Search results for: anti-granulocyte antibodies

Authors: Sergio Alejandro Cuevas, Catherine Etchebest, Fernando Luis Barroso Da Silva

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The flavivirus genus has several organisms responsible for generating various diseases in humans. Special in Brazil, Zika (ZIKV), Dengue (DENV) and Yellow Fever (YFV) viruses have raised great health concerns due to the high number of cases affecting the area during the last years. Diagnostic is still a difficult issue since the clinical symptoms are highly similar. The understanding of their common structural/dynamical and biomolecular interactions features and differences might suggest alternative strategies towards differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV, DENV and YFV non-structural proteins 1 (NS1) protein. By means of computational studies, we calculated the main physical chemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil that could suggest possible molecular mechanisms for the increase of the virulence of ZIKV. It is interesting to note that despite the small changes in the protein sequence due to the high sequence identity among the studied strains, the electrostatic properties are strongly impacted by the pH which also impact on their biomolecular interactions with partners and, consequently, the molecular viral biology. African and Asian strains are distinguishable. Exploring the interfaces used by NS1 to self-associate in different oligomeric states, and to interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can explain the different immunological response. By the comparison with the known antibody structure available for the West Nile virus, we demonstrated that the antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high specificity antibodies.

Keywords: zika, biomolecular interactions, electrostatic interactions, molecular mechanisms

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Authors: Christen Kutz

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Objective: The aim of this study is to review a case series of 4 high-level Olympic and Division 1 wrestlers who experienced debilitating posterior circulation ischemic strokes during or after a competitive wrestling event and to identify risk factors, etiology and outcomes of stroke in young, healthy elite wrestlers. Background: Stroke occurs in one in 10,000 people under age 64. In young adults, the most common causes of stroke are cardiac embolism, hypercoagulable state, and vasculopathy. One-third of these strokes occur in young, fit individuals. There is little published literature about ischemic strokes that occur in wrestlers. Based on the nature of wrestling, the risk of injury or dissection to neurovascular structures may be a possible theory, but very few case reports exist. Methodology: 4 wrestlers under the age of 44 with a known history of ischemic stroke participated in individual interviews either in person or virtually. Each of the wrestlers provided their demographic information, wrestling background, clinical presentation at the time of stroke, imaging results, identification of potential risk factors, acute treatment and recovery. Results: 3 white male Division 1 wrestlers (2 Lehigh University, 1 Lock Haven University) and 1 black male 2008 Olympian experienced posterior circulation strokes. Case #1 felt a “pop” while wrestling (lateral medullary infarct, possible vertebral artery dissection); Case #2 awoke with severe vertigo, sweating, and vomiting after wrestling the previous day (left cerebellar infarct, (+) protein S deficiency); Case #3 severe vertigo, ataxia, and sensation of impending doom after wrestling earlier that week (left cerebellar infarct, hypoplastic left vertebral artery (+) anti-cardiolipin antibodies). Case #4 severe dizziness, confusion (left cerebellar stroke, vertebral artery dissection, small PFO). Conclusion: 3 wrestlers were started on anti-platelet therapy, risk factors were modified, and returned to their sport. 1 wrestler was placed on anti-coagulation and retired from competition.

Keywords: stroke, wrestling, Olympic, posterior circulation

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Authors: Soumaya Mrabet, Taieb Ach, Imen Akkari, Amira Atig, Neirouz Ghannouchi, Koussay Ach, Elhem Ben Jazia

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Introduction: Celiac disease (CD) and type 1 diabetes mellitus (T1D) are complex disorders with shared genetic components. The association between CD and T1D has been reported in many pediatric series. The aim of our study is to describe the epidemiological, clinical and evolutive characteristics of adult patients presenting this association. Material and Methods: This is a retrospective study including patients diagnosed with CD and T1D, explored in Internal Medicine, Gastroenterology and Endocrinology and Diabetology Departments of the Farhat Hached University Hospital, between January 2005 and June 2016. Results: Among 57 patients with CD, 15 patients had also T1D (26.3%). There are 11 women and 4 men with a median age of 27 years (16-48). All patients developed T1D prior to the diagnosis of CD with an average duration of 47 months between the two diagnosis (6 months-5 years). CD was revealed by recurrent abdominal pain in 11 cases, diarrhea in 10 cases, bloating in 8 cases, constipation in 6 cases and vomiting in 2 cases. Three patients presented cycle disorders with secondary amenorrhea in 2 patients. Anti-Endomysium, anti-transglutaminase and Anti-gliadin antibodies were positive respectively in 57, 54 and 11 cases. The biological tests revealed anemia in 10 cases, secondary to iron deficiency in 6 cases and folate and vitamin B12 deficiency in 4 cases, hypoalbuminaemia in 4 cases, hypocalcemia in 3 cases and hypocholesterolemia in 1 patient. Upper gastrointestinal endoscopy showed an effacement of the folds of the duodenal mucosa in 6 cases and a congestive duodenal mucosa in 3 cases. The macroscopic appearance was normal in the others cases. Microscopic examination showed an aspect of villous atrophy in 57 cases, which was partial in 10 cases and total in 47 cases. After an average follow-up of 3 years 2 months, the evolution was favorable in all patients under gluten-free diet with the necessity of less important doses of insulin in 10 patients. Conclusion: In our study, the prevalence of T1D in adult patients with CD was 26.3%. This association can be attributed to overlapping genetic HLA risk loci. In recent studies, the role of gluten as an important player in the pathogenesis of CD and T1D has been also suggested.

Keywords: celiac disease, gluten, prevalence, type 1 diabetes

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Authors: Rania Hanafi Mahmoud Said, Rasha Mohamed Taha

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Background: A recent study has explored the potential of an approach to treating infected root canals using a combination of Mineral Trioxide Aggregate (MTA) and Tetracycline-loaded Nanochitosan. Material and methods: Forty dogs were included in the study, with infected periapical areas induced by leaving access openings in their teeth for four months. Bacteriological samples from the infected root canals were collected and managed anaerobically to identify and count the different microorganisms present. The most common microorganisms detected were Prevotella oris, Fusobacterium nucleatum, Streptococcus viridans, Enterococcus faecalis, Clostridium subterminale, Porphyromonas gingivalis, and Peptostreptococcus anaerobius. The dogs were divided into four groups based on the sealant used to treat the infected periapical areas: Group I: Negative control (no treatment) Group II: Positive control (MTA only) Group III: MTA + tetracycline Group IV: MTA + tetracycline loaded on nanochitosan Results: Periapical areas in Group IV showed significantly more bone healing than those in Groups I, II, and III. The newly formed bone was evaluated radiographically, histologically, and immunohistochemically using Osteopontin (OSP) antibodies. Data collected was statistically analysed using SPSS software at a 0.05 significance level. Conclusion: The study concluded that the combined use of Tetracycline-loaded Nanochitosan and MTA presents a promising approach for the treatment of infected root canals. The potent antimicrobial activity of Tetracycline-loaded Nanochitosan, along with the biocompatibility and desirable properties of MTA, may synergistically contribute to improved clinical outcomes in endodontic therapy. This study has important implications for the clinical management of infected root canals. The combination of Tetracycline-loaded Nanochitosan and MTA could provide a more effective and efficient means of treating these challenging cases. Further research is needed to confirm these findings in humans and to optimize the treatment protocol.

