Search results for: selective targeting
1137 Immunoliposome-Mediated Drug Delivery to Plasmodium-Infected and Non-Infected Red Blood Cells as a Dual Therapeutic/Prophylactic Antimalarial Strategy
Authors: Ernest Moles, Patricia Urbán, María Belén Jiménez-Díaz, Sara Viera-Morilla, Iñigo Angulo-Barturen, Maria Antònia Busquets, Xavier Fernàndez-Busquets
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Bearing in mind the absence of an effective vaccine against malaria and its severe clinical manifestations causing nearly half a million deaths every year, this disease represents nowadays a major threat to life. Besides, the basic rationale followed by currently marketed antimalarial approaches is based on the administration of drugs on their own, promoting the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the parasitized erythrocyte high enough to kill the intracellular pathogen while minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the specific delivery of immunoliposome (iLP)-encapsulated antimalarials to Plasmodium falciparum-infected red blood cells (pRBCs). Unfortunately, this strategy has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here, we show that encapsulation efficiencies reaching >96% can be achieved for the weakly basic drugs chloroquine (CQ) and primaquine using the pH gradient active loading method in liposomes composed of neutrally charged, saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the intracellular delivery of drugs not affecting the erythrocytic metabolism. Using this strategy, we have obtained unprecedented nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Polyethylene glycol-coated liposomes conjugated with monoclonal antibodies specific for the erythrocyte surface protein glycophorin A (anti-GPA iLP) were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added iLPs retained into the cells. When exposed for only 15 min to P. falciparum in vitro cultures synchronized at early stages, free CQ had no significant effect over parasite viability up to 200 nM drug, whereas iLP-encapsulated 50 nM CQ completely arrested its growth. Furthermore, when assayed in vivo in P. falciparum-infected humanized mice, anti-GPA iLPs cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg. In comparison, free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement in drug antimalarial efficacy is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.Keywords: immunoliposomal nanoparticles, malaria, prophylactic-therapeutic polyvalent activity, targeted drug delivery
Procedia PDF Downloads 3681136 Sports and Exercise Medicine: A Public Health Tool in Combating and Preventing the Side Effects of a Sedentary Lifestyle
Authors: Shireen Ibish
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Physical inactivity and unhealthy diets have contributed to a global burden of disease with increased relation to non-communicable diseases, increased risk of colon and breast cancer, high prevalence of depression, reduced quality of life and early death. The World Health Organisation’s facts on Obesity show a tripling in prevalence across the European Region since the 1980s. This has lead to a huge public health burden, being responsible for and 10-13% of deaths (fourth largest cause of global mortality) and 2-8% of health costs in the Region. In the UK alone, the present cost of physical inactivity has been estimated to be £8.2 billion. In 2002 a paper published in the International Journal of Epidemiology on ‘sedentary’ lifestyle, put into figures the increasingly worrying statistics across European countries. “Percentages of sedentary lifestyles across European countries ranged between 43.3% (Sweden) and 87.8% (Portugal)”. This was especially so amongst obese subjects, less- educated people, and smokers. While in the UK’s “50% of adult population in the UK is predicted to be obese by 2050.” Sports and Exercise Medicine, as a specialty, has a lot to offer in targeting this globally increasing epidemic. The worrying figures and the increasing knowledge of combating and preventing this issue have lead to increased awareness amongst the medical profession and more targeted interventions to reduce the burden of disease. “The public health element of the specialty is critical – this is not simply a specialty for the management of elite athletes’ medical conditions – it is central to the promotion of exercise as a means of disease prevention, to enhance well-being and in the management of disease.” WHO advised on creating National policies, encouraging and providing opportunities for greater physical activity, and improve the affordability, availability and accessibility of healthy foods. In the UK various different movements have been established to target this problem. The Motivate2Move, Move Eat Treat and guidelines advising specialties on targeting and encouraging exercise in the population (Sport and Exercise Medicine A Fresh Approach).Keywords: sedentary lifestyle, obesity, public health burden, medicine
Procedia PDF Downloads 5621135 Application of Regularized Low-Rank Matrix Factorization in Personalized Targeting
Authors: Kourosh Modarresi
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The Netflix problem has brought the topic of “Recommendation Systems” into the mainstream of computer science, mathematics, and statistics. Though much progress has been made, the available algorithms do not obtain satisfactory results. The success of these algorithms is rarely above 5%. This work is based on the belief that the main challenge is to come up with “scalable personalization” models. This paper uses an adaptive regularization of inverse singular value decomposition (SVD) that applies adaptive penalization on the singular vectors. The results show far better matching for recommender systems when compared to the ones from the state of the art models in the industry.Keywords: convex optimization, LASSO, regression, recommender systems, singular value decomposition, low rank approximation
Procedia PDF Downloads 4481134 Exploring Coordination between Monetary and Macroprudential Policies Using a Monetary Policy Procyclicality Ratio
Authors: Lukasz Kurowski, Paweł Smaga
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We explore the procyclicality of monetary policy decisions towards the financial cycle in the 1995−2015 period on a sample of six central banks. Using interest rate paths and the credit-to-GDP gap to construct a monetary policy procyclicality ratio, we provide evidence that monetary policy procyclicality was high in BoE and CNB and low in Riksbank and ECB. The results support the need for coordination between macroprudential and monetary policies, for example, by including financial stability considerations to the inflation targeting strategy.Keywords: central bank, financial stability, macroprudential policy, monetary policy
Procedia PDF Downloads 3671133 Exploring Fluoroquinolone-Resistance Dynamics Using a Distinct in Vitro Fermentation Chicken Caeca Model
Authors: Bello Gonzalez T. D. J., Setten Van M., Essen Van A., Brouwer M., Veldman K. T.
