In vitro effects of exercise on the heart

Pathologic and physiologic factors acting on the heart can produce consistent pressure changes, volume overload, or increased cardiac output. These changes may then lead to cardiac remodeling, ultimately resulting in cardiac hypertrophy. Exercise can also induce hypertrophy, primarily physiologic in nature. To determine the mechanisms responsible for each type of remodeling, it is important to examine the heart at the functional unit, the cardiomyocyte. Tests of individual cardiomyocyte function in vitro provide a deeper understanding of the changes occurring within the heart during hypertrophy. Examination of cardiomyocyte function during exercise primarily follows one of two pathways: the addition of hypertrophic inducing agents in vitro to normal cardiomyocytes, or the use of trained animal models and isolating cells following the development of hypertrophy in vivo. Due to the short lifespan of adult cardiomyocytes, a proportionately scant amount of research exists involving the direct stimulation of cells in vitro to induce hypertrophy. These attempts provide the only current evidence, as it is difficult to gather extensive data demonstrating cell growth as a result of in vitro physical stimulation. Researchers have created ways to combine skeletal myocytes with cardiomyocytes to produce functional muscle cells used to repair pathologic heart tissue, but continue to struggle with the short lifespan of these cells. While there have been promising findings regarding the mechanisms that surround cardiac hypertrophy in vitro, the translation of in vitro findings to in vivo function is not consistent. Therefore, the focus of this review is to highlight recent studies that have investigated the effect of exercise on the heart, both in vitro and in vivo.     

Effects of petrosaspongiolide R on the surface topology of bee venom PLA(2): a limited proteolysis and mass spectrometry analysis

Petrosaspongiolides are sponge metabolites belonging to the family of the γ-hydroxybutenolide marine terpenoids. They possess a remarkable in vitro and in vivo anti-inflammatory profile, due to the specific inhibition of group II and III secretory phospholipase A₂ enzymes, and for this reason can be considered as potential lead for the development of anti-inflammatory drugs. The molecular mechanism of bee venom phospholipase A₂ inactivation has been identified, and the ligand-enzyme complex formation is guided by either non-covalent and covalent interactions. In this work we have analyzed the conformational changes induced by petrosaspongiolide R on the bee venom phospholipase A₂ topology during the molecular recognition process, through the application of limited proteolysis and mass spectrometric methodologies. The results are indicative of structural changes at the N- and C-terminal domains producing a more compact conformational arrangement of the enzyme.     

Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus

Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora.     

Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy

White spruce is an important commercial species for reforestation. The success in its propagation through somatic embryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a “metabolic footprinting” study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D ¹H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis.     

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.     

Netrin-4 regulates angiogenic responses and tumor cell growth

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas.Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.     

Activated Brain Endothelial Cells Cross-Present Malaria Antigen

In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8⁺ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8⁺ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Cepharanthine inhibits hepatocellular carcinoma cell growth and proliferation by regulating amino acid metabolism and suppresses tumorigenesis in vivo

Cepharanthine (CEP), a natural compound extracted from Stephania cepharantha Hayata, has been found to have the potential to treat a variety of tumors in recent years. This study aims to evaluate the anti-hepatocellular carcinoma (HCC) effect of CEP and determine its in-depth mechanism. In this study, Hep3B and HCCLM3 cells were selected to evaluate the antitumor effects of CEP in vitro, whereas tumor xenograft in nude mice was performed to make in vivo anti-tumor assessment. RNA-sequence (RNA-seq) was used to identify possible molecular targets and pathways. Further, gas chromatography mass spectrometry (GC-MS) was performed to assess the differential metabolites involved in mediating the effect of CEP on the HCC cell line. Our results showed that CEP treatment resulted in the dose-dependent inhibition of cell viability, migration, and proliferation and could also induce apoptosis in HCC cells. RNA-seq following CEP treatment identified 168 differentially expressed genes (DEGs), which were highly enriched in metabolism-associated pathways. In addition, CEP down-regulated many metabolites through the amino acid metabolism pathway. In vivo experiment showed that CEP significantly suppressed tumor growth. Our results indicate that CEP has significant antitumor effects and has the potential to be a candidate drug for HCC treatment.     

Clinical trials with haemopoietic growth factors

Five glycoprotein growth factors capable of stimulating the proliferation and differentiation of haemopoietic progenitor cells in vitro have been identified and sequenced over the past ten years. Recombinant DNA technology has recently enabled the production of sufficient amounts of these agents for preclinical testing. Erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) have already entered clinical studies in humans. Interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) should soon be available for use in humans. EPO corrects the anaemia of end stage renal failure, improving the quality of life for such patients and preventing the need for red cell transfusions. At high dose it increases platelet production in vitro and in vivo and may be of value in humans to prevent the thrombocytopaenia associated with chemotherapy. G-CSF and GM-CSF have been used in several clinical studies. Administration of both growth factors results in a leucocytosis, G-CSF predominantly increasing neutrophil production and GM-CSF increasing production of neutrophils, eosinophils and monocytes. The optimal administration of these agents is via continuous intravenous infusion or daily subcutaneous injections at doses of 3–10μg/kg/24 h. GM-CSF has shown promising results in patients with AIDS and the myelodysplastic syndrome and both G-CSF and GM-CSF have reduced the duration of neutropaenia and incidence of infection associated with chemotherapy. These agents may allow an escalation of the dose-intensity of chemotherapy in the future and thereby, hopefully, increase the response rate and survival for patients with a variety of neoplasms. Several other potential roles for these haemopoietic growth factor are discussed.     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

