Search results for: tumor suppressor gene

Authors: Mukolwe D. Lubembe, Odongo O. David, Githaka Naftali, Kanduma Esther, Marinda Oosthuizen, Kgomotso P. Sibeko

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Theileriosis is a tick-borne disease of cattle caused by an apicomplexan protozoan parasite of the genus Theileria. In eastern and southern Africa, Theileria infections in cattle are caused by the species Theileria parva whose natural reservoir is the African buffalo (Syncerus caffer). Currently, East Coast Fever (ECF) caused by the cattle-derived Theileria parva is still a major problem in eastern Africa and some parts of southern Africa but not in South Africa following its eradication in the 1950s. However, Corridor disease (CD) caused by the buffalo-derived Theileria parva still remains a concern in South Africa. The diversity of Theileria parva in South Africa in comparison to other affected countries is poorly defined yet its known to be the survival strategy of this parasite. We assessed the antigenic diversity of Theileria parva isolates from Buffalo and cattle at the wildlife-livestock interface comparing samples from South Africa, Zimbabwe, Kenya, Tanzania, and Uganda. Antigenic epitopes of eight schizont antigen genes (Tp1, Tp3, Tp4, Tp5, Tp6, Tp7, Tp8 and Tp10) were amplified by PCR from genomic DNA extracted from blood samples collected from cattle and buffalo at the wildlife-livestock interface. Amplicons were purified and then sequenced on NGS platform. Full length open reading frames (ORFs) of two schizont antigen genes (Tp2 and Tp9) and one sporozoite antigen gene, p67 were also amplified from genomic DNA. Amplicons were then purified and cloned for sequencing. Analysis was based on sequence differences in the genes. Preliminary results show an extensively diverse population of Theileria parva circulating in buffalo and cattle populations at the wildlife-livestock interface. Diversity of the antigen genes contributes to the evasion of the immune system of the host by Theileria parva. This possess a concern in that, some of the Theileria parva populations may re-assort and become adapted to cattle to cause a form of theileriosis that is as fatal as ECF in areas where ECF was eradicated or is absent

Keywords: Theileria parva, east coast fever, corridor diseases, antigen genes, diversity

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Authors: Shih-Ping Liu, Cheng-Hsuan Chang, Yu-Chuen Huang, Shih-Yin Chen, Woei-Cherng Shyu

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Embryonic stem cells (ES) and induced pluripotnet stem cells (iPS) are both pluripotent stem cells. For mouse stem cells culture technology, leukemia inhibitory factor (LIF) was used to maintain the pluripotency of stem cells in vitro. However, LIF is an expensive reagent. The goal of this study was to find out a pure compound extracted from Chinese herbal medicine that could maintain stem cells pluripotency to replace LIF and improve the iPS generation efficiency. From 20 candidates traditional Chinese medicine we found that Cordycepsmilitaris triggered the up-regulation of stem cells activating genes (Oct4 and Sox2) expression levels in MEF cells. Cordycepin, a major active component of Cordycepsmilitaris, also could up-regulate Oct4 and Sox2 gene expression. Furthermore, we used ES and iPS cells and treated them with different concentrations of Cordycepin (replaced LIF in the culture medium) to test whether it was useful to maintain the pluripotency. The results showed higher expression levels of several stem cells markers in 10 μM Cordycepin-treated ES and iPS cells compared to controls that did not contain LIF, including alkaline phosphatase, SSEA1, and Nanog. Embryonic body formation and differentiation confirmed that 10 μM Cordycepin-containing medium was capable to maintain stem cells pluripotency after four times passages. For mechanism analysis, microarray analysis indicated extracellular matrix and Jak/Stat signaling pathway as the top two deregulated pathways. In ECM pathway, we determined that the integrin αVβ5 expression levels and phosphorylated Src levels increased after Cordycepin treatment. In addition, the phosphorylated Jak2 and phosphorylated Sat3 protein levels were increased after Cordycepin treatment and suppressed with the Jak2 inhibitor, AG490. The expression of cytokines associated with Jak2/Stat3 signaling pathway were also up-regulated by Q-PCR and ELISA assay. Lastly, we used Oct4-GFP MEF cells to test iPS generation efficiency following Cordycepin treatment. We observed that 10 Μm Cordycepin significantly increased the iPS generation efficiency in day 21. In conclusion, we demonstrated Cordycepin could maintain the pluripotency of stem cells through both of ECM and Jak2/Stat3 signaling pathway and improved iPS generation efficiency.

Keywords: cordycepin, iPS cells, Jak2/Stat3 signaling pathway, molecular biology

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Authors: Feixiong Chen, Naoufel Haddour, Marie Frenea-Robin, Yves MéRieux, Yann Chevolot, Virginie Monnier

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Neonatal thrombopenia occurs when the mother generates antibodies against her baby’s platelet antigens. It is particularly critical for newborns because it can cause coagulation troubles leading to intracranial hemorrhage. In this case, diagnosis must be done quickly to make platelets transfusion immediately after birth. Before transfusion, platelet antigens must be tested carefully to avoid rejection. The majority of thrombopenia (95 %) are caused by antibodies directed against Human Platelet Antigen 1a (HPA-1a) or 5b (HPA-5b). The common method for antigen platelets detection is polymerase chain reaction allowing for identification of gene sequence. However, it is expensive, time-consuming and requires significant blood volume which is not suitable for newborns. We propose to develop a point-of-care device based on double functionalized magnetic colloids with 1) antibodies specific to antigen platelets and 2) highly sensitive electroactive molecules in order to be detected by an electrochemical microsensor. These magnetic colloids will be used first to isolate platelets from other blood components, then to capture specifically platelets bearing HPA-1a and HPA-5b antigens and finally to attract them close to sensor working electrode for improved electrochemical signal. The expected advantages are an assay time lower than 20 min starting from blood volume smaller than 100 µL. Our functionalization procedure based on amine dendrimers and NHS-ester modification of initial carboxyl colloids will be presented. Functionalization efficiency was evaluated by colorimetric titration of surface chemical groups, zeta potential measurements, infrared spectroscopy, fluorescence scanning and cyclic voltammetry. Our results showed that electroactive molecules and antibodies can be immobilized successfully onto magnetic colloids. Application of a magnetic field onto working electrode increased the detected electrochemical signal. Magnetic colloids were able to capture specific purified antigens extracted from platelets.

