Hr Of Incubation Research Articles

Ultrastructural changes observed in Colletotrichum alienum conidia following treatment with cold plasma or plasma‐activated water

AbstractPrevious studies demonstrated that a reduction in germination of Colletotrichum alienum conidia could be achieved following treatment with cold plasma (CP) or incubation with plasma‐activated water (PAW). In this study, the mode of action of CP and PAW on C. alienum conidia was explored using transmission electron microscopy. Following treatment of the conidia, noticeable ultrastructural changes were observed, including cell wall maceration, disorganization of the cytoplasm and vacuole, and changes to the nuclei and mitochondria. Disorganization of the cytoplasm was exhibited by 85% of conidia after CP or PAW treatment. Also, 85% of conidia were observed with disorganized vacuoles after 6 min of CP treatment and 65% after 3 hr incubation in PAW. Further, 45% or more conidia had modified cell walls after PAW incubation or CP treatment. Deformation of conidia was observed in 23% of conidia after PAW incubation. These ultrastructural changes are a likely reason for the reduced germination of C. alienum conidia following CP or PAW treatment.

In-vitro Study on the Efficacy of Entomopathogenic Nematodes (Heterorhabditidae and Steinernematidae) for the Control of the Indian Cabbage White, Pieris canidia

Pieris canidia is one of the serious pests of cruciferous crops causing extensive damage to agricultural crops. Entomopathogenic nematodes (EPNs) therefore represent ideal candidates for biological control of the pest. In this study, the efficacy of four indigenous EPNs species, Heterorhabditis indica, Heterorhabditis baujardi, Steinernema sangi, and Steinernema surkhetense from Mizoram was evaluated against P. canidia under laboratory condition. Different concentrations of nematodes, 10, 25, 50, 100, and 200 infective juveniles per larva (IJs/larva) were used to evaluate larval mortality rate, host penetration rate, and progeny production in insect cadaver. All the four EPN species showed high pathogenicity against the pest for different nematode concentrations and observation periods. At a concentration of 200 IJs/larva, cent percent mortality of the pest was recorded at 72 hr post-incubation for S. surkhetense, 96 hr for S. sangi and H. indica, and 120 hr post-incubation for H. baujardi. Based on the values of LC50 and LT50, H. indica was the most pathogenic among the four nematode species while S. sangi was the least pathogenic. After 24 hr of incubation, the LC50 values of H. indica, H. baujardi, S. surkhetense, and S. sangi were 88.60, 98.29, 113.26 and 95.61 IJs/larva, respectively. At 10 IJs/larva, the LT50 values of H. indica, H. baujardi, S. sangi, and S. surkhetense were 88.12, 90.69, 88.102.30, and 88.11 hr, respectively. The study also disclosed that all the four EPN species successfully infect the pest and showed high rate of penetration into the host. Furthermore, all the nematode isolates were capable of producing large numbers of IJs in larval cadaver of P. canidia that significantly varies between the EPN species. The isolate H. baujardi produced the highest number of IJs followed by H. indica, S. surkhetense, and S. sangi. Our findings therefore reveal that all the four EPN isolates have the potential to be developed as biological control agents for P. canidia.

Abstract 3057: Development of radiation-triggered phosphor platform for localized activation in combinatory cancer treatment