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Resistance to fluoroquinolones (FQ) has evolved increasingly over the years, posing a significant challenge for the treatment of human infections, particularly gastrointestinal tract infections caused by zoonotic bacteria transmitted through the food chain and environment. In broiler chickens, a relatively high proportion of FQ resistance has been observed in Escherichia coli indicator, Salmonella and Campylobacter isolates. We hypothesize that flumequine (Flu), used as a secondary choice for the treatment of poultry infections, could potentially be associated with a high proportion of FQ resistance. To evaluate this hypothesis, we used an in vitro fermentation chicken caeca model. Two continuous single-stage fermenters were used to simulate in real time the physiological conditions of the chicken caeca microbial content (temperature, pH, caecal content mixing, and anoxic environment). A pool of chicken caecal content containing FQ-resistant E. coli obtained from chickens at slaughter age was used as inoculum along with a spiked FQ-susceptible Campylobacter jejuni strain isolated from broilers. Flu was added to one of the fermenters (Flu-fermenter) every 24 hours for two days to evaluate the selection and maintenance of FQ resistance over time, while the other served as a control (C-Fermenter). The experiment duration was 5 days. Samples were collected at three different time points: before, during and after Flu administration. Serial dilutions were plated on Butzler culture media with and without Flu (8mg/L) and enrofloxacin (4mg/L) and on MacConkey culture media with and without Flu (4mg/L) and enrofloxacin (1mg/L) to determine the proportion of resistant strains over time. Positive cultures were identified by mass spectrometry and matrix-assisted laser desorption/ionization (MALDI). A subset of the obtained isolates were used for Whole Genome Sequencing analysis. Over time, E. coli exhibited positive growth in both fermenters, while C. jejuni growth was detected up to day 3. The proportion of Flu-resistant E. coli strains recovered remained consistent over time after antibiotic selective pressure, while in the C-fermenter, a decrease was observed at day 5; a similar pattern was observed in the enrofloxacin-resistant E. coli strains. This suggests that Flu might play a role in the selection and persistence of enrofloxacin resistance, compared to C-fermenter, where enrofloxacin-resistant E. coli strains appear at a later time. Furthermore, positive growth was detected from both fermenters only on Butzler plates without antibiotics. A subset of C. jejuni strains from the Flu-fermenter revealed that those strains were susceptible to ciprofloxacin (MIC < 0.12 μg/mL). A selection of E. coli strains from both fermenters revealed the presence of plasmid-mediated quinolone resistance (PMQR) (qnr-B19) in only one strain from the C-fermenter belonging to sequence type (ST) 48, and in all from Flu-fermenter belonged to ST189. Our results showed that Flu selective impact on PMQR-positive E. coli strains, while no effect was observed in C. jejuni. Maintenance of Flu-resistance was correlated with antibiotic selective pressure. Further studies into antibiotic resistance gene transfer among commensal and zoonotic bacteria in the chicken caeca content may help to elucidate the resistance spread mechanisms.Keywords: fluoroquinolone-resistance, escherichia coli, campylobacter jejuni, in vitro model
Procedia PDF Downloads 531132 The Impact of Treatment of Latent Tuberculosis on the Incidence: The Case of Algeria
Authors: Schehrazad Selmane
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We present a deterministic model which describes the dynamics of tuberculosis in Algerian population where the vaccination program with BCG is in place since 1969 and where the WHO recommendations regarding the DOTS (directly observed treatment, short course) strategy are in application. The impact of an intervention program, targeting recently infected people among all close contacts of active cases and their treatment to prevent endogenous reactivation, on the incidence of tuberculosis, is investigated. We showed that a widespread treatment of latently infected individuals for some years is recommended to shift from higher to lower equilibrium state and thereafter relaxation is recommended.Keywords: deterministic model, reproduction number, stability, tuberculosis
Procedia PDF Downloads 3241131 Esophageal Premalignant and Malignant Epithelial Lesions: Pathological Characteristics and Value of Cyclooxygenase-2 Expression.
Authors: Hanan Mohamed Abd Elmoneim, Rawan Saleh AlJawi, Razan Saleh AlJawi, Aseel Abdullah AlMasoudi , Zyad Adnan Turkistani, Anas Abdulkarim Alkhoutani , Ohood Musaed AlJuhani , Hanan Attiyah AlZahrani
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Background Esophageal cancer is the eighth most common cancer worldwide. More than 90% of esophageal cancers are either squamous cell carcinoma or adenocarcinoma. Squamous dysplasia is a precancerous lesion for squamous cell carcinoma and Barrett's esophagus is the precancerous lesion for adenocarcinoma. Gastro-esophageal reflux disease (GERD) is the initiation factor for Barrett's esophagus. Cyclooxygenase-2 (COX-2) is a key enzyme in arachidonic metabolism. It appears to play an important role in gastrointestinal carcinogenesis. COX-2 activity may be a potential target for the prevention of cancer progression by selective COX-2 inhibitors, which decrease proliferation and increase apoptosis. Objectives To assess COX-2 expression in premalignant and malignant esophageal epitheliums changes and detect its roles in progression of these lesions. Materials and Methods We analyzed the expression of COX-2 immunohistochemically in 40 esophageal biopsies utilizing the streptavidin-biotin-peroxidase complex method on archival formalin fixed-paraffin embedded blocks. Histopathologically, 17 (42.5%) of cases were non-malignant cases which included GERD, Barrett's esophagus and squamous dysplasia. The malignant cases were 23 (57.5%) squamous cell carcinoma, adenocarcinoma and undifferentiated carcinoma. Results In non-malignant cases 7 (41.2%) out of 17 cases had high COX-2 expression. In squamous cell carcinoma 10 (83.3%) out of 12 cases had high COX-2 expression. The expression of COX-2 was high in all 9 (100%) cases of adenocarcinoma. COX-2 expression is significantly increased (P=0.005 and P=0.0001) in squamous cell carcinoma and adenocarcinoma respectively. There was a significant difference in COX-2 immunoreactivity between malignant and non-malignant lesions (P=0.0003). Conclusion COX-2 is responsible for the progression of esophageal diseases from benign to malignant. We recommend that COX-2 immunohistochemistry should be done routinely for premalignant and malignant esophageal lesions as selective COX-2 inhibitors will be helpful in the treatment. Further studies on molecular and genetic basis of COX-2 expression are needed to unmask its role and relation to progression of esophageal lesions.Keywords: Cox-2, Esophageal adinocarcinoma, Esophageal squamous cell carcinoma, Immunohistochemistry.
