Single Culture System Research Articles

Efficient Method for Liposome-Based Gene Transfer in Cattle Embryos.

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.

Deciphering the potential role of quorum quenching in efficient aerobic denitrification driven by a synthetic microbial community

Low efficiency is one of the main challenges for the application of aerobic denitrification technology in wastewater treatment. To improve denitrification efficiency, a synthetic microbial community (SMC) composed of denitrifiers Acinetobacter baumannii N1 (AC), Pseudomonas aeruginosa N2 (PA) and Aeromonas hydrophila (AH) were constructed. The nitrate (NO3−-N) reduction efficiency of the SMC reached 97 % with little nitrite (NO2−-N) accumulation, compared to the single-culture systems and co-culture systems. In the SMC, AH proved to mainly contribute to NO3−-N reduction with the assistance of AC, while PA exerted NO2−-N reduction. AC and AH secreted N-hexanoyl-DL-homoserine lactone (C6-HSL) to promote the electron transfer from the quinone pool to nitrate reductase. The declined N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), resulting from quorum quenching (QQ) by AH, stimulated the excretion of pyocyanin, which could improve the electron transfer from complex III to downstream denitrifying enzymes for NO2−-N reduction. In addition, C6-HSL mainly secreted by PA led to the up-regulation of TCA cycle-related genes and provided sufficient energy (such as NADH and ATP) for aerobic denitrification. In conclusion, members of the SMC achieved efficient denitrification through the interactions between QQ, electron transfer, and energy metabolism induced by N-acyl-homoserine lactones (AHLs). This study provided a theoretical basis for the engineering application of synthetic microbiome to remove nitrate wastewater.

CRIOTOLERANCIA DE EMBRIONES BOVINOS CULTIVADOS INDIVIDUALMENTE EN UN MEDIO SUPLEMENTADO CON L-CARNITINA

<p><strong>Background:</strong> L-carnitine is a lipid metabolism enhancer and a potent antioxidant that prevents oxidative damage and improves cryotolerance of bovine embryos. <strong>Objective:</strong> To determine the effect of L-carnitine during oocyte maturation on developmental competence and cryotolerance of single bovine embryo cultured. <strong>Methodology:</strong> Embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). In experiment 1, two individual maturation, fertilization and culture systems were used in 24-well plates with 20 µL drops of medium covered with mineral oil and 96-well plates with 30 µL drops of medium. In experiment 2, oocytes were randomly distributed into two groups and single matured in 96-well plates in medium supplemented or not with 0.6mg/mL L-carnitine. On day 7 post fertilization, blastocysts were vitrified on solid surface in Fiberplug. Non-vitrified blastocysts were used as control. Embryonic survival after devitrification was determined by blastocysts re-expansion and hatching rate at 24 and 48 hours of post-devitrification culture. Total cell number and apoptotic rate by TUNEL-DAPI staining were used as quality and cryotolerance indicator. In both cases, cleavage and blastocyst rates were evaluated at 48 hours and 7 days post fertilization, respectively. <strong>Results:</strong> No significant differences were found for embryonic development between single culture systems. There was no effect of L-carnitine supplementation during maturation on embryo development, but embryo survival had increased (P < 0.05) at 24- and 48-hours post devitrification. <strong>Implications:</strong> Treatment with L-carnitine had increased (P < 0.05) post-thaw re-expansion rates (86.8 ± 3.0 vs 70.0 ± 4.4) and it was similar to non-vitrified control (89.7 ± 2.6). Mean cell number and apoptotic cell index, were similar for all treatment groups. <strong>Conclusion:</strong> L-carnitine supplementation during maturation, does not improve division rate and subsequent development of single cultured embryos, however increases cryotolerance post devitrification.</p>

Nicotinamide ameliorates mitochondria-related neuronal apoptosis and cognitive impairment via the NAD+/SIRT3 pathway

