Search results for: fluorescent staining

Authors: John Sodeau, David O'Connor, Shane Daly, Stig Hellebust

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This study is the first to employ the online WIBS (Waveband Integrated Biosensor Sensor) technique for the monitoring of bioaerosol emissions and non-fluorescing “dust” released from a composting/green waste site. The purpose of the research was to provide a “proof of principle” for using WIBS to monitor such a location continually over days and nights in order to construct comparative “bioaerosol site profiles”. Current impaction/culturing methods take many days to achieve results available by the WIBS technique in seconds.The real-time data obtained was then used to assess variations of the bioaerosol counts as a function of size, “shape”, site location, working activity levels, time of day, relative humidity, wind speeds and wind directions. Three short campaigns were undertaken, one classified as a “light” workload period, another as a “heavy” workload period and finally a weekend when the site was closed. One main bioaerosol size regime was found to predominate: 0.5 micron to 3 micron with morphologies ranging from elongated to elipsoidal/spherical. The real-time number-concentration data were consistent with an Andersen sampling protocol that was employed at the site. The number-concentrations of fluorescent particles as a proportion of total particles counted amounted, on average, to ~1% for the “light” workday period, ~7% for the “heavy” workday period and ~18% for the weekend. The bioaerosol release profiles at the weekend were considerably different from those monitored during the working weekdays.

Keywords: bioaerosols, composting, fluorescence, particle counting in real-time

Procedia PDF Downloads 331

Authors: N. Varsamis, M. Gerasimou, P. Christodoulou, S. Mantzoukis, G. Kolliopoulou, N. Zotos

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Purpose: Body fluids (pleural, peritoneal, synovial, pericardial, cerebrospinal) are an important element in the detection of microorganisms. For this reason, it is important to examine them in the Intensive Care Unit (ICU) patients. Material and Method: Body fluids are transported through sterile containers and enriched as soon as possible with Tryptic Soy Broth (TSB). After one day of incubation, the broth is poured into selective media: Blood, Mac Conkey No. 2, Chocolate, Mueller Hinton, Chapman and Saboureaud agar. The above selective media are incubated directly for 2 days. After this period, if any number of microbial colonies are detected, gram staining is performed. After that, the isolated organisms are identified by biochemical techniques in the automated Microscan system (Siemens) and followed by a sensitivity test on the same system using the minimum inhibitory concentration MIC technique. The sensitivity test is verified by Kirby Bauer-based plate test. Results: In 2017 the Laboratory of Microbiology received 60 samples of body fluids from the ICU. More specifically the Microbiology Department received 6 peritoneal fluid specimens, 18 pleural fluid specimens and 36 cerebrospinal fluid specimens. 36 positive cultures were tested. S. epidermidis was identified in 18 specimens, S. haemolyticus in 6, and E. faecium in 12. Conclusions: The results show low detection of microorganisms in body fluid cultures.

Keywords: body fluids, culture, intensive care unit, microorganisms

Procedia PDF Downloads 182

Authors: Nunthawan Nowwarote, Waleerat Sukarawan, Prasit Pavasant, Thanaphum Osathanon

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Interleukin 6 (IL-6) is a multifunctional cytokine, regulating various biological responses in several tissues. A Recent study shows that IL-6 plays a role in stemness maintenance in stem cells isolated from human exfoliated deciduous teeth (SHEDs). However, the role of IL-6 on cell differentiation in SHEDs remains unknown. The present study investigated the effect of IL-6 on SHEDs differentiation. Cells were isolated from dental pulp tissues of human deciduous teeth. Flow cytometry was used to determined mesenchymal stem cell marker expression, and the multipotential differentiation (osteogenic, adipogenic and neurogenic lineage ) was also determined. The mRNA was determined using real-time quantitative polymerase chain reaction, and the phenotypes were confirmed by chemical and immunofluorescence staining. Results demonstrated that SHEDs expressed CD44, CD73, CD90, CD105 but not CD45. Further, the up-regulation of osteogenic, adipogenic and neurogenic marker genes was observed upon maintaining cells in osteogenic, adipogenic and neurogenic induction medium, respectively. The addition of IL-6 induced osteogenic by up-regulated osteogenic marker gene also increased in vitro mineralization. Under neurogenic medium supplement with IL-6, up-regulated neurogenic marker. Whereas, an addition of IL-6 attenuated adipogenic differentiation by SHEDs. In conclusion, this evidence implies that IL-6 may participate in cells differentiation ability of SHEDs.

Keywords: SHEDs, IL-6, cell differentiations, dental pulp

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Authors: Choo Hong Ang

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The most popular approach to save energy for large commercial buildings in Malaysia is to replace the existing chiller plant of high kW/ton to one of lower kW/ton. This approach, however, entails large capital outlay with a long payment period of up to 7 years. This paper shows that by using multiple approaches, other than replacing the existing chiller plant, an energy saving of up to 20 %, is possible. The main methodology adopted was to identify and then plugged all heat ingress paths into a building, including putting up glass structures to prevent mixing of internal air-conditioned air with the ambient environment, and replacing air curtains with glass doors. This methodology could save up to 10 % energy bill. Another methodology was to change fixed speed motors of air handling units (AHU) to variable speed drive (VSD) and changing escalators to motion-sensor type. Other methodologies included reducing heat load by blocking air supply to non-occupied parcels, rescheduling chiller plant operation, changing of fluorescent lights to LED lights, and conversion from tariff B to C1. A case example of Komtar, the tallest building in Penang, is given here. The total energy bill for Komtar was USD2,303,341 in 2016 but was reduced to USD 1,842,927.39 in 2018, a significant saving of USD460,413.86 or 20 %. In terms of kWh, there was a reduction from 18, 302,204.00 kWh in 2016 to 14,877,105.00 kWh in 2018, a reduction of 3,425,099.00 kWh or 18.71 %. These methodologies used were relatively low cost and the payback period was merely 24 months. With this achievement, the Komtar building was awarded champion of the Malaysian National Energy Award 2019 and second runner up of the Asean Energy Award. This experience shows that a strong commitment to energy saving is the key to effective energy saving.

