Terms Of Metabolic Activity Research Articles

Gaseous Micro-Embolic Activity and Goal-Directed Perfusion Management in a Closed System for Cardiopulmonary Bypass and Minimally Invasive Extracorporeal Circulation during Coronary Artery Bypass Grafting.

Cardiopulmonary bypass (CPB) techniques are becoming minimally invasive in clinical practice. The literature describes various extracorporeal techniques which seek to eliminate air-blood contact and reduce both hemodilution and the contact surface such as in Minimally invasive Extracorporeal Circulation (MiECC) and closed systems for CPB. However, the delivery of micro-embolic activity in the circuit and metabolic activity in terms of oxygen delivery for Goal-Directed Perfusion (GDP) management, in relation to the patient's blood volume and central venous pressure, have never been related and correlated. In this report, we present a cohort study that investigated these aspects between the closed SVR2000 System and modular MiECC (both from Eurosets SRL, Medolla, Italy). Data were collected retrospectively and used to compare 60 consecutive patients who underwent isolated coronary artery bypass grafting (CABG) surgery by two surgeons using an SVR2000 oxygenator in 30 procedures, with a matched cohort of patients from the same period who underwent isolated CABG surgery by two other surgeons using a modular MiECC in 30 procedures. The primary endpoints collected were data on micro-embolic activity, including the number of gaseous micro-emboli in the circuit during the procedure, the mean maintenance value of oxygen delivery (DO2) and data relating to venous return volume and central venous pressure (CVP). During the CPB procedures, the following values were recorded for the closed SVR2000 and MiECC groups, respectively: the average number of gaseous micro-emboli (GME) in the venous line, 833 ± 23 vs 1221 ± 45 (p = 0.028); GME in the outlet of the pump, 375 ± 45 vs 429 ± 76 (p = 0.89; GME in the arterial line, 189 ± 36 vs 205 ± 27 (p = 0.92), and the volume of GME in the arterial line (mL), 0.32± 12 vs 0.49± 17 (p = 0.93). The mean Indexed Oxygen Delivery (DO2i) during cross-clamp (ml/min/m2) was 319 ±12 vs 278 ±9 (p = 0.0019), respectively. The maximum mean volume of venous return in the soft-shell venous reservoir (ml) was 1801 ±128 vs 824 ±192 (p = 0.038). The mean central venous pressure (CVP) during cross-clamp (mmHg) was 0 ± 2 vs 6 ± 2 (p = 0.019). In this study, the results in the closed SVR2000 group were not statistically inferior to those in the modular MiECC group in terms of gaseous micro-embolic activity during CPB. Our analysis showed an important reduction of GME delivery in both systems. The closed SVR2000 group showed better management for GDP in terms of DO2i, associated with the flexibility of dynamic volume management and the absence of cavitation and regulation of the rate per minute and pump flow, which were reported in the MiECC group. The SVR2000 and modular MiECC systems were both safe and effective in perioperative practice without iatrogenic problems.

MODERN ASPECTS OF PHARMACOGENETICS: FROM THEORY TO PRACTICE IN PERINATOLOGY AND PEDIATRICS

Pharmacogenetic testing (PT) is a modern tool in the doctor’s practice, which allows to make the right clinical decision indiffi cult cases, when the expected result of medical measures is not achieved. It is clear that certain metabolic processes in the human body, as well as a number of diseases, are genetically programmed. Therefore, despite the large number of unexplained mechanisms of individual response to drugs, genetic testing occupies one of the leading positions among methods of selecting drug therapy in complex clinical cases. However, the successful implementation of this promising method must overcome a number of obstacles, including limited evidence of eff ectiveness, ethical, legal, and social factors. The purpose of this review is to highlight modern concepts and practical aspects of the use of PT. The article addresses the problem of expanding the indications for PT when it is not limited to preventive use only. PT allows to identify drugs associated with an increased risk of causing side eff ects, with a narrow therapeutic index, to reduce the number of drugs in treatment, to choose the dosage of the drug. A variety of PT platforms can be used in a physician’s offi ce, which can be broadly divided into two categories – genotyping- based tests and sequencing- based tests. Depending on the gene being tested, diff erent algorithms can be used to generate results. Some gene variants can be described in terms of metabolic activity or general function, while others can only be described as present or absent. Results for gene variants can also be reported as normal, intermediate or low function for the corresponding gene. Pharmacogenetic clinical decision support systems (CDSS) are computer- based systems that assist healthcare providers in prescribing medications at the point of care. These systems provide physicians and other healthcare providers with appropriately fi ltered pharmacogenetic information, such as drug-gene interaction alerts or patient- specifi c treatment recommendations. A pharmacogenetic CDSS can either be integrated into a local hospital information system or used as a stand- alone application such as a web service or mobile application. Pharmacogenetics can increase the quantity and quality of information available to pregnant women and their physicians about medication use during pregnancy. Implementation of PT recommendations intoroutine pediatric practice requires carefully coordinated strategies at the national, regional, and health system levels. To date, pharmacogenetics provides mosaic information on the association between response to drug therapy and genetic background. It is expected that the next step will be a study in a larger group of participants to investigate the contribution of epigenetic factors and to provide clinical recommendations for adjusting or selecting therapy based on the personal characteristics of the patient.