Emerging evidence suggests that mitochondria play a central role in mental health disorders including schizophrenia. Here we investigated whether nicotinamide (NAM) normalized cognitive impairment via a mechanism involving the mitochondrial Sirtuin 3 (SIRT3) pathway. The 24 h maternal separation (MS) rat model was used to mimic schizophrenia-associate phenotypes. Schizophrenia-like behaviors and memory impairments were detected using the pre-pulse inhibition test, novel object recognition test, and Barnes maze test, and neuronal apoptosis was characterized using multiple assays. SIRT3 activity was inhibited pharmacologically or by knockdown in HT22 cells, and BV2 microglia and SIRT3-knockdown HT22 cells were co-cultured in vitro. Mitochondrial molecules were measured by western blotting, and mitochondrial damage was measured with reactive oxygen species and mitochondrial membrane potential assays. Proinflammatory cytokines were assayed by ELISA and microglial activation was detected by immunofluorescence. MS animals showed behavioral and cognitive impairment and increased neuronal apoptosis. Supplementation with NAM or administration of honokiol, a SIRT3 activator, reversed all of the changes in behavioral and neuronal phenotypes. Administration of the SIRT3 inhibitor 3-TYP in control and NAM-treated MS rats caused behavioral and neuronal phenotypes similar to MS. In vitro, inhibition of SIRT3 activity with 3-TYP or by knockdown in HT22 cells increased ROS accumulation and caused neuronal apoptosis in a single-culture system. In co-culture systems, SIRT3 knockdown in HT22 cells activated BV2 microglia and increased levels of TNF-α, IL-6, and IL-1β. The administration of NAM blocked these alterations. Taken together, these data suggest that NAM can rescue neuronal apoptosis and microglial over-activation through the nicotinamide adenine dinucleotide (NAD+)-SIRT3-SOD2 signaling pathway, furthering our understanding of the pathogenesis of schizophrenia and providing avenues for novel treatments.

Untargeted Metabolomics Sensitively Differentiates Gut Bacterial Species in Single Culture and Co-Culture Systems.

Gut microbiome plays a vital role in human health, and its characteristic has been widely identified through next-generation sequencing techniques. Although with great genomic insights into gut microbiome, its functional information is not clearly elaborated through metagenomic techniques. On the other hand, it is suggested that fecal metabolome can be used as a functional readout of the microbiome composition; therefore, we designed a proof-of-concept study to first characterize the metabolome of different gut microbes and then investigate the relationship between bacterial metabolomes and their compositions in co-culture systems. We selected eight representative bacteria species from Bifidobacterium (2), Bacteroides (1), Lactobacillus (4), and Akkermansia (1) genera as our model microbes. Liquid chromatography coupled mass spectrometry-based untargeted metabolomics was utilized to explore the microbial metabolome of bacteria single cultures and co-culture systems. Through spectral comparisons, our results showed that untargeted metabolomics could capture the similarity and differences in metabolic profiles from eight representative gut bacteria. Also, untargeted metabolomics could sensitively differentiate gut bacterial species based on our statistical analyses. For example, citrulline and histamine levels were significantly different among four Lactobacillus species. In addition, in the co-culture systems with different bacteria population ratios, gut bacterial metabolomes can be used to quantitatively reflect bacterial population in a mixed culture. For instance, the relative abundance of 2-hydroxybutyric acid changed proportionately with the changed population ratio of Lactobacillus reuteri in the co-culture system. In summary, we proposed a workflow that could demonstrate the capability of untargeted metabolomics in differentiating gut bacterial species and detecting their characteristic metabolites proportionally to the microbial population in co-culture systems.

Yi Shen Juan Bi Pill Regulates the Bone Immune Microenvironment via the JAK2/STAT3 Signaling Pathway in Vitro.

