Search results for: giant cancer cells

Authors: J. Barilla, M. Lokajíček, H. Pisaková, P. Simr

Abstract:

The final biological effect of ionizing particles may be influenced strongly by some chemical substances present in cells mainly in the case of low-LET radiation. The influence of oxygen may be particularly important because oxygen is always present in living cells. The corresponding processes are then running mainly in the chemical stage of radio biological mechanism. The radical clusters formed by densely ionizing ends of primary or secondary charged particles are mainly responsible for final biological effect. The damage effect depends then on radical concentration at a time when the cluster meets a DNA molecule. It may be strongly influenced by oxygen present in a cell as oxygen may act in different directions: at small concentration of it the interaction with hydrogen radicals prevails while at higher concentrations additional efficient oxygen radicals may be formed. The basic radical concentration in individual clusters diminishes, which is influenced by two parallel processes: chemical reactions and diffusion of corresponding clusters. The given simultaneous evolution may be modeled and analyzed well with the help of Continuous Petri nets. The influence of other substances present in cells during irradiation may be studied, too. Some results concerning the impact of oxygen content will be presented.

Keywords: radiobiological mechanism, chemical phase, DSB formation, Petri nets

Procedia PDF Downloads 308

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

Abstract:

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

Procedia PDF Downloads 143

Authors: Hawraa Zbeeb, Hala Khalifeh, Mohamad Khalil, Francesca Storace, Francesca Baldini, Giulio Lupidi, Laura Vergani

Abstract:

Sarcopoterium spinosum, a widely distributed spiny shrub belonging to the Rosaceae family, is rich in essential and beneficial constituents. In fact, S. spinosum fruits and roots are traditionally used as herbal medicine in the eastern Mediterranean landscape, and this shrub is mentioned as a medicinal plant in a large number of ethnobotanical surveys. Aqueous root extracts from S. spinosum are used by traditional medicinal practitioners for weight loss treatment of diabetes and pain. Moreover, the anti-diabetic activity of S. spinosum root extract has been reported in different studies, but the beneficial effects of aerial parts, especially fruits, have not been elucidated yet. The aim of the present study was to investigate the in vitro antioxidant and lipid-lowering properties of an ethanolic extract from S. spinosum fruits using both hepatic (FaO) and endothelial (HECV) cells in an attempt to evaluate its possible employment as a nutraceutical supplement. First of all, in vitro spectrophotometric assays were employed to characterize the extract. The total phenol content (TPC) was evaluated by Folin–Ciocalteu spectrophotometric method and the radical scavenging activity was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. After that, the beneficial effects of the extract were tested on cells. FaO cells treated for 3 hours with 0.75 mM oleate/palmitate mix (1:2 molar ratio) mimic in vitro a moderate hepato-steatosis. HECV cells exposed for 1 hour to 100 µM H₂O₂ mimic an oxidative insult leading to oxidative stress conditions. After the metabolic and oxidative insult, both cell lines were treated with increasing concentrations of the S. spinosum extract (1, 10, 25 µg/mL) for 24 hours. The results showed the S. spinosum ethanolic extract is rather rich in phenols (TPC of 18.6 mgGAE/g dry extracts). Moreover, the extract showed a good scavenging ability in vitro (IC₅₀ 15.9 µg/ml and 10.9 µg/ml measured by DPPH and ABTS assays, respectively). When the extract was tested on cells, the results showed that it could ameliorate some markers of cell dysfunction. The three concentrations of the extract led to a significant decrease in the intracellular triglyceride (TG) content in steatotic FaO cells measured by spectrophotometric assay. On the other hand, HECV cells treated with increasing concentrations of the extract did not result in a significant decrease in both lipid peroxidation measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay, and in reactive oxygen species (ROS) production measured by fluorometric analysis after DCF staining. Interestingly, the ethanolic extract was able to accelerate the wound repair of confluent HECV cells with respect to H₂O₂-insulted cells as measured by T-scratch assay. Taken together, these results seem to indicate that the ethanol extract from S. spinosum fruits is rich in phenol compounds and plays considerable lipid-lowering activity in vitro on steatotic hepatocytes and accelerates wound healing repair on endothelial cells. In light of that, the ethanolic extract from S. spinosum fruits could be a potential candidate for nutraceutical applications.

Keywords: antioxidant activity, ethanolic extract, lipid-lowering activity, phenolic compounds, Sarcopoterium spinosum fruits

Procedia PDF Downloads 173

Authors: Agajie Likie Bogale, Tilahun Teklehaymanot, Getnet Mitike Kassie, Girmay Medhin, Jemal Haidar Ali, Nega Berhe Belay

Abstract:

Objectives: The aim of this study is to evaluate the performance of visual inspection using acetic acid compared with HPV DNA testing among women living with HIV in Ethiopia. Methods: Acomparative cross-sectional study was conducted to address the aforementioned objective. Data were collected from January to October 2021 to compare the performance of these two screening modalities. Trained clinicians collected cervical specimens and immediately applied acetic acid for visual inspection. The HPV DNA testing was done using Abbott m2000rt/SP by trained laboratory professionals in accredited laboratories. A total of 578 HIV positive women with age 25-49 years were included. Results: Test positivity was 8.9% using VIA and 23.3% using HPV DNA test. The sensitivity and specificity of the VIA test were 19.2% and 95.1%, respectively, while the positive and negative predictive values of the VIA test were 54.4% and 79.4%, respectively. The strength of agreement between the two screening methods was poor (k=0.184), and the area under the curve was 0.572. The burden of genetic distribution of high risk HPV16 was 3.8%, and mixed HPV16& other HR HPV was 1.9%. Other high risk HPV types were predominant in this study (15.7%). Conclusion: The high positivity result using HPV DNA testing compared with VIA, and low sensitivity of VIA are indicating that the implementation of HPV DNA testing as the primary screening strategy is likely to reduce cervical cancer cases and deaths of women in the country.

