Search results for: in vitro metabolites

Authors: Bahaa Eldin A. Fouda, Khaled N. Yossef, Mohamed Elhosseny, Ahmed Lotfy, Mohamed Salama, Mohamed Sobh

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Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on the brain during the early childhood period. As human brains are vulnerable during this period, various chemicals would have their maximum effects on brains during early childhood. Some toxicants have been confirmed to induce developmental toxic effects on CNS e.g. lead, however; most of the agents cannot be identified with certainty due the defective nature of predictive toxicology models used. A novel alternative method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system. This in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects. In the present study, we verified the possible DNT of epoxomicin which is a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. The cortices were aseptically dissected out from the brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was, then, transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neuropsheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0, 4, 5, 11, and 14 days, sphere size was determined by software analyses. The diameter of each neurosphere was measured and exported to excel file further to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Epoxomicin was found to affect proliferation and viability of neuropsheres, these effects were positively correlated to doses and progress of time. This study confirms the DNT effects of epoxomicin on 3D neurospheres model. The effects on proliferation suggest possible gross morphologic changes while the decrease in viability propose possible focal lesion on exposure to epoxomicin during early childhood.

Keywords: neural progentor cells, epoxomicin, neurosphere, medical and health sciences

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Authors: Raghuram Kandimalla, Sanjeeb Kalita, Bhaswati Choudhury, Jibon Kotoky

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The quest for developing an ideal suture material prompted our interest to develop a novel suture with advantageous characteristics to market available ones. We developed novel suture biomaterial from muga silk (Antheraea assama) and ramie (Boehmeria nivea) plant fiber. Field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) and thermo gravimetric analysis (TGA) results revealed the physicochemical properties of the fibers which supports the suitability of fibers for suture fabrication. Tensile properties of the prepared sutures were comparable with market available sutures and it found to be biocompatible towards human erythrocytes and nontoxic to mammalian cells. The prepared sutures completely healed the superficial deep wound incisions within seven days in adult male wister rats leaving no rash and scar. Histopathology studies supports the wound healing ability of sutures, as rapid synthesis of collagen, connective tissue and other skin adnexal structures were observed within seven days of surgery. Further muga suture surface modified by exposing the suture to oxygen plasma which resulted in formation of nanotopography on suture surface. Broad spectrum antibiotic amoxicillin was functionalized on the suture surface to prepare an advanced antimicrobial muga suture. Surface hydrophilicity induced by oxygen plasma results in an increase in drug-impregnation efficiency of modified muga suture by 16.7%. In vitro drug release profiles showed continuous and prolonged release of amoxicillin from suture up to 336 hours. The advanced muga suture proves to be effective against growth inhibition of Staphylococcus aureus and Escherichia coli, whereas normal muga suture offers no antibacterial activity against both types of bacteria. In vivo histopathology studies and colony-forming unit count data revealed accelerated wound healing activity of advanced suture over normal one through rapid synthesis and proliferation of collagen, hair follicle and connective tissues.

Keywords: sutures, biomaterials, silk, Ramie

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Authors: Recep Kesli, Huseyin Bilgin, Yasar Unlu, Gokhan Gungor

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Aim: The aim of this study, was to investigate the presence of Helicobacter pylori (H. pylori) infection by culture, histology, and RUT (Rapid Urease Test) in gastric antrum biopsy samples taken from patients presented with dyspeptic complaints and to determine resistance rates of amoxicillin, clarithromycin, levofloxacin and metronidazole against the H. pylori strains by E-test. Material and Methods: A total of 278 patients who admitted to Konya Education and Research Hospital Department of Gastroenterology with dyspeptic complaints, between January 2011-July 2013, were included in the study. Microbiological and histopathological examinations of biopsy specimens taken from antrum and corpus regions were performed. The presence of H. pylori in biopsy samples was investigated by culture (Portagerm pylori-PORT PYL, Pylori agar-PYL, GENbox microaer, bioMerieux, France), histology (Giemsa, Hematoxylin and Eosin staining), and RUT(CLOtest, Cimberly-Clark, USA). Antimicrobial resistance of isolates against amoxicillin, clarithromycin, levofloxacin, and metronidazole was determined by E-test method (bioMerieux, France). As a gold standard in the diagnosis of H. pylori; it was accepted that the culture method alone was positive or both histology and RUT were positive together. Sensitivity and specificity for histology and RUT were calculated by taking the culture as a gold standard. Sensitivity and specificity for culture were also calculated by taking the co-positivity of both histology and RUT as a gold standard. Results: H. pylori was detected in 140 of 278 of patients with culture and 174 of 278 of patients with histology in the study. H. pylori positivity was also found in 191 patients with RUT. According to the gold standard criteria, a false negative result was found in 39 cases by culture method, 17 cases by histology, and 8 cases by RUT. Sensitivity and specificity of the culture, histology, and RUT methods of the patients were 76.5 % and 88.3 %, 87.8 % and 63 %, 94.2 % and 57.2 %, respectively. Antibiotic resistance was investigated by E-test in 140 H. pylori strains isolated from culture. The resistance rates of H. pylori strains to the amoxicillin, clarithromycin, levofloxacin, and metronidazole was detected as 9 (6.4 %), 22 (15.7 %), 17 (12.1 %), 57 (40.7 %), respectively. Conclusion: In our study, RUT was found to be the most sensitive, culture was the most specific test between culture, histology, and RUT methods. Although we detected the specificity of the culture method as high, its sensitivity was found to be quite low compared to other methods. The low sensitivity of H. pylori culture may be caused by the factors affect the chances of direct isolation such as spoild bacterium, difficult-to-breed microorganism, clinical sample retrieval, and transport conditions.

Keywords: antimicrobial resistance, culture, histology, H. pylori, RUT

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Authors: Frans J. Smit, Wisdom A. Munzeiwa, Hermanus C. M. Vosloo, Lyn-Marie Birkholtz, Richard K. Haynes

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Malaria and antimicrobial infections remain significant global health concerns, necessitating the continuous search for novel therapeutic approaches. This abstract presents an overview of the potential use of triazenes as effective agents against malaria and various antimicrobial pathogens. Triazenes are a class of compounds characterized by a linear arrangement of three nitrogen atoms, rendering them structurally distinct from their cyclic counterparts. This study investigates the efficacy of triazenes against malaria and explores their antimicrobial activity. Preliminary results revealed significant antimalarial activity of the triazenes, as evidenced by in vitro screening against P. falciparum, the causative agent of malaria. Furthermore, the compounds exhibited broad-spectrum antimicrobial activity, indicating their potential as effective antimicrobial agents. These compounds have shown inhibitory effects on various essential enzymes and processes involved in parasite survival, replication, and transmission. The mechanism of action of triazenes against malaria involves interactions with critical molecular targets, such as enzymes involved in the parasite's metabolic pathways and proteins responsible for host cell invasion. The antimicrobial activity of the triazenes against bacteria and fungi was investigated through disc diffusion screening. The antimicrobial efficacy of triazenes has been observed against both Gram-positive and Gram-negative bacteria, as well as multidrug-resistant strains, making them potential candidates for combating drug-resistant infections. Furthermore, triazenes possess favourable physicochemical properties, such as good stability, solubility, and low toxicity, which are essential for drug development. The structural versatility of triazenes allows for the modification of their chemical composition to enhance their potency, selectivity, and pharmacokinetic properties. These modifications can be tailored to target specific pathogens, increasing the potential for personalized treatment strategies. In conclusion, this study highlights the potential of triazenes as promising candidates for the development of novel antimalarial and antimicrobial therapeutics. Further investigations are necessary to determine the structure-activity relationships and optimize the pharmacological properties of these compounds. The results warrant additional research, including MIC studies, to further explore the antimicrobial activity of the triazenes. Ultimately, these findings contribute to the development of more effective strategies for combating malaria and microbial infections.

