Peroxide-doped apatites: Preparation and effect of synthesis parameters

This contribution focused on the preparation of peroxide-doped calciumphosphate apatites—in viewof potential uses as bioactive bioceramics with antimicrobial functions, and on their main physico-chemical characteristics. Two synthesis routeswere investigated. First, the hydrolysis of β-TCP in the presence ofH2O2 was followed. However, only elevated concentrations inH2O2 in themediumor temperatures around 150 °C allowed us to reach the complete β-TCP-to-apatite hydrolysis process, and the obtained samples exhibited a high crystallinity state with no non-apatitic chemical environments. The second protocol tested consisted in the direct apatite precipitation in the presence of H2O2 in themedium (at room temperature). This protocol led to single-phased nanocrystalline apatites, and our data indicate that part of the apatitic OH− ions were substituted by oxygenated species, and typically by peroxide ions (quantified). Physico-chemical modifications in the form of an improvement in crystallinity state, an increase in unit cell volume, and the presence of additional Raman bands were noticed and discussed

Nanocrystalline apatites: From powders to biomaterials

Non-stoichiometric nanocrystalline apatite powders are used to elaborate highly-bioactive biomaterials. Their exceptional surface reactivity arises from a structured but rather unstable hydrated layer involving ions in nonapatitic chemical environments, like in bone mineral. The initial powder characteristics can be tailored through precipitation parameters (pH, temperature, maturation time in solution). The drying of nanocrystalline apatite suspensions at very low temperature (4 °C) leads to ceramic-like materials exhibiting average mechanical properties (compressive strength 54 MPa) and a high porosity which could be exploited to entrap active organic compounds (e.g. growth factors). The consolidation at 150–200 °C of nanocrystalline apatite powders has also been studied using uni-axial pressing and spark plasma sintering (SPS). The results indicate only a limited alteration of the initial nanocrystals, and the bioceramics obtained show mechanical properties close to those reached with sintered stoichiometric HA. The high ion mobility in the hydrated layer of the nanocrystals can lead to “crystal fusion” processes. This capability to favor crystal–crystal interactions at low temperature, while preserving the non-stoichiometry and nanometer dimensions of apatite crystals, opens interesting perspectives for the elaboration of new resorbable and highly-bioactive bioceramics

STOP Proteins are Responsible for the High Degree of Microtubule Stabilization Observed in Neuronal Cells

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation

Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin: Microtubule and calmodulin binding on Mn modules of MAP6

International audienceMicrotubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin

MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.: Microtubule stabilization by MAP6

International audienceMicrotubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II.: Phosphorylation of STOP by CaMKII

International audienceSTOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process

Evidence for new C-terminally truncated variants of α- and β-tubulins

New