Keywords: mineral trioxide aggregate, tetracycline-loaded nanochitosan, periapical infection, osteopontine

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Authors: Zakieh Rostamzadeh, Zahra Shirmohammadi

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Human cytomegalovirus is a species of the cytomegalovirus family of viruses, which in turn is a member of the viral family known as herpesviridae or herpesviruses. Although they may be found throughout the body, HCMV infections are frequently associated with the salivary glands. HCMV infection is typically unnoticed in healthy people, but can be life-threatening for the immunocompromised such as HIV-infected persons, organ transplant recipients, or newborn infants. After infection, HCMV has an ability to remain latent within the body over long periods. Cytomegalovirus (CMV) causes infection in immunocompromised, hemophilia patients and those who received blood transfusion frequently. This study aimed at determining the prevalence of cytomegalovirus (CMV) antibodies in hemophilia patients. Materials and Methods: A retrospective observational study was carried out in Urmia, North West of Iran. The study population comprised a sample of 50 hemophilic patients born after 1985 and have received blood factors in West Azerbaijan. The exclusion criteria include: drug abusing, high risk sexual contacts, vertical transmission of mother to fetus and suspicious needling. All samples were evaluated with the method of ELISA, with a certain kind of kit and by a certain laboratory. Results: Fifty hemophiliacs from 250 patients registered with Urmia Hemophilia Society were enrolled in the study including 43 (86%) male, and 7 (14%) female. The mean age of patients was 10.3 years, range 3 to 25 years. None of patients had risk factors mentioned above. Among our studied population, 34(68%) had hemophilia A, 1 (2%) hemophilia B, 8 (16%) VWF, 3(6%) factor VII deficiency, 1 (2%) factor V deficiency, 1 (2%) factor X deficiency, 1 (2%). Sera of 50 Hemodialysis patients were investigated for CMV-specific immunoglobulin G (IgG) and IgM. % 91.89 patients were anti-CMV IgG positive and %40.54 was seropositive for anti-CMV IgM. 37.8% patient had serological evidence of reactivation and 2.7% of patients had the primary infection. Discussion: There was no relationship between the antibody titer and: drug abusing, high risk sexual contacts, vertical transmission of mother to fetus and suspicious needling.

Keywords: bioinformatics, biomedicine, cytomegalovirus, immunocompromise

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Authors: P. Annapurna, Cecil Ross, Sudhir Krishna, Sweta Srivastava

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Irradiation plays a pivotal role in cervical cancer treatment, however some tumors exhibit resistance to therapy while some exhibit relapse, due to better repair and enhanced resistance mechanisms operational in their cells. The present study aims to understand the signaling mechanism operational in resistance phenotype and in the present study we report the role of Rho GTPase associated protein kinase (ROCK) signaling in cervical carcinoma radio-resistance. ROCK signaling has been implicated in several tumor progressions and is important for DNA repair. Irradiation of spheroid cultures of SiHa cervical carcinoma derived cell line at 6Gy resulted in generation of resistant cells in vitro which had better clonogenic abilities and formed larger and more colonies, in soft agar colony formation assay, as compared to the non-irradiated cells. These cells also exhibited an enhanced motility phenotype. Cell cycle profiling showed the cells to be blocked in G2M phase with enhanced pCDC2 levels indicating onset of possible DNA repair mechanism. Notably, 3 days post-irradiation, irradiated cells showed increased ROCK2 translocation to the nucleus with enhanced protein expression as compared to the non-irradiated cells. Radio-sensitization of the resistant cells was enhanced using Y27632, an inhibitor to ROCK signaling. The treatment of resistant cells with Y27632 resulted in increased cell death upon further irradiation. This observation has been confirmed using inhibitory antibodies to ROCK1/2. Result show that both ROCK1/2 have a functional contribution in radiation resistance of cervical cancer cells derived from cell lines. Interestingly enrichment of stem like cells (Hoechst negative cells) was also observed upon irradiation and these cells were markedly sensitive to Y27632 treatment. Our results thus suggest the role of ROCK signaling in radio-resistance in cervical carcinoma. Further studies with human biopsies, mice models and mechanistic of ROCK signaling in the context of radio-resistance will clarify the role of this molecule further and allow for therapeutics development.

Keywords: cervical carcinoma, radio-resistance, ROCK signaling, cancer treatment

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Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

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Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

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Authors: Emanuela Zilli, Antonio Madia, Milvia Marchiori, Paola Anello, Chiara Cabbia, Emanuela Velo, Delia Campagnolo, Michele Scomazzon, Emanuela Salvatico, S. Tikvina, Antonio Miotti

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Hepatitis C is an inflammation of the liver caused by the hepatitis C virus (HCV). Antiviral medicines can cure more than 95% of cases of hepatitis C infection, but access to diagnosis and treatment remains low. The ULSS 6 Euganea – Health Trust has implemented a campaign to eradicate hepatitis C in the province of Padua (North-East of Italy), which can be subdivided into three areas: North (300.000 inhabitants), Centre (400.000) and South (300.000). In September 2021, the project was launched in the Northern area; a set of brochures was distributed in outpatient services, general practitioners’ clinics and offices, community pharmacy services, social health districts, and through social networks. The Hepatology Service contacted 460 patients selected by the Clinical Laboratory (positivity for HCV antibodies): 83 patients (18.0%) had been already cured of HCV infection, missing or deceased; 377 patients (82.0%) met the criteria to be eligible for HCV eradication therapy and were therefore included in a Day Service specific agenda and followed by a multidisciplinary team of healthcare professionals, with a dedicated telephone line. Haemato-chemical tests, general medical check-ups and ultrasound tests with fibroscan were performed. Patients were tested for Sars-CoV-2 positivity; those not yet vaccinated against Covid-19 were encouraged to complete the vaccination scheme. All 377 patients (100%) received HCV eradication therapy at the community pharmacy service; a detailed explanation of how to take their medication was provided. At the end of the first phase, Covid-19 vaccination rate was 100% (377/377), including patients already vaccinated and new-vaccinated. Check-up appointments were arranged after 2 or 3 months, according to the treatment plan. The awareness campaign and the organization of HCV eradication therapy service by ULSS 6 Euganea are proving to be effective; the project is now going to be applied to Central and Southern areas of the province (1.132 patients).

Keywords: public health, HCV-eradication, Covid-19 emergency, health communication strategies

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Authors: Charles Bijjah Nkhata, Memory Nekati Mvula, Milton Masautso Kalongonda, Martha Masamba, Isaac Thom Shawa

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Viral Hepatitis is a serious public health concern globally with deaths estimated at 1.4 million annually due to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis B and C are the most common viruses that cause liver damage. However, the majority of infected individuals are unaware of their serostatus. Viral Hepatitis has contributed to maternal and neonatal morbidity and mortality. There is no updated data on the Epidemiology of hepatitis B and C among pregnant mothers in Malawi. To assess the epidemiology of Hepatitis B and C viruses among pregnant women at Queen Elizabeth Central Hospital. Specific Objectives • To determine sero-prevalence of HBsAg and Anti-HCV in pregnant women at QECH. • To investigate risk factors associated with HBV and HCV infection in pregnant women. • To determine the distribution of HBsAg and Anti-HCV infection among pregnant women of different age group. A descriptive cross-sectional study was conducted among pregnant women at QECH in last quarter of 2021. Of the 114 pregnant women, 96 participants were consented and enrolled using a convenient sampling technique. 12 participants were dropped due to various reasons; therefore 84 completed the study. A semi-structured questionnaire was used to collect socio-demographic and behavior characteristics to assess the risk of exposure. Serum was processed from venous blood samples and tested for HBsAg and Anti-HCV markers utilizing Rapid screening assays for screening and Enzyme Linked Immunosorbent Assay for confirmatory. A total of 84 pregnant consenting pregnant women participated in the study, with 1.2% (n=1/84) testing positive for HBsAg and nobody had detectable anti-HCV antibodies. There was no significant link between HBV and HCV in any of the socio-demographic data or putative risk variables. The findings indicate a viral hepatitis prevalence lower than the set range by the WHO. This suggests that HBV and HCV are rare in pregnant women at QECH. Nevertheless, accessible screening for all pregnant women should be provided. The prevention of MTCT is key for reduction and prevention of the global burden of chronic viral Hepatitis.