Abstract A defined spatial and temporal control of induction of toxicity in response to external ionizing radiation such as X-rays has unique clinical benefits over conventional systems such as passive drug delivery or its activation. Here, we have combined X-rays with well characterized nanoscintilators emitting light that activates paired photosensitizers in photodynamic therapy (PDT) that improves the prowess of PDT to deep seated tumor treatment. Yttrium oxide nanoparticle construct doped with Europium (YOE) and coated with a silica layer entrapping methylene blue (MB) as the photosensitizer was fabricated. The nanoconstruct (YSM, for YOE, silica, MB) was decorated with polyethylene glycol (PEG) to make it biocompatible and characterized extensively for physicochemically parameters. Transmission electron microscopy (TEM) revealed a resultant average nanoparticle diameter of 24.1 ± 5.7 nm, and dynamic light scattering (DLS) a hydrodynamic diameter of 135.3 ± 17.9 nm. This nanoconstruct was evaluated for its ability to generate singlet oxygen reactive oxygen species upon irradiation using singlet oxygen sensor green (SOSG) fluorescence assay. Further, demonstrated that this nanoconstruct was taken up by cancer cells in vitro and by tumors in vivo. The uptake of YSM particles by Panc02 murine pancreatic cancer cells was tested by inductively coupled plasma – mass spectrometry. This demonstrated robust uptake in cells after 24hr incubation. No noticeable cell death occurred with doses of 0 Gy and 4 Gy, cell viability decreased significantly after a dose of 8 Gy irradiation in cells cultured with YSM when compared to controls. Since 8 Gy is within the range of a clinically applicable dose used routinely in stereotactic radiotherapy regimens, these results verified the potential of YSM to be used as scintillator-photosensitizer complexes for x-ray activated photodynamic therapy. Finally the biodistribution of YSM nanoconstructs in vivo in C57BL6 mice harboring subcutaneous Panc02 tumors show YSM accumulation in tumors. A versatile, biocompatible Yttrium oxide scintillator-photosensitizer complex (YSM) that can generate x-ray induced cytotoxic reactive oxygen species is fabricated. This is capable of facilitating x-ray activated photodynamic therapy (XPDT (external beam) and/or iXPDT (internal x-ray source)). It shows promise in maximizing the efficacy of the combinatory treatment and enhanced improvement of the quality of life and overall survival of cancer patients. Citation Format: Rao V. Papineni, Sunil Krishnan, Ajayan Pulickel, Onur Sahin. Development of radiation-triggered phosphor platform for localized activation in combinatory cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3057.

EVALUATION OF ANTIOXIDANT AND ANTIBACTERIAL POTENTIAL OF ZANTHOXYLUM ALATUM FRUIT AND LEAVES EXTRACTS AGAINST SELECTED PATHOGENIC BACTERIA

Zanthoxylum alatum (Z. alatum), an important medicinal plant is used for various ailments including chest infection, cough, cholera, fever, stomach disorders, gas problems, indigestion, piles, toothache, gum problems, dyspepsia and stomachic worldwide. Keeping in view the medicinal potential of this plant, fruit and leaves methanolic (MeOH) extracts were prepared, evaluated for antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and for antibacterial potential by well diffusion and macrodilution methods in-vitro. Our results confirmed that Z. alatum fruit and leaves extracts have significant antioxidant activity with IC50 values 0.28 ± 0.07 mg/ml and 0.34 ± 0.05 mg/ml, respectively. The inhibitory trend at highest tested concentration (120 mg/ml) at 24 hr incubation in well diffusion method was recorded as 0.39, 0.30, 0.28, 0.27 and 0.18 cm against S. pyogenes, B. cereus, E. coli, S. aureus and S. enterica for leaf extract, respectively. In case of fruit extract inhibitory trend at highest tested concentration was observed as 0.33, 0.32, 0.31, 0.30 and 0.28 cm against S. aureus, S. enterica, S. pyogenes, B. cereus and E. coli, respectively. The fruit extract showed higher zones of inhibition than leaves extracts against all the test bacteria except S. pyogenes. Moreover, highest zones of inhibition were observed at lowest incubation (24 hr) and lowest zones were observed at highest incubation period (72 hr) for all tested concentrations. Macrodilution method showed antibacterial susceptibility in liquid medium with different levels of IC50 values ranging from 1.6±0.13 mg/ml to 10.3±0.7 5mg/ml. Interestingly, none of the tested bacteria showed resistance against any of the test extract in well diffusion or macrodilution method expressing the Z. alatum as potent candidates to kill bacteria in semisolid or in liquid medium to fulfill medical needs in future.

Results of a Non-Interventional Clinical Trial of Apograft — a Novel Technology for GvHD Prevention

