Search results for: in vitro biofilm formation

Authors: Jyh-Ping Chen, Yu-Tin Lai

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Wound healing is a complicated process involving overlapping hemostasis, inflammation, proliferation, and maturation phases. Ideal wound dressings can replace native skin functions in full thickness skin wounds through faster healing rate and also by reducing scar formation. Poly(lactic-co-glycolic acid) (PLGA) is an U.S. FDA approved biodegradable polymer to be used as ideal wound dressing material. Several in vitro and in vivo studies have demonstrated the effectiveness of curcumin in decreasing the release of inflammatory cytokines, inhibiting enzymes associated with inflammations, and scavenging free radicals that are the major cause of inflammation during wound healing. Heparin has binding affinities to various growth factors. With the unique and beneficial features offered by those molecules toward the complex process of wound healing, we postulate a composite wound dressing constructed from PLGA, curcumin and heparin would be a good candidate to accelerate scarless wound healing. In this work, we use electrospinning to prepare curcumin-loaded aligned PLGA nanofibrous membranes (PC NFMs). PC NFMs were further subject to oxygen plasma modification and surfaced-grafted with heparin through carbodiimide-mediated covalent bond formation to prepare curcumin-loaded PLGA-g-heparin (PCH) NFMs. The nanofibrous membranes could act as three-dimensional scaffolds to attract fibroblast migration, reduce inflammation, and increase wound-healing related growth factors concentrations at wound sites. From scanning electron microscopy analysis, the nanofibers in each NFM are with diameters ranging from 456 to 479 nm and with alignment angles within  0.5°. The NFMs show high tensile strength and good water absorptivity and provide suitable pore size for nutrients/wastes transport. Exposure of human dermal fibroblasts to the extraction medium of PC or PCH NFM showed significant protective effects against hydrogen peroxide than PLGA NFM. In vitro wound healing assays also showed that the extraction medium of PCH NFM showed significantly better migration ability toward fibroblasts than PC NFM, which is further better than PLGA NFM. The in vivo healing efficiency of the NFMs was further evaluated by a full thickness excisional wound healing diabetic rat model. After 14 days, PCH NFMs exhibits 86% wound closure rate, which is significantly different from other groups (79% for PC and 73% for PLGA NFM). Real-time PCR analysis indicated PC and PCH NFMs down regulated anti-oxidative enzymes like glutathione peroxidase (GPx) and superoxide dismutase (SOD), which are well-known transcription factors involved in cellular inflammatory responses to stimuli. From histology, the wound area treated with PCH NFMs showed more vascular lumen formation from immunohistochemistry of α-smooth muscle actin. The wound site also had more collagen type III (65.8%) expression and less collagen type I (3.5%) expression, indicating scar-less wound healing. From Western blot analysis, the PCH NFM showed good affinity toward growth factors from increased concentration of transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2) at the wound site to accelerate wound healing. From the results, we suggest PCH NFM as a promising candidate for wound dressing applications.

Keywords: Curcumin, heparin, nanofibrous membrane, poly(lactic-co-glycolic acid) (PLGA), wound dressing

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Authors: B. S. Muddukrishna, Karthik Aithal, Aravind Pai

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Introduction: Preparation of binary cocrystals of Etraverine (ETR) by using Tartaric Acid (TAR) as a conformer was the main focus of this study. Etravirine is a Class IV drug, as per the BCS classification system. Methods: Cocrystals were prepared by slow evaporation technique. A mixture of total 500mg of ETR: TAR was weighed in molar ratios of 1:1 (371.72mg of ETR and 128.27mg of TAR). Saturated solution of Etravirine was prepared in Acetone: Methanol (50:50) mixture in which tartaric acid is dissolved by sonication and then this solution was stirred using a magnetic stirrer until the solvent got evaporated. Shimadzu FTIR – 8300 system was used to acquire the FTIR spectra of the cocrystals prepared. Shimadzu thermal analyzer was used to achieve DSC measurements. X-ray diffractometer was used to obtain the X-ray powder diffraction pattern. Shake flask method was used to determine the equilibrium dynamic solubility of pure, physical mixture and cocrystals of ETR. USP buffer (pH 6.8) containing 1% of Tween 80 was used as the medium. The pure, physical mixture and the optimized cocrystal of ETR were accurately weighed sufficient to maintain the sink condition and were filled in hard gelatine capsules (size 4). Electrolab-Tablet Dissolution tester using basket apparatus at a rotational speed of 50 rpm and USP phosphate buffer (900 mL, pH = 6.8, 37 ˚C) + 1% Tween80 as a media, was used to carry out dissolution. Shimadzu LC-10 series chromatographic system was used to perform the analysis with PDA detector. An Hypersil BDS C18 (150mm ×4.6 mm ×5 µm) column was used for separation with mobile phase comprising of a mixture of ace¬tonitrile and phosphate buffer 20mM, pH 3.2 in the ratio 60:40 v/v. The flow rate was 1.0mL/min and column temperature was set to 30°C. The detection was carried out at 304 nm for ETR. Results and discussions: The cocrystals were subjected to various solid state characterization and the results confirmed the formation of cocrystals. The C=O stretching vibration (1741cm-1) in tartaric acid was disappeared in the cocrystal and the peak broadening of primary amine indicates hydrogen bond formation. The difference in the melting point of cocrystals when compared to pure Etravirine (265 °C) indicates interaction between the drug and the coformer which proves that first ordered transformation i.e. melting endotherm has disappeared. The difference in 2θ values of pure drug and cocrystals indicates the interaction between the drug and the coformer. Dynamic solubility and dissolution studies were also conducted by shake flask method and USP apparatus one respectively and 3.6 fold increase in the dynamic solubility were observed and in-vitro dissolution study shows four fold increase in the solubility for the ETR: TAR (1:1) cocrystals. The ETR: TAR (1:1) cocrystals shows improved solubility and dissolution as compared to the pure drug which was clearly showed by solid state characterization and dissolution studies.