Rheumatoid arthritis (RA) is characterized by an impaired articular bone immune microenvironment, which is associated with regulatory T cells (Tregs) hypofunction and osteoclasts (OCs) hyperfunction and leads to articular bone erosion and systemic bone loss. Studies have shown that Tregs slow bone loss in RA by regulating the bone resorption function of OCs and the JAK/STAT signaling pathway can regulate the immunosuppressive function of Tregs and reduce the bone erosion function of OCs. Yi Shen Juan Bi Pill (YSJB) is a classic Chinese herbal compound for the treatment of RA. However, whether YSJB regulates bone immune microenvironment homeostasis through JAK/STAT signaling pathway remains unclear. Based on in vitro OC single culture, Treg single culture and OC-Treg coculture systems, treatments were performed using drug-containing serum, AG490 and JAK2 siRNA to explore whether YSJB-containing serum regulates the homeostasis of the bone immune microenvironment through the JAK/STAT signaling pathway. In vitro, YSJB treatment decreased the number of TRAP+ cells and the areas of bone resorption and inhibited the expression of RANK, NFATc1, c-fos, JAK2, and STAT3 in both the OC single culture system and the OC-Treg coculture system. Tregs further reduced the number of TRAP+ cells and the areas of bone resorption in the coculture system. YSJB promoted the secretion of IL-10 while inhibiting the expression of JAK2 and STAT3 in Tregs. Moreover, inhibiting the expression of JAK2 with the JAK2 inhibitor AG490 and JAK2 siRNA improved the immunosuppressive functions of Treg, inhibited OC differentiation and bone resorption. Our study demonstrates that YSJB can regulate OC-mediated bone resorption and Treg-mediated bone immunity through the JAK2/STAT3 signaling pathway. This study provides a new strategy for regulating the bone immune microenvironment in RA with traditional Chinese medicine.

IGF-I Medium Supplementation Improves Singly Cultured Cat Oocyte Maturation and Embryo Development In Vitro

Simple SummaryRoutine embryo production is the cornerstone for several research topics such as assisted reproduction techniques. For this, suitable culture media are needed to achieve satisfactory results, but the optimal culture medium is yet to be developed for felids. In this study, the supplementation of the medium with insulin-like growth factors (IGF-I, IGF-II) and granulocyte-macrophage colony-stimulating factor (GM-CSF) coordinated with other items related to in vitro procedures, such as type of fertilisation and type of culture, are analysed. Finally, the addition of IGF-I and GM-CSF to the culture medium improved embryo production in a single-culture system. The supplementation with IGF-I increased the maturation rate of feline oocytes, as well as the morula and blastocyst rates of embryos, independently of the fertilisation method used. As only a small number of oocytes can be retrieved from wild species, this conclusion may be of special relevance for protocols for endangered felids.Embryo production is a routine procedure in several species. However, in felids, the effectiveness of this approach is far behind that in the majority of laboratory species. The development of a suitable environment starts with the proper composition of culture media. Therefore, for the improvement of assisted reproduction techniques and their outcome in cats, this is an urgent task. As the addition of insulin-like growth factors (IGF-I, IGF-II) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was beneficial in other mammalian species, this study aims to check whether these components, combined with other factors (such as type of fertilisation or type of culture) can provide a benefit in the felid culture system in current use. Thus, these supplements, in different concentrations and combinations, were merged with the use of two fertilisation techniques and randomly assigned to single or group culturing. The results showed that the addition of IGF-I and/or GM-CSF produced an increase in morula and blastocyst rate in a single culture system. In particular, the supplementation with 20 ng/mL of IGF-I incremented the maturation rate by 10% and significantly increased the morula and blastocyst rates in single culturing. This result is especially remarkable for wild felids, where only a few oocytes and/or embryos are available.

Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus

The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions are complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: (i) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and (ii) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

Elimination of stress factors by continuous embryo culture and its influence on in vitro fertilization outcomes

