Early Incubation Times Research Articles

Resolving the Maturation of Elastomeric Protein Condensates

Tropoelastin is a key protein in the formation of connective tissue such as lungs, arteries, and cartilage. The assembly and further cross‐linking process of tropoelastin culminates in the formation of elastin fibers, a resilient biomaterial capable of withstanding numerous cycles of stress and strain. Like other intrinsically disordered proteins, tropoelastin can undergo liquid‐liquid phase separation in vitro and in the extracellular space. This event is thought to aid in the self‐assembly and subsequent maturation of elastin fibers. Although the mechanical properties and morphology of mature elastin fibers have been extensively studied, the properties of elastin liquid droplets and their subsequent maturation into a solid remains poorly understood. Here, we use a model mini‐elastin polypeptide that mimics the domain architecture of naturally occurring tropoelastin to characterize this transition. We use fluorescence recovery after photobleaching (FRAP) and microrheology to capture the transition of elastin droplets from a liquid to a solid‐like state. We find that elastin droplets behave as viscous fluids at early incubation times, however, a rapid liquid‐to‐solid transition is observed in a timeframe of 80 minutes when held at constant temperature, even in the absence of cross‐linker. We further resolve the changes in dynamics, diffusion, and material properties of elastin condensates over the course of this transition. This work, which reveals the material transition from within elastin condensates, lends new insight into the early steps of the self‐assembly process of elastin while also contributing to the expanding repertoire of condensate maturation models in biological systems.

Effect of nitrogen on the structure evolution and biological properties of mesoporous bioactive glass nanospheres: Experiments and simulations

Mesoporous bioactive glasses (MBGs) have displayed great potential in dental restorations and bone regeneration for their controllable structure and great bioactivity. In this work, we have systematically investigated the effect of nitrogen on structure evolution, apatite-forming ability and cytotoxicity in MBG by successfully preparing the N-containing MBG nanospheres by a modified sol-gel method. It is interesting to demonstrate that all obtained samples exhibit uniform interstitial mesoporous nanospheres microstructure, with the particles size increases as the nitrogen addition increases from 5 to 15 mol.%. Notably, a large amount of blooming flower-like hydroxyapatite deposited on the surface of 15N-MBG after immersed in simulated body fluid for 7 days, exhibiting excellent apatite-forming ability. It is highlighted that the incorporation of nitrogen can stimulate cell proliferation at early incubation time. Moreover, all the MBGs have no obvious inhibitory impact on the hPDLCs growth at extracted concentration. Furthermore, via glass network connectivity analysis from molecular dynamics simulations, the decrease of network connectivity caused by the incorporation of nitrogen could provide looser glass network and higher bioactivity, which explains the experimental results well.

Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

Temporal, Spatial, and Temperature Controls on Organic Carbon Mineralization and Methanogenesis in Arctic High-Centered Polygon Soils.

Warming temperatures in continuous permafrost zones of the Arctic will alter both hydrological and geochemical soil conditions, which are strongly linked with heterotrophic microbial carbon (C) cycling. Heterogeneous permafrost landscapes are often dominated by polygonal features formed by expanding ice wedges: water accumulates in low centered polygons (LCPs), and water drains outward to surrounding troughs in high centered polygons (HCPs). These geospatial differences in hydrology cause gradients in biogeochemistry, soil C storage potential, and thermal properties. Presently, data quantifying carbon dioxide (CO2) and methane (CH4) release from HCP soils are needed to support modeling and evaluation of warming-induced CO2 and CH4 fluxes from tundra soils. This study quantifies the distribution of microbial CO2 and CH4 release in HCPs over a range of temperatures and draws comparisons to previous LCP studies. Arctic tundra soils were initially characterized for geochemical and hydraulic properties. Laboratory incubations at −2, +4, and +8°C were used to quantify temporal trends in CO2 and CH4 production from homogenized active layer organic and mineral soils in HCP centers and troughs, and methanogen abundance was estimated from mcrA gene measurements. Results showed that soil water availability, organic C, and redox conditions influence temporal dynamics and magnitude of gas production from HCP active layer soils during warming. At early incubation times (2–9 days), higher CO2 emissions were observed from HCP trough soils than from HCP center soils, but increased CO2 production occurred in center soils at later times (>20 days). HCP center soils did not support methanogenesis, but CH4-producing trough soils did indicate methanogen presence. Consistent with previous LCP studies, HCP organic soils showed increased CO2 and CH4 production with elevated water content, but HCP trough mineral soils produced more CH4 than LCP mineral soils. HCP mineral soils also released substantial CO2 but did not show a strong trend in CO2 and CH4 release with water content. Knowledge of temporal and spatial variability in microbial C mineralization rates of Arctic soils in response to warming are key to constraining uncertainties in predictive climate models.

