM-1 (Tm 78.8°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. Conclusions: The new assay was able to detect the presence of two different carbapenemase gene type by real-time PCR.

Keywords: resistance, b-lactamases, E. coli, real-time PCR

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Authors: E. Rahimi, S. Shaigannia

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Salmonella typhimurium and Salmonella enteritidis are important infectious agents causing food poisoning and food-borne gastrointestinal diseases. This study was carried out in order to investigate the distribution of virulence genes and antimicrobial resistance properties of S. typhimurium and S. enteritidis isolated from ruminant milk and dairy products in Iran. Overall 360 raw and pasteurized milk and traditional and commercial dairy products were purchased from random selected supermarkets and retail stories of Isfahan province, Iran. Samples were cultured immediately and those found positive for Salmonella were analyzed for the presence of S. typhimurium, S. enteritidis and several putative genes using PCR. Totally, 13 (3.61%), 8 (2.22%), 1 (0.27%) and 4 (1.11%) samples were found to be contaminated with Salmonella spp., S. typhimurium, S. enteritidis and other species of Salmonella, respectively. PCR results showed that invA, rfbJ, fliC and spv were the detected virulence genes in S. typhimurium and S. enteritidis positive samples. To the authors’ knowledge, the present study is the first prevalence report of virulence genes of S. typhimurium and S. enteritidis isolated from ruminant milk and traditional and commercial dairy products in Iran.

Keywords: Salmonella typhimurium, Salmonella enteritidis, virulence genes, ruminant milk, dairy products

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Authors: Ashraf Ali, Kriti Upadhyay, Puja Sohal, Anant Mohan, Randeep Guleria

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Background: Gene silencing by aberrant promoter hypermethylation is common in lung cancer and is an initiating event in its development. Aim: To evaluate the gene promoter hypermethylation frequency in serum and tissue of lung cancer patients. Method: 95 newly diagnosed untreated advance stage lung cancer patients and 50 cancer free matched controls were studied. Bisulfite modification of tissue and serum DNA was done; modified DNA was used as a template for methylation-specific PCR analysis. Survival was assessed for one year. Results: Of 95 patients, 82% were non-small cell lung cancer (34% squamous cell carcinoma, 34% non-small cell lung cancer and 14% adenocarcinoma) and 18% were small cell lung cancer. Biopsy revealed that tissue of 89% and 75% of lung cancer patients and 85% and 52% of controls had promoter hypermethylated for MGMT (p=0.35) and p16(p<0.001) gene, respectively. In serum, 33% and 49% of lung cancer patients and 28% and 43% controls were positive for MGMT and p16 gene. No significant correlation was found between survival and clinico-pathological parameters. Conclusion: High gene promoter methylation frequency of p16 gene in tissue biopsy may be linked with early stages of carcinogenesis. Appropriate follow-up is required for confirmation of this finding.

Keywords: lung cancer, MS- PCR, methylation, molecular biology

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Authors: Jie Lin, Yiwen Pan, Li Zhang, Zhangyong Xia

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Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids, immune cells, and extracellular matrix in the arterial walls. This pathological process can lead to the formation of plaques that can obstruct blood flow and trigger various cardiovascular diseases such as heart attack and stroke. The underlying molecular mechanisms still remain unclear, although many studies revealed the dysfunction of endothelial cells, recruitment and activation of monocytes and macrophages, and the production of pro-inflammatory cytokines and chemokines in atherosclerosis. This study aimed to identify hub genes involved in the progression of atherosclerosis and to analyze their biological function in silico, thereby enhancing our understanding of the disease’s molecular mechanisms. Through the analysis of microarray data, we examined the gene expression in media and neo-intima from plaques, as well as distant macroscopically intact tissue, across a cohort of 32 hypertensive patients. Initially, 112 differentially expressed genes (DEGs) were identified. Subsequent immune infiltration analysis indicated a predominant presence of 27 immune cell types in the atherosclerosis group, particularly noting an increase in monocytes and macrophages. In the Weighted gene co-expression network analysis (WGCNA), 10 modules with a minimum of 30 genes were defined as key modules, with blue, dark, Oliver green and sky-blue modules being the most significant. These modules corresponded respectively to monocyte, activated B cell, and activated CD4 T cell gene patterns, revealing a strong morphological-genetic correlation. From these three gene patterns (modules morphology), a total of 2509 key genes (Gene Significance >0.2, module membership>0.8) were extracted. Six hub genes (CD36, DPP4, HMOX1, PLA2G7, PLN2, and ACADL) were then identified by intersecting 2509 key genes, 102 DEGs with lipid-related genes from the Genecard database. The bio-functional analysis of six hub genes was estimated by a robust classifier with an area under the curve (AUC) of 0.873 in the ROC plot, indicating excellent efficacy in differentiating between the disease and control group. Moreover, PCA visualization demonstrated clear separation between the groups based on these six hub genes, suggesting their potential utility as classification features in predictive models. Protein-protein interaction (PPI) analysis highlighted DPP4 as the most interconnected gene. Within the constructed key gene-drug network, 462 drugs were predicted, with ursodeoxycholic acid (UDCA) being identified as a potential therapeutic agent for modulating DPP4 expression. In summary, our study identified critical hub genes implicated in the progression of atherosclerosis through comprehensive bioinformatic analyses. These findings not only advance our understanding of the disease but also pave the way for applying similar analytical frameworks and predictive models to other diseases, thereby broadening the potential for clinical applications and therapeutic discoveries.

Keywords: atherosclerosis, hub genes, drug prediction, bioinformatics

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Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Marija Ratkova Manovska, Mirko Prodanov, Dean Jankuloski, Katerina Blagoevska

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Staphylococci, which are widely found in the environment, animals, humans, and food products, include Staphylococcus aureus (S. aureus), the most significant pathogenic species in this genus. The virulence and toxicity of S. aureus are primarily attributed to the presence of specific genes responsible for producing toxins, biofilms, invasive components, and antibiotic resistance. Staphylococcal food poisoning, caused by the production of staphylococcal enterotoxins (SEs) by these strains in food, is a common occurrence. Globally, S. aureus food intoxications are typically ranked as the third or fourth most prevalent foodborne intoxications. For this study, a total of 333 milk samples and 1160 dairy product samples were analyzed between 2016 and 2020. The strains were isolated and confirmed using the ISO 6888-1:1999 "Horizontal method for enumeration of coagulase-positive staphylococci." Molecular analysis of the isolates, conducted using conventional PCR, involved detecting the 23s gene of S. aureus, the nuc gene, the mecA gene, and 11 genes responsible for producing enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, ser, sej, and sep). The 23s gene was found in 93 (75.6%) out of 123 isolates of Staphylococcus spp. obtained from milk. Among the 76 isolates from dairy products, either S. aureus or the 23s gene was detected in 49 (64.5%) of them. The mecA gene was identified in three isolates from raw milk and five isolates from cheese samples. The nuc gene was present in 98.9% of S. aureus strains from milk and 97.9% from dairy products. Other Staphylococcus strains carried the nuc gene in 26.7% of milk strains and 14.8% of dairy product strains. Genes associated with SEs production were detected in 85 (69.1%) strains from milk and 38 (50%) strains from dairy products. In this study, 10 out of the 11 SEs genes were found, with no isolates carrying the see gene. The most prevalent genes detected were seg and sei, with some isolates containing up to five different SEs genes. These findings indicate the presence of enterotoxigenic staphylococci strains in the tested samples, emphasizing the importance of implementing proper sanitation and hygienic practices, utilizing safe raw materials, and ensuring adequate handling of finished products. Continued monitoring for the presence of SEs is necessary to ensure food safety and prevent intoxication.

Keywords: dairy products, milk, Staphylococci, enterotoxins, SE genes

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Authors: Fahimeh Palizban, Farshad Noravesh, Amir Hossein Saeidian, Mahya Mehrmohamadi

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In the recent decade, the emergence of liquid biopsy has significantly improved cancer monitoring and detection. Dying cells, including those originating from tumors, shed their DNA into the blood and contribute to a pool of circulating fragments called cell-free DNA. Accordingly, identifying the tissue origin of these DNA fragments from the plasma can result in more accurate and fast disease diagnosis and precise treatment protocols. Open chromatin regions are important epigenetic features of DNA that reflect cell types of origin. Profiling these features by DNase-seq, ATAC-seq, and histone ChIP-seq provides insights into tissue-specific and disease-specific regulatory mechanisms. There have been several studies in the area of cancer liquid biopsy that integrate distinct genomic and epigenomic features for early cancer detection along with tissue of origin detection. However, multimodal analysis requires several types of experiments to cover the genomic and epigenomic aspects of a single sample, which will lead to a huge amount of cost and time. To overcome these limitations, the idea of predicting OCRs from WGS is of particular importance. In this regard, we proposed a computational approach to target the prediction of open chromatin regions as an important epigenetic feature from cell-free DNA whole genome sequence data. To fulfill this objective, local sequencing depth will be fed to our proposed algorithm and the prediction of the most probable open chromatin regions from whole genome sequencing data can be carried out. Our method integrates the signal processing method with sequencing depth data and includes count normalization, Discrete Fourie Transform conversion, graph construction, graph cut optimization by linear programming, and clustering. To validate the proposed method, we compared the output of the clustering (open chromatin region+, open chromatin region-) with previously validated open chromatin regions related to human blood samples of the ATAC-DB database. The percentage of overlap between predicted open chromatin regions and the experimentally validated regions obtained by ATAC-seq in ATAC-DB is greater than 67%, which indicates meaningful prediction. As it is evident, OCRs are mostly located in the transcription start sites (TSS) of the genes. In this regard, we compared the concordance between the predicted OCRs and the human genes TSS regions obtained from refTSS and it showed proper accordance around 52.04% and ~78% with all and the housekeeping genes, respectively. Accurately detecting open chromatin regions from plasma cell-free DNA-seq data is a very challenging computational problem due to the existence of several confounding factors, such as technical and biological variations. Although this approach is in its infancy, there has already been an attempt to apply it, which leads to a tool named OCRDetector with some restrictions like the need for highly depth cfDNA WGS data, prior information about OCRs distribution, and considering multiple features. However, we implemented a graph signal clustering based on a single depth feature in an unsupervised learning manner that resulted in faster performance and decent accuracy. Overall, we tried to investigate the epigenomic pattern of a cell-free DNA sample from a new computational perspective that can be used along with other tools to investigate genetic and epigenetic aspects of a single whole genome sequencing data for efficient liquid biopsy-related analysis.

Keywords: open chromatin regions, cancer, cell-free DNA, epigenomics, graph signal processing, correlation clustering

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Authors: Soumya S., Manju Nidagodu Jayakumar, Vellore Kannan Gopinath

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Dental pulp inflammation resulting from dental caries often leads to a pathologic condition known as irreversible pulpitis and the currently managed by root canal treatment. Extirpation of the entire pulp tissue is done during this procedure, and the canal space is filled with synthetic materials. Recent studies in the stem cell biology state that some portion of the irreversibly inflamed pulp tissue could be viable with progenitor cells, having the properties similar to that of Mesenchymal stem cells. Hence, we aim to isolate Dental Pulp Stem Cells (DPSCs) from patients diagnosed with severe irreversible pulpitis and characterize the cells for the MSC specific markers. The pulp tissue was collected from the dental clinic and subjected to collagenase/dispase digestion. The isolated cells were expanded in culture, and the phenotypic characterization was done using flow cytometry. MSC specific markers such as CD-90, CD-73, and CD-105 were analysed along with negative markers such as CD-14 and CD-45. The isolated cells expressed positive expression for CD markers with CD90 and CD105 ( > 95%) and CD73 (19%). The cells did not express the negative markers CD-14 and CD-45. The commercially available DPSCs from vital extracted teeth, preferably molar/wisdom teeth with large pulp cavity or incomplete root growth in young patients (aged 15-30 years) showed more than 90% expression for all the CD markers such as CD-90, 73 and 105, whereas negative for CD-14 and CD-45. The DPSCs isolated from inflamed pulp tissue showed a less expression for CD-73 compared to the commercially available DPSCs whereas, as the other two markers were found to show similar percentage of positive expression. This could be attributed to the fact that the pulp population is very heterogeneous and we used the pooled tissue from different patients. Hence the phenotypic characterization and comparison with the commercially available DPSCs proved that the inflamed pulp tissue is a good source of MSC like cells which can be utilized further for regenerative application.

Keywords: collagenase/dispase, dental pulp stem cells, flow cytometry, irreversible pulpitis

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Authors: Naomi Seidu, Edward Poluyi, Chibuikem Ikwuegbuenyi, Eghosa Morgan

Abstract:

The current poor prognosis of glioblastoma has called for the need for an improvement in treatment methods in order to improve its survival rate. Despite the different interventions currently available for this tumor, the average survival is still only a few months. (12-15). The aim is to create a more favorable prognosis and have a reduction in the resistance to treatment currently being experienced, even with surgical interventions and chemotherapy. From the available literature, there is a relationship between the presence of HOX genes (Homeobox genes) and glioblastoma, which could be attributable to the increasing treatment resistance. Hence silencing these genes can be a key to improving survival rates of glioblastoma. A series of studies have highlighted the role that HOX genes play in glioblastoma prognosis. Promotion of human glioblastoma initiation, aggressiveness, and resistance to Temozolomide has been associated with HOXA9. The role of HOX gene expression in cancer stem cells should be studied as it could provide a means of designing CSC-targeted therapies, as CSCs play a part in the initiation and progression of solid tumors.

Keywords: GBM- glioblastoma, HOXA gene- homeobox genes cluster, signaling pathways, temozolomide

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Authors: Kara J. Coffman-Rea, Karen E. Samonds

Abstract:

Microplastic pollution is an urgent global threat to our planet and human health. Microplastic particles have been detected within our food, water, and atmosphere, and found within the human stool, placenta, and lung tissue. However, most spectrometric microplastic detection methods require chemical digestion which can alter or destroy microplastic particles and makes it impossible to acquire information about their in-situ distribution. MALDI TOF MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) is an analytical method using a soft ionization technique that can be used for polymer analysis. This method provides a valuable opportunity to both acquire information regarding the in-situ distribution of microplastics and also minimizes the destructive element of chemical digestion. In addition, MALDI TOF MS allows for expanded analysis of the microplastics including detection of specific additives that may be present within them. MALDI TOF MS is particularly sensitive to sample preparation and has not yet been used to analyze environmental microplastics within their specific location (e.g., biological tissues, sediment, water). In this study, microplastics were created using polyethylene gloves, polystyrene micro-foam, and polyethylene terephthalate cable sleeving. Plastics were frozen using liquid nitrogen and ground to obtain small fragments. An artificial tissue was created using a cellulose sponge as scaffolding coated with a MaxGel Extracellular Matrix to simulate human lung tissue. Optimal preparation techniques (e.g., matrix, cationization reagent, solvent, mixing ratio, laser intensity) were first established for each specific polymer type. The artificial tissue sample was subsequently spiked with microplastics, and specific polymers were detected using MALDI-TOF-MS. This study presents a novel method for the detection of environmental polyethylene, polyethylene terephthalate, and polystyrene microplastics within a complex sample. Results of this study provide an effective method that can be used in future microplastics research and can aid in determining the potential threats to environmental and human health that they pose.

Keywords: environmental plastic pollution, MALDI-TOF MS, microplastics, polymer identification

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Authors: Naseem Fatima, A. N. Srivastava, Tasleem Raza, Vijay Kumar

Abstract:

Introduction: Carcinoma gallbladder is third most common gastrointestinal lethal disease with the highest incidence and mortality rate among women in Northern India. Scientists have found several risk factors that make a person more likely to develop gallbladder cancer; among these risk factors, deregulation of miRNAs has been demonstrated to be one of the most crucial factors. The changes in the expression of specific miRNA genes result in the control of inflammation, cell cycle regulation, stress response, proliferation, differentiation, apoptosis and invasion thus mediate the process in tumorgenesis. The aim of this study was to investigate the role of MiRNA-335 and may as a molecular marker in early detection of gallbladder cancer in suspected cases. Material and Methods: A total of 20 consecutive patients with gallbladder cancer aged between 30-75 years were registered for the study. Total RNA was extracted from tissue by using the mirVANA MiRNA isolation Kit according to the manufacturer’s protocol. The MiRNA- 335 and U6 snRNA-specific cDNA were reverse-transcribed from total RNA using Taqman microRNA reverse-transcription kit according to the manufacturer’s protocol. TaqMan MiRNA probes hsa-miR-335 and Taqman Master Mix without AmpEase UNG, Individual real-time PCR assays were performed in a 20 μL reaction volume on a Real-Time PCR system (Applied Biosystems StepOnePlus™) to detect MiRNA-335 expression in tissue. Relative quantification of target MiRNA expression was evaluated using the comparative cycle threshold (CT) method. The correlation was done in between cycle threshold (CT Value) of target MiRNA in gallbladder cancer with respect to non-cancerous Cholelithiasis gallbladder. Each sample was examined in triplicate. The Newman-Keuls Multiple Comparison Test was used to determine the expression of miR-335. Results: MiRNA335 was found to be significantly downregulated in the gallbladder cancer tissue (P<0.001), when compared with non-cancerous Cholelithiasis gallbladder cases. Out of 20 cases, 75% showed reduced expression of MiRNA335, were at last stage of disease with low overall survival rate and remaining 25% were showed up-regulated expression of MiRNA335 with high survival rate. Conclusion: The present study showed that reduced expression of MiRNA335 is associated with the advancement of the disease, and its deregulation may provide important clues to understanding it as a prognostic marker and opportunities for future research.

Keywords: carcinoma gallbladder, downregulation, MiRNA-335, RT-PCR assay

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Authors: Ilya B. Tsyrlov, Dmitry Y. Oshchepkov

Abstract:

A computational search for dioxin response elements (DREs) in genes of proteins comprising the Ah receptor (AhR) cytosolic core complex was performed by highly efficient tool SITECON. Eventually, the following number of new DREs in 5’flanking region was detected by SITECON: one in AHR gene, five in XAP2, eight in HSP90AA1, and three in HSP90AB1 genes. Numerous DREs found in genes of AhR and AhR cytosolic complex members would shed a light on potential mechanisms of expression, the stoichiometry of unliganded AhR core complex, and its degradation vs biosynthesis dynamics resulted from treatment of target cells with the AhR most potent ligand, 2,3,7,8-TCDD. With human viruses, reduced susceptibility to TCDD of geneencoding HIV-1 P247 was justified by the only potential DRE determined in gag gene encoding HIV-1 P24 protein, whereas the regulatory region of CMV genes encoding IE gp/UL37 has five potent DRE, 1.65 kb/UL36 – six DRE, pp65 and pp71 – each has seven DRE, and pp150 – ten DRE. Also, from six to eight DRE were determined with SITECON in the regulatory region of HSV-1 IE genes encoding tegument proteins, UL36 and UL37, and of UL19 gene encoding bindingglycoprotein C (gC). So, TCDD in the low picomolar range may activate in human cells AhR: Arnt transcription pathway that triggers CMV and HSV-1 reactivation by binding to numerous promoter DRE within immediate-early (IE) genes UL37 and UL36, thus committing virus to the lytic cycle.

Keywords: dioxin response elements, Ah receptor, AhR: Arnt transcription pathway, human and viral genes

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Authors: Abu Salim Mustafa

Abstract:

The comparisons of mycobacterial genomes have identified several Mycobacterium tuberculosis-specific genomic regions that are absent in other mycobacteria and are known as regions of differences. Due to M. tuberculosis-specificity, the peptides encoded by these regions could be useful in the specific diagnosis of tuberculosis. To explore this possibility, overlapping synthetic peptides corresponding to 39 proteins predicted to be encoded by genes present in regions of differences were tested for antibody-reactivity with sera from tuberculosis patients and healthy subjects. The results identified four immunodominant peptides corresponding to four different proteins, with three of the peptides showing significantly stronger antibody reactivity and rate of positivity with sera from tuberculosis patients than healthy subjects. The fourth peptide was recognized equally well by the sera of tuberculosis patients as well as healthy subjects. Predication of antibody epitopes by bioinformatics analyses using ABCpred server predicted multiple linear epitopes in each peptide. Furthermore, peptide sequence analysis for sequence identity using BLAST suggested M. tuberculosis-specificity for the three peptides that had preferential reactivity with sera from tuberculosis patients, but the peptide with equal reactivity with sera of TB patients and healthy subjects showed significant identity with sequences present in nob-tuberculous mycobacteria. The three identified M. tuberculosis-specific immunodominant peptides may be useful in the serological diagnosis of tuberculosis.

Keywords: genomic regions of differences, Mycobacterium tuberculossis, peptides, serodiagnosis

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Authors: C. Miranda, S. Cunha, R. Soares, M. Maia, G. Igrejas, F. Silva, P. Poeta

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The worldwide inappropriate use of antibiotics has increased the emergence of antimicrobial-resistant microorganisms isolated from animals, humans, food, and the environment. To combat this complex and multifaceted problem is essential to know the prevalence in livestock animals and possible ways of transmission among animals and between these and humans. Enterococci species, in particular E. faecalis and E. faecium, are the most common nosocomial bacteria, causing infections in animals and humans. Thus, the aim of this study was to characterize resistance and virulence factors genes among two enterococci species isolated from calf colostrums in Portuguese dairy farms. The 55 enterococci isolates (44 E. faecalis and 11 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB, and ermC), tetracycline (tetL, tetM, tetK, and tetO), quinupristin/dalfopristin (vatD and vatE) and vancomycin (vanB). Of which, 25 isolates (15 E. faecalis and 10 E. faecium) were tested until now for 8 virulence factors genes (esp, ace, gelE, agg, cpd, cylA, cylB, and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. All enterococci isolates showed resistance to erythromicyn and tetracycline through the presence of the genes: ermB (n=29, 53%), ermC (n=10, 18%), tetL (n=49, 89%), tetM (n=39, 71%) and tetK (n=33, 60%). Only two (4%) E. faecalis isolates showed the presence of tetO gene. No resistance genes for vancomycin were found. The virulence genes detected in both species were cpd (n=17, 68%), agg (n=16, 64%), ace (n=15, 60%), esp (n=13, 52%), gelE (n=13, 52%) and cylLL (n=8, 32%). In general, each isolate showed at least three virulence genes. In three E. faecalis isolates was not found virulence genes and only E. faecalis isolates showed virulence genes for cylA (n=4, 16%) and cylB (n=6, 24%). In conclusion, these colostrum samples that were consumed by calves demonstrated the presence of antibiotic-resistant enterococci harbored virulence genes. This genotypic characterization is crucial to control the antibiotic-resistant bacteria through the implementation of restricts measures safeguarding public health. Acknowledgements: This work was funded by the R&D Project CAREBIO2 (Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics - towards innovative theragnostic biomarkers), with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Keywords: antimicrobial resistance, calf, colostrums, enterococci

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Authors: M. Raissy, M. Shahrani

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The present study was done to evaluate the presence of tetracycline resistance genes in Lactococcus garvieae isolated from cultured rainbow trout, West Iran. The isolates were examined for antimicrobial resistance using disc diffusion method. Of the 49 strains tested, 19 were resistant to tetracycline (38.7%), 32 to enrofloxacin (65.3%), 21 to erythromycin (42.8%), 20 to chloramphenicol and trimetoprim-sulfamethoxazole (40.8%). The strains were then characterized for their genotypic resistance profiles. The results revealed that all 49 isolates contained at least one of the tetracycline resistance genes. Tet (A) was found in 89.4% of tetracycline resistant isolates and the frequency of other gene were as follow: tet (E) 42.1%, tet (B) 47.3%, tet (D) 15.7%, tet (L) 26.3%, tet (K) 52.6%, tet (G) 36.8%, tet (34) 21%, tet (S) 63.1%, tet (C) 57.8%, tet (M) 73.6%, tet (O) 42.1%. The results revealed high levels of antibiotic resistance in L. garvieae strains which is a potential danger for trout culture as well as for public health.

Keywords: Lactococcus garvieae, tetracycline resistance genes, rainbow trout, antimicrobial resistance

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Authors: Rahul Agarwal, Ashutosh Singh

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Colorectal cancer (CRC) also refers as bowel cancer or colon cancer. It involves the development of abnormal growth of cells in colon or rectum part of the body. This work leads to the development of a miRNA database in colorectal cancer. We named this database- miCoRe. This database comprises of all validated colon-rectal cancer miRNAs information from various published literature with an effectual knowledge based information retrieval system. miRNAs have been collected from various published literature reports. MySQL is used for main-framework of miCoRe while the front-end was developed in PHP script. The aim of developing miCoRe is to create a comprehensive central repository of colorectal carcinoma miRNAs with all germane information of miRNAs and their target genes. The current version of miCoRe consists of 238 miRNAs which are known to be implicated in malignancy of CRC. Alongside with miRNA information, miCoRe also contains the information related to the target genes of these miRNA. miCoRe furnishes the information about the mechanism of incidence and progression of the disease, which would further help the researchers to look for colorectal specific miRNAs therapies and CRC specific targeted drug designing. Moreover, it will also help in development of biomarkers for the better and early detection of CRC and will help in better clinical management of the disease.

