Search results for: fibernectin expression

Authors: Karthick Dharmalingam, Stalin Ramakrishnan, Haseena Banu Hedayathullah Khan, Sachidanandanam Thiruvaiyaru Panchanadham, Shanthi Palanivelu

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Background: Breast cancer is arising as the most dreadful cancer affecting women worldwide. Hence, there arises a need to search and test for new drugs. Herbal formulations used in Siddha preparations are proved to be effective against various types of cancer. They also offer advantage through synergistic amplification and diminish any possible adverse effects. Tridham (TD) is a herbal formulation prepared in our laboratory consisting of Terminalia chebula, Elaeocarpus ganitrus and Prosopis cineraria in a definite ratio and has been used for the treatment of mammary carcinoma. Objective: To study the restorative effect of Tridham and penta galloyl glucose (a component of TD) on DMBA induced mammary carcinoma in female Sprague Dawley rats. Materials and Methods: Rats were divided into seven groups of six animals each. Group I (Control) received corn oil. Group II– mammary carcinoma was induced by DMBA dissolved in corn oil single dose orally. Group III and Group IV were induced with DMBA and subsequently treated with Tridham and penta galloyl glucose, respectively for 48 days. Group V was treated with DMBA and subsequently with a standard drug, cyclophosphamide. Group VI and Group VII were given Tridham and penta galloyl glucose alone, respectively for 48 days. After the experimental period, the animals were sacrificed by cervical decapitation. The mammary gland tissue was excised and levels of antioxidants were determined by biochemical assay. p53 and PCNA expression were accessed using immunohistochemistry. Nrf-2, Cox-2 and caspase-3 protein expression were studied by Western Blotting analysis. p21, Bcl-2, Bax, Bad and caspase-8 gene expression were studied by RT-PCR. Results: Histopathological studies confirmed induction of mammary carcinoma in DMBA induced rats and treatment with TD and PGG resulted in regression of tumour. The levels of enzymic and non-enzymic antioxidants were decreased in DMBA induced rats when compared to control rats. The levels of cell cycle inhibitory markers and apoptotic markers were decreased in DMBA induced rats when compared to control rats. These parameters were restored to near normal levels on treatment with Tridham and PGG. Conclusion: The results of the present study indicate the antineoplastic effect of Tridham and PGG are exerted through the modulation of antioxidant status and expression of cell cycle regulatory markers as well as apoptotic markers. Acknowledgment: Financial assistance provided in the form of ICMR-SRF by Indian Council of Medical Research (ICMR), India is gratefully acknowledged here.

Keywords: antioxidants, Mammary carcinoma, pentaGalloyl glucose, Tridham

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Authors: Pelin Balcik Ercin

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Introduction: ZEB transcription factor family member ZEB2, has a role in epithelial to mesenchymal transition during development and metastasis. The altered circulating extracellular miRNAs expression is observed in diseases, and extracellular miRNAs have an important role in cancer cell microenvironment. In ChIP-Seq study, the expression of miR-2110 was found to be regulated by ZEB2. In this study, the effects of miR2110 on cell proliferation and migration of hepatocellular carcinoma (HCC) cells were examined. Material and Methods: SNU398 cells transfected with mimic miR2110 (20nM) (HMI0375, Sigma-Aldrich) and negative control miR (HMC0002, Sigma-Aldrich). MicroRNA isolation was accomplished with miRVANA isolation kit according to manufacturer instructions. cDNA synthesis was performed expression, respectively, and calibrated with Ct of controls. The real-time quantitative PCR (RT-qPCR) reaction was performed using the TaqMan Fast Advanced Master Mix (Thermo Sci.). Ct values of miR2110 were normalized to miR-186-5p and miR16-5p for the intracellular gene. Cell proliferation analysis was analyzed with the xCELLigence RTCA System. Wound healing assay was analyzed with the ImageJ program and relative fold change calculated. Results: The mimic-miR-2110 transfected SNU398 cells nearly nine-fold (log2) more miR-2110 expressed compared to negative control transfected cells. The mimic-miR-2110 transfected HCC cell proliferation significantly inhibited compared to the negative control cells. Furthermore, miR-2110-SNU398 cell migration capacity was relatively four-fold decreased compared to negative control-miR-SNU398 cells. Conclusion: Our results suggest the miR-2110 inhibited cell proliferation and also miR-2110 negatively affect cell migration compared to control groups in HCC cells. These data suggest the complexity of microRNA EMT transcription factors regulation. These initial results are pointed out the predictive biomarker capacity of miR-2110 in HCC.

Keywords: epithelial to mesenchymal transition, EMT, hepatocellular carcinoma cells, micro-RNA-2110, ZEB2

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Authors: Hung-Chieh Chen, Kuo-Hui Wang, Tsu-Lin Yeh

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Proteomics represent the performance of genomic complement proteins and the protein level on functional genomics. This study adopted proteomic strategies for comparing serum proteins among three groups: elite sprinter (sprint runner group, SR), long-distance runners (long-distance runner group, LDR), and the untrained control group (control group, CON). Purposes: This study aims to identify elite sprinters and long-distance runners’ serum protein and to provide a comparison of their serum proteome’ composition. Methods: Serum protein fractionations that separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by a quantitative nano-LC-MS/MS-based proteomic profiling. The one-way analysis of variance (ANOVA) and Scheffe post hoc comparison (α= 0.05) was used to determine whether there is any significant difference in each protein level among the three groups. Results: (1) After analyzing the 307 identified proteins, there were 26 unique proteins in the SR group, and 18 unique proteins in the LDR group. (2) For the LDR group, 7 coagulation function-associated proteins’ expression levels were investigated: vitronectin, serum paraoxonase/arylesterase 1, fibulin-1, complement C3, vitamin K-dependent protein, inter-alpha-trypsin inhibitor heavy chain H3 and von Willebrand factor, and the findings show the seven coagulation function-associated proteins were significantly lower than the group of SR. (3) Comparing to the group of SR, this study found that the LDR group’s expression levels of the 2 antioxidant proteins (afamin and glutathione peroxidase 3) were also significantly lower. (4) The LDR group’s expression levels of seven immune function-related proteins (Ig gamma-3 chain C region, Ig lambda-like polypeptide 5, clusterin, complement C1s subcomponent, complement factor B, complement C4-A, complement C1q subcomponent subunit A) were also significantly lower than the group of SR. Conclusion: This study identified the potential serum protein markers for elite sprinters and long-distance runners. The changes in the regulation of coagulation, antioxidant, or immune function-specific proteins may also provide further clinical applications for these two different track athletes.