Keywords: Magnetic Nanoparticles , Electroactive Molecules, Antibody, Platelet

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Authors: Esam Mohammed Al-shaebi

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One of the most crucial approaches for treating human diseases, particularly parasite infections, is nanomedicine. One of the most significant protozoan diseases that impact farm and domestic animals is coccidiosis. While, amprolium is one of the traditional anticoccidial medication, the advent of drug-resistant strains of Eimeria necessitates the development of novel treatments. The goal of the current investigation was to determine whether biosynthesized selenium nanoparticles (Bio-SeNPs) using Azadirachta indica leaves extract might treat mice with Eimeria papillata infection in the jejunal tissue. Five groups of seven mice each were used, as follows: Group 1: Non-infected-non-treated (negative control). Group 2: Non-infected treated group with Bio-SeNPs (0.5 mg/kg of body weight). Groups 3-5 were orally inoculated with 1×103 sporulated oocysts of E. papillata. Group 3: Infected-non-treated (positive control). Group 4: Infected and treated group with Bio-SeNPs (0.5 mg/kg). Group 5: Infected and treated group with the Amprolium. Groups 4 and 5 daily received oral administration (for 5 days) of Bio-SeNPs and anticoccidial medication, respectively, after infection. Bio-SeNPs caused a considerable reduction in oocyst output in mice feces (97.21%). This was also accompanied by a significant reduction in the number of developmental parasitic stages in the jejunal tissues. Glutathione reduced (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were dramatically reduced by the Eimeria parasite, whereas, nitric oxide (NO) and malonaldehyde (MDA) levels were markedly elevated. The amount of goblet cells and MUC2 gene expression were used as apoptotic indicators, and both were considerably downregulated by infection. However, infection markedly increased the expression of inflammatory cytokines (IL-6 and TNF-α) and the apoptotic genes (Caspase-3 and BCL2). Bio-SeNPs were administrated to mice to drastically lower body weight, oxidative stress, and inflammatory and apoptotic indicators in the jejunal tissue. Our research thus showed the involvement of Bio-SeNPs in protecting mice with E. papillata infections against jejunal damage.

Keywords: coccidiosis, nanoparticles, azadirachta indica, oxidative stress

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Authors: Xiaoyuan Chen

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Background: This study aimed to characterize the epidemiological and clinical features of five rare subtypes of hepatocellular carcinoma (HCC) and to create a competing risk nomogram for predicting cancer-specific survival. Methods: This study used the Surveillance, Epidemiology, and End Results database to analyze the clinicopathological data of 50,218 patients with classic HCC and five rare subtypes (ICD-O-3 Histology Code=8170/3-8175/3) between 2004 and 2018. The annual percent change (APC) was calculated using Joinpoint regression, and a nomogram was developed based on multivariable competing risk survival analyses. The prognostic performance of the nomogram was evaluated using the Akaike information criterion, Bayesian information criterion, C-index, calibration curve, and area under the receiver operating characteristic curve. Decision curve analysis was used to assess the clinical value of the models. Results: The incidence of scirrhous carcinoma showed a decreasing trend (APC=-6.8%, P=0.025), while the morbidity of other rare subtypes remained stable from 2004 to 2018. The incidence-based mortality plateau in all subtypes during the period. Clear cell carcinoma was the most common subtype (n=551, 1.1%), followed by fibrolamellar (n=241, 0.5%), scirrhous (n=82, 0.2%), spindle cell (n=61, 0.1%), and pleomorphic (n=17, ~0%) carcinomas. Patients with fibrolamellar carcinoma were younger and more likely to have non-cirrhotic liver and better prognoses. Scirrhous carcinoma shared almost the same macro clinical characteristics and outcomes as classic HCC. Clear cell carcinoma tended to occur in the Asia-Pacific elderly male population, and more than half of them were large HCC (Size>5cm). Sarcomatoid (including spindle cell and pleomorphic) carcinoma was associated with larger tumor size, poorer differentiation, and more dismal prognoses. The pathological subtype, T stage, M stage, surgery, alpha-fetoprotein, and cancer history were identified as independent predictors in patients with rare subtypes. The nomogram showed good calibration, discrimination, and net benefits in clinical practice. Conclusion: The rare subtypes of HCC had distinct clinicopathological features and biological behaviors compared with classic HCC. Our findings could provide a valuable reference for clinicians. The constructed nomogram could accurately predict prognoses, which is beneficial for individualized management.

Keywords: hepatocellular carcinoma, pathological subtype, fibrolamellar carcinoma, scirrhous carcinoma, clear cell carcinoma, spindle cell carcinoma, pleomorphic carcinoma

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Authors: Omar Nouri, Wafa Mnejja, Nejla Fourati, Fatma Dhouib, Wicem Siala, Ilhem Charfeddine, Afef Khanfir, Jamel Daoud

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Background and objective: Induction chemotherapy (IC) followed by concomitant chemo radiotherapy with intensity modulated radiation (IMRT) technique is actually the recommended treatment modality for locally advanced nasopharyngeal carcinomas (NPC). The aim of this study was to evaluate the prognostic factors predicting loco regional relapse with this new treatment protocol. Patients and methods: A retrospective study of 52 patients with NPC treated between June 2016 and July 2019. All patients received IC according to the protocol of the Head and Neck Radiotherapy Oncology Group (Gortec) NPC 2006 (3 TPF courses) followed by concomitant chemo radiotherapy with weekly cisplatin (40 mg / m2). Patients received IMRT with integrated simultaneous boost (SIB) of 33 daily fractions at a dose of 69.96 Gy for high-risk volume, 60 Gy for intermediate risk volume and 54 Gy for low-risk volume. Median age was 49 years (19-69) with a sex ratio of 3.3. Forty five tumors (86.5%) were classified as stages III - IV according to the 2017 UICC TNM classification. Loco regional relapse (LRR) was defined as a local and/or regional progression that occurs at least 6 months after the end of treatment. Survival analysis was performed according to Kaplan-Meier method and Log-rank test was used to compare anatomy clinical and therapeutic factors that may influence loco regional free survival (LRFS). Results: After a median follow up of 42 months, 6 patients (11.5%) experienced LRR. A metastatic relapse was also noted for 3 of these patients (50%). Target volumes coverage was optimal for all patient with LRR. Four relapses (66.6%) were in high-risk target volume and two (33.3%) were borderline. Three years LRFS was 85,9%. Four factors predicted loco regional relapses: histologic type other than undifferentiated (UCNT) (p=0.027), a macroscopic pre chemotherapy tumor volume exceeding 100 cm³ (p=0.005), a reduction in IC doses exceeding 20% (p=0.016) and a total cumulative cisplatin dose less than 380 mg/m² (p=0.0.34). TNM classification and response to IC did not impact loco regional relapses. Conclusion: For nasopharyngeal carcinoma, tumors with initial high volume and/or histologic type other than UCNT, have a higher risk of loco regional relapse. Therefore, they require a more aggressive therapeutic approaches and a suitable monitoring protocol.