Procedia PDF Downloads 3441130 Use of Metamaterials Structures to Reduce the SAR in the Human Head
Authors: Hafawa Messaoudi, Taoufik Aguili
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Due to the rapid growth in the use of wireless communication systems, there has been a recent increase in public concern regarding the exposure of humans to Radio Frequency (RF) electromagnetic radiation. This is particularly evident in the case of mobile telephone handsets. Previously, the insertion of a ferrite sheet between the antenna and the human head, the use of conductive materials (such as aluminum), the use of metamaterials (SRR), frequency selective surface (FSS), and electromagnetic band gap (EBG) structures to design high performance devices were proposed as methods of reducing the SAR value. This paper aims to provide an investigation of the effectiveness of various available Specific Absorption Rate (SAR) reduction solutions.Keywords: EBG, HIS, metamaterials, SAR reduction
Procedia PDF Downloads 5181129 Acoustic Analysis of Psycho-Communication Disorders within Moroccan Students
Authors: Brahim Sabir
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Psycho-Communication disorders negatively affect the academic curriculum for students in higher education. Thus, understanding these disorders, their causes and effects will give education specialists a tool for the decision, which will lead to the resolution of problems related to the integration of students with Psycho-Communication disorders. It is in this context that a statistical study was conducted, targeting the population object of study, namely Moroccan students. Pathological voice samples were recorded and analyzed acoustically with PRAAT software, in order to build a model that will be the basis for the objective diagnostic.Keywords: psycho-communication disorders, acoustic analysis, PRAAT
Procedia PDF Downloads 3861128 Let-7 Mirnas Regulate Inflammatory Cytokine Production in Bovine Endometrial Cells after Lipopolysaccharide Challenge by Targeting TNFα
Authors: S. Ibrahim, D. Salilew-Wondim, M. Hoelker, C. Looft, E. Tholen, C. Grosse-Brinkhaus, K. Schellander, C. Neuhoff, D. Tesfaye
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Bovine endometrial cells appear to have a key role in innate immune defense of the female genital tract. A better understanding of molecular changes in microRNAs (miRNAs) and their target genes expression may identify reliable prognostic indicators for cows that will resolve inflammation and resume cyclicity. In the current study, we hypothesized that let-7 miRNAs family has a primary role in the innate immune defence of the endometrium tissue against bacterial infection, which is partly achieved via regulating mRNA stability of pro-inflammatory cytokines at the post-transcriptional level. Therefore, we conducted two experiments. In the first experiment, primary bovine endometrial cells were challenged with clinical (3.0 μg/ml) and sub-clinical (0.5 μg/ml) doses of lipopolysaccharide (LPS) for 24h. In the 2nd experiment, we have investigated the potential role of let-7 miRNAs (let-7a and let-7f) using gain and loss of function approaches. Additionally, tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 induced transcript 1 (TGFB1I1) and serum deprivation response (SDPR) genes were validated using reporter assay. Here we addressed for the first time that let-7 miRNAs have a precise role in bovine endometrium, where LPS dysregulated let-7 miRNAs family expression was associated with an increased pro-inflammatory cytokine level by directly/indirectly targeting the TNFα, interleukin 6 (IL6), nuclear factor kappa-light-chain enhancer of activated B cells (NFκB), TGFβ1I1 and SDPR genes. To our knowledge, this is the first study showing that TNFα, TGFβ1I1 and SDPR were identified and validated as novel let-7 miRNAs targets and could have a distinct role in inflammatory immune response of LPS challenged bovine endometrial cells. Our data represent a new finding by which uterine homeostasis is maintained through functional regulation of let-7a by down-regulation of pro-inflammatory cytokines expression (TNFα and IL6) at the mRNA and protein levels. These findings suggest that LPS serves as a negative regulator of let-7 miRNAs expression and provides a mechanism for the persistent pro-inflammatory phenotype, which is a hallmark of bovine subclinical endometritis.Keywords: bovine endometrial cells, let-7, lipopolysaccharide, pro-inflammatory cytokines
Procedia PDF Downloads 3711127 Computer-Aided Drug Repurposing for Mycobacterium Tuberculosis by Targeting Tryptophanyl-tRNA Synthetase
Authors: Neslihan Demirci, Serdar Durdağı
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Mycobacterium tuberculosis is still a worldwide disease-causing agent that, according to WHO, led to the death of 1.5 million people from tuberculosis (TB) in 2020. The bacteria reside in macrophages located specifically in the lung. There is a known quadruple drug therapy regimen for TB consisting of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). Over the past 60 years, there have been great contributions to treatment options, such as recently approved delamanid (OPC67683) and bedaquiline (TMC207/R207910), targeting mycolic acid and ATP synthesis, respectively. Also, there are natural compounds that can block the tryptophanyl-tRNA synthetase (TrpRS) enzyme, chuangxinmycin, and indolmycin. Yet, already the drug resistance is reported for those agents. In this study, the newly released TrpRS enzyme structure is investigated for potential inhibitor drugs from already synthesized molecules to help the treatment of resistant cases and to propose an alternative drug for the quadruple drug therapy of tuberculosis. Maestro, Schrodinger is used for docking and molecular dynamic simulations. In-house library containing ~8000 compounds among FDA-approved indole-containing compounds, a total of 57 obtained from the ChemBL were used for both ATP and tryptophan binding pocket docking. Best of indole-containing 57 compounds were subjected to hit expansion and compared later with virtual screening workflow (VSW) results. After docking, VSW was done. Glide-XP docking algorithm was chosen. When compared, VSW alone performed better than the hit expansion module. Best scored compounds were kept for ten ns molecular dynamic simulations by Desmond. Further, 100 ns molecular dynamic simulation was performed for elected molecules according to Z-score. The top three MMGBSA-scored compounds were subjected to steered molecular dynamic (SMD) simulations by Gromacs. While SMD simulations are still being conducted, ponesimod (for multiple sclerosis), vilanterol (β₂ adrenoreceptor agonist), and silodosin (for benign prostatic hyperplasia) were found to have a significant affinity for tuberculosis TrpRS, which is the propulsive force for the urge to expand the research with in vitro studies. Interestingly, top-scored ponesimod has been reported to have a side effect that makes the patient prone to upper respiratory tract infections.Keywords: drug repurposing, molecular dynamics, tryptophanyl-tRNA synthetase, tuberculosis
Procedia PDF Downloads 1111126 3D Label-Free Bioimaging of Native Tissue with Selective Plane Illumination Optical Microscopy
Authors: Jing Zhang, Yvonne Reinwald, Nick Poulson, Alicia El Haj, Chung See, Mike Somekh, Melissa Mather
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Biomedical imaging of native tissue using light offers the potential to obtain excellent structural and functional information in a non-invasive manner with good temporal resolution. Image contrast can be derived from intrinsic absorption, fluorescence, or scatter, or through the use of extrinsic contrast. A major challenge in applying optical microscopy to in vivo tissue imaging is the effects of light attenuation which limits light penetration depth and achievable imaging resolution. Recently Selective Plane Illumination Microscopy (SPIM) has been used to map the 3D distribution of fluorophores dispersed in biological structures. In this approach, a focused sheet of light is used to illuminate the sample from the side to excite fluorophores within the sample of interest. Images are formed based on detection of fluorescence emission orthogonal to the illumination axis. By scanning the sample along the detection axis and acquiring a stack of images, 3D volumes can be obtained. The combination of rapid image acquisition speeds with the low photon dose to samples optical sectioning provides SPIM is an attractive approach for imaging biological samples in 3D. To date all implementations of SPIM rely on the use of fluorescence reporters be that endogenous or exogenous. This approach has the disadvantage that in the case of exogenous probes the specimens are altered from their native stage rendering them unsuitable for in vivo studies and in general fluorescence emission is weak and transient. Here we present for the first time to our knowledge a label-free implementation of SPIM that has downstream applications in the clinical setting. The experimental set up used in this work incorporates both label-free and fluorescent illumination arms in addition to a high specification camera that can be partitioned for simultaneous imaging of both fluorescent emission and scattered light from intrinsic sources of optical contrast in the sample being studied. This work first involved calibration of the imaging system and validation of the label-free method with well characterised fluorescent microbeads embedded in agarose gel. 3D constructs of mammalian cells cultured in agarose gel with varying cell concentrations were then imaged. A time course study to track cell proliferation in the 3D construct was also carried out and finally a native tissue sample was imaged. For each sample multiple images were obtained by scanning the sample along the axis of detection and 3D maps reconstructed. The results obtained validated label-free SPIM as a viable approach for imaging cells in a 3D gel construct and native tissue. This technique has the potential use in a near-patient environment that can provide results quickly and be implemented in an easy to use manner to provide more information with improved spatial resolution and depth penetration than current approaches.Keywords: bioimaging, optics, selective plane illumination microscopy, tissue imaging