Keywords: malaria, anti-microbials, triazene, resistance

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Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Nadine khadraoui, Rym Essid, Olfa Tabbene, Imen Chniba, Safa Boujemaa, Selim Jallouli, Nadia Fares, Behija Mlik, Boutheina Ben Abdelmoumen Mardassi

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Background and aim: Mycoplasma hominis is an opportunistic pathogen that can cause various gynecological infections such cervicitis, infertility, and, less frequently, extra-genital infections. Previous studies on the antimicrobial susceptibility of Mycoplasma hominis Tunisian strains have highlighted a significant resistance, even multi-resistance, to the most used antibiotic in the therapy of consequential infections. To address this concern, the present study aimed for the alternative of phytotherapy. Peganum harmala seed extract was tested as an antibacterial agent against multidrug-resistant M.hominis clinical strains. Material and Methods: Peganum harmala plant was collected from Ain Sebaa, Tabarka, North West region of Tunisia in April 2018, air-dried, grounded and extracted by different solvents.The crude methanolic extract was further partitioned with n-HEX, DCM, EtOAC and n-BuOl. Antibacterial activity was evaluated against M. hominis ATCC 23114 and 20 M. hominis clinical strains.The antimycoplasmal activity was tested by the microdilution method, and MIC values were determined. Phytochemical analysis and hemolytic activity on human erythrocytes were also performed. The active fraction was then subjected to purification, and the chemical identification of the active compound was investigated. Results: Among the tested fractions, the n-BuOH extract was the most active fraction since it exhibited an inhibitory effect against M. hominis ATCC 23114 and 80% of the tested clinical strains with MIC between 125 and 1000 µg/ml. The phytochemical analysis of the n-BuOH revealed its metabolic abundance in polyphenols, flavonoids and condensed tannin with levels of 257.37 mg AGE/g, 172.27 mg EC/g and 58.27 mg EC/g, respectively. In addition, P. harmala n-BuOH extract exhibited potent bactericidal activity against all M. hominis isolates with CMB values ranging between 125 and 4000 µg/ml. Further, the active fraction exhibited weak cytotoxicity effect at active concentrations when tested on human erythrocytes. The active compound was identified by gas chromatography–mass spectrometry as an indole alkaloid harmaline. Conclusion: In summary, Peganum harmala extract demonstrated an interesting anti-mycoplasmal activity against M. hominis Tunisian strains. Therefore, it could be considered as a potential candidate for the treatment of consequential infections. However, further studies are necessary to evaluate its mechanism of action in mycoplasmas.

Keywords: mycoplasma hominis, peganum harmala, antibioresistance, phytotherapy, phytochemical analysis

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Authors: Priya Sepaha

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Commercial Surrogacy refers to a contract in which a woman carries a pregnancy for intended parents. There are two types of surrogacy; first, Traditional Surrogacy, in which, sperm of the donor or father is artificially inseminated in the women and carries the fetus till birth. Second, Gestational Surrogacy, in which the egg and sperm of the intended parent are collected for artificial fertilization through In Vitro Fertilization (IVF) technique and after the embryo formation, it is transferred into the womb of a surrogate mother with the help of Assisted Reproductive Technique. Surrogacy has become so widespread in India that it has now been nicknamed the "rent-a-womb" capital of the world due to relatively low cost and lack of stringent regulatory legalisation. The legal aspects surrounding surrogacy are complex, diverse and mostly unsettled. Although this appears to be beneficial for the parties concerned, there are certain sensitive issues which need to be addressed to ensure ample protection to all stakeholders. Commercial surrogacy is an emerging business and a new means of human trafficking particularly in India. Poor and illiterate women are often lured in such deals by their spouse or broker for earning easy money. Traffickers also use force, fraud, or coercion at times to intimidate the probable surrogate mothers. A major chunk of money received from covert surrogacy agreement is taken away by the brokers. The Law Commission of India has specifically reviewed the issue as India is emerging as a major global surrogacy destination. The Supreme Court of India held in the Manji's case in 2008, that commercial surrogacy can be permitted with certain restrictions but had directed the Legislature to pass an appropriate Law for governing Surrogacy in India. The draft Assisted Reproductive Technique (ART) Bill, 2010 is still pending for approval. At present, the Surrogacy Contract between the parties and the ART Clinics Guidelines are perhaps the only guiding force. The Immoral Trafficking Prevention Act (ITPA), 1956 and Sections 366(A) and 372 of the Indian Penal Code, 1860 are perhaps the only existing laws, which deal with human trafficking. Yet, none of these provisions specifically deal with the serious issue of trafficking for the purpose of Commercial Surrogacy. India remains one of the few countries that still allow commercial surrogacy. International Surrogacy involves bilateral issues, where the laws of both the nations have to be at par in order to ensure that the concerns and interests of parties involved get amicably resolved. There is urgent need to pass a comprehensive law by incorporating the latest developments in this field in order to make it ethical on the one hand and to curb disguised human trafficking on the other.

Keywords: business, human trafficking, legal, surrogacy

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Authors: Ziani Borhane Eddine Cherif, Hazzi Mohamed, Mouhouche Fazia

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The vegetal extracts of aromatic and medicinal plants start to have much of interest like potential sources of natural bioactive molecules. Many features are conferred by the nature of the chemical function of their major constituents (phenol, alcohol, aldehyde, cetone). This biopotential lets us to focalize on the study of three main biological activities, the antioxidant, antibiotic and insecticidal activities of six Algerian aromatic plants in the aim of making in evidence by the chromatographic analysis (CPG and CG/SM) the phytochemical compounds implicating in this effects. The contents of Oxygenated monoterpenes represented the most prominent group of constituents in the majority of plants. However, the α-Terpineol (28,3%), Carvacrol (47,3%), pulégone (39,5%), Chrysanthenone (27,4%), Thymol 23,9%, γ-Terpinene 23,9% and 2-Undecanone(94%) were the main components. The antioxyding activity of the Essential oils and no-volatils extracts was evaluated in vitro using four tests: inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical-scavenging activity (ABTS•+), the thiobarbituric acid reactive substances (TBARS) assays and the reducing power. The measures of the IC50 of these natural compounds revealed potent activity (between 254.64-462.76mg.l-1), almost similar to that of BHT, BHA, Tocopherol and Ascorbic acid (126,4-369,1 mg.l-1) and so far than the Trolox one (IC50= 2,82mg.l-1). Furthermore, three ethanol extracts were found to be remarkably effective toward DPPH and ABTS inhibition, compared to chemical antioxidant BHA and BHT (IC = 9.8±0.1 and 28±0.7 mg.l-1, respectively); for reducing power test it has also exhibited high activity. The study on the insecticidal activity effect by contact, inhalation, fecundity and fertility of Callosobruchus maculatus and Tribolium confusum showed a strong potential biocide reaching 95-100% mortality only after 24 hours. The antibiotic activity of our essential oils were evaluated by a qualitative study (aromatogramme) and quantitative (MIC, MBC and CML) on four bacteria (Gram+ and Gram-) and one strain of pathogenic yeast, the results of these tests showed very interesting action than that induced by the same reference antibiotics (Gentamycin, and Nystatin Ceftatidine) such that the inhibition diameters and MIC values for tested microorganisms were in the range of 23–58 mm and 0.015–0.25%(v/v) respectively.

Keywords: aromatic plants, essential oils, no-volatils extracts, bioactive molecules, antioxidant activity, insecticidal activity, antibiotic activity

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Authors: A. M. Tarola, R. Preti, A. M. Girelli, P. Campana

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Safeguarding, studying and enhancing biodiversity play an important and indispensable role in re-launching agriculture. The ancient local varieties are therefore a precious resource for genetic and health improvement. In order to protect biodiversity through the recovery and valorization of autochthonous varieties, in this study we analyzed 12 samples of four ancient apple cultivars representative of Friuli Venezia Giulia, selected by local farmers who work on a project for the recovery of ancient apple cultivars. The aim of this study is to evaluate the polyphenolic profile and the antioxidant capacity that characterize the organoleptic and functional qualities of this fruit species, besides having beneficial properties for health. In particular, for each variety, the following compounds were analyzed, both in the skins and in the pulp: gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, coumaric acid, ferulic acid, rutin, phlorizin, phloretin and quercetin to highlight any differences in the edible parts of the apple. The analysis of individual phenolic compounds was performed by High Performance Liquid Chromatography (HPLC) coupled with a diode array UV detector (DAD), the antioxidant capacity was estimated using an in vitro essay based on a Free Radical Scavenging Method and the total phenolic compounds was determined using the Folin-Ciocalteau method. From the results, it is evident that the catechins are the most present polyphenols, reaching a value of 140-200 μg/g in the pulp and of 400-500 μg/g in the skin, with the prevalence of epicatechin. Catechins and phlorizin, a dihydrohalcone typical of apples, are always contained in larger quantities in the peel. Total phenolic compounds content was positively correlated with antioxidant activity in apple pulp (r² = 0,850) and peel (r² = 0,820). Comparing the results, differences between the varieties analyzed and between the edible parts (pulp and peel) of the apple were highlighted. In particular, apple peel is richer in polyphenolic compounds than pulp and flavonols are exclusively present in the peel. In conclusion, polyphenols, being antioxidant substances, have confirmed the benefits of fruit in the diet, especially as a prevention and treatment for degenerative diseases. They demonstrated to be also a good marker for the characterization of different apple cultivars. The importance of protecting biodiversity in agriculture was also highlighted through the exploitation of native products and ancient varieties of apples now forgotten.