Keywords: viral hepatitis, hepatitis B, hepatitis C, pregnancy, malawi, liver disease, mother to child transmission

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Authors: Pavel Damborsky, Martina Zamorova, Jaroslav Katrlik

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Prostate cancer is annually the most common newly diagnosed cancer among men. An extensive number of evidence suggests that traditional serum Prostate-specific antigen (PSA) assay still suffers from a lack of sufficient specificity and sensitivity resulting in vast over-diagnosis and overtreatment. Thus, the early-stage detection of prostate cancer (PCa) plays undisputedly a critical role for successful treatment and improved quality of life. Over the last decade, particular altered glycans have been described that are associated with a range of chronic diseases, including cancer and inflammation. These glycans differences enable a distinction to be made between physiological and pathological state and suggest a valuable biosensing tool for diagnosis and follow-up purposes. Aberrant glycosylation is one of the major characteristics of disease progression. Consequently, the aim of this study was to develop a more reliable tool for early-stage PCa diagnosis employing lectins as glyco-recognition elements. Biosensor and biochip technology putting to use lectin-based glyco-profiling is one of the most promising strategies aimed at providing fast and efficient analysis of glycoproteins. The proof-of-concept experiments based on sandwich assay employing anti-PSA antibody and an aptamer as a capture molecules followed by lectin glycoprofiling were performed. We present a lectin-based biosensing assay for glycoprofiling of serum biomarker PSA using different biosensor and biochip platforms such as label-free surface plasmon resonance (SPR) and microarray with fluorescent label. The results suggest significant differences in interaction of particular lectins with PSA. The antibody-based assay is frequently associated with the sensitivity, reproducibility, and cross-reactivity issues. Aptamers provide remarkable advantages over antibodies due to the nucleic acid origin, stability and no glycosylation. All these data are further step for construction of highly selective, sensitive and reliable sensors for early-stage diagnosis. The experimental set-up also holds promise for the development of comparable assays with other glycosylated disease biomarkers.

Keywords: biomarker, glycosylation, lectin, prostate cancer

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Authors: M. Bolacali

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The aim of the study is to determine the effect of immunocastration vaccine administration at different doses on fattening performance of feedlot Holstein bulls. Bopriva® is a vaccine that stimulates the animals' own immune system to produce specific antibodies against gonadotropin releasing factor (GnRF). Ninety four Holstein male calves (309.5 ± 2.58 kg body live weight and 267 d-old) assigned to the 4 treatments. Control group; 1 mL of 0.9% saline solution was subcutaneously injected to intact bulls on 1st and 60th days of the feedlot as placebo. On the same days of the feedlot, Bopriva® at two doses of 1 mL and 1 mL for Trial-1 group, 1.5 mL, and 1.5 mL for Trial-2 group, 1.5 mL, and 1 mL for Trial-3 group were subcutaneously injected to bulls. The study was conducted in a private establishment in the Sirvan district of Siirt province and lasted 180 days. The animals were weighed at the beginning of fattening and at 30-day intervals to determine their live weights at various periods. The statistical analysis for normal distribution data of the treatment groups was carried out with the general linear model procedure of SPSS software. The fattening initial live weight in Control, Trial-1, Trial-2 and Trial-3 groups was respectively 309.21, 306.62, 312.11, and 315.39 kg. The fattening final live weight was respectively 560.88, 536.67, 548.56, and 548.25 kg. The daily live weight gain during the trial was respectively 1.40, 1.28, 1.31, and 1.29 kg/day. The cold carcass yield was respectively 51.59%, 50.32%, 50.85%, and 50.77%. Immunocastration vaccine administration at different doses did not affect the live weights and cold carcass yields of Holstein male calves reared under intensive conditions (P > 0.05). However, it was determined to reduce fattening performance between 61-120 days (P < 0.05) and 1-180 days (P < 0.01). In addition, it was determined that the best performance among the vaccine-treated groups occurred in the group administered a 1.5 mL of vaccine on the 1st and 60th study days. In animals, castration is used to control fertility, aggressive and sexual behaviors. As a result, the fact that stress is induced by physical castration in animals and active immunization against GnRF maintains performance by maximizing welfare in bulls improves carcass and meat quality and controls unwanted sexual and aggressive behavior. Considering such features, it may be suggested that immunocastration vaccine with Bopriva® can be administered as a 1.5 mL dose on the 1st and 60th days of the fattening period in Holstein bulls.

Keywords: anti-GnRF, fattening, growth, immunocastration

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Authors: Engin Berber, Nurettin Canakoglu, Ibrahim Sozdutmaz, Merve Caliskan, Shaikh Terkis Islam Pavel, Hazel Yetiskin, Aykut Ozdarendeli

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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The CCHFV genome consists of three molecules of negative-sense single-stranded RNA, each encapsulated separately. The virion particle contains viral RNA polymerase (L segment), surface glycoproteins Gn and Gc (Msegment), and a nucleocapsid protein NP (S segment). CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Eastern Europe. Clinical CCHF was first recognized in Turkey in 2002. The numbers of CCHF cases have gradually increased in Turkey making the virus a public health concern. Between 2002 and 2014, more than 8000 the CCHF cases have been reported in Turkey and mortality rate is around 5%. So, Turkey is one of the countries where the epidemy has become spread to the wider geography and the biggest outbreaks of CCHF have occurred in the world. We have recently developed an inactivated cell-culture based vaccine against CCHF. We have showed that the Balb/c mice immunized with the CCHF vaccine induced the high level of neutralizing antibodies. In this study, we aimed to determine the immunodominant regions of nucleoprotein (NP) CCHFV Kelkit06 strain which stimulate T cells. For this purpose, pools of overlapping NP were used for an IFN- γ ELISPOT assay. Balb/c mice were divided into two groups for the experiment. Two groups (n = 10 each) were immunized via the intraperitoneal route with 5, or 10μg of the cell culture-based vaccine. The control group (n = 6) was mock immunized with PBS. Booster injections with the same formulation were given on days 21 and 42 after the first immunization. The higher reactivity against the CCHFV NP pools 31-40 and 80-90 was determined in the two dose groups. In order to analyze the vaccine-induced T cell responses in Balb/c mice immunized with varying doses of the vaccine, we have been also currently working on CD4+, CD8+ and CD3 + T cells by flow cytometry.

Keywords: Crimean-Congo hemorrhagic fever virus, immunodominant regions of NP, T cell response, vaccine

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Authors: Jamboor K. Vishwanatha

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Among the potent anti-cancer agents, curcumin has been found to be very efficacious against various cancer cells. Despite multiple medicinal benefits of curcumin, poor water solubility, poor physiochemical properties and low bioavailability continue to pose major challenges in developing a formulation for clinical efficacy. To improve its potential application in the clinical area, we formulated poly lactic-co-glycolic acid (PLGA) nanoparticles. The PLGA nanoparticles were formulated using solid-oil/water emulsion solvent evaporation method and then characterized for percent yield, encapsulation efficiency, surface morphology, particle size, drug distribution within nanoparticles and drug polymer interaction. Our studies showed the successful formation of smooth and spherical curcumin loaded PLGA nanoparticles with a high percent yield of about 92.01±0.13% and an encapsulation efficiency of 90.88±0.14%. The mean particle size of the nanoparticles was found to be 145nm. The in vitro drug release profile showed 55-60% drug release from the nanoparticles over a period of 24 hours with continued sustained release over a period of 8 days. Exposure to curcumin loaded nanoparticles resulted in reduced cell viability of cancer cells compared to normal cells. We used a novel non-covalent insertion of a homo-bifunctional spacer for targeted delivery of curcumin to various cancer cells. Functionalized nanoparticles for antibody/targeting agent conjugation was prepared using a cross-linking ligand, bis(sulfosuccinimidyl) suberate (BS3), which has reactive carboxyl group to conjugate efficiently to the primary amino groups of the targeting agents. In our studies, we demonstrated successful conjugation of antibodies, Annexin A2 or prostate specific membrane antigen (PSMA), to curcumin loaded PLGA nanoparticles for targeting to prostate and breast cancer cells. The percent antibody attachment to PLGA nanoparticles was found to be 92.8%. Efficient intra-cellular uptake of the targeted nanoparticles was observed in the cancer cells. These results have emphasized the potential of our multifunctional curcumin nanoparticles to improve the clinical efficacy of curcumin therapy in patients with cancer.