The Protective Role of Oleuropein Aglycone against Pesticide-Induced Toxicity in a Human Keratinocytes Cell Model.

The extensive use of agricultural pesticides to improve crop quality and yield significantly increased the risk to the public of exposure to small but repeated doses of pesticides over time through various routes, including skin, by increasing the risk of disease outbreaks. Although much work was conducted to reduce the use of pesticides in agriculture, little attention was paid to prevention, which could reduce the toxicity of pesticide exposure by reducing its impact on human health. Extra virgin olive oil (EVOO), a major component of the Mediterranean diet, exerts numerous health-promoting properties, many of which are attributed to oleuropein aglycone (OleA), the deglycosylated form of oleuropein, which is the main polyphenolic component of EVOO. In this work, three pesticides with different physicochemical and biological properties, namely oxadiazon (OXA), imidacloprid (IMID), and glyphosate (GLYPHO), were compared in terms of metabolic activity, mitochondrial function and epigenetic modulation in an in vitro cellular model of human HaCaT keratinocytes to mimic the pathway of dermal exposure. The potential protective effect of OleA against pesticide-induced cellular toxicity was then evaluated in a cell pre-treatment condition. This study showed that sub-lethal doses of OXA and IMID reduced the metabolic activity and mitochondrial functionality of HaCaT cells by inducing oxidative stress and altering intracellular calcium flux and caused epigenetic modification by reducing histone acetylation H3 and H4. GLYPHO, on the other hand, showed no evidence of cellular toxicity at the doses tested. Pretreatment of cells with OleA was able to protect cells from the damaging effects of the pesticides OXA and IMID by maintaining metabolic activity and mitochondrial function at a controlled level and preventing acetylation reduction, particularly of histone H3. In conclusion, the bioactive properties of OleA reported here could be of great pharmaceutical and health interest, as they could be further studied to design new formulations for the prevention of toxicity from exposure to pesticide use.

Role of cytokine in malignant T-cell metabolism and subsequent alternation in T-cell tumor microenvironment.

T cells are an important component of adaptive immunity and T-cell-derived lymphomas are very complex due to many functional sub-types and functional elasticity of T-cells. As with other tumors, tissues specific factors are crucial in the development of T-cell lymphomas. In addition to neoplastic cells, T- cell lymphomas consist of a tumor micro-environment composed of normal cells and stroma. Numerous studies established the qualitative and quantitative differences between the tumor microenvironment and normal cell surroundings. Interaction between the various component of the tumor microenvironment is crucial since tumor cells can change the microenvironment and vice versa. In normal T-cell development, T-cells must respond to various stimulants deferentially and during these courses of adaptation. T-cells undergo various metabolic alterations. From the stage of quiescence to attention of fully active form T-cells undergoes various stage in terms of metabolic activity. Predominantly quiescent T-cells have ATP-generating metabolism while during the proliferative stage, their metabolism tilted towards the growth-promoting pathways. In addition to this, a functionally different subset of T-cells requires to activate the different metabolic pathways, and consequently, this regulation of the metabolic pathway control activation and function of T-cells. So, it is obvious that dynamic, and well-regulated metabolic pathways are important for the normal functioning of T-cells and their interaction with the microenvironment. There are various cell signaling mechanisms of metabolism are involved in this regulation and more and more studies have suggested the involvement of additional signaling in the development of the overall metabolic phenotype of T cells. These important signaling mediators include cytokines and hormones. The impact and role of these mediators especially the cytokines on the interplay between T-cell metabolism and the interaction of T-cells with their micro-environments in the context of T-cells lymphomas are discussed in this review article.

