Helena L. A. Vieira

Marketed Marine Natural Products in the Pharmaceutical and Cosmeceutical Industries: Tips for Success

The marine environment harbors a number of macro and micro organisms that have developed unique metabolic abilities to ensure their survival in diverse and hostile habitats, resulting in the biosynthesis of an array of secondary metabolites with specific activities. Several of these metabolites are high-value commercial products for the pharmaceutical and cosmeceutical industries. The aim of this review is to outline the paths of marine natural products discovery and development, with a special focus on the compounds that successfully reached the market and particularly looking at the approaches tackled by the pharmaceutical and cosmetic companies that succeeded in marketing those products. The main challenges faced during marine bioactives discovery and development programs were analyzed and grouped in three categories: biodiversity (accessibility to marine resources and efficient screening), supply and technical (sustainable production of the bioactives and knowledge of the mechanism of action) and market (processes, costs, partnerships and marketing). Tips to surpass these challenges are given in order to improve the market entry success rates of highly promising marine bioactives in the current pipelines, highlighting what can be learned from the successful and unsuccessful stories that can be applied to novel and/or ongoing marine natural products discovery and development programs.

Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species

Low oxygen concentrations (hypoxia) occur in several physiological and pathological cellular situations such as embryogenesis and stem cell modulation (in terms of differentiation/proliferation), or ischemic stroke and cancer. On the other side of the coin, the generation of reactive oxygen species (ROS) is tightly controlled by the cell. ROS control redox sensitive signaling pathways and thus regulate cell physiology, such as programmed cell death, inflammation and/or stem cell modulation. Herein we analyze the role of hypoxia and ROS in the modulation of neuronal differentiation focusing on: (i) in vivo neurogenesis and (ii) in vitro neuronal differentiation from neural stem/precursor cells. In vivo, hypoxia promotes neurogenesis in embryos, newborns and adults, as well as in response to noxious stimuli such as ischemia. On the other hand, oxygen and ROS also play a role in in vitro neuronal differentiation. They further impact tumor growth by influencing cell proliferation and differentiation, such as in neuroblastoma development. Therefore, manipulating hypoxia and ROS production represents a useful therapeutic tool if one needs either to enhance or to modulate neurogenesis and neuronal differentiation, such as in cell replacement or in malignant cell proliferation.

Glutathionylation of Adenine Nucleotide Translocase Induced by Carbon Monoxide Prevents Mitochondrial Membrane Permeabilization and Apoptosis

The present work demonstrates the ability of CO to prevent apoptosis in a primary culture of astrocytes. For the first time, the antiapoptotic behavior can be clearly attributed to the inhibition of mitochondrial membrane permeabilization (MMP), a key event in the intrinsic apoptotic pathway. In isolated non-synaptic mitochondria, CO partially inhibits (i) loss of potential, (ii) the opening of a nonspecific pore through the inner membrane, (iii) swelling, and (iv) cytochrome c release, which are induced by calcium, diamide, or atractyloside (a ligand of ANT). CO directly modulates ANT function by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally, CO induces reactive oxygen species (ROS) generation, and its prevention by β-carotene decreases CO cytoprotection in intact cells as well as in isolated mitochondria, revealing the key role of ROS. On the other hand, CO induces a slight increase in mitochondrial oxidized glutathione, which is essential for apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP, and (iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG trigger ANT glutathionylation, a post-translational process regulating protein function in response to redox cellular changes. In conclusion, CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG.

Pre‐conditioning induced by carbon monoxide provides neuronal protection against apoptosis

Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme‐oxygenase, presenting anti‐apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme‐oxygenase product critical for its anti‐apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre‐conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP‐dependent mitochondrial K channel (mitoKATP) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoKATP abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoKATP, which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies.

Carbon Monoxide Modulates Apoptosis by Reinforcing Oxidative Metabolism in Astrocytes: ROLE OF Bcl-2*

Background: Low doses of carbon monoxide (CO) prevent apoptosis in several cell models, including astrocytes. Results: CO improves cytochrome c oxidase (COX) activity and induces mitochondrial biogenesis. Bcl-2 expression and interaction with COX is involved in CO signaling. Conclusion: CO stimulates oxidative phosphorylation, improves metabolism, and prevents astrocytic apoptosis. Significance: Metabolism modulation can be a potential strategy against cerebral ischemia. Modulation of cerebral cell metabolism for improving the outcome of hypoxia-ischemia and reperfusion is a strategy yet to be explored. Because carbon monoxide (CO) is known to prevent cerebral cell death; herein the role of CO in the modulation of astrocytic metabolism, in particular, at the level of mitochondria was investigated. Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization, and plasmatic membrane permeability. CO exposure enhanced intracellular ATP generation, which was accompanied by an increase on specific oxygen consumption, a decrease on lactate production, and a reduction of glucose use, indicating an improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c oxidase (COX) enzymatic specific activity and stimulated mitochondrial biogenesis. In astrocytes, COX interacts with Bcl-2, which was verified by immunoprecipitation; this interaction is superior after 24 h of CO treatment. Furthermore, CO enhanced Bcl-2 expression in astrocytes. By silencing Bcl-2 expression with siRNA transfection, CO effects in astrocytes were prevented, namely: (i) inhibition of apoptosis, (ii) increase on ATP generation, (iii) stimulation of COX activity, and (iv) mitochondrial biogenesis. Thus, Bcl-2 expression is crucial for CO modulation of oxidative metabolism and for conferring cytoprotection. In conclusion, CO protects astrocytes against oxidative stress-induced apoptosis by improving metabolism functioning, particularly mitochondrial oxidative phosphorylation.