Keywords: loco regional relapse, modulation intensity radiotherapy, nasopharyngeal carcinoma, prognostic factors

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Authors: Anyimc, C. J. Aneke, J. O. Orji, O. Nworie, U. C. C. Egbule

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The microbial examination and antimicrobial susceptibility profile of microorganism isolated from the salt mining site in Ebonyi state were evaluated in the present study using a standard microbiological technique. A total of 300 samples were randomly collected in three sample groups (A, B, and C) of 100 each. Isolation, Identification and characterization of organization present on the soil samples were determined by culturing, gram-staining and biochemical technique. The result showed the following organisms were isolated with their frequency as follow: Bacillus species (37.3%) and Staphylococcus species(23.5%) had the highest frequency in the whole Sample group A and B while Klebsiella specie (15.7%), Pseudomonas species(13.7%), and Erwinia species (9.8%) had the least. Rhizopus species (42.0%) and Aspergillus species (26.0%) were the highest fungi isolated, followed by Penicillum species (20.0%) while Mucor species (4.0%), and Fusarium species (8.0%) recorded the least. Sample group C showed high microbial population of all the microbial isolates when compared to sample group A and B. Disc diffusion method was used to determine the susceptibility of isolated bacteria to various antibiotics (oxfloxacin, pefloxacin, ciprorex, augumentin, gentamycin, ciproflox, septrin, ampicillin), while agar well diffusion method was used to determine the susceptibility of isolated fungi to some antifungal drugs (metronidazole, ketoconazole, itraconazole fluconazole). The antibacterial activity of the antibiotics used showed that ciproflux has the best inhibitory effect on all the test bacteria. Ketoconazole showed the highest inhibitory effect on the fungal isolates, followed by itraconazole, while metronidazole and fluconazole showed the least inhibitory effect on the entire test fungal isolates. Hence, the multiple drug resistance of most isolates to appropriate drugs of choice are of great public health concern and cells for periodic monitoring of antibiograms to detect possible changing patterns. Microbes isolated in the salt mining site can also be used as a source of gene(s) that can increase salt tolerance in different crop species through genetic engineering.

Keywords: microorganisms, antibacterial, antifungal, resistance, salt mining site, Ebonyi State

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Karol Rodriguez-Peña, Martha L. Macias-Rubalcava, Leticia Rocha-Zavaleta, Sergio Sanchez

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A bioassay-guided study for anti-cancer compounds from endophytes of the Mexican medicinal plant Amphipteryygium adstringens resulted in the isolation of a streptomycete capable of producing a group of compounds with high cytotoxic activity. Microorganisms from surface sterilized samples of various sections of the plant were isolated and all the actinomycetes found were evaluated for their potential to produce compounds with cytotoxic activity against cancer cell lines MCF7 (breast cancer) and HeLa (cervical cancer) as well as the non-tumoural cell line HaCaT (keratinocyte). The most active microorganism was picked for further evaluation. The identification of the microorganism was carried out by 16S rDNA gene sequencing, finding the closest proximity to Streptomyces scabrisporus, but with the additional characteristic that the strain isolated in this study was capable of producing colorful compounds never described for this species. Crude extracts of dichloromethane and ethyl acetate showed IC50 values of 0.29 and 0.96 μg/mL for MCF7, 0.51 and 1.98 μg/mL for HeLa and 0.96 and 2.7 μg/mL for HaCaT. Scaling the fermentation to 10 L in a bioreactor generated 1 g of total crude extract, which was fractionated by silica gel open column to yield 14 fractions. Nine of the fractions showed cytotoxic activity. Fraction 4 was chosen for subsequent purification because of its high activity against cancerous cell lines, lower activity against keratinocytes. HPLC-UV-MS/ESI was used for the evaluation of this fraction, finding at least 10 different compounds with high values of m/z (≈588). Purification of the compounds was carried out by preparative thin-layer chromatography. The prevalent compound was Steffimycin B, a molecule known for its antibiotic and cytotoxic activities and also for its low solubility in aqueous solutions. Along with steffimycin B, another five compounds belonging to the steffimycin family were isolated and at this moment their structures are being elucidated, some of which display better solubility in water: an attractive property for the pharmaceutical industry. As a conclusion to this study, the isolation of endophytes resulted in the discovery of a strain capable of producing compounds with high cytotoxic activity that need to be studied for their possible utilization.

Keywords: amphipterygium adstringens, cytotoxicity, streptomyces scabrisporus, steffimycin

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

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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

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Authors: Rostyslav Horbay, Nick Korolis, Vahid Anvari, Rostyslav Stoika

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Introduction: Giant cancer cells (GCC) are common in all types of cancer, especially after poor therapy. Some specific features of such cells include ~10-fold enlargement, drug resistance, and the ability to propagate similar daughter cells. We used murine NK/Ly lymphoma, an aggressive and fast growing lymphoma model that has already shown drastic changes in GCC comparing to parental cells (chromatin condensation, nuclear fragmentation, tighter OXPHOS/cellular respiration coupling, multidrug resistance). Materials and methods: In this study, we compared morpho-functional changes of GCC that predominantly show either a cytostatic or a cytotoxic effect after treatment with drugs. We studied the effect of a combined cytostatic/cytotoxic drug treatment to determine the correlation of drug efficiency and GCC formation. Doses of G1/S-specific drug paclitaxel/PTX (G2/M-specific, 50 mg/mouse), vinblastine/VBL (50 mg/mouse), and DNA-targeting agents doxorubicin/DOX (125 ng/mouse) and cisplatin/CP (225 ng/mouse) on C57 black mice. Several tests were chosen to estimate morphological and physiological state (propidium iodide, Rhodamine-123, DAPI, JC-1, Janus Green, Giemsa staining and other), which included cell integrity, nuclear fragmentation and chromatin condensation, mitochondrial activity, and others. A single and double factor ANOVA analysis were performed to determine correlation between the criteria of applied drugs and cytomorphological changes. Results: In all cases of treatment, several morphological changes were observed (intracellular vacuolization, membrane blebbing, and interconnected mitochondrial network). A lower gain in ascites (49.97% comparing to control group) and longest lifespan (22+9 days) after tumor injection was obtained with single VBL and single DOX injections. Such ascites contained the highest number of GCC (83.7%+9.2%), lowest cell count number (72.7+31.0 mln/ml), and a strong correlation coefficient between increased mitochondrial activity and percentage of giant NK/Ly cells. A high number of viable GCC (82.1+9.2%) was observed compared to the parental forms (15.4+11.9%) indicating that GCC are more drug resistant than the parental cells. All this indicates that the giant cell formation and its ability to obtain drug resistance is an expanding field in cancer research.