Recently, infertility has become one of the most important endemic conditions, affecting approximately 15–20 % of couples worldwide. Among others, the careerist lifestyle, the increasing maternal age and the parallel increment in the aneuploidy rate of embryos play a crucial role in this phenomenon. In this study, embryological parameters and pregnancy outcomes were investigated in IVF cycles using either sequential embryo culture or a single step culture system. By sequential media, oocytes/embryos are needlessly exposed to the potentially negative effects of light exposure, temperature decrement and altered oxygen tension. In comparison with sequential media, single step media induced 1.28, 1.21 and 1.21-fold increments in implantation, biochemical pregnancy and clinical pregnancy rates, respectively. Pregnancy outcomes showed strong maternal age-dependency, so the difference between the two investigated culture systems was equalized by the increasing maternal ages (35–44 years) and the supposed incidence of embryo aneuploidy. Nevertheless, the significant enlargements in the outcomes of the younger ages (25–34) induced by the single step cultures suggest that, beside the resultant maternal aneuploidy, aneuploidy (reduced pregnancy rates) may evolve from exposure to the mentioned environmental stress factors.

Kinetic analysis of sequential metabolism of triazolam and its extrapolation to humans using an entero-hepatic two-organ microphysiological system.

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling.

Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system.

Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3β inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.

Differentiation of human pluripotent stem cells toward pharyngeal endoderm derivatives: Current status and potential.

The pharyngeal apparatus, a transient embryological structure, includes diverse cells from all three germ layers that ultimately contribute to a variety of adult tissues. In particular, pharyngeal endoderm produces cells of the inner ear, palatine tonsils, the thymus, parathyroid and thyroid glands, and ultimobranchial bodies. Each of these structures and organs contribute to vital human physiological processes, including central immune tolerance (thymus) and metabolic homeostasis (parathyroid and thyroid glands, and ultimobranchial bodies). Thus, improper development or damage to pharyngeal endoderm derivatives leads to complicated and severe human maladies, such as autoimmunity, immunodeficiency, hypothyroidism, and/or hypoparathyroidism. To study and treat such diseases, we can utilize human pluripotent stem cells (hPSCs), which differentiate into functionally mature cells in vitro given the proper developmental signals. Here, we discuss current efforts regarding the directed differentiation of hPSCs toward pharyngeal endoderm derivatives. We further discuss model system and therapeutic applications of pharyngeal endoderm cell types produced from hPSCs. Finally, we provide suggestions for improving hPSC differentiation approaches to pharyngeal endoderm derivatives with emphasis on current single cell-omics and 3D culture system technologies.

Ocimum basilicum suspension culture as resource for bioactive triterpenoids: yield enrichment by elicitation and bioreactor cultivation

The competence of Ocimum basilicum suspension culture for the production of pharmacologically distinguished triterpenoids had been explored to meet their escalating demand with inequitable natural resources. The production of three pentacyclic triterpenes—betulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA), could be achieved for the first time in the O. basilicum suspension culture, together with the prevalently occurring rosmarinic acid (RA). The production of RA was most abundant (15.73 ± 0.28 mg/g DW) followed by that of BA (14.63 ± 0.21 mg/g DW), UA (4.71 ± 0.23 mg/g DW) and OA (0.91 ± 0.02 mg/g DW) in reducing order on their respective optimum cultivation period. Compared to the in-vivo control leaves, the suspension cell revealed almost 3.25-, 2.89-, and 1.79-folds higher OA, UA and RA contents, respectively. The BA synthesizing power of suspension cells exemplified notable trend as its presence in the control leaves remained obscured. By and large, cultivation in a 10 l bioreactor and elicitation with methyl jasmonate in bioreactor and shake flask improved the cumulative productivities of the targeted metabolites with reciprocated divergence in the amounts of the two isomers, i.e., UA and OA. The overall findings of the present study bear special merit in realising the global demand of these multiple terpenoids in a single culture system of O. basilicum, which remained unrevealed so far. First report towards the in vitro production of multiple pharmacologically distinguished, high demand triterpenoids—betulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA), along with rosmarinic acid (RA) in suspension culture of Ocimum basilicum. Notable yield enhancement through bioreactor cultivation and strategic elicitation with methyl jasmonate in shake flask and bioreactor added additional merit to this study in realizing the global demand of these multiple terpenoids in a single culture system of O. basilicum.

Ginsenoside Rg3 and Rh2 protect trimethyltin-induced neurotoxicity via prevention on neuronal apoptosis and neuroinflammation.