Keywords: cervical cancer screening, HPV DNA, VIA, Ethiopia

Procedia PDF Downloads 133

Authors: Aditya Manna

Abstract:

Introduction: As in any developing countries state of West Bengal in India has a huge burden of cancer patients in advanced stage coming from rural area where awareness regarding the usefulness of palliative care in rather poor. Objective: Our goal is to give a pain free good quality of life in these advanced stage cancer patients. Objective of this study is to identify the main difficulties in achieving the above goal in a rural village setting in India. Method: Advanced cancer patients in need of palliative care in various villages in of rural India were selected for this study. Their symptoms and managements in that rural surroundings were evaluated by an NGO (under the guidance of a senior palliative care specialist) working in that area. An attempt was made to identify the main obstacles in getting proper palliative care in a rural setting. Results: Pain, fatigue are the main symptoms effecting these patients. In most patients pain and other symptoms control were grossly inadequate due to lack of properly trained manpower in the rural India. However regular homecare visits by a group of social workers were of immense help in the last few months of life. NGO team was well guided by a palliative care specialist. Conclusion: There is a wide gap of trained manpower in this filled in rural areas of India. Dedicated groups from rural area itself need encouragement and proper training, so that difficult symptoms can be managed locally along with necessary social and psychological support to these patients.

Keywords: palliative care, NGO, rural India, home care

Procedia PDF Downloads 292

Authors: Olena Grygorieva

Abstract:

Nowadays mechanisms of thymocytes emigration from the thymus to the periphery are investigated actively. We have proposed a hypothesis of thymocytes’ migration from the thymus through lymphatic vessels during periodical short-term local edema. By morphological, hystochemical methods we have examined quantity of lymphocytes, epitelioreticulocytes, mast cells, blood and lymphatic vessels in morpho-functional areas of rats’ thymuses during the first week after birth in 4 hours interval. In newborn and beginning from 8 hour after birth every 12 hours specific density of the thymus, absolute quantity of microcirculatory vessels, especially of lymphatic ones, lymphcyte-epithelial index, quantity of mast cells and their degranulative forms increase. Structure of extracellular matrix, intrathymical microenvironment and lymphocytes’ adhesive properties change. Absolute quantity of small lymphocytes in thymic cortex changes wavy. All these changes are straightly expressed from 0 till 2, from 12 till 16, from 108 till 120 hours of postnatal life. During this periods paravasal lymphatic vessels are stuffed with lymphocytes, i.e. discrete migration of lymphocytes from the thymus occurs. After rapid edema reduction, quantity of lymphatic vessels decrease, they become empty. Therefore, in the thymus of newborn periodical short-term local edema is observed, on its top discrete migration of lymphocytes from the thymus occurs.

Keywords: lymphocytes, lymphatic vessels, mast cells, thymus

Procedia PDF Downloads 221

Authors: N. Selami, M. Kaid Harche

Abstract:

A pink-pigmented, aerobic, facultatively methylotrophic bacterium, was isolated from Retama monosperma root nodules and identified as a member of the genus Methylobacterium. Inoculation of R. monosperma plants by a pure culture of isolate strain under a hydroponic condition, resulted, 10 dpi, the puffiness at lateral roots. The observation in detail the anatomy and ultra-structure of infection sites by light and electron microscopy show that the bacteria induce stimulation of the division of cortical cells and digestion of epidermis cells then, Methylobacterium was observed in the inter and intracellular spaces of the outer cortex root. These preliminary results allow us to suggest the establishment of an epi-endosymbiotic interaction between Methylobacterium sp and R. monosperma.

Keywords: endophytic colonization, Methylobacterium, microscopy, nodule, pink pigmented, Retama monosperma

Procedia PDF Downloads 361

Authors: Bayan Almofty, Yuto Yamaki, Tadamasa Terai, Sadahito Uto

Abstract:

Myopathy (muscles disease) treatment are expected in the field of regenerative medicine and applied research of cultured muscle to bio actuator is performed in Biomedical Engineering as applied research of cultured muscle. This study is about cultured myoblast C2C12 from mouse skeletal muscle and a mechanism of cultured muscle contraction by electric stimulation is investigated. Grayanotoxins (GTXs) belong to neurotoxins known to enhance the permeability of cell membrane for Na ions. Grayanotoxins are extracted from a famous Pieris japonica and Ericaceae as a phytotoxin. We investigated the functional role of GTXs on muscle cells (C2C12) contraction and membrane potential. A change in membrane potential is measured using a micro glass tube electrode contraction of myotubes is induced by applying an external electrical stimulation. The contraction and membrane potential change induced by injection of current using the micro glass electrode are also measured. From the result, contraction and membrane potential of muscle cells was affected by GTXs treatment, suggesting that the diverse chemical structures of GTXs are responsible for contraction and membrane potential of muscle cells.