Keywords: ANOVA, cisplatin, doxorubicin, drug resistance, giant cancer cells, NK/Ly lymphoma, paclitaxel, vinblastine

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Authors: Ali Bahkali, Rayan Yousef Booq, Mohammad Khiyami

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Soil samples were collected from five different regions in the Kingdom of Saudi Arabia. Microbiological methods included dilution methods and pour plates to isolate and purify bacteria soil. The ability of isolates to develop biopolymer was investigated on petri dishes containing elements and substance concentrations stimulating developing biopolymer. Fluorescent stains, Nile red and Nile blue were used to stain the bacterial cells developing biopolymers. In addition, Sudan black was used to detect biopolymers in bacterial cells. The isolates which developed biopolymers were identified based on their gene sequence of 1 6sRNA and their ability to grow and synthesize PHAs on mineral medium supplemented with 1% dates molasses as the only carbon source under nitrogen limitation. During the study 293 bacterial isolates were isolated and detected. Through the initial survey on the petri dishes, 84 isolates showed the ability to develop biopolymers. These bacterial colonies developed a pink color due to accumulation of the biopolymers in the cells. Twenty-three isolates were able to grow on dates molasses, three strains of which showed the ability to accumulate biopolymers. These strains included Bacillus sp., Ralstonia sp. and Microbacterium sp. They were detected by Nile blue A stain with fluorescence microscopy (OLYMPUS IX 51). Among the isolated strains Ralstonia sp. was selected after its ability to grow on molasses dates in the presence of a limited nitrogen source was detected. The optimum conditions for formation of biopolymers by isolated strains were investigated. Conditions studied included, best incubation duration (2 days), temperature (30°C) and pH (7-8). The maximum PHB production was raised by 1% (v1v) when using concentrations of dates molasses 1, 2, 3, 4 and 5% in MSM. The best inoculated with 1% old inoculum (1= OD). The ideal extraction method of PHA and PHB proved to be 0.4% sodium hypochlorite solution, producing a quantity of polymer 98.79% of the cell's dry weight. The maximum PHB production was 1.79 g/L recorded by Ralstonia sp. after 48 h, while it was 1.40 g/L produced by R.eutropha ATCC 17697 after 48 h.

Keywords: bacteria forming polyhydroxyalkanoate, detection, molecular, Saudi Arabia

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Authors: Vanitha Varadharaj, Vijalakshmi Krishnamurthy

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Annona squamosa Linn, commonly known as Sugar apple, belonging to the family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. The alkaloid and flavonoids present in Annona squamosa leaf has proved to have antioxidant activity. The present work has been planned to investigate the effect of ethanolic leaf extract of Annona squamosa leaf on Den Induced wistar albino rats. The study was carried out to analyze the biochemical Parmeters like Total Proteins, Bilirubin, Enzymatic and Non –Enzymatic enzymes, Marker enzymes and Tumor markers in serum and also the histopathological studies in liver is carried out in control and DEN induced rats. Supplementation of ELAS (Ethanolic Leaf Extract Of Annona squamosa) reduced the liver weight and also reduced the tumour incidence. Chemoprevention group showed near normal values of bilirubin when compared with the control rats. Total protein was decreased in the cancer bearing group and on treatment with the extract the levels of protein were restored. Both in pre and post treatment group, the activities of enzymatic antioxidants such as superoxide dismutase, catalase, and Glutathione peroxidase were increased but in pre treated animals it was more effective than post treated animals. The non- enzymatic antioxidants such as vitamin C and vitamin E were brought back to normal level significantly in post and pre treated animals. Activities of marker enzymes such as SGOT, SGPT, ALP, γ GT were significantly elevated in the serum of cancer animals and the values returned to normal after treatment with the extract suggesting the hepato protective effect of the extract. Lipid peroxide was found to be elevated in the cancer induced group. This condition was brought back to the normal in the pre and post treated animals with ELAS. Histological examination also confirmed the anti- carcinogenic potential of ELAS, Cancer induced groups had a triple fold increase in their AFP values when compared to other groups. DEN treatment increased the level of AFP expression while ELAS partially counteracted the effect of it. So the scientific validation obtained from this study may pave way to many budding scientists to find new drugs from Annona squamosa for various ailments.

Keywords: annona squamosa, biochemical parmeters, cancer, leaf extract

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Authors: Nigatu Tadesse, Li Juan, Liuhong Ming

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Gastric cancer (GC) is the fifth most common and fourth deadly cancer in the world with limited treatment options at late advanced stage in which surgical therapy is not recommended with chemotherapy remain as the mainstay of treatment. However, the occurrence of chemoresistance as well as intera-tumoral and inter-tumoral heterogeneity of response to targeted and immunotherapy underlined a clear unmet treatment need in gastroenterology. Several molecular and cellular alterations ascribed for chemo resistance in GC including cancer stem cells (CSC) and tumor microenvironment (TME) remodeling. Cancer cells including CSC bears higher metabolic demand and major changes in TME involves alterations of gut microbiota interacting with nutrients metabolism. Metabolic upregulation in lipids, carbohydrates, amino acids, fatty acids biosynthesis pathways identified as a common hall mark in GC. Metabolic addiction to methionine metabolism occurs in many cancer cells to promote the biosynthesis of S-Adenosylmethionine (SAM), a universal methyl donor molecule for high rate of transmethylation in GC and promote cell proliferation. Targeting methionine metabolism found to promotes chemo-sensitivity with treatment non-heterogeneity. Methionine restriction (MR) promoted the arrest of cell cycle at S/G2 phase and enhanced downregulation of GC cells resistance to apoptosis (including ferroptosis), which suggests the potential of synergy with chemotherapies acting at S-phase of the cell cycle as well as inducing cell apoptosis. Accumulated evidences showed both the biogenesis as well as intracellular metabolism of exogenous methionine could be safe and effective target for therapy either alone or in combination with chemotherapies. This review article provides an over view of the upregulation in methionine biosynthesis pathway and the molecular signaling through the PI3K/Akt/mTOR-c-MYC axis to promote metabolic reprograming through activating the expression of L-type aminoacid-1 (LAT1) transporter and overexpression of Methionine adenosyltransferase 2A(MAT2A) for intercellular metabolic conversion of exogenous methionine to SAM in GC, and the potential of targeting with novel therapeutic agents such as methioninase (METase), Methionine adenosyltransferase 2A (MAT2A), c-MYC, methyl like transferase 16 (METTL16) inhibitors that are currently under clinical trial development stages and future perspectives.

Keywords: gastric cancer, methionine metabolism, pi3k/akt/mtorc1-c-myc axis, gut microbiota, MAT2A, c-MYC, METTL16, methioninase

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: Holm Zerbe, Laura Macias, Hans-Joachim Schuberth, Wolfram Petzl

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Mastitis is among the most important production diseases in cows. It accounts for large parts of antimicrobial drug use in the dairy industry worldwide. Due to the imminent normative to reduce the use of antimicrobial drugs in livestock, new ways for therapy and prophylaxis of mastitis are needed. Recently epigenetic regulation of inflammation by chromatin modifications has increasingly drawn attention. Currently, some epigenetic modifiers have already been approved for the use in humans, however little is known about their actions in the bovine system. The aim of our study was to investigate whether three selected epigenetic modifiers (Vitamin D3, SAHA and S2101) influence the initial immune response towards mastitis pathogens in bovine udder tissue in vitro. Tissue explants of the teat cistern and udder parenchyma were collected from 21 cows and were incubated for 36 hours in the absence and presence of epigenetic modifiers. Additionally, the tissue was stimulated with heat-inactivated particles of Escherichia coli and Staphylococcus aureus, which are regarded as two of the most important mastitis pathogens. After incubation, the explants were tested by RT-qPCR for transcript abundances of immune-related candidate genes. Gene expression was validated in culture supernatants by an AlphaLISA assay. Furthermore, the culture supernatants were analyzed for their chemotactic capacity through a chemotaxis assay. Statistical analysis of data was performed with the program ‘R’ version 3.2.3. Vitamin D3 had no effect on the immune response of udder tissue in vitro after stimulation with mastitis pathogens. The epigenetic modifiers SAHA and S2101 however significantly blocked the pathogen-induced upregulation of CXCL8, TNFα, S100A9 and LAP (P < 0.05). The regulation of IL10 was not affected by treatment with SAHA and S2101. Transcript abundances for CXCL8 were reflected by IL8 contents and chemotactic activity in culture supernatants. In conclusion, these data show the potential of epigenetic modifiers (SAHA and S2101) to block overshooting inflammation in the udder. Thus epigenetic modifiers may serve in future as immune modulators for the treatment and/or prophylaxis of clinical mastitis. (Funded by Deutsche Forschungsgemeinschaft PE 1495/2-1).

