Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

Role of Bicaudal C1 in renal gluconeogenesis and its novel interaction with the CTLH complex.

Altered glucose and lipid metabolism fuel cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Renal cysts also associate with mutations in Bicaudal C1 (Bicc1) or in its self-polymerizing sterile alpha motif (SAM). Here, we found that Bicc1 maintains normoglycemia and the expression of the gluconeogenic enzymes FBP1 and PEPCK in kidneys. A proteomic screen revealed that Bicc1 interacts with the C-Terminal to Lis-Homology domain (CTLH) complex. Since the orthologous Gid complex in S. cerevisae targets FBP1 and PEPCK for degradation, we mapped the topology among CTLH subunits and found that SAM-mediated binding controls Bicc1 protein levels, whereas Bicc1 inhibited the accumulation of several CTLH subunits. Under the conditions analyzed, Bicc1 increased FBP1 protein levels independently of the CTLH complex. Besides linking Bicc1 to cell metabolism, our findings reveal new layers of complexity in the regulation of renal gluconeogenesis compared to lower eukaryotes.

Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate.

In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent.

SF3B1 mutations provide genetic vulnerability to copper ionophores in human acute myeloid leukemia.

In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.

The role of pore size and mechanical properties on the accumulation, retention and distribution of F98 glioblastoma cells in macroporous hydrogels.

Glioblastoma (GBM) accounts for half of all central nervous system tumors. Once the tumor is removed, many GBM cells remain present near the surgical cavity and infiltrate the brain up to a distance of 20-30 mm, resulting in recurrence a few months later. GBM remains incurable due to the limited efficiency of current treatments, a result of the blood-brain barrier and sensitivity of healthy brain tissues to chemotherapy and radiation. A new therapeutic paradigm under development to treat GBM is to attract and accumulate GBM cells in a cancer cell trap inserted in the surgical cavity after tumor resection. In this work, porous gels were prepared using porous polylactide molds obtained from melt-processed co-continuous polymer blends of polystyrene and polylactide, with an average pore size ranging from 5 µm to over 500 µm. In order to efficiently accumulate and retain GBM brain cancer cells within a macroporous sodium alginate-based hydrogel trap, the pores must have an average diameter superior to 100 µm, with the best results obtained at 225 µm. In that case, the accumulation and retention of F98 GBM cells were more homogeneous, especially when functionalized with RGD adhesion peptides. At an alginate concentration of 1% w/v, the compression modulus reaches 15 kPa, close to the average value of 1-2 kPa reported for brain tissues, while adhesion and retention were also superior compared to 2% w/v gels. Overall, 1% w/v gels with 225 µm pores functionalized with the RGD peptide display the best performances.

Genome-wide interrogation of Mammalian stem cell fate determinants by nested chromosome deletions.

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP-based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.

Antagonistic interactions among structured domains in the multivalent Bicc1-ANKS3-ANKS6 protein network govern phase transitioning of target mRNAs.

The growing number of diseases linked to aberrant phase transitioning of ribonucleoproteins highlights the need to uncover how the interplay between multivalent protein and RNA interactions is regulated. Cytoplasmic granules of the RNA binding protein Bicaudal-C (Bicc1) are regulated by the ciliopathy proteins ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6, but whether and how target mRNAs are affected is unknown. Here, we show that head-to-tail polymers of Bicc1 nucleated by its SAM domain are interconnected by K homology (KH) domains in a protein meshwork that mediates liquid-to-gel transitioning of client transcripts. Moreover, while the dispersion of these granules by ANKS3 concomitantly released bound mRNAs, co-recruitment of ANKS6 by ANKS3 reinstated Bicc1 condensation and ribonucleoparticle assembly. RNA-independent Bicc1 polymerization and its dual regulation by ANKS3 and ANKS6 represent a new mechanism to couple the reversible immobilization of client mRNAs to controlled protein phase transitioning between distinct metastable states.

Comparison between systemic analgesia, continuous wound catheter analgesia and continuous thoracic paravertebral block: a randomised, controlled trial of postthoracotomy pain management.