Keywords: polymeric nanoparticles, cancer therapy, sustained release, curcumin

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Authors: Katerina H. Takova, Ivan N. Minkov, Gergana G. Zahmanova

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Molecular farming is the production of recombinant proteins in plants with the aim to use the protein as a purified product, crude extract or directly in the planta. Plants gain more attention as expression systems compared to other ones due to the cost effective production of pharmaceutically important proteins, appropriate post-translational modifications, assembly of complex proteins, absence of human pathogens to name a few. In addition, transient expression in plant leaves enables production of recombinant proteins within few weeks. Hepatitis E virus (HEV) is a causative agent of acute hepatitis. HEV causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. Presently, all efforts for development of Hepatitis E vaccine are focused on the Open Read Frame 2 (ORF2) capsid protein as it contains epitopes that can induce neutralizing antibodies. For our purpose, we used the CMPV-based vector-pEAQ-HT for transient expression of HEV ORF2 in Nicotiana benthamina. Different molecular analysis (Western blot and ELISA) showed that HEV ORF2 capsid protein was expressed in plant tissue in high-yield up to 1g/kg of fresh leaf tissue. Electron microscopy showed that the capsid protein spontaneously assembled in low abundance virus-like particles (VLPs), which are highly immunogenic structures and suitable for vaccine development. The expressed protein was recognized by both human and swine HEV positive sera and can be used as a diagnostic reagent for the detection of HEV infection. Production of HEV capsid protein in plants is a promising technology for further HEV vaccine investigations. Here, we reported for a rapid high-yield transient expression of a recombinant protein in plants suitable for vaccine production as well as a diagnostic reagent. Acknowledgments -The authors’ research on HEV is supported with grants from the Project PlantaSYST under the Widening Program, H2020 as well as under the UK Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1). The authors want to thank Prof. George Lomonossoff (JIC, Norwich, UK) for his contribution.

Keywords: hepatitis E virus, plant molecular farming, transient expression, vaccines

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Authors: J. J. Lee, S. R. Sung, E. J. Yum, S. C. Kim, B. H. Hyun, M. Her, H. S. Lee

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Canine brucellosis is a critical problem in dogs leading to reproductive diseases which are mainly caused by Brucella canis. There are, nonetheless, not clear symptoms so that it may go unnoticed in most of the cases. Serodiagnosis for canine brucellosis has not been confirmed. Moreover, it has substantial difficulties due to broad cross-reactivity between the rough cell wall antigens of B. canis and heterospecific antibodies present in normal, uninfected dogs. Thus, this study was conducted to characterize the immunogenic proteins in cytoplasmic antigen (CPAg) of B. canis, which defined the antigenic sensitivity of the humoral antibody responses to B. canis-infected dogs. In analysis of B. canis CPAg, first, we extracted and purified the cytoplasmic proteins from cultured B. canis by hot-saline inactivation, ultrafiltration, sonication, and ultracentrifugation step by step according to the sonicated antigen extract method. For characterization of this antigen, we checked the sort and range of each protein on SDS-PAGE and verified the immunogenic proteins leading to reaction with antisera of B. canis-infected dogs. Selected immunodominant proteins were identified using MALDI-MS/MS. As a result, in an immunoproteomic assay, several polypeptides in CPAg on one or two-dimensional electrophoresis (DE) were specifically reacted to antisera from B. canis-infected dogs but not from non-infected dogs. The polypeptides with approximate 150, 80, 60, 52, 33, 26, 17, 15, 13, 11 kDa on 1-DE were dominantly recognized by antisera from B. canis-infected dogs. In the immunoblot profiles on 2-DE, ten immunodominant proteins in CPAg were detected with antisera of infected dogs between pI 3.5-6.5 at approximate 35 to 10 KDa, without any nonspecific reaction with sera in non-infected dogs. Ten immunodominant proteins identified by MALDI-MS/MS were identified as superoxide dismutase, bacteroferritin, amino acid ABC transporter substrate-binding protein, extracellular solute-binding protein family3, transaldolase, 26kDa periplasmic immunogenic protein, Rhizopine-binding protein, enoyl-CoA hydratase, arginase and type1 glyceraldehyde-3-phosphate dehydrogenase. Most of these proteins were determined by their cytoplasmic or periplasmic localization with metabolism and transporter functions. Consequently, this study discovered and identified the prominent immunogenic proteins in B. canis CPAg, highlighting that those antigenic proteins may accomplish a specific serodiagnosis for canine brucellosis. Furthermore, we will evaluate those immunodominant proteins for applying to the advanced diagnostic methods with high specificity and accuracy.

Keywords: Brucella canis, Canine brucellosis, cytoplasmic antigen, immunogenic proteins

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Authors: Olarinde Olaniran, Oluyomi A. Sowemimo

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Toxoplasmosis is frequently misdiagnosed or underdiagnosed, and it is the third most common cause of hospitalization due to food-borne infection. Intra-uterine infection with Toxoplasma gondii due to active parasitaemia during pregnancy can cause severe and often fatal cerebral damage, abortion, and stillbirth of a fetus. The aim of the study was to investigate the prevalence of T. gondii infection in women of childbearing age in selected communities of Osun State with a view to determining the risk factors which predispose to the T. gondii infection. Five (5) ml of blood was collected by venopuncture into a plain blood collection tube by a medical laboratory scientist. Serum samples were separated by centrifuging the blood samples at 3000 rpm for 5 mins. The sera were collected with Eppendorf tubes and stored at -20°C analysis for the presence of IgG and IgM antibodies against T. gondii by commercially available enzyme-linked immunosorbent assay (ELISA) kit (Demeditec Diagnostics GmbH, Germany) conducted according to the manufacturer’s instructions. The optical densities of wells were measured by a photometer at a wavelength of 450 nm. Data collected were analysed using appropriate computer software. The overall seroprevalence of T. gondii among the women of child-bearing age in selected seven communities in Osun state was 76.3%. Out of 76.3% positive for Toxoplasma gondii infection, 70.0% were positive for anti- T. gondii IgG, and 32.3% were positive for IgM, and 26.7% for both IgG and IgM. The prevalence of T. gondii was lowest (58.9%) among women from Ile Ife, a peri-urban community, and highest (100%) in women residing in Alajue, a rural community. The prevalence of infection was significantly higher (P= 0.000) among Islamic women (87.5%) than in Christian women (70.8%). The highest prevalence (86.3%) was recorded in women with primary education, while the lowest (61.2%) was recorded in women with tertiary education (p =0.016). The highest prevalence (79.7%) was recorded in women that reside in rural areas, and the lowest (70.1%) was recorded in women that reside in peri-urban area (p=0.025). The prevalence of T. gondii infection was highest (81.4%) in women with one miscarriage, while the prevalence was lowest in women with no miscarriages (75.9%). The age of the women (p=0.042), Islamic religion (p=0.001), the residence of the women (p=0.001), and water source were all positively associated with T. gondii infection. The study concluded that there was a high seroprevalence of T. gondii recorded among women of child-bearing age in the study area. Hence, there is a need for health education and create awareness of the disease and its transmission to women of reproductive age group in general and pregnant women in particular to reduce the risk of T. gondii in pregnant women.

