A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Highlighting the potential of Synechococcus elongatus PCC 7942 as platform to produce α-linolenic acid through an updated genome-scale metabolic modeling.

Cyanobacteria are prokaryotic organisms that capture energy from sunlight using oxygenic photosynthesis and transform CO2 into products of interest such as fatty acids. Synechococcus elongatus PCC 7942 is a model cyanobacterium efficiently engineered to accumulate high levels of omega-3 fatty acids. However, its exploitation as a microbial cell factory requires a better knowledge of its metabolism, which can be approached by using systems biology tools. To fulfill this objective, we worked out an updated, more comprehensive, and functional genome-scale model of this freshwater cyanobacterium, which was termed iMS837. The model includes 837 genes, 887 reactions, and 801 metabolites. When compared with previous models of S. elongatus PCC 7942, iMS837 is more complete in key physiological and biotechnologically relevant metabolic hubs, such as fatty acid biosynthesis, oxidative phosphorylation, photosynthesis, and transport, among others. iMS837 shows high accuracy when predicting growth performance and gene essentiality. The validated model was further used as a test-bed for the assessment of suitable metabolic engineering strategies, yielding superior production of non-native omega-3 fatty acids such as α-linolenic acid (ALA). As previously reported, the computational analysis demonstrated that fabF overexpression is a feasible metabolic target to increase ALA production, whereas deletion and overexpression of fabH cannot be used for this purpose. Flux scanning based on enforced objective flux, a strain-design algorithm, allowed us to identify not only previously known gene overexpression targets that improve fatty acid synthesis, such as Acetyl-CoA carboxylase and ß-ketoacyl-ACP synthase I, but also novel potential targets that might lead to higher ALA yields. Systematic sampling of the metabolic space contained in iMS837 identified a set of ten additional knockout metabolic targets that resulted in higher ALA productions. In silico simulations under photomixotrophic conditions with acetate or glucose as a carbon source boosted ALA production levels, indicating that photomixotrophic nutritional regimens could be potentially exploited in vivo to improve fatty acid production in cyanobacteria. Overall, we show that iMS837 is a powerful computational platform that proposes new metabolic engineering strategies to produce biotechnologically relevant compounds, using S. elongatus PCC 7942 as non-conventional microbial cell factory.

Circulating Endothelial Cells From Septic Shock Patients Convert to Fibroblasts Are Associated With the Resuscitation Fluid Dose and Are Biomarkers for Survival Prediction.

OBJECTIVES: To determine whether circulating endothelial cells from septic shock patients and from nonseptic shock patients are transformed in activated fibroblast by changing the expression level of endothelial and fibrotic proteins, whether the level of the protein expression change is associated with the amount of administered resuscitation fluid, and whether this circulating endothelial cell protein expression change is a biomarker to predict sepsis survival. DESIGN: Prospective study. SETTING: Medical-surgical ICUs in a tertiary care hospital. PATIENTS: Forty-three patients admitted in ICU and 22 healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Circulating mature endothelial cells and circulating endothelial progenitor cells from septic shock and nonseptic shock patients showed evidence of endothelial fibrosis by changing the endothelial protein expression pattern. The endothelial proteins were downregulated, whereas fibroblast-specific markers were increased. The magnitude of the expression change in endothelial and fibrotic proteins was higher in the septic shock nonsurvivors patients but not in nonseptic shock. Interestingly, the decrease in the endothelial protein expression was correlated with the administered resuscitation fluid better than the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in the septic shock nonsurvivors patients but not in nonseptic shock. Notably, the significant difference between endothelial and fibrotic protein expression indicated a nonsurvival outcome in septic shock but not in nonseptic shock patients. Remarkably, area under the receiver operating characteristic curve analysis showed that endothelial protein expression levels predicted the survival outcome better than the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in septic shock but not in nonseptic shock patients. CONCLUSIONS: Circulating endothelial cells from septic shock patients are acutely converted into fibroblasts. Endothelial and fibrotic protein expression level are associated with resuscitation fluid administration magnitude and can be used as biomarkers for an early survival diagnosis of sepsis.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+]i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Endothelial fibrosis induced by suppressed STAT3 expression mediated by signaling involving the TGF-ß1/ALK5/Smad pathway.