Keywords: skeletal muscle, C2C12, myoblast, myotubes, contraction, Grayanotoxins, membrane potential, neurotoxins, phytotoxin

Procedia PDF Downloads 463

Authors: Subrata Kar, Banani Kundu, Papiya Nandy, Ruma Basu, Sukhen Das

Abstract:

Natural montmorilollite clay is a common ingredient in pharmaceutical products, both as excipients and active support; hence considered as suitable candidate for Drug Delivery System. In this work, cationic detergent CTAB is used to increase the interlayer spacing of Na+-Montmoriollite clay to intercalate curcumin and methotrexate. Methotrexate is a folic acid antagonist, anti-proliferative and immunosuppressive agent; while curcumin is a bioactive constituent of rhizomes of Curcuma longa, possessing remarkable chemo-preventive and anti-inflammatory properties. The resultant inorganic-organic hybrids are characterized by X-ray diffraction (XRD), Infrared spectroscopy (FTIR) and Thermo Gravimetric Analysis (TGA) to confirm successful intercalation of curcumin and Methotrexate within clay layers. Pharmaceutical investigation of the hybrids is explored by studying the drug loading (%), encapsulation efficiency and release kinetics. Finally in-vitro studies are performed using cancer cells to find the effect of released curcumin to improve the sensitivity of clay bound methotrexate to ameliorate cell death compared to their effectiveness when used without the inorganic aluminosilicate vehicle.

Keywords: montmorillonite, methotrexate, curcumin, loading efficiency, release kinetics, anticancer activity

Procedia PDF Downloads 511

Authors: Tanima Chatterjee, Maitree Bhattacharyya

Abstract:

The global challenge of type 2 diabetes mellitus is a major health concern in this millennium, and researchers are continuously exploring new targets to develop a novel therapeutic strategy. Type 2 diabetes mellitus (T2DM) is often coupled with dyslipidemia increasing the risks for cardiovascular (CVD) complications. Enhanced oxidative and nitrosative stresses appear to be the major risk factors underlying insulin resistance, dyslipidemia, β-cell dysfunction, and T2DM pathogenesis. Autophagy emerges to be a promising defense mechanism against stress-mediated cell damage regulating tissue homeostasis, cellular quality control, and energy production, promoting cell survival. In this study, we have attempted to explore the pivotal role of autophagy in T2DM subjects with or without dyslipidemia in peripheral blood mononuclear cells and insulin-resistant HepG2 cells utilizing flow cytometric platform, confocal microscopy, and molecular biology techniques like western blotting, immunofluorescence, and real-time polymerase chain reaction. In the case of T2DM with dyslipidemia higher population of autophagy, positive cells were detected compared to patients with the only T2DM, which might have resulted due to higher stress. Autophagy was observed to be triggered both by oxidative and nitrosative stress revealing a novel finding of our research. LC3 puncta was observed in peripheral blood mononuclear cells and periphery of HepG2 cells in the case of the diabetic and diabetic-dyslipidemic conditions. Increased expression of ATG5, LC3B, and Beclin supports the autophagic pathway in both PBMC and insulin-resistant Hep G2 cells. Upon blocking autophagy by 3-methyl adenine (3MA), the apoptotic cell population increased significantly, as observed by caspase‐3 cleavage and reduced expression of Bcl2. Autophagy has also been evidenced to control oxidative stress-mediated up-regulation of inflammatory markers like IL-6 and TNF-α. To conclude, this study elucidates autophagy to play a protective role in the case of diabetes mellitus with dyslipidemia. In the present scenario, this study demands to have a significant impact on developing a new therapeutic strategy for diabetic dyslipidemic subjects by enhancing autophagic activity.

Keywords: autophagy, apoptosis, dyslipidemia, reactive oxygen species, reactive nitrogen species, Type 2 diabetes

Procedia PDF Downloads 127

Authors: Abu Salim Mustafa

Abstract:

Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.

Keywords: mycobacterium tuberculosis, PPE68, peptides, vaccine

Procedia PDF Downloads 129

Authors: Dong-An Wang

Abstract:

All kinds of microspheres have been extensively employed as carriers for drug, gene and therapeutic cell delivery. Most therapeutic cell delivery microspheres rely on a two-step methodology: fabrication of microspheres and subsequent seeding of cells onto them. In this study, we have developed a novel one-step cell encapsulation technique using a convenient and instant water-in-oil single emulsion approach to form cell-encapsulated gelatin microspheres. This technology is adopted for hyaline cartilage tissue engineering, in which autologous chondrocytes are used as therapeutic cells. Cell viability was maintained throughout and after the microsphere formation (75-100 µm diameters) process that avoids involvement of any covalent bonding reactions or exposure to any further chemicals. Further encapsulation of cell-laden microspheres in alginate gels were performed under 4°C via a prompt process. Upon the formation of alginate constructs, they were immediately relocated into CO2 incubator where the temperature was maintained at 37°C; under this temperature, the cell-laden gelatin microspheres dissolved within hours to yield similarly sized cavities and the chondrocytes were therefore suspended within the cavities inside the alginate gel bulk. Hence, the gelatin cell-laden microspheres served two roles: as cell delivery vehicles which can be removable through temperature curing, and as porogens within an alginate hydrogel construct to provide living space for cell growth and tissue development as well as better permeability for mutual diffusions. These cell-laden microspheres, namely “temperature-cured dissolvable gelatin microsphere based cell carriers” (tDGMCs), were further encapsulated in a chondrocyte-laden alginate scaffold system and analyzed by WST-1, gene expression analyses, biochemical assays, histology and immunochemistry stains. The positive results consistently demonstrated the promise of tDGMC technology in delivering these non-anchorage dependent cells (chondrocytes). It can be further conveniently translated into delivery of other non-anchorage dependent cell species, including stem cells, progenitors or iPS cells, for regeneration of tissues in internal organs, such as engineered hepatogenesis or pancreatic regeneration.