Keywords: dynamic solubility, Etraverine, in vitro dissolution, slurry method

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Authors: Wanbao Chen, Qianqian Yao, Zhenming Zhou

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Whole grains with intact pericarp are largely resistant to digestion by ruminants because entire kernels are not conducive to bacterial attachment. But processing methods makes the starch more accessible to microbes, and increases the rate and extent of starch degradation in the rumen. To estimate the feasibility of applying a steam-flaking as the processing technique of grains for ruminants, cereal grains (maize, wheat, barley and sorghum) were processed by steam-flaking (steam temperature 105°C, heating time, 45 min). And chemical analysis, in vitro gas production, volatile fatty acid concentrations, and energetic values were adopted to evaluate the effects of steam-flaking. In vitro cultivation was conducted for 48h with the rumen fluid collected from steers fed a total mixed ration consisted of 40% hay and 60% concentrates. The results showed that steam-flaking processing had a significant effect on the contents of neutral detergent fiber and acid detergent fiber (P < 0.01). The concentration of starch gelatinization degree in all grains was also great improved in steam-flaking grains, as steam-flaking processing disintegrates the crystal structure of cereal starch, which may subsequently facilitate absorption of moisture and swelling. Theoretical maximum gas production after steam-flaking processing showed no great difference. However, compared with intact grains, total gas production at 48 h and the rate of gas production were significantly (P < 0.01) increased in all types of grain. Furthermore, there was no effect of steam-flaking processing on total volatile fatty acid, but a decrease in the ratio between acetate and propionate was observed in the current in vitro fermentation. The present study also found that steam-flaking processing increased (P < 0.05) organic matter digestibility and energy concentration of the grains. The collective findings of the present study suggest that steam-flaking processing of grains could improve their rumen fermentation and energy utilization by ruminants. In conclusion, the utilization of steam-flaking would be practical to improve the quality of common cereal grains.

Keywords: cereal grains, gas production, in vitro rumen fermentation, steam-flaking processing

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Authors: Er-Yuan Chuang

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Bio-mimetic matters have biological functionalities. This might be valuable in the development of versatile biomaterials. At biological fields, protein-based materials might be components to form a 3D network of extracellular biomolecules, containing growth factors. Also, the protein-based biomaterial provides biochemical and structural assistance of adjacent cells. In this study, we try to prepare protein based biomaterial, which was harvested from living animal. We analyzed it’s chemical, physical and biological property in vitro. Besides, in vivo bio-interaction of the prepared biomimetic matrix was tested in an animal model. The protein-based biomaterial has degradability and biocompatibility. This development could be used for tissue regenerations and be served as platform technologies.

Keywords: protein based, in vitro study, in vivo study, biomaterials

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Authors: Abdul Matin, Salik Nawaz, Suk-Yul Jung

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Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated > 90% binding and > 70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore, macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall, to our best knowledge, we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Keywords: Balamuthia mandrillaris, macrophages, cytokines, human brain microvascular endothelial cells, Balamuthia amoebic encephalitis

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Authors: Rajasekhar Reddy Poonuru, Anusha Parnem

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Purpose: To formulate proliposome powder of agomelatine, an antipsychotic drug, and to evaluate physicochemical, in vitro characters and effect of surface charge on ex vivo intestinal permeation. Methods: Film deposition technique was employed to develop proliposomal powders of agomelatin with varying molar ratios of lipid Hydro Soy PC L-α-phosphatidylcholine (HSPC) and cholesterol with fixed sum of drug. With the aim to derive free flowing and stable proliposome powder, fluid retention potential of various carriers was examined. Liposome formation and number of vesicles formed for per mm3 up on hydration, vesicle size, and entrapment efficiency was assessed to deduce an optimized formulation. Sodium cholate added to optimized formulation to induce surface charge on formed vesicles. Solid-state characterization (FTIR, DSC, and XRD) was performed with the intention to assess native crystalline and chemical behavior of drug. The in vitro dissolution test of optimized formulation along with pure drug was evaluated to estimate dissolution efficiency (DE) and relative dissolution rate (RDR). Effective permeability co-efficient (Peff(rat)) in rat and enhancement ratio (ER) of drug from formulation and pure drug dispersion were calculated from ex vivo permeation studies in rat ileum. Results: Proliposomal powder formulated with equimolar ratio of HSPC and cholesterol ensued in higher no. of vesicles (3.95) with 90% drug entrapment up on hydration. Neusilin UFL2 was elected as carrier because of its high fluid retention potential (4.5) and good flow properties. Proliposome powder exhibited augmentation in DE (60.3 ±3.34) and RDR (21.2±01.02) of agomelation over pure drug. Solid state characterization studies demonstrated the transformation of native crystalline form of drug to amorphous and/or molecular state, which was in correlation with results obtained from in vitro dissolution test. The elevated Peff(rat) of 46.5×10-4 cm/sec and ER of 2.65 of drug from charge induced proliposome formulation with respect to pure drug dispersion was assessed from ex vivo intestinal permeation studies executed in ileum of wistar rats. Conclusion: Improved physicochemical characters and ex vivo intestinal permeation of drug from charge induced proliposome powder with Neusilin UFL2 unravels the potentiality of this system in enhancing oral delivery of agomelatin.