Procedia PDF Downloads 2431125 Improving Binding Selectivity in Molecularly Imprinted Polymers from Templates of Higher Biomolecular Weight: An Application in Cancer Targeting and Drug Delivery
Authors: Ben Otange, Wolfgang Parak, Florian Schulz, Michael Alexander Rubhausen
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The feasibility of extending the usage of molecular imprinting technique in complex biomolecules is demonstrated in this research. This technique is promising in diverse applications in areas such as drug delivery, diagnosis of diseases, catalysts, and impurities detection as well as treatment of various complications. While molecularly imprinted polymers MIP remain robust in the synthesis of molecules with remarkable binding sites that have high affinities to specific molecules of interest, extending the usage to complex biomolecules remains futile. This work reports on the successful synthesis of MIP from complex proteins: BSA, Transferrin, and MUC1. We show in this research that despite the heterogeneous binding sites and higher conformational flexibility of the chosen proteins, relying on their respective epitopes and motifs rather than the whole template produces highly sensitive and selective MIPs for specific molecular binding. Introduction: Proteins are vital in most biological processes, ranging from cell structure and structural integrity to complex functions such as transport and immunity in biological systems. Unlike other imprinting templates, proteins have heterogeneous binding sites in their complex long-chain structure, which makes their imprinting to be marred by challenges. In addressing this challenge, our attention is inclined toward the targeted delivery, which will use molecular imprinting on the particle surface so that these particles may recognize overexpressed proteins on the target cells. Our goal is thus to make surfaces of nanoparticles that specifically bind to the target cells. Results and Discussions: Using epitopes of BSA and MUC1 proteins and motifs with conserved receptors of transferrin as the respective templates for MIPs, significant improvement in the MIP sensitivity to the binding of complex protein templates was noted. Through the Fluorescence Correlation Spectroscopy FCS measurements on the size of protein corona after incubation of the synthesized nanoparticles with proteins, we noted a high affinity of MIPs to the binding of their respective complex proteins. In addition, quantitative analysis of hard corona using SDS-PAGE showed that only a specific protein was strongly bound on the respective MIPs when incubated with similar concentrations of the protein mixture. Conclusion: Our findings have shown that the merits of MIPs can be extended to complex molecules of higher biomolecular mass. As such, the unique merits of the technique, including high sensitivity and selectivity, relative ease of synthesis, production of materials with higher physical robustness, and higher stability, can be extended to more templates that were previously not suitable candidates despite their abundance and usage within the body.Keywords: molecularly imprinted polymers, specific binding, drug delivery, high biomolecular mass-templates
Procedia PDF Downloads 481124 Candida antartica Lipase Assisted Enrichment of n-3 PUFA in Indian Sardine Oil
Authors: Prasanna Belur, P. R. Ashwini, Sampath Charanyaa, I. Regupathi
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Indian oil sardine (Sardinella longiceps) are one of the richest and cheapest sources of n-3 polyunsaturated fatty acids (n-3 PUFA) such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). The health benefits conferred by n-3 PUFA upon consumption, in the prevention and treatment of coronary, neuromuscular, immunological disorders and allergic conditions are well documented. Natural refined Indian Sardine oil generally contain about 25% (w/w) n-3 PUFA along with various unsaturated and saturated fatty acids in the form of mono, di, and triglycerides. Having high concentration of n-3 PUFA content in the glyceride form is most desirable for human consumption to avail maximum health benefits. Thus, enhancing the n-3 PUFA content while retaining it in the glyceride form with green technology is the need of the hour. In this study, refined Indian Sardine oil was subjected to selective hydrolysis by Candida antartica lipase to enhance n-3 PUFA content. The degree of hydrolysis and enhancement of n-3 PUFA content was estimated by determining acid value, Iodine value, EPA and DHA content (by Gas Chromatographic methods after derivitization) before and after hydrolysis. Various reaction parameters such as pH, temperature, enzyme load, lipid to aqueous phase volume ratio and incubation time were optimized by conducting trials with one parameter at a time approach. Incubating enzyme solution with refined sardine oil with a volume ratio of 1:1, at pH 7.0, for 60 minutes at 50 °C, with an enzyme load of 60 mg/ml was found to be optimum. After enzymatic treatment, the oil was subjected to refining to remove free fatty acids and moisture content using previously optimized refining technology. Enzymatic treatment at the optimal conditions resulted in 12.11 % enhancement in Degree of hydrolysis. Iodine number had increased by 9.7 % and n-3 PUFA content was enhanced by 112 % (w/w). Selective enhancement of n-3 PUFA glycerides, eliminating saturated and unsaturated fatty acids from the oil using enzyme is an interesting preposition as this technique is environment-friendly, cost effective and provide natural source of n-3 PUFA rich oil.Keywords: Candida antartica, lipase, n-3 polyunsaturated fatty acids, sardine oil
Procedia PDF Downloads 2191123 Targeting APP IRE mRNA to Combat Amyloid -β Protein Expression in Alzheimer’s Disease
Authors: Mateen A Khan, Taj Mohammad, Md. Imtaiyaz Hassan
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Alzheimer’s disease is characterized by the accumulation of the processing products of the amyloid beta peptide cleaved by amyloid precursor protein (APP). Iron increases the synthesis of amyloid beta peptides, which is why iron is present in Alzheimer's disease patients' amyloid plaques. Iron misregulation in the brain is linked to the overexpression of APP protein, which is directly related to amyloid-β aggregation in Alzheimer’s disease. The APP 5'-UTR region encodes a functional iron-responsive element (IRE) stem-loop that represents a potential target for modulating amyloid production. Targeted regulation of APP gene expression through the modulation of 5’-UTR sequence function represents a novel approach for the potential treatment of AD because altering APP translation can be used to improve both the protective brain iron balance and provide anti-amyloid efficacy. The molecular docking analysis of APP IRE RNA with eukaryotic translation initiation factors yields several models exhibiting substantial binding affinity. The finding revealed that the interaction involved a set of functionally active residues within the binding sites of eIF4F. Notably, APP IRE RNA and eIF4F interaction were stabilized by multiple hydrogen bonds with residues of APP IRE RNA and eIF4F. It was evident that APP IRE RNA exhibited a structural complementarity that tightly fit within binding pockets of eIF4F. The simulation studies further revealed the stability of the complexes formed between RNA and eIF4F, which is crucial for assessing the strength of these interactions and subsequent roles in the pathophysiology of Alzheimer’s disease. In addition, MD simulations would capture conformational changes in the IRE RNA and protein molecules during their interactions, illustrating the mechanism of interaction, conformational change, and unbinding events and how it may affect aggregation propensity and subsequent therapeutic implications. Our binding studies correlated well with the translation efficiency of APP mRNA. Overall, the outcome of this study suggests that the genomic modification and/or inhibiting the expression of amyloid protein by targeting APP IRE RNA can be a viable strategy to identify potential therapeutic targets for AD and subsequently be exploited for developing novel therapeutic approaches.Keywords: Alzheimer's disease, Protein-RNA interaction analysis, molecular docking simulations, conformational dynamics, binding stability, binding kinetics, protein synthesis.