Keywords: apple, biodiversity, polyphenols, antioxidant activity, HPLC-DAD, characterization

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Authors: Kersch-Counet C., Fransen K. H. S., Broyd M., Nyakayiru J. D. O. A., Schoemaker M. H., Mallee L. F., Bovee-Oudenhoven I. M. J.

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Background: Around one third of people, including athletes, suffer from feelings of gut discomfort. Fermentation of dairy is a process that has been associated with products that can improve gut health. However, insight in (potential) health benefits of most fermented foods is limited to chemical analyses and in-vitro models. Objective: The aim of this open-label, single-arm explorative trial was to investigate in a real life setting the effect of consumption of a fermented whey product for 3 weeks on self-perceived physical and mental wellbeing and digestive issues in 150 US recreational athletes (20-50 years of age) with self-reported gut complaints at enrolment. Methods: Participants living at the West-Coast of the US received for 3 weeks a daily powder of 15 g of BiotisTM Fermentis to be mixed in water using a supplied shaker. Weekly questionnaires were conducted by MMR research to study the effect on physical/mental health issues and self-perceived gut complaints. Non-parametric tests (e.g., Friedman test) were used to assess statistical differences over time while the Kruskal-Wallis and Wilcoxon signed-rank tests were used for sub-groups analysis. Results: Bloating, stress and anxiety were the top 3 issues of the US recreational athletes. Satisfaction of physical wellbeing increased significantly throughout the 3-weeks of fermented whey product consumption (p<0.0005). Combined digestive issues decreased significantly after 2- and 3-weeks of product consumption, with bloating showing a significant reduction (p<0.05). There was a trend that self-reported stress levels reduced after 3 weeks and participants said to significantly feel more active, energetic, and vital (p<0.05). Subgroup analysis showed that gender and habitual protein supplement consumption were associated with specific health issues and modulated the response to the fermented dairy product. Conclusion: Daily consumption of the fermented BiotisTM Fermentis product is associated with a reduction in self-perceived gastrointestinal symptoms and improved overall wellbeing and mood state in US recreational athletes. This large nutrition and health consumer study brings valuable insights in self-reported gut complaints of recreational athletes in the US and their response to a fermented dairy product. A controlled clinical trial in a targeted population is recommended to scientifically substantiate the product effect as observed in this explorative study.

Keywords: real-life study, digestive health, fermented whey, sports

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Authors: Z. Terzopoulou, I. Koliakou, D. Bikiaris

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Tissue engineering offers a new approach to regenerate diseased or damaged tissues such as bone. Great effort is devoted to eliminating the need of removing non-degradable implants at the end of their life span, with biodegradable polymers playing a major part. Poly(ε-caprolactone) (PCL) is one of the best candidates for this purpose due to its high permeability, good biodegradability and exceptional biocompatibility, which has stimulated extensive research into its potential application in the biomedical fields. However, PCL degrades much slower than other known biodegradable polymers and has a total degradation of 2-4 years depending on the initial molecular weight of the device. This is due to its relatively hydrophobic character and high crystallinity. Consequently, much attention has been given to the tunable degradation of PCL to meet the diverse requirements of biomedicine. Poly(ε-caprolactone) (PCL) is a biodegradable polyester that lacks bioactivity, so when used in bone tissue engineering, new bone tissue cannot bond tightly on the polymeric surface. Therefore, it is important to incorporate reinforcing fillers into PCL matrix in order to result in a promising combination of bioactivity, biodegradability, and strength. Natural clay halloysite nanotubes (HNTs) were incorporated into PCL polymeric matrix, via in situ ring-opening polymerization of caprolactone, in concentrations 0.5, 1 and 2.5 wt%. Both unmodified and modified with aminopropyltrimethoxysilane (APTES) HNTs were used in this study. The effect of nanofiller concentration and functionalization with end-amino groups on the physicochemical properties of the prepared nanocomposites was studied. Mechanical properties were found enhanced after the incorporation of nanofillers, while the modification increased further the values of tensile and impact strength. Thermal stability of PCL was not affected by the presence of nanofillers, while the crystallization rate that was studied by Differential Scanning Calorimetry (DSC) and Polarized Light Optical Microscopy (POM) increased. All materials were subjected to enzymatic hydrolysis in phosphate buffer in the presence of lipases. Due to the hydrophilic nature of HNTs, the biodegradation rate of nanocomposites was higher compared to neat PCL. In order to confirm the effect of hydrophilicity, contact angle measurements were also performed. In vitro biomineralization test confirmed that all samples were bioactive as mineral deposits were detected by X-ray diffractometry after incubation in SBF. All scaffolds were tested in relevant cell culture using osteoblast-like cells (MG-63) to demonstrate their biocompatibility

Keywords: biomaterials, nanocomposites, scaffolds, tissue engineering

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Authors: Paresh Patel, Priya Patel, Vaidehi Sorathiya, Navin Sheth

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The aim of present work was to fabricate Salmeterol Xinafoate (SX) metered dose inhaler (MDI) for asthma and to evaluate the SX loaded solid lipid nanoparticles (SLNs) for pulmonary delivery. Solid lipid nanoparticles can be used to deliver particles to the lungs via MDI. A modified solvent emulsification diffusion technique was used to prepare Salmeterol Xinafoate loaded solid lipid nanoparticles by using compritol 888 ATO as lipid, tween 80 as surfactant, D-mannitol as cryoprotecting agent and L-leucine was used to improve aerosolization behaviour. Box-Behnken design was applied with 17 runs. 3-D surface response plots and contour plots were drawn and optimized formulation was selected based on minimum particle size and maximum % EE. % yield, in vitro diffusion study, scanning electron microscopy, X-ray diffraction, DSC, FTIR also characterized. Particle size, zeta potential analyzed by Zetatrac particle size analyzer and aerodynamic properties was carried out by cascade impactor. Pre convulsion time was examined for control group, treatment group and compare with marketed group. MDI was evaluated for leakage test, flammability test, spray test and content per puff. By experimental design, particle size and % EE found to be in range between 119-337 nm and 62.04-76.77% by solvent emulsification diffusion technique. Morphologically, particles have spherical shape and uniform distribution. DSC & FTIR study showed that no interaction between drug and excipients. Zeta potential shows good stability of SLNs. % respirable fraction found to be 52.78% indicating reach to the deep part of lung such as alveoli. Animal study showed that fabricated MDI protect the lungs against histamine induced bronchospasm in guinea pigs. MDI showed sphericity of particle in spray pattern, 96.34% content per puff and non-flammable. SLNs prepared by Solvent emulsification diffusion technique provide desirable size for deposition into the alveoli. This delivery platform opens up a wide range of treatment application of pulmonary disease like asthma via solid lipid nanoparticles.