Keywords: biomarkers, coagulation, immune response, oxidative stress

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Authors: Ali D. Omur, Betul Apaydin Yildirim, Yavuz S. Saglam, Selim Comakli, Mustafa Ozkaraca

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Twenty-eight male Sprague Dawley rats (aged 3 months) were used in the study. The animals were given feed and water as ad libitum. Sprague Dawley rats were randomly divided into 4 groups as 7 rats in each group. Aflatoxin B1 (7.5 μg/200 g), resveratrol (60 mg/kg) was administered to rats in groups other than the control group. At the end of the 16th day, blood, semen and tissue specimens were taken by decapitation under ether anesthesia. The effects of aflatoxin B1 and resveratrol on spermatological, pathological and biochemical parameters were determined in rats. When we evaluate the spermatological parameters, it is understood that resveratrol has a statistically significant difference in terms of sperm motility and viability (membrane integrity) compared to the control group and aflatoxin B1 administration groups, indicating a protective effect on spermatological parameters (groups: control, resveratrol, aflatoxin B1 and Afb1 + res; respectively, values of motility: 71,42 ± 0,52b, 72,85 ± 1, 48c , 60,71 ± 1,30a, 57,14 ± 2, 40a; values of viability: 63,85 ± 1,33b, 70,42 ± 2,61c, 55,00 ± 1,54a, 56,57 ± 0,89a. In terms of pathological parameters -histopathological examination- in the control and resveratrol groups, seminiferous tubules were observed to be in normal structure. In the group treated with aflatoxin, the regular structure of the spermatogenic cells deteriorated, and the seminiferous tubules became necrotic and degenerative. In the group treated with Afb1 + res, the decreasing of necrotic and degenerative changes were determined compared with in the group treated with aflatoxin. As immunohistochemical examination, cleaved caspase 3 expression was found to be very low in the control and resveratrol groups. Cleaved caspase 3 expression was severely exacerbated in seminiferous tubules in aflatoxin group but cleaved caspase 3 expression level decreased in Afb1 + res. In the biochemical direction, resveratrol has been shown to inhibit the adverse effects of aflatoxin on antioxidant levels (GSH-mmol/L, CAT-kU/L, GPx-U/mL, SOD-EU/mL) and to show a protective effect. For this purpose, the use of resveratrol with antioxidant activity was investigated in preventing or ameliorating damage to aflatoxin B1. It has been concluded that resveratrol effectively prevents the aflatoxin-induced testicular damage and lipid peroxidation. It has also been shown that resveratrol has protective effects on sperm motility and viability.

Keywords: Aflatoxin B1, rat, resveratrol, sperm

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Authors: B. González-Yebra, B. Flores-Nieto, P. Aguilar-Salinas, M. Preciado Puga, A. L. González Yebra

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The monitoring of populations occupationally exposed to organic solvents has been an important issue for several shoe factories for years since the International Agency for Research on Cancer (IARC) has advised on the potential carcinogenic risk of chemicals related to occupations. In order to detect if exposition to organic solvents used in some Mexican shoe factories contributes to oral carcinogenesis, we performed monitoring in three factories. Occupational exposure was determined by using monitors 3M. Organic solvents were assessed by gas chromatography. Then, we recruited 30 shoe workers (30.2 ± 8.4 years) and 10 unexposed subjects (43.3 ± 11.2 years) for the micronuclei (MN) test and immunodetection of some cancer biomarkers (ki-67, p16, caspase-3) in scraped oral epithelial cells. Monitored solvents detected were acetone, benzene, hexane, methyl ethyl ketone, and toluene in acceptable levels according to Official Mexican Norm. We found by MN test higher incidence of nuclear abnormalities (karyorrhexis, pycnosis, karyolysis, condensed chromatin, and macronuclei) in the exposed group than the non-exposed group. On the other hand, we found, a negative expression for Ki-67 and p16 in exfoliated epithelial cells from exposed and non-exposed to organic solvents subjects. Only caspase-3 shown positive patter of expression in 9/30 (30%) exposed subjects, and we detected high karyolysis incidence in caspase-3 subjects (p = 0.021). The absence of expression of proliferation markers p16 and ki-67 and presence of apoptosis marker caspase-3 are indicating the absence of malignancy in oral epithelial cells and low risk for oral cancer. It is a fact that the MN test is a very effective method to detect nuclear abnormalities in exfoliated buccal cells from subjects that have been exposed to organic solvents in the shoe industry. However, in order to improve this tool and predict cancer risk is it is mandatory to implement complementary tests as other biomarkers that can help to detect malignancy in individuals occupationally exposed.