The acute exposure of trimethyltin (TMT) develops clinical syndrome characterized by amnesia, aggressive behavior, and complex seizures. This neurotoxicant selectively induces hippocampal neuronal injury and glial activation accompanied with resultant neuroinflammation. Here we report two candidates ginsenosides Rg3 and Rh2 as neuroprotection agents using a mouse model of TMT intoxication via a single injection (2mg/kg) and primary neuronal culture systems. Four-week administration of Rg3 or Rh2 significantly reduced TMT-induced seizures and behavioral changes. Rg3 and Rh2 significantly attenuated the oxidative stress evidenced by improvement on antioxidant enzymes and neuronal loss and astrocytic activation in mouse brain. In primary cultures, TMT induced significant neuronal death after 24-h intoxication and vigorous secretion of inflammatory cytokines (IL-1α/β, IL-6, TNF-α, and MCP-1) in astrocytes. Pretreatment with Rg3 or Rh2 not only reduced cell death but efficiently suppressed above mentioned inflammatory cytokines confirmed by antibody array test. The underlying protective mechanism by Rg3 and Rh2 was delineated through selective upregulation of PI3K/Akt and suppression of ERK activation. Intriguingly, Rg3 and Rh2 protected oligodendrocyte progenitor cells (O-2A) from TMT intoxication via promoting type 2 astrocytic differentiation without further inflammatory activation. Collectively, Rg3 and Rh2 interventions aimed at reducing oxidative stress and neuroinflammation neurotoxicity therefore are of therapeutic benefit in TMT-induced neurodegeneration.

Modeling of nanoparticle transport through the female reproductive tract for the treatment of infectious diseases

The secreted mucus layer in the vaginal epithelium presents a formidable barrier to the transport of active agents for the prevention and treatment of female reproductive tract (FRT) infections. Nanoparticle-mediated drug delivery has been proposed to help facilitate the transport and release of active agents through the cervicovaginal mucus (CVM) and underlying mucosa. However, both nanoparticles (NPs) and free active agents face a variety of challenges, often requiring the administration of high localized doses to circumvent leakage and poor penetration to targeted intravaginal tissue compartments. To address these challenges, “stealth” NP modifications have been investigated, due to their favorable mucus-penetrating properties, resulting in improved intravaginal active agent retention and transport. A number of other NP characteristics including size, surface modification type, ligand density, and co-modification, as well as the complexity of the FRT tissue are involved in obtaining adequate tissue penetration and, if needed, cell internalization. Studies that systematically investigate variations of these characteristics have yet to be conducted, with the goal to obtain a better understanding of what properties most impact prophylactic and therapeutic benefit. To complement the progress made with experimental evaluation of active agent transport in in vitro and in vivo, mathematical modeling has recently been applied to analyze the transport performance of agents and delivery vehicles in the FRT. Here, we build upon this work to simulate NP transport through mucus gel, epithelial, and stromal compartments, with the goal to provide a platform that can systematically evaluate transport based on NP and tissue characteristics. Model parameters such as PEG density and NP release (decay) rate from mucus gel into the epithelium, are set from previous in vitro and in vivo experimental work that assessed the transport of poly(lactic-co-glycolic acid (PLGA) NPs. The modeling results show that while unmodified and 2% PEG-modified NPs were retained in mucus for ∼1–4 h, dependent upon decay constant values, and traverse to the epithelium, no NP penetration was observed in the stroma. In contrast, NPs modified with 3% PEG, exhibited prolonged retention in each compartment, remaining for ∼4–6 h. Moreover, a significant concentration of NPs is observed in the stroma, indicating a transition in transport behavior. For NPs modified with 5, 8, or 25% PEG, steady retention profiles were noted, which gradually decline over 24 h. To supplement this modeling study and to develop a more representative experimental system that may be useful in future work, we report on the feasibility of constructing single and multicellular layered (MCL) culture systems to represent the epithelial and stromal tissue of the FRT. We anticipate that a combined mathematical/experimental approach may longer term enable prediction and customization of patient tissue-specific approaches to attain effective NP-mediated drug delivery and release for the treatment of infectious disease.