Keywords: salmeterol xinafoate, solid lipid nanoparticles, box-behnken design, solvent emulsification diffusion technique, pulmonary delivery

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Authors: Luisa Barreira, Viana Castaneda, Maria J. Rodrigues, Florinda Gama, Tamara Santos, Marta Oliveira, Catarina Pereira, Maribela Pestana, Pedro Correia, Miguel Salazar, Carla Nunes, Luisa Custodio, Joao Varela

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In the last century, the world witnessed an exponential demographic increase that has put an enormous pressure on agriculture and food production. Associated also with climate changes, there has been a decrease in the amount of available freshwater and an increased salinization of soils which can affect the production of most food crops. Halophytes, however, are plants able to withstand high salinities while maintaining a good growth productivity. To cope with the excess salt, they produce secondary metabolites (e.g. vitamins and phenolic compounds) which, along with the natural presence of some minerals, makes them not only nutritionally rich but also functional foods. Some halophytes, as quinoa or salicornia, are already used in some countries, mostly as gourmet food. Hydroponic cultivation of halophytes using seawater or diluted seawater for watering can decrease the pressure on freshwater resources while producing a nutritional and functional food. The XtremeGourmet project funded by the EU aims to develop and optimize the production of different halophytes by hydroponics. One of the more specific objectives of this project is the study of halophytes’ productivity and chemical composition under different abiotic conditions, e.g. salt and nutrient concentration and light intensity. Three species of halophytes commonly occurring in saltmarshes of the South of Portugal (Inula chrithmoides, Salicornia ramosissima and Mesembryanthemum nodiflorum) were cultivated using hydroponics under different salinities, ranging from 5 to 45 dS/m. For each condition, several parameters were assessed namely: total and commercial productivity, electrical conductivity, total soluble solids, proximal composition, mineral profile, total phenolics, flavonoids and condensed tannins content and antioxidant activity. Results show that productivity was significantly reduced for all plants with increasing salinity up to salinity 29 dS/m and remained low onwards. Oppositely, the electrical conductivity and the total soluble solids content of the produced plants increased with salinity, reaching a plateau at 29 dS/m. It seems that plants reflect the salt concentration of the water up to some point, being able to regulate their salt content for higher salinities. The same tendency was observed for the ash content of these plants, which is related to the mineral uptake from the cultivating media and the plants’ capacity to both accumulate and regulate ions’ concentration in their tissues. Nonetheless, this comes with a metabolic cost which is observed by a decrease in productivity. The mineral profile of these plants shows high concentrations of sodium but also high amounts of potassium. In what concerns the microelements, these plants appear to be a good source of manganese and iron and the low amounts of toxic metals account for their safe consumption in moderate amounts. Concerning the phenolics composition, plants presented moderate concentrations of phenolics but high amounts of condensed tannins, particularly I. crithmoides which accounts for its characteristic sour and spicy taste. Contrary to some studies in which higher amounts of phenolics were found in plants cultivated under higher salinities, in this study, the highest amount of phenolic compounds were found in plants grown at the lowest or intermediate salinities. Nonetheless, there was a positive correlation between the concentration of these compounds and the antioxidant capacity of the plants’ extracts.

Keywords: functional properties, halophytes, hydroponics, nutritional composition, salinity effect

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Authors: D. Pradhan, G. Tripathy, S. Pradhan

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Objectives: Imperviousness gainst estrogen treatments is a noteworthy reason for infection backslide and mortality in estrogen receptor alpha (ERα)- positive breast diseases. Tamoxifen or estrogen withdrawal builds the reliance of breast malignancy cells on INT3 flagging. Here, we researched the commitment of Quercetin and INT3 motioning in endocrine-safe breast tumor cells. Methods: We utilized two models of endocrine treatments safe (ETR) breast tumor: Tamoxifen-safe (TamR) and long haul estrogen-denied (LTED) MCF7 cells. We assessed the transitory and intrusive limit of these cells by Transwell cells. Articulation of epithelial to mesenchymal move (EMT) controllers and in addition INT3 receptors and targets were assessed by constant PCR and western smudge investigation. Besides, we tried in-vitro hostile to Quercetin monoclonal Antibodies (mAbs) and Gamma Secretase Inhibitors (GSIs) as potential EMT inversion remedial specialists. At last, we created stable Quercetin overexpressing MCF7 cells and assessed their EMT components and reaction to Tamoxifen. Results: We found that ETR cells procured an Epithelial to Mesenchymal move (EMT) phenotype and showed expanded levels of Quercetin and INT3 targets. Interestingly, we distinguished more elevated amount of INT3 however lower levels of INT1 and INT3 proposing a change to motioning through distinctive INT3 receptors after obtaining of resistance. Against Quercetin monoclonal antibodies and the GSI PF03084014 were powerful in obstructing the Quercetin/INT3 pivot and in part repressing the EMT process. As a consequence of this, cell relocation and attack were weakened and the immature microorganism like populace was essentially decreased. Hereditary hushing of Quercetin and INT3 prompted proportionate impacts. At long last, stable overexpression of Quercetin was adequate to make MCF7 lethargic to Tamoxifen by INT3 initiation. Conclusions: ETR cells express abnormal amounts of Quercetin and INT3, whose actuation eventually drives intrusive conduct. Hostile to Quercetin mAbs and GSI PF03084014 lessen articulation of EMT particles decreasing cell obtrusiveness. Quercetin overexpression instigates Tamoxifen resistance connected to obtaining of EMT phenotype. Our discovering propose that focusing on Quercetin and INT3 warrants further clinical Correlation as substantial restorative methodologies in endocrine-safe breast.

Keywords: endocrine, epithelial, mesenchymal, INT3, quercetin, MCF7

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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: S. Pradhan, D. Pradhan, G. Tripathy

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Anti-estrogen treatment resistant is a noteworthy reason for disease relapse and mortality in estrogen receptor alpha (ERα)- positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependance of breast malignancy cells on INT3 signaling. Here, we researched the contribution of Quercetin and INT3 signaling in endocrine resistant breast cancer cells. Methods: We utilized two models of endocrine therapies resistant (ETR-) breast cancer: tamoxifen-resistant (TamR) and long term estrogen-deprived (LTED) MCF7 cells. We assessed the migratory and invasive limit of these cells by Transwell assay. Expression of epithelial to mesenchymal transition (EMT) controllers and in addition INT3 receptors and targets were assessed by real-time PCR and western blot analysis. Besides, we tried in vitro anti-Quercetin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. At last, we created stable Quercetin over expessing MCF7 cells and assessed their EMT features and response to tamoxifen. Results:We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and showed expanded levels of Quercetin and INT3 targets. Interestingly, we detected higher level of INT3 however lower levels of INT31 and INT32 proposing a switch to targeting through distinctive INT3 receptors after obtaining of resistance. Anti-Quercetin monoclonal antibodies and the GSI PF03084014 were effective in obstructing the Quercetin/INT3 axis and in part inhibiting the EMT process. As a consequence of this, cell migration and invasion were weakened and the stem cell like population was considerably decreased. Genetic hushing of Quercetin and INT3 prompted proportionate impacts. Finally, stable overexpression of Quercetin was adequate to make MCF7 lethargic to tamoxifen by INT3 activation. Conclusions: ETR cells express abnormal amounts of Quercetin and INT3, whose actuation eventually drives invasive conduct. Anti-Quercetin mAbs and GSI PF03084014 lessen expression of EMT molecules decreasing cellular invasiveness. Quercetin overexpression instigates tamoxifen resistance connected to obtaining of EMT phenotype. Our discovering propose that focusing on Quercetin and/or INT3 warrants further clinical assessment as substantial therapeutic methodologies in endocrine-resistant breast cancer.

Keywords: quercetin, INT3, mesenchymal transition, MCF7 breast cancer cells

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Authors: Elena Maria Scalisi