Keywords: seroepidemiology, Toxoplasma gondii, women, child-bearing, age, communities, Ile -Ife, Nigeria

Procedia PDF Downloads 149

Authors: Olubukayo-Opeyemi Oyetayo, Oscar Mendez-Lucio, Andreas Bender, Hans Kiefer

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The thermal melting curve of a protein provides information on its conformational stability and could provide cues on its aggregation behavior. Naturally-occurring osmolytes have been shown to improve the thermal stability of most proteins in a concentration-dependent manner. They are therefore commonly employed as additives in therapeutic protein purification and formulation. A number of intertwined and seemingly conflicting mechanisms have been put forward to explain the observed stabilizing effects, the most prominent being the preferential exclusion mechanism. We attempted to probe and summarize molecular mechanisms for thermal stabilization of a monoclonal antibody (mAb) by developing quantitative structure-activity relationships using a rationally-selected library of 120 osmolyte-like compounds in the polyhydric alcohols, amino acids and methylamines classes. Thermal stabilization potencies were experimentally determined by thermal shift assays based on differential scanning fluorimetry. The cross-validated QSAR model was developed by partial least squares regression using descriptors generated from Molecular Operating Environment software. Careful evaluation of the results with the use of variable importance in projection parameter (VIP) and regression coefficients guided the selection of the most relevant descriptors influencing mAb thermal stability. For the mAb studied and at pH 7, the thermal stabilization effects of tested compounds correlated positively with their fractional polar surface area and inversely with their fractional hydrophobic surface area. We cannot claim that the observed trends are universal for osmolyte-protein interactions because of protein-specific effects, however this approach should guide the quick selection of (de)stabilizing compounds for a protein from a chemical library. Further work with a large variety of proteins and at different pH values would help the derivation of a solid explanation as to the nature of favorable osmolyte-protein interactions for improved thermal stability. This approach may be beneficial in the design of novel protein stabilizers with optimal property values, especially when the influence of solution conditions like the pH and buffer species and the protein properties are factored in.

Keywords: thermal stability, monoclonal antibodies, quantitative structure-activity relationships, osmolytes

Procedia PDF Downloads 298

Authors: Akterono Budiyati, Gita Kusumo, Teguh Putra, Fritzie Rexana, Antonius Kurniawan, Aru Sudoyo, Ahmad Utomo, Andi Utama

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The presence of the programmed death ligand-1 (PD-L1) has been used in multiple clinical trials and approved as biomarker for selecting patients more likely to respond to immune checkpoint inhibitors. However, the expression of PD-L1 is regulated in different ways, which leads to a different significance of its presence. Positive PD-L1 within tumors may result from two mechanisms, induced PD-L1 expression by T-cell presence or genetic mechanism that lead to constitutive PD-L1 expression. Amplification of PD-L1 genes was found as one of genetic mechanism which causes an increase in PD-L1 expression. In case of colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). Although the correlation between PD-L1 expression and MSI status has been widely studied, so far the precise mechanism of PD-L1 gene activation in CRC patients, particularly in MSI population have yet to be clarified. In this present study we have profiled 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. PD-L1 expression was measured on tumor cells with cut off of 1% whereas loss of nuclear MMR protein expressions in tumor cells but not in normal or stromal cells indicated presence of MSI. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using single Tube TaqMan Copy Number Assays Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) (15%; 8/53) (P=0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low-level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). Additionally, we found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlate with PD-L1 expression. Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.

Keywords: colorectal cancer, gene amplification, microsatellite instable, programmed death ligand-1

Procedia PDF Downloads 189

Authors: Nicholas Fletcher, Zachary Houston, Yongmei Zhao, Christopher Howard, Kristofer Thurecht

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One promising approach to enhance nanomedicine therapeutic efficacy is to include a targeting agent, such as an antibody, to increase accumulation at the tumor site. However, the application of such targeted nanomedicines remains limited, in part due to difficulties involved with biomolecule conjugation to synthetic nanomaterials. One approach recently developed to overcome this has been to engineer bispecific antibodies (BsAbs) with dual specificity, whereby one portion binds to methoxy polyethyleneglycol (mPEG) epitopes present on synthetic nanomedicines, while the other binds to molecular disease markers of interest. In this way, noncovalent complexes of nanomedicine core, comprising a hyperbranched polymer (HBP) of primarily mPEG, decorated with targeting ligands are able to be produced by simple mixing. Further work in this area has now demonstrated such complexes targeting the breast cancer marker epidermal growth factor receptor (EGFR) to show enhanced binding to tumor cells both in vitro and in vivo. Indeed the enhanced accumulation at the tumor site resulted in improved therapeutic outcomes compared to untargeted nanomedicines and free chemotherapeutics. The current work on these BsAb-HBP conjugates focuses on further probing antibody-nanomaterial interactions and demonstrating broad applicability to a range of cancer types. Herein are reported BsAb-HBP materials targeted towards prostate-specific membrane antigen (PSMA) and study of their behavior in vivo using ⁸⁹Zr positron emission tomography (PET) in a dual-tumor prostate cancer xenograft model. In this model mice bearing both PSMA+ and PSMA- tumors allow for PET imaging to discriminate between nonspecific and targeted uptake in tumors, and better quantify the increased accumulation following BsAb conjugation. Also examined is the potential for formation of these targeted complexes in situ following injection of individual components? The aim of this approach being to avoid undesirable clearance of proteinaceous complexes upon injection limiting available therapeutic. Ultimately these results demonstrate BsAb functionalized nanomaterials as a powerful and versatile approach for producing targeted nanomedicines for a variety of cancers.

Keywords: bioengineering, cancer, nanomedicine, polymer chemistry

Procedia PDF Downloads 113

Authors: Merilin Ivanova Ivanova, Kalin Dimitrov Atanasov, Stefan Petrov Enchev

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Backgroud: Neuromyelitis optica spectrum disorder, also known as Devic’s disease, is a relapsing demyelinating autoimmune inflammatory disorder of the central nervous system associated with anti-aquaporin 4 (AQP4) antibodies that can manifest with devastating secondary neurological deficits. Most commonly affected are the optic nerves and the spinal cord-clinically this is often presented with optic neuritis (loss of vision), transverse myelitis(weakness or paralysis of extremities),lack of bladder and bowel control, numbness. APS is a core clinical entity of NMOSD and adds to the clinical representation the following symptoms: intractable nausea, vomiting and hiccup, it usually occurs isolated at onset, and can lead to a significant delay in the diagnosis. The condition may have features similar to multiple sclerosis (MS) but the episodes are worse in NMO and it is treated differently. It could be relapsing or monophasic. Possible complications are visual field defects and motor impairment, with potential blindness and irreversible motor deficits. In severe cases, myogenic respiratory failure ensues. The incidence of reported cases is approximately 0.3–4.4 per 100,000. Paediatric cases of NMOSD are rare but have been reported occasionally, comprising less than 5% of the reported cases. Objective: The case serves to show the difficulty when it comes to the diagnostic processes regarding a rare autoimmune disease with non- specific symptoms, taking large interval of rimes to reveal as complete clinical manifestation of the aforementioned syndrome, as well as the necessity of multidisciplinary approach in the setting of а general paediatric department in аn emergency hospital. Methods: itpatient's history, clinical presentation, and information from the used diagnostic tools(MRI with contrast of the central nervous system) lead us to the conclusion .This was later on confirmed by the positive results from the anti-aquaporin 4 (AQP4) antibody serology test. Conclusion: APS is a common symptom of NMOSD and is considered a challenge in a differential-diagnostic plan. Gaining an increased awareness of this disease/syndrome, obtaining a detailed patient history, and performing thorough physical examinations are essential if we are to reduce and avoid misdiagnosis.