Keywords: mastitis, cattle, epigenetics, immunomodulation

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Authors: Hailemeleak Regassa

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Traditional medicines use medicinal plants as a source of ingredients, and many modern medications are indirectly derived from plants. Consumers in affluent nations are growing disenchanted with contemporary healthcare and looking for alternatives. Oxidative stress is the primary cause of multiple diseases, and exogenous antioxidant supplementation or strengthening the body's endogenous antioxidant defenses are potential ways to counteract the negative effects of oxidative damage. Plants can biosynthesize non-enzymatic antioxidants that can reduce ROS-induced oxidative damage. Aging often aids the propagation and development of carcinogenesis, and older animals and older people exhibit increased vulnerability to tumor promoters. Cancer is a major public health issue, with several anti-cancer medications in clinical use. Potential drugs such as flavopiridol, roscovitine, combretastatin A-4, betulinic acid, and silvestrol are in the clinical or preclinical stages of research. Methodology: Microbial Growth media, Dimethyl sulfoxide (DMSO), methanol, ethyl acetate, and n-hexane were obtained from Himedia Labs, Mumbai, India. plant were collected from the Herbal Garden of Shoolini University campus, Solan, India (Latitude - 30.8644° N and longitude - 77.1184° E). The identity was confirmed by Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.), India, and documented in Voucher specimens - UHF- Herbarium no. 13784; vide book no. 3818 Receipt No. 086. The plant materials were washed with tap water, and 0.1% mercury chloride for 2 minutes, rinsed with distilled water, air dried, and kept in a hot air oven at 40ºc on blotting paper until all the water evaporated and became well dried for grinding. After drying, the plant materials were grounded using a mixer grinder into fine powder transferred into airtight containers with proper labeling, and stored at 4ºc for future use (Horablaga et al., 2023). The extraction process was done according to Altemimi et al., 2017. The 5g powder was mixed with 15 ml of the respective solvents (n-hexane, ethyl acetate, and methanol), and kept for 4-5 days on the platform shaker. The solvents used are based on their increasing polarity index. Then the extract was centrifuged at 10,000rpm for 5 minutes and filtered using No.1 Whatman filter paper.

Keywords: cancer, phytomedicine, medicinal plants, oncology

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Authors: Puneet Goyal, Aarti Agarwal

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Background and Objective: Nutritional support is an integral part of palliative care of advanced non-resectable abdominal malignancy patients, though is frequently neglected aspect. Non-Healing high output Entero-cutaneous fistulas sometimes require long term parenteral nutrition, to take care of catabolism and replacement of nutrients. We present a case of inoperable pancreatic malignancy with high output entero-cutaneous fistula, which was provided parenteral nutritional support with the use of Totally Implantable Venous Access Device (TIVAD). Method and Results: 55 year old man diagnosed with carcinoma pancreas had developed high entero-cutaneous fistula. His tumor was found to be inoperable and was on total parenteral nutrition through routine central line. This line was difficult to maintain as he required it for a long term TPN. He was planned to undergo Totally Implantable Venous Access Device (TIVAD) implantation. 8Fr single lumen catheter with Groshong non-return Valve (Bard Access Systems, Inc. USA) was inserted through right internal jugular vein, under fluoroscopic guidance. The catheter was tunneled subcutaneously and brought towards infraclavicular pocket, cut at appropriate length and connected to port and locked. Port was sutured in floor of pocket. Free flow of blood aspirated, flushed with heparinized saline. There was no kink observed in entire length of catheter under fluoroscopy. Skin over infraclavicular pocket was sutured. Long term catheter care and associated risks were explained to patient and relatives. Patient continued to receive total parenteral nutrition as well as other supportive therapy though TIVAD for next 6 weeks, till his demise. Conclusion: TIVADs are standard of care for long term venous access solutions in cancer patients requiring chemotherapy. In this case, we extended its use for providing parenteral nutrition and other supportive therapy. TIVADs can be implanted in advanced cancer patients for providing venous access solution required for various palliative treatments and medications. This will help in improving quality of life and satisfaction amongst terminally ill cancer patients.

Keywords: parenteral nutrition, totally implantable venous access device, long term venous access, interventions in anesthesiology

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Authors: G. Krishnamoorthy, S. Anandhakumar

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The effect of D-Lysine (D-Lys) on collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide(NHS) initiated cross linking using experimental and modelling tools are evaluated. The results of the Coll-D-Lys-EDC/NHS scaffold also indicate an increase in the tensile strength (TS), percentage of elongation (% E), denaturation temperature (Td), and decrease the decomposition rate compared to L-Lys-EDC/NHS. Scanning electron microscopic (SEM) and atomic force microscopic (AFM) analyses revealed a well ordered with properly oriented and well-aligned structure of scaffold. The D-Lys stabilizes the scaffold against degradation by collagenase than L-Lys. The cell assay showed more than 98% fibroblast viability (NIH3T3) and improved cell adhesions, protein adsorption after 72h of culture when compared with native scaffold. Cell attachment after 74h was robust, with cytoskeletal analysis showing that the attached cells were aligned along the fibers assuming a spindle-shape appearance, despite, gene expression analyses revealed no apparent alterations in mRNA levels, although cell proliferation was not adversely affected. D-Lysine (D-Lys) plays a pivotal role in the self-assembly and conformation of collagen fibrils. The D-Lys assisted EDC/NHS initiated cross-linking induces the formation of an carboxamide by the activation of the side chain -COOH group, followed by aminolysis of the O-iso acylurea intermediates by the -NH2 groups are directly joined via an isopeptides bond. This leads to the formation of intra- and inter-helical cross links. Modeling studies indicated that D-Lys bind with collagen-like peptide (CLP) through multiple H-bonding and hydrophobic interactions. Orientational changes in collagenase on CLP-D-Lys are observed which may decrease its accessibility to degradation and stabilize CLP against the action of the former. D-Lys has lowest binding energy and improved fibrillar-assembly and staggered alignment without the undesired structural stiffness and aggregations. The proteolytic machinery is not well equipped to deal with Coll-D-Lys than Coll-L-Lys scaffold. The information derived from the present study could help in designing collagenolytically stable heterochiral collagen based scaffold for biomedical applications.