The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226.

Background and purpose:Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay.Experimental approach:Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity.Findings/Results:Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 μg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK.Conclusion and implication:Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.

Modulatory effect of heat stress on viability of primary cultured chicken satellite cells and expression of heat shock proteins ex vivo

Satellite cells promote muscle repairing and muscle growth. Thereby the intention of the present study was to investigate the beneficial effects of heat stress at different time intervals on chicken satellite cells’ viability. Satellite cells were isolated from 1-day-old chicks and treated at two different temperatures (37 °C and 41 °C) for various time periods (6 h, 12 h, 24 h, 48 h, and 72 h). Both temperatures significantly increased cell viability after 24 h and 48 h. After 12 h, cell viability was significantly increased at 41 °C compared to 37 °C. However, more apoptotic cells were observed at end of the experiment of 41 °C compared to 37 °C. In addition, more live cells were found at early of experimental period at 41 °C than 37 °C. Additionally, protein and mRNA expression of HSP70, HP60 and HSP47 were significantly upregulated throughout the experimental period at temperature of 41 °C compared to those at 37 °C. These results indicate that cell viability and expression of heat stress related proteins/genes are induced by high temperature of 41 °C via heat stress pathway whereas activation of heat stress related proteins/genes are lower at 37 °C. Thus, 41 °C can trigger satellite cells’ viability essential for better cell survival than 37 °C at early incubation time.

Hydrophobic Domain Structure of Linear-Dendritic Poly(ethylene glycol) Lipids Affects RNA Delivery of Lipid Nanoparticles.

In this work, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids (PEG-GnCm) were synthesized through a strategy using sequential aza- and sulfa-Michael addition reactions. The effect of modulating the hydrophobic domain of linear-dendritic PEG lipids was systematically investigated for in vitro and in vivo small RNA delivery as the surface-stabilizing component of 5A2-SC8 dendrimer lipid-based nanoparticles (DLNPs). The lipid alkyl lengths (C8, C12, and C16) and dendrimer generations (G1, G2, and G3) were altered to create PEG-GnCm with different physical properties and anchoring potential. The tail chemical structure of PEG-GnCm did not affect the formulation of 5A2-SC8 DLNPs, including the nanoparticle size, RNA encapsulation, and stability. However, the tail chemical structure did dramatically affect the RNA delivery efficacy of the formed 5A2-SC8 DLNPs with different PEG-GnCm. First-generation PEG lipids (PEG-G1C8, PEG-G1C12, and PEG-G1C16) and a second-generation PEG lipid (PEG-G2C8) formed 5A2-SC8 DLNPs that could deliver siRNAs effectively in vitro and in vivo. 5A2-SC8 DLNPs formulated with second-generation PEG lipids (PEG-G2C12 and PEG-G2C16) and all three third-generation PEG lipids (PEG-G3C8, PEG-G3C12, and PEG-G3C16) lost the ability to deliver siRNA effectively in vitro and in vivo. Overall, we determined that the hydrophobic domain chemical structure of linear-dendritic poly(ethylene glycol) lipids affected the RNA delivery of DLNPs by impacting the escape of 5A2-SC8 DLNPs from endosomes at early cell incubation times, thereby indicating how PEG lipid anchoring and chemical structure can modulate in vitro and in vivo siRNA delivery efficacies.