Keywords: chiller plant, energy saving measures, heat ingress, large building

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Authors: Shivani Kuttuva, James Sampson, Timothy Simmons, Vinayak Thattaruparambil, Holly Burton, Peter Coyne

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Anogenital mammary-like glands were established to be embryological remnants of breast tissue due to failed resolution of the ectodermal mammary ridge. However, recent studies are now considering this to represent normal constituents of the anogenital area with histological resemblance to the orthotopic breast tissue with multiple benign and malignant lesions arising from it. The incidence of the above has been predominant in females in the vulval region. Due to the paucity of cases reported in men, this poses a diagnostic and therapeutic challenge resulting in a delay in treatment and, thereby, poor outcomes. Our patient presented to the dermatology clinic with an itchy, purplish lesion in the peri-anal region which, on punch biopsy, was diagnosed to be Extra-mammary Paget’s disease and taken up for Wide local excision. Immunochemically, staining was positive for HER2, ER and Cytokeratin 7, keeping with the presence of actual breast tissue with no primary breast carcinoma. Due to the invasive nature of the disease, he required Abdominoperineal resection with flap reconstruction. Despite complete surgical clearance and adjuvant radiotherapy, the disease progressed to adjacent inguinal and obturator lymph nodes with origin resembling anogenital type mammary glands but histology negative for hormonal receptors of the breast.

Keywords: anogenital mammary-like glands, abdominoperineal resection, ectopic breast tissue, ectopic male breast carcinoma, peri-anal skin lesion

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Authors: Saravanan Govindaraju, Kyusik Yun

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The purpose of this study is to develop the chitosan doped curcumin gold cluster nanofiber for wound healing and skin cancer drug delivery applications. Chitosan is a typical marine polysaccharide composed of glucosamine and n-acetyl glucosamine biodegradable and biocompatible polymer. Curcumin is a natural bioactive molecule obtained from Curcuma longo, it mostly occurs in some Asian countries like India and China. It has naturally antioxidant, antimicrobial, wound healing and anticancer property. Due to this advantage, we prepared a combination of natural polymer chitosan with Curcumin and gold nanocluster nanofiber (CH-CUR-AuNCs nanofibers). The prepared nanofiber was characterized by using Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Antibacterial studies were performed with E.coli and S.aureus. Antioxidant assay, drug release test, and cytotoxicity will be evaluated. Prepared nanofiber emits low intensity of red fluorescent. The FTIR confirm the presence of chitosan and Curcumin in the nanofiber. In vitro study clearly shows the antibacterial activity against the gram negative and gram positive bacteria. Particularly, synthesised nanofibers provide better antibacterial activity against gram negative than gram positive. Cytotoxicity study also provides better killing rate in cancer cell, biocompatible with normal cell. Prepared CH-CUR-AuNCs nanofibers provide the better killing rate to bacterial strains and cancer cells. Finally, prepared nanofiber can be possible to use for wound healing dressing, patch for skin cancer and other biomedical applications.

Keywords: curcumin, chitosan, gold clusters, nanofibers

Procedia PDF Downloads 239

Authors: Abhilasha Dadhich, Manisha Mathur, Sanjay Kumar, Hemant Dadhich

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The camel (Camelus dromedarius) is an important animal component of the fragile desert eco-system of India. Apart from others, impaired milk and meat production decrease in performance and even death are some of the major consequences of parasitic disease like coccidiosis in camel. Coccidiosis which is an acute invasion and destruction of intestinal mucosa by protozoa of the genera Eimeria or isospora spp. Post-Mortem examinations of 5 carcasses of dromedary of different age groups aged from 2 to 5 years were conducted. The history indicated that the camels were suffering from diarrhoea, dysentery, pyrexia, inappetence, weight loss, and emaciation. Post mortem examinations showed macroscopic and microscopic alterations in the small intestine, particularly in jejunum and ileum regions. The mucosae were congested, and haemorrhagic on which there were numerous whitish-grey nodular foci were observed. The affected intestinal tissue specimens were preserved in 10% formal saline and processed mechanically for paraffin embedding by acetone and benzene technique. The sections were stained with haematoxylin and eosin method of staining for histopathological examinations. Histologically, typical lesions such as congestion and haemorrhages were present. The intestinal villi were oedematous; mucosa degenerated and desquamated, along with infiltration of eosinophils and macrophages. Crypts of lieberkuhn were obliterated due to presence of schizonts in lamina propria. Older camels served as the source of spread of coccidial infection and were also predisposed to secondary infections.

Keywords: camel, coccidiosis, Eimeria, histopathology

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Authors: S. V. Patil, P. S. Dounde, S. S. Patil

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Emulgels have emerged as one of the most interesting topical delivery system as it has dual release control system that is gel and emulsion. The major objective behind this formulation is delivery of hydrophobic drugs to systemic circulation via skin. In fact presence of a gelling agent in water phase converts a classical emulsion in to emulgel. The emulgel for dermatological use has several favorable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, non-staining, water-soluble, longer shelf life, bio-friendly, transparent and pleasing appearance. Various penetration enhancers can potentiate the effect. So this can be used as better topical drug delivery systems over present conventional systems available in market. Piroxicam is a non-steroidal anti-inflammatory drug that has major problems when administered orally; it is an insoluble drug and has irritant effect on gastro intestinal tract lead to ulceration and bleeding. The aim of this study was to overcoming these problems through preparation of topical emulgel of this drug. Emulgel of Piroxicam was prepared using Carbopol 940 along with mentha oil and clove oil as permeation enhancer. The prepared emulgel were evaluated for their physical appearance, pH determination, viscosity, spreadability, in vitro drug release, ex vivo permeation studies. All the prepared formulations showed acceptable physical properties, homogeneity, consistency, spreadability, viscosity and pH value. The emulgel was found to be stable with respect to physical appearance, pH, rheological properties and drug content at all temperature and conditions for three month.

Keywords: emulgel, piroxicam, menthe oil, clove oil

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Authors: Lucian Mocan, Flaviu Tabaran, Teodora Mocan, Cristian Matea, Cornel Iancu

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We present method of Gold Nanoparticle enhanced laser thermal ablation of HepG2 cells (Human hepatocellular liver carcinoma cell line), based on a simple gold nanoparticle carrier system, such as serum albumin (BSA), and demonstrate its selective therapeutic efficacy. Hyperspectral, contrast phase, and confocal microscopy combined immunochemical staining were used to demonstrate the selective internalization of HSA-GNPs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. We examined the ability of laser-activated carbon nanotubes to induce Hsp70 expression using confocal microscopy. Hep G2 cells heat-shocked (laser activated BSA-GNPs) to 42°C demonstrated an up-regulation of Hsp70 compared with control cells (BSA-GNPs treated cells without laser), which showed no detectable constitutive expression of Hsp70. We observed a time-dependent induction in Hsp70 expression in Hep G2 treated with BSA-GNPs and LASER irradiated. The post-irradiation apoptotic rate of HepG2 cells treated with HSA-GNPs ranged from 88.24% (for 50 mg/L) at 60 seconds, while at 30 minute the rate increased to 92.34% (50 mg/L). These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

Keywords: gold nanoparticles, liver cancer, albumin, laser irradiation

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Authors: Samaneh Nabavi, Hadi Shirzad, Somayeh Khanjani, Shirin Jalili

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Quantum dots (QDs) are semiconductor nanocrystals that exhibit intrinsic optical and electrical properties that are size dependent due to the quantum confinement effect. Quantum confinement is brought about by the fact that in bulk semiconductor material the electronic structure consists of continuous bands, and that as the size of the semiconductor material decreases its radius becomes less than the Bohr exciton radius (the distance between the electron and electron-hole) and discrete energy levels result. As a result QDs have a broad absorption range and a narrow emission which correlates to the band gap energy (E), and hence QD size. QDs can thus be tuned to give the desired wavelength of fluorescence emission.Due to their unique properties, QDs have attracted considerable attention in different scientific areas. Also, they have been considered for forensic applications in recent years. The ability of QDs to fluoresce up to 20 times brighter than available fluorescent dyes makes them an attractive nanomaterial for enhancing the visualization of latent fingermarks, or poorly developed fingermarks. Furthermore, the potential applications of QDs in the detection of nitroaromatic explosives, such as TNT, based on directive fluorescence quenching of QDs, electron transfer quenching process or fluorescence resonance energy transfer have been paid to attention. DNA analysis is associated tightly with forensic applications in molecular diagnostics. The amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. Accordingly, highly sensitive detection of human genomic DNA is an essential issue for forensic study. QDs have also a variety of advantages as an emission probe in forensic DNA quantification.

Keywords: forensic science, quantum dots, DNA typing, explosive sensor, fingermark analysis

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Authors: Lina T. Al Kury, Oleg I. Voitychuk, Ramiz M. Ali, Sehamuddin Galadari, Keun-Hang Susan Yang, Frank Christopher Howarth, Yaroslav M. Shuba, Murat Oz

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A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier studies. In the present work, we have hypothesized that the antiarrhythmic effects reported for AEA are due to its negative inotropic effect and altered action potential (AP) characteristics. Therefore, we tested the effects of AEA on contractility and electrophysiological properties of rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) caused a significant decrease in the amplitudes of electrically-evoked myocyte shortening and Ca2+ transients and significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 µg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists). Furthermore, AEA inhibited voltage-activated inward Na+ (INa) and Ca2+ (IL,Ca) currents; major ionic currents shaping the APs in ventricular myocytes, in a voltage and PTX-independent manner. Collectively, the results suggest that AEA depresses ventricular myocyte contractility, by decreasing the action potential duration (APD), and inhibits the function of voltage-dependent Na+ and L-type Ca2+ channels in a manner independent of cannabinoid receptors. This mechanism may be importantly involved in the antiarrhythmic effects of anandamide.

Keywords: action potential, anandamide, cannabinoid receptor, endocannabinoid, ventricular myocytes

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Authors: Ae Ra Han, Taek Hoo Lee, Sun Zoo Kim, Hwa Young Lee

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Introduction: Ovarian endometrioma is one of the important causes of poor ovarian reserve and up to half of them have recurred. However, the treatment for recurrence prevention has limited efficiency and repeated surgical management makes worsen the ovarian reserve. To find better management for recurrence prevention, we investigated risk factors and biomarkers for the recurrence of ovarian endometrioma. Methods: The medical records of women with the history of surgical dissection for ovarian endometrioma were collected. After exclusion of the cases with concurrent hysterectomy, been menopaused during follow-up, incomplete medical record, and loss of follow-up, a total of 134 women were enrolled. Immunohistochemical staining for progesterone receptor isoform B (PR-B) and nuclear factor kappa B (NFκB) was done with the fixed tissue blocks of their endometriomas which were collected at the time of surgery. Results: Severity of dysmenorrhea and co-existence of adenomyosis had significant correlation with recurrence of endometrioma. Increased PR-B (P = .041) and decreased NFκB (P = .036) immunoreactivity were found in recurrent group. Serum CA-125 level at the time of recurrence was higher than the highest level of CA-125 during follow-up in unrecurred group (55.6 vs. 21.3 U/mL, P = .014). Conclusion: We found that the severity of dysmenorrhea and coexistence of adenomyosis are risk factors for recurrence of ovarian endometrioma, and serial follow-up of CA-125 is effective to detect and prevent the recurrence. However, to determine the possibility of immunoreactivity of PR-B and NFκB as biomarkers for ovarian endometrioma, further studies of various races and large numbers with prospective design are needed.

Keywords: endometriosis, recurrence, biomarker, risk factor

Procedia PDF Downloads 533

Authors: Guoqiang Yu, Mehrana Mohtasebi, Jinghong Sun, Thomas Pittman

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Malignant glioma (MG) is the most common type of primary malignant brain tumor. Surgical resection of MG remains the cornerstone of therapy, and the extent of resection correlates with patient survival. A limiting factor for resection, however, is the difficulty in differentiating the tumor from normal tissue during surgery. Fluorescence imaging is an emerging technique for real-time intraoperative visualization of MGs and their boundaries. However, most clinical-grade neurosurgical operative microscopes with fluorescence imaging ability are hampered by low adoption rates due to high cost, limited portability, limited operation flexibility, and lack of skilled professionals with technical knowledge. To overcome the limitations, we innovatively integrated miniaturized light sources, flippable filters, and a recording camera to the surgical eye loupes to generate a wearable fluorescence eye loupe (FLoupe) device for intraoperative imaging of fluorescent MGs. Two FLoupe prototypes were constructed for imaging of Fluorescein and 5-aminolevulinic acid (5-ALA), respectively. The wearable FLoupe devices were tested on tumor-simulating phantoms and patients with MGs. Comparable results were observed against the standard neurosurgical operative microscope (PENTERO® 900) with fluorescence kits. The affordable and wearable FLoupe devices enable visualization of both color and fluorescence images with the same quality as the large and expensive stationary operative microscopes. The wearable FLoupe device allows for a greater range of movement, less obstruction, and faster/easier operation. Thus, it reduces surgery time and is more easily adapted to the surgical environment than unwieldy neurosurgical operative microscopes.

Keywords: fluorescence guided surgery, malignant glioma, neurosurgical operative microscope, wearable fluorescence imaging device

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Authors: M. R. Akbari, H. Yousefnia, A. Ghasemi

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Al2O3:C,Mg fluorescent nuclear track detector (FNTD) and Al2O3:C optically stimulated luminescence detector (OSLD) are becoming two of the applied detectors in ion dosimetry. Therefore, the response of these detectors to hadron beams is highly of interest in radiation therapy (RT) using ion beams. In this study, these detectors' responses to proton and Helium-4 ion beams were compared using Monte Carlo simulations. The calculated data for proton beams were compared with Markus ionization chamber (IC) measurement (in water phantom) from M.D. Anderson proton therapy center. Monte Carlo simulations were performed via the FLUKA code (version 2011.2-17). The detectors were modeled in cylindrical shape at various depths of the water phantom without shading each other for obtaining relative depth dose in the phantom. Mono-energetic parallel ion beams in different incident energies (100 MeV/n to 250 MeV/n) were collided perpendicularly on the phantom surface. For proton beams, the results showed that the simulated detectors have over response relative to IC measurements in water phantom. In all cases, there were good agreements between simulated ion ranges in the water with calculated and experimental results reported by the literature. For proton, maximum peak to entrance dose ratio in the simulated water phantom was 4.3 compared with about 3 obtained from IC measurements. For He-4 ion beams, maximum peak to entrance ratio calculated by both detectors was less than 3.6 in all energies. Generally, it can be said that FLUKA is a good tool to calculate Al2O3:C,Mg FNTD and Al2O3:C OSLD detectors responses to therapeutic proton and He-4 ion beams. It can also calculate proton and He-4 ion ranges with a reasonable accuracy.

Keywords: comparison, FNTD and OSLD detectors response, light ion beams, Monte Carlo simulations

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Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

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Authors: Alessandro Poma, Kal Karim, Sergey Piletsky, Giuseppe Battaglia

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The recent increasing emergency threat to public health of infectious influenza diseases has prompted interest in the detection of avian influenza virus (AIV) H5N1 in humans as well as animals. A variety of technologies for diagnosing AIV infection have been developed. However, various disadvantages (costs, lengthy analyses, and need for high-containment facilities) make these methods less than ideal in their practical application. Molecularly Imprinted Polymeric Nanoparticles (MIP NPs) are suitable to overcome these limitations by having high affinity, selectivity, versatility, scalability and cost-effectiveness with the versatility of post-modification (labeling – fluorescent, magnetic, optical) opening the way to the potential introduction of improved diagnostic tests capable of providing rapid differential diagnosis. Here we present our first results in the production and testing of MIP NPs for the detection of AIV H5N1. Recent developments in the solid-phase synthesis of MIP NPs mean that for the first time a reliable supply of ‘soluble’ synthetic antibodies can be made available for testing as potential biological or diagnostic active molecules. The MIP NPs have the potential to detect viruses that are widely circulating in farm animals and indeed humans. Early and accurate identification of the infectious agent will expedite appropriate control measures. Thus, diagnosis at an early stage of infection of a herd or flock or individual maximizes the efficiency with which containment, prevention and possibly treatment strategies can be implemented. More importantly, substantiating the practicability’s of these novel reagents should lead to an initial reduction and eventually to a potential total replacement of animals, both large and small, to raise such specific serological materials.

Keywords: influenza virus, molecular imprinting, nanoparticles, polymers

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Authors: Hoa Thi Hoang, Stephan Sass, Michael U. Kumke

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Concanavalin A (conA) is a protein has been widely used in sensor system based on its specific binding to α-D-Glucose or α-D-Manose. For glucose sensor using conA, either fluoresence based techniques with intensity based or lifetime based are used. In this research, liposomes made from phospholipids were used as a biomimetic membrane system. In a first step, novel building blocks containing perylene labeled glucose units were added to the system and used to decorate the surface of the liposomes. Upon the binding between rhodamine labeled con A to the glucose units at the biomimetic membrane surface, a Förster resonance energy transfer system can be formed which combines unique fluorescence properties of perylene (e.g., high fluorescence quantum yield, no triplet formation) and its high hydrophobicity for efficient anchoring in membranes to form a novel probe for the investigation of sugar-driven binding reactions at biomimetic surfaces. Two glucose-labeled perylene derivatives were synthesized with different spacer length between the perylene and glucose unit in order to probe the binding of conA. The binding interaction was fully characterized by using high-end fluorescence techniques. Steady-state and time-resolved fluorescence techniques (e.g., fluorescence depolarization) in combination with single-molecule fluorescence spectroscopy techniques (fluorescence correlation spectroscopy, FCS) were used to monitor the interaction with conA. Base on the fluorescence depolarization, the rotational correlation times and the alteration in the diffusion coefficient (determined by FCS) the binding of the conA to the liposomes carrying the probe was studied. Moreover, single pair FRET experiments using pulsed interleaved excitation are used to characterize in detail the binding of conA to the liposome on a single molecule level avoiding averaging out effects.

Keywords: concanavalin A, FRET, sensor, biomimetic membrane

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Authors: K. Swapna

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A Series of Alkali-Earth Boro Tellurite (AEBT) glasses doped with different concentrations of Dy³⁺ ions have been prepared by using melt quenching technique and characterized through spectroscopic techniques such as optical absorption, excitation, emission and photoluminescence decay to understand their utility in optoelectronic devices such as lasers and white light emitting diodes (w-LEDs). Raman spectrum recorded for an undoped glass is used to measure the phonon energy of the host glass and various functional groups present in the host glass (AEBT). The intensities of the electronic transitions and the ligand environment around the Dy³⁺ ions were studied by applying Judd-Ofelt (J-O) theory to the recorded absorption spectra of the glasses. The evaluated J-O parameters are subsequently used to measure various radiative parameters such as transition probability (AR), radiative branching ratio (βR) and radiative lifetimes (τR) for the prominent fluorescent levels of Dy³⁺ ions in the as-prepared glasses. The luminescence spectra recorded at 387 nm excitation show three emission transitions (⁴F9/2→⁶H15/2 (blue), ⁴F9/2→⁶H13/2 (yellow) and ⁴F9/2 → ⁶H11/2 (red)) of which the yellow transition observed at 575 nm is found to be highly intense. The experimental branching ratio (βexp) and stimulated emission crosssection (σse) were measured from luminescence spectra. The experimental lifetimes (τexp) measured from the decay spectral profiles are combined with radiative lifetimes to measure quantum efficiencies of the as-prepared glasses. The yellow to blue intensity ratios and chromaticity color coordinates are found to vary with Dy³⁺ ion concentrations. The aforementioned results reveal that these glasses are aptly suitable for w-LEDs and laser devices.

Keywords: glasses, J-O parameters, photoluminescence, I-H model

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Authors: Vidhula Ahire, Amit Kumar, K. P. Mishra, Gauri Kulkarni

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Herbal polyphenols have gained significance because of their increasing promise in prevention and treatment of cancer. Therefore, development of a dietary compound as an effective radiosensitizer and a radioprotector is highly warranted for cervical cancer patients undergoing therapy. This study describes the cytotoxic effects of the flavonoid, ellagic acid (EA) when administered either alone or in combination with gamma radiation on cervical cancer HeLa cells in vitro. Apoptotic index and proliferation were measured by using trypan blue assay. Reproductive cell death was analyzed by clonogenic assay. Propidium iodide staining for flowcytometry was performed to analyze cell cycle modulation. Nuclear and mitochondrial changes were studied with specific dyes. DNA repair kinetics was analyzed by immunofluorescence assay. Evaluation and comparison of EA effects were performed with other clinically used breast cancer drugs. When tumor cells were exposed to 2 and 4 Gy of irradiation in presence of EA (10 μM), it yielded a synergistic cytotoxic effect on cervical cancer cells whereas in NIH3T3 cells it reversed the injury caused by irradiation and abetted in the regaining of normal healthy cells. At 24h ~25foci/cell was observed and 2.6 fold decrease in the mitochondrial membrane potential. Up to 40% cell were arrested in the G1 phase and 20-36% cells exhibited apoptosis. Our results demonstrate the role of increased apoptosis and cell cycle modulation in the mechanism of EA mediated radiosensitization of cervical cancer cells and thus advocating EA as an adjuvant for preclinical trials in cancer chemo- radiotherapy.

Keywords: cervical cancer, ellagic acid, sensitization, radiation therapy

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Authors: Qing Cao, Fei Wang, Yu-Qiang Wang, Li-Ya Huang, Tian-Tian Sang, Shu-Yan Chen

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Aims: TNF Receptor-Associated Factor 6 (TRAF6) acts as a multifunctional regulator of the Transforming Growth Factor (TGF)-β signaling pathway, and mediates Smad-independent JNK and p38 activation via TGF-β. This study was performed to test the hypothesis that TGF-β/TRAF6 is essential for angiotensin-II (Ang II)-induced differentiation of rat c-kit+ Cardiac Stem Cells (CSCs). Methods and Results: c-kit+ CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-β type I receptor inhibitor (SB431542) or the small interfering RNA (siRNA)-mediated knockdown of TRAF6 were used to investigate the role of TRAF6 in TGF-β signaling. Rescue of TRAF6 siRNA transfected cells with a 3'UTR deleted siRNA insensitive construct was conducted to rule out the off target effects of the siRNA. TRAF6 dominant negative (TRAF6DN) vector was constructed and used to infect c-kit+ CSCs, and western blotting was used to assess the expression of TRAF6, JNK, p38, cardiac-specific proteins, and Wnt signaling proteins. Physical interactions between TRAF6 and TGFβ receptors were studied by coimmunoprecipitation. Cardiac differentiation was suppressed in the absence of TRAF6. Forced expression of TRAF6 enhanced the expression of TGF-β-activated kinase1 (TAK1), and inhibited Wnt signaling. Furthermore, TRAF6 increased the expression of cardiac-specific proteins (cTnT and Cx-43) but inhibited the expression of Wnt3a. Conclusions: Our data suggest that TRAF6 plays an important role in Ang II induced differentiation of c-kit+ CSCs via the non-canonical signaling pathway.

Keywords: cardiac stem cells, differentiation, TGF-β, TRAF6, ubiquitination, Wnt

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Authors: Ruphi Naz, Altaf Ahmad, Mohammad Anis

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Sialic acid (5-Acetylneuraminic acid, Neu5Ac) is a common sugar found as a terminal residue on glycoconjugates in many animals. Humans brain and the central nervous system contain the highest concentration of sialic acid (as N-acetylneuraminic acid) where these acids play an important role in neural transmission and ganglioside structure in synaptogenesis. Due to its important biological function, sialic acid is attracting increasing attention. To understand metabolic networks, fluxes and regulation, it is essential to be able to determine the cellular and subcellular levels of metabolites. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. Taking this, we developed a genetically encoded FRET (fluorescence resonance energy transfer) based nanosensor to analyse the sialic acid level in living cells. Sialic acid periplasmic binding protein (sia P) from Haemophilus influenzae was taken and ligated between the FRET pair, the cyan fluorescent protein (eCFP) and Venus. The chimeric sensor protein was expressed in E. coli BL21 (DE3) and purified by affinity chromatography. Conformational changes in the binding protein clearly confirmed the changes in FRET efficiency. So any change in the concentration of sialic acid is associated with the change in FRET ratio. This sensor is very specific to sialic acid and found stable with the different range of pH. This nanosensor successfully reported the intracellular level of sialic acid in bacterial cell. The data suggest that the nanosensors may be a versatile tool for studying the in vivo dynamics of sialic acid level non-invasively in living cells

Keywords: nanosensor, FRET, Haemophilus influenzae, metabolic networks

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Authors: M. Gerasimou, S. Mantzoukis, P. Christodoulou, N. Varsamis, G. Kolliopoulou, N. Zotos

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Purpose: Microbial infection of the blood is a serious condition where bacteria invade the bloodstream and cause systemic disease. In such cases, blood cultures are performed. Blood cultures are a key diagnostic test for intensive care unit (ICU) patients. Material and method: The BacT/Alert system, which measures the production of carbon dioxide with metabolic organisms, is used. The positive result in the BacT/Alert system is followed by culture in the following selective media: Blood, Mac Conkey No 2, Chocolate, Mueller Hinton, Chapman and Sabaureaud agar. Gram staining method was used to differentiate bacterial species. The microorganisms were identified by biochemical techniques in the automated Microscan (Siemens) system and followed by a sensitivity test on the same system using the minimum inhibitory concentration MIC technique. The sensitivity test is verified by a Kirby Bauer-based test. Results: In 2017 the Laboratory of Microbiology received 3347 blood cultures. Of these, 170 came from the ICU. 116 found positive. Of these S. epidermidis was identified in 42, A. baumannii in 27, K. pneumoniae in 12 (4 of these KPC ‘Klebsiella pneumoniae carbapenemase’), S. hominis in 8, E. faecium in 7, E. faecalis in 5, P. aeruginosa in 3, C. albicans in 3, S. capitis in 2, K. oxytoca in 2, P. mirabilis in 2, E. coli in 1, S. intermidius in 1 and S. lugdunensis in 1. Conclusions: The study of epidemiological data and microbial resistance phenotypes is essential for the choice of therapeutic regimen for the early treatment and limitation of multivalent strains, while it is a crucial factor to solve diagnostic problems.

Keywords: blood culture, bloodstream, infection, intensive care unit

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Authors: Murad Ghanim

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The citrus greening disease, also known as Huanglongbing, caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) has resulted in tremendous losses and the death of millions of citrus trees worldwide. CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The closely-related bacterium Candidatus Liberibacter solanacearum (CLso), which is associated with vegetative disorders in carrots and the zebra chips disease in potatoes, is transmitted by other psyllid species including Bactericera trigonica in carrots and B. ckockerelli in potatoes. Chemical sprays are currently the prevailing method for managing these diseases for limiting psyllid populations; however, they are limited in their effectiveness. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is very limited. CLas induces changes in the nuclear architecture in the midgut of ACP and activates programmed cell death (apoptosis) in this organ. Strikingly, CLso displayed an opposite effect in the gut of B. trigonica, showing limited apoptosis, but widespread necrosis. Electron and fluorescent microscopy further showed that CLas induced the formation of Endoplasmic reticulum (ER) inclusion- and replication-like bodies, in which it increases and multiplies. ER involvement in bacterial replication is hypothesized to be the first stage of an immune response leading to the apoptotic and necrotic responses. ER exploitation and the subsequent events that lead to these cellular and stress responses might activate a cascade of molecular responses ending up with apoptosis and necrosis. Understanding the molecular interactions that underlay the necrotic/apoptotic responses to the bacteria will increase our knowledge of ACP-CLas, and BT-CLso interactions, and will set the foundation for developing novel, and efficient strategies to disturb these interactions and inhibit the transmission.

Keywords: Liberibacter, psyllid, transmission, apoptosis, necrosis

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Authors: Arefeh Jafarian, Mohammad Taghikani, Saied Abroun, Amir Allahverdi, Masoud Soleimani

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Introduction: The miRNAs have key roles in control of pancreatic islet development and insulin secretion. In this regards, current study investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. Findings: After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose as well as extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. In derived IPCs by miR-375 alone are capable to express insulin and other endocrine specific transcription factors, the cells lack the machinery to respond to glucose. The differentiated hMSCs by miR-375 and anti-miR-9 lentiviruses could secrete insulin and c-peptide in a glucose-regulated manner. Conclusion: It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

Keywords: diabetes, differentiation, MSCs, insulin producing cells, miR-375, miR-9

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Authors: Aminu Bashir Mohammad, Agwu Ezera, Abdulrazaq G. Habib, Garba Iliyasu

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Background: Drug resistance tuberculosis (DR-TB) continues to be a challenge in developing countries with poor resources. Routine screening for primary DR-TB before commencing treatment is not done in public hospitals in Nigeria, even with the large body of evidence that shows a high prevalence of primary DR-TB. Data on drug resistance and its genetic determinant among follow up TB patients is lacking in Nigeria. Hence the aim of this study was to determine the prevalence and genetic determinant of drug resistance among follow up TB patients in a tertiary hospital in Nigeria. Methods: This was a cross-sectional laboratory-based study conducted on 384 sputum samples collected from consented follow-up tuberculosis patients. Standard microbiology methods (Zeil-Nielsen staining and microscopy) and PCR (Line Probe Assay)] were used to analyze the samples collected. Person’s Chi-square was used to analyze the data generated. Results: Out of three hundred and eighty-four (384) sputum samples analyzed for mycobacterium tuberculosis (MTB) and DR-TB twenty-five 25 (6.5%) were found to be AFB positive. These samples were subjected to PCR (Line Probe Assay) out of which 18(72%) tested positive for DR-TB. Mutations conferring resistance to rifampicin (rpo B) and isoniazid (katG, and or inhA) were detected in 12/18(66.7%) and 6/18(33.3%), respectively. Transmission dynamic of DR-TB was not significantly (p>0.05) dependent on demographic characteristics. Conclusion: There is a need to strengthened the laboratory capacity for diagnosis of TB and drug resistance testing and make these services available, affordable, and accessible to the patients who need them.

Keywords: drug resistance tuberculosis, genetic determinant, intensive phase, Nigeria

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Authors: Gupta Renu, T. S. Roy

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Introduction: For the prospect of successful replacement therapies in treatment of Diabetes mallitus it is necessary to know events occurring during normal human pancreas development. Literature of human pancreas development are few in number as well as mainly related to first trimester because of ethical and technical difficulties. So the study was conducted on 12 fetuses from 12 gestational weeks (GW) to 5 months of infant to know normal development of exocrine and endocrine part of human pancreas. Material and Methods: Human fetalpancreases were screened by haematoxyline and eosin staining and done electron microscopy for suitable specimens to know ultrastructural detail of fetal pancreas. Results:It was observed arborized tubules, the cells budding out from these tubules differentiated into primitive acini and islets in 12thGW. At 14 weeks scanty granules were observed in the endocrine cells which coincided with the capillary invasion of the islets. The ducts and acini were surrounded by well-organized connective tissue. The acinihad elongated cells, small amount of cytoplasm and large open face euchromatic nuclei with single nucleolus. The mature form of islets of Langerhans was observed close to the acini and duct in 20 GW fetus. Connective tissue around the duct was well organized.No significant developmental change was observed early postnatal, infant. Conclusion: The development of both component exocrine as well as endocrine part of human fetal pancreas was studied by light and electron microscopy. Observations suggested that the fetal pancreas contained mainly ducts, few acini, many centroacinar cells, and large undifferentiated tissue.