Keywords: biomarkers, oral cancer, organic solvents, shoe industries

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Authors: Iddrisu Ibrahim, Joseph Atia Ayariga, Junhuan Xu, Daniel A. Abugri, Robertson K. Boakai, Olufemi S. Ajayi

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The recalcitrance of pathogenic bacteria indicates that millions of people who are at risk of infection arising from chronic diseases, surgery, organ transplant, diabetes, and several other debilitating diseases present an aura of potentially untreatable illness due to resistance development. Antimicrobial resistance has successfully become a global health menace, and resistances are often acquired by bacteria through health-care-related incidence (HRI) orchestrated by multi-drug resistant (MDR) and extended drug-resistant pathogens (EDRP). To understand the mechanisms S. Typhimurium uses to resist CDB, we study the abundance of LPS modification, Ergosterols, Mysristic palmitic resistance, Oleic acid resistance of susceptible and resistant S. Typhimurium. Using qPCR, we also analyzed the expression of selected genes known for enabling resistance in S. Typhimurium. We found high abundance of LPS, Ergosterols, Mysristic palmitic resistance, Oleic acid resistance of and high expression of resistant genes in S. Typhimurium compared to the susceptible strain. LPS modification, Ergosterols, Mysristic palmitic resistance, Oleic acid and genes such as Fims, integrons, blaTEM are important indicators of resistance development of S. typhimurium.

Keywords: antimicrobials, resistance, Cannabidiol, Salmonella, blaTEM, fimA, Lipopolysaccharide, Ergosterols

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Authors: Badr Alsulami, Ahmed S. Elamary

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This paper summarizes and presents main results of an in-depth numerical analysis dealing with the shear buckling resistance of aluminum plate girders. The studies conducted have permitted the development of a simple design expression to determine the critical shear buckling stress in aluminum web panels. This expression takes into account the effects of reduction of strength in aluminum alloys due to the welding process. Ultimate shear resistance (USR) of plate girders can be obtained theoretically using Cardiff theory or Hӧglund’s theory. USR of aluminum alloy plate girders predicted theoretically using BS8118 appear inconsistent when compared with test data. Theoretical predictions based on Hӧglund’s theory, are more realistic. Cardiff theory proposed to predict the USR of steel plate girders only. Welded aluminum alloy plate girders studied experimentally by others; the USR resulted from tests are reviewed. Comparison between the test results with the values obtained from Hӧglund’s theory, BS8118 design method, and Cardiff theory performed theoretically. Finally, a new equation based on Cardiff tension-field theory proposed to predict theoretically the USR of aluminum plate girders.

Keywords: shear resistance, aluminum, Cardiff theory, Hӧglund's theory, plate girder

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Authors: Chin-Tsu Ma, Yi-Jhen Wu, Hsia Ying Cheng, Han Hsiang Huang, Shyh Ming Kuo

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The use of specific molecules including nutrients and pharmacological agents has been tried in modulation of stem cells differentiation (MSCs) to insulin producing cells. The aim of this study is to investigate the ability of nano collagen molecules (nutrient or scaffold) to enhance the MSCs differentiation into insulin-producing cells in combination with nicotinamide and exendin-4 (pharmacological agents) in vitro and in vivo. The results demonstrated that the cells exhibit morphologically islet-like clusters after treatment with nano collagen molecules, nicotinamide and exendin-4. MSCs extra treated with nano collagen molecules showed significant increases in Nkx6.1 and insulin mRNA expression at 14-d and 21-d culture compared with those merely treated with nicotinamide and exendin-4. Early 7-day elevation in PDX-1 mRNA expression was observed. Furthermore, the MSCs exposed to nano collagen molecules produced the highest secretion of insulin (p ＜ 0.05). Type-2 diabetes induced by high-fat diet and low dose of streptozotocin in rat model was built in this study. This rat exhibited higher food intake, water intake, lower glucose tolerance, lower-insulin tolerance, and higher HbA1C (significant increases, p ＜ 0.01) as compared with the normal rat that demonstrated the model of type-2 diabetes was successfully built. Biopsy examinations also showed that obvious destruction of islet. After injection of differentiated MSCs into the destructed pancreas of diabetes rat, more regenerated islet were observed at the rats that treated with nano collagen molecules and exhibited much lower HbA1C as compared with the normal rat and diabetes rat after 4 weeks (significant deceases, p ＜ 0.001). These results indicate that the culturing MSCs with nano collagen molecules, nicotinamide, and exendin-4 are beneficial for MSCs differentiation into islet-like cells. These nano collagen molecules may lead to alternations or up-regulation of gene expression and influence the differentiated outcomes induced by nicotinamide and exendin-4.

Keywords: nano collagen molecules, nicotinamide, MSCs, diabetes

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Authors: Vladimir Ryabov, Mikhail Baryshevs, Mikhail Voskresenskey, Boris Popov

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The human prostate gland (PG) samples were obtained from patients who had undergone radical prostatectomy for prostate cancer (PC) and used to extract total RNA and prepare the prostate stromal cell cultures (PSCC) and patients-derived organoids (PDO). Growth of the cell cultures was accessed under microscopic evaluation in transmitted light and the marker expression by reverse polymerase chain reaction (RT-PCR), immunofluorescence, and immunoblotting. Some PCR products from prostate tissue, PSCC, and PDO were cloned and sequenced. We found that the cells of early and late passages of PSCC and corresponding PDO expressed luminal (androgen receptor, AR; cytokeratin 18, CK18) and basal (CK5, p63) epithelial markers, the production of which decreased or disappeared in late PSCC and PDO. The PSCC and PDO of early passages from cancer tissue additionally produced cancer markers AMACR, TMPRSS2-ERG, and Ezh2. The expression of TMPRSS2-ERG fusion transcripts was verified by cloning and sequencing the PCR products. The results obtained suggest that early passages of PSCC might be used as a pre-clinical model for the evaluation of early markers of prostate cancer.