Media evaporation in a dry culture incubator; effect of dish, drop size and oil on media osmolality

Modern benchtop incubators typically lack humidity, a significant difference compared to traditional big box incubators. Combined with the recent popularity of single step culture systems, media evaporation resulting in an increase in osmolality during the culture period is a legitimate concern. The objective of this study was to evaluate variables that may impact evaporation over the time course of normal human embryo culture in a dry benchtop incubator. Prospective Study. : Cleavage medium (Sage) + 10% SPS was used for all experiments. Dishes for each treatment were prepared and placed into the same chamber of a K Systems G185 incubator. After 6 days, media from microdrops in each treatment was recovered and osmolality measured on a Wescor 5600 Vapor Pressure osmometer. All experiments were repeated 3 times, with a minimum of 13 drops measured per treatment. Media prior to incubation was used as a control. In experiment 1, 30 uL drops were prepared under 3.5 mL of oil (OvOil) in a Falcon 35 mm dish and under 4.5 mL of oil in a mini-GPS dish. In experiment 2, drop volume was examined in the mini-GPS dish (20uL, 40uL and 60uL). In experiment 3, 30uL drops in a mini-GPS dish were covered with 3, 4, or 5 mL oil. In experiment 4, 30 uL drops in a mini-GPS dish were covered with 4.5ml OvOil, light oil, or heavy oil. Results are presented as mean ± SEM in mOsm. Significance was determined at p<0.01. Uninterrupted culture for 6 days in a benchtop incubator resulted in a significant increase in medium osmolality regardless of dish type (Falcon control: 275.8 ± 1.5; Falcon 6 day: 300.7 ± 2.0; GPS control: 275.9 ± 1.7; GPS 6 day: 293.1 ± 1.5). Although evaporation occurred in both types of dishes, GPS dishes resulted in a significant improvement in osmolality on D6. Evaporation resulting in increased osmolality occurred in all drop sizes (control: 271.0 ± 1.4; 20 uL: 306.6 ± 2.6; 40 uL: 292.4 ± 2.6; 60 uL: 302.4 ± 3.3), although 40 uL drops resulted in lower osmolality than 20 or 60 uL drops. Elevated osmolality also resulted regardless of oil volume (control: 272.9 ± 1.5; 3 mL: 300.6 ± 2.8; 4 mL: 298.1 ± 1.9; 5 mL: 290.6 ± 2.1). However, 5 mL of oil overlay resulted in a lower osmolality than 3 mL of oil. Finally, the type of oil had no impact on media evaporation (control: 271.3 ± 1.5; OvOil: 291.6 ± 1.5; heavy: 291.8 ± 2.2; light: 294.9 ± 2.4). Significant evaporation occurs during continuous culture for 6 days in a dry benchtop incubator. None of the dishes, drop sizes, or types of oil tested prevented evaporation, although a combination of 40 uL drops in mini-GPS dishes under 5 ml oil may result in the lowest osmolality. However, even this combination in dry incubator conditions results in osmolality (∼291) that is elevated compared to the intended osmolality of this culture medium (∼272), which may be detrimental to embryo development.

Carbon, nitrogen, and phosphorus removal, and lipid production by three saline microalgae grown in synthetic wastewater irradiated with different photon fluxes