Keywords: biomaterials, tissue engineering, microsphere, hydrogel, porogen, anchorage dependence

Procedia PDF Downloads 391

Authors: Mayuva Youngsabanant-Areekijseree, Chanikarn Srinark, S. Sengsai, Mayuree Pumipaiboon

Abstract:

Our project was aimed at morphology of oocytes, follicle cells and follicular fluid proteins study of Large White pig (at local slaughter house in Nakhon Pathom Province). The porcine oocytes and follicular fluid of healthy small follicles (1-2 mm), medium follicles (3-6 mm in diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected from the ovary by sterile technique. Then, the oocytes and the follicle cells were separated from the fluid. The oocytes were round shape and surrounded by zona pellucida with numerous layers of cumulus cells. Based on the number of cumulus cell layers surrounding oocytes, the oocytes were classified into 5 types, which were intact-, multi-, partial-cumulus layer oocyte, completely denuded oocyte and degenerative oocyte. The collected oocytes showed high percentages of intact- and multi- cumulus cell layers in the small follicles (53.48%) medium follicles (56.94%) and large follicles (56.52%) which have high potential to develop into mature oocytes in vitro. Proteins from follicular fluid of 3 size follicles were separated by SDS-PAGE and LC/MS/MS. The molecular weight of follicular fluid proteins from the small follicles were 24, 60-65, 79, 110, 140, 160, and > 220 kDa. Meanwhile, the follicular fluid protein from medium and large follicle contained 52, 65, 79, 90, 110, 120, 160, 190 and > 220 kDa. Almost all proteins played important roles in promoting and regulating growth and development of oocytes and ovulation. This finding was an initial tool for in vitro testing and applied biotechnology research. Acknowledgements: The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE, reproductive biology

Procedia PDF Downloads 229

Authors: Taleb Lotfi, Benarbia Maheidine, Allam Hamza, Boutira Fatima, Boukerche Abdelbaki

Abstract:

Introduction and purpose of the study: This is a retrospective single-center study with an analytical aim to determine the prognostic factors for relapse in patients treated with radiotherapy after total laryngectomy with lymph node dissection for laryngeal cancer at the Emir Abdelkader cancer center in Oran (Algeria). Material and methods: During the study period from January 2014 to December 2018, eighty-nine patients (n=89) with squamous cell carcinoma of the larynx were treated with postoperative radiotherapy. Relapse-free survival was studied in the univariate analysis according to pre-treatment criteria using Kaplan-Meier survival curves. We performed a univariate analysis to identify relapse factors. Statistically significant factors have been studied in the multifactorial analysis according to the Cox model. Results and statistical analysis: The average age was 62.7 years (40-86 years). It was a squamous cell carcinoma in all cases. Postoperatively, the tumor was classified as pT3 and pT4 in 93.3% of patients. Histological lymph node involvement was found in 36 cases (40.4%), with capsule rupture in 39% of cases, while the limits of surgical excision were microscopically infiltrated in 11 patients (12.3%). Chemotherapy concomitant with radiotherapy was used in 67.4% of patients. With a median follow-up of 57 months (23 to 104 months), the probabilities of relapse-free survival and five-year overall survival are 71.2% and 72.4%, respectively. The factors correlated with a high risk of relapse were locally advanced tumor stage pT4 (p=0.001), tumor site in case of subglottic extension (p=0.0003), infiltrated surgical limits R1 (p=0.001), l lymph node involvement (p=0.002), particularly in the event of lymph node capsular rupture (p=0.0003) as well as the time between surgery and adjuvant radiotherapy (p=0.001). However, in the subgroup analysis, the major prognostic factors for disease-free survival were subglottic tumor extension (p=0.001) and time from surgery to adjuvant radiotherapy (p=0.005). Conclusion: Combined surgery and postoperative radiation therapy are effective treatment modalities in the management of laryngeal cancer. Close cooperation of the entire cervicofacial oncology team is essential, expressed during a multidisciplinary consultation meeting, with the need to respect the time between surgery and radiotherapy.

Keywords: laryngeal cancer, laryngectomy, postoperative radiotherapy, survival

Procedia PDF Downloads 98

Authors: K. Rawojć, J. Miszczyk, A. Możdżeń, A. Panek, J. Swakoń, M. Rydygier

Abstract:

Due to the mitotic delay, poor mitotic index and disappearance of lymphocytes from peripheral blood circulation, assessing the DNA damage after high dose exposure is less effective. Conventional chromosome aberration analysis or cytokinesis-blocked micronucleus assay do not provide an accurate dose estimation or radiosensitivity prediction in doses higher than 6.0 Gy. For this reason, there is a need to establish reliable methods allowing analysis of biological effects after exposure in high dose range i.e., during particle radiotherapy. Lately, Premature Chromosome Condensation (PCC) has become an important method in high dose biodosimetry and a promising treatment modality to cancer patients. The aim of the study was to evaluate the usefulness of drug-induced PCC scoring procedure in an experimental mode, where 100 G2/M cells were analyzed in different dose ranges. To test the consistency of obtained results, scoring was performed by 3 independent persons in the same mode and following identical scoring criteria. Whole-body exposure was simulated in an in vitro experiment by irradiating whole blood collected from healthy donors with 60 MeV protons and 250 keV X-rays, in the range of 4.0 – 20.0 Gy. Drug-induced PCC assay was performed on human peripheral blood lymphocytes (HPBL) isolated after in vitro exposure. Cells were cultured for 48 hours with PHA. Then to achieve premature condensation, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers. The dose-effect curves were derived by counting the excess chromosome fragments. The results indicated adequate dose estimates for the whole-body exposure scenario in the high dose range for both studied types of radiation. Moreover, compared results revealed no significant differences between scores, which has an important meaning in reducing the analysis time. These investigations were conducted as a part of an extended examination of 60 MeV protons from AIC-144 isochronous cyclotron, at the Institute of Nuclear Physics in Kraków, Poland (IFJ PAN) by cytogenetic and molecular methods and were partially supported by grant DEC-2013/09/D/NZ7/00324 from the National Science Centre, Poland.