Mesenchymal stem cell engineering by ARCA analog-capped mRNA

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for invitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. β-S-ARCA D1 and β-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that β-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based invitro transfection of HPLC-purified and ARCA- or β-S-ARCA D1-capped mRNA is optimal for MSC engineering.

Zn-based Metal-Organic Frameworks (MOFs) Incorporated into Collagen-Polysaccharide-based Composite Hydrogels for Their Use in Wound Healing

Based on the physicochemical properties of metal-organic frameworks (MOFs), and the need of having an anti-inflammatory and antibacterial effect at the same time in a material for wound healing applications, a multicomponent polymer system was synthesized. Zn-based MOFs having different amino acids acting as ligands (L-tryptophan (Trp), L-phenylalanine (Phe), and L-histidine (His)) in their coordination sphere were successfully prepared and validated by FTIR and XRD. Each MOF was incorporate d into a semi-interpenetrated polymeric network (semi-IPN) based on collagen (C)-guar gum (GG)-polyurethane (PU), given composite hydrogels as end products. The physicochemical properties of these novel composite hydrogels, their in vitro biocompatibility, and their use as cell carriers were studied. The maximum swelling capacity was shown by CGG-Zn(Trp) while the crosslinking index was higher for CGG-Zn(Phe) and CGG-Zn(His), indicating that the chemical structure of the amino acid of the Zn-based MOF tailors these physicochemical properties. All composite hydrogels were resistant to a hydrolysis degradation process (at pH=5 and 7.4) being not entirely degraded after 14 days and having residual masses above 57%. The incorporation of Zn-based MOFs increased the biocompatibility in terms of metabolic activity and proliferation of porcine fibroblast in contact with the composite hydrogels. The low release capacity of fibroblast encapsula ted inside hydrogels evaluated for 10 days indicated the non-efficient capacity of these matrixes as cell carriers, regardless of the structure of the Zn-based MOF. The strong adhesion of porcine fibroblast to the composite hydrogels is related to improving metabolic activity al lowing its proliferation, making these multicomponent polymeric systems useful as efficient materials in biomedical applications such as wound dressing and tissue engineering.

Performance of Two Extracts Derived from Propolis on Mature Biofilm Produced by Candida albicans.

Species of the Candida genus represent the third most common cause of onychomycosis, the most frequent and difficult to treat nail infection. Onychomycosis has been attributed to fungi organized in biofilm and some natural products have proved promising for its treatment. This study aimed to evaluate the antibiofilm activity of propolis extract (PE) and its by-product (WPE) on 7-day preformed biofilms produced by Candida albicans in polystyrene microplates, as well as in an ex vivo model on human nail fragments. The cytotoxicity and permeation capacity were also assessed. Firstly, multiple parameters were evaluated over 7 days to elucidate the dynamics of biofilm formation by C. albicans. The cell viability and total biomass did not vary much from the beginning; however, days 3 and 4 were crucial in terms of metabolic activity, which was significantly increased, and the levels of extracellular matrix components, wherein proteins and nucleic acids experienced an increase, but polysaccharide levels dropped. Architecturally, one-day biofilm showed a monolayer of organized cells (blastoconidia, hyphae, and pseudohyphae), while in the seven-day biofilm there was a three-dimensional well-structured and complex biofilm. This yeast was also able to form a biofilm on both surfaces of the nail, without an additional nutritional source. Both extracts showed excellent antibiofilm activity against the 7-day preformed biofilm and were not toxic to Vero cells at concentrations compatible with the antifungal and antibiofilm activities. Both extracts permeated the experimentally infected nail, with WPE being more efficient. The results of this study, taken together, reinforce the potential of these natural products, containing propolis, as a safe option for the topical treatment of onychomycosis.

Photocurable Thiol-yne Alginate Hydrogels for Regenerative Medicine Purposes.

Every year millions of people worldwide undergo surgical interventions, with the occurrence of mild or severe post-treatment consequences meaning that rehabilitation plays a key role in modern medicine. Considering the cases of burns and plastic surgery, the pressing need for new materials that can be used for wound patches or body fillers and are able to sustain tissue regeneration and promote cell adhesion and proliferation is clear. The challenges facing next-generation implant materials also include the need for improved structural properties for cellular organization and morphogenic guidance together with optimal mechanical, rheological, and topographical behavior. Herein, we propose for the first time a sodium alginate hydrogel obtained by a thiol-yne reaction, easily synthesized using carbodiimide chemistry in a two-step reaction. The hydrogels were formed in all cases within a few minutes of light irradiation, showing good self-standing properties under solicitation. The mechanical, rheological, topographical, and swelling properties of the gels were also tested and reported. Lastly, no cytotoxicity was detected among the hydrogels. Soluble extracts in culture media allowed cell proliferation, and no differences between samples were detected in terms of metabolic activity and DNA content. These results suggest the potential use of these cytocompatible hydrogels in tissue engineering and regenerative medicine.

Bioactive Interpenetrating Hydrogel Networks Based on 2-Hydroxyethyl Methacrylate and Gelatin Intertwined with Alginate and Dopped with Apatite as Scaffolding Biomaterials

Our goal was to create bioimitated scaffolding materials for biomedical purposes. The guiding idea was that we used an interpenetrating structural hierarchy of natural extracellular matrix as a “pattern” to design hydrogel scaffolds that show favorable properties for tissue regeneration. Polymeric hydrogel scaffolds are made in a simple, environmentally friendly way without additional functionalization. Gelatin and 2-hydroxyethyl methacrylate were selected to prepare interpenetrating polymeric networks and linear alginate chains were added as an interpenetrant to study their influence on the scaffold’s functionalities. Cryogelation and porogenation methods were used to obtain the designed scaffolding biomaterials. The scaffold’s structural, morphological, and mechanical properties, in vitro degradation, and cell viability properties were assessed to study the effects of the preparation method and alginate loading. Apatite as an inorganic agent was incorporated into cryogelated scaffolds to perform an extensive biological assay. Cryogelated scaffolds possess superior functionalities essential for tissue regeneration: fully hydrophilicity, degradability and mechanical features (2.08–9.75 MPa), and an optimal LDH activity. Furthermore, cryogelated scaffolds loaded with apatite showed good cell adhesion capacity, biocompatibility, and non-toxic behavior. All scaffolds performed equally in terms of metabolic activity and osteoconductivity. Cryogelated scaffolds with/without HAp could represent a new advance to promote osteoconductivity and enhance hard tissue repair. The obtained series of scaffolding biomaterials described here can provide a wide range of potential applications in the area of biomedical engineering.

Time-keeping and decision-making in living cells: Part I

To survive and reproduce, a cell must process information from its environment and its own internal state and respond accordingly, in terms of metabolic activity, gene expression, movement, growth, division and differentiation. These signal–response decisions are made by complex networks of interacting genes and proteins, which function as biochemical switches and clocks, and other recognizable information-processing circuitry. This theme issue of Interface Focus (in two parts) brings together articles on time-keeping and decision-making in living cells—work that uses precise mathematical modelling of underlying molecular regulatory networks to understand important features of cell physiology. Part I focuses on time-keeping: mechanisms and dynamics of biological oscillators and modes of synchronization and entrainment of oscillators, with special attention to circadian clocks.

Perfluorooctane sulfonic acid (PFOS) inhibits vessel formation in a human 3D co-culture angiogenesis model (NCFs/HUVECs)

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous environmental pollutant. In humans, PFOS exposure has been associated with a number of adverse health outcomes, including reduced birth weight. Whether PFOS is capable of affecting angiogenesis and thus possibly fetal development is unknown. Therefore, we investigated 1) the metabolic activity of PFOS-exposed endothelial cells (human umbilical vein endothelial cells, HUVECs), fibroblasts (normal colon fibroblasts, NCFs), and epithelial cells (human colorectal carcinoma cells, HCT116), 2) PFOS-specific inhibition of vascular endothelial growth factor receptor (VEGFR)2 stimulation in KDR/NFAT-RE HEK293 cells, and 3) the antiangiogenic potential of PFOS in a 3D in vitro angiogenesis model of HUVECs and NCFs. In terms of metabolic activity, endothelial cells (HUVECs) were much more sensitive to PFOS than fibroblasts (NCFs) or epithelial cells (HCT116). VEGFR2 signaling in KDR/NFAT-RE HEK293 cells decreased with increasing PFOS concentrations. In co-culture (angiogenesis assay), PFOS treatment resulted in a dose-dependent reduction in tip and branch formation, tip length (μm), and total structural area (μm2) with stable metabolic activity of HUVECs up to high concentrations. We conclude that PFOS possesses antiangiogenic properties. Inhibition of VEGFR2 signaling indicates a possible mechanism of action that can be linked to an existing Adverse Outcome Pathway (AOP43) containing the AO reduced birth weight. Further studies are needed to confirm PFOS-specific adverse effects on angiogenesis, placental perfusion, and fetal growth.