Mariculture production has increased in the last decades, with untreated wastewater discharged directly into the sea, impacting coastal ecosystems. There is a need for mariculture wastewater treatment systems that are cost-effective. This can be met by the implementation of wastewater treatment systems that in addition to removing pollutants are capable of producing valuable by-products such as biomass for the biofuel industry. In this study, Dunaliella sp., Nannochloropsis sp. and Tetraselmis sp. microalgae were cultivated in controlled environments simulating mariculture wastewaters. Single stage culture systems were used to grow these microalgae, the growing conditions included inducing stress with different photon flux densities (900, 1500 and 2000 μmol m−2 s−1), and low carbon, nitrogen and phosphorus concentrations obtained at the stationary phase, in order to force these microalgae to increase their lipid content. The best results were obtained with Tetraselmis sp., which achieved 132.8 mg L−1 day−1 of biomass productivity at 900 μmol m−2 s−1. Nevertheless the best lipid productivity was reached at 1500 μmol m−2 s−1, also by Tetraselmis sp., being 29.5 mg L−1 day−1, where biomass productivity was of 124.5 mg L−1 day−1. All three microalgae species were able to remove >90% of nitrogen and orthophosphates, and 80% of carbon, which makes them suitable for treating mariculture wastewater, and in addition, represent a valuable high lipid content biomass byproduct usable as raw material for biodiesel synthesis.

Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

Impact of Mummy Substance on the Proliferation and Migration of Human Adipose-derived Stem Cells and Fibroblasts in Separate or Co-culture Model

Wound healing is critical to the regeneration of various organs and tissues. Traditional medicines, notably mummy, could serve as an alternative stimulator for the treatment of wounds. Till date, mummy has been used for the modulation of inflammation, articular injuries, and bone fractures, as well as wound healing. This study aims to evaluate the effect of mummy on the proliferation and migration of human adipose-derived stem cells and fibroblasts in single or co-culture in vitro conditions. The effective concentration of mummy substance was determined on fibroblasts and adipose-derived stem cells. The cells were treated with mummy separately or co-cultured over a period of 96 h. In vitro scratch assay was used to monitor cell migration rate. Effectiveness of mummy in inducing proliferation was evaluated by measuring the Ki-67 expression using flow cytometry analysis. Based on the data obtained, mummy was found to generate significant increase in the migration of fibroblasts as compared to untreated control in single culture system. On the other hand, an increased migration rate was recorded for adipose-derived stem cells, but it did not reach significant levels. Interestingly, the migration rate of co-cultured fibroblast and adipose-derived stem cells improved after exposure to mummy compared to matched controls. Incubation of adipose-derived stem cells, but not fibroblasts with mummy profoundly increased the expression of Ki-67 as compared to untreated cells. No significant effects were observed in the co-culture system. These data suggested that mummy altered the dynamics of stem cells and mature fibroblasts in vitro. Distinct cell responses could possibly be affected with regard to different cell types.

Diarylheptanoid Hirsutenone Attenuates Osteoclastogenesis by Suppressing IFNγ and NF-κB Signaling in Th1 and Preosteoclastic Cells.

The aim of the present study was to examine the inhibitory roles and mechanisms of hirsutenone (HTN) in the regulation of osteoclastogenesis. Gene levels were compared to assure the effects of HTN on osteoclastogenesis in mouse splenocytes/CD4+ T cells, mouse macrophage-like cell line RAW264.7 (preosteoclast), MG63 (osteoblast), and RPMI1788 (B cell) cells. The mechanism by which HTN regulates the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits inhibitor of kappaB (IκB) and nuclear factor-kappaB (NF-κB) signaling was examined by Western blotting and luciferase reporter assays. Our results demonstrated that HTN effectively downregulated the expression of interferon γ (IFNγ), interleukin-22 (IL-22), IL-1β, and tartrate-resistant acid phosphatase (TRAP) in splenocyte-/CD4+-RAW264.7 co-culture system. Moreover, receptor activator of nuclear factor-κB ligand (RANKL) and CD25 expression were also significantly inhibited in MG63 and CD4+ single culture system, suggesting an additional independent effect of HTN on osteoclastogenesis. Notably, TRAF6 was markedly degraded along with a decrease in nuclear factor of activated T-cells (NFATc) and NF-κB activities in RAW264.7 cells. Finally, we concluded that HTN directly or indirectly inhibits osteoclastogenesis via the inhibition of NF-κB signaling by promoting TRAF6 degradation, and plays a crucial role in suppressing the expression of RANKL and cytokines expressed in IFNγ-producing T-helper 1 (Th1) cells. These findings suggest that HTN may be a promising therapeutic candidate for diseases resulting from bone loss.