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The increasing production and the use of TiO₂ nanoparticles (NPs) have inevitably led to their release into the environment, thereby posing a threat to organisms and also for human. Human exposure to TiO₂-NPs may occur during both manufacturing and use. TiO₂-NPs are common in consumer products for dermal application, toothpaste, food colorants, and nutritional supplements, then oral exposure may occur during use of such products. Into the body, TiO₂-NPs thanks to their small size (<100 nm), can, through testicular blood barrier inducing effect on testis and then on male reproductive health. The nanoscale size of TiO₂ increase the surface-to-volume ratio making them more reactive in a cell, then TiO₂ NPs increase their ability to produce reactive oxygen species (ROS). In male germ cells, ROS may have important implications in maintaining the normal functions of mature spermatozoa at physiological levels, moreover, in spermatozoa they are important signaling molecules for their hyperactivation and acrosome reaction. Nevertheless, an excess of ROS by external inputs such as NPs can increased the oxidative stress (OS), which results in damage DNA and apoptosis. The aim of our study has been investigate the impact of TiO₂ NPs on human spermatozoa, evaluating DNA damage and the expression of proteins involved in cell stress. According WHO guidelines 2021, we have exposed human spermatozoa in vitro to TiO₂ NP at concentrations 50 ppm, 100 ppm, 250 ppm, and 500 ppm for 1 hour (at 37°C and CO₂ at 5%). DNA damage was evaluated by Sperm Chromatin Dispersion Test (SCD) and TUNEL assay; moreover, we have evaluated the expression of biomarkers of oxidative stress like Heat Shock Protein 70 (HSP70) and Metallothioneins (MTs). Also, sperm parameters as motility viability have been evaluated. Our results not report a significant reduction in motility of spermatozoa at the end of the exposure. On the contrary, the progressive motility was increased at the highest concentration (500 ppm) and was statistically significant compared to control (p <0.05). Also, viability was not changed by exposure to TiO₂-NPs (p <0.05). However, increased DNA damage was observed at all concentrations, and the TUNEL assay highlighted the presence of single strand breaks in the DNA. The spermatozoa responded to the presence of TiO₂-NPs with the expression of Hsp70, which have a protective function because they allow the maintenance of cellular homeostasis in stressful/ lethal conditions. A positivity for MTs was observed mainly for the concentration of 4 mg/L. Although the biological and physiological function of the metallothionein (MTs) in the male genital organs is unclear, our results highlighted that the MTs expressed by spermatozoa maintain their biological role of detoxification from metals. Our results can give additional information to the data in the literature on the toxicity of TiO₂-NPs and reproduction.

Keywords: human spermatozoa, DNA damage, TiO₂-NPs, biomarkers

Procedia PDF Downloads 129

Authors: R. Zhankina, U. Zhanbyrbekuly, A. Tamadon, M. Askarov, R. Sherkhanov, D. Akhmetov, D. Saipiyeva, N. Keulimzhaev

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Male infertility affects about 15% of couples of reproductive age. Approximately 10–15% have azoospermia who have previously been diagnosed with male infertility. Azoospermia is regarded as the absence of spermatozoa in the ejaculate and is found in 10-15% of infertile men. Non-obstructive azoospermia is considered a cause of male infertility that is not amenable to drug therapy. Patients with non-obstructive azoospermia are unable to have their "own" children and have only options for adoption or use of donor sperm. Advances in assisted reproductive technologies such as intracytoplasmic sperm injection in vitro fertilization have significantly changed the management of patients with non-obstructive azoospermia. Advances in biotechnology have increased the options for treating patients with non-obstructive azoospermia. Mesenchymal stem cell therapy has been recognized as a new option for infertility treatment. Material and methods of the study: After obtaining informed consent, 5 patients diagnosed with non-obstructive azoospermia were included in an open, non-randomized study. The age of the patients ranged from 24 to 35 years. The examination was carried out before the start of treatment, which included biochemical blood tests, hormonal profile levels (luteinizing hormone, follicle-stimulating hormone, testosterone, prolactin, inhibin B); tests for tumor markers; genetic research. All studies were carried out in compliance with the requirements of Protocol No. 8 dated 06/09/20, approved by the Local Ethical Commission of NJSC "Astana Medical University". The control examination of patients was carried out after 6 months, by re-taking the program and hormonal profile (testosterone, luteinizing hormone, follicle-stimulating hormone, prolactin, inhibin B). Before micro-TESE of the testis, all 5 patients underwent myeloexfusion in the operating room. During the micro-TESE, autotransplantation of mesenchymal stem cells into the testicular network, previously cultured in a cell technology laboratory for 2 weeks, was performed. Results of the study: in all patients, the levels of total testosterone increased, the level of follicle-stimulating hormone decreased, the levels of luteinizing hormone returned to normal, the level of inhibin B increased. IVF with a positive result; another patient (20%) had spermatogenesis cells. Non-obstructive azoospermia and mesenchymal stem cells Conclusions: The positive results of this work serve as the basis for the application of a new cellular therapeutic approach for the treatment of non-obstructive azoospermia using mesenchymal stem cells.

Keywords: cell therapy, regenerative medicine, male infertility, mesenchymal stem cells

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Authors: Emine Aydin, Duygu Gocmen

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Pumpkin flour (PF) has a long shelf life and can be used as a nutritive, functional (antioxidant properties, phenolic contents, etc.) and coloring agent in many food items, especially in bakery products, sausages, instant noodles, pasta and flour mixes. Pre-treatment before drying is one of the most important factors affecting the quality of a final powdered product. Pretreatment, such as soaking in a bisulfite solution, provides that total carotenoids in raw materials rich in carotenoids, especially pumpkins, are retained in the dried product. This is due to the beneficial effect of antioxidant additives in the protection of carotenoids in the dehydrated plant foods. The oxygen present in the medium is removed by the radical SO₂, and thus the carotene degradation caused by the molecular oxygen is inhibited by the presence of SO₂. In this study, pumpkin flours (PFs) produced by two different applications (with or without metabisulfite pre-treatment) and then dried in a freeze dryer. The phenolic contents and antioxidant activities of pumpkin flour were determined. In addition to this, the compound of bioavailable phenolic substances which is obtained by PF has also been investigated using in vitro methods. As a result of researches made in recent years, it has been determined that all nutrients taken with foodstuffs are not bioavailable. Bioavailability changes depending on physical properties, chemical compounds, and capacities of individual digestion of foods. Therefore in this study; bioaccessible phenolics and phenolic bioaccessibility were also determined. The phenolics of the samples with metabisulfite application were higher than those of the samples without metabisulfite pre-treatment. Soaking in metabisulfite solution might have a protective effect for phenolic compounds. Phenolics bioaccessibility of pumpkin flours was investigated in order to assess pumpkin flour as sources of accessible phenolics. The higher bioaccessible phenolics (384.19 mg of GAE 100g⁻¹ DW) and phenolic bioaccessibility values (33.65 mL 100 mL⁻¹) were observed in the pumpkin flour with metabisulfite pre-treatment. Metabisulfite application caused an increase in bioaccessible phenolics of pumpkin flour. According to all assay (ABTS, CUPRAC, DPPH, and FRAP) results, both free and bound phenolics of pumpkin flour with metabisulfite pre-treatment had higher antioxidant activity than those of the sample without metabisulfite pre-treatment. The samples subjected to MS pre-treatment exhibited higher antioxidant activities than those of the samples without MS pre-treatment, this possibly due to higher phenolic contents of the samples with metabisulfite applications. As a result, metabisulfite application caused an increase in phenolic contents, bioaccessible phenolics, phenolic bioaccessibility and antioxidant activities of pumpkin flour. It can be said that pumpkin flour can be used as an alternative functional and nutritional ingredient in bakery products, dairy products (yoghurt, ice-cream), soups, sauces, infant formulae, confectionery, etc.

Keywords: pumpkin flour, bioaccessible phenolics, phenolic bioaccessibility, antioxidant activity

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Authors: Jos Olijve

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Introduction and Purpose: Endotoxins are found in the outer membrane of gram-negative bacteria and have profound in vitro and in vivo responses. They can trigger strong immune responses and negatively affect various cellar activities particular cells expressing toll-like receptors. They are therefore unwanted contaminants of biomaterials sourced from natural raw materials, and their activity must be as low as possible. Collagen and gelatin are natural extracellular matrix components and have, due to their low allergenic potential, suitable biological properties, and tunable physical characteristics, high potential in biomedical applications. The purpose of this study was to determine the influence of autoclave sterilization of gelatin on physical properties and endotoxin level compared to surfactant purified gelatin. Methods: Type A gelatin from Sigma-Aldrich (G1890) with endotoxin level of 35000 endotoxin units (EU) per gram gelatin and type A gelatins from Rousselot Gent with endotoxin activity of 30000 EU per gram were used. A 10 w/w% G1890 gelatin solution was autoclave sterilized during 30 minutes at 121°C and 1 bar over pressure. The physical properties and the endotoxin level of the sterilized G1890 gelatin were compared to a type A gelatin from Rousselot purified with Triton X100 surfactant. The Triton X100 was added to a concentration of 0.5 w/w% which is above the critical micellar concentration. The gelatin surfactant mixtures were kept for 30-45 minutes under constant stirring at 55-60°C. The Triton X100 was removed by active carbon filtration. The endotoxin levels of the gelatins were measured using the Endozyme recombinant factor C method from Hyglos GmbH (Germany). Results and Discussion: Autoclave sterilization significantly affect the physical properties of gelatin. Molecular weight of G1890 decreased from 140 to 50kDa, and gel strength decreased from 300 to 40g. The endotoxin level of the gelatin reduced after sterilization from 35000 EU/g to levels of 400-500 EU/g. These endotoxin levels are however still far above the upper endotoxin level of 0.05 EU/ml, which resembles 5 EU/g gelatin based on a 1% gelatin solution, to avoid cell proliferation alteration. Molecular weight and gel strength of Rousselot gelatin was not altered after Triton X100 purification and remained 150kDa and 300g respectively. The endotoxin levels of Triton X100 purified Rousselot gelatin was < 5EU/g gelatin. Conclusion: Autoclave sterilization of gelatin is, in comparison to Triton X100 purification, not efficient to inactivate endotoxin levels in gelatin to levels below the upper limit to avoid cell proliferation alteration. Autoclave sterilization gave a significant decrease in molecular weight and gel strength which makes autoclave sterilized gelatin, in comparison to Triton X100 purified gelatin, not suitable for 3D printing.

