Concentration In Culture Medium Research Articles

Antiviral effect of plant-based drug on flu virus production in human fetal lung fibroblasts culture

Introduction . The number of complications in flu and acute respiratory viral infection, especially during epidemics, reaches 20–30%. The main causes of complications are immune protection disorders, leading to a sharp decrease in the antibacterial resistance of the body. Of particular importance is the prevention and treatment of flu in risk groups, especially in young children and elderly people. Aim of the study . Experimental study of possible antiinfluenza action of plant-based antiviral drug (EPs 7630) on the model of influenza A virus adapted to the human fetal lung fibroblast culture in conditions as close as possible to the lung tissue lesion in influenza infection. Materials and methods . The drug (EPs 7630) in the form of 11% solution in ethanol was diluted with sterile nutrient medium for application in the culture medium in the appropriate concentration. Transformed diploid culture of human fetal lung fibroblasts was used. Before the research 10 passages of the culture were made using growth medium with 10% embryonic bovine serum. Influenza virus A/Aichi/1/68 (H3N2) was also used, which was preliminarily adapted to the reproduction of human fetal lung fibroblasts in the cell culture. The cytotoxicity of the drug and its antiviral activity regarding the influenza virus were studied. Results. The presence of the drug in the human fetal lung fibroblasts culture in 3 tested concentrations had dose-dependent antiviral effect on the production of influenza virus A/Aichi/1/68 (H3N2), which also depends on the multiplicity of infection and the scheme of the drug use. Conclusion. The obtained data indicate that in case of low multiplicity of infection, the preparation has both preventive and therapeutic antiviral effect on the reproduction of influenza virus. The combination of antiviral and immunomodulatory action determines the bifunctionality of the drug, which contributes both to the suppression and elimination of influenza virus from the organ – the target and the body as a whole, and increase its non-specific congenital resistance, which makes it possible to consider the plant-based drug (EPs 7630) as the preferred tool in the prevention and comprehensive treatment of influenza and other respiratory viral infections in children and adults.

Metformin attenuates steroidogenesis in ovarian follicles of the broiler breeder hen.

The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

Determination of Dissolved CO2 Concentration in Culture Media: Evaluation of pH Value and Mathematical Data

Carbon dioxide is the most influential gas in greenhouse gasses and its amount in the atmosphere reached 412 µmol/mol in August 2020, which increased rapidly, by 48%, from preindustrial levels. A brand-new chemical industry, namely organic chemistry and catalysis science, must be developed with carbon dioxide (CO2) as the source of carbon. Nowadays, many techniques are available for controlling and removing carbon dioxide in different chemical processes. Since the utilization of CO2 as feedstock for a chemical commodity is of relevance today, this study will focus on how to increase CO2 solubility in culture media used for growing microbes. In this work, the CO2 solubility in a different medium was investigated. Sodium hydroxide (NaOH) and monoethanolamine (MEA) were added to the culture media (3.0 g/L dipotassium phosphate (K2HPO4), 0.2 g/L magnesium chloride (MgCl2), 0.2 g/L calcium chloride (CaCl2), and 1.0 g/L sodium chloride (NaCl)) for growing microbes in order to observe the difference in CO2 solubility. Factors of temperature and pressure were also studied. The determination of CO2 concentration in the solution was measured by gas analyzer. The result obtained from optimization revealed a maximum CO2 concentration of 19.029 mol/L in the culture media with MEA, at a pressure of 136.728 kPa, operating at 20.483 °C.

Modulation of sugar and nitrogen in callus induction media alter PAL pathway, SA and biomass accumulation in rice callus

In this study, the effect of varying nitrogen and sucrose concentrations in culture media was evaluated with respect to biomass production, accumulation of flavonoids, anthocyanin, and associated gene expression in rice callus. The callus was induced on control MS (M1), sugar-deficient (M2), sugar-excessive (M3), nitrogen-deficient (M4), and nitrogen-excessive (M5) media. The results indicated that the callus induction percentage (CIP) as well as the size and fresh weight of the callus were inhibited by all types of media compared with control media. Varying the sucrose and nitrogen concentration significantly affected callus morphology and caused a browning effect. Genes related to flavonoid biosynthesis (CHS, CHI, F3H, FLS and DFR) were upregulated in the callus cultured in all four media types compared with control media. Likewise, flavonoid and anthocyanin accumulation were higher in callus grown in excessive sugar- and nitrogen-containing media compared with control media. Unlike flavonoids, salicylic acid (SA) regulation was significantly higher in callus grown in sugar- and nitrogen-deficient media compared with control media. Sugar content was significantly higher in callus cultured in sugar-excessive media, whereas it was reduced in the callus cultured in the other media types compared with control media. Finally, chlorophyll was reduced in all callus media compared with the control media. The current study concluded that, varying concentration of sugar and nitrogen inhibit callus development and morphology via alteration of secondary metabolites and their related genes.

Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system

Background: High stocking density and intensive feeding in aquaculture systems lead to the accumulation of organic waste, which results in an increase in ammonia, nitrite, and nitrite concentrations in culture media. Biofloc is a potential technology to overcome this problem. The starter is a crucial carbon source for bacteria in the formation of biofloc. The objective of the present study aimed to explore the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Methods: A completely randomized design with four levels of treatment was used in this study. The tested starter was (A) control treatment, biofloc without starter, (B) biofloc with molasses starter, (C) biofloc with tapioca starter, and (D) biofloc with sucrose starter. The floc was cultured in 100-L tanks with a salinity of 17 ppt. The tanks were stocked with O. niloticus with a size of 3.71±0.11 cm at a stocking density of 30 fish per tank. The fish were fed on a commercial diet two times a day at satiation for 40 days. The ammonia, nitrite, and nitrite concentrations were measured for an interval of 8 days. Results: The study showed that the NH3-N range was 0.02–0.07 mg L−1 (mean, 0.03 ± 0.02 mg L−1), NO2-N range was 0.20–0.43 mg L−1 (mean, 0.25 ± 0.12 mg L−1), and NO3-N range was 0.90–3.20 mg L−1 (mean, 1.42 ± 1.19 mg L−1). Conclusion: Among the starters tested, molasses was found to be the best for biofloc in tilapia culture.

Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system

Background: High stocking density and intensive feeding in aquaculture systems lead to the accumulation of organic waste, which results in an increase in ammonia, nitrite, and nitrite concentrations in culture media. Biofloc is a potential technology to overcome this problem. The starter is a crucial carbon source for bacteria in the formation of biofloc. The objective of the present study aimed to explore the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Methods: A completely randomized design with four levels of treatment was used in this study. The tested starter was (A) control treatment, biofloc without starter, (B) biofloc with molasses starter, (C) biofloc with tapioca starter, and (D) biofloc with sucrose starter. The floc was cultured in 100-L tanks with a salinity of 17 ppt. The tanks were stocked with O. niloticus with a size of 3.71±0.11 cm at a stocking density of 30 fish per tank. The fish were fed on a commercial diet two times a day at satiation for 40 days. The ammonia, nitrite, and nitrite concentrations were measured for an interval of 8 days. Results: The study showed that the NH3-N range was 0.02–0.07 mg L−1 (mean, 0.03 ± 0.02 mg L−1), NO2-N range was 0.20–0.43 mg L−1 (mean, 0.25 ± 0.12 mg L−1), and NO3-N range was 0.90–3.20 mg L−1 (mean, 1.42 ± 1.19 mg L−1). Conclusion: Among the starters tested, molasses was found to be the best for biofloc in tilapia culture.

Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system

Background: High stocking density and intensive feeding in aquaculture systems lead to the accumulation of organic waste, which results in an increase in ammonia, nitrite, and nitrite concentrations in culture media. Biofloc is a potential technology to overcome this problem. The starter is a crucial carbon source for bacteria in the formation of biofloc. The objective of the present study aimed to explore the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Methods: A completely randomized design with four levels of treatment was used in this study. The tested starter was (A) control treatment, biofloc without starter, (B) biofloc with molasses starter, (C) biofloc with tapioca starter, and (D) biofloc with sucrose starter. The floc was cultured in 100-L tanks with a salinity of 17 ppt. The tanks were stocked with O. niloticus with a size of 3.71±0.11 cm at a stocking density of 30 fish per tank. The fish were fed on a commercial diet two times a day at satiation for 40 days. The ammonia, nitrite, and nitrite concentrations were measured for an interval of 8 days. Results: The study showed that the NH 3-N range was 0.02–0.07 mg L −1 (mean, 0.03 ± 0.02 mg L −1), NO 2-N range was 0.20–0.43 mg L −1 (mean, 0.25 ± 0.12 mg L −1), and NO 3-N range was 0.90–3.20 mg L −1 (mean, 1.42 ± 1.19 mg L −1). Conclusion: Among the starters tested, molasses was found to be the best for biofloc in tilapia culture.

Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system.

Background: High stocking density and intensive feeding in aquaculture systems lead to the accumulation of organic waste, which results in an increase in ammonia, nitrite, and nitrite concentrations in culture media. Biofloc is a potential technology to overcome this problem. The starter is a crucial carbon source for bacteria in the formation of biofloc. The objective of the present study aimed to explore the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Methods: A completely randomized design with four levels of treatment was used in this study. The tested starter was (A) control treatment, biofloc without starter, (B) biofloc with molasses starter, (C) biofloc with tapioca starter, and (D) biofloc with sucrose starter. The floc was cultured in 100-L tanks with a salinity of 17 ppt. The tanks were stocked with O.niloticus with a size of 3.71±0.11 cm at a stocking density of 30 fish per tank. The fish were fed on a commercial diet two times a day at satiation for 40 days. The ammonia, nitrite, and nitrite concentrations were measured for an interval of 8 days. Results: The study showed that the NH 3-N range was 0.02-0.07 mg L -1 (mean, 0.03 ± 0.02 mg L -1), NO 2-N range was 0.20-0.43 mg L -1 (mean, 0.25 ± 0.12 mg L -1), and NO 3-N range was 0.90-3.20 mg L -1 (mean, 1.42 ± 1.19 mg L -1). Conclusion: Among the starters tested, molasses was found to be the best for biofloc in tilapia culture.

A novel culture medium for freshwater diatoms promotes efficient photoautotrophic batch production of biomass, fucoxanthin, and eicosapentaenoic acid

Diatoms have recently emerged as new promising sources of fucoxanthin and eicosapentaenoic acid (EPA). Most applied studies in this area have focused on marine diatoms whereas the characterization of freshwater diatom metabolism has been restricted to ecophysiological studies. Here, we describe a novel culture medium for freshwater diatoms, the FDMed medium, and we demonstrate that it supports high biomass, fucoxanthin, and EPA production yields in photoautotrophic batch cultures of Sellaphora minima and Nitzschia palea. Growth was monitored for 21 days, fucoxanthin and EPA concentrations were assayed all along cultivation processes, and results were compared to those obtained for two other media (WC and Modified COMBO (MCOMBO)). The FDMed medium was found to increase biomass yields comparatively to MCOMBO medium (5–7-fold higher algal concentration at the stationary phase in S. minima and N. palea, respectively) while WC medium enables nearly no growth. The evolution of nitrate, silicate, and phosphate concentrations in culture media indicated a much better balance of macroelements in the FDMed medium. Fucoxanthin contents within the biomass remained very stable in the FDMed medium compared to the other media, in which it gradually decreases, and the best results (around 7.5 mg g-1 dry weight ) were obtained for S. minima in the FDMed medium. EPA could only be measured in the FDMed medium and the highest content (14.0 mg EPA g-1 dry weight) was recorded in N. palea. This work demonstrates the great industrial potential of freshwater diatoms and provides an efficient culture medium for those organisms.

Application of Central Composite Design to Optimize Spawns Propagation

Despite the increased recognition of the nutritional value of the Oyster mushroom, coupled with its ability to tolerate a wide range of climatic conditions, its production is still at infancy stage with low adoption rate in Kenya. The low uptake could be attributed to the cost of spawns or lack of skills for spawns preparations coupled with poor knowledge on oyster mushroom consumption benefits. The objective of this study was to optimize Pleurotus ostreatus (Oyster mushroom) spawns production. To achieve the objective, the spawns propagation was optimized by varying the temperature level, sterilization time and culture media concentration in order to establish the feasible levels which minimized the days of mycelium full development using central composite designs. Based on the study findings, 26.29˚C, 17.36 minutes and 60.95 g/L of temperature level, sterilization time and culture media concentration levels respectively minimized the days to full coverage of mycelium in a petri dish. Central composite designs for controlling temperature, sterilization time and culture media concentration were recommended for spawns maximum production. A further research on multiple response optimizations aimed at achieving resistance to bacterial diseases and yield by varying the strain in the culture were recommended.

Callus culture as a new approach for the production of high added value compounds in Ilex paraguariensis: genotype influence, medium optimization and compounds identification.

Ilex paraguariensis (yerba mate) is a native species from South America and is a rich source of bioactive compounds. There is a lack of research efforts on the phytochemical investigation of callus culture from this species. In the present study, an effort was made to optimize callus culture conditions and to identify secondary compounds. Calli were induced from 10 genotypes using leaf explants and the best genotype was selected to evaluate the effects of cytokinin types and concentrations on callus induction and biomass accumulation. The best genotype and cytokinin treatment were used to conduct one last experiment with sucrose concentrations in culture media and its effects on calli biomass, antioxidant activity and secondary compounds accumulation. Callus initiation was genotype dependent, and the 6-156-6 line had the best response. Zeatin supplemented medium showed higher callus induction rate (82%) and higher biomass accumulation after 120 days (328.2 mg). Higher biomass and secondary compounds accumulation were observed for calli on 3% sucrose medium. Antioxidant activity was not affected by sucrose concentrations. Yerba mate callus culture allowed the accumulation of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, theobromine and caffeine.

Physiological and biochemical response of Phoenix dactylifera L. Hayani cv. embryogenic callus and somatic embryos to salt stress under in vitro conditions

Salinity stress as a major abiotic stress has influenced more than 6% of the world land which isincreasing daily due to climate changes. The effect of salinity on callus regeneration and organogenesis of datepalm was tested under in vitro conditions. This study was conducted to investigate the effect of salt stress oncallus proliferation, somatic embryo formation of ‘Hayani’ date palm in vitro. The embryogenic callus wassubjected to MS medium with BA at 0.05 mg/l and NAA at 0.1 mg/l supplied with different sodium chloride(NaCl) concentrations (0, 500, 1000, 1500 and 2000 ppm) for 2 subcultures. Salinity affects severalphysiological and biochemical processes. The investigated parameters include embryogenic callus growth,differentiation to form somatic embryos, fresh weight of both callus and embryos, biochemical analysis such astotal carbohydrates, proline, protein content and minerals in embryogenic callus and somatic embryos. Lowlevels of abiotic stress factors can induce favorable responses in the growth and differentiation. However, highlevels are detrimental resulting in reduced growth and some cell death. The callus fresh weight and the numberof embryos were enhanced by adding 500 and 1000 ppm NaCl to the proliferation medium; higher saltconcentrations affected the callus regeneration negatively, where at 2000 ppm NaCl the less somatic embryoswere formed. High concentrations of NaCl (1500 and 2000 ppm) decreased total soluble carbohydrates in bothembryogenic callus and somatic embryos, proline content increased gradually as the external concentration ofNaCl increased and the protein content decreased in both embryogenic callus and somatic embryos with theincrease of NaCl concentration in culture media to 2000 ppm. Salinity inhibits water uptake and increases theconcentration of toxic ions such as Na+and reduced the K+ content. Tissue culture with the use of this techniquewould allow selection for salt tolerance in the date palm. In fact, somatic embryos subjected to 500 and 1000ppm NaCl concentrations were successfully regenerated into plantlets.

Serotonin-evoked cytosolic Ca2+ release and opioid receptor expression are upregulated in articular cartilage chondrocytes from osteoarthritic joints in horses

Osteoarthritis is a pain-associated progressive disease and pain mediators, such as opioid receptors, expressed in articular cartilage could represent novel therapeutic targets. Acute and chronic stages of OA indicate different metabolic abilities of the chondrocytes depending on inflammatory state.This study aimed to investigate the response of healthy and osteoarthritic chondrocytes and their expression and release of pain mediators in response to acute inflammation.Interleukin-1 beta (IL-1β) and lipopolysaccharide (LPS) were used to induce an acute inflammatory response in cultured equine chondrocytes harvested from healthy joints (HC) and osteoarthritic joints (OAC), the latter representing acute exacerbation of a chronic inflammatory state. Intracellular Ca2+ release was determined after exposure to serotonin (5-hydroxytryptamine (5-HT), glutamate or ATP. Protein expression levels of F- and G-actin, representing actin rearrangement, and opioid receptors were investigated. Glutamate concentrations in culture media were measured. Cartilage was immunohistochemically stained for µ (MOR), κ (KOR), and δ (DOR) opioid receptors.Upon exposure to acute inflammatory stimuli, OAC showed increased intracellular Ca2+ release after 5-HT stimulation and increased expression of MOR and KOR. When cells were stimulated by inflammatory mediators, glutamate release was increased in both HC and OAC. Immunostaining for MOR was strong in OA cartilage, whereas KOR was less strongly expressed. DOR was not expressed by cultured HC and OAC and immunostaining of OA cartilage equivocal.We show that chondrocytes in different inflammatory stages react differently to the neurotransmitter 5-HT with respect to intracellular Ca2+ release and expression of peripheral pain mediators.Our findings suggest that opioids and neurotransmitters are important in the progression of equine OA. The inflammatory stage of OA (acute versus chronic) should be taken into consideration when therapeutic strategies are being developed.

Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β-galactosidase modification.

3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2'-fucosyllactose (2'-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2'-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2'-FL, and lower digestibility of 3-FL than 2'-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in kcat and the decrease in Km . We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP- L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D-lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L-1 ·hr-1 and the specific 3-FL yield of 0.5 g/g dry cell weight.

Valorization of sugarcane straw to produce highly conductive bacterial cellulose / graphene nanocomposite films through in situ fermentation: Kinetic analysis and property evaluation

Abstract Bacterial cellulose based nanocomposites have found a growing interest in recent decades due to their impressive inherent characteristics with potential applications in diverse sectors. However, there remain several challenges due to increased production cost, lower yield, and sustainability or biocompatibility issues after chemical-based modifications. This study demonstrates the fabrication of bacterial cellulose-reduced graphene oxide films via in-situ fermentation approach using abundantly available agricultural waste (sugarcane straw) as a feedstock. The presence of reduced graphene oxide at different concentrations in culture media, significantly altered the fermentation kinetics, as evident from kinetic parameter and yield coefficients. Higher yields of bacterial cellulose-reduced graphene oxide nanocomposites, with presence of strongly integrated network-like structures between bacterial cellulose nanofibers and reduced graphene oxide nanosheets were observed at 2 wt % reduced graphene oxide loadings. Formation of such percolated networks was confirmed from improved mechanical properties and enhanced electrical conductivity, through both experimental and modeling investigations. The proposed in-situ fermentation technique to produce highly conductive bacterial cellulose-reduced graphene oxide films provides an alternative approach to meet the growing demands of biomass-derived renewable and sustainable biomaterials with commercial significance.

Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Inhibition of Pro-Inflammatory Cytokine Secretion by Select Antioxidants in Human Coronary Artery Endothelial Cells.

Inflammatory and oxidative stress in endothelial cells are implicated in the pathogenesis of premature atherosclerosis in diabetes. To determine whether high-dextrose concentrations induce the expression of pro-inflammatory cytokines, human coronary artery endothelial cells (HCAEC) were exposed to either 5.5 or 27.5 mM dextrose for 24-hours and interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor α (TNF α) levels were measured by enzyme immunoassays. To determine the effect of antioxidants on inflammatory cytokine secretion, cells were also treated with α-tocopherol, ascorbic acid, and the glutathione peroxidase mimetic ebselen. Only the concentration of IL-1β in culture media from cells exposed to 27.5 mM dextrose increased relative to cells maintained in 5.5 mM dextrose. Treatment with α-tocopherol (10, 100, and 1,000 μM) and ascorbic acid (15, 150, and 1,500 μM) at the same time that the dextrose was added reduced IL-1β, IL-6, and IL-8 levels in culture media from cells maintained at 5.5 mM dextrose but had no effect on IL-1β, IL-6, and IL-8 levels in cells exposed to 27.5 mM dextrose. However, ebselen treatment reduced IL-1β, IL-6, and IL-8 levels in cells maintained in either 5.5 or 27.5 mM dextrose. IL-2 and TNF α concentrations in culture media were below the limit of detection under all experimental conditions studied suggesting that these cells may not synthesize detectable quantities of these cytokines. These results suggest that dextrose at certain concentrations may increase IL-1β levels and that antioxidants have differential effects on suppressing the secretion of pro-inflammatory cytokines in HCAEC.

A silk fibroin/decellularized extract of Wharton\u2019s jelly hydrogel intended for cartilage tissue engineering

A hybrid hydrogel was obtained from decellularized extract from Wharton’s jelly (DEWJ) and silk fibroin (SF) and characterized for cartilage tissue engineering. Wharton’s jelly was used due to its similarity with articular cartilage in extracellular matrix composition. Also, silk fibroin has good mechanical properties which make this construct appropriate for cartilage repair. Decellularization of Wharton’s jelly was verified by DAPI staining, DNA quantification, and PCR analysis. Then, the biochemical composition of DEWJ was determined by ELISA kits for total proteins, collagens, sulfated glycosaminoglycans (sGAG), and transforming growth factor β1 (TGF-β1). After fabricating pure SF and SF/DEWJ hybrid hydrogels, their physical and mechanical properties were characterized by FESEM, Fourier-transform infrared spectroscopy (FTIR) and rheological assays (amplitude and frequency sweeps). Furthermore, cell viability and proliferation were assessed by MTT assay. The results have shown that DEWJ in hybrid hydrogels enhances mechanical properties of the construct relative to pure SF hydrogels. Also, this extract at its 40% concentration in culture media and 20% or 40% concentrations in SF/DEWJ hybrid hydrogels significantly increases population of the cells compared to control and pure SF hydrogel after 7 days. In conclusion, this study proposes the potential of SF/DEWJ hybrid hydrogels for cartilage tissue engineering applications.

Clinical application of immunomagnetic reduction for quantitative analysis of beta-subunit of human chorionic gonadotropin in blastocyst culture media to differentiate embryo quality

BackgroundThe most important factor for a successful pregnancy after in vitro fertilization is embryo quality. The aim of this study was to explore the possibility that using the immunomagnetic reduction (IMR) assay to quantitatively measure β-subunit of human chorionic gonadotropin (β-hCG) in blastocyst culture media to differentiate embryo quality. MethodsThis was a prospective case-control study including 28 samples of blastocyst culture media. We used single-step blastocyst culture and IMR assay to analyze β-hCG concentrations in culture media. We also explored the relationship between IMR signals of β-hCG and morphological grading of blastocysts. Resultsβ-hCG concentration-dependent IMR signals were highly correlated with blastocyst morphological quality (Spearman correlation coefficient: 0.731). Receiver-operating characteristic curve analysis showed a cut-off IMR value to differentiate embryo quality of 0.873%, with an area under the curve of 0.947, sensitivity of 0.882 and specificity of 0.818. Furthermore, subanalysis also revealed a positive correlation between β-hCG concentration-dependent IMR signals and trophectoderm grading, with a Spearman correlation coefficient of 0.576. ConclusionsAn IMR assay can quantitatively measure β-hCG in blastocyst culture media, and may be a potential clinical tool to assist in the assessment of good blastocyst quality before embryo transfer.

144 Sex ratio of in vitro-produced goat embryos

Goats are important livestock species because they produce meat, milk, and fibre and are also easily maintainable on small farms. Although goats provide many products and consumption of goat meat is increasing in the United States, the industry lags compared with many species with regard to IVF techniques to enhance goat production. It has been demonstrated in other species that male IVF embryos tend to develop faster than those of females. This may be due to increased tolerance of male embryos to inadequate conditions, particularly, glucose concentrations in culture media. However, the sex ratio of goat embryos produced utilising IVF remains unknown. The aim of this study was to determine the sex ratio of goat embryos utilising a commercially available media suite (IVF Biosciences, Falmouth, UK). Oocytes were harvested from ovaries obtained from 2 local abattoirs and matured in vitro. Frozen sperm from 1 of 2 billy goats were randomly assigned for each round of IVF. Embryos were evaluated daily from Days (D) 6 through 9 of in vitro culture. On the day an embryo reached the expanded blastocyst stage, it was removed from culture and placed into DNA extraction buffer (PicoPureTM DNA Extraction Kit, Applied Biosystems, Waltham, MA, USA) and stored at −20 for PCR analysis, typically within one month of collection. In all unknown samples, positive male (sperm) and female (uterus) controls, the amelogenin gene was amplified and products were evaluated on a 1.5% agarose gel with ethidium bromide. Embryos with 2 bands (202 and 262bp) were classified as male, and those with 1 band (262bp) were classified as female. Embryos with no bands were not included in analysis. Embryos reached the expanded blastocyst stage on D6 (n=29), D7 (n=39), and D8/9 (n=35, combined for evaluation). A chi-squared analysis comparing the percentage of male and female embryos to the expected 50% was completed for each time point (D6, D7, D8/9), as well as overall ratios (D6-9). In total, 350 oocytes were utilised in 6 rounds of IVF resulting in a mean blastocyst rate of 32% (range 17-47%). There was no significant difference in the number of embryos that were male on D6 (55%) and D7 (46%). However, on D8/9 significantly fewer embryos were male (29% male; P=0.01). Overall, there was no significant difference (P=0.14) in the sex ratio, with 41% male and 59% female embryos. Our findings are somewhat consistent with other species, in that male goat embryos produced via IVF develop more quickly in culture conditions; however, female embryos were still able to tolerate culture conditions. Delayed blastocyst development may not necessarily be an indication of a reduced quality embryo but one that is slower to develop based on its sex. This could be due to expression of X-linked genes being unbalanced during pre-implantation embryo development stages and warrants further study. One influencer of sex ratio we are currently investigating is the impact of glucose during culture, to further understand metabolism in IVF embryos.