Keywords: neuromyelitis, devic's disease, hiccup, autoimmune, MRI

Procedia PDF Downloads 20

Authors: Pei-Ying Lo, Jing-Hao Ciou, Kai-Cheng Yang, Jia-Huei Zheng, Shih-Hsiang Huang, Kuen-Chan Lee, Er-Chieh Cho

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As-produced CNTs are insoluble in all organic solvents and aqueous solutions have imposed limitations to the use of CNTs. Therefore, how to debundle carbon nanotubes and to modify them for further uses is an important issue. There are several methods for the dispersion of CNTs in water using covalent attachment of hydrophilic groups to the surface of tubes. These methods, however, alter the electronic structure of the nanotubes by disrupting the network of sp2 hybridized carbons. In order to keep the nanotubes’ intrinsic mechanical and electrical properties intact, non-covalent interactions are increasingly being explored as an alternative route for dispersion. Apart from conventional surfactants such as sodium dodecylsulfate (SDS) or sodium dodecylbenzenesulfonate (SDBS) which are highly effective in dispersing CNTs, biopolymers have received much attention as dispersing agents due to the anticipated biocompatibility of the dispersed CNTs. Also, The pyrenyl group is known to interact strongly with the basal plane of graphene via π-stacking. In this study, a highly re-dispersible biopolymer is reported for the synthesis of pyrene-modified poly-L-lysine (PBPL) and poly(D-Glu, D-Lys) (PGLP). To provide the evidence of the safety of the PBPL/CNT & PGLP/CNT materials we use in this study, H1299 and HCT116 cells were incubated with PBPL/CNT & PGLP/CNT materials for toxicity analysis, MTS assays. The results from MTS assays indicated that no significant cellular toxicity was shown in H1299 and HCT116 cells. Furthermore, the fluorescence marker fluorescein isothiocyanate (FITC) was added to PBPL & PGLP dispersions. From the fluorescent measurements showed that the chemical functionalisation of the PBPL/CNT & PGLP/CNT conjugates with the fluorescence marker were successful. The fluorescent PBPL/CNT & PGLP/CNT conjugates could find application in medical imaging. In the next step, the GFP gene is immobilized onto PBPL/CNT conjugates by introducing electrostatic interaction. GFP-transfected cells that emitted fluorescence were imaged and counted under a fluorescence microscope. Due to the unique biocompatibility of PBPL modified CNTs, the GFP gene could be transported into H1299 cells without using antibodies. The applicability of such soluble and chemically functionalised polypeptide/CNT conjugates in biomedicine is currently investigated. We expect that this polypeptide/CNT system will be a safe and multi-functional nanomedical delivery platform and contribute to future medical therapy.

Keywords: carbon nanotube, nanotoxicology, GFP transfection, polypeptide/CNT hybrids

Procedia PDF Downloads 319

Authors: Jianuo Sun, Jinghan Wang, Sirui Zhang, Chenhan Xu, Hongxia Hao, Hong Zhou

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In recent years, the diversification and industrialization of drug-related crimes have posed significant threats to public health and safety globally. The widespread and increasingly younger demographics of drug users and the persistence of drug-impaired driving incidents underscore the urgency of this issue. Drug detection, a specialized forensic activity, is pivotal in identifying and analyzing substances involved in drug crimes. It relies on pharmacological and chemical knowledge and employs analytical chemistry and modern detection techniques. However, current drug detection methods are limited by their inability to perform semi-quantitative, real-time field analyses. They require extensive, complex laboratory-based preprocessing, expensive equipment, and specialized personnel and are hindered by long processing times. This study introduces an alternative approach using nucleic acid aptamers and Aggregation-Induced Emission (AIE) technology. Nucleic acid aptamers, selected artificially for their specific binding to target molecules and stable spatial structures, represent a new generation of biosensors following antibodies. Rapid advancements in AIE technology, particularly in tetraphenyl ethene-based luminous, offer simplicity in synthesis and versatility in modifications, making them ideal for fluorescence analysis. This work successfully synthesized, isolated, and purified an AIE molecule and constructed a probe comprising the AIE molecule, nucleic acid aptamers, and exonuclease for cocaine detection. The probe demonstrated significant relative fluorescence intensity changes and selectivity towards cocaine over other drugs. Using 4-Butoxytriethylammonium Bromide Tetraphenylethene (TPE-TTA) as the fluorescent probe, the aptamer as the recognition unit, and Exo I as an auxiliary, the system achieved rapid detection of cocaine within 5 mins in aqueous and urine, with detection limits of 1.0 and 5.0 µmol/L respectively. The probe-maintained stability and interference resistance in urine, enabling quantitative cocaine detection within a certain concentration range. This fluorescent sensor significantly reduces sample preprocessing time, offers a basis for rapid onsite cocaine detection, and promises potential for miniaturized testing setups.

Keywords: drug detection, aggregation-induced emission (AIE), nucleic acid aptamer, exonuclease, cocaine

Procedia PDF Downloads 27

Authors: Michio Kurosu

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Alterations in glycosylation not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Identification of cell type-specific glycoconjugates (tumor markers) has led to the discovery of new assay systems for certain cancers via immunodetection reagents. N- and O-linked glycans are the most abundant forms of glycoproteins. Recent studies of cancer immunotherapy are based on the immunogenicity of truncated O-glycan chains (e.g., Tn, sTn, T, and sLea/x). The prevalence of N-linked glycan changes in the development of tumor cells is known; however, therapeutic antibodies against N-glycans have not yet been developed. This is due to the lack of specificity of N-linked glycans between normal/healthy and cancer cells. Abnormal branching of N-linked glycans has been observed, particularly in solid cancer cells. While the discovery of drug-like glycosyltransferase inhibitors that block the biosynthesis of specific branching has a very low likelihood of success, altered glycosylation levels can be exploited by suppressing N-glycan biosynthesis through the inhibition of dolichyl-phosphate N-acetylglucosaminephosphotransferase1 (DPAGT1) activity. Inhibition of DPAGT1 function leads to changes of O-glycosylation on proteins associated with mitochondria and zinc finger binding proteins (indirect effects). On the basis of dynamic crosstalk between DPAGT1 and Snail/Slung/ZEB1 (a family of transcription factors that promote the repression of the adhesion molecules), we have developed pharmacologically acceptable selective DPAGT1 inhibitors. Tunicamycin kills a wide range of cancer and healthy cells in a non-selective manner. In sharp contrast, our DPAGT1 inhibitors display strong cytostatic effects against 16 solid cancers, which require the overexpression of DPAGT1 in their progression but do not affect the cell viability of healthy cells. The identified DPAGT1 inhibitors possess impressive anti-metastatic ability in various solid cancer cell lines and induce their mitochondrial structural changes, resulting in apoptosis. A prototype DPAGT1 inhibitor, APPB has already been proven to shrink solid tumors (e.g., pancreatic cancers, triple-negative breast cancers) in vivo while suppressing metastases and has strong synergistic effects when combined with current cytotoxic drugs (e.g., paclitaxel). At this conference, our discovery of selective DPAGT1 inhibitors with drug-like properties and proof-of-pharmaceutical concept studies of a novel DPAGT1 inhibitor are presented.

Keywords: DPAGT1 inhibitors, anti-metastatic drugs, natural product based drug designs, cytostatic effects

Procedia PDF Downloads 44

Authors: K. Graniczkowska, N. Beloglazova, S. De Saeger

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The threat of synthetic drugs is one of the most significant current drug problems worldwide. The use of drugs of abuse has increased dramatically during the past three decades. Among others, Amphetamine-Type Stimulants (ATS) are globally the second most widely used drugs after cannabis, exceeding the use of cocaine and heroin. ATS are potent central nervous system (CNS) stimulants, capable of inducing euphoric static similar to cocaine. Recreational use of ATS is widespread, even though warnings of irreversible damage of the CNS were reported. ATS pose a big problem and their production contributes to the pollution of the environment by discharging big volumes of liquid waste to sewage system. Therefore, there is a demand to develop robust and sensitive sensors that can detect ATS and their intermediates in environmental water samples. A rapid and simple test is required. Analysis of environmental water samples (which sometimes can be a harsh environment) using antibody-based tests cannot be applied. Therefore, molecular imprinted polymers (MIPs), which are known as synthetic antibodies, have been chosen for that approach. MIPs are characterized with a high mechanical and thermal stability, show chemical resistance in a broad pH range and various organic or aqueous solvents. These properties make them the preferred type of receptors for application in the harsh conditions imposed by environmental samples. To the best of our knowledge, there are no existing MIPs-based sensors toward amphetamine and its intermediates. Also not many commercial MIPs for this application are available. Therefore, the aim of this study was to compare different techniques to obtain MIPs with high specificity towards ATS and characterize them for following use in a sensing unit. MIPs against amphetamine and its intermediates were synthesized using a few different techniques, such as electro-, thermo- and UV-initiated polymerization. Different monomers, cross linkers and initiators, in various ratios, were tested to obtain the best sensitivity and polymers properties. Subsequently, specificity and selectivity were compared with commercially available MIPs against amphetamine. Different linkers, such as lipoic acid, 3-mercaptopioponic acid and tyramine were examined, in combination with several immobilization techniques, to select the best procedure for attaching particles on sensor surface. Performed experiments allowed choosing an optimal method for the intended sensor application. Stability of MIPs in extreme conditions, such as highly acidic or basic was determined. Obtained results led to the conclusion about MIPs based sensor applicability in sewage system testing.