CONTEXT: Thoracotomy is the surgical procedure that creates the greatest demand for postoperative analgesia. OBJECTIVE: We aimed to compare the efficacy of systemic analgesia, continuous wound catheter (CWC) analgesia and thoracic paravertebral block (TPVB) for pain management after thoracotomy, assessed by Visual Analogue Scale (VAS) pain score and morphine consumption. DESIGN: Prospective, randomised study. SETTING: University teaching hospital. Inclusions from April 2007 to February 2010. PATIENTS: 153 adult patients scheduled for pulmonary surgery. INTERVENTIONS: All three groups received systemic analgesia with paracetamol and morphine (patient-controlled analgesia, PCA). The PCA group received systemic analgesia only. The TPVB group underwent insertion of a paravertebral catheter and the CWC group underwent CWC catheter insertion at the end of the intervention. MAIN OUTCOME MEASURES: Pain score at rest as assessed by VAS. RESULTS: One hundred and fifty-three patients were included, of whom 140 were included in the final analysis (50 PCA, 44 TPVB, 46 CWC). Baseline and surgical characteristics were comparable in the three groups. VAS scores were statistically different between the TPVB and PCA groups at rest (at 0, 1, 3, 6 h; P < 0.0026) and after coughing (0, 1, 3, 6, 12 h; P < 0.003). In recovery room care, titrated morphine doses were significantly lower (P = 0.00001) in the TPVB group than in the other two. Morphine consumption was statistically lower in the TPVB group than in the PCA group at 24 h (P = 0.0036). There was no difference between CWC and PCA groups in terms of VAS scores or morphine consumption. No signs of toxicity or local complications were observed. CONCLUSION: Our results support the efficacy of TPVB for pain management after thoracotomy, at rest and after coughing. These results confirm the preference for TPVB over epidural analgesia in postthoracotomy pain care. CWC failed to decrease pain and morphine consumption and performed no better than placebo.

Effect of Chitosan on Alginate-Based Macroporous Hydrogels for the Capture of Glioblastoma Cancer Cells.

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction.

We demonstrate that the secretome of mesenchymal stromal cells (MSCs) suppresses plasma cell (PC) immunoglobulin (Ig) production, induces plasmablast proliferation, and leads to interleukin-10-mediated blockade in vitro. We found that these effects are the result of MSC-derived CC chemokine ligands CCL2 and CCL7. More specifically, MSCs further processed these CC chemokines by the activity of matrix metalloproteinases (MMPs), leading to the generation of proteolytically processed antagonistic CCL2 variant. Neutralizing CCL2 or inhibiting MMP enzymatic activity abolished the PC-suppressive effect of MSCs. We also observed that MMP-processed CCL2 suppresses signal transducer and activator of transcription 3 (STAT3) activation in PC. As a result, the transcription factor PAX5 is induced, thus explaining the inhibition of Ig synthesis. The absence of inhibitory effects by MSC on the humoral response of CCR2(-/-) mice to xenoantigen suggests that MMP-cleaved CCL2/CCR2 interaction as well as downstream phosphatase activity is necessary for antagonistic effect. We tested syngeneic MSCs in hemophilic B6 mice with predeveloped antihuman factor VIII (hFVIII) antibodies and demonstrated a robust decrease in hFVIII-specific IgG levels. Thus, MSCs may play a role in modulating Ig production by PCs via MMP processing of CCL2 and may represent an appealing cell therapy approach for pathologic humoral responses.

Tetra- and hexavalent mannosides inhibit the pro-apoptotic, antiproliferative and cell surface clustering effects of concanavalin-A: impact on MT1-MMP functions in marrow-derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) mobilization and recruitment by experimental vascularizing tumors involves membrane type 1-matrix metalloproteinase (MT1-MMP) functions. Given that the mannose-specific lectin Concanavalin-A (ConA) induces MT1-MMP expression and mimics biological lectins/carbohydrate interactions, we synthesized and tested the potential of 11 mannoside clusters to block ConA activities on MSC. We found that tetra- and hexavalent mannosides reversed ConA-mediated changes in MSC morphology and antagonized ConA-induced caspase-3 activity and proMMP-2 activation. Tetra- and hexavalent mannosides also inhibited ConA- but not the cytoskeleton disrupting agent Cytochalasin-d-induced MT1-MMP cell surface proteolytic processing mechanisms, and effects on cell cycle phase progression. The antiproliferative and pro-apoptotic impact of ConA on the MT1-MMP/glucose-6-phosphate transporter signaling axis was also reversed by these mannosides. In conclusion, we designed and identified glycocluster constructions that efficiently interfered with carbohydrate-binding proteins (lectins) interaction with oligosaccharide moieties of glycoproteins at the cell surface of MSC. These glycoclusters may serve in carbohydrate-based anticancer strategies through their ability to specifically target MT1-MMP pleiotropic functions in cell survival, proliferation, and extracellular matrix degradation.

Silencing of the MT1-MMP/ G6PT axis suppresses calcium mobilization by sphingosine-1-phosphate in glioblastoma cells.

The contributions of membrane type-1 matrix metalloproteinase (MT1-MMP) and of the glucose-6-phosphate transporter (G6PT) in sphingosine-1-phosphate (S1P)-mediated Ca(2+) mobilization were assessed in glioblastoma cells. We show that gene silencing of MT1-MMP or G6PT decreased the extent of S1P-induced Ca(2+) mobilization, chemotaxis, and extracellular signal-related kinase phosphorylation. Chlorogenic acid and (-)-epigallocatechin-3-gallate, two diet-derived inhibitors of G6PT and of MT1-MMP, respectively, reduced S1P-mediated Ca(2+) mobilization. An intact MT1-MMP/G6PT signaling axis is thus required for efficient Ca(2+) mobilization in response to bioactive lipids such as S1P. Targeted inhibition of either MT1-MMP or G6PT may lead to reduced infiltrative and invasive properties of brain tumor cells.

Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6.

Head-to-tail polymers of sterile alpha motifs (SAM) can scaffold large macromolecular complexes. Several SAM-domain proteins that bind each other are mutated in patients with cystic kidneys or laterality defects, including the Ankyrin (ANK) and SAM domain-containing proteins ANKS6 and ANKS3, and the RNA-binding protein Bicc1. To address how their interactions are regulated, we first determined a high-resolution crystal structure of a Bicc1-SAM polymer, revealing a canonical SAM polymer with a high degree of flexibility in the subunit interface orientations. We further mapped interactions between full-length and distinct domains of Bicc1, ANKS3, and ANKS6. Neither ANKS3 nor ANKS6 alone formed macroscopic homopolymers in vivo. However, ANKS3 recruited ANKS6 to Bicc1, and the three proteins together cooperatively generated giant macromolecular complexes. Thus, the giant assemblies are shaped by SAM domains, their flanking sequences, and SAM-independent protein-protein and protein-mRNA interactions.

Effect of tetrahydrobiopterin on selective endothelial dysfunction of epicardial porcine coronary arteries induced by cardiopulmonary bypass.

BACKGROUND: We hypothesized that cardiopulmonary bypass induces a selective alteration of the coronary arterial endothelial cell signal transduction which could be explained by a state of depletion and/or decreased activity of endogenous tetrahydrobiopterin (BH(4)). The aim of this study was to assess the effects of cardiopulmonary bypass and BH(4) on the endothelial function of epicardial coronary arteries in a swine model of cardiopulmonary bypass. METHODS: Swine underwent 90 min of cardiopulmonary bypass alone (N=19) or in association with a brief cardioplegic arrest with (N = 6) or without (N = 5) in vivo BH(4) administration, followed by a 60-min period following weaning from cardiopulmonary bypass and were compared to a control group (N = 7). Endothelium-dependent relaxations of epicardial coronary artery rings were studied using standard organ chamber experiments in the presence or absence of in vitro BH(4) or superoxide dismutase (SOD) and catalase. RESULTS: Cardiopulmonary bypass caused a statistically significant reduction of endothelium-dependent relaxations to serotonin (p < 0.0001), bradykinin (p < 0.001), UK14304 (p < 0.0001) and calcium ionophore (p < 0.01) in epicardial porcine coronary arteries. In vitro and in vivo BH(4) supplementation improved endothelium-dependent relaxations to serotonin and bradykinin, which were left unchanged by SOD-catalase administration. Cardiopulmonary bypass was associated with a decrease in nitric oxide availability (p = 0.002) and increased oxidative stress (p < 0.001), which were both restored by in vivo BH(4) administration (p < 0.001). CONCLUSION: Treatment with BH(4) improves the endothelial dysfunction of porcine epicardial coronary arteries, restores nitric oxide availability and reduces the oxidative stress associated with cardiopulmonary bypass.

Reduced mortality and strokes with off-pump coronary artery bypass grafting surgery in octogenarians.