IntroductionGvHD is a threat to the wellbeing and survival of patients following hematopoietic stem cell transplantation (HSCT). In-vivo and ex-vivo T cell depletion are the most effective methods for GvHD prevention, but attendant risks of infection, graft failure and leukemic recurrence offset the advantages of these manipulations. Less extensive T cell depletion leaves many patients at risk for the ravages of treatment-resistant GvHD. Alternative approaches to eliminate GvHD-causing cells are needed. Fas ligand (FasL), a member of the tumor necrosis factor (TNF) family, can selectively induce apoptosis of mature T cells while sparing stem and progenitor cells. We have developed a novel FasL-mediated selection process which eliminates mature T cell subsets in hematopoietic stem cell grafts and effectively prevents GvHD in pre-clinical models without impairing engraftment and immune system recovery. We report here data from ex-vivo and in-vivo studies supporting the use of FasL-mediated depletion of mature T-cells, a process we have named ApoGraft, as a novel effective method for GvHD prophylaxis in the context of allogeneic HSCT.Methods:Aliquots of mobilized peripheral blood cells (MPBC) were collected from 104 consenting healthy donors and were used ex-vivo for optimization of the ApoGraft manufacturing process. ApoGraft is manufactured under GMP guidelines, and includes two washing steps, a 2 hr incubation of the graft at 37oC with human recombinant different concentrations of FasL (MegaFasL, Adipogen), and 2 additional washing steps to remove unbound FasL. Aliquots from 25 MPBC apheresis that had been subjected to the complete ApoGraft process were analyzed ex-vivo for cell yield, viability and cellular constitution by FACS and for clonogenicity potential by colony forming unit (CFU) assays. Additionally, engraftment and GvHD prevention of ApoGraft were evaluated with 7 aliquots of MPBC apheresis in NOD-SCID IL2-Rgamma-null (NSG) mice (n=349). Here we report results from representative studies were unfractionated ApoGraft (2.5x10^6 cells) or CD34+ purified cells (1.0x10^5 cells) from ApoGraft were transplanted into irradiated (2-2.75Gy) NSG mice. The mice were monitored clinically for GVHD occurrence post transplantation; engraftment and differentiation were evaluated using FACS analysis.Results:Cellular viability was 98% (±1%) in the ApoGraft samples analyzed. Comparison of ApoGraft and Starting Material (SM) showed a mean 19.9% (±4.3%) reduction of total CD3+ T cells; early apoptosis of CD3+ cell increased by 5-fold as compared to Starting Material. Importantly, the percentage of viable CD34+ stem and progenitor cells was not affected by ApoGraft processing. Proliferation and differentiation of hematopoietic progenitors in colony forming unit (CFU) assay was not affected. In NSG mice infused with unfractionated ApoGraft or CD34+ cells purified from it, chimerism level and differentiation were similar to those of mice infused with control MPBC (figure 1A-1B). Most importantly, ApoGraft infused mice had lower GvHD clinical score and prolonged survival as compared to mice infused with control MPBC (maximal score 1.7±0.7 vs. 8.8±0.8) (Figure 1C).Conclusion:ApoGraft is an apoptosis-based selection process that eliminates mature T cells from stem cells grafts, while preserving CD34+ cells quantity, quality and functionality. In our preclinical murine model, ApoGraft reduced GvHD without impairing engraftment. Ongoing animal studies are evaluating the graft vs. leukemic capabilities of ApoGraft and the preservation of anti-viral immunity after ApoGraft processing. Additionally, ApoGraft is currently being evaluated in a Phase I/IIa study (NCT02828878) in subjects with hematological malignancies undergoing matched related allogeneic HSCT. [Display omitted] DisclosuresZuckerman:BioSight Ltd.: Consultancy. Yarkoni:Cellect Biotechnology Ltd.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nechushtan:Cellect Biotechnology Ltd.: Employment. Rodionov:Cellect Biotechnology Ltd.: Employment. Gez:Cellect Biotechnology Ltd.: Employment. Rosenzwaig:Cellect Biotechnology Ltd.: Employment. Rodin:Cellect Biotechnology Ltd.: Employment. Pereg:Cellect Biotechnology Ltd.: Employment.

Colistin Susceptibility Testing by Rapid Colistin Disk Elution Test Among Enterobacteriaceae in Low-Resource Setting.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-μg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 μg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-μL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Safety, probiotic properties, antimicrobial activity, and technological performance of Lactobacillus strains isolated from Iranian raw milk cheeses.