Keywords: amphetamine type stimulants, environment, molecular imprinted polymers, MIPs, sensor

Procedia PDF Downloads 220

Authors: Ainur Sharip, Jose E. Perez, Nouf Alsharif, Aldo I. M. Bandeas, Enzo D. Fabrizio, Timothy Ravasi, Jasmeen S. Merzaban, Jürgen Kosel

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Bone marrow-derived human mesenchymal stem cells (MSCs) are attractive candidates for tissue engineering and regenerative medicine, due to their ability to differentiate into osteoblasts, chondrocytes or adipocytes. Differentiation is influenced by biochemical and biophysical stimuli provided by the microenvironment of the cell. Thus, altering the mechanical characteristics of a cell culture scaffold can directly influence a cell’s microenvironment and lead to stem cell differentiation. Mesenchymal stem cells were cultured on densely packed, vertically aligned magnetic iron nanowires (NWs) and the effect of NWs on the cell cytoskeleton rearrangement and differentiation were studied. An electrochemical deposition method was employed to fabricate NWs into nanoporous alumina templates, followed by a partial release to reveal the NW array. This created a cell growth substrate with free-standing NWs. The Fe NWs possessed a length of 2-3 µm, with each NW having a diameter of 33 nm on average. Mechanical stimuli generated by the physical movement of these iron NWs, in response to a magnetic field, can stimulate osteogenic differentiation. Induction of osteogenesis was estimated using an osteogenic marker, osteopontin, and a reduction of stem cell markers, CD73 and CD105. MSCs were grown on the NWs, and fluorescent microscopy was employed to monitor the expression of markers. A magnetic field with an intensity of 250 mT and a frequency of 0.1 Hz was applied for 12 hours/day over a period of one week and two weeks. The magnetically activated substrate enhanced the osteogenic differentiation of the MSCs compared to the culture conditions without magnetic field. Quantification of the osteopontin signal revealed approximately a seven-fold increase in the expression of this protein after two weeks of culture. Immunostaining staining against CD73 and CD105 revealed the expression of antibodies at the earlier time point (two days) and a considerable reduction after one-week exposure to a magnetic field. Overall, these results demonstrate the application of a magnetic NW substrate in stimulating the osteogenic differentiation of MSCs. This method significantly decreases the time needed to induce osteogenic differentiation compared to commercial biochemical methods, such as osteogenic differentiation kits, that usually require more than two weeks. Contact-free stimulation of MSC differentiation using a magnetic field has potential uses in tissue engineering, regenerative medicine, and bone formation therapies.

Keywords: cell substrate, magnetic nanowire, mesenchymal stem cell, stem cell differentiation

Procedia PDF Downloads 169

Authors: Srinivas Bathini, Duraichelvan Raju, Simona Badilescu, Muthukumaran Packirisamy

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A bio-sensing method, based on the plasmonic property of gold nano-islands, has been developed for detection of exosomes in a clinical setting. The position of the gold plasmon band in the UV-Visible spectrum depends on the size and shape of gold nanoparticles as well as on the surrounding environment. By adsorbing various chemical entities, or binding them, the gold plasmon band will shift toward longer wavelengths and the shift is proportional to the concentration. Exosomes transport cargoes of molecules and genetic materials to proximal and distal cells. Presently, the standard method for their isolation and quantification from body fluids is by ultracentrifugation, not a practical method to be implemented in a clinical setting. Thus, a versatile and cutting-edge platform is required to selectively detect and isolate exosomes for further analysis at clinical level. The new sensing protocol, instead of antibodies, makes use of a specially synthesized polypeptide (Vn96), to capture and quantify the exosomes from different media, by binding the heat shock proteins from exosomes. The protocol has been established and optimized by using a glass substrate, in order to facilitate the next stage, namely the transfer of the protocol to a microfluidic environment. After each step of the protocol, the UV-Vis spectrum was recorded and the position of gold Localized Surface Plasmon Resonance (LSPR) band was measured. The sensing process was modelled, taking into account the characteristics of the nano-island structure, prepared by thermal convection and annealing. The optimal molar ratios of the most important chemical entities, involved in the detection of exosomes were calculated as well. Indeed, it was found that the results of the sensing process depend on the two major steps: the molar ratios of streptavidin to biotin-PEG-Vn96 and, the final step, the capture of exosomes by the biotin-PEG-Vn96 complex. The microfluidic device designed for sensing of exosomes consists of a glass substrate, sealed by a PDMS layer that contains the channel and a collecting chamber. In the device, the solutions of linker, cross-linker, etc., are pumped over the gold nano-islands and an Ocean Optics spectrometer is used to measure the position of the Au plasmon band at each step of the sensing. The experiments have shown that the shift of the Au LSPR band is proportional to the concentration of exosomes and, thereby, exosomes can be accurately quantified. An important advantage of the method is the ability to discriminate between exosomes having different origins.

Keywords: exosomes, gold nano-islands, microfluidics, plasmonic biosensing

Procedia PDF Downloads 145

Authors: Chrislaura Carmo, Luca Deiana, Mafalda Laranjo, Abilio Sobral, Armando Cordova

Abstract:

Photodynamic therapy (PDT) is currently used for the treatment of malignancies and premalignant tumors. It is based on the capture of a photosensitizing molecule (PS) which, when excited by light at a certain wavelength, reacts with oxygen and generates oxidizing species (radicals, singlet oxygen, triplet species) in target tissues, leading to cell death. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indaceno) derivatives are emerging as important candidates for photosensitizer in photodynamic therapy of cancer cells due to their high triplet quantum yield. Today these dyes are relevant molecules in photovoltaic materials and fluorescent sensors. In this study, it will be demonstrated the possibility that BODIPY can be covalently linked to thioglycolic acid through the click reaction. Thiol−ene click chemistry has become a powerful synthesis method in materials science and surface modification. The design of biobased allyl-terminated precursors with high renewable carbon content for the construction of the thiol-ene polymer networks is essential for sustainable development and green chemistry. The work aims to synthesize the BODIPY (10-(4-(allyloxy) phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2] diazaborinin-4-ium-5-uide) and to click reaction with Thioglycolic acid. BODIPY was synthesized by the condensation reaction between aldehyde and pyrrole in dichloromethane, followed by in situ complexation with BF3·OEt2 in the presence of the base. Then it was functionalized with allyl bromide to achieve the double bond and thus be able to carry out the click reaction. The thiol−ene click was performed using DMPA (2,2-Dimethoxy-2-phenylacetophenone) as a photo-initiator in the presence of UV light (320–500 nm) in DMF at room temperature for 24 hours. Compounds were characterized by standard analytical techniques, including UV-Vis Spectroscopy, 1H, 13C, 19F NMR and mass spectroscopy. The results of this study will be important to link BODIPY to polymers through the thiol group offering a diversity of applications and functionalization. This new molecule can be tested as third-generation photosensitizers, in which the dye is targeted by antibodies or nanocarriers by cells, mainly in cancer cells, PDT and Photodynamic Antimicrobial Chemotherapy (PACT). According to our studies, it was possible to visualize a click reaction between allyl BODIPY and thioglycolic acid. Our team will also test the reaction with other thiol groups for comparison. Further, we will do the click reaction of BODIPY with a natural polymer linked with a thiol group. The results of the above compounds will be tested in PDT assays on various lung cancer cell lines.