Procedia PDF Downloads 501122 Soybean Lecithin Based Reverse Micellar Extraction of Pectinase from Synthetic Solution
Authors: Sivananth Murugesan, I. Regupathi, B. Vishwas Prabhu, Ankit Devatwal, Vishnu Sivan Pillai
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Pectinase is an important enzyme which has a wide range of applications including textile processing and bioscouring of cotton fibers, coffee and tea fermentation, purification of plant viruses, oil extraction etc. Selective separation and purification of pectinase from fermentation broth and recover the enzyme form process stream for reuse are cost consuming process in most of the enzyme based industries. It is difficult to identify a suitable medium to enhance enzyme activity and retain its enzyme characteristics during such processes. The cost effective, selective separation of enzymes through the modified Liquid-liquid extraction is of current research interest worldwide. Reverse micellar extraction, globally acclaimed Liquid-liquid extraction technique is well known for its separation and purification of solutes from the feed which offers higher solute specificity and partitioning, ease of operation and recycling of extractants used. Surfactant concentrations above critical micelle concentration to an apolar solvent form micelles and addition of micellar phase to water in turn forms reverse micelles or water-in-oil emulsions. Since, electrostatic interaction plays a major role in the separation/purification of solutes using reverse micelles. These interaction parameters can be altered with the change in pH, addition of cosolvent, surfactant and electrolyte and non-electrolyte. Even though many chemical based commercial surfactant had been utilized for this purpose, the biosurfactants are more suitable for the purification of enzymes which are used in food application. The present work focused on the partitioning of pectinase from the synthetic aqueous solution within the reverse micelle phase formed by a biosurfactant, Soybean Lecithin dissolved in chloroform. The critical micelle concentration of soybean lecithin/chloroform solution was identified through refractive index and density measurements. Effect of surfactant concentrations above and below the critical micelle concentration was considered to study its effect on enzyme activity, enzyme partitioning within the reverse micelle phase. The effect of pH and electrolyte salts on the partitioning behavior was studied by varying the system pH and concentration of different salts during forward and back extraction steps. It was observed that lower concentrations of soybean lecithin enhanced the enzyme activity within the water core of the reverse micelle with maximizing extraction efficiency. The maximum yield of pectinase of 85% with a partitioning coefficient of 5.7 was achieved at 4.8 pH during forward extraction and 88% yield with a partitioning coefficient of 7.1 was observed during backward extraction at a pH value of 5.0. However, addition of salt decreased the enzyme activity and especially at higher salt concentrations enzyme activity declined drastically during both forward and back extraction steps. The results proved that reverse micelles formed by Soybean Lecithin and chloroform may be used for the extraction of pectinase from aqueous solution. Further, the reverse micelles can be considered as nanoreactors to enhance enzyme activity and maximum utilization of substrate at optimized conditions, which are paving a way to process intensification and scale-down.Keywords: pectinase, reverse micelles, soybean lecithin, selective partitioning
Procedia PDF Downloads 3651121 Microstructures of Si Surfaces Fabricated by Electrochemical Anodic Oxidation with Agarose Stamps
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This paper investigates the fabrication of microstructures on Si surfaces by using electrochemical anodic oxidation with agarose stamps. The fabricating process is based on a selective anodic oxidation reaction that occurs in the contact area between a stamp and a Si substrate. The stamp which is soaked in electrolyte previously acts as a current flow channel. After forming the oxide patterns as an etching mask, a KOH aqueous is used for the wet etching of Si. A complicated microstructure array of 1 cm2 was fabricated by the method with high accuracy.Keywords: microstructures, anodic oxidation, silicon, agarose stamps
Procedia PDF Downloads 2981120 Molecular Alterations Shed Light on Alteration of Methionine Metabolism in Gastric Intestinal Metaplesia; Insight for Treatment Approach
Authors: Nigatu Tadesse, Ying Liu, Juan Li, Hong Ming Liu
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Gastric carcinogenesis is a lengthy process of histopathological transition from normal to atrophic gastritis (AG) to intestinal metaplasia (GIM), dysplasia toward gastric cancer (GC). The stage of GIM identified as precancerous lesions with resistance to H-pylori eradication and recurrence after endoscopic surgical resection therapies. GIM divided in to two morphologically distinct phenotypes such as complete GIM bearing intestinal type morphology whereas the incomplete type has colonic type morphology. The incomplete type GIM considered to be the greatest risk factor for the development of GC. Studies indicated the expression of the caudal type homeobox 2 (CDX2) gene is responsible for the development of complete GIM but its progressive downregulation from incomplete metaplasia toward advanced GC identified as the risk for IM progression and neoplastic transformation. The downregulation of CDX2 gene have promoted cell growth and proliferation in gastric and colon cancers and ascribed in chemo-treatment inefficacies. CDX2 downregulated through promoter region hypermethylation in which the methylation frequency positively correlated with the dietary history of the patients, suggesting the role of diet as methyl carbon donor sources such as methionine. However, the metabolism of exogenous methionine is yet unclear. Targeting exogenous methionine metabolism has become a promising approach to limits tumor cell growth, proliferation and progression and increase treatment outcome. This review article discusses molecular alterations that could shed light on the potential of exogenous methionine metabolisms, such as gut microbiota alteration as sources of methionine to host cells, metabolic pathway signaling via PI3K/AKt/mTORC1-c-MYC to rewire exogenous methionine and signature of increased gene methylation index, cell growth and proliferation in GIM, with insights to new treatment avenue via targeting methionine metabolism, and the need for future integrated studies on molecular alterations and metabolomics to uncover altered methionine metabolism and characterization of CDX2 methylation in gastric intestinal metaplasia for potential therapeutic exploitation.Keywords: altered methionine metabolism, Intestinal metaplesia, CDX2 gene, gastric cancer
Procedia PDF Downloads 691119 Building Envelope Engineering and Typologies for Complex Architectures: Composition and Functional Methodologies
Authors: Massimiliano Nastri
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The study examines the façade systems according to the constitutive and typological characters, as well as the functional and applicative requirements such as the expressive, constructive, and interactive criteria towards the environmental, perceptive, and energy conditions. The envelope systems are understood as instruments of mediation, interchange, and dynamic interaction between environmental conditions. The façades are observed for the sustainable concept of eco-efficient envelopes, selective and multi-purpose filters, adaptable and adjustable according to the environmental performance.Keywords: typologies of façades, environmental and energy sustainability, interaction and perceptive mediation, technical skins