In vitro evaluation of Álcool desidrogenase kinetics in Ziziphus joazeiro fruits to alleviate the harmful effects of alcohol

Ziziphus joazeiro is endemic to the brazilian Caatinga and its fruits can be used as a food supplement by accelerating the ethanol metabolism in the body and reducing the alcohol harmful effects due to high alcohol dehydrogenase (ADH) activity. The objective was to determine the kinetics of ADH activity, in different incubation times, of Z. joazeiro mature fruits as a food supplement. Ziziphus joazeiro fruits, at the fourth maturation stage, were incubated for 0 (no incubation), 3, 6, 12, 24 and 48 hours at room temperature. The ADH activity was determined. ADH activity was higher in fruits incubated for 0 and 3 h. The ADH activity was higher in the early incubation times, probably due to the greater availability of NAD+, which after being reduced to NADH delayed regeneration. Without the cofactor in the oxidized form, the enzymatic activity decreases. Ziziphus joazeiro fruit has the potential to be used as a food supplement accelerating the alcohol metabolism and reducing the harmful effects.

Tentative breakpoints and areas of technical uncertainty for early reading automated disc diffusion for Enterobacterales.

Disc diffusion is a reliable, accurate and cost-efficient procedure for antimicrobial susceptibility testing (AST) but requires long (18-24 h) incubation times. Reading of disc diffusion after short incubation times (6-8 h) by automated systems is feasible but should be categorized with time-adapted breakpoints to reduce errors. This study systematically compared early readings (6 and 8 h) of disc diffusion using an automated system with that of the standard 18 h EUCAST method. Time-adapted tentative breakpoints were proposed to discriminate susceptible from resistant isolates and areas of technical uncertainty were defined to minimize the risk of errors. A total of 1106 Enterobacterales isolates with a wide variety of resistance mechanisms and resistance profiles were included. All isolates were analysed for susceptibility to amoxicillin/clavulanic acid, ceftriaxone, cefepime, meropenem, ciprofloxacin and gentamicin using the automated WASPLabTM system. Part of the collection (515 isolates) was also analysed for susceptibility to an additional 10 antibiotics. Separation between WT and non-WT populations was poorer at early incubation times than following standard incubation. Editing of rapid automated AST results after 6 and 8 h incubation with time-adapted breakpoints resulted in 84.0% and 88.5% interpretable results with assignment to the resistant or susceptible category. Major error and very major error rates for the 6 h readings were only 0.4% and 0.3%, virtually identical to those of 18 h AST reading. Time-adapted clinical breakpoints in disc diffusion testing for Enterobacterales allow for accurate automated AST interpretation after shortened incubation times for a large number of antibiotics, with the additional possibility of subsequent confirmation after 18 h incubation.

Luminescent switch of polysaccharide-peptide-quantum dot nanostructures for targeted-intracellular imaging of glioblastoma cells