Keywords: collagen, collagenase, collagen like peptide, D-lysine, heterochiral collagen scaffold

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Authors: Ghada Mahmoud El Kassas, Maged Atta El Wakeel

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Background: Environmental enteric dysfunction (EED) is impending trouble that flared up in the last decades to be pervasive in infants and children. EED is asymptomatic villous atrophy of the small bowel that is prevalent in the developing world and is associated with altered intestinal function and integrity. Evidence has suggested that supplementary zinc might ameliorate this damage by reducing gastrointestinal inflammation and may also benefit cognitive development. Objective: We tested whether zinc supplementation improves intestinal integrity, growth, and cognitive function in stunted children predicted to have EED. Methodology: This case–control prospective interventional study was conducted on 120 Egyptian Stunted children aged 1-10 years who recruited from the Nutrition clinic, the National research center, and 100 age and gender-matched healthy children as controls. At the primary phase of the study, Full history taking, clinical examination, and anthropometric measurements were done. Standard deviation score (SDS) for all measurements were calculated. Serum markers as Zonulin, Endotoxin core antibody (EndoCab), highly sensitive C-reactive protein (hsCRP), alpha1-acid glycoprotein (AGP), Tumor necrosis factor (TNF), and fecal markers such as myeloperoxidase (MPO), neopterin (NEO), and alpha-1-anti-trypsin (AAT) (as predictors of EED) were measured. Cognitive development was assessed (Bayley or Wechsler scores). Oral zinc at a dosage of 20 mg/d was supplemented to all cases and followed up for 6 months, after which the 2ry phase of the study included the previous clinical, laboratory, and cognitive assessment. Results: Serum and fecal inflammatory markers were significantly higher in cases compared to controls. Zonulin (P < 0.01), (EndoCab) (P < 0.001) and (AGP) (P < 0.03) markedly decreased in cases at the end of 2ry phase. Also (MPO), (NEO), and (AAT) showed a significant decline in cases at the end of the study (P < 0.001 for all). A significant increase in mid-upper arm circumference (MUAC) (P < 0.01), weight for age z-score, and skinfold thicknesses (P< 0.05 for both) was detected at end of the study, while height was not significantly affected. Cases also showed significant improvement of cognitive function at phase 2 of the study. Conclusion: Intestinal inflammatory state related to EED showed marked recovery after zinc supplementation. As a result, anthropometric and cognitive parameters showed obvious improvement with zinc supplementation.

Keywords: stunting, cognitive function, environmental enteric dysfunction, zinc

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Authors: Mohammed Abdullah Alshahrani

Abstract:

The research focuses on investigating the use of partial least squares (PLS) methodology for addressing challenges associated with high-dimensional correlated data. Recent technological advancements have led to experiments producing data characterized by a large number of variables compared to observations, with substantial inter-variable correlations. Such data patterns are common in chemometrics, where near-infrared (NIR) spectrometer calibrations record chemical absorbance levels across hundreds of wavelengths, and in genomics, where thousands of genomic regions' copy number alterations (CNA) are recorded from cancer patients. PLS serves as a widely used method for analyzing high-dimensional data, functioning as a regression tool in chemometrics and a classification method in genomics. It handles data complexity by creating latent variables (components) from original variables. However, applying PLS can present challenges. The study investigates key areas to address these challenges, including unifying interpretations across three main PLS algorithms and exploring unusual negative shrinkage factors encountered during model fitting. The research presents an alternative approach to addressing the interpretation challenge of predictor weights associated with PLS. Sparse estimation of predictor weights is employed using a penalty function combining a lasso penalty for sparsity and a Cauchy distribution-based penalty to account for variable dependencies. The results demonstrate sparse and grouped weight estimates, aiding interpretation and prediction tasks in genomic data analysis. High-dimensional data scenarios, where predictors outnumber observations, are common in regression analysis applications. Ordinary least squares regression (OLS), the standard method, performs inadequately with high-dimensional and highly correlated data. Copy number alterations (CNA) in key genes have been linked to disease phenotypes, highlighting the importance of accurate classification of gene expression data in bioinformatics and biology using regularized methods like PLS for regression and classification.

Keywords: partial least square regression, genetics data, negative filter factors, high dimensional data, high correlated data

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Authors: Binaya Tamang, Buddhhi Raj Pokhrel, Narayan Gautam

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Background: There may be a connection between thyroid function and vitamin D status since both bind to similar nuclear hormone receptors and have similar response regions on gene promoters. The purpose of the current study was to investigate the relationship between thyroid hormones and vitamin D levels in females who were attending a tertiary care center in western Nepal and were either hypothyroid or euthyroid. Methods: This hospital-based cross-sectional study was carried out between March 2020 and March 2021 by the Biochemistry department of the Universal College of Medical Sciences (UCMS), Bhairahawa, Province No. 5, Nepal, in cooperation with Internal medicine. Prior to the study, institutional review committee approval (UCMS/IRC/008/20) was acquired from UCMS. Women who visited the Internal Medicine OPD of UCMS and were advised to get a thyroid function test (TFT) were included in the study population. Only those who were willing to participate in the study were enrolled after the goals and advantages of the study had been explained to them. Participants who had recently used vitamin D supplements and medications that affected thyroid hormones were excluded. The participants gave their consent verbally and in writing. After getting the consent, a convenient sample technique was applied. Serum was isolated after drawing 3 ml of blood in a plain vial. Chemiluminescence assay was used to analyze vitamin D and thyroid hormones (MAGLUMI 2000). SPSS version 16.0 for Windows was used to conduct the statistical analysis. Statistical significance was defined as a P-value < 0.05. Results: Majority of the study population (n=214, 71%) had insufficient serum vitamin D levels. Among the thyroid groups, the median Vitamin D levels were significantly lower in hypothyroid (16.88 ng/ml) as compared to the euthyroid groups (25.01 ng/ml) (P<0.001). Similarly, serum Vitamin D levels were considerably lower in the obese population (16.86 ng/ml) as compared to the normal BMI group (24.90 ng/ml) (P<0.001) as well as in the vegetarian (15.43 ng.ml) than mixed diet consumer (24.89 ng/ml) (P<0.01). Even after the adjustment for these variables, the Vitamin D levels were significantly lower in the hypothyroid population than in the euthyroid group (P<0.001). Conclusion: Comparing the hypothyroid population to the euthyroid, the median serum vitamin D levels were considerably lower. We were alarmed to see that the majority of euthyroid participants also had low levels of vitamin D. Therefore if left untreated, low vitamin D levels in hypothyroid patients could worsen their health further.