Effect of Escherichia coli Morphogene bolA on Biofilms

ABSTRACT Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.

Mitochondria as Targets of Apoptosis Regulation by Nitric Oxide

In addition to their vital role as the cells power stations, mitochondria exert an important function in apoptosis. In response to most if not all apoptosis inducers, mitochondrial membranes are permeabilized, leading to the release of potentially toxic proteins, mostly from the intermembrane space to the rest of the cells. Such pro‐apoptotic intermembrane proteins include the caspase‐independent death effector AIF, as well as cytochrome c, which can trigger the activation of caspases, once it has reached the cytosol. The mitochondrial permeabilization process can be induced by a variety of different xenobiotics, via a direct effect on mitochondrial membranes. Alternatively, mitochondrial permeabilization can be induced by endogenous second messengers, which are elicited in response to stress. The permeabilization process is controlled by the mitochondrial permeability transition pore complex (PTPC), by proteins of the Bcl‐2/Bax family, as well as by lipids and metabolites. Nitric oxide (NO) is one of the second messengers that can trigger apoptosis by inducing mitochondrial membrane permeabilization. This effect may involve a direct effect on the PTPC and/or indirect effects secondary to the NO‐mediated inhibition of oxidative phosphorylation. This has far‐reaching implications for the pathophysiology of NO. IUBMB Life, 55: 613‐616, 2003

Neuroprotective effects of digested polyphenols from wild blackberry species

PurposeBlackberry ingestion has been demonstrated to attenuate brain degenerative processes with the benefits ascribed to the (poly)phenolic components. The aim of this work was to evaluate the neuroprotective potential of two wild blackberry species in a neurodegeneration cell model and compare them with a commercial variety.MethodsThis work encompasses chemical characterization before and after an in vitro digestion and the assessment of neuroprotection by digested metabolites. Some studies targeting redox/cell death systems were also performed to assess possible neuroprotective molecular mechanisms.ResultsThe three blackberry extracts presented some quantitative differences in polyphenol composition that could be responsible for the different responses in the neurodegeneration cell model. Commercial blackberry extracts were ineffective but both wild blackberries, Rubus brigantinus and Rubus vagabundus, presented neuroprotective effects. It was verified that a diminishment of intracellular ROS levels, modulation of glutathione levels and activation of caspases occurred during treatment. The last effect suggests a preconditioning effect since caspase activation was not accompanied by diminution in cell death and loss of functionality.ConclusionsThis is the first time that metabolites obtained from an in vitro digested food matrix, and tested at levels approaching the concentrations found in human plasma, have been described as inducing an adaptative response.

New method to assess mitophagy flux by flow cytometry

Mitochondrial autophagy, also known as mitophagy, is an autophagosome-based mitochondrial degradation process that eliminates unwanted or damaged mitochondria after cell stress. Most studies dealing with mitophagy rely on the analysis by fluorescence microscopy of mitochondrial-autophagosome colocalization. However, given the fundamental role of mitophagy in the physiology and pathology of organisms, there is an urgent need for novel quantitative methods with which to study this process. Here, we describe a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red, a widely used mitochondria-selective probe. Used in combination with selective inhibitors it may allow for the determination of mitophagy flux. Here, we test the validity of the use of this method in cell lines and in primary cell and tissue cultures.

Preconditioning Triggered by Carbon Monoxide (CO) Provides Neuronal Protection Following Perinatal Hypoxia-Ischemia

Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a major role. Increased resistance against major damage can be achieved by preconditioning triggered by subtle insults. CO, a toxic molecule that is also generated endogenously, may have a role in preconditioning as low doses can protect against inflammation and apoptosis. In this study, the role of CO-induced preconditioning on neurons was addressed in vitro and in vivo. The effect of 1 h of CO treatment on neuronal death (plasmatic membrane permeabilization and chromatin condensation) and bcl-2 expression was studied in cerebellar granule cells undergoing to glutamate-induced apoptosis. COs role was studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common carotid artery ligature +75 min at 8% oxygen). Apoptotic cells, assessed by Nissl staining were counted with a stereological approach and cleaved caspase 3-positive profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in hippocampal extracts by Western Blot. CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced apoptosis in the hippocampus, limited cytochrome c released from mitochondria and reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in hippocampus of CO pre-treated rat pups. Our results show that CO preconditioning elicits a molecular cascade that limits neuronal apoptosis. This could represent an innovative therapeutic strategy for high-risk cerebral hypoxia-ischemia patients, in particular neonates.