Procedia PDF Downloads 1421118 Room Temperature Ionic Liquids Filled Mixed Matrix Membranes for CO2 Separation
Authors: Asim Laeeq Khan, Mazhar Amjad Gilani, Tayub Raza
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The use of fossil fuels for energy generation leads to the emission of greenhouse gases particularly CO2 into the atmosphere. To date, several techniques have been proposed for the efficient removal of CO2 from flue gas mixtures. Membrane technology is a promising choice due to its several inherent advantages such as low capital cost, high energy efficiency, and low ecological footprint. One of the goals in the development of membranes is to achieve high permeability and selectivity. Mixed matrix membranes comprising of inorganic fillers embedded in polymer matrix are a class of membranes that have showed improved separation properties. One of the biggest challenges in the commercialization if mixed matrix membranes are the removal of non-selective voids existing at the polymer-filler interface. In this work, mixed matrix membranes were prepared using polysulfone as polymer matrix and ordered mesoporous MCM-41 as filler materials. A new approach to removing the interfacial voids was developed by introducing room temperature ionic (RTIL) at the polymer-filler interface. The results showed that the imidazolium based RTIL not only provided wettability characteristics but also helped in further improving the separation properties. The removal of interfacial voids and good contact between polymer and filler was verified by SEM measurement. The synthesized membranes were tested in a custom built gas permeation set-up for the measurement of gas permeability and ideal gas selectivity. The results showed that the mixed matrix membranes showed significantly higher CO2 permeability in comparison to the pristine membrane. In order to have further insight into the role of fillers, diffusion and solubility measurements were carried out. The results showed that the presence of highly porous fillers resulted in increasing the diffusion coefficient while the solubility showed a slight drop. The RTIL filled membranes showed higher CO2/CH4 and CO2/N2 selectivity than unfilled membranes while the permeability dropped slightly. The increase in selectivity was due to the highly selective RTIL used in this work. The study revealed that RTIL filled mixed matrix membranes are an interesting candidate for gas separation membranes.Keywords: ionic liquids, CO2 separation, membranes, mixed matrix membranes
Procedia PDF Downloads 4721117 Fluorescence Gold Nanoparticles: Sensing Properties and Cytotoxicity Studies in MCF-7 Human Breast Cancer Cells
Authors: Cristina Núñez, Rufina Bastida, Elena Labisbal, Alejandro Macías, María T. Pereira, José M. Vila
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A highly selective quinoline-based fluorescent sensor L was designed in order to functionalize gold nanoparticles (GNPs@L). The cytotoxicity of compound L and GNPs@L on the MCF-7 breast cancer cells was explored and it was observed that L and GNPs@L compounds induced apoptosis in MCF-7 cancer cells. The cellular uptake of the hybrid system GNPs@L was studied using confocal laser scanning microscopy (CLSM).Keywords: cytotoxicity, fluorescent probes, nanoparticles, quinoline
Procedia PDF Downloads 3751116 Efficiently Dispersed MnOx on Mesoporous 3D Cubic Support for Cyclohexene Epoxidation
Authors: G. Imran, A. Pandurangan
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Epoxides constitute important intermediates for the production of fine and bulk chemicals as well as valuable building blocks for the synthesis of a variety of bioactive molecules. Manganese oxides are used as selective catalyst for various redox type reactions and also effectively used in the field of catalytic disposal of pollutants. Non-toxic, cost efficient factor and more over existence of wide range of oxidation state (+2 to +7) makes catalyst more interesting for both academic research and industrial applications. However, the serious drawback lying is the lower surface area. Exceedingly dispersed manganese oxide grafted over mesoporous solid material KIT-6 through ALD (Atomic Layer Deposition) technique effectively catalyze cyclohexene with H2O2 (30% in water) to corresponding epoxides. Highly selective epoxide >99% with 55.7% conversion of cyclohexene was achieved using huge dispersed active sites of MnOx species containing catalysts. Various weight percent such as (1, 3, 5, 7 & 10 wt %) of manganese (II) acetylacetonate complex was employed as Mn source to post-graft via active silanol groups of KIT-6 and are designated as (Mn-G-KIT-6). XRD, N2 sorption, HR-TEM, DRS-UV-VIS, EPR and H2-TPR were employed for structural and textural properties. Immense Mn species of about 95% proportion on silica matrix obtained was evident from ICP-OES.The resulting materials exhibited Type IV adsorption isotherms indiacting mesopore in nanorange. Si-KIT-6 and Mn-G-KIT-6 materials exhibited surface area of 519-289 m2/g and with decrease in pore volume of 0.96-0.49 cm3/g with pore diameter ranging 7.9- 7.2 with increase in wt%. DRS-UV-VIS spectroscopy and EPR studies reveal that manganese coexists as Mn2+/3+ species as extra-framework sites and frame-work sites that result in dispersion on surface of silica matrix of KIT-6 and incorporated manganese sites with silanol groups along with small sized MnO cluster, evident from HR-TEM which increase with Mn content. Conventional production of epoxides by the intramolecular etherification of chlorohydrins formed by the reaction of alkenes with hypochlorous acid is the major drawbacks obtained recently. The most efficient synthesis of oxiranes (epoxides) is obtained by mesoporous catalysts (Mn-G-KIT-6) are presented here and discussed.Keywords: ALD, epoxidation, mesoporous, MnOx
Procedia PDF Downloads 1781115 Khaya Cellulose Supported Copper Nanoparticles for Chemo Selective Aza-Michael Reactions
Authors: M. Shaheen Sarkar, M. Lutfor Rahman, Mashitah Mohd Yusoff
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We prepared a highly active Khaya cellulose supported poly(hydroxamic acid) copper nanoparticles by the surface modification of Khaya cellulose through graft co-polymerization and subsequently amidoximation. The Cu-nanoparticle (0.05 mol% to 50 mol ppm) was selectively promoted Aza-Michael reaction of aliphatic amines to give the corresponding alkylated products at room temperature in methanol. The supported nanoparticle was easy to recover and reused seven times without significance loss of its activity.Keywords: Aza-Michael, copper, cellulose, nanoparticles, poly(hydroxamic acid)
Procedia PDF Downloads 3361114 In vitro and in vivo Infectivity of Coxiella burnetii Strains from French Livestock
Authors: Joulié Aurélien, Jourdain Elsa, Bailly Xavier, Gasqui Patrick, Yang Elise, Leblond Agnès, Rousset Elodie, Sidi-Boumedine Karim