Keywords: vitamin D, thyroid hormones, euthyroid, hypothyroid, Nepal

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Authors: Md. Fazlul Karim, Ashik Sharfaraz, Aysha Ferdoushi

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Background: Computational medicinal chemistry approaches are used for designing and identifying new drug-like molecules, predicting properties and pharmacological activities, and optimizing lead compounds in drug development. SIRT7, a nicotinamide adenine dinucleotide (NAD+)-dependent deacylase which regulates aging, is an emerging target for cancer therapy with mounting evidence that SIRT7 downregulation plays important roles in reversing cancer phenotypes and suppressing tumor growth. Activation or altered expression of SIRT7 is associated with the progression and invasion of various cancers, including liver, breast, gastric, prostate, and non-small cell lung cancer. Objectives: The goal of this work was to identify potent and selective bioactive candidate inhibitors of SIRT7 by in silico screening of small molecule compounds obtained from Nigella sativa (N. sativa). Methods: SIRT7 structure was retrieved from The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), and its active site was identified using CASTp and metaPocket. Molecular docking simulation was performed with PyRx 0.8 virtual screening software. Drug-likeness properties were tested using SwissADME and pkCSM. In silico toxicity was evaluated by Osiris Property Explorer. Bioactivity was predicted by Molinspiration software. Antitumor activity was screened for Prediction of Activity Spectra for Substances (PASS) using Way2Drug web server. Molecular dynamics (MD) simulation was carried out by Desmond v3.6 package. Results: A total of 159 bioactive compounds from the N. Sativa were screened against the SIRT7 enzyme. Five bioactive compounds: chrysin (CID:5281607), pinocembrin (CID:68071), nigellidine (CID:136828302), nigellicine (CID:11402337), and epicatechin (CID:72276) were identified as potent SIRT7 anti-cancer candidates after docking score evaluation and applying Lipinski's Rule of Five. Finally, MD simulation identified Chrysin as the top SIRT7 anti-cancer candidate molecule. Conclusion: Chrysin, which shows a potential inhibitory effect against SIRT7, can act as a possible anti-cancer drug candidate. This inhibitor warrants further evaluation to check its pharmacokinetics and pharmacodynamics properties both in vitro and in vivo.

Keywords: SIRT7, antitumor, molecular docking, molecular dynamics simulation

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Authors: Inês N. Peça , A. Bicho, Rui Gardner, M. Margarida Cardoso

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Inorganic/organic nanocomplexes offer tremendous scope for future biomedical applications, including imaging, disease diagnosis and drug delivery. The combination of Fe3O4 with biocompatible polymers to produce smart drug delivery systems for use in pharmaceutical formulation present a powerful tool to target anti-cancer drugs to specific tumor sites through the application of an external magnetic field. In the present study, we focused on the evaluation of the effect of the magnetic field application time on the rate of drug release from iron oxide polymeric nanoparticles. Doxorubicin, an anticancer drug, was selected as the model drug loaded into the nanoparticles. Nanoparticles composed of poly(d-lactide-co-glycolide (PLGA), a biocompatible polymer already approved by FDA, containing iron oxide nanoparticles (MNP) for magnetic targeting and doxorubicin (DOX) were synthesized by the o/w solvent extraction/evaporation method and characterized by scanning electron microscopy (SEM), by dynamic light scattering (DLS), by inductively coupled plasma-atomic emission spectrometry and by Fourier transformed infrared spectroscopy. The produced particles yielded smooth surfaces and spherical shapes exhibiting a size between 400 and 600 nm. The effect of the magnetic doxorubicin loaded PLGA nanoparticles produced on cell viability was investigated in mammalian CHO cell cultures. The results showed that unloaded magnetic PLGA nanoparticles were nontoxic while the magnetic particles without polymeric coating show a high level of toxicity. Concerning the therapeutic activity doxorubicin loaded magnetic particles cause a remarkable enhancement of the cell inhibition rates compared to their non-magnetic counterpart. In vitro drug release studies performed under a non-permanent magnetic field show that the application time and the on/off cycle duration have a great influence with respect to the final amount and to the rate of drug release. In order to determine the mechanism of drug release, the data obtained from the release curves were fitted to the semi-empirical equation of the the Korsmeyer-Peppas model that may be used to describe the Fickian and non-Fickian release behaviour. Doxorubicin release mechanism has shown to be governed mainly by Fickian diffusion. The results obtained show that the rate of drug release from the produced magnetic nanoparticles can be modulated through the magnetic field time application.

Keywords: drug delivery, magnetic nanoparticles, PLGA nanoparticles, controlled release rate

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Authors: Mwafaq Ibdah, Mossab, Yahyaa, Dor Rachmany, Yoram Gerchman, Doron Holland, Liora Shaltiel-Harpaz

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Pear Psylla is the most important pest of pear in all pear-growing regions, in Asian, European, and the USA. Pear psylla damages pears in several ways: high-density populations of these insects can cause premature leaf and fruit drop, diminish plant growth, and reduce fruit size. In addition, their honeydew promotes sooty mold on leaves and russeting on fruit. Pear psyllas are also considered vectors of pear pathogens such as Candidatus Phytoplasma pyri causing pear decline that can lead to loss of crop and tree vigor, and sometimes loss of trees. Psylla control is a major obstacle to efficient integrated pest management. Recently we have identified two naturally resistance pear accessions (Py.760-261 and Py.701-202) in the Newe Ya’ar live collection. GC-MS volatile metabolic profiling identified several volatile compounds common in these accessions but lacking, or much less common, in a sensitive accession, the commercial Spadona variety. Among these volatiles were styrene and its derivatives. When the resistant accessions were used as inter-stock, the volatile compounds appear in commercial Spadona scion leaves, and it showed reduced susceptibility to pear psylla. Laboratory experiments and applications of some of these volatile compounds were very effective against psylla eggs, nymphs, and adults. The genes and enzymes involved in the specific reactions that lead to the biosynthesis of styrene in plant are unknown. We have identified a phenolic acid decarboxylase that catalyzes the formation of p-hydroxystyrene, which occurs as a styrene analog in resistant pear genotypes. The His-tagged and affinity chromatography purified E. coli-expressed pear PyPAD1 protein could decarboxylate p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene. In addition, PyPAD1 had the highest activity toward p-coumaric acid. Expression analysis of the PyPAD gene revealed that its expressed as expected, i.e., high when styrene levels and psylla resistance were high.