The objective of this study was to investigate probiotic, antimicrobial, technological and safety properties of lactobacillus strains isolated from local Iranian cheese made from raw milk. Six different samples were prepared, after serial dilution, culture was performed on MRS culture medium. The gram‐positive and catalase‐negative lactobacillus strains were subjected to grouping and identifying using biochemical tests, carbohydrates fermentation profiles, and 16S rDNA analysis. The results of sequence analysis showed the Lactobacillus spp. belonged to Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei. After 3 hr incubation at pH=2, 3–6 log units of strains decreased which Lactobacillus acidophilus (B14) and Lactobacillus brevis (B2) showed highest resistance to low pH as well as simulated GIT juices. The highest and lowest hydrophobicity degree was belonged to L. acidophilus (B14) (65.9%) and L. casei (B22) (25.6%), respectively. Also, the highest auto‐aggregation and coaggregation were observed in L. acidophilus (B14) (51.3%) and L. plantarum (B20) (43.6%). The adhered percentage of strains varied from 2.5% to 14.6%. L. plantarum (B20) showed highest proteolytic activity followed by L. acidophilus (B14). Also, the highest autolytic activity belonged to L. acidophilus (B14). All of the strains showed low acidifying potential, except for L. acidophilus (B17) which decreased 2.05 unit of pH after 24 hr. The isolates did not show lipolytic activity as well as biogenic amines production (except L. brevis B3). All of the strains were sensitive to chloramphenicol and erythromycin except L. acidophilus (B15) and L. casei (B22). All strains showed no hemolysis activity which make them safe for consumption. Based on the obtained results, L. acidophilus (B14) presented the best probiotic and technological characteristics and is proposed for using as coculture in the dairy industrial.

Cyto-genotoxicity, antibacterial, and antibiofilm properties of green synthesized silver nanoparticles using Penicillium toxicarium.

The fungi are becoming the distinguished organisms utilized in the biological synthesis of metallic nanoparticles because of their metal bioaccumulation ability. Addressed herein, the extracellular synthesis of silver nanoparticles (AgNPs) was carried out by using the cell-free filtrate of Penicillium toxicarium KJ173540.1. P. toxicarium was locally isolated and identified using both classical and molecular methods according to ribosomal internal transcribed spacer area of 18S rDNA. The optimum conditions for the AgNPs synthesis were found as 0.25 mM AgNO3 concentrations with pH 12 values at 45°C after 64 hr incubation in dark. Biosynthesized AgNPs were characterized via microscopic and spectroscopic techniques such as transmission electron microscopy, scanning electron microscopy, energy dispersive X-ray analysis, Fourier transform infrared spectrophotometer, and ultraviolet-visible spectroscopy. Zetasizer measurements presented that the high negative potential value (-18.1 mV) and PDI (0.495) supported the excellent colloidal nature of AgNPs with long-range stability and high dispersity. AgNPs exhibited cyto-genotoxicity in Allium cepa root meristem cells by decreasing mitotic index and increasing chromosome aberrations in a dose-dependent manner. Then, 100 and 50% concentration of biosynthesized AgNPs showed antibacterial activity on Staphylococcus aureus and Bacillus subtilis. A decreasing biofilm formation of Pseudomonas aeruginosa 80.69, 48.32, and 28.41% was also observed at 100, 50, and 25% of mycosynthesized AgNP, respectively.

Effect of radio frequency glow-discharge treatment of titanium on human gingival fibroblasts as a function of distance.

The mechanical stability and long-term success of an implant depends on the early healing phase and osseointegration of the bone around it. In addition, a healthy gingival tissue around the implant acts as a barrier that prevents bacteria and pathological byproducts from reaching the implant site. This study investigated the in-vitro attachment and spreading of human gingival fibroblasts (HGF) on bacterial grade polystyrene (PS) at different distances from radio-frequency glow-discharge (RFGD)-treated commercially pure titanium (cpTi) specimens. Controls included sterile cpTi specimens without RFGD treatment. A second set of experiments utilized media transferred to new bacterial grade polystyrene dishes (no cpTi) after the medium was conditioned by exposure to cpTi, either with or without RFGD treatment, for 24 hr. Surface characterization of the dishes was conducted through contact angle measurements and infrared spectroscopy. Cell numbers and surface areas were determined from Image J analysis of multiple microscopic images of fixed, stained cells. The results showed significantly greater numbers and surface areas on bacterial grade PS dishes at distances up to 15 mm from the RFGD-treated cpTi groups than for the controls. Moreover, a significant effect of the conditioned medium from RFGD-treated cpTi versus control cultures was shown on the numbers of fibroblasts attached to bacterial grade polystyrene dishes after 24 hr (p< 0.005) and 48 hr (p= 0.002) incubation. Surface areas of cells exposed to conditioned medium were not significantly different (p≥ 0.05). Surface characterization of the PS dishes showed a higher value of the critical surface tensions of the treated group when compared to the control group.

The Effect Allium sativum (Garlic Extract) as Prebiotic Substance on the Activity of Probiotic Bacteria Lactobacillus acidophilus Against Some Locally Isolates of Pathogenic Bacteria

The aim of this study was to improve the growth and inhibitory activity of the selected Lactobacillus isolate.A total of twenty samples of dairy products (fresh yogurt) were collected. Seven isolates of Lactobacillusspecies . were obtained, and identified as three Lactobacillus fermentum, three Lactobacillus acidophilus andone of Lactobacillus brevis. The growth of lactobacillus acidophilus was counted , also inhibitory activity ofLactobacillus acidophilus against some pathogenic bacteria was tested) for two different incubation periodsbefore the addition of Allium sativum(garlic extract) as prebiotic substances. three different concentrationsof it were used by addition of it toDe Man, Rogosa and Sharpe agar (MRS) medium as prebiotic substances , and two incubation periods(24, 48) hour. The effect of Allium sativum on the growth of probiotic bacteria was evaluated, measurementof Lactobacillus acidophilus growth curve was done after and before adding garlic extract. For analyzinginhibitory activity of Lactobacillus acidophilus with the Allium ,results showed that Lactobacillus acidophiluspropagated in MRS fortified with 20% and 30% v/v garlic extract with incubation period 24 hr by giving thehighest inhibition zone.It was concluded that Lactobacillus.acidophilus isolate can grow well and exhibit good effect against Gramnegative bacteria in a medium containing garlic extract (that is added as a substrate supporting probioticgrowth and activity). also it was clear that filtrate of Lactobacillus .acidophilus isolate that propagatingin spesific broth fortified with 20% garlic extract at 48 hr incubation period produce antibacterial effectbetter than 10% , it was concluded that prebiotic treatment enhance the growth of Lactobacillus acidophilusespecially at 24 hr. this may due to fructooligosaccharide content in garlic , also when propagated in MRSwithout prebiotic treatment, and which may due to highly decrease in pH which cause inhibition the growthof Lactobacillus acidophillus.

In vitro analyses of Artemisia extracts on Plasmodium falciparum suggest a complex antimalarial effect.

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA’s effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro.

A Cross-sectional Study on Chronic Fungal Rhinosinusitis in a Tertiary Care Hospital in Central Delhi, India

Chronic Rhinosinusitis is a common disorder, and its prevalence vary from 1-20% globally. The incidence of fungal sinusitis has increased to such extent in recent years that fungal infection is a common diagnosis in patients with Chronic Rhinosinusitis. The objectives of this current research were objectives of estimating the prevalence of Fungal aetiology in chronic sinusitis patients and their drug sensitivity pattern with common antifungal drugs. A total of 61 Cases present with Chronic Rhinosinusitis (CRS), visited in a tertiary care hospital based in Central Delhi, were included in our study. Excision of sinus tissue, including polyps and masses, were collected in the operation theatre during Functional Endoscopic Sinus Surgery (FESS) procedure in a sterile manner. All the tissues brought in sterile normal saline were processed for bacteriological and mycological examination. Tissues, obtained in 10% formalin were processed for histopathological and cytological analysis. A total of 14 (22.9%) cases of Chronic Rhinosinusitis were affected by fungal etiologies. By E test, the MIC range for isolates of Rhizopus arrhizus after 24 hr of incubation was 1-2 μg/mL, and the mean was 1.5 μg/Ml. Similarly, the MIC range for isolates of Aspergillus flavus after 48 hr of incubation was 0.5-16 μg/mL, and the mean was 4.09μg/mL. By the M38-A broth dilution method, the MIC range for the isolates of Rhizopus arrhizus after 24 hr of incubation was 0.5-2 μg/mL, and the mean was 1.25 μg/ml. Similarly, the MIC range for isolates of Aspergillus flavus after 48 hr of incubation was 0.5-4 μg/mL, and the mean was 1.95 μg/mL.

Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein‐free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II‐S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris‐based extender, containing either 20% egg yolk, 1% soybean lecithin Type II‐S or 1% soybean lecithin Type IV‐S. The samples were then frozen. Sperm motility was evaluated by computer‐assisted sperm analysis (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (SYBR‐14/PI) post‐thaw, as well as after 2 and 4 hr incubation at 37°C. Post‐thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin‐based extenders. Individual motility post‐thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean‐based extenders. The number of high green stained spermatozoa were significantly higher in Type IV‐S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin‐based extenders to cryopreserve canine semen.

Multi‐objective optimization of enzyme‐assisted juice extraction from custard apple: An integrated approach using RSM and ANN coupled with sensory acceptance

The enzyme-assisted juice extraction from custard apple (Annona squamosa L.) puree was optimized within the domain of 0.3%–5% (w/w) enzyme concentration (EC), 0.5–5 hr incubation period (t), and 30–60°C temperature (T). A multi-objective optimization was carried out using two techniques; response surface methodology (RSM) and artificial neural network with genetic algorithm (ANN-GA). The models showed an overall R2 and mean squared error (MSE) of 0.97 and 2.20 for RSM and 0.96 and 1.86 for ANN, respectively. Sensory evaluation was performed for the two best conditions from both the techniques. The final optimum condition (EC: T: t) of 2.21%: 47°C: 4.47 hr was selected having the maximum overall acceptability of 6.02. The corresponding experimental response was 88% (w/w) yield, 32.8 mg GE/ml total reducing sugar, and 60.9% clarity. The optimally extracted custard apple beverage showed 94.0 µg GAE/ml of phenolic content and 34.7 μg GAEAC/ml of antioxidant capacity. Practical applications Custard apple is an underexplored tropical fruit. Despite having good nutritional values and attractive flavor, its availability as a processed product is very limited, mainly because custard apple is highly perishable. Manufacturing juice or beverage from custard apple can reduce its wastage and promote its utilization. Enzyme-assisted juice extraction is one of the most efficient and environmentally friendly ways to obtain better yield and clarity. Pectinase enzyme was used to enhance juice extraction, and a multi-objective optimization was followed using two techniques; response surface methodology (RSM) and artificial neural network with genetic algorithm (ANN-GA). The best solutions given by RSM and ANN-GA were compared based on sensory evaluation and beverage receiving maximum overall acceptability was finally selected. The current study will undoubtedly help in promoting the utilization of custard apple, its enzyme treatment, reduce wastage, and thereby pave the pathway for similar underexplored fruits.

TOPICAL DRUG DELIVERY OF GOSSYPIN FROM PRONIOSOMAL GEL FORMULATIONS: IN VITRO EFFICACY AGAINST HUMAN MELANOMA CELLS

Objective: This work aimed to establish and formulate the gossypinproniosomal gel drug delivery system.&#x0D; Methods: Gossypin-loaded proniosomal gels (GPG) was prepared using specific non-ionic surfactants (Spans), followed by particle size (PS), entrapment efficiency (percent EE), in vitro, ex-vivo drug release, and in vitro efficacy of Gossypin against human melanoma cells (A-375).&#x0D; Results: The results showed that the percentage EE for the GPG is appropriate (81.3 %–95.5 %) and they are Nano-sized (189.3–912.0 nm) and the gels diffusion provided the desired sustaining effect for GPG-F7 formulation (75.5 percent). The GPG reported cell viability of 14.9±2.3 percent compared with 16.1±1.1 percent for free Gossypin at the maximum dose of 100 μg/ml for A-375 human melanoma cells after 24 hr incubation time. No major changes were seen in the percentage EE, PS of GPG after storage for 90d, in the physical stability report.&#x0D; Conclusion: The results obtained suggest that the proniosomal drug delivery system can enhance the flux to the skin and achieve the ideal Gossypin sustainability effect. Consequently, the use of proniosomal gel may be advantageous with regard to the topical delivery of Gossypin for melanoma treatment management.

Study of growth kinetic and gastrointestinal stability of acid-bile resistant Lactobacillus plantarum strains isolated from Siahmazgi traditional cheese.

In this study, 22 Lactobacillus plantarum strains isolated from Siahmazgi traditional cheese were evaluated using different tests including resistance to low pH (1.50 and 2.50) and bile salt (0.50 and 1.00%), growth kinetic at low pH values and survival under simulated gastric and intestinal fluids. All the strains retained their viability at pH 2.50. However, the survival of all of the isolates was decreased at pH 1.50. Ten out of 22 strains which were able to tolerate low pH were selected for further investigations. All the selected isolates were able to grow at low pH. Strain F2 showed the highest specific growth rate. Five out of 10 isolates showed a significant decrease in bacterial count varied from 2.00 to 7.00 log CFU mL-1 during 3 hr exposure to 0.50% bile salt, while five isolates represented resistance to 0.50% bile during 3 hr. A significant reduction was observed in survival of all of the isolate at 1.00% bile salt concentration. Furthermore, viability of the selected isolates was lowered during 1 hr incubation under gastric conditions, while it remained unchanged within next 2 hr. Although, no significant changes were seen in bacterial count of the selected isolates during 1 hr of exposure to simulated intestinal condition, the survival of the isolates was relatively reduced after 3 hr. In conclusion, five out of 22 examined L. plantarum isolates showed appropriate resistance properties, therefore, could be good candidates for further examinations including functional and safety evaluation supporting their use as probiotics.

Platelet Activating Factor Receptor Binding and Contractile Protein Expression by Ovine Fetal Pulmonary Vascular Smooth Muscle Cells: Role in Rho Kinase Mediation of PAF Receptor-Linked Physiological Responses

We studied effects of hypoxia and some modulators of PAFR–mediated signaling in fetal ovine arterial and venous smooth muscle cells to aid in explaining PAF regulation of fetal pulmonary hemodynamics in utero. PAFR, a G protein coupled receptor, is characteristically measured in membrane proteins. Profile of binding to soluble and nuclear fraction proteins from SMC have not been studied. Rho kinase (ROCK) inhibitors decrease PAFR-linked smooth muscle growth, suggesting that PAFR internalization may not be only the pathway to downregulate PAFR-mediated responses. We hypothesized that PAF binds to SMC subcellular protein fractions and together with ROCK contributes to physiological effects of PAF. Protein fractions were prepared from cells cultured in normoxia or hypoxia. PAF binding, PAFR protein and gene expression were studied. In normoxia, PAFR density (Bmax) in arterial and venous SMC were 487±37 and 624±17 respectively, which increased by 30-35% in hypoxia, and was greater in venous than arterial SMC in normoxia and hypoxia. The KD for PAFR binding to arterial cells was higher than for venous, and then hypoxia decreased the KD of PAFR binding in both cell types. PAFR binding to subcellular fractions were detectable, but were significantly lower than for membrane fractions in all conditions; e.g., normoxia: venous versus membrane, soluble fraction, 40%; nuclear fraction, 20%. ROCK inhibitor (Y-27632) attenuated PAFR binding after 24 hr incubation comparable to effects of cycloheximide and CV-3988. Unlike CV-3988, Y-27632 had no effect on PAF binding to pre-synthesized receptors within 30 min incubation. Y-27632 and HA-1077 increased MLCK and Calponin expression, although oxygen-related effects in venous SMC were different. PAFR gene expression was higher in hypoxia and effect of PAF and CV-3988 treatment on gene expression was paradoxically higher in arterial SMC supporting a translational control of PAFR activity in lung SMC. This study shows that PAFR binding to the SMC is not exclusive to membranes and that hypoxia in conjunction with ROCK modulate PAFR-mediated signaling in pulmonary vascular SMC in vitro with different effects on cells from arteries and veins. Different effects of ROCK on contractile proteins expression in hypoxia suggest involvement of these proteins in PAF-induced lung SMC reactivity in utero.

99m Tc-HYNIC/EDDA]-MccJ25 antimicrobial peptide analog as a potential radiotracer for detection of infection.

Bacterial infections are a serious risk to human health, and therefore techniques for early detection of infectious foci need to be further developed to begin treatment quickly and achieve better results. Antimicrobial peptides labeled with gamma-emission radio nuclides are important diagnostic radiotracers in nuclear medicine. This study was conducted to evaluate the potential of a 99m Tc-labeled MicrocinJ25 (MccJ25) antimicrobial peptide analog for early detection of infection. For this purpose, a HYNIC conjugated cyclic peptide derivative based on the primary structure of MccJ25 peptide was prepared and labeled by 99m Tc with tricine and EDDA as coligands. The [99m Tc-HYNIC/EDDA]-MccJ25 peptide analog showed high radiochemical purity (˃90% (n=5)) which was stable up to 24hr after labeling. The radiotracer showed specific uptake to the Escherichia coli (E.coli) bacterial (40.45±5.21%) at 1hr incubation. High kidneys uptake of radioactivity (4.71±0.84% and 3.76±0.45% ID/g at 1 and 4hr after injection respectively) demonstrates that most of the whole body clearance was proceeded via the urinary system. Significant radioactivity uptake (1.71±0.34%ID/g) was observed in thigh muscle of mouse with E.coli induced infection at 1hr after injection. In the blocking test, due to the significant decrease of radioactivity uptake in the infection site (0.62±0.21%ID/g after 1hr), the specificity of infection uptake was reviled. Despite the high activity of the bladder due to urinary excretion, the infected area was somewhat visible. Hence, the results indicate the potential of this new radiotracer to be used as a diagnostic agent in E.coli infections.

Evaluation of nutrient content andin Vitrogas fermentation of some crop residues as feed supplements for ruminants

The objective of this study was to evaluate the nutritive value of Yam peel (YP), Maize chaff (MC) and Rice straw (RS). Evaluation of the nutrient and anti-nutrient content of the crop residues involved determination of the proximate, mineral and phytochemical contents of YP, MC and RS. In vitro gas production (IVGP) of crop residues was carried out over a period of 24 hr incubation to predict Metabolizable energy (ME), organic matter digestibility (OMD) and short chain fatty acids (SCFA). Methane (CH4) wasmeasured by introducing 10 M NaOH into the inoculums after incubation. Result revealed significant (p&lt;0.05) differences in the values of the proximate composition. The dry matter ranged between 85.55 and 92.18 g/100 g in YP and MC respectively. The highest crude protein (5.01 g/100 g) was obtained in MC, while the lowest (4.00 g/100 g) was recorded for YP. The crude fibre ranged between 25.05 g/100g and 41.49 g/100g (YP). The mineral content of YP, MC and RS were not significantly different from each other, but within acceptable limit for optimum metabolism. YP, MC and RS were implicated for anti-nutrients, though no significant. The IVGP varied significantly (p&lt; 0.05) with the MC recording the highest (30.10ml) value, while the lowest (23.96ml) value was obtained in YP. Same trend was observed for ME, OMD, SCFA and CH4ranging from 5.32–11.12 MJ/Kg DM; 35.13 –64.14 %; 0.50-1.12 μmol and 9.05 –11.00ml respectively.It can be concluded that YP, MC and RShas potential in livestock nutrition. Also the problem associated with disposal of crop residues can be conveniently reduced by incorporating them into ruminant feed.

PSIX-35 Quebracho Tannin Influences Lipolytic Activity in Mature Porcine Adipocytes

Abstract Fat deposition in pork enhances flavor of meat; however, too much fat is an undesirable commodity in a health-conscious society. Therefore, manipulating the nutritional components of a swine diet to aid in the deliberate deposition of fat for the purpose of flavor while avoiding overconditioning is an aim in production. Nutrient additives, such as condensed polyphenolic tannins, inhibit pre-adipocyte maturation, but the role on lipid metabolism in mature adipocytes (MA) remains unclear. Therefore, it is hypothesized that quebracho tannin will alter lipid metabolism in porcine MA. Subcutaneous adipose tissue was collected from 5 ± 0 month old (n = 3) barrows weighing 37.7 ± 1.84kg. Tissue was enzymatically dispersed (collagenase type II) to isolate lipid filled adipocytes. After enzymatic separation the cells were rinsed and divided into 2 groups for separate incubation periods plus tannin treatment: 1) 2 hr incubation time with/without tannin (Quebracho Schinopsis lorentzii; 0M, 0.1mg, 0.5mg, and 1mg) or 2) 24 hr incubation time with/without tannin (0M, 0.1mg, 0.5mg, and 1mg). Approximately 4x105 cells/well were cultured in triplicate/treatment dose at 37 °C with 5% CO2 in atmosphere. Upon termination of the culture period, media was processed for analysis of glycerol content to determine lipolytic activity using an enzymatic colorimetric assay. The MIXED procedure of SAS for factorial treatment design was utilized to determine the effect of time and tannin treatment on lipolytic activity in cultured MA. Glycerol content was significantly higher (P£0.001) in tannin treated cultures. Time tended (P = 0.1) to influence the magnitude of lipolytic activity. Hence, quebracho tannin appears to augment lipolytic activity in cultured porcine MA. Determining the effect of tannin on lipolytic regulators will support the supposition that tannins influence MA lipid metabolism.