Keywords: cell response to radiation exposure, drug induced premature chromosome condensation, premature chromosome condensation procedure, proton therapy

Procedia PDF Downloads 349

Authors: Mohammed A. Abutalib

Abstract:

Papillary thyroid carcinoma (PTC) accounts for the most common type of thyroid cancer, a well-differentiated type. PTC is featured by biologically low-grade and less aggressive tumors with a survival rate of 10 years in most of the diagnosed cases. PTC can be presented with the involvement of cervical lymph nodes in about 50% of the patients, yet the distant spread is very uncommon. Herein, we discussed an early 50-year-old male patient with a history of PTC that presented to the emergency department complaining of shortness of breath and a radiological finding of hydrothorax. Cytologic examination, together with immune-histochemical staining and molecular studies of pleural effusion aspiration, concluded the definitive diagnosis of metastatic papillary thyroid carcinoma in the pleural space. PTC seldom causes metastatic niches in the pleural space, and this is a rare clinical presentation; nevertheless, a differential diagnosis of thyroid metastasis needs to be excluded.

Keywords: thyroid cancer, malignant pleural effusion, cytology aspiration, papillary thyroid carcinoma

Procedia PDF Downloads 101

Authors: Maarja Karbus, Elsbet Lippmaa, Kadri Kööp, Mare Tupits

Abstract:

Aim: The aim is to describe the experiences and needs of mothers of children with cancer in coping with the child's illness. Background: Cancer affects different life areas. Especially if it is a child, in this case the whole family is involved. Loved ones are mentally affected, there are limitations, and life changes need to be made to make the whole treatment regimen and recovery as comfortable as possible. Also, the whole process is expensive and time consuming. The research is part of a larger project that covers the experiences and needs of parents of children with chronic illness and coping strategies related to the child's illness. Design: Qualitative, empirical, descriptive research. Method: Semi-structured interviews were used to collect data and inductive content analysis was used to analyze the data. The interviews were conducted in the autumn of 2020, 5 respondents participated in the research. Results and Conclusions: The research revealed that the mothers' experiences of coping with a child's disease included health-related experiences, material aspects, changes in lifestyle, support systems and contact with professionals. Regarding the organizational and material aspects of life, the subjects presented experiences with economic problems, adaptation of changes in lifestyle, access to information and changes in the treatment process. With regard to health, the respondents identified experiences with the mother's physical and mental health and experiences with the health of an ill child. The experience of different support systems was related to the support of family, friends, acquaintances, various organizations and specialists. Experiences with specialist support included experiences with family relationships and positive and negatiive experiences with staff. The mothers' needs in dealing with the child's disease included the mother's emotional needs, the support of other family members, and the need for various support systems and services. The needs of coping with the child were the need for understanding, support, confidence, the need to be strong and courageous, the need to ignore one's own needs, and the need for personal time and rest. The needs of other family members included the needs of an ill child and the need to pay attention to other children in the family. The needs of different supporters and services were related to different helpers and different services.

Keywords: cancer, mother, coping, child, need, experience, illness

Procedia PDF Downloads 145

Authors: Alexandru Grigorescu, Alexandra Protesanu

Abstract:

Background: Approximately 34-67 percent of cancer patients experience an episode of uncontrolled pain during the course of their disease, depending on the stage. The aim is to provide evidence-based data for pain prevalence, diagnosis and treatment recommendations on an integrative model of medical oncology and palliative care for patients with cancer diagnostic in a day hospital. Patients and method: Consultation registers and electronic records of 166 Patients (Pts) were studied from April 2022 to March 2023. Pts with pain syndrome were selected. The pain was objectified by the visual pain scale. To elucidate the causes of the pain, investigations were carried out: bone scintigraphy, CT scan, and PET-CT. The analgesic treatments were represented by weak and strong morphine, radiotherapy, and bisphosphonates. Result: During the mentioned period, 166 oncological patients (74 women and 92 men) were treated in the oncology day hospitalization service. There were 1,500 consultations, 40 of which were only for pain. The neoplastic locations were: gynecological, malignant melanoma, breast, gastric, bronchopulmonary, colorectal, liver, pancreatic, bladder, and kidney. 70 Pts presented pain syndrome. The causes of the pain were represented by bone metastases, compressive tumors, and post-surgical status. Drug treatment: Tramadol 47 Pts, of which 10 switched to a major opioid (Oxycodonum, Morphine sulfate), 20 Pts were treated with Oxycodonum as the first intention. In 5 patients ry to rotated morphine, 20 Pts received palliative radiotherapy, 10 Pts were treated with bisphosphonates. 2 Pts required neurosurgery consultation for an antalgic intervention. 5 Pts had important adverse reactions to morphine. All patients and their families were advised by a medical oncologist and psychologist for a lifestyle change. Conclusions: The prevalence of pain was similar to that described in the literature. In most cases, the pain could be managed in the day hospital. Weak and strong morphine represented the main pain therapy. Palliative radiotherapy was the second most effective therapy. Treatment with bisphosphonates was useful. Surgical interventions were rarely indicated. Discussions with patients and their families regarding the lifestyle change were important.