Keywords: pear Psylla, volatile, GC-MS, resistance

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Authors: Vichayada Laohapiboolkul

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Introduction: As the legal use of cannabis is being widely accepted throughout the world, medical cannabis has been explored in order to become an alternative cure for patients. Tetrahydrocannabinol (THC) and Cannabidiol (CBD) are natural cannabinoids found in the Cannabis plant which is proved to have positive treatment for various diseases and symptoms such as chronic pain, neuropathic pain, spasticity resulting from multiple sclerosis, reduce cancer-associated pain, autism spectrum disorders (ASD), dementia, cannabis and opioid dependence, psychoses/schizophrenia, general social anxiety, posttraumatic stress disorder, anorexia nervosa, attention-deficit hyperactivity disorder, and Tourette's disorder. Regardless of all the medical benefits, THC, if not metabolized, can lead to mild up to severe adverse drug reactions (ADR). The enzyme CYP2C19 was found to be one of the metabolizers of THC. However, the suballele CYP2C19*2 manifests as a poor metabolizer which could lead to higher levels of THC than usual, possibly leading to various ADRs. Objective: The aim of this study was to investigate the distribution of CYP2C19, specifically CYP2C19*2, genes in Thai patients treated with medical cannabis along with adverse drug reactions. Materials and Methods: Clinical data and EDTA whole blood for DNA extraction and genotyping were collected from patients for this study. CYP2C19*2 (681G>A, rs4244285) genotyping was conducted using the Real-time PCR (ABI, Foster City, CA, USA). Results: There were 42 medical cannabis-induced ADRs cases and 18 medical cannabis tolerance controls who were included in this study. A total of 60 patients were observed where 38 (63.3%) patients were female and 22 (36.7%) were male, with a range of age approximately 19 - 87 years. The most apparent ADRs for medical cannabis treatment were dry mouth/dry throat (76.7%), followed by tachycardia (70%), nausea (30%) and a few arrhythmias (10%). In the total of 27 cases, we found a frequency of 18 CYP2C19*1/*1 alleles (normal metabolizers, 66.7%), 8 CYP2C19*1/*2 alleles (intermediate metabolizers, 29.6%) and 1 CYP2C19*2/*2 alleles (poor metabolizers, 3.7%). Meanwhile, 63.6% of CYP2C19*1/*1, 36.3% and 0% of CYP2C19*1/*2 and *2/*2 in the tolerance controls group, respectively. Conclusions: This is the first study to confirm the distribution of CYP2C19*2 allele and the prevalence of poor metabolizer genes in Thai patients who received medical cannabis for treatment. Thus, CYP2C19 allele might serve as a pharmacogenetics marker for screening before initiating treatment.

Keywords: medical cannabis, adverse drug reactions, CYP2C19, tetrahydrocannabinol, poor metabolizer

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Authors: Gulnaz Khan, Meha F. Aftab, Munazza Murtaza, Rizwana S. Waraich

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Advanced glycation end products (AGEs) precursor and its abnormal accumulation cause damage to various tissues and organs. AGEs have pathogenic implication in several diseases including diabetes. Existing AGEs inhibitors are not in clinical use, and there is a need for development of novel inhibitors. The present investigation aimed at identifying the novel AGEs inhibitors and assessing their mechanism of action for treating insulin resistance in mice model of diabetes. Novel derivatives of benzylidene of indan-1,3-dione were synthesized. The compounds were selected to study their action mechanism in improving insulin resistance, in vitro, in human hepatocytes and murine adipocytes and then, in vivo, in mice genetic model of diabetes (db/db). Mice were treated with novel derivatives of benzylidene of indane 1,3-dione. AGEs mediated ROS production was measured by dihydroethidium fluorescence assay. AGEs level in the serum of treated mice was observed by ELISA. Gene expression of receptor for AGEs (RAGE), PPAR-gamma, TNF-alpha and GLUT-4 was evaluated by RT-PCR. Glucose uptake was measured by fluorescent method. Microscopy was used to analyze glycogen synthesis in muscle. Among several derivatives of benzylidene of indan-1,3-dione, IDD-24, demonstrated highest inhibition of AGESs. IDD-24 significantly reduced AGEs formation and expression of receptor for advanced glycation end products (RAGE) in fat, liver of db/db mice. Suppression of AGEs mediated ROS production was also observed in hepatocytes and fat cell, after treatment with IDD-24. Glycogen synthesis was increased in muscle tissue of mice treated with IDD-24. In adipocytes, IDD-24 prevented AGEs induced reduced glucose uptake. Mice treated with IDD-24 exhibited increased glucose tolerance, serum adiponectin levels and decreased insulin resistance. The result of present study suggested that IDD-24 can be a possible treatment target to address glycotoxins induced insulin resistance.

Keywords: advance glycation end product, hyperglycemia, indan-1, 3-dione, insulin resistance

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Authors: Nima Samie, Batoul Sadat Haerian, Sekaran Muniandy, M. S. Kanthimathi

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Colon and breast cancers are categorized as the most prevalent types of cancer worldwide. Recently, the broad clinical application of metal complex compounds has led to the discovery of potential therapeutic drugs. The aim of this study was to evaluate the cytotoxic action of a selected nickel complex compound (NCC) against human colon and breast cancer cells. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, MCF-7 and Hs 190.T cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content , measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results showed that our nickel complex compound displayed a potent suppressive effect on HT-29, WiDr, MCF-7 and Hs 190.T after 24 h of treatment with IC50 value of 2.02±0.54, 2.13±0.65, 3.76±015 and 3.14±0.45 µM respectively. This cytotoxic effect on normal cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the nickel complex compound. Activation of this pathway was further evidenced by significant activation of caspase 9 and 3/7.The nickel complex compound (NCC) was also shown activate the extrinsic pathways of apoptosis by activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. The results of this study suggest that the nickel complex compound is a potent anti-cancer agent inducing both intrinsic and extrinsic pathways as well as cell cycle arrest in colon and breast cancer cells.

Keywords: nickel complex, apoptosis, cytoskeletal rearrangement, colon cancer, breast cancer

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae causes a wide range of infections, including urinary tract infections, sepsis, bacteremia, pneumonia, and liver abscesses. The emergence of multi-drug resistance in this bacterium led to a major setback for clinical management. WHO also endorsed a need for finding alternative therapy to antibiotics for the treatment of these infections. Development of vaccines and passive antibody therapy has been proven as a potent alternative to antibiotics in the case of MDR, XDR, and PDR Klebsiella infections. Siderophore receptors have been demonstrated to be overexpressed for the internalization of iron siderophore complexes during infections in most Gram-negative bacteria. For the present study, immune response to siderophore receptors to establish this protein as a potential immunogen for the development of therapeutic leads was explored. Clinical strains of Klebsiella pneumoniae were grown in iron-deficient conditions, and the iron-regulated outer membrane proteins were extracted and characterized through mass spectrometry for specific identification. The gene for identified protein was cloned in pET- 28a vector and expressed in E. coli. The native protein and the recombinant protein were isolated and purified and used as antigens for the generation of immune response in BALB/c mice. The native protein of Klebsiella pneumoniae grown in iron-deficient conditions was identified as FepA (Ferrienterobactin receptor) and other siderophore receptors. This 80 kDa protein generated an immune response in BALB/c mice. The antiserum from mice after subsequent booster doses was collected and showed binding with FepA protein in western blot and phagocytic uptake of the K. pneumoniae in the presence antiserum from immunized mice also observed from the animal studies after bacterial challenge post immunisation in mice have shown bacterial clearance. The antiserum from mice showed binding and clearance of the Klebsiella pneumoniae bacteria in vitro and in vivo. These antigens used for generating an active immune response in mice can further be used for therapeutic monoclonal antibody development against Klebsiella pneumoniae infections.

Keywords: antiserum, FepA, Klebsiella pneumoniae, multi drug resistance, siderophore receptor

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