Synthesis, structure-activity relationships and molecular docking studies of phenyldiazenyl sulfonamides as aromatase inhibitors.

The exploration of innovative aromatase inhibitors represents an important approach for the identification of new therapeutic treatments of breast cancer. In this respect, a series of phenyldiazenyl sulfonamides was designed, synthesized and tested. Compounds 3b, 3f and 5f showed an aromatase inhibition in the micromolar range and were evaluated invitro on the human breast cancer cell line MCF7 by MTT assay, cytotoxicity assay (LDH release), cell cycle analysis and apoptosis, revealing a dose-dependent inhibition profile. In particular, 3f displayed the best reduction in terms of metabolic activity and an anti-proliferative effect on MCF7 cells, being blocked in the G1/S phase checkpoint. Moreover, computational studies were carried out to better understand at a molecular level of detail the rationale behind the effective binding to the active site of aromatase of the more active inhibitor 3f. The obtained results allow to consider this compound as an interesting lead for the development of a new class of non-steroidal aromatase inhibitors.

Catch-22 in specialized metabolism: balancing defense and growth.

Plants are unsurpassed biochemists that synthesize a plethora of molecules in response to an ever-changing environment. The majority of these molecules, considered as specialized metabolites, effectively protect the plant against pathogens and herbivores. However, this defense most probably comes at a great expense, leading to reduction of growth (known as the 'growth-defense trade-off'). Plants employ several strategies to reduce the high metabolic costs associated with chemical defense. Production of specialized metabolites is tightly regulated by a network of transcription factors facilitating its fine-tuning in time and space. Multifunctionality of specialized metabolites-their effective recycling system by re-using carbon, nitrogen, and sulfur, thus re-introducing them back to the primary metabolite pool-allows further cost reduction. Spatial separation of biosynthetic enzymes and their substrates, and sequestration of potentially toxic substances and conversion to less toxic metabolite forms are the plant's solutions to avoid the detrimental effects of metabolites they produce as well as to reduce production costs. Constant fitness pressure from herbivores, pathogens, and abiotic stressors leads to honing of specialized metabolite biosynthesis reactions to be timely, efficient, and metabolically cost-effective. In this review, we assess the costs of production of specialized metabolites for chemical defense and the different plant mechanisms to reduce the cost of such metabolic activity in terms of self-toxicity and growth.

Microbial Diversity of Terrestrial Geothermal Springs in Armenia and Nagorno-Karabakh: A Review.

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8–70 °C and pH range 6.0–7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960–2090 m above sea level and are located on the Alpide (Alpine–Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus–Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91–97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.

PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

Priming as a strategy to overcome detrimental pH effects on cells for intervertebral disc regeneration.

Disc disease is characterised by degeneration of the nucleus pulposus (NP), the central gelatinous tissue of the intervertebral disc (IVD). As degeneration progresses, the microenvironment of the IVD becomes more hostile (i.e. decrease in oxygen, glucose and pH), providing a significant challenge for regeneration using cell-based therapies. Tissue engineering strategies such as priming cells or micro tissues with growth factors prior to implantation may overcome some of these issues by providing a pre-formed protective niche composed of extracellular matrix. The present study investigated the effect of priming on bone-marrow-derived stem cells (BMSCs) and articular chondrocytes (ACs) using transforming growth factor β3 (TGF-β3), cultured at different pH levels (pH 7.1, 6.8 and 6.5) representative of the in vivo disc microenvironment. Low pH was found to have a detrimental effect on both cell viability and matrix accumulation, which could be mitigated by priming cells using TGF-β3. Investigating the activation of the transmembrane acid-sensing ion channels (ASIC-1 and -3) showed an increased expression of ASIC-1 in BMSCs and ASIC-3 in ACs at lower pH levels post-priming. Metabolic activity in terms of lactic acid production was also found to be affected significantly by priming, whereas oxygen and glucose consumptions did not change considerably. Overall, the study demonstrated that cells could be equipped to sustain the harsh environment of the IVD and promote accumulation of NP-like matrix through priming. Such an approach may open new avenues to engineer tissues capable of sustaining challenging microenvironments such as those found in the IVD.