Keywords: cancer pain, opioids, medical oncology, palliative care

Procedia PDF Downloads 62

Authors: Salwa Hagag Abdelaziz, Naglaa Fathy Youssef, Nadia Abdellatif Hassan, Rasha Wesam Abdelrahman

Abstract:

Breast cancer is considered as a substantial health concern and practicing mammography screening [MS] is important in minimizing its related morbidity. So it is essential to have a better understanding of breast cancer screening behaviors of women and factors that influence utilization of them. The aim of this study is to identify the factors that are linked to MS behaviors among the Egyptian women. A cross-sectional descriptive design was carried out to provide a snapshot of the factors that are linked to MS behaviors. A convenience sample of 311 women was utilized and all eligible participants admitted to the Women Imaging Unit who are 40 years of age or above, coming for mammography assessment, not pregnant or breast feeding and who accepted to participate in the study were included. A structured questionnaire was developed by the researchers and contains three parts; Socio-demographic data; Motivating factors associated with MS; and association between MS and model of behavior change. The analyzed data indicated that most of the participated women (66.6 %) belonged to the age group of 40-49.A high proportion of participants (58.1%) of group having previous MS influenced by their neighbors to practice MS, whereas 32.7 % in group not having previous MS were influenced by family members which indicated significant differences (P <0.05). Doctors and media are shown to be the least influence of others to practice MS. Women with intention to have a future mammogram had higher OR (1.404) for practicing MS compared with women with no intention. Further studies are needed to examine the relation between Trans-theoretical Model [TTM] and practicing MS.

Keywords: breast cancer, mammography, screening behaviors, morbidity

Procedia PDF Downloads 437

Authors: T. Wresdiyati, S. Sa’diah, A. Winarto

Abstract:

Diabetes mellitus (DM) is a metabolic disease that can be indicated by the high level of blood glucose. The objective of this study was to observe the antidiabetic properties of ethanolic extract of Indonesian Swietenia mahagoni Jacq. seed on the profile of pancreatic superoxide dismutase and β-cells in the alloxan- experimental diabetic rats. The Swietenia mahagoni seed was obtained from Leuwiliang-Bogor, Indonesia. Extraction of Swietenia mahagoni was done by using ethanol with maceration methods. A total of 25 male Sprague dawley rats were divided into five groups; (a) negative control group, (b) positive control group (DM), (c) DM group that was treated with Swietenia mahagoni seed extract, (d) DM group that was treated with acarbose, and (e) non-DM group that was treated with Swietenia mahagoni seed extract. The DM groups were induced by alloxan (110 mg/kgBW). The extract was orally administrated to diabetic rats 500 mg/kg/BW/day for 28 days. The extract showed hypoglycemic effect, increased body weight, increased the content of superoxide dismutase in the pancreatic tissue, and delayed the rate of β-cells damage of experimental diabetic rats. These results suggested that the ethanolic extract of Indonesian Swietenia mahagoni Jacq. seed could be proposed as a potential anti-diabetic agent.

Keywords: beta cells, diabetes, hypoglycemic, rat, Swietenia mahagoni

Procedia PDF Downloads 290

Authors: Yuanyuan Zhang, Yujuan Zheng, Giuseppina Barutello, Sumako Kameishi, Kungchun Chiu, Katharina Hennig, Martial Balland, Federica Cavallo, Lars Holmgren

Abstract:

Angiogenesis describes that new blood vessels migrate from pre-existing ones to form 3D lumenized structure and remodeling. During directional migration toward the gradient of pro-angiogenic factors, the endothelial cells, especially the tip cells need filopodia to sense the environment and exert the pulling force. Of particular interest are the integrin proteins, which play an essential role in focal adhesion in the connection between migrating cells and extracellular matrix (ECM). Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving angiogenesis. We have previously identified Angiomotin (Amot), a member of Amot scaffold protein family, as a promoter for endothelial cell migration in vitro and zebrafish models. Hence, we established inducible endothelial-specific Amot knock-out mice to study normal retinal angiogenesis as well as tumor angiogenesis. We found that the migration ratio of the blood vessel network to the edge was significantly decreased in Amotec- retinas at postnatal day 6 (P6). While almost all the Amot defect tip cells lost migration advantages at P7. In consistence with the dramatic morphology defect of tip cells, there was a non-autonomous defect in astrocytes, as well as the disorganized fibronectin expression pattern correspondingly in migration front. Furthermore, the growth of transplanted LLC tumor was inhibited in Amot knockout mice due to fewer vasculature involved. By using MMTV-PyMT transgenic mouse model, there was a significantly longer period before tumors arised when Amot was specifically knocked out in blood vessels. In vitro evidence showed that Amot binded to beta-actin, Integrin beta 1 (ITGB1), Fibronectin, FAK, Vinculin, major focal adhesion molecules, and ITGB1 and stress fibers were distinctly induced by Amot transfection. Via traction force microscopy, the total energy (force indicater) was found significantly decreased in Amot knockdown cells. Taken together, we propose that Amot is a novel partner of the ITGB1/Fibronectin protein complex at focal adhesion and required for exerting force transition between endothelial cell and extracellular matrix.

Keywords: angiogenesis, angiomotin, endothelial cell migration, focal adhesion, integrin beta 1

Procedia PDF Downloads 230

Authors: Amit Kamra, V. K. Jain, Pragya

Abstract:

Mammography is widely used technique for breast cancer screening. There are various other techniques for breast cancer screening but mammography is the most reliable and effective technique. The images obtained through mammography are of low contrast which causes problem for the radiologists to interpret. Hence, a high quality image is mandatory for the processing of the image for extracting any kind of information from it. Many contrast enhancement algorithms have been developed over the years. In the present work, an efficient morphology based technique is proposed for contrast enhancement of masses in mammographic images. The proposed method is based on Multiscale Morphology and it takes into consideration the scale of the structuring element. The proposed method is compared with other state-of-the-art techniques. The experimental results show that the proposed method is better both qualitatively and quantitatively than the other standard contrast enhancement techniques.

