A Tale Of Two Sirtuins: The Impact Of Sirt1 And Sirt3 On The Pathophysiology Of Shock

Both acute blood loss and severe infection activate common cellular pathways leading to shock – a pathologic condition characterized by systemic inflammation, oxidative stress, and mitochondrial dysfunction. Sirtuins, a highly conserved group of NAD-dependent enzymes, play a critical role in cellular survival and many of the benefits associated with sirtuin activation are thought to be secondary to decreased inflammation, reduced oxidative stress, and improved mitochondrial physiology. As such, we hypothesized that sirtuin pathways play a crucial role in shock and could be could be targeted to improve outcomes following acute blood loss and severe infection. In a series of in vivo and in vitro experiments recapitulating hemorrhagic shock and severe sepsis, we explored the impact of sirtuin activation on inflammation, mitochondrial function, and survival. Following decompensated hemorrhagic shock, resuscitation with resveratrol, a SIRT1 activator, significantly improved renal mitochondrial function and decreased oxidative damage. Similarly, resuscitation with nicotinamide monononucleotide (NMN), a key biosynthetic NAD precursor, was found to mitigate inflammation, support cellular energetics and improve both physiologic resilience and survival. In contrast, impaired expression of either SIRT1 or SIRT3 resulted in a pro-inflammatory phenotype with accelerated mortality in sepsis. Interestingly, deletion of SIRT1 did not significantly worsen the degree of mitochondrial dysfunction observed in septic liver, but was associated with decreased CI and CII respiration in kidney. Deletion of SIRT3 did not significantly impact the degree of mitochondrial dysfunction observed in either liver or kidney. Taken together, these data strongly suggest that SIRT1 and SIRT3 play a key role in the pathophysiology of shock. Although further research is needed to determine if SIRT1 and SIRT3 overexpression improves outcomes or if pharmacologically manipulating NAD metabolism in conjunction with sirtuin activation provides added benefit, targeting sirtuins appears beneficial in hemorrhagic and septic shock

Ex Vivo Recapitulation of Trauma-Induced Coagulopathy and Preliminary Assessment of Trauma Patient Platelet Function Under Flow Using Microfluidic Technology

Background: Relevant to trauma induced coagulopathy (TIC) diagnostics, microfluidic assays allow controlled hemodynamics for testing of platelet and coagulation function using whole blood.Methods: Hemodilution or hyperfibrinolysis was studied under flow with modified healthy whole blood. Furthermore, platelet function was also measured using whole blood from trauma patients admitted to a Level 1 Trauma center. Platelet deposition was measured with PPACK-inhibited blood perfused over collagen surfaces at a wall shear rate of 200 s-1, while platelet/fibrin deposition was measured with corn trypsin inhibitor (CTI)-treated blood perfused over TF/collagen.Results: In hemodilution studies, PPACK-treated blood displayed almost no platelet deposition when diluted to 10% Hct with saline, platelet poor plasma (PPP), or platelet rich plasma (PRP). Using similar dilutions, platelet/fibrin deposition was essentially absent for CTI-treated blood perfused over TF/collagen. To mimic hyperfibrinolysis during trauma, exogenous tPA (50 nM) was added to blood prior to perfusion over TF/collagen. At both venous and arterial flows, the generation and subsequent lysis of fibrin was detectable within 6 min, with lysis blocked by addition of the plasmin inhibitor, [epsilon]-aminocaproic acid. Microfluidic assay of PPACK-inhibited whole blood from trauma patients revealed striking defects in collagen response and secondary platelet aggregation in 14 of 21 patients, while platelet hyperfunction was detected in 3 of 20 patients.Conclusions: Rapid microfluidic detection of (i) hemodilution-dependent impairment of clotting, (ii) clot instability due to lysis, (iii) blockade of fibrinolysis, or (iv) platelet dysfunction during trauma may provide novel diagnostic opportunities to predict TIC risk.Level of Evidence: Level IVStudy type: Diagnostic Tes

Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene

BACKGROUND: Runx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo. RESULTS: The developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D. CONCLUSIONS: SpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation

Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

BACKGROUND: Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. RESULTS: We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. CONCLUSIONS: Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis

CBFbeta is a facultative Runx partner in the sea urchin embryo

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation

CBFbeta is a facultative Runx partner in the sea urchin embryo

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation

Evolution of anterior Hox regulatory elements among chordates

<p>Abstract</p> <p>Background</p> <p>The <it>Hox </it>family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the <it>Hox </it>paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed.</p> <p>Results</p> <p>To investigate the degree of conservation in <it>cis</it>-regulatory components that form the basis of <it>Hox </it>expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the <it>CiHox1, CiHox2 </it>and <it>CiHox4 </it>genes of the ascidian <it>Ciona intestinalis </it>which direct neural specific domains of expression. Using functional assays in <it>Ciona </it>and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to <it>Hox </it>paralogy group genes, we compared the activity of these four <it>Ciona cis</it>-elements with a series of neural specific enhancers from the amphioxus <it>Hox1-3 </it>genes and from mouse <it>Hox </it>paralogous groups 1-4.</p> <p>Conclusions</p> <p>This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as <it>Hox </it>response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these <it>Hox </it>response <it>cis</it>-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, <it>cis</it>-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental <it>Hox </it>expression in neural tissue and appeared with urochordates after cephalochordate divergence.</p

Sea urchin vault structure, composition, and differential localization during development

BACKGROUND: Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis. RESULTS: The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven ~50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14–55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis. CONCLUSIONS: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development

CBFbeta is a facultative Runx partner in the sea urchin embryo

Background: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. Results: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. Conclusion: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation

Coral assemblages at higher latitudes favor short-term potential over long-term performance

Funding for this research was provided by a Natural Environment Research Council (NERC) Doctoral Training Programme Scholarship to JC, a Royal Geographical Society Ralph Brown Expedition Award (RBEA 03/19) to MB and JC, the Australian Research Council Centre of Excellence for Coral Reef Studies (CE140100020) to JMP and others, the Australian Research Council Centre of Excellence for Environmental Decisions (CE110001014), a British Ecological Society small grant, the Winifred Violet Scott Charitable Trust, and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement TRIM-DLV-747102 to MB. BS was supported by an Australian Research Council Discovery Early CareerResearch Award (DE230100141), a University of Sydney Fellowship and a Chancellor’s Postdoctoral Research Fellowship from the University of Technology Sydney.The persistent exposure of coral assemblages to more variable abiotic regimes is assumed to augment their resilience to future climatic variability. Yet, while the determinants of coral population resilience across species remain unknown, we are unable to predict the winners and losers across reef ecosystems exposed to increasingly variable conditions. Using annual surveys of 3171 coral individuals across Australia and Japan (2016-2019), we explore spatial variation across the short- and long-term dynamics of competitive, stress-tolerant, and weedy assemblages to evaluate how abiotic variability mediates the structural composition of coral assemblages. We illustrate how, by promoting short-term potential over long-term performance, coral assemblages can reduce their vulnerability to stochastic environments. However, compared to stress-tolerant, and weedy assemblages, competitive coral taxa display a reduced capacity for elevating their short-term potential. Accordingly, future climatic shifts threaten the structural complexity of coral assemblages in variable environments, emulating the degradation expected across global tropical reefs.Publisher PDFPeer reviewe