Effects of colon-targeted vitamins on the composition and metabolic activity of the human gut microbiome– a pilot study

ABSTRACT An increasing body of evidence has shown that gut microbiota imbalances are linked to diseases. Currently, the possibility of regulating gut microbiota to reverse these perturbations by developing novel therapeutic and preventive strategies is being extensively investigated. The modulatory effect of vitamins on the gut microbiome and related host health benefits remain largely unclear. We investigated the effects of colon-delivered vitamins A, B2, C, D, and E on the gut microbiota using a human clinical study and batch fermentation experiments, in combination with cell models for the assessment of barrier and immune functions. Vitamins C, B2, and D may modulate the human gut microbiome in terms of metabolic activity and bacterial composition. The most distinct effect was that of vitamin C, which significantly increased microbial alpha diversity and fecal short-chain fatty acids compared to the placebo. The remaining vitamins tested showed similar effects on microbial diversity, composition, and/or metabolic activity in vitro, but in varying degrees. Here, we showed that vitamins may modulate the human gut microbiome. Follow-up studies investigating targeted delivery of vitamins to the colon may help clarify the clinical significance of this novel concept for treating and preventing dysbiotic microbiota-related human diseases. Trial registration: ClinicalTrials.gov, NCT03668964. Registered 13 September 2018 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03668964.

Nitrogen repletion favors cellular metabolism and improves eicosapentaenoic acid production in the marine microalga Isochrysis sp. CASA CC 101

Isochrysis sp. is a marine microalga and is a potential source of eicosapentaenoic acid and docosahexaenoic acid, which possess several health benefits. In general, Isochrysis sp. grow well in nitrate-rich culture medium which influences fatty acid biosynthesis; therefore, this study focused on how nitrate influences cellular metabolism in the marine microalga Isochrysis sp. CASA CC 101. Initially, response surface methodology was performed to optimize the level of major nutrients: nitrate and phosphate. The results showed that Isochrysis sp. when grown with nitrate at a concentration of 593 mg.L−1 produced higher eicosapentaenoic acid (7.3 mg.L1), whereas Isochrysis sp. when grown with higher concentration of phosphate did not produce higher levels of eicosapentaenoic acid. The influence of nitrate on cellular metabolism was further confirmed by culturing Isochrysis sp. at higher nitrate conditions. The cellular metabolic activity was analyzed by reactive oxygen species staining assay and the results showed that the cells exhibited reduced oxidative stress with lower green fluorescence under nitrate-repleted conditions. Furthermore, live/dead staining and cell viability assay revealed that live cell population was higher in nitrate-repleted conditions. Nile red and scanning electron microscopy analysis revealed that the cell size and intracellular lipid droplets of microalgal cells grown under nitrate-repleted cells were low, whereas the cell size and intracellular lipid droplets of the microalgal cells grown under nitrogen-depleted conditions were high. Further, the fatty acid profile of Isochrysis sp. grown under nitrogen-repleted conditions showed an increased eicosapentaenoic acid (22.52 mg.g−1 biomass). The mRNA expression of polyunsaturated fatty acid biosynthetic genes such as Δ5-desaturase (Δ5Des) was significantly elevated in nitrate-repleted conditions, whereas the expression of the Δ6-desaturase (Δ6Des) remained unaltered under all the conditions. This is the first study to demonstrate the correlation between nitrate repletion and cellular metabolic activity in terms of eicosapentaenoic acid production in Isochrysis sp. CASA CC 101.

Bevacizumab Induces Upregulation of Keratin 3 and VEGFA in Human Limbal Epithelial Cells in Vitro.

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.

In Vitro Effects of Bisphenol A and Tetrabromobisphenol A on Cell Viability and Reproduction-Related Gene Expression in Pituitaries from Sexually Maturing Atlantic Cod (Gadus morhua L.)

Bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) are widely used industrial chemicals, ubiquitously present in the environment. While BPA is a well-known endocrine disruptor and able to affect all levels of the teleost reproductive axis, information regarding TBBPA on this subject is very limited. Using primary cultures from Atlantic cod (Gadus morhua), the present study was aimed at investigating potential direct effects of acute (72 h) BPA and TBBPA exposure on cell viability and the expression of reproductive-relevant genes in the pituitary. The results revealed that both bisphenols stimulate cell viability in terms of metabolic activity and membrane integrity at environmentally relevant concentrations. BPA had no direct effects on gonadotropin gene expression, but enhanced the expression of gonadotropin-releasing hormone (GnRH) receptor 2a, the main gonadotropin modulator in Atlantic cod. In contrast, TBBPA increased gonadotropin transcript levels but had no effect on GnRH receptor mRNA. In conclusion, both anthropogenic compounds display endocrine disruptive properties and are able to directly interfere with gene expression related to reproductive function in cod pituitary cells at environmentally relevant concentrations in vitro.