Keywords: agomelatin, proliposome, sodium cholate, neusilin

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Authors: D. R. Masvodza, G. Coetzer, E. van der Watt

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Microorganisms usually have a prolific growth nature and may cause major problems on in-vitro cultures. For in vitro propagation to be successful explants need to be sterile. In order to determine the best sterilization method for potato explants cv. Amerthyst, five sterilization methods were applied separately to 24 shoots. The first sterilization method was the use of 20% sodium hypochlorite with 1 ml Tween 20 for 15 minutes. The second, third and fourth sterilization methods were the immersion of explants in 70% ethanol in a beaker for either 30 seconds, 1 minute or 2 minutes, followed by 1% sodium hypochlorite with 1 ml Tween 20 for 5 minutes. For the control treatment, no chemicals were used. Finally, all the explants were rinsed three times with autoclaved distilled water and trimmed to 1-2 cm. Explants were then cultured on MS medium with 0.01 mg L-1 NAA and 0.1 mg L-1 GA3 and supplemented with 2 mg L-1 D-calcium pentothenate. The trial was laid out as a complete randomized design, and each treatment combination was replicated 24 times. At 7, 14 and 21 days after culture, data on explant color, survival, and presence or absence of contamination was recorded. Best results were obtained when 20% sodium hypochlorite was used with 1 ml Tween 20 for 15 minutes which is sterilization method 1. Method 2 was comparable to method 1 when explants were cultured in glass vessels. Explants in glass vessels were significantly less contaminated than explants in polypropylene vessel. Therefore at times, ideal methods for sterilization should be coupled with ideal culture conditions such as good quality culture vessel, rather than the addition of more stringent sterilants.

Keywords: culture containers, explants, sodium hypochlororite, sterilization

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Authors: Mardiati Zain, W. S. N. Rusmana, Erpomen, Malik Makmur, Ezi Masdia Putri

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Background and Aim: The leaves of the Leucaenaleucocephala tree have potential as a nitrogen source for ruminants. Leucaena leaf meal as protein supplement has been shown to improve the feed quality of ruminants. The effects of different levels of Leucaena leucocephala supplementation as substitute of concentrate on fiber digestibility and in vitro rumen fermentation characteristics were investigated. This research was conducted in vitro. The study used a randomized block design consisting of 3 treatments and 5 replications. The treatments were A. 40% rice straw ammoniated + 60% concentrate, B. 40% rice straw ammoniated + 50% concentrate + 10% Leucaena leuchephala, C. 40% rice straw ammoniated + 40% concentrate + 20% Leucaena leuchephala, Result: The results showed that the addition of Leucaena leucocephala increased the digestibility of Neutral detergent Fiber NDF and Acid Detergent Fiber (ADF) (p < 0.05). In this study, rumen NH3, propionate, amount of escape protein and total Volatyl Fatty Acid (VFA) were found increased significantly at treatment B. No significant difference was observed in acetate and butyrate production. The populations of total protozoa and methane production had significantly decreased (P < .05) in supplemented group. Conclusion: Supplementation of leuchaena leucochepala on completed feed based on ammoniated rice straw in vitro can increase fiber digestibility, VFA production and decreased protozoa pupulataion and methane production. Supplementation of 10% and 20% L. leucochepala were suitable to be used for further studies, therefore in vivo experiment is required to study the effects on animal production.

Keywords: digestibility, Leucaena leucocephala, complete feed, rice straw ammoniated

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Authors: Ž. Vaštag, Lj. Popović, S. Popović

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After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilized as raw material for processing of bio-functional hydrolysates. In this study, the in vitro bioactivity of an alcalase (AH) and a pepsin hydrolysate (PH) prepared from the major pumpkin 12S globulin (cucurbitin) are compared. The hydrolysates were produced at optimum reaction conditions (temperature, pH) for the enzymes, during 60min. The bioactivity testing included antioxidant and angiotensin I converting enzyme inhibitory activity assays. The hydrolysates showed high potential as natural antioxidants and possibly antihypertensive agents in functional food or nutraceuticals. Additionally, preliminary studies have shown that both hydrolysates could exhibit modest α-amylase inhibitory activity, which indicates on their hypoglycemic potential.

Keywords: cucurbitin, alcalase, pepsin, protein hydrolysates, in vitro bioactivity

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Authors: Samir Dekali, David Crouzier

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Due to their new physico-chemical properties engineered nanoparticles (ENPs) are increasingly employed in numerous industrial sectors (such as electronics, textile, aerospace, cosmetics, pharmaceuticals, food industry, etc). These new physico-chemical properties can also represent a threat for the human health. Consumers can notably be exposed involuntarily by different routes such as inhalation, ingestion or through the skin. Several studies recently reported a possible biodistribution of these ENPs on the blood-brain barrier (BBB). Consequently, there is a great need for developing BBB in vitro models representative of the in vivo situation and capable of rapidly and accurately assessing ENPs toxic effects and their potential translocation through this barrier. In this study, several in vitro models established with micro-endothelial brain cell lines of different origins (bEnd.3 mouse cell line or a new human cell line) co-cultivated or not with astrocytic cells (C6 rat or C8-B4 mouse cell lines) on Transwells® were compared using different endpoints: trans-endothelial resistance, permeability of the Lucifer yellow and protein junction labeling. Impact of NIST diesel exhaust particles on BBB cell viability is also discussed.