Keywords: endotoxin, gelatin, molecular weight, sterilization, Triton X100

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Authors: Danna Jia, Bin Li

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Background: Atopic dermatitis (AD) is a chronic and refractory inflammatory skin disease characterized by relapsing eczematous and pruritic skin lesions. The global prevalence of AD ranges from 1~ 20%, and its incidence rates are increasing. It affects individuals from infancy to adulthood, significantly impacting their daily lives and social activities. Despite its major health burden, the precise mechanisms underlying AD remain unknown. Understanding the genetic differences associated with AD is crucial for advancing diagnosis and targeted treatment development. This study aims to identify candidate genes of AD by using bioinformatics analysis. Methods: We conducted a comprehensive analysis of four pooled transcriptomic datasets (GSE16161, GSE32924, GSE130588, and GSE120721) obtained from the Gene Expression Omnibus (GEO) database. Differential gene expression analysis was performed using the R statistical language. The differentially expressed genes (DEGs) between AD patients and normal individuals were functionally analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Furthermore, a protein-protein interaction (PPI) network was constructed to identify candidate genes. Results: Among the patient-level gene expression datasets, we identified 114 shared DEGs, consisting of 53 upregulated genes and 61 downregulated genes. Functional analysis using GO and KEGG revealed that the DEGs were mainly associated with the negative regulation of transcription from RNA polymerase II promoter, membrane-related functions, protein binding, and the Human papillomavirus infection pathway. Through the PPI network analysis, we identified eight core genes: CD44, STAT1, HMMR, AURKA, MKI67, and SMARCA4. Conclusion: This study elucidates key genes associated with AD, providing potential targets for diagnosis and treatment. The identified genes have the potential to contribute to the understanding and management of AD. The bioinformatics analysis conducted in this study offers new insights and directions for further research on AD. Future studies can focus on validating the functional roles of these genes and exploring their therapeutic potential in AD. While these findings will require further verification as achieved with experiments involving in vivo and in vitro models, these results provided some initial insights into dysfunctional inflammatory and immune responses associated with AD. Such information offers the potential to develop novel therapeutic targets for use in preventing and treating AD.

Keywords: atopic dermatitis, bioinformatics, biomarkers, genes

Procedia PDF Downloads 62

Authors: K. J. Keerthi, Vasundhara Kamineni, A. Ravi Shanker, T. Rammurthy, A. Vijaya Lakshmi, Q. Hasan

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Pancreatic β-cells are the predominant insulin-producing cell types within the Islets of Langerhans and insulin is the primary hormone which regulates carbohydrate and fat metabolism. Apoptosis of β-cells or insufficient insulin production leads to Diabetes Mellitus (DM). Current therapy for diabetes includes either medical management or insulin replacement and regular monitoring. Replacement of β- cells is an attractive treatment option for both Type-1 and Type-2 DM in view of the recent paper which indicates that β-cells apoptosis is the common underlying cause for both the Types of DM. With the development of Edmonton protocol, pancreatic β-cells allo-transplantation became possible, but this is still not considered as standard of care due to subsequent requirement of lifelong immunosuppression and the scarcity of suitable healthy organs to retrieve pancreatic β-cell. Fetal pancreatic cells from abortuses were developed as a possible therapeutic option for Diabetes, however, this posed several ethical issues. Hence, in the present study Mesenchymal stem cells (MSCs) were differentiated into insulin producing cells which were isolated from Human Umbilical cord (HUC) tissue. MSCs have already made their mark in the growing field of regenerative medicine, and their therapeutic worth has already been validated for a number of conditions. HUC samples were collected with prior informed consent as approved by the Institutional ethical committee. HUC (n=26) were processed using a combination of both mechanical and enzymatic (collagenase-II, 100 U/ml, Gibco ) methods to obtain MSCs which were cultured in-vitro in L-DMEM (Low glucose Dulbecco's Modified Eagle's Medium, Sigma, 4.5 mM glucose/L), 10% FBS in 5% CO2 incubator at 37°C. After reaching 80-90% confluency, MSCs were characterized with Flowcytometry and Immunocytochemistry for specific cell surface antigens. Cells expressed CD90+, CD73+, CD105+, CD34-, CD45-, HLA-DR-/Low and Vimentin+. These cells were differentiated to β-cells by using H-DMEM (High glucose Dulbecco's Modified Eagle's Medium,25 mM glucose/L, Gibco), β-Mercaptoethanol (0.1mM, Hi-Media), basic Fibroblast growth factor (10 µg /L,Gibco), and Nicotinamide (10 mmol/L, Hi-Media). Pancreatic β-cells were confirmed by positive Dithizone staining and were found to be functionally active as they released 8 IU/ml insulin on glucose stimulation. Isolating MSCs from usually discarded, abundantly available HUC tissue, expanding and differentiating to β-cells may be the most feasible cell therapy option for the millions of people suffering from DM globally.

Keywords: diabetes mellitus, human umbilical cord, mesenchymal stem cells, differentiation

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Authors: Kevin De Castro Cogle, Mirian Kubo, Maria Anastasiadi, Fady Mohareb, Claire Rossi

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Background: The bioaccessibility of bioactive compounds is a critical determinant of the nutritional quality of various food products. Despite its importance, there is a limited number of comprehensive studies aimed at assessing how the composition of a food matrix influences the bioaccessibility of a compound of interest. This knowledge gap has prompted a growing need to investigate the intricate relationship between food matrix formulations and the bioaccessibility of bioactive compounds. One such class of bioactive compounds that has attracted considerable attention is curcuminoids. These naturally occurring phytochemicals, extracted from the roots of Curcuma longa, have gained popularity owing to their purported health benefits and also well known for their poor bioaccessibility Project aim: The primary objective of this research project is to systematically assess the influence of matrix composition on the bioaccessibility of curcuminoids. Additionally, this study aimed to develop a series of predictive models for bioaccessibility, providing valuable insights for optimising the formula for functional foods and provide more descriptive nutritional information to potential consumers. Methods: Food formulations enriched with curcuminoids were subjected to in vitro digestion simulation, and their bioaccessibility was characterized with chromatographic and spectrophotometric techniques. The resulting data served as the foundation for the development of predictive models capable of estimating bioaccessibility based on specific physicochemical properties of the food matrices. Results: One striking finding of this study was the strong correlation observed between the concentration of macronutrients within the food formulations and the bioaccessibility of curcuminoids. In fact, macronutrient content emerged as a very informative explanatory variable of bioaccessibility and was used, alongside other variables, as predictors in a Bayesian hierarchical model that predicted curcuminoid bioaccessibility accurately (optimisation performance of 0.97 R2) for the majority of cross-validated test formulations (LOOCV of 0.92 R2). These preliminary results open the door to further exploration, enabling researchers to investigate a broader spectrum of food matrix types and additional properties that may influence bioaccessibility. Conclusions: This research sheds light on the intricate interplay between food matrix composition and the bioaccessibility of curcuminoids. This study lays a foundation for future investigations, offering a promising avenue for advancing our understanding of bioactive compound bioaccessibility and its implications for the food industry and informed consumer choices.

Keywords: bioactive bioaccessibility, food formulation, food matrix, machine learning, probabilistic modelling

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Authors: Sanjay Shukla

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Excessive use of antibiotics is a main problem in the treatment of wounds and other chronic infections and antibiotic treatment is frequently non-curative, thus alternative treatment is necessary. Phage therapy is considered one of the most effective approaches to treat multi-drug resistant bacterial pathogens. Infections caused by Staphylococcus aureus are very efficiently controlled with phage cocktails, containing a different individual phages lysate infecting a majority of known pathogenic S. aureus strains. The aim of current study was to investigate the efficiency of a purified phage cocktail for prophylactic as well as therapeutic application in mouse model and in large animals with chronic septic infection of wounds. A total of 150 sewage samples were collected from various livestock farms. These samples were subjected for the isolation of bacteriophage by double agar layer method. A total of 27 sewage samples showed plaque formation by producing lytic activity against S. aureus in double agar overlay method out of 150 sewage samples. In TEM recovered isolates of bacteriophages showed hexagonal structure with tail fiber. In the bacteriophage (ØVS) had an icosahedral symmetry with the head size 52.20 nm in diameter and long tail of 109 nm. Head and tail were held together by connector and can be classified as a member of the Myoviridae family under the order of Caudovirale. Recovered bacteriophage had shown the antibacterial activity against the S. aureus in vitro. Cocktail (ØVS1, ØVS5, ØVS9 and ØVS 27) of phage lysate were tested to know in vivo antibacterial activity as well as the safety profile. Result of mice experiment indicated that the bacteriophage lysate was very safe, did not show any appearance of abscess formation which indicates its safety in living system. The mice were also prophylactically protected against S. aureus when administered with cocktail of bacteriophage lysate just before the administration of S. aureus which indicates that they are good prophylactic agent. The S. aureus inoculated mice were completely recovered by bacteriophage administration with 100% recovery which was very good as compere to conventional therapy. In present study ten chronic cases of wound were treated with phage lysate and follow up of these cases was done regularly up to ten days (at 0, 5 and 10 d). Result indicated that the six cases out of ten showed complete recovery of wounds within 10 d. The efficacy of bacteriophage therapy was found to be 60% which was very good as compared to the conventional antibiotic therapy in chronic septic wounds infections. Thus, the application of lytic phage in single dose proved to be innovative and effective therapy for treatment of septic chronic wounds.

Keywords: phage therapy, phage lysate, antimicrobial resistance, S. aureus

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Authors: G. A. Oladosu1, P. O. Ogbodogbo, C. I. Makinde1, M. O. Tijani, O. A. Adegboyega

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Saprolegniasis is a major pathogenic infection that contributes significantly to poor hatching rates in incubated fish eggs in the Africa catfish hatchery in Nigeria. Malachite green known to be very effective against this condition has been banned because it is carcinogenic. There is, therefore, the need for other effective yet safer methods of controlling saprolegniasis in incubated fish eggs. A total of 50 ml crude, chloroform extract of pyocyanin from which solvent was removed to attain 30 ml, having a concentration of 12.16 ug/ml was produced from 700 ml broth culture of Pseudomonas aeruginosa isolated from a previous study. In-vitro susceptibility of the fungus was investigated by exposing fungal infected eggs to two different time-concentration ratios of pyocyanin; 0.275 ug/ml and 2.75 ug/ml for 1 and 24 hours, and 5 mg/L malachite green as positive control while normal saline was the control. The efficacy of pyocyanin was evaluated using the degree of mycelial growth inhibition in different treatments. Fertilized Clarias gariepinus eggs (between 45 to 64 eggs) were then incubated in 20 ml of medium containing similar concentrations of pyocyanin and malachite green, with freshwater as a control for 24 hours. Hatching rates of the incubated eggs were observed. Three samples of un-hatched eggs were taken from each medium and observed for the presence of fungal pathogens using microscopy. Another batch of three samples of un-hatched eggs from each treatment was also inoculated on Sabourand dextrose agar (SDA) using Egg-Agar Transfer Technique to observe for fungal growth. Mycelial growth was inhibited in fungal infected eggs treated with 2.75 ug/ml for 24 hrs and the 5 mg/L malachite green for both 1 hr and 24 hrs. The mortality rate was 100% in fertilized C. gariepinus eggs exposed for 24 hrs to 0.275 and 2.75 ug/ml of pyocyanin. The mortality rate was least in malachite green followed by the control treatment. Embryonic development was observed to be arrested in the eggs treated with the two pyocyanin concentrations as they maintain their colour but showed no development beyond the gastrula stage, whereas viable eggs in the control and malachite green treatments developed fully into healthy hatchlings. Furthermore, microscopy of the un-hatched eggs revealed the presence of a protozoan ciliate; Colpidium sp, (Tetrahymenidae), as well as a pathogenic fungus; Saprolegnia sp. in the control but not in the malachite green and pyocyanin treatments. Growth of Saprolegnia sp was also observed in SDA culture of un-hatched eggs from the control, but not from pyocyanin and malachite green treated eggs. Pyocyanin treatment of incubated eggs of Clarias gariepinus effectively prevented fungal infection in the eggs, but also arrested the development of the embryo. Therefore, crude chloroform extract of pyocyanin from Pseudomonas aeruginosa cannot be used in the control of Saprolegniasis in incubated Clarias gariepinus eggs at the concentration and duration tested in this study.

Keywords: African catfish, bioprophylaxis, catfish embryo, Saprolegniasis

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Authors: Denis V. Axenov-Gribanov, Irina V. Voytsekhovskaya, Evgenii S. Protasov, Maxim A. Timofeyev

Abstract:

Isolated ecosystems existing under specific environmental conditions have been shown to be promising sources of new strains of actinobacteria. The taiga forest of Baikal Siberia has not been well studied, and its actinobacterial population remains uncharacterized. The proximity between the huge water mass of Lake Baikal and high mountain ranges influences the structure and diversity of the plant world in Siberia. Here, we report the isolation of eighteen actinobacterial strains from male cones of Pinus sylvestris trees growing on the shore of the ancient Lake Baikal in Siberia. The actinobacterial strains were isolated on solid nutrient MS media and Czapek agar supplemented with cycloheximide and phosphomycin. Identification of actinobacteria was carried out by 16S rRNA gene sequencing and further analysis of the evolutionary history. Four different liquid and solid media (NL19, DNPM, SG and ISP) were tested for metabolite production. The metabolite extracts produced by the isolated strains were tested for antibacterial and antifungal activities. Also, antiradical activity of crude extracts was carried out. Strain Streptomyces sp. IB 2014 I 74-3 that active against Gram-negative bacteria was selected for dereplication analysis with using the high-yield liquid chromatography with mass-spectrometry. Mass detection was performed in both positive and negative modes, with the detection range set to 160–2500 m/z. Data were collected and analyzed using Bruker Compass Data Analysis software, version 4.1. Dereplication was performed using the Dictionary of Natural Products (DNP) database version 6.1 with the following search parameters: accurate molecular mass, absorption spectra and source of compound isolation. Thus, in addition to more common representative strains of Streptomyces, several species belonging to the genera Rhodococcus, Amycolatopsis, and Micromonospora were isolated. Several of the selected strains were deposited in the Russian Collection of Agricultural Microorganisms (RCAM), St. Petersburg, Russia. All isolated strains exhibited antibacterial and antifungal activities. We identified several strains that inhibited the growth of the pathogen Candida albicans but did not hinder the growth of Saccharomyces cerevisiae. Several isolates were active against Gram-positive and Gram-negative bacteria. Moreover, extracts of several strains demonstrated high antioxidant activity. The high proportion of biologically active strains producing antibacterial and specific antifungal compounds may reflect their role in protecting pollen against phytopathogens. Dereplication of the secondary metabolites of the strain Streptomyces sp. IB 2014 I 74-3 was resulted in the fact that a total of 59 major compounds were detected in the culture liquid extract of strain cultivated in ISP medium. Eight compounds were preliminarily identified based on characteristics described in the Dictionary of Natural Products database, using the search parameters Streptomyces sp. IB 2014 I 74-3 was found to produce saframycin A, Y3 and S; 2-amino-3-oxo-3H-phenoxazine-1,8-dicarboxylic acid; galtamycinone; platencin A4-13R and A4-4S; ganefromycin d1; the antibiotic SS 8201B; and streptothricin D, 40-decarbamoyl, 60-carbamoyl. Moreover, forty-nine of the 59 compounds detected in the extract examined in the present study did not result in any positive hits when searching within the DNP database and could not be identified based on available mass-spec data. Thus, these compounds might represent new findings.

Keywords: actinobacteria, Baikal Lake, biodiversity, male cones, Pinus sylvestris

Procedia PDF Downloads 209

Authors: Kobauri Sophio, Kantaria Tengiz, Tugushi David, Puiggali Jordi, Katsarava Ramaz

Abstract:

Biodegradable biomaterials (BB) are of high interest for numerous applications in modern medicine as resorbable surgical materials and drug delivery systems. This kind of materials can be cleared from the body after the fulfillment of their function that excludes a surgical intervention for their removal. One of the most promising BBare amino acids based biodegradable poly(ester amide)s (PEAs) which are composed of naturally occurring (α-amino acids) and non-toxic building blocks such as fatty diols and dicarboxylic acids. Key bis-nucleophilic monomers for synthesizing the PEAs are diamine-diesters-di-p-toluenesulfonic acid salts of bis-(α-amino acid)-alkylenediesters (TAADs) which form the PEAs after step-growth polymerization (polycondensation) with bis-electrophilic counter-partners - activated diesters of dicarboxylic acids. The PEAs combine all advantages of the 'parent polymers' – polyesters (PEs) and polyamides (PAs): Ability of biodegradation (PEs), a high affinity with tissues and a wide range of desired mechanical properties (PAs). The scopes of applications of thePEAs can substantially be expanded by their functionalization, e.g. through the incorporation of hydrophobic fragments into the polymeric backbones. Hydrophobically modified PEAs can form non-covalent adducts with various compounds that make them attractive as drug carriers. For hydrophobic modification of the PEAs, we selected so-called 'Azlactone Method' based on the application of p-phenylene-bis-oxazolinons (bis-azlactones, BALs) as active bis-electrophilic monomers in step-growth polymerization with TAADs. Interaction of BALs with TAADs resulted in the PEAs with low MWs (Mw2,800-19,600 Da) and poor material properties. The high-molecular-weight PEAs (Mw up to 100,000) with desirable material properties were synthesized after replacement of a part of BALs with activated diester - di-p-nitrophenylsebacate, or a part of TAAD with alkylenediamine – 1,6-hexamethylenediamine. The new hydrophobically modified PEAs were characterized by FTIR, NMR, GPC, and DSC. It was shown that after the hydrophobic modification the PEAs retain the biodegradability (in vitro study catalyzed by α-chymptrypsin and lipase), and are of interest for constructing resorbable surgical and pharmaceutical devices including drug delivering containers such as microspheres. The new PEAs are insoluble in hydrophobic organic solvents such as chloroform or dichloromethane (swell only) that allowed elaborating a new technology of fabricating microspheres.

Keywords: amino acids, biodegradable polymers, bis-azlactones, microspheres

Procedia PDF Downloads 162

Authors: G. J. Fan, T. T. Lee, M. H. Chen, T. F. Shiao, B. Yu, C. F. Lee

Abstract:

Rice straw with poor feed quality and spent mushroom substrate are both the most abundant agricultural residues in Taiwan. Edible mushrooms from white rot fungi possess lignocellulase activity. It was expected to improve the feeding value of rice straw for ruminant by solid-state fermentation pretreatment using spent mushroom substrate. Six varieties or subspecies of spent edible mushrooms (Pleurotus ostreatus (blue or white color), P. sajor-caju, P. citrinopileatus, P. eryngii and Ganoderma lucidium) substrate were evaluated in solid-state fermentation process with rice straw for 8 wks. Quality improvement of fermented rice straw was determined by its in vitro digestibility, lignocellulose degradability, and cell wall breakdown checked by scanning electron microscope. Results turned out that Pleurotus ostreatus (white color) and P. sajor-caju had the better lignocellulose degradation effect than the others and was chosen for advance in vivo study. Rice straw fermented with spent Pleurotus ostreatus or Pleurotus sajor-caju mushroom substrate 8 wks was prepared for growing and lactating feeding trials of dairy goat, respectively. Pangolagrass hay at 15% diet dry matter was the control diet. Fermented or original rice straw was added to substitute pangolagrass hay in both feeding trials. A total of 30 head of Alpine castrated ram were assigned into three groups for 11 weeks, 5 pens (2 head/pen) each group. A total of 21 head of Saanen and Alpine goats were assigned into three treatments and individually fed in two repeat lactating trials with 28-d each. In castrated ram study, results showed that fermented rice straw by spent Pleurotus ostreatus mushroom substrate attributed the higher daily dry matter intakes (DMI, 1.53 vs. 1.20 kg) and body weight gain (138 vs. 101 g) than goats fed original rice straw. DMI (2.25 vs. 1.81 kg) and milk yield (3.31 vs. 3.02 kg) of lactating goats fed control pangolagrass diet and fermented rice straw by spent Pleurotus sajor-caju mushroom substrate were also higher than those fed original rice straw diet (P < 0.05). Milk compositions, milk fat, protein, total solid and lactose, were similar among treatments. In conclusion, solid-state fermentation by spent Pleurotus ostreatus or Pleurotus sajor-caju mushroom substrate could effectively improve the feeding value of rice straw. Fermented rice straw is a good alternative fiber feed resource for growing and lactating dairy goats and 15% in diet dry matter is recommended.

Keywords: feeding value, fermented rice straw, growing and lactating dairy goat, spent Pleurotus ostreatus and Pleurotus sajor-caju mushroom substrate

Procedia PDF Downloads 152

Authors: Shraddha Rane, Richard Warren, Stephen Eyre

Abstract:

L-23 is a pro-inflammatory molecule that signals T cells to release cytokines such as IL-17A and IL-22. Psoriasis is driven by a dysregulated immune response, within which IL-23 is now thought to play a key role. Genome-wide association studies (GWAS) have identified a number of genetic risk loci that support the involvement of IL-23 signalling in psoriasis; in particular a robust susceptibility locus at a gene encoding a subunit of the IL-23 receptor (IL23R) (Stuart et al., 2015; Tsoi et al., 2012). The lead psoriasis-associated SNP rs9988642 is located approximately 500 bp downstream of IL23R but is in tight linkage disequilibrium (LD) with a missense SNP rs11209026 (R381Q) within IL23R (r2 = 0.85). The minor (G) allele of rs11209026 is present in approximately 7% of the population and is protective for psoriasis and several other autoimmune diseases including IBD, ankylosing spondylitis, RA and asthma. The psoriasis-associated missense SNP R381Q causes an arginine to glutamine substitution in a region of the IL23R protein between the transmembrane domain and the putative JAK2 binding site in the cytoplasmic portion. This substitution is expected to affect the receptor’s surface localisation or signalling ability, rather than IL23R expression. Recent studies have also identified a psoriatic arthritis (PsA)-specific signal at IL23R; thought to be independent from the psoriasis association (Bowes et al., 2015; Budu-Aggrey et al., 2016). The lead PsA-associated SNP rs12044149 is intronic to IL23R and is in LD with likely causal SNPs intersecting promoter and enhancer marks in memory CD8+ T cells (Budu-Aggrey et al., 2016). It is therefore likely that the PsA-specific SNPs affect IL23R function via a different mechanism compared with the psoriasis-specific SNPs. It could be hypothesised that the risk allele for PsA located within the IL23R promoter causes an increase IL23R expression, relative to the protective allele. An increased expression of IL23R might then lead to an exaggerated immune response. The independent genetic signals identified for psoriasis and PsA in this locus indicate that different mechanisms underlie these two conditions; although likely both affecting the function of IL23R. It is very important to further characterise these mechanisms in order to better understand how the IL-23 receptor and its downstream signalling is affected in both diseases. This will help to determine how psoriasis and PsA patients might differentially respond to therapies, particularly IL-23 biologics. To investigate this further we have developed an in vitro model using CD4 T cells which express either wild type IL23R and IL12Rβ1 or mutant IL23R (R381Q) and IL12Rβ1. Model expressing different isotypes of IL23R is also underway to investigate the effects on IL23R expression. We propose to further investigate the variants for Ps and PsA and characterise key intracellular processes related to the variants.

Keywords: IL23R, psoriasis, psoriatic arthritis, SNP

Procedia PDF Downloads 145