Keywords: localized prostate cancer, prostate epithelial markers, prostate cancer markers, AMACR, TMPRSS2-ERG, prostate stromal cell cultures, PDO

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Authors: Sonam Kumari

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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, possesses a remarkable feature of entering into and emerging from a persistent state. The mechanism by which Mtb switches from the dormant state to the replicative form is still poorly characterized. Proteome studies have given us an insight into the role of certain proteins in giving stupendous virulence to Mtb, but numerous dotsremain unconnected and unaccounted. The WhiB family of proteins is one such protein that is associated with developmental processes in actinomycetes.Mtb has seven such proteins (WhiB1 to WhiB7).WhiB proteins are transcriptional regulators; their conserved C-terminal HTH motif is involved in DNA binding. They regulate various essential genes of Mtbby binding to their promoter DNA. Biophysical Analysis of the effect of DNA binding on WhiB proteins has not yet been appropriately characterized. Interaction with DNA induces conformational changes in the WhiB proteins, confirmed by steady-state fluorescence and circular dichroism spectroscopy. ITC has deduced thermodynamic parameters and the binding affinity of the interaction. Since these transcription factors are highly unstable in vitro, their stability and solubility were enhanced by the co-expression of molecular chaperones. The present study findings help determine the conditions under which the WhiB proteins interact with their interacting partner and the factors that influence their binding affinity. This is crucial in understanding their role in regulating gene expression in Mtbandin targeting WhiB proteins as a drug target to cure TB.

Keywords: tuberculosis, WhiB proteins, mycobacterium tuberculosis, nucleic acid binding

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Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

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Authors: Chuanpeng Zhou

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The effect of dietary carbohydrate (CHO) level on serum physiological response, hepatic antioxidant abilities and heat shock protein 70 (HSP70) expression of Wuchang bream (Megalobrama amblycephala) was studied. Two isonitrogenous (28.56% crude protein) and isolipidic (5.28% crude lipid) diets were formulated to contain 30% or 53% wheat starch. Diets were fed for 90 days to fish in triplicate tanks (28 fish per tank). At the end of feeding trial, significantly higher serum triglyceride level, insulin level, cortisol level, malondialdehyde (MDA) content were observed in fish fed the 53% CHO diet, while significantly lower serum total protein content, alkaline phosphatase (AKP) activity, superoxide dismutase (SOD) activity and total antioxidative capacity (T-AOC) were found in fish fed the 53% CHO diet compared with those fed the 30% diet. The relative level of hepatic heat shock protein 70 mRNA was significantly higher in the 53% CHO group than that in the 30% CHO at 6, 12, and 48 h after feeding. The results of this study indicated that ingestion of 53% dietary CHO impacted the nonspecific immune ability and caused metabolic stress of Megalobrama amblycephala.

Keywords: Megalobrama amblycephala, carbohydrate, fasted and postprandial response, immunity, Hsp70

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Authors: Mei Yuin Joanne Wee, Rosli Md. Illias

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Cell surface display is the expression of a protein with an anchoring motif on the surface of the cell. This approach offers advantages when used in bioconversion in terms of easier purification steps and more efficient enzymatic reaction. A surface display system using ice nucleation protein (InaA) from Erwina ananas as an anchoring motif has been constructed to display xylanase (xyl) on the surface of Escherischia coli. The InaA was truncated so that it is made up of the N- and C-terminal domain (INPANC-xyl) and it has successfully directed xylanase to the surface of the cell. A study was also done on xylanase fused to two other ice nucleation proteins, InaK (INPKNC-xyl) and InaZ (INPZNC-xyl) from Pseudomonas syringae KCTC 1832 and Pseudomonas syringae S203 respectively. Surface localization of the fusion protein was verified using SDS-PAGE and Western blot on the cell fractions and all anchoring motifs were successfully displayed on the outer membrane of E. coli. Upon comparison, whole-cell activity of INPANC-xyl was more than six and five times higher than INPKNC-xyl and INPZNC-xyl respectively. Furthermore, the expression of INPANC-xyl on the surface of E. coli did not inhibit the growth of the cell. This is the first report of surface display system using ice nucleation protein, InaA from E. ananas. From this study, this anchoring motif offers an attractive alternative to the current surface display systems.

Keywords: cell surface display, Escherischia coli, ice nucleation protein, xylanase

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Authors: Komal Vig, Syamala Soumyakrishnan, Yadav Baral

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Bypass surgery, using the autologous vein has been one of the most effective treatments for cardiovascular diseases (CVD). More recently tissue engineering including engineered vascular grafts to synthesize blood vessels is gaining usage. Dacron and ePTFE has been employed for vascular grafts, however, these does not work well for small diameter grafts (<6 mm) due to intimal hyperplasia and thrombosis. In the present study PTFE was treated with LTP to improve the endothelialization of intimal surface of graft. Scaffolds were also modified with polyvinylpyrrolidone coated silver nanoparticles (Ag-PVP) and the antimicrobial peptides, p753 and p359. Human umbilical vein endothelial cells (HUVEC) were plated on the developed scaffolds and cell proliferation was determined by the MTT assay. Cells attachment on scaffolds was visualized by microscopy. mRNA expressions levels of different cell markers were investigated using quantitative real-time PCR (qPCR). X ray photoelectron spectroscopic confirmed the introduction of oxygenated functionalities from LTP air plasma. Microscopic and MTT assays indicated increase in cell viability in LTP treated scaffolds. Gene expression studies shows enhanced expression of cell adhesion marker Integrin- α 5 gene after LTP treatment. The KB test displayed a zone of inhibition for Ag-PVP, p753 and p359 of 19mm, 14mm, and 12mm respectively. To determine toxicity of antimicrobial agents to cells, MTT Assay was performed using HEK293 cells. MTT Assay exhibited that Ag-PVP and the peptides were non-toxic to cells at 100μg/mL and 50μg/mL, respectively. Live/dead analysis and plate count of treated bacteria exhibited bacterial inhibition on develop scaffold compared to non-treated scaffold. SEM was performed to analyze the structural changes of bacteria after treatment with antimicrobial agents. Gene expression studies were conducted on RNA from bacteria treated with Ag-PVP and peptides using qRT-PCR. Based on our initial results, more scaffolds alternatives will be developed and investigated for cell growth and vascularization studies.

Keywords: low temperature plasma, vascular graft, HUVEC cells, antimicrobial

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Authors: Simona Perga, Chiara Beltramo, Floriana Fruscione, Isabella Martini, Federica Cavallo, Federica Riccardo, Paolo Buracco, Selina Iussich, Elisabetta Razzuoli, Katia Varello, Lorella Maniscalco, Elena Bozzetta, Angelo Ferrari, Paola Modesto

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Introduction: Human and canine melanoma have common clinical, histologic characteristics making dogs a good model for comparative oncology. The identification of specific genes and a better understanding of the genetic landscape, signaling pathways, and tumor–microenvironmental interactions involved in the cancer onset and progression is essential for the development of therapeutic strategies against this tumor in both species. In the present study, the differential expression of genes in spontaneously occurring canine melanoma and in paired normal tissue was investigated by targeted RNAseq. Material and Methods: Total RNA was extracted from 17 canine malignant melanoma (CMM) samples and from five paired normal tissues stored in RNA-later. In order to capture the greater genetic variability, gene expression analysis was carried out using two panels (Qiagen): Human Immuno-Oncology (HIO) and Mouse-Immuno-Oncology (MIO) and the miSeq platform (Illumina). These kits allow the detection of the expression profile of 990 genes involved in the immune response against tumors in humans and mice. The data were analyzed through the CLCbio Genomics Workbench (Qiagen) software using the Canis lupus familiaris genome as a reference. Data analysis were carried out both comparing the biologic group (tumoral vs. healthy tissues) and comparing neoplastic tissue vs. paired healthy tissue; a Fold Change greater than two and a p-value less than 0.05 were set as the threshold to select interesting genes. Results and Discussion: Using HIO 63, down-regulated genes were detected; 13 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Eighteen genes were up-regulated, 14 of those were also down-regulated comparing neoplastic sample vs. paired healthy tissue. Using the MIO, 35 down regulated-genes were detected; only four of these were down-regulated, also comparing neoplastic sample vs. paired healthy tissue. Twelve genes were up-regulated in both types of analysis. Considering the two kits, the greatest variation in Fold Change was in up-regulated genes. Dogs displayed a greater genetic homology with humans than mice; moreover, the results have shown that the two kits are able to detect different genes. Most of these genes have specific cellular functions or belong to some enzymatic categories; some have already been described to be correlated to human melanoma and confirm the validity of the dog as a model for the study of molecular aspects of human melanoma.

Keywords: animal model, canine melanoma, gene expression, spontaneous tumors, targeted RNAseq

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Authors: Kyle Phillips, Ndiko Ludidi

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Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22 which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extracellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of up-regulation and silencing of the maize RD22 gene on the tolerance of maize to drought. The potential role of the maize RD22 gene in maize drought tolerance can be used as a tool to improve food security.

Keywords: abscisic acid, drought-responsive cis-acting elements, maize drought tolerance, RD22

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Authors: Rafia S. Al-lamki, Jun Wang, Simon Pacey, Jordan Pober, John R. Bradley

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Tumor necrosis factor alpha (TNF), signaling through TNFR2, may act an autocrine growth factor for renal tubular epithelial cells. Clear cell renal carcinomas (ccRCC) contain cancer stem cells (CSCs) that give rise to progeny which form the bulk of the tumor. CSCs are rarely in cell cycle and, as non-proliferating cells, resist most chemotherapeutic agents. Thus, recurrence after chemotherapy may result from the survival of CSCs. Therapeutic targeting of both CSCs and the more differentiated bulk tumor populations may provide a more effective strategy for treatment of RCC. In this study, we hypothesized that TNFR2 signaling will induce CSCs in ccRCC to enter cell cycle so that treatment with ligands that engage TNFR2 will render CSCs susceptible to chemotherapy. To test this hypothesis, we have utilized wild-type TNF (wtTNF) or specific muteins selective for TNFR1 (R1TNF) or TNFR2 (R2TNF) to treat either short-term organ cultures of ccRCC and adjacent normal kidney (NK) tissue or cultures of CD133+ cells isolated from ccRCC and adjacent NK, hereafter referred to as stem cell-like cells (SCLCs). The effect of cyclophosphamide (CP), currently an effective anticancer agent, was tested on CD133+SCLCs from ccRCC and NK before and after R2TNF treatment. Responses to TNF were assessed by flow cytometry (FACS), immunofluorescence, and quantitative real-time PCR, TUNEL, and cell viability assays. Cytotoxic effect of CP was analyzed by Annexin V and propidium iodide staining with FACS. In addition, we assessed the effect of TNF on isolated SCLCs differentiation using a three-dimensional (3D) culture system. Clinical samples of ccRCC contain a greater number SCLCs compared to NK and the number of SCSC increases with higher tumor grade. Isolated SCLCs show expression of stemness markers (oct4, Nanog, Sox2, Lin28) but not differentiation markers (cytokeratin, CD31, CD45, and EpCAM). In ccRCC organ cultures, wtTNF and R2TNF increase CD133 and TNFR2 expression and promote cell cycle entry whereas wtTNF and R1TNF increase TNFR1 expression and promote cell death of SCLCs. Similar findings are observed in SCLCs isolated from NK but the effect was greater in SCLCs isolated from ccRCC. Application of CP distinctly triggered apoptotic and necrotic cell death in SLCSs pre-treatment with R2TNF as compared to CP treatment alone, with SCLCs from ccRCC more sensitive to CP compared to SLCS from NK. Furthermore, TNF promotes differentiation of SCLCs to an epithelial phenotype in 3D cultures, confirmed by cytokeratin expression and loss of stemness markers Nanog and Sox2. The differentiated cells show positive expression of TNF and TNFR2. These findings provide evidence that selective engagement of TNFR2 drive CSCs to cell proliferation/differentiation, and targeting of cycling cells with TNFR2 agonist in combination with anti-cancer agents may be a potential therapy for RCC.

Keywords: cancer stem cells, ccRCC, cell cycle, cell death, TNF, TNFR1, TNFR2, CD133

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Authors: Lali Shanshiashvili, Marika Chikviladze, Nino Mamulashvili, Maia Sepashvili, Nana Narmania, David Mikeladze

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Purpose: During demyelinating inflammatory diseases and after the damage of the myelin sheet, myelin-derived proteins, including myelin basic protein (MBP), are secreted into the extracellular space. MBP shows extensive post-translational modifications, including the deimination of arginine residues. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than the unmodified one. It is hypothesized that MBP could change the inflammatory response in astrocytes. Methods: MBP was isolated and purified from bovine brain white matter. Primary astrocyte cultures were prepared from whole brains of 2-day-old Wistar rats. For evaluation of glutamate uptake/release in astrocytes following treatment of cells with MBP charge isomers, Glutamate Assay Kit was used. The expression of EAAT-2 (excitatory amino acid transporters), peroxisome proliferator-activated receptor gamma (PPAR- γ), inhibitor of nuclear factor kappa B (IkB), and high mobility group protein B1 (HMGB1) in astrocytes were assayed by Western Blot analysis. Results: This study investigated the action of deiminated isomer (C8) on the cultured primary astrocytes and compared its effects with the effects of unmodified C1 isomers. The study found that C8 and C1 MBP differently act on the uptake and release of glutamate in astrocytes: nonmodified C1 MBP increases the uptake of glutamate and does not change the release, whereas C8 decreases the release of glutamate but does not alter the uptake. Nevertheless, both isomers increased the expression of PPAR-γ and EAAT2 in the same intensity. However, immunostaining and Western Blots of cell lysates showed a decrease of IkB and increased expression of HMGB1 after the treatment of astrocytes by C8. Moreover, in the presence of C8, astrocytes release more nitric oxide than unmodified C1 isomers. Conclusion: These data suggest that the deiminated isomer of MBP evokes an inflammatory response and enhances the ability of astrocytes to release proinflammatory mediators through activation of NF-kB after the breakdown of myelin sheets. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.

Keywords: myelin basic protein, glutamate, deimination, astrocytes, inflammation

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Authors: Sina Mahdavi

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Background and Objective: Multiple sclerosis (MS) is an inflammatory autoimmune disease of the CNS that affects the myelination process in the central nervous system (CNS). Complex interactions of various "environmental or infectious" factors may act as triggers in autoimmunity and disease progression. The association between viral infections, especially Human Herpesvirus 6 (HHV-6), and MS is one potential cause that is not well understood. In this study, we aim to summarize the available data on HHV-6 infection in MS disease progression. Materials and Methods: For this study, the keywords "Multiple sclerosis", " Human Herpesvirus 6 ", and "central nervous system" in the databases PubMed and Google Scholar between 2017 and 2022 were searched, and 12 articles were chosen, studied, and analyzed. Results: HHV 6 tends towards TCD 4+ lymphocytes and enters the CNS due to the weakening of the blood-brain barrier due to inflammatory damage. Following the observation that the HHV-6 U24 protein has a seven amino acid sequence with myelin basic protein, which is one of the main components of the myelin sheath, it could cause a molecular mimicry mechanism followed by cross-reactivity. Reactivation of HHV-6 in the CNS can cause the release of proinflammatory cytokines, including TNF-α, leading to immune-mediated demyelination in patients with MS. Conclusion: There is a high expression of endogenous retroviruses during the course of MS, which indicates the relationship between HHV-6 and MS, and that this virus can play a role in the development of MS by creating an inflammatory state. Therefore, measures to modulate the expression of HHV-6 may be effective in reducing inflammatory processes in demyelinated areas of MS patients.

Keywords: multiple sclerosis, human herpesvirus 6, central nervous system, autoimmunity

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Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais

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The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.

Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology

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Authors: Qing Zhang, Janak L. Pathak, Macro N. Helder, Richard T. Jaspers, Yin Xiao

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Introduction: Nanomaterial-based bone regeneration is greatly influenced by the immune microenvironment. Tissue-engineered nanomaterials mediate the inflammatory response of macrophages to regulate bone regeneration. Silica nanoparticles have been widely used in tissue engineering-related preclinical studies. However, the effect of topological features on the surface of silica nanoparticles on the immune response of macrophages remains unknown. Purposes: The aims of this research are to compare the influences of normal and pollen-like silica nano-surface topography on macrophage immune responses and to obtain insight into their potential regulatory mechanisms. Method: Macrophages (RAW 264.7 cells) were exposed to mesoporous silica nanoparticles with normal morphology (MSNs) and pollen-like morphology (PMSNs). RNA-seq, RT-qPCR, and LSCM were used to assess the changes in expression levels of immune response-related genes and proteins. SEM and TEM were executed to evaluate the contact and adherence of silica nanoparticles by macrophages. For the assessment of the immunomodulation-mediated osteogenic potential, BMSCs were cultured with conditioned medium (CM) from LPS pre-stimulated macrophage cultures treated with MSNs or PMSNs. Osteoimmunomodulatory potential of MSNs and PMSNs in vivo was tested in a mouse cranial bone osteolysis model. Results: The results of the RNA-seq, RT-qPCR, and LSCM assays showed that PMSNs inhibited the expression of pro-inflammatory genes and proteins in macrophages. SEM images showed distinct macrophage membrane surface binding patterns of MSNs and PMSNs. MSNs were more evenly dispersed across the macrophage cell membrane, while PMSNs were aggregated. PMSNs-induced macrophage anti-inflammatory response was associated with upregulation of the cell surface receptor CD28 and inhibition of ERK phosphorylation. TEM images showed that both MSNs and PMSNs could be phagocytosed by macrophages, and inhibiting nanoparticle phagocytosis did not affect the expression of anti-inflammatory genes and proteins. Moreover, PMSNs-induced conditioned medium from macrophages enhanced BMP-2 expression and osteogenic differentiation mBMSCs. Similarly, PMSNs prevented LPS-induced bone resorption via downregulation of inflammatory reaction. Conclusions: PMSNs can promote bone regeneration by modulating osteoimmunological processes through surface topography. The study offers insights into how surface physical contact cues can modulate the regulation of osteoimmunology and provides a basis for the application of nanoparticles with pollen-like morphology to affect immunomodulation in bone tissue engineering and regeneration.