BACKGROUND: Off-pump coronary artery bypass surgery (OPCAB) has been revived and has gained popularity, although the exact subsets of patients who might benefit most from this technique are unknown. The aim of this retrospective study was to compare the results of coronary artery bypass grafting surgery (CABG) in octogenarians using cardiopulmonary bypass (CPB) or OPCAB techniques. METHODS AND RESULTS: Over a 5-year period (1995-1999), 125 patients older than 80 years of age were operated for isolated myocardial revascularization (63 using CPB and 62 with OPCAB). There was no statistically significant difference in preoperative comorbidities between groups or in mean left ventricular ejection fraction (54.5+/-15.3% in the CPB group and 50.9+/-13.5% in the OPCAB group, respectively). The mean number of distal anastomosis per patient was 2.9 in CPB group and 2.6 in OPCAB group (P=ns). The majority of patients in both groups had unstable angina and were operated on an urgent basis. The operative mortality was 15.9% in the CPB group and 4.8% in the OPCAB group (P=0.04). There were 4 postoperative strokes (6.3%) in the CPB group and none (0%) in the OPCAB group (P=0.04). The percentage of patients transfused was 92.1% in the CPB group and 72.6% in the OPCAB group (P<0.01). Postoperative myocardial infarction occurred in 11.3% in the CPB group and 14.5% in the OPCAB group (P=NS). For all the parameters entered in the multivariate analysis with logistic regression model, the type of surgery (CPB or OPCAB) was an independent predictor of operative mortality and stroke (P=0.0375). The odds ratio (OR) indicates that operative mortality and stroke occur 4 times (OR=4.171) more often in CPB patients than in OPCAB patients. Follow-up showed no significant difference between the 2 groups in terms of cardiac events and mortality. CONCLUSIONS: This retrospective study suggests a benefit of OPCAB in terms of operative mortality and stroke for octogenarian patients when compared with CPB in our institution.

Influence of intracoronary shunt size on coronary endothelial function during off-pump coronary artery bypass.

OBJECTIVE: The insertion of intracoronary shunts during off-pump coronary artery bypass surgery may induce severe endothelial dysfunction in the target arteries. The purpose of this study was to determine the role of the ratio of the shunt diameter to the target artery diameter in the development of endothelial dysfunction and to develop guidelines for selecting the most appropriate shunt size. METHODS: Nine swine (25 +/- 4 kg) were used for this study. Three ClearView shunts 3 mm, 2 mm, and 1.25 mm in diameter were inserted proximally to distally via 3 arteriotomies into the right coronary artery to obtain oversizing with the 3-mm shunt, undersizing with the 1.25-mm shunt, and a satisfying congruence with the 2-mm shunt. The shunts were left in place for 15 minutes, and the levels of bleeding were quantified. Coronary rings upstream and downstream from the arteriotomies were then harvested and placed in organ chambers to study endothelium-dependent relaxations to serotonin and bradykinin. Noninstrumented coronary arteries served as controls. RESULTS: A decrease of endothelium-dependent relaxation occurred with the 3-mm shunts (P <.005), which were associated with an adequate hemostasis. A decrease of endotheliumdependent relaxation occurred with the 2-mm shunts (P <.05), which were associated with intermittent bleeding, and no significant decrease of endothelium-dependent relaxation occurred with 1.25-mm shunts (P >.05), which were associated with continuous bleeding. CONCLUSIONS: Intracoronary shunts are associated with different disadvantages, depending on the mismatch to the target coronary artery, and whatever their size, shunts are not the ideal device for safely obtaining a satisfactory hemostasis. These results support our clinical attitude of the selective use of intracoronary shunts.

Interrupted coalescent nitinol clip versus continuous suture coronary anastomosis: a comparative endothelial function study.

BACKGROUND: A new penetrating stapled anastomotic system using nitinol microclips (Coalescent Surgical U-Clip) has been developed to facilitate the construction of compliant interrupted sutures for minimally invasive and robotic surgery as well as for conventional procedures. The purpose of this study was to determine the effect of nitinol U-Clips used for coronary anastomosis in the development of endothelial dysfunction, compared with conventional running sutures. METHODS: In a porcine model, both internal thoracic arteries were harvested, and the heart was removed. In a Krebs solution, 2 anastomoses were realized ex vivo between internal thoracic arteries and the left anterior descending artery. One was carried out with 12 Coalescent microclips, and the other used conventional running 7-0 polypropylene suture material (Prolene). Coronary rings on the anastomotic sites were then placed in organ chambers filled with oxygenated Krebs solution. Vascular reactivity studies were performed in standard organ chamber experiments. After the contraction of the coronary arteries in response to prostaglandin F2alpha, the endothelium-dependent relaxation response to bradykinin was studied. The other coronary arteries served as controls. RESULTS: There was no statistically significant difference among the groups (P >.05) in the amplitude of the contraction response to KCl and prostaglandin F2alpha. There were no statistically significant differences in endothelium-dependent relaxation response to bradykinin between the nitinol microclip group and controls, between the suture group and controls, and between the nitinol microclip and suture groups. CONCLUSIONS: Coalescent nitinol U-Clips used as anastomotic devices do not induce an endothelial dysfunction and allow a compliant anastomosis under satisfactory conditions.