Keywords: nanoparticles, blood-brain barrier, diesel exhaust particles, toxicology

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Authors: Abu Hasan, Rochmadi, Hary Sulistyo, Suharto Honggokusumo

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This research purpose is to study the effect of Ingredients mixing sequence in rubber compounding onto the formation of bound rubber and cross link density of natural rubber and also the relationship of bound rubber and cross link density. Analysis of bound rubber formation of rubber compound and cross link density of rubber vulcanizates were carried out on a natural rubber formula having masticated and mixing, followed by curing. There were four methods of mixing and each mixing process was followed by four mixing sequence methods of carbon black into the rubber. In the first method of mixing sequence, rubber was masticated for 5 min and then rubber chemicals and carbon black N 330 were added simultaneously. In the second one, rubber was masticated for 1 min and followed by addition of rubber chemicals and carbon black N 330 simultaneously using the different method of mixing then the first one. In the third one, carbon black N 660 was used for the same mixing procedure of the second one, and in the last one, rubber was masticated for 3 min, carbon black N 330 and rubber chemicals were added subsequently. The addition of rubber chemicals and carbon black into masticated rubber was distinguished by the sequence and time allocated for each mixing process. Carbon black was added into two stages. In the first stage, 10 phr was added first and the remaining 40 phr was added later along with oil. In the second one to the fourth one, the addition of carbon black in the first and the second stage was added in the phr ratio 20:30, 30:20, and 40:10. The results showed that the ingredients mixing process influenced bound rubber formation and cross link density. In the three methods of mixing, the bound rubber formation was proportional with crosslink density. In contrast in the fourth one, bound rubber formation and cross link density had contradictive relation. Regardless of the mixing method operated, bound rubber had non linear relationship with cross link density. The high cross link density was formed when low bound rubber formation. The cross link density became constant at high bound rubber content.

Keywords: bound-rubber, cross-link density, natural rubber, rubber mixing process

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Authors: Chih-Hsiang Chang, Fang-Jyun Su

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This paper explores whether stock characteristics influence the herding formation among investors in the US equity market. To extend the research scope of the existing literature, this paper further examines the role that stock risk characteristics play in the US equity market, and the way they influence investors’ decision-making. First, empirical results show that whether general stocks or high-risk stocks, there are no herding behaviors among the investors in the US equity market during the whole research period or during four great events. Moreover, stock characteristics have great influence on investors’ trading decisions. Finally, there is a bidirectional lead-lag relationship of the herding formation between high-risk stocks and low-risk stocks, but the influence of high-risk stocks on the low-risk stocks is stronger than that of low-risk stocks on the high-risk stocks.

Keywords: stock characteristics, herding formation, investment decision, US equity market, lead-lag relationship

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Authors: Hatem K. Hamed, Mohamed S. Hrouda

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The Middle Paleocene Lower Beda Formation was deposited in a ramp system with local shoaling. The main constituent is limestone, with subordinate dolomites and Shales. Reservoir quality is largely influenced by depositional environments and diagenesis processes. Generally the reservoir quality of Lower Beda Formation is low risk on the Inferred Horst and in the Southern Shelf where the Lower Beda formation comprises mainly of calcarenties. In the vicinity of the well GG1 the Lower Beda comprise mainly of argillaceous calcilutites and shale. The reservoir quality gradually improves from high risk to moderate risk towards KK1, LL1 and NN1 wells. The average gross thickness of Lower Beda Formation is about 300 ft. The net thickness varies from about 270 ft. in the E1-71 well to about 30 ft. in the vicinity of GG1-71 well. The net thickest of Lower Beda form a NNW-SSW trend with an average of 250 ft. the change in facies is due to change in the depositional environment, from lagoonal to shoal barrier to open marine affected the reservoir quality. The Upper Cretaceous Sirte Shale is the main source rock. It is developed within the three troughs surrounding the study area. S-Marada Trough to the N- E, Gerad Trough to the N N-W, and Abu Tummym Sub-basin to the S-W of the Inferred Horst. Sirte shale reaches 1000ft, of organically rich section. It has good organic contents over large area 2% to 3%. Hydrocarbon shows were encountered in several wells in Beda Formation this is an indication of vertical and lateral migration of hydrocarbon. The overlying Upper Paleocene Khalifa Formation is a transgressive shale, it is an effective regional top seal. Lithofacies variations in Khalifa Shale, from shales to limestones in the southern shelf in R1-71 well approximately 50-75% of the secession is limestone. About 47 million barrel of hydrocarbon recoverable reserves is expected to be trapped in structural and stratigraphic traps in Beda Formation in the study area.

Keywords: Sirte basin, Beda formation, concession 71, petroleum play fairway analysis

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Authors: Athanasios Triantafyllou, Georgios Papagiannis, Vassilios Nikolaou, Panayiotis J. Papagelopoulos, George C. Babis

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In vitro measurements are widely used in order to predict THAs wear rate implementing gait kinematic and kinetic parameters. Clinical tests of materials and designs are crucial to prove the accuracy and validate such measurements. The purpose of this study is to examine the affection of THA gait kinematics and kinetics on wear during gait, the essential functional activity of humans, by comparing in vivo gait data to in vitro results. Our study hypothesis is that both implants will present the same hip joint kinematics and kinetics during gait. 127 unilateral primary cementless total hip arthroplasties were included in the research. Independent t-tests were used to identify a statistically significant difference in kinetic and kinematic data extracted from 3D gait analysis. No statistically significant differences observed at mean peak abduction, flexion and extension moments between the two groups (P.abduction= 0,125, P.flexion= 0,218, P.extension= 0,082). The kinematic measurements show no statistically significant differences too (Prom flexion-extension= 0,687, Prom abduction-adduction= 0,679). THA kinematics and kinetics during gait are important biomechanical parameters directly associated with implants wear. In vitro studies report less wear in CoC than CoXLPE when tested with the same gait cycle kinematic protocol. Our findings confirm that both implants behave identically in terms of kinematics in the clinical environment, thus strengthening in vitro results of CoC advantage. Correlated to all other significant factors that affect THA wear could address in a complete prism the wear on CoC and CoXLPE.