Keywords: physical contact, osteoimmunology, macrophages, silica nanoparticles, surface morphology, membrane receptor, osteogenesis, inflammation

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Authors: Georgi Bebrov, Rozalina Dimova

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In the field of quantum secure communication, there is no evaluation that characterizes quantum secure communication (QSC) protocols in a complete, general manner. The current paper addresses the problem concerning the lack of such an evaluation for QSC protocols by introducing an optimality evaluation, which is expressed as the average over the three main parameters of QSC protocols: efficiency, security, and practicality. For the efficiency evaluation, the common expression of this parameter is used, which incorporates all the classical and quantum resources (bits and qubits) utilized for transferring a certain amount of information (bits) in a secure manner. By using criteria approach whether or not certain criteria are met, an expression for the practicality evaluation is presented, which accounts for the complexity of the QSC practical realization. Based on the error rates that the common quantum attacks (Measurement and resend, Intercept and resend, probe attack, and entanglement swapping attack) induce, the security evaluation for a QSC protocol is proposed as the minimum function taken over the error rates of the mentioned quantum attacks. For the sake of clarity, an example is presented in order to show how the optimality is calculated.

Keywords: quantum cryptography, quantum secure communcation, quantum secure direct communcation security, quantum secure direct communcation efficiency, quantum secure direct communcation practicality

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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: Peter Jean-Paul, Shanaz Wahid

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The problem of division by zero can be stated as: “what is the value of 0 x 1/0?” This expression has been considered undefined by mathematicians because it can have two equally valid solutions either 0 or 1. Recently semi-structured complex number set was invented to solve “division by zero”. However, whilst the number set had some merits it was considered to have a poor theoretical foundation and did not provide a quality solution to “division by zero”. Moreover, the set lacked consistency in simple algebraic calculations producing contradictory results when dividing by zero. To overcome these issues this research starts by treating the expression " 0 x 1/0" as a quantum mechanical system that produces two tangled results 0 and 1. Dirac Notation (a tool from quantum mechanics) was then used to redefine the unstructured unit p in semi-structured complex numbers so that p represents the superposition of two results (0 and 1) and collapses into a single value when used in algebraic expressions. In the process, this paper describes a new number set called Quantum Semi-structured Complex Numbers that provides a valid solution to the problem of “division by zero”. This research shows that this new set (1) forms a “Field”, (2) can produce consistent results when solving division by zero problems, (3) can be used to accurately describe systems whose mathematical descriptions involve division by zero. This research served to provide a firm foundation for Quantum Semi-structured Complex Numbers and support their practical use.

Keywords: division by zero, semi-structured complex numbers, quantum mechanics, Hilbert space, Euclidean space

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Authors: A. Alyami, S. Christmas, K. Neeltje, G. Pollakis, B. Paxton, Z. Al-Bayati

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The Human leukocyte antigen-G (HLA-G) molecule plays an important role in immunomodulation. To date, 16 untranslated regions (UTR) HLA-G haplotypes have been previously defined by sequenced SNPs in the coding region. From these, UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-6 and UTR-7 are the most frequent 3’UTR haplotypes at the global level. UTR-1 is associated with higher levels of soluble HLA-G and HLA-G expression, whereas UTR-5 and UTR-7 are linked with low levels of soluble HLA-G and HLA-G expression. Human immunodeficiency virus type 1 (HIV-1) infection results in the progressive loss of immune function in infected individuals. The virus escape mechanism typically includes T lymphocytes and NK cell recognition and lyses by classical HLA-A and B down-regulation, which has been associated with non-classical HLA-G molecule up-regulation, respectively. We evaluated the haplotypes of the HLA-G 3′ untranslated region frequencies observed in three HIV-1 groups from the Netherlands and their susceptibility to develop infection. The three groups are made up of mainly men who have sex with men (MSM), injection drug users (IDU) and a high-risk-seronegative (HRSN) group. DNA samples were amplified with published primers prior sequencing. According to our results, the low expresser frequencies show higher in HRSN compared to other groups. This is indicating that 3’UTR polymorphisms may be identified as potential prognostic biomarkers to determine susceptibility to HIV.

Keywords: Human leukocyte antigen-G (HLA-G) , men who have sex with men (MSM), injection drug users (IDU), high-risk-seronegative (HRSN) group, high-untranslated region (UTR)

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Authors: Naif Al-Jomah, Falah H Al-Mohanna, Abdelilah Aboussekhra

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Active breast cancer-associated fibroblasts (CAFs), the most influential cells in breast tumor microenvironment, express/secrete high levels of the proinvasive/metastatic interleukin-6 (IL-6). Therefore, we have tested here the effect of the IL-6 receptor (IL-6R) inhibitor tocilizumab (TCZ; Actemra) on different active breast CAFs. We have shown that TCZ potently and persistently suppresses the expression of various CAF biomarkers, namely α-SMA, SDF-1 as well as the STAT3 pathway and its downstream target AUF1. TCZ also inhibited the proliferation, migration and invasion abilities of active breast CAF cells. Additionally, TCZ repressed the ability of CAF cells in promoting epithelial-to-mesenchymal transition, and enhancing the migratory/invasive and proliferative capacities of breast cancer cells in vitro. Importantly, these findings were confirmed in orthotopic humanized breast tumors in mice. Furthermore, TCZ suppressed the expression of the pro-angiogenic factor VEGF-A and its transactivator HIF-1α in CAF cells, and consequently inhibited the angiogenic-promoting effect of active CAFs both in vitro and in orthotopic tumor xenografts. These results indicate that inhibition of the IL-6/STAT3/AUF1 pathway by TCZ can normalize active breast CAFs and suppress their paracrine pro-carcinogenic effects, which paves the way toward development of specific CAF-targeting therapy, badly needed for more efficient breast cancer treatments.