Anchoring a Plant Cytochrome P450 via PsaM to the Thylakoids in Synechococcus sp. PCC 7002: Evidence for Light-Driven Biosynthesis

Plants produce an immense variety of specialized metabolites, many of which are of high value as their bioactive properties make them useful as for instance pharmaceuticals. The compounds are often produced at low levels in the plant, and due to their complex structures, chemical synthesis may not be feasible. Here, we take advantage of the reducing equivalents generated in photosynthesis in developing an approach for producing plant bioactive natural compounds in a photosynthetic microorganism by functionally coupling a biosynthetic enzyme to photosystem I. This enables driving of the enzymatic reactions with electrons extracted from the photosynthetic electron transport chain. As a proof of concept, we have genetically fused the soluble catalytic domain of the cytochrome P450 CYP79A1, originating from the endoplasmic reticulum membranes of Sorghum bicolor, to a photosystem I subunit in the cyanobacterium Synechococcus sp. PCC 7002, thereby targeting it to the thylakoids. The engineered enzyme showed light-driven activity both in vivo and in vitro, demonstrating the possibility to achieve light-driven biosynthesis of high-value plant specialized metabolites in cyanobacteria.     

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.     

The role of extracellular ions in the pathogenesis of myonecrosis induced by a myotoxin isolated from Broad-Banded Copperhead (Agkistrodon contortrix laticinctus) venom

Pathalogical changes in murine skeletal muscle cells induced by ACL (Agkistrodon contortix laticinctus, Broad-Banded Copperhead) myotoxin in vivo were compared to pathological changes induced by an influx of Ca²⁺ and other ions into cut skeletal muscle cells in vitro in the absence of myotoxin. In vivo, ACL myotoxin induced a rapid myonecrosis characterized by densely clumped myofibrils in the cytoplasm. In vitro, this pathological change was not produced by incubating skeletal muscle cells in Ca²⁺ concentrations as high as 200 mM, whereas skeletal muscle cells incubated in concentrations of 150 mM and 300 mM NaCl contained densely clumped myofibrils similar in morphology to muscle cells damaged by ACL myotoxin in vivo. Treatments of 300 mM KCl did not produce densely clumped myofibrils in muscle cells. These results suggest that an influx of Na⁺, possibly through disrupted regions of sarcolemma, be may primarily responsible for the pathological changes, including clumped myofibrils, induced by ACL myotoxin in vivo. However, an influx of extracellular Ca²⁺ which has been proposed to produce densely clumped myofibrils in muscle cells damaged by other snake venom myotoxins, may not be responsible for this pathological change since extracellular Ca²⁺ concentrations much higher than physiological levels did not produce this change in skeletal muscle cells in vitro.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondria and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling. Channeling presents challenges but also opportunities for metabolic engineering applications. It rigidifies fluxes in native pathways by trapping or excluding metabolites for bioconversions, causing substrate catabolite repressions or inferior efficiencies in engineered pathways. Channeling is an overlooked regulatory mechanism used to control flux responses under environmental/genetic perturbations. The heterogeneous distribution of intracellular enzymes also confounds kinetic modeling and multiple-omics analyses. Understanding the scope and mechanisms of channeling in central pathways may improve our interpretation of robust fluxomic topology throughout metabolic networks and lead to better design and engineering of heterologous pathways.     

The use of Aspergillus niger cultures for biotransformation of terpenoids

Aspergillus niger is a well-known fungus that has been used for many different biotransformations of organic compounds. The terpenoids include a large variety of natural hydrocarbons and their derivatives, mostly obtained from plant essential oils, but some obtained from animals or fungi. They may be acyclic or have one or more rings of various sizes, and they show a variety of biological activities that include antibacterial, antifungal, antiparasitic, antiviral, and anticancer activities. Terpenoids are classified as monoterpenoids (C₁₀), sesquiterpenoids (C₁₅), diterpenoids (C₂₀), triterpenoids (C₃₀), and others. This review summarizes experimental processes that use cultures of various A. niger strains to carry out stereoselective biochemical reactions in terpenoids, including related epoxides, lactones, N-phenylcarbamates, and saponins, to produce metabolites that may be useful as flavors and fragrances or as new experimental drug candidates. Cultures of A. niger that add hydroxyl, carbonyl, and other groups at specific positions or reduce double bonds have resulted in the production of valuable new compounds.     

Immunobiology of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from almost all tissues and effectively expanded in vitro. Although their true in situ properties and biological functions remain to be elucidated, these in vitro expanded cells have been shown to possess potential to differentiate into specific cell lineages. It is speculated that MSCs in situ have important roles in tissue cellular homeostasis by replacing dead or dysfunctional cells. Recent studies have demonstrated that in vitro expanded MSCs of various origins have great capacity to modulate immune responses and change the progression of different inflammatory diseases. As tissue injuries are often accompanied by inflammation, inflammatory factors may provide cues to mobilize MSCs to tissue sites with damage. Before carrying out tissue repair functions, MSCs first prepare the microenvironment by modulating inflammatory processes and releasing various growth factors in response to the inflammation status. In this review, we focus on the crosstalk between MSCs and immune responses and their potential clinical applications, especially in inflammatory diseases.