Keywords: total hip arthroplasty biomechanics, THA gait analysis, ceramic on ceramic kinematics, ceramic on XLPE kinetics, total hip replacement wear

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Authors: Nursyafiqah Abdul Kahar, Niraku Rosmawati Ahmad, Hisham Mohamad, Siti Nuruljannah Mohd Marzuki

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Assessment of geotechnical properties of the subsoil is necessary for generating relevant input for the design and construction of a foundation. It is significant for the future development in the area. The focus of this research is to investigate the preliminary geotechnical properties of the uncemented sandstone from Kati formation at Puncak Iskandar, Seri Iskandar. A series of basic soil tests, oedometer and direct shear box tests were carried out to obtain the soil parameters. The uncemented sandstone of Kati Formation was found to have well-graded and poorly graded sand distribution, depending on the location where the samples were obtained. The sand grains distribution was in a range of 82%-100% while, the specific gravity of the uncemented sandstone is in the range 2.65-2.86. The preconsolidation pressure for USB3 was 990 kPa indicating that the sandstone at USB3 sample had undergone 990 kPa of overburden pressure. The angle of friction for uncemented sandstone was ranging between 23.34°-32.92°.

Keywords: geotechnical properties, Kati formation, uncemented sandstone, oedometer test; shear box test

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Authors: A. C. Rana, Abhinav Singh Rana

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Layer by layer coating of biocompatible polyelectrolytes converts the liposomes into stable version i.e 'layersomes'. This system was further used to deliver the Amphotericin B through the oral route. Extensive optimization of different process variables resulted in the formation of layersomes with the particle size of 238.4±5.1, PDI of 0.24±0.16, the zeta potential of 34.6±1.3, and entrapment efficiency of 71.3±1.2. TEM analysis further confirmed the formation of spherical particles. Trehalose (10% w/w) resulted in the formation of fluffy and easy to redisperse cake in freeze dried layersomes. Controlled release up to 50 % within 24 h was observed in the case of layersomes. The layersomes were found stable in simulated biological fluids and resulted in the 3.59 fold higher bioavailability in comparison to free Amp-B. Furthermore, the developed formulation was found to be safe in comparison to Fungizone as indicated by blood urea nitrogen (BUN) and creatinine level.

Keywords: amphotericin B, layersomes, liposomes, toxicity

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Authors: Manjula Weerasekera, Chris Sissons, Lisa Wong, Sally Anderson, Ann Holmes, Richard Cannon

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The aim of this study was to characterize bacterial population and yeasts in saliva by Polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and measure yeast levels by culture. PCR-DGGE was performed to identify oral bacteria and yeasts in 24 saliva samples. DNA was extracted and used to generate DNA amplicons of the V2–V3 hypervariable region of the bacterial 16S rDNA gene using PCR. Further universal primers targeting the large subunit rDNA gene (25S-28S) of fungi were used to amplify yeasts present in human saliva. Resulting PCR products were subjected to denaturing gradient gel electrophoresis using Universal mutation detection system. DGGE bands were extracted and sequenced using Sanger method. A potential relationship was evaluated between groups of bacteria identified by cluster analysis of DGGE fingerprints with the yeast levels and with their diversity. Significant interpersonal variation of salivary microbiome was observed. Cluster and principal component analysis of the bacterial DGGE patterns yielded three significant major clusters, and outliers. Seventeen of the 24 (71%) saliva samples were yeast positive going up to 10³ cfu/mL. Predominately, C. albicans, and six other species of yeast were detected. The presence, amount and species of yeast showed no clear relationship to the bacterial clusters. Microbial community in saliva showed a significant variation between individuals. A lack of association between yeasts and the bacterial fingerprints in saliva suggests the significant ecological person-specific independence in highly complex oral biofilm systems under normal oral conditions.

Keywords: bacteria, denaturing gradient gel electrophoresis, oral biofilm, yeasts

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Authors: Sihem Benmimoune, Abdelbaki Lemgharbi, Ahmed Ait Yahia, Abdelkrim Kameli

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Our study is based on chemical and phytochemical characterization of Artemisia absinthium L and in vitro tests to demonstrate the biological activities of essential oil and natural extract. A qualitative and quantitative comparison of the essential oil extracted by two extraction procedures was performed by analysis of CG/SM and the yield calculation. The method of hydrodistillation has a chemical composition and provides oil content than the best training water vapor. These oils are composed mainly of thujone followed chamazulene and ρ-cymene. The antimicrobial activity of wormwood oil was tested in vitro by two methods (agar diffusion and microdilution) on four plant pathogenic fungi (Aspergillus sp, Botrytis cinerea, Fusarium culmorum and Helminthosporium sp). The study of the antifungal effect showed that this oil has an inhibitory effect counterpart the microorganisms tested in particular the strain Botrytis cinerea. Otherwise, this activity depends on the nature of the oil and the germ itself. The antioxidant activity in vitro was studied with the DPPH method. The activity test shows that the oil and extract of Artemisia absinthium have a very low antioxidant capacity compared to the antioxidants used as a reference. The extract has a potentially high antiradical power not from its oil. The quantitative determinations of phenolic compounds by the Folin-Ciocalteu revealed that absinthe is low in total polyphenols and tannins.