Keywords: angiogenesis, interleukin-6, paracrine, cancer-associated fibroblasts

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Authors: Seunghwan Seo, Woo-Seok Lee, Ji-Sun Shin, Young Kyoung Rhee, Chang-Won Cho, Hee-Do Hong, Kyung-Tae Lee

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Doenjang, known as traditional Korean food, is product of a natural mixed fermentation process carried out by lactic acid bacteria (LAB). Lactobacillus sakei K040706 (K040706) has been accepted as the most populous LAB in over ripened doenjang. Recently, we reported the immunostimulatory effects of K040706 in RAW 264.7 macrophages and in a cyclophosphamide-induced mouse model. In this study, we investigated the ameliorative effects of K040706 in a dextran sulfate sodium (DSS)-induced colitis mouse model. We induced colitis using DSS in 5-week-ICR mice over 14 days with or without 0.1, 1 g/kg/day K040706 orally. The body weight, stool consistency, and gross bleeding were recorded for determination of the disease activity index (DAI). At the end of treatment, animals were sacrificed and colonic tissues were collected and subjected to histological experiments and myeloperoxidase (MPO) accumulation, cytokine determination, qRT-PCR and Western blot analysis. Results showed that K040706 significantly attenuated DSS-induced DAI score, shortening of colon length, enlargement of spleen and immune cell infiltrations into colonic tissues. Histological examinations indicated that K040706 suppressed edema, mucosal damage, and the loss of crypts induced by DSS. These results were correlated with the restoration of tight junction protein expression, such as, ZO-1 and occludin in K040706-treated mice. Moreover, K040706 reduced the abnormal secretions and mRNA expressions of pro-inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). DSS-induced mRNA expression of intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) in colonic tissues was also downregulated by K040706 treatment. Furthermore, K040706 suppressed the protein and mRNA expression of toll-like receptor 4 (TLR4) and phosphorylation of NF-κB and signal transducer and activator of transcription 3 (STAT3). These results suggest that K040706 has an anti-colitic effect by inhibition of intestinal inflammatory responses in DSS-induced colitic mice.

Keywords: Lactobacillus sakei, NF-κB, STAT3, ulcerative colitis

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Authors: Ricardo de Oliveira Orsi, Aline Astolfi, Daniel Diego Mendes, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, Yan Souza Lima, Juliana Lunardi, Giovanna do Padro Ribeiro, Samir Moura Kadri

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NECTAR Brazilian group (Center of Education, Science, and Technology in Rational Beekeeping) conducted studies on the pesticides honey bees effects using the transcriptome sequencing (RNA-Seq) analyzes for gene expression studies. In this way, we analyzed the effects of Pyraclostrobin and Fipronil on the honey bees with 21 old-days (forager) in laboratory conditions. For this, frames containing sealed brood were removed from the beehives and maintenance on the stove (32°C and 75% humidity) until the bees were born. So, newly emerged workers were marked on the pronotum with a non-toxic pen and reintroduced into their original hives. After 21 days, 120 marked bees were collected with an entomological forces and immediately stored in Petri dishes, perforated to ensure ventilation, and kept fasted for 3 hours. These honeybees were exposed to food contaminated or not with the sublethal dose of Pyraclostrobin (850 ppb/bee) or Fipronil (2.5 ppb/bee). After four hours of exposure, 15 bees from each treatment were referred to transcriptome analysis. Total RNA analysis was extracted from the brain pools (03 brains per pool) using the TRIzol® reagent protocol according to the manufacturer's instructions. cDNA libraries were constructed, and the FASTQC program was used to check adapter content and assess the quality of raw reads. Differential expression analysis was performed with the DESeq2 package. Genes that had an adjusted value of less than 0.05 were considered to be significantly up-regulated. Regarding the Pyraclostrobin, alterations were observed in the pattern of 17 gene related to of antioxidant system, cellular respiration, glucose metabolism, and regulation of juvenile hormone and the hormone insulin. Glyphosate altered the 10 gene related to the digestive system, exoskeleton composition, vitamin E transport, and antioxidant system. The results indicate that the necessity of studies using the sublethal doses to evaluate the pesticides uses and risks on crops and its effects on the honey bees.

Keywords: beekeeping, honey bees, pesticides, transcriptome

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Authors: Dina Fatmawati, Sofia Mubarika, Mae Sri Wahyuningsih

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Chemotherapy for breast cancer is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous study, the combination of Doxorubicin (Dox) and ethanolic extract of Dioscorea esculenta tuber ((EED) was found to have a synergistic effect on T47D human breast cancer cell line. In this study, we investigated the apoptotic effect of the combination on T47D human breast cancer cells and normal fibroblasts cell line and its effects on the expression of Caspase-3 and cleaved poly (ADP-Ribose) Polymerase-1 (cPARP-1) protein. T47D cell lines and fibroblasts cells were treated with the combination of Dox and EED. Apoptotic effect of the combination was determined using flow cytrometry assay. Protein expressions were determined by immunocytochemistry staining. The percentage of apoptotic cells were significantly higher in T47D cell lines (75%) than that of in fibroblast cells (23%). The expression of Caspase 3 (84.53%) and cPARP-1 (83.36%) were significantly higher in the cancer cell lines than those of normal cells. These results indicate that the combination of doxorubicin and Dioscorea esculenta is a promising candidate for the treatment of breast cancer cells.

Keywords: Dioscorea esculenta, Doxorubicin, apoptosis, immunocytochemistry, cancer cells

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Authors: P. Narrima, C. Y. Looi, M. A. Mohd, H. M. Ali

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Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development.

Keywords: antiproliferative, apoptosis, MCF-7 human breast cancer, Persea declinata

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