During systemic inflammatory pathologies, mediators of inflammation circulate in the bloodstream and interact with endothelial cells (ECs), resulting in endothelial dysfunction that maintains and enhances the pathological condition. Inflammatory mediators change the protein expression profile of ECs, which become activated fibroblasts via endothelial-to-mesenchymal transition. This process is characterized by downregulated endothelial proteins and strongly upregulated fibrotic-specific genes and extracellular matrix-forming proteins. The main inductor of endothelial fibrosis is transforming growth factor-ß1 (TGF-ß1), which acts through the TGF-ß1/activin receptor-like kinase 5 (ALK5)/Smads intracellular signaling pathway. The signal transducer and activator of transcription 3 (STAT3) is also involved in fibrosis in several tissues (e.g. heart and vascular system), where STAT3 signaling decreases TGF-ß1-induced responses by directly interacting with Smad proteins, suggesting that decreased STAT3 could induce TGF-ß1-mediated fibrosis. However, it is unknown if suppressed STAT3 expression induces EC fibrosis through a mechanism involving the TGF-ß signaling pathway. The present study evaluated the fibrotic actions of STAT3 suppression in ECs and investigated TGF-ß1/ALK5/Smad4 signaling pathway participation. Suppressed STAT3 expression stimulated fibrotic conversion in ECs, as mediated by protein expression reprograming that decreased endothelial marker expression and increased fibrotic and extracellular matrix protein levels. The potential mechanism underlying these changes was dependent on TGF-ß1 secretion, the ALK5 activation pathway, and Smad4 translocation into the nucleus. We conclude that suppressed STAT3 expression converts ECs into activated fibroblasts via TGF-ß1/ALK5/Smad4 signaling pathway involvement.

Excess iodide induces an acute inhibition of the sodium/iodide symporter in thyroid male rat cells by increasing reactive oxygen species.

Na+/I- symporter (NIS) mediates iodide (I-) uptake in the thyroid gland, the first and rate-limiting step in the biosynthesis of the thyroid hormones. The expression and function of NIS in thyroid cells is mainly regulated by TSH and by the intracellular concentration of I-. High doses of I- for 1 or 2 days inhibit the synthesis of thyroid hormones, a process known as the Wolff-Chaikoff effect. The cellular mechanisms responsible for this physiological response are mediated in part by the inhibition of I- uptake through a reduction of NIS expression. Here we show that inhibition of I- uptake occurs as early as 2 hours or 5 hours after exposure to excess I- in FRTL-5 cells and the rat thyroid gland, respectively. Inhibition of I- uptake was not due to reduced NIS expression or altered localization in thyroid cells. We observed that incubation of FRTL-5 cells with excess I- for 2 hours increased H2O2 generation. Furthermore, the inhibitory effect of excess I- on NIS-mediated I- transport could be recapitulated by H2O2 and reverted by reactive derived oxygen species scavengers. The data shown here support the notion that excess I- inhibits NIS at the cell surface at early times by means of a posttranslational mechanism that involves reactive derived oxygen species.

Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel.

A hallmark of severe inflammation is reactive oxygen species (ROS) overproduction induced by increased inflammatory mediators secretion. During systemic inflammation, inflammation mediators circulating in the bloodstream interact with endothelial cells (ECs) raising intracellular oxidative stress at the endothelial monolayer. Oxidative stress mediates several pathological functions, including an exacerbated EC migration. Because cell migration critically depends on calcium channel-mediated Ca(2+) influx, the molecular identification of the calcium channel involved in oxidative stress-modulated EC migration has been the subject of intense investigation. The transient receptor potential melastatin 4 (TRPM4) protein is a ROS-modulated non-selective cationic channel that performs several cell functions, including regulating intracellular Ca(2+) overload and Ca(2+) oscillation. This channel is expressed in multiple tissues, including ECs, and contributes to the migration of certain immune cells. However, whether the TRPM4 ion channel participates in oxidative stress-mediated EC migration is not known. Herein, we investigate whether oxidative stress initiates or enhances EC migration and study the role played by the ROS-modulated TRPM4 ion channel in oxidative stress-mediated EC migration. We demonstrate that oxidative stress enhances, but does not initiate, EC migration in a dose-dependent manner. Notably, we demonstrate that the TRPM4 ion channel is critical in promoting H2O2-enhanced EC migration. These results show that TRPM4 is a novel pharmacological target for the possible treatment of severe inflammation and other oxidative stress-mediated inflammatory diseases.

Oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a TGF-ß1 and TGF-ß2-dependent pathway.

During the pathogenesis of systemic inflammation, reactive oxygen species (ROS) circulate in the bloodstream and interact with endothelial cells (ECs), increasing intracellular oxidative stress. Although endothelial dysfunction is crucial in the pathogenesis of systemic inflammation, little is known about the effects of oxidative stress on endothelial dysfunction. Oxidative stress induces several functions, including cellular transformation. A singular process of cell conversion is tendothelial-to-mesenchymal transition, in which ECs become myofibroblasts, thus losing their endothelial properties and gaining fibrotic behavior. However, the participation of oxidative stress as an inductor of conversion of ECs into myofibroblasts is not known. Thus, we studied the role played by oxidative stress in this conversion and investigated the underlying mechanism. Our results show that oxidative stress induces conversion of ECs into myofibroblasts through decreasing the levels of endothelial markers and increasing those of fibrotic and ECM proteins. The underlying mechanism depends on the ALK5/Smad3/NF-κB pathway. Oxidative stress induces the expression and secretion of TGF-ß1 and TGF-ß2 and p38 MAPK phosphorylation. Downregulation of TGF-ß1 and TGF-ß2 by siRNA technology abolished the H2O2-induced conversion. To our knowledge, this is the first report showing that oxidative stress is able to induce conversion of ECs into myofibroblasts via TGF-ß secretion, emerging as a source for oxidative stress-based vascular dysfunction. Thus, oxidative stress emerges as a decisive factor in inducing conversion of ECs into myofibroblasts through a TGF-ß-dependent mechanism, changing the ECs protein expression profile, and converting normal ECs into pathological ones. This information will be useful in designing new and improved therapeutic strategies against oxidative stress-mediated systemic inflammatory diseases.

Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons.

Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription.

Lipopolysaccharide induces a fibrotic-like phenotype in endothelial cells.

Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor-like kinase 5 (ALK5) activity-dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.

Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala.

Recent in vitro evidence indicates that astrocytes can modulate synaptic plasticity by releasing neuroactive substances (gliotransmitters). However, whether gliotransmitter release from astrocytes is necessary for higher brain function in vivo, particularly for memory, as well as the contribution of connexin (Cx) hemichannels to gliotransmitter release, remain elusive. Here, we microinfused into the rat basolateral amygdala (BLA) TAT-Cx43L2, a peptide that selectively inhibits Cx43-hemichannel opening while maintaining synaptic transmission or interastrocyte gap junctional communication. In vivo blockade of Cx43 hemichannels during memory consolidation induced amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short-term memory, locomotion, or shock reactivity. The amnesic effect was transitory, specific for memory consolidation, and was confirmed after microinfusion of Gap27, another Cx43-hemichannel blocker. Learning capacity was recovered after coinfusion of TAT-Cx43L2 and a mixture of putative gliotransmitters (glutamate, glutamine, lactate, d-serine, glycine, and ATP). We propose that gliotransmitter release from astrocytes through Cx43 hemichannels is necessary for fear memory consolidation at the BLA. Thus, the present study is the first to demonstrate a physiological role for astroglial Cx43 hemichannels in brain function, making these channels a novel pharmacological target for the treatment of psychiatric disorders, including post-traumatic stress disorder.

Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells.

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes in different tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several muscular dystrophies are characterized by extensive fibrosis. However, the exact role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that myoblasts responded to Ang-II by increasing protein levels of connective tissue growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced CTGF and fibronectin levels by DPI and apocynin. We also show that Ang-II-induced ROS production and fibrosis require PKC activity as indicated by the generic PKC inhibitor chelerythrine. These results strongly suggest that the fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires NAD(P)H-induced ROS in skeletal muscle cells.

Increased expression of the transient receptor potential melastatin 7 channel is critically involved in lipopolysaccharide-induced reactive oxygen species-mediated neuronal death.