Keywords: enhancement, mammography, multi-scale, mathematical morphology

Procedia PDF Downloads 414

Authors: Abderrahmane Hemmani, Hamid Khachab, Dennai Benmoussa, Hassane Benslimane, Abderrachid Helmaoui

Abstract:

Back surface field GaAs with n -p-p+ structures are found to have better characteristics than the conventional solar cells. A theory, based on the transport of both minority carriers under the charge neutrality condition, has been developed in the present paper which explains behavior of the back surface field solar cells. That is reported with an efficiency of 25,05% (Jsc=33.5mA/cm2, Vco=0.87v and fill factor 86% under AM1.5 global conditions). We present the effect of technological parameters of the p+ layer on the conversion efficiency on the solar cell. Good agreement is achieved between our results and the simulation results given the variation of the equivalent recombination velocity to p+ layer as a function of BSF thickness and BSF doping.

Keywords: back surface field, GaAs, solar cell, technological parameters

Procedia PDF Downloads 428

Authors: Ayyaz Hussain, Tariq Sadad

Abstract:

Breast cancer (BC) is one of the widespread ailments among females globally. The early prognosis of BC can decrease the mortality rate. Exact findings of benign tumors can avoid unnecessary biopsies and further treatments of patients under investigation. However, due to variations in images, it is a tough job to isolate cancerous cases from normal and benign ones. The machine learning technique is widely employed in the classification of BC pattern and prognosis. In this research, a deep convolution neural network (DCNN) called AlexNet architecture is employed to get more discriminative features from breast tissues. To achieve higher accuracy, K-nearest neighbor (KNN) classifiers are employed as a substitute for the softmax layer in deep learning. The proposed model is tested on a widely used breast image database called MIAS dataset for experimental purposes and achieved 99% accuracy.

Keywords: breast cancer, DCNN, KNN, mammography

Procedia PDF Downloads 131

Authors: Tedman Cheuk-Yiu Chau, Xavier Harvey, Hung Nguyen

Abstract:

Defunctioning ileostomies are frequently used as an adjunct to low anterior resection in the surgical treatment of rectal cancer. Despite reducing the rate of clinically relevant anastomotic leak, the burden of defunctioning ileostomy is significant, with up to two-thirds of patients reporting stoma-related morbidity. International data have demonstrated an increased risk of bowel dysfunction and lower quality of life in patients with delayed closure (greater than six months post-surgery). While timely reversal is safe and cost-effective, the time to the reversal in Australian and New Zealand public hospitals is not described in the published literature. Thus, it is important to assess the current timeliness of ileostomy closure in the Australian regional context and examine the reasons for the delay. A retrospective analysis of ileostomy closure in Launceston General Hospital (LGH) patients treated with low/ultra low anterior resection for rectal cancer between 2012 and 2019 was undertaken. 94 cases of rectal adenocarcinoma undergoing ultralow anterior resection were examined over the years between 2012-2019. Amongst these, 21 cases (22.3%) were not reversed due to disease progress, death prior to reversal, or surgical complication. Demographics, disease status, surgical technique, and hospital inpatient events of these cases were examined. An average waiting time of 213.2 days was noted. Reasons for the delay include non-specified/prolonged hospital waiting time (54%), delayed or complicated chemotherapy course (13%), surgical complication (11%), advanced age, and frailty(5%). Complication of a delayed ileostomy reversal includes post-operation ileus and the development of an incisional hernia. We conclude that a delayed reversal of ileostomy can contribute to a higher incidence of stoma-related co-morbidities and contribute to a longer hospital stay and therefore use of public hospital resources.

Keywords: anterior resection, colorectal surgery, ileostomy reversal, rectal cancer

Procedia PDF Downloads 88

Authors: Vijaya Madhuri Devraj, Swarnalatha Guditi, Kiran Kumar Bokara, Gangadhar Taduri

Abstract:

Cell-based strategies may open therapeutic approaches that promote tolerance through manipulation of macrophages to increase long-term transplant survival rates and minimize side effects of the current immune suppressive regimens. The aim of the present study was, therefore, to test and compare the therapeutic potential of MSC and Tregs on macrophage polarization to develop an alternate cell-based treatment option in kidney transplantation. In the current protocol, macrophages from kidney transplant recipients with graft dysfunction were co-cultured with MSCs and Treg cells with and without cell-cell contact on transwell plates, further to quantitatively assess macrophage polarization in response to MSC and Treg treatment over time, M1 and M2 cell surface markers were used. Additionally, multiple soluble analytes were analyzed in cell supernatant by using bead-based immunoassays. Furthermore, to confirm our findings, gene expression analysis was done. MSCs induced the formation of M2 macrophages more than Tregs when macrophages M0 were cultured in transwell without cell contact. From this, we deduced the mechanism that soluble factors present in the MSCs condition media are involved in skewing of macrophages towards type 2 macrophages; similarly, in co-culture with cell-cell contact, MSCs resulted in more M2 type macrophages than Tregs. And an important finding of this study is the combination of both MSC-Treg showed significantly effective and consistent results in both with and without cell contact setups. Hence, it is suggestive to prefer MSCs over Tregs for secretome-based therapy and a combination of both for either therapy for effective transplantation outcomes. Our findings underline a key role of Tregs and MSCs in promoting macrophage polarization towards anti-inflammatory type. The study has great importance in prolongation of allograft and patient survival without any rejection by cell-based therapy, which induce self-tolerance and controlling infection.