Keywords: colorectal cancer, database, miCoRe, miRNAs

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Authors: A. Reum Son, Jin Seon Kwon, Seung Hun Park, Hai Bang Lee, Moon Suk Kim

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These days, stem cell therapy is focused on for promising source of treatment in clinical human disease. As a supporter of stem cells, in situ-forming hydrogels with growth factors and cells appear to be a promising approach in tissue engineering. To examine osteogenic differentiation of hTMSCs which is one of mesenchymal stem cells in vivo in an injectable hydrogel, we use a methoxy polyethylene glycol-polycaprolactone blockcopolymer (MPEG-PCL) solution with osteogenic factors. We synthesized MPEG-PCL hydrogel and measured viscosity to check sol-gel transition. In order to demonstrate osteogenic ability of hTMSCs, we conducted in vitro osteogenesis experiment. Then, to confirm the cell cytotoxicity, we performed WST-1 with hTMSCs and MPEG-PCL. As the result of in vitro experiment, we implanted cell and hydrogel mixture into animal model and checked degree of osteogenesis with histological analysis and amount of expression genes. Through these experimental data, MPEG-PCL hydrogel has sol-gel transition in temperature change and is biocompatible with stem cells. In histological analysis and gene expression, hTMSCs are very good source of osteogenesis with hydrogel and will use it to tissue engineering as important treatment method. hTMSCs could be a good adult stem cell source for usability of isolation and high proliferation. When hTMSCs are used as cell therapy method with in situ-formed hydrogel, they may provide various benefits like a noninvasive alternative for bone tissue engineering applications.

Keywords: injectable hydrogel, stem cell, osteogenic differentiation, tissue engineering

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Authors: Oshma Chakoory, Sophie Comtet-Marre, Pierre Peyret

Abstract:

Metagenomic classifiers are widely used for the taxonomic profiling of metagenomic data and estimation of taxa relative abundance. Small subunit rRNA genes are nowadays a gold standard for the phylogenetic resolution of complex microbial communities, although the power of this marker comes down to its use as full-length. We benchmarked the performance and accuracy of rRNA-specialized versus general-purpose read mappers, reference-targeted assemblers and taxonomic classifiers. We then built a pipeline called RiboTaxa to generate a highly sensitive and specific metataxonomic approach. Using metagenomics data, RiboTaxa gave the best results compared to other tools (Kraken2, Centrifuge (1), METAXA2 (2), PhyloFlash (3)) with precise taxonomic identification and relative abundance description, giving no false positive detection. Using real datasets from various environments (ocean, soil, human gut) and from different approaches (metagenomics and gene capture by hybridization), RiboTaxa revealed microbial novelties not seen by current bioinformatics analysis opening new biological perspectives in human and environmental health. In a study focused on corals’ health involving 20 metagenomic samples (4), an affiliation of prokaryotes was limited to the family level with Endozoicomonadaceae characterising healthy octocoral tissue. RiboTaxa highlighted 2 species of uncultured Endozoicomonas which were dominant in the healthy tissue. Both species belonged to a genus not yet described, opening new research perspectives on corals’ health. Applied to metagenomics data from a study on human gut and extreme longevity (5), RiboTaxa detected the presence of an uncultured archaeon in semi-supercentenarians (aged 105 to 109 years) highlighting an archaeal genus, not yet described, and 3 uncultured species belonging to the Enorma genus that could be species of interest participating in the longevity process. RiboTaxa is user-friendly, rapid, allowing microbiota structure description from any environment and the results can be easily interpreted. This software is freely available at https://github.com/oschakoory/RiboTaxa under the GNU Affero General Public License 3.0.

Keywords: metagenomics profiling, microbial diversity, SSU rRNA genes, full-length phylogenetic marker

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

Abstract:

Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

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