AIMS: To assess the mechanisms involved in lipopolysaccharide (LPS)-induced neuronal cell death, we examined the cellular consequences of LPS exposure in differentiated PC12 neurons and primary hippocampal neurons. RESULTS: Our data show that LPS is able to induce PC12 neuronal cell death without the participation of glial cells. Neuronal cell death was mediated by an increase in cellular reactive oxygen species (ROS) levels. Considering the prevalent role of specific ion channels in mediating the deleterious effect of ROS, we assessed their contribution to this process. Neurons exposed to LPS showed a significant intracellular Ca(2+) overload, and nonselective cationic channel blockers inhibited LPS-induced neuronal death. In particular, we observed that both LPS and hydrogen peroxide exposure strongly increased the expression of the transient receptor protein melastatin 7 (TRPM7), which is an ion channel directly implicated in neuronal cell death. Further, both LPS-induced TRPM7 overexpression and LPS-induced neuronal cell death were decreased with dithiothreitol, dipheniliodonium, and apocynin. Finally, knockdown of TRPM7 expression using small interference RNA technology protected primary hippocampal neurons and differentiated PC12 neurons from the LPS challenge. INNOVATION: This is the first report showing that TRPM7 is a key protein involved in neuronal death after LPS challenge. CONCLUSION: We conclude that LPS promotes an abnormal ROS-dependent TRPM7 overexpression, which plays a crucial role in pathologic events, thus leading to neuronal dysfunction and death.

Transient receptor potential melastatin 4 inhibition prevents lipopolysaccharide-induced endothelial cell death.

AIMS: Endothelial dysfunction is decisive in the progression of cardiovascular diseases. Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS)-mediated endothelial cell death is a main feature observed in inflammation secondary to endotoxaemia, emerging as a leading cause of death among critically ill patients in intensive care units. However, the molecular mechanism underlying LPS-induced endothelial cell death is not well understood. Transient receptor protein melastatin 4 (TRPM4) is an ion channel associated with cell death that is expressed in endothelium and modulated by ROS. Here, we investigate the role of TRPM4 in LPS-induced endothelial cell death, testing whether suppression of the expression of TRPM4 confers endothelial cell resistance to LPS challenge. METHODS AND RESULTS: Using primary cultures of human umbilical vein endothelial cells (HUVEC), we demonstrate that TRPM4 is critically involved in LPS-induced endothelial cell death. HUVEC exposed to LPS results in Na(+)-dependent cell death. Pharmacological inhibition of TRPM4 with 9-phenanthrol or glibenclamide protects endothelium against LPS exposure for 48 h. Furthermore, TRPM4-like currents increase in cells pre-treated with LPS and inhibited with glibenclamide. Of note, suppression of TRPM4 expression by siRNA or suppression of its activity in a dominant negative mutant is effective in decreasing LPS-induced endothelial cell death when cells are exposed to LPS for 24-30 h. CONCLUSION: TRPM4 is critically involved in LPS-induced endothelial cell death. These results demonstrate that either pharmacological inhibition of TRPM4, suppression of TRPM4 expression, or inhibition of TRPM4 activity are able to protect endothelium against LPS injury. These results are useful in sepsis drug design and development of new strategies for sepsis therapy.

Lipopolysaccharide signaling in the carotid chemoreceptor pathway of rats with sepsis syndrome.

In addition to their role in cardiorespiratory regulation, carotid body (CB) chemoreceptors serve as sensors for inflammatory status and as a protective factor during sepsis. However, lipopolysaccharide-induced sepsis (LPS) reduces CB responsiveness to excitatory or depressant stimuli. We tested whether LPS exerts a direct effect on the carotid chemoreceptor pathway, the CB and its sensory ganglion. We determined that the rat CB and nodose-petrosal-jugular ganglion complex (NPJgc) express TLR4, TNF-α and its receptors (TNF-R1 and TNF-R2). LPS administration (15mg/kg intraperitoneally) evoked MyD88-mechanism pathway activation in CB and NPJgc, with NF-κB p65, p38 MAPK, and ERK activation. Consistently, LPS increased TNF-α and TNF-R2. Double-labeling studies showed that the aforementioned pathway occurs in TH-containing glomus cells and NPJgc neurons, components of the chemosensitive neural pathway. Thus, our results suggest that LPS acting directly through TLR4/MyD88-mechanism pathways increases TNF-α and TNF-R2 expression in the carotid chemoreceptor pathway. These results show a novel afferent pathway to the central nervous system during endotoxemia, and could be relevant in understanding sepsis pathophysiology and therapy.