Keywords: artemisia absinthium, biological activities, essential oil, extraction processes

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Authors: Marek Halenár, Eva Tvrdá, Tomáš Slanina, Ľubomír Ondruška, Eduard Kolesár, Peter Massányi, Adriana Kolesárová

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The present in vitro study was designed to reveal whether amygdalin (AMG) is able to cause changes to the motility, viability and mitochondrial activity of rabbit spermatozoa. New Zealand White rabbits (n = 10) aged four months were used in the study. Semen samples were collected from each animal and used for the in vitro incubation. The samples were divided into five equal parts and diluted with saline supplemented with 0, 0.5, 1, 2.5 and 5 mg/mL AMG. At times 0h, 3h and 5h spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system, cell viability was examined with the metabolic activity (MTT) assay, and the eosin-nigrosin staining technique was used to evaluate the viability of rabbit spermatozoa. All AMG concentrations exhibited stimulating effects on the spermatozoa activity, as shown by a significant preservation of the motility (P<0.05 with respect to 0.5 mg/mL and 1 mg/mL AMG; Time 5 h) and mitochondrial activity (P< 0.05 in case of 0.5 mg/mL AMG; P< 0.01 in case of 1 mg/mL AMG; P < 0.001 with respect to 2.5 mg/mL and 5 mg/mL AMG; Time 5 h). None of the AMG doses supplemented had any significant impact of the spermatozoa viability. In conclusion, the data revealed that short-term co-incubation of spermatozoa with AMG may result in a higher preservation of the sperm structural integrity and functional activity.

Keywords: amygdalin, CASA, mitochondrial activity, motility, rabbits, spermatozoa, viability

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Authors: Mojtaba Heydarizad

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Andarokh basin is one of the main karstic regions in Khorasan Razavi province NE Iran. This basin is part of Kopeh-Dagh mega zone extending from Caspian Sea in the east to northern Afghanistan in the west. This basin is covered by Mozdooran Formation, Ngr evaporative formation and quaternary alluvium deposits in descending order of age. Mozdooran carbonate formation is notably karstified. The main surface karstic features in Mozdooran formation are Groove karren, Cleft karren, Rain pit, Rill karren, Tritt karren, Kamintza, Domes, and Table karren. In addition to surface features, deep karstic feature Andarokh Cave also exists in the region. Studying Ca, Mg, Mn, Sr, Fe concentration and Sr/Mn ratio in Mozdooran formation samples with distance to main faults and joints system using PCA analyses demonstrates intense meteoric digenesis role in controlling carbonate rock geochemistry. The karst evaluation in Andarokh basin varies from early stages 'deep seated karst' in Mesozoic to mature karstic system 'Exhumed karst' in quaternary period. Andarokh cave (the main cave in Andarokh basin) is rudimentary branch work consists of three passages of A, B and C and two entrances Andarokh and Sky.

Keywords: Andarokh basin, Andarokh cave, geochemical analyses, karst evaluation

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Authors: S. Ghasemi, K. Khorasani

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In this paper, the problem of fault detection and isolation in the attitude control subsystem of spacecraft formation flying is considered. In order to design the fault detection method, an extended Kalman filter is utilized which is a nonlinear stochastic state estimation method. Three fault detection architectures, namely, centralized, decentralized, and semi-decentralized are designed based on the extended Kalman filters. Moreover, the residual generation and threshold selection techniques are proposed for these architectures.

Keywords: component, formation flight of satellites, extended Kalman filter, fault detection and isolation, actuator fault

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Authors: D. H. Tejavathi

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Medicinal plants are globally valuable sources of herbal products. Wild populations of many medicinal plants are facing threat of extinction because of their narrow distribution, endemicity, and degradation of specific habitats. Micropropagation is an established in vitro technique by which large number of clones can be obtained from a small bit of explants in a short span of time within a limited space. Mycorrhization can minimize the transient transplantation shock, experienced by the micropropagated plants when they are transferred from lab to land. AM fungal association improves the physiological status of the host plants through better uptake of water and nutrients, particularly phosphorus. Consequently, the growth performance and biosynthesis of active principles are significantly enhanced in AM fungal treated plants. Bacopa monnieri, Andrographis paniculata, Agave vera-curz, Drymaria cordata and Majorana hortensis, important medicinal plants used in various indigenous systems of medicines, are selected for the present study. They form the main constituents of many herbal formulations. Standard in vitro techniques were followed to obtain the micropropagated plants. Shoot tips and nodal segments were used as explants. Explants were cultured on Murashige and Skoog, and Phillips and Collins media supplemented with various combinations of growth regulators. Multiple shoots were obtained on a media containing both auxins and cytokinins at various concentrations and combinations. Multiple shoots were then transferred to rooting media containing auxins for root induction. Thus, obtained in vitro regenerated plants were subjected to brief acclimatization before transferring them to land. One-month-old in vitro plants were treated with AM fungi, and the symbiotic effect on the overall growth parameters was analyzed. It was found that micropropagation coupled with mycorrhization has significant effect on the enhancement of biomass and biosynthesis of active principles in these selected medicinal plants. In vitro techniques coupled with mycorrhization have opened a possibility of obtaining better clones in respect of enhancement of biomass and biosynthesis of active principles. Beneficial effects of AM fungal association with medicinal plants are discussed.

Keywords: cultivation, medicinal plants, micropropagation, mycorrhization

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Authors: Ovye Yohanna Musah

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Shale formations of the Middle Benue Trough in North Central Nigeria present a variety of opportunities for the exploration, development and exploitation of unconventional natural gas. Prospective formations range in age from Albian through Coniacian; they include the Asu River Group, Awe, Ezeaku and the Awgu formations, however, the Keana and Lafia formations are thought to be of lesser importance. The Awgu formation presents the best prospect when compared to the Barnett Shales of Fort Worth Basin in Texa, United States with regards to the organic matter maturition, TOC content of formation and shale thicknesses which are key attributes that aid in determining the economic viability of any shale gas play. The vitrinite reflectance value from Rock Eval pyrolysis for Awe and Awgu formations are 0.89—1.34(%) and 0.83—1.13(%) respectively and are good and sufficiently mature to generate gas from the Benue Trough. The TOC value are good for Awgu formation which is 0.83—6.54(%) and closest to that of the Barnett at 1—4.5(%). Asu River and Ezeaku are less viable. Furthermore, the High to Medium Volatile bituminous coals found in the Awgu formation are characterized by high TOC contents which may enhance gas generation and this is good for further examination and possible development.

Keywords: shale gas, resource, unconventional, benue, TOC

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Authors: Khaled S. Al Salhen

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Aldehyde oxidase is molybdo-flavoenzyme involved in the oxidation of hundreds of endogenous and exogenous and N-heterocyclic compounds and environmental pollutants. Uncharged N-heterocyclic aromatic compounds such phenanthridine are commonly distributed pollutants in soil, air, sediments, surface water and groundwater, and in animal and plant tissues. Phenanthridine as uncharged N-heterocyclic aromatic compound was incubated with partially purified aldehyde oxidase from rainbow trout fish liver. Reversed-phase HLPC method was used to separate the oxidation products from phenanthridine and the metabolite was identified. The 6(5H)-phenanthridinone was identified the major metabolite by partially purified aldehyde oxidase from fish liver. Kinetic constant for the oxidation reactions were determined spectrophotometrically and showed that this substrate has a good affinity (Km = 78 ± 7.6 µM) for hepatic aldehyde oxidase, coupled with a relatively high oxidation rate (0.77± 0.03 nmol/min/mg protein). In addition, the kinetic parameters of hepatic fish aldehyde oxidase towards the phenanthridine substrate indicate that in vitro biotransformation by hepatic fish aldehyde oxidase will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase.

Keywords: aldehyde oxidase, fish, phenanthridine, specificity

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Authors: Qiuyue Peng, Vladimir Zachar

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The application of adipose-derived stromal/stem cells (ASCs) in regenerative medicine is gaining more awareness due to their advanced translational potential and abundant source preparations. However, ASC-based translation has been confounded by high subpopulation heterogeneity, causing ambiguity about its precise therapeutic value. Some phenotypes defined by a unique combination of positive and negative surface markers have been found beneficial to the required roles. Therefore, the immunophenotypic repertoires of cultured ASCs and temporal changes of distinct subsets were investigated in this study. ASCs from three donors undergoing cosmetic liposuction were cultured in standard culturing methods, and the co-expression patterns based on the combination of selected markers at passages 1, 4, and 8 were analyzed by multi-chromatic flow cytometry. The results showed that the level of heterogeneity of subpopulations of ASCs became lower by in vitro expansion. After a few passages, most of the CD166⁺/CD274⁺/CD271⁺ based subpopulations converged to CD166 single positive cells. Meanwhile, these CD29⁺CD201⁺ double-positive cells, in combination with CD36/Stro-1 expression or without, feathered only the major epitopes and maintained prevailing throughout the whole process. This study suggested that, upon in vitro expansion, the phenotype repertoire of ASCs redistributed and stabilized in a way that cells co-expressing exclusively the strong markers remained dominant. These preliminary findings provide a general overview of the distribution of heterogeneous subsets residents within human ASCs during expansion in vitro. It is a critical step to fully characterize ASCs before clinical application, although the biological effects of heterogeneous subpopulations still need to be clarified.

Keywords: adipose-derived stromal/stem cells, heterogeneity, immunophenotype, subpopulations

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Authors: Abdulmalek Ahmed

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Formation pressure is the main function that affects drilling operation economically and efficiently. Knowing the pore pressure and the parameters that affect it will help to reduce the cost of drilling process. Many empirical models reported in the literature were used to calculate the formation pressure based on different parameters. Some of these models used only drilling parameters to estimate pore pressure. Other models predicted the formation pressure based on log data. All of these models required different trends such as normal or abnormal to predict the pore pressure. Few researchers applied artificial intelligence (AI) techniques to predict the formation pressure by only one method or a maximum of two methods of AI. The objective of this research is to predict the pore pressure based on both drilling parameters and log data namely; weight on bit, rotary speed, rate of penetration, mud weight, bulk density, porosity and delta sonic time. A real field data is used to predict the formation pressure using five different artificial intelligence (AI) methods such as; artificial neural networks (ANN), radial basis function (RBF), fuzzy logic (FL), support vector machine (SVM) and functional networks (FN). All AI tools were compared with different empirical models. AI methods estimated the formation pressure by a high accuracy (high correlation coefficient and low average absolute percentage error) and outperformed all previous. The advantage of the new technique is its simplicity, which represented from its estimation of pore pressure without the need of different trends as compared to other models which require a two different trend (normal or abnormal pressure). Moreover, by comparing the AI tools with each other, the results indicate that SVM has the advantage of pore pressure prediction by its fast processing speed and high performance (a high correlation coefficient of 0.997 and a low average absolute percentage error of 0.14%). In the end, a new empirical correlation for formation pressure was developed using ANN method that can estimate pore pressure with a high precision (correlation coefficient of 0.998 and average absolute percentage error of 0.17%).