Abstract Glioblastoma multiforme (or GBM) remains one of the deadliest types of brain cancers. Nanomedicine can offer new strategies for fighting against GBM by combining the earliest possible diagnosis with multiple options of therapy. Hence, in this work, cysteine (Cys) and Poly-L-Arginine (PA) moieties were grafted to carboxymethyl cellulose (CMC) to produce biofunctional hybridized macromolecules (CMC_Cys and CMC_PA). These polymer-peptide conjugates were used simultaneously as surface capping ligands and biofunctional modifiers for the synthesis of ternary Ag-In-S (AIS) quantum dots (AIS QDs) via a green chemical process in aqueous medium and room temperature. These core-shell supramolecular nanostructures (AIS@CMC, AIS@CMC_Cys, and AIS@CMC_PA) were tested as fluorescent nanoprobes (“OFF-ON”) for targeted bioimaging and in vitro intracellular tracking of glioblastoma cells (GBM, U-87 MG). The nanosystems were characterized for physicochemical, structural, and morphological properties by NMR, UV–Vis, PL, FTIR, TEM/EDX/SAED, zeta potential, and DLS. Cytocompatibility was evaluated by mitochondrial activity assay, and confocal laser scanning microscopy was performed for investigating the kinetics of cellular uptake. The grafting caused a noticeable reduction of surface charges, associated with a drastic photoluminescence quenching (i.e., “OFF-state”) of AIS@CMC_Cys and AIS@CMC_PA compared to unmodified AIS@CMC. This effect was smartly applied for bioimaging GBM cells and for monitoring the internalization process by intracellular tracking, which underwent strong “de-quenching” at very early incubation times (~5 min). Thus, these novel hybrid nanocolloids produced via eco-friendly scalable aqueous process show potential as responsive fluorescent bioprobes for bioimaging and tracking intracellular pathways and mechanisms as a powerful weapon for fighting against brain cancer cells.

Bone Mineral Crystallinity Governs the Orchestration of Ossification and Resorption during Bone Remodeling.

The circularly causal orchestration of bone production and destruction is a part of the standard model of bone remodeling, but the crystallinity of the bone mineral, which naturally alternates during this process, has not had a steady place in it. Here we show that osteoclasts and osteoblasts, the cells resorbing and building bone, respectively, can sense the crystallinity of the bone mineral and adjust their activity thereto. Specifically, osteoblastic MC3T3-E1 cells secreted mineral nodules more copiously when they were brought into contact with amorphous calcium phosphate (ACP) nanoparticles than when they were challenged with their crystalline, hydroxyapatite (HAp) analogues. Moreover, the gene expressions of osteogenic markers BGLAP, ALP, BSP-1, and RUNX2 in MC3T3-E1 cells were higher in the presence of ACP than in the presence of HAp. At the same time, the dental pulp stem cells differentiated into an osteoblastic phenotype to a degree that was inversely proportional to the amount and the crystallinity of the mineral added to their cultures. In contrast, the resorption of HAp nanoparticles was more intense than the resorption of ACP, as concluded by the greater retention of the latter particles inside the osteoclastic RAW264.7 cells after 10 days of incubation and also by the time-dependent free Ca2+ concentration measurements in the cell culture media at early incubation time points (<1 week), prior to the spontaneous crystallization of the amorphous phase. A detailed morphological, compositional, and microstructural characterization of ACP and HAp is provided too, and it is shown that although ACP transforms to HAp in the cell culture media, some microstructural properties are retained in the powder following this transformation, influencing the resorption rate. On the basis of these findings, a model of bone remodeling at the level of individual biogenic apatite nanoparticles was proposed, taking into account the effects of hydration and lattice strain. According to this model, apatite is a "living" mineral, undergoing fluctuations in crystallinity within a closed ossifying/resorptive feedback loop in a way that buffers against potential runaway effects. A finite degree of amorphousness of every apatite crystal in bone is seen as a vital prerequisite for a healthy, dynamic bone remodeling process, and the best bone mineral, from this standpoint, is the living mineral, the one undergoing a constant process of structural change in response to biochemical stimuli thanks to its partially amorphous microstructure and unique interfacial dynamics.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Promoting the treatment of crude oil alkane pollution through the study of enzyme activity