Keywords: gestational weeks (GW), acini, islets of Langerhans, ducts

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Authors: Sizhu Wang, Cuisong Tang, Lin Zhang, Guangyu Tang

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Objective: We aimed to detect the composition of bone marrow fatty acids early after ovariectomized (OVX) surgery and explore the potential mechanism. Methods: Thirty-two female Sprague-Dawley (SD) rats (12 weeks) were randomly divided into OVX group and Sham group (N=16/group), and received ovariectomy or sham surgery respectively. After 3 and 28 days, eight rats in each group were sacrificed to detect the composition of bone marrow fatty acids by gas chromatography–mass spectrometry (GC–MS) and evaluate the trabecular bone microarchitecture by micro-CT. Significant different fatty acids in the early stage of post-menopausal osteoporosis were selected by OPLS-DA and t test. Then selected fatty acids were further studied in the process of osteogenic differentiation through RT-PCR and Alizarin Red S staining. Results: An apparent sample clustering and group separation were observed between OVX group and sham group three days after surgery, which suggested the role of bone marrow fatty acids in the early stage of postmenopausal osteoporosis. Specifically, myristate, palmitoleate and arachidonate were found to play an important role in classification between OVX group and sham group. We further investigated the effect of palmitoleate and arachidonate on osteogenic differentiation and found that palmitoleate promoted the osteogenic differentiation of MC3T3-E1 cells while arachidonate inhibited this process. Conclusion: Profound bone marrow fatty acids changes have taken place in the early stage of post-menopausal osteoporosis. Bone marrow fatty acids may begin to affect osteogenic differentiation shortly after deficiency of estrogen.

Keywords: bone marrow fatty acids, GC-MS, osteoblast, osteoporosis, post-menopausal

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Authors: Alaa E. El-sisi, Sherin Zakaria

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Rebamipide is an antiulcer drug with unique properties such as anti-inflammatory action. It induces endogenous prostaglandin e2 (PGE2). PGE2 is considered as a potent physiological suppressor of liver fibrosis. Aim of study: This study investigated the effect of rebamipide on hepatic fibrosis. Material and Method: Hepatic fibrosis was induced by intraperitoneal injections (IP) injection of CCl4 (0.45 mL/kg) in corn oil 1:5 twice a week for 4 weeks. Rats were divided into four groups as follow: Group 1 treated with CCL4 only, group 2 and 3 treated with CCL4 and rebamipide 60 mg/kg/day (group2) or 100 mg/kg/day (group3), and the fourth group was considered as control group and treated with vehicles. ALT, AST, and Bilirubin were assayed in serum. Antioxidant markers such as malondialdhyde (MDA) and superoxide dismutase (SOD) and fibrotic markers such as hyaluronic acid (HA) and procollagen-III (procol-III) were evaluated in liver tissues. IL-10 as well as PGE2 were also assayed in liver tissues. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen precipitation in liver tissues was visualized using masson trichrom stain. Results: Rebamipide inhibit CCL4 induced increase in ALT and AST significantly (p < 0.05). Rebamipide exerted an antioxidant effect as it inhibits CCL4 induced increased MDA level and decreased SOD activity. Fibrotic markers assay revealed that repamipide (60 or 100 mg/kg/day) decreased the level of procol-III and HA compared to CCl4 (p < 0.05). Oral administration of Rebamipide was associated with a significant increase (p < 0.05) of PGE2 and IL-10. Rebamipide especially at the dose of (100 mg/kg/day) restores liver histology structure and abolish collagen precipitation in liver tissues. Conclusion: Rebamipide retards hepatic fibrosis induced by CCL4 may be through the induction of PGE2 level.

Keywords: fibrotic markers, hepatic fibrosis, PGE2, rebamipide

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Authors: Xiao Wei, Dandan Sheng, Xiaoyan Jiang, Lili Gui, Huizhu Wang, Xi Yu, Hailiang Liu, Min Hong

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Objective: Mice Type2 allergic contact dermatitis was utilized in this study to explore the effect of AS-IV on Type 2 allergic inflammatory. Methods: The mice were topically sensitized on the shaved abdomens with 1.5% FITC solution on abdominal skin in the day 1 and day 2 and elicited on the right ear with 0.5% FITC solution at day 6. Mice were treated with either AS-IV or normal saline from day 1 to day 5 (induction phase). Auricle swelling was measured 24 h after the elicitation. Ear pathohistological examination was carried out by HE staining. IL-4\IL-13, and IL-9 levels of ear tissue were detected by ELISA. Mice were treated with AS-IV at the initial stage of induction phase, ear tissue was taked at day 3.TSLP level of ear tissue was detected by ELISA and TSLPmRNA\NF-kBmRNA\TLRs（TLR2\TLR3\TLR8\TLR9）mRNA were detected by PCR. Results: AS-IV induction phase evidently inhibited the auricle inflam-mation of the models; pathohistological results indicated that AS-IV induction phase alleviated local edema and angiectasis of mice models and reduced lymphocytic infiltration. AS-IV induction phase markedly decreased IL-4\IL-13, and IL-9 levels in ear tissue. Moreover, at the initial stage of induction pha-se, AS-IV significantly reduced TSLP\TSLPmRNA\NF-kBmRNA\TLR2mRNA\TLR8 mRNA levels in ear tissue. Conclusion: Administration with AS-IV in induction phase could inhibit Type 2 allergic contact dermatitis in mice significantly, and the mechanism may be related with regulating TSLP through TLRs-NF-kB pathway.

Keywords: Astragaioside IV, allergic contact dermatitis, TSLP, interleukin-4, interleukin-13, interleukin-9

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Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

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Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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