Keywords: Artificial Intelligence (AI), Formation pressure, Artificial Neural Networks (ANN), Fuzzy Logic (FL), Support Vector Machine (SVM), Functional Networks (FN), Radial Basis Function (RBF)

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Authors: Pakize Neslihan Taslı, Alev Cumbul, Gul Merve Yalcın, Fikrettin Sahin

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A key experimental trial in the regeneration of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. A different approach combining the gel-casting method with Hydroxy Apatite HA-based scaffold and different cell lineages as a hybrid system leads to successively mimic the early stage of tooth development, in vitro. HA is widely accepted as a bioactive material for guided bone and tooth regeneration. In this study, it was reported that, HA porous scaffold preparation, characterization and evaluation of structural and chemical properties. HA is the main factor that exists in tooth and it is in harmony with structural, biological, and mechanical characteristics. Here, this study shows mimicking immature tooth at the late bell stage design and construction of HA scaffolds for cell transplantation of human Adipose Stem Cells (hASCs), human Bone Marrow Stem Cells (hBMSCs) and Gingival Epitelial cells for the formation of human tooth dentin-pulp-enamel complexes in vitro. Scaffold characterization was demonstrated by SEM, FTIR and pore size and density measurements. The biological contraction of dental tissues against each other was demonstrated by mRNA gene expressions, histopatologic observations and protein release profile by ELISA tecnique. The tooth shaped constructs with a pore size ranging from 150 to 300 µm arranged by gathering right amounts of materials provide interconnected macro-porous structure. The newly formed tissue like structures that grow and integrate within the HA designed constructs forming tooth cementum like tissue, pulp and bone structures. These findings are important as they emphasize the potential biological effect of the hybrid scaffold system. In conclusion, this in vitro study clearly demonstrates that designed 3D scaffolds shaped as a immature tooth at the late bell stage were essential to form enamel-dentin-pulp interfaces with an appropriate cell and biodegradable material combination. The biomimetic architecture achieved here is providing a promising platform for dental tissue engineering.

Keywords: tooth regeneration, tissue engineering, adipose stem cells, hydroxyapatite tooth engineering, porous scaffold

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Authors: Saziye Ugur

Abstract:

In this study, optical, film formation, morphological and the magnetic properties of a nanocomposite system, composed of polystyrene (PS) latex polymer and core-shell magnetic nanoparticles (MNPs) is presented. Nine different mixtures were prepared by mixing of PS latex dispersion with different amount of MNPs in the range of (0- 100 wt%). PS/MNPs films were prepared from these mixtures on glass substrates by drop casting method. After drying at room temperature, each film sample was separately annealed at temperatures from 100 to 250 °C for 10 min. In order to monitor film formation process, the transmittance of these composites was measured after each annealing step as a function of MNPs content. Below a critical MNPs content (30 wt%), it was found that PS percolates into the MNPs hard phase and forms an interconnected network upon annealing. The transmission results showed above this critical value, PS latexes were no longer film forming at all temperatures. Besides, the PS/MNPs composite films also showed excellent magnetic properties. All composite films showed superparamagnetic behaviors. The saturation magnetisation (Ms) first increased up to 0.014 emu in the range of (0-50) wt% MNPs content and then decreased to 0.010 emu with increasing MNPs content. The highest value of Ms was approximately 0.020 emu and was obtained for the film filled with 85 wt% MNPs content. These results indicated that the optical, film formation and magnetic properties of PS/MNPs composite films can be readily tuned by varying loading content of MNPs nanoparticles.

Keywords: composite film, film formation, magnetic nanoparticles, ps latex, transmission

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Authors: M. Areekijseree, W. Pongsawat, M. Pumipaiboon, C. Thepsithar, S. Sengsai, T. Chuen-Im

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Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by zona pellucida with layer of cumulus cells ranging between 59.29-202.14 µm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 µg/mL porcine follicle-stimulating hormone, 1 µg/mL LH, 1µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: cumulus cells, electron microscopy, in vitro, porcine oocyte

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Authors: Abhishek Kumar Sah, Preeti K. Suresh

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Effective drug delivery to the eye is a massive challenge, due to complicated physiological ocular barriers, rapid washout by tear and nasolachrymal drainage. Thus, most of the conventional ophthalmic formulations face the problem of low ocular bioavailability. Ophthalmic drug therapy can be improved by enhancing the precorneal drug retention along with improved drug penetration. The aim of the present investigation was to develop and evaluate a biodegradable polymer poly (D, L-lactide-co-glycolide) (PLGA) coated nanoparticulate carrier of loteprednol etabonate. PLGA nanoparticles were prepared by modified emulsification/solvent diffusion method using high-speed homogenizer followed by sonication. The nanoparticles were characterized for various parameters such as particle size, zeta potential, polydispersity index, X-ray powder diffraction (XRD), Transmission electron microscopy (TEM), in vitro drug release profile and stability. The prepared nanocarriers displayed mean particle size in the range of 271.7 to 424.4 nm, with zeta potential less than –10 mV. In vitro release in simulated tear fluid (STF) nanocarrier showed an extended release profile of loteprednol etabonate. TEM confirmed the spherical morphology and smooth surface of the particles. All the prepared formulations were found to be stable at varying temperatures.

Keywords: drug delivery, ocular delivery, polymeric nanoparticles, loteprednol etabonate

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