Keywords: graft rejection, graft tolerance, macrophage polarization, mesenchymal stem cells, regulatory T cells, transplant immunology

Procedia PDF Downloads 113

Authors: Gizachew Belay Adugna, Yu-Tai Tao

Abstract:

Perovskite solar cells (PSCs) have been gaining impressive progress with excellent power conversion efficiency (PCE) of 25.5% in small-area devices. However, the conventional film coating approach is not applicable to large-area module fabrication. Meniscus-guided coating, including blade coating, slot-die coating, and bar coating, is solution processing and promising for large-area and cost-effective film coating to industrial-scale PSCs. Here, we develop simple and scalable solution shearing (SS) and bar coating (BC) methods to coat all layers on large-area (10x10 cm²) substrate in FTO/c-TiO₂/mp-TiO₂/ CH₃NH₃PbI₃/Spiro-OMeTAD/Ag device structure, except the Ag electrode. All solution-sheared PSC exhibited a champion power conversion efficiency of 15.89% in the conational DMF/DMSO solvent. Whereas a very high PCE of 20.30% compared to the controlled spin-coated device (SC, 17.60%) was achieved from the large area sheared perovskite film in a green ACN/MA solvent. Similarly, a remarkable PCE of 18.50% was achieved for a device fabricated from a large-area perovskite film in a simpler and more compatible Bar-coating system. This strategy demonstrates the huge potential for module fabrication and future PSC commercialization.

Keywords: Perovskite solar cells, larger area film coating, meniscus-guided film coating, solution-shearing, bar-coating, power conversion efficiency

Procedia PDF Downloads 67

Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

Abstract:

The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

Procedia PDF Downloads 139

Authors: Katie Kilgour, Brendan Turner, Carly Catella, Michael Daniele, Stefano Menegatti

Abstract:

In vivo, the extracellular matrix (ECM) provides biochemical and mechanical properties that are instructional to resident cells to form complex tissues with characteristics to develop and support vascular networks. In vitro, the development of vascular networks can be guided by biochemical patterning of substrates via spatial distribution and display of peptides and growth factors to prompt cell adhesion, differentiation, and proliferation. We have developed a technique utilizing peptide ligands that specifically bind vascular endothelial growth factor (VEGF), erythropoietin (EPO), or angiopoietin-1 (ANG1) to spatiotemporally distribute growth factors to cells. This allows for the controlled release of each growth factor, ultimately enhancing the formation of a vascular network. Our engineered tissue constructs (ETCs) are fabricated out of gelatin methacryloyl (GelMA), which is an ideal substrate for tailored stiffness and bio-functionality, and covalently patterned with growth factor specific peptides. These peptides mimic growth factor receptors, facilitating the non-covalent binding of the growth factors to the ETC, allowing for facile uptake by the cells. We have demonstrated in the absence of cells the binding affinity of VEGF, EPO, and ANG1 to their respective peptides and the ability for each to be patterned onto a GelMA substrate. The ability to organize growth factors on an ETC provides different functionality to develop organized vascular networks. Our results demonstrated a method to incorporate biochemical cues into ETCs that enable spatial and temporal control of growth factors. Future efforts will investigate the cellular response by evaluating gene expression, quantifying angiogenic activity, and measuring the speed of growth factor consumption.

Keywords: growth factor, hydrogel, peptide, angiogenesis, vascular, patterning

Procedia PDF Downloads 157

Authors: Walter Vera-Ortega, Idoia Busnadiego, Sam J. Wilson

Abstract:

Introduction: Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the acquired immunodeficiency syndrome (AIDS), a leading cause of death worldwide infecting millions of people each year. Despite intensive research in vaccine development, therapies against HIV-1 infection are not curative, and the huge genetic variability of HIV-1 challenges to drug development. Current animal models for HIV-1 research present important limitations, impairing the progress of in vivo approaches. Macaques require a CD8+ depletion to progress to AIDS, and the maintenance cost is high. Mice are a cheaper alternative but need to be 'humanized,' and breeding is not possible. The development of an HIV-1 clone able to replicate in mice is a challenging proposal. The lack of human co-factors in mice impedes the function of the HIV-1 accessory proteins, Tat and Rev, hampering HIV-1 replication. However, Tat and Rev function can be replaced by constitutive/chimeric promoters, codon-optimized proteins and the constitutive transport element (CTE), generating a novel HIV-1 clone able to replicate in mice without disrupting the amino acid sequence of the virus. By minimally manipulating the genomic 'identity' of the virus, we propose the generation of an HIV-1 clone able to replicate in mice to assist in antiviral drug development. Methods: i) Plasmid construction: The chimeric promoters and CTE copies were cloned by PCR using lentiviral vectors as templates (pCGSW and pSIV-MPCG). Tat mutants were generated from replication competent HIV-1 plasmids (NHG and NL4-3). ii) Infectivity assays: Retroviral vectors were generated by transfection of human 293T cells and murine NIH 3T3 cells. Virus titre was determined by flow cytometry measuring GFP expression. Human B-cells (AA-2) and Hela cells (TZMbl) were used for infectivity assays. iii) Protein analysis: Tat protein expression was determined by TZMbl assay and HIV-1 capsid by western blot. Results: We have determined that NIH 3T3 cells are able to generate HIV-1 particles. However, they are not infectious, and further analysis needs to be performed. Codon-optimized HIV-1 constructs are efficiently made in 293T cells in a Tat and Rev independent manner and capable of packaging a competent genome in trans. CSGW is capable of generating infectious particles in the absence of Tat and Rev in human cells when 4 copies of the CTE are placed preceding the 3’LTR. HIV-1 Tat mutant clones encoding different promoters are functional during the first cycle of replication when Tat is added in trans. Conclusion: Our findings suggest that the development of an HIV-1 Tat-Rev independent clone is challenging but achievable aim. However, further investigations need to be developed prior presenting our HIV-1 clone as a candidate model for research.

Keywords: codon-optimized, constitutive transport element, HIV-1, long terminal repeats, research model

Procedia PDF Downloads 305