Keywords: bodipy, click reaction, thioglycolic acid, allyl, thiol-ene click

Procedia PDF Downloads 92

Authors: Rajish Shil, Amal Alduhoori, Vipin Thomachan, Jamal Teir, Radhakrishnan Renganathan

Abstract:

Background: Antiphospholipid antibody syndrome (APS) has a broad spectrum of thrombotic and non-thrombotic clinical manifestations. We present a case of APS presenting with seizure, stroke, and atrial mass. Case Description: A 38-year-old male presented with headache of 10 days duration and tonic-clonic seizure. The neurological examination was normal. Magnetic resonance imaging of brain showed small acute right cerebellar infarct. Magnetic resonance angiography of brain and neck showed a focal narrowing in the origin of the internal carotid artery bilaterally. Electroencephalogram was normal. He was started on aspirin, atorvastatin, and carbamazepine. Transthoracic and trans-esophageal echocardiography showed a pedunculated and lobular atrial mass, measuring 1 X 1.5 cm, which was freely mobile across mitral valve opening across the left ventricular inflow. Autoimmune screening showed positive Antiphospholipid antibodies in high titer (Cardiolipin IgG > 120 units/ml, B2 glycoprotein IgG 90 units/mL). Anti-nuclear antibody was negative. Erythrocyte sedimentation rate and C-reactive protein levels were normal. Platelet count was low (111 x 109/L). The patient underwent successful surgical removal of the mass, which looked like a thrombotic clot, and Histopathological analysis confirmed it as a fibrinous clot, with no evidence of tumor cells. The patient was started on full anticoagulation treatment and was followed up regularly in the clinic, where our patient did not have any further complications from the disease. Discussion: Our patient was diagnosed to have APS based on the features of high positive anticardiolipin antibody IgG and B2 glycoprotein IgG levels, Stroke, thrombocytopenia, and abnormal echo findings. Thrombotic vegetation can mimic an atrial myxoma on echo. Conclusion: APS can present with neurological and cardiac manifestations, and therefore a high index of suspicion is necessary for a diagnosis of the disease as it can affect both short and long term treatment plans and prognosis. Therefore, in patients presenting with neurological symptoms like seizures, weakness and radiological diagnosis of stroke in a young patient, where atrial masses could be thought to be the cause of stroke, they should be screened for any concomitant findings of thrombocytopenia and/or activated partial thromboplastin time prolongation, which should raise the suspicion of vasculitis, specifically APS to be the primary cause of the clinical presentation.

Keywords: antiphospholipid syndrome, seizures, atrial mass, stroke

Procedia PDF Downloads 90

Authors: Markus Bredel, Sindhu Nair, Hoa Q. Trummell, Rajani Rajbhandari, Christopher D. Willey, Lewis Z. Shi, Zhuo Zhang, William J. Placzek, James A. Bonner

Abstract:

Purpose: EGFR-targeted monoclonal antibodies (mAbs) provide clinical benefit in some patients with H&N squamous cell carcinoma (HNSCC), but others progress with minimal response. Missense mutations in the EGFR ectodomain (ECD) can be acquired under mAb therapy by mimicking the effect of large deletions on receptor untethering and activation. Little is known about the contribution of EGFR ECD mutations to EGFR activation and anti-EGFR response in HNSCC. Methods: We selected patient-derived HNSCC cells (UM-SCC-1) for resistance to mAb Cetuximab (CTX) by repeated, stepwise exposure to mimic what may occur clinically and identified two concurrent EGFR ECD mutations (UM-SCC-1R). We examined the competence of the mutants to bind EGF ligand or CTX. We assessed the potential impact of the mutations through visual analysis of space-filling models of the native sidechains in the original structures vs. their respective side-chain mutations. We performed CRISPR in combination with site-directed mutagenesis to test for the effect of the mutants on ligand-independent EGFR activation and sorting. We determined the effects on receptor internalization, endocytosis, downstream signaling, and radiation sensitivity. Results: UM-SCC-1R cells carried two non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD. Structural modeling predicted that these mutants restrict the adoption of a tethered, inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed a reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation. Single and double-mutant models demonstrated that the G33S mutant is dominant over the N56K mutant in its effect on EGFR activation and EGF binding. CTX-resistant UM-SCC-1R cells demonstrated cross-resistance to mAb Panitumuab but, paradoxically, remained sensitive to the reversible receptor tyrosine kinase inhibitor Erlotinib. Conclusions: HNSCC cells can select for EGFR ECD mutations under EGFR mAb exposure that converge to trap the receptor in an open, constitutively activated state. These mutants impede the receptor’s competence to bind mAbs and EGF ligand and alter its endosomal trafficking, possibly explaining certain cases of clinical mAb and radiation resistance.

Keywords: head and neck cancer, EGFR mutation, resistance, cetuximab

Procedia PDF Downloads 58

Authors: B. T. Naveen Kumar, Anuj Tyagi, Niraj Kumar Singh, Visanu Boonyawiwat, A. H. Shanthanagouda, Orawan Boodde, K. M. Shankar, Prakash Patil, Shubhkaramjeet Kaur

Abstract:

Early mortality syndrome (EMS)/Acute Hepatopancreatic Necrosis Disease (AHPND) has emerged as a major obstacle for the shrimp farming around the world. It is caused by a strain of Vibrio parahaemolyticus. The possible preventive and control measure is, early and rapid detection of the pathogen in the broodstock, post-larvae and monitoring the shrimp during the culture period. Polymerase chain reaction (PCR) based early detection methods are good, but they are costly, time taking and requires a sophisticated laboratory. The present study was conducted to develop a simple, sensitive and rapid diagnostic farmer level kit for the reliable detection of AHPND in shrimp. A panel of monoclonal antibodies (MAbs) were raised against the recombinant Pir B protein (rPirB). First, an immunodot was developed by using MAbs G3B8 and Mab G3H2 which showed specific reactivity to purified r-PirB protein with no cross-reactivity to other shrimp bacterial pathogens (AHPND free Vibrio parahaemolyticus (Indian strains), V. anguillarum, WSSV, Aeromonas hydrophila, and Aphanomyces invadans). Immunodot developed using Mab G3B8 is more sensitive than that with the Mab G3H2. However, immunodot takes almost 2.5 hours to complete with several hands-on steps. Therefore, the flow-through assay (FTA) was developed by using a plastic cassette containing the nitrocellulose membrane with absorbing pads below. The sample was dotted in the test zone on the nitrocellulose membrane followed by continuos addition of five solutions in the order of i) blocking buffer (BSA) ii) primary antibody (MAb) iii) washing Solution iv) secondary antibody and v) chromogen substrate (TMB) clear purple dots against a white background were considered as positive reactions. The FTA developed using MAbG3B8 is more sensitive than that with MAb G3H2. In FTA the two MAbs showed specific reactivity to purified r-PirB protein and not to other shrimp bacterial pathogens. The FTA is simple to farmer/field level, sensitive and rapid requiring only 8-10 min for completion. Tests can be developed to kits, which will be ideal for use in biosecurity, for the first line of screening (at the port or pond site) and during monitoring and surveillance programmes overall for the good management practices to reduce the risk of the disease.

Keywords: acute hepatopancreatic necrosis disease, AHPND, flow-through assay, FTA, farmer level, immunodot, pond site, shrimp

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