Microbes appear to play a key role in bioremediation of petroleum hydrocarbons pollution and little attention has been paid to the enzyme activity in the process of alkane bioremediation. Oil field bacterium identified as Pseudomonas synxantha LSH-7′ was chosen as the tested strain. Periodically collected samples were analyzed by GC-FID (Gas Chromatography- Flame Ionization Detector) and RT-qPCR (Quantitative-Real-Time-PCR). GC-FID results showed this bacterial strain has great degradation ability on crude oil n-alkanes and RT-qPCR data indicated the differences between the three genes expression including AlkB-, Cytochromes P450-, and almA- related when grown on different-chain alkanes. Meanwhile, enzyme activity like alkane hydroxylase, alcohol dehydrogenase, dehydrogenase, protease, phosphatase, catalase and lipase were measured. Extracellular alkane hydroxylase was induced in a higher degree than intracellular in the early incubation time, alcohol dehydrogenase increased/decreased along with alkane hydroxylase, and the pH of the medium obviously decreased. Other enzymes were also described including dehydrogenase activity that reached a highest point that was slower than alcohol dehydrogenase, protease activity started multiplying after a period of culture while biomass was immediately increased, catalase activity dramatically enhanced in the presence of alkanes, phosphatase activity was closely linked to pH approximately but lipase activity was found to be moderate.

P-31 - Impact of peroxynitrite-mediated nitrosative stress on human sperm cells

Nitrosative stress is caused by high levels of reactive nitrogen species (RNS) and among the most toxic RNS is peroxynitrite. In infertile men there is an association between high levels of peroxynitrite and decreased sperm quality; however, less is known about the specific cellular impairment caused by peroxynitrite in sperm cells. The aim here was to characterize the in vitro effect of peroxynitrite on human sperm. For this, spermatozoa were exposed to 3-morpholinosydnonimine, a compound that generates peroxynitrite, and several parameters of sperm quality and biochemical markers of cell death were assessed. Untreated sperm were included as controls. At early incubation times peroxynitrite exposure caused a decrease in mitochondrial membrane potential, ATP level and motility while thiol oxidation and tyrosine nitration increased. At prolonged incubation time viability decreased and DNA oxidation, DNA fragmentation and ultrastructural damage increased. Mitochondrial permeability transition (MPT) was induced while caspase activation and phosphatidylserine externalization were not evidenced. These results indicate that peroxynitrite exposure impairs sperm function and ultrastructure and lead to the appearance of biochemical markers of cell death compatible with MPT-driven necrosis

Effect of nitrogen fertilization on the fate of rice residue-C in paddy soil depending on depth: 13C amino sugar analysis

A 100-day incubation experiment was conducted to (i) trace the fate of rice residue-derived 13C in the amino sugar (AS) pool in 0–1-cm (oxic) and 1–5-cm (anoxic) layers of paddy soil and (ii) evaluate the effects of inorganic N ((NH4)2SO4) fertilization on the formation of AS at early and late incubation times (5 and 100 days, respectively). The accumulation of rice residue-derived AS occurred at 5 and 100 days in both soil layers as a result of AS stabilization. Inorganic N addition increased the contents of rice residue-derived muramic acid, glucosamine, and galactosamine in the 0–1-cm soil layer for both incubation times by average on 14.7–20.8%, 23.7–31.8%, and 11.6–23.3%, respectively. In contrast, no effects of N fertilization on AS content in the 1–5-cm soil layer were found. The amount of rice residue-derived AS was higher in the 1–5-cm than in the 0–1-cm soil layer at early incubation time, probably due to the higher contents of ammonium here compared to the upmost oxic layer where nitrate was the dominated N form. Thus, the preferential uptake of ammonium but not nitrate by microorganisms led to the higher formation of rice residue-derived AS in the anoxic soil layer. The ratio of fungal to bacterial residues (fungal glucosamine/muramic acid) ranged between 1.0 and 1.7 for rice residue-derived AS and was 12.5–14.6 for total AS, indicating that fungi and bacteria have similar contributions to the decomposition of fresh rice residue whereas native soil organic matter (SOM) is a fungi-predominated process. This study emphasized that coupling of C and N cycles in paddy soils is different in oxic and anoxic layers, resulting in variation of plant residue decomposition and formation of SOM.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BackgroundThe increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.ObjectivesHere, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and MethodsThe encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.ResultsIn fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.ConclusionsOur founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