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Q fever is a worldwide zoonosis caused by the gram-negative obligate intracellular bacterium Coxiella burnetii. Following the recent outbreaks in the Netherlands, a hyper virulent clone was found to be the cause of severe human cases of Q fever. In livestock, Q fever clinical manifestations are mainly abortions. Although the abortion rates differ between ruminant species, C. burnetii’s virulence remains understudied, especially in enzootic areas. In this study, the infectious potential of three C. burnetii isolates collected from French farms of small ruminants were compared to the reference strain Nine Mile (in phase II and in an intermediate phase) using an in vivo (CD1 mice) model. Mice were challenged with 105 live bacteria discriminated by propidium monoazide-qPCR targeting the icd-gene. After footpad inoculation, spleen and popliteal lymph node were harvested at 10 days post-inoculation (p.i). The strain invasiveness in spleen and popliteal nodes was assessed by qPCR assays targeting the icd-gene. Preliminary results showed that the avirulent strains (in phase 2) failed to pass the popliteal barrier and then to colonize the spleen. This model allowed a significant differentiation between strain’s invasiveness on biological host and therefore identifying distinct virulence profiles. In view of these results, we plan to go further by testing fifteen additional C. burnetii isolates from French farms of sheep, goat and cattle by using the above-mentioned in vivo model. All 15 strains display distant MLVA (multiple-locus variable-number of tandem repeat analysis) genotypic profiles. Five of the fifteen isolates will bee also tested in vitro on ovine and bovine macrophage cells. Cells and supernatants will be harvested at day1, day2, day3 and day6 p.i to assess in vitro multiplication kinetics of strains. In conclusion, our findings might help the implementation of surveillance of virulent strains and ultimately allow adapting prophylaxis measures in livestock farms.Keywords: Q fever, invasiveness, ruminant, virulence
Procedia PDF Downloads 3561113 Isolation and Identification of Salmonella spp and Salmonella enteritidis, from Distributed Chicken Samples in the Tehran Province using Culture and PCR Techniques
Authors: Seyedeh Banafsheh Bagheri Marzouni, Sona Rostampour Yasouri
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Salmonella is one of the most important common pathogens between humans and animals worldwide. Globally, the prevalence of the disease in humans is due to the consumption of food contaminated with animal-derived Salmonella. These foods include eggs, red meat, chicken, and milk. Contamination of chicken and its products with Salmonella may occur at any stage of the chicken processing chain. Salmonella infection is usually not fatal. However, its occurrence is considered dangerous in some individuals, such as infants, children, the elderly, pregnant women, or individuals with weakened immune systems. If Salmonella infection enters the bloodstream, the possibility of contamination of tissues throughout the body will arise. Therefore, determining the potential risk of Salmonella at various stages is essential from the perspective of consumers and public health. The aim of this study is to isolate and identify Salmonella from chicken samples distributed in the Tehran market using the Gold standard culture method and PCR techniques based on specific genes, invA and ent. During the years 2022-2023, sampling was performed using swabs from the liver and intestinal contents of distributed chickens in the Tehran province, with a total of 120 samples taken under aseptic conditions. The samples were initially enriched in buffered peptone water (BPW) for pre-enrichment overnight. Then, the samples were incubated in selective enrichment media, including TT broth and RVS medium, at temperatures of 37°C and 42°C, respectively, for 18 to 24 hours. Organisms that grew in the liquid medium and produced turbidity were transferred to selective media (XLD and BGA) and incubated overnight at 37°C for isolation. Suspicious Salmonella colonies were selected for DNA extraction, and PCR technique was performed using specific primers that targeted the invA and ent genes in Salmonella. The results indicated that 94 samples were Salmonella using the PCR technique. Of these, 71 samples were positive based on the invA gene, and 23 samples were positive based on the ent gene. Although the culture technique is the Gold standard, PCR is a faster and more accurate method. Rapid detection through PCR can enable the identification of Salmonella contamination in food items and the implementation of necessary measures for disease control and prevention.Keywords: culture, PCR, salmonella spp, salmonella enteritidis
Procedia PDF Downloads 651112 Development of Peptide Inhibitors against Dengue Virus Infection by in Silico Design
Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus
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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor
Procedia PDF Downloads 3511111 Screening for Hit Identification against Mycobacterium abscessus
Authors: Jichan Jang
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Mycobacterium abscessus is a rapidly growing life-threatening mycobacterium with multiple drug-resistance mechanisms. In this study, we screened the library to identify active molecules targeting Mycobacterium abscessus using resazurin live/dead assays. In this screening assay, the Z-factor was 0.7, as an indication of the statistical confidence of the assay. A cut-off of 80% growth inhibition in the screening resulted in the identification of four different compounds at a single concentration (20 μM). Dose-response curves identified three different hit candidates, which generated good inhibitory curves. All hit candidates were expected to have different molecular targets. Thus, we found that compound X, identified, may be a promising candidate in the M. abscessus drug discovery pipeline.Keywords: Mycobacterium abscessus, antibiotics, drug discovery, emerging Pathogen
Procedia PDF Downloads 2011110 Improving the Bioprocess Phenotype of Chinese Hamster Ovary Cells Using CRISPR/Cas9 and Sponge Decoy Mediated MiRNA Knockdowns
Authors: Kevin Kellner, Nga Lao, Orla Coleman, Paula Meleady, Niall Barron
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Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. To improve yields and find beneficial bioprocess phenotypes genetic engineering plays an essential role in recent research. The miR-23 cluster, specifically miR-24 and miR-27, was first identified as differentially expressed during hypothermic conditions suggesting a role in proliferation and productivity in CHO cells. In this study, we used sponge decoy technology to stably deplete the miRNA expression of the cluster. Furthermore, we implemented the CRISPR/Cas9 system to knockdown miRNA expression. Sponge constructs were designed for an imperfect binding of the miRNA target, protecting from RISC mediated cleavage. GuideRNAs for the CRISPR/Cas9 system were designed to target the seed region of the miRNA. The expression of mature miRNA and precursor were confirmed using RT-qPCR. For both approaches stable expressing mixed populations were generated and characterised in batch cultures. It was shown, that CRISPR/Cas9 can be implemented in CHO cells with achieving high knockdown efficacy of every single member of the cluster. Targeting of one miRNA member showed that its genomic paralog is successfully targeted as well. The stable depletion of miR-24 using CRISPR/Cas9 showed increased growth and specific productivity in a CHO-K1 mAb expressing cell line. This phenotype was further characterized using quantitative label-free LC-MS/MS showing 186 proteins differently expressed with 19 involved in proliferation and 26 involved in protein folding/translation. Targeting miR-27 in the same cell line showed increased viability in late stages of the culture compared to the control. To evaluate the phenotype in an industry relevant cell line; the miR-23 cluster, miR-24 and miR-27 were stably depleted in a Fc fusion CHO-S cell line which showed increased batch titers up to 1.5-fold. In this work, we highlighted that the stable depletion of the miR-23 cluster and its members can improve the bioprocess phenotype concerning growth and productivity in two different cell lines. Furthermore, we showed that using CRISPR/Cas9 is comparable to the traditional sponge decoy technology.Keywords: Chinese Hamster ovary cells, CRISPR/Cas9, microRNAs, sponge decoy technology
Procedia PDF Downloads 1911109 Vascular Targeted Photodynamic Therapy Monitored by Real-Time Laser Speckle Imaging
Authors: Ruth Goldschmidt, Vyacheslav Kalchenko, Lilah Agemy, Rachel Elmoalem, Avigdor Scherz
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Vascular Targeted Photodynamic therapy (VTP) is a new modality for selective cancer treatment that leads to the complete tumor ablation. A photosensitizer, a bacteriochlorophyll derivative in our case, is first administered to the patient and followed by the illumination of the tumor area, by a near-IR laser for its photoactivation. The photoactivated drug releases reactive oxygen species (ROS) in the circulation, which reacts with blood cells and the endothelium leading to the occlusion of the blood vasculature. If the blood vessels are only partially closed, the tumor may recover, and cancer cells could survive. On the other hand, excessive treatment may lead to toxicity of healthy tissues nearby. Simultaneous VTP monitoring and image processing independent of the photoexcitation laser has not yet been reported, to our knowledge. Here we present a method for blood flow monitoring, using a real-time laser speckle imaging (RTLSI) in the tumor during VTP. We have synthesized over the years a library of bacteriochlorophyll derivatives, among them WST11 and STL-6014. Both are water soluble derivatives that are retained in the blood vasculature through their partial binding to HSA. WST11 has been approved in Mexico for VTP treatment of prostate cancer at a certain drug dose, and time/intensity of illumination. Application to other bacteriochlorophyll derivatives or other cancers may require different treatment parameters (such as light/drug administration). VTP parameters for STL-6014 are still under study. This new derivative mainly differs from WST11 by its lack of the central Palladium, and its conjugation to an Arg-Gly-Asp (RGD) sequence. RGD is a tumor-specific ligand that is used for targeting the necrotic tumor domains through its affinity to αVβ3 integrin receptors. This enables the study of cell-targeted VTP. We developed a special RTLSI module, based on Labview software environment for data processing. The new module enables to acquire raw laser speckle images and calculate the values of the laser temporal statistics of time-integrated speckles in real time, without additional off-line processing. Using RTLSI, we could monitor the tumor’s blood flow following VTP in a CT26 colon carcinoma ear model. VTP with WST11 induced an immediate slow down of the blood flow within the tumor and a complete final flow arrest, after some sporadic reperfusions. If the irradiation continued further, the blood flow stopped also in the blood vessels of the surrounding healthy tissue. This emphasizes the significance of light dose control. Using our RTLSI system, we could prevent any additional healthy tissue damage by controlling the illumination time and restrict blood flow arrest within the tumor only. In addition, we found that VTP with STL-6014 was the most effective when the photoactivation was conducted 4h post-injection, in terms of tumor ablation success in-vivo and blood vessel flow arrest. In conclusion, RTSLI application should allow to optimize VTP efficacy vs. toxicity in both the preclinical and clinical arenas.Keywords: blood vessel occlusion, cancer treatment, photodynamic therapy, real time imaging
Procedia PDF Downloads 2181108 Particle Deflection in a PDMS Microchannel Caused by a Plane Travelling Surface Acoustic Wave
Authors: Florian Keipert, Hagen Schmitd
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The size selective separation of different species in a microfluidic system is an actual task in biological or medical research. Former works dealt with the utilisation of the acoustic radiation force (ARF) caused by a plane travelling Surface Acoustic Wave (tSAW). In literature the ARF is described by a dimensionless parameter κ, depending on the wavelength and the particle diameter. To our knowledge research was done for values 0.2 < κ < 5.8 showing that the ARF is dominating the acoustic streaming force (ASF) for κ > 1.2. As a consequence the particle separation is limited by κ. In addition the dependence on the electrical power level was examined but only for κ > 1 pointing out an increased particle deflection for higher electrical power levels. Nevertheless a detailed study on the ASF and ARF especially for κ < 1 is still missing. In our setup we used a tSAW with a wavelength λ = 90 µm and 3 µm PS particles corresponding to κ = 0.3. Herewith the influence of the applied electrical power level on the particle deflection in a polydimethylsiloxan micro channel was investigated. Our results show an increased particle deflection for an increased electrical power level, which coincides with the reported results for κ > 1. Therefore particle separation is in contrast to literature also possible for lower κ values. Thereby the experimental setup can be generally simplified by a coordinated electrical power level for the specific particle size. Furthermore this raises the question of whether this particle deflection is caused only by the ARF as adopted so far or by the ASF or the sum of both forces. To investigate this fact a 0% - 24% saline solution was used and thus the mismatch between the compressibility of the PS particle and the working fluid could be changed. Therefore it is possible to change the relative strength between ARF and ASF and consequently the particle deflection. We observed a decreasing in the particle deflection for an increased NaCl content up to a 12% saline solution and subsequently an increasing of the particle deflection. Our observation could be explained by the acoustic contrast factor Φ, which depends on the compressibility mismatch. The compressibility of water is increased by the NaCl and the range of a 0% - 24% saline solution covers the PS particle compressibility. Hence the particle deflection reaches a minimum value for the accordance between compressibility of PS particle and saline solution. This minimum value can be estimated as the particle deflection only caused by the ASF. Knowing the particle deflection due to the ASF the particle deflection caused by the ARF can be calculated and thus finally the relation between both forces. Concluding, the particle deflection and therefore the size selective particle separation generated by a tSAW can be achieved for values κ < 1, simplifying actual setups by adjusting the electrical power level. Beyond we studied for the first time the relative strength between ARF and ASF to characterise the particle deflection in a microchannel.Keywords: ARF, ASF, particle separation, saline solution, tSAW
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