Degradation profile of synthetic coral scaffold in cell culture media

&lt;p&gt;The scaffold is one of the factors in tissue engineering that determine the success of bone regeneration. The important characteristic of the scaffold is able to degrade gradually. In vitro study using cells, the scaffold will be exposed to culture media. Therefore, degradation profile for scaffold needs to be examined. This study aims to investigate the degradation profile of synthetic coral scaffold in cell culture media using pH measurement. The method used the synthetic coral scaffolds were prepared from denaturalized collagen (gelatin) and calcium carbonate (calcite) with a concentration of 5:5 and 4:6 weight % in aqua dest. The scaffold&lt;span lang="IN" style=""&gt;s&lt;/span&gt; were fabricated in membrane thick film which was then physically crosslinked.&amp;nbsp; &lt;span lang="IN" style=""&gt;The &lt;/span&gt;10 % &lt;span lang="IN" style=""&gt;of gelatin scaffold was used as a control.&lt;/span&gt;&amp;nbsp; The scaffolds were incubated in cell culture media (non-phenol red Dulbecco’s Modified Eagle Medium) for 1 until 8 days, and pH changes of the medium were measured. As the result, Profile of degradation on day 1 to day 4 showed the 5:5 scaffold had the smallest degradation. The results indicated the significant different between scaffold concentration in day 1 (p=0.005), 5th(p=0.03), and 6th day (p=0.011). At the end of incubated days, the pH changed but not significantly different. LSD showed the significant differences between scaffold (5:5 and 4:6) with control and no significant difference between 2 concentrations of the scaffold. The conclusion of this study is the synthetic coral scaffold degraded gradually until the end incubation time and between concentration had different degradation profile in the early incubation time using pH measurement.&lt;br&gt;&lt;/p&gt;

Gold Nanoparticle Size and Shape Effects on Cellular Uptake and Intracellular Distribution of siRNA Nanoconstructs.

Gold nanoparticles (AuNPs) show potential for transfecting target cells with small interfering RNA (siRNA), but the influence of key design parameters such as the size and shape of the particle core is incomplete. This paper describes a side-by-side comparison of the in vitro response of U87 glioblastoma cells to different formulations of siRNA-conjugated gold nanoconstructs targeting the expression of isocitrate dehydrogenase 1 (IDH1) based on 13 nm spheres, 50 nm spheres, and 40 nm stars. 50 nm spheres and 40 nm stars showed much higher uptake efficiency compared to 13 nm spheres. Confocal fluorescence microscopy showed that all three formulations were localized in the endosomes at early incubation times (2 h), but after 24 h, 50 nm spheres and 40 nm stars were neither in endosomes nor in lysosomes while 13 nm spheres remained in endosomes. Transmission electron microscopy images revealed that the 13 nm spheres were enclosed and dispersed within endocytic vesicles while 50 nm spheres and 40 nm stars were aggregated, and some of these NPs were outside of endocytic vesicles. In our comparison of nanoconstructs with different sizes and shapes, while holding siRNA surface density and nanoparticle concentration constant, we found that larger particles (50 nm spheres and 40 nm stars) showed higher potential as carriers for the delivery of siRNA.

Sex-specific foraging during parental care in a size-monomorphic seabird, the Australasian Gannet (Morus serrator)

ABSTRACT Sex differences in foraging behavior are typically studied in size-dimorphic taxa. Data on sex-specific behavior in monomorphic taxa are needed to test theories of reproductive investment. It has been suggested that in seabirds foraging niche separation may be related to decreased intersexual competition for food between cooperating pair-bonded individuals. Alternatively, sex differences in foraging niches may be driven by different nutritional requirements of females associated with the reproductive costs of egg production and oviposition. To assess these possibilities, we studied a size-monomorphic colonial seabird, the Australasian Gannet (Morus serrator) at the Cape Kidnappers gannetry, New Zealand. We recorded maximum dive depths, and distinct diet composition of incubating females as indicated by stable isotopic signatures. Results suggested greater female foraging effort during early times of incubation, indicated by significantly deeper maximum dives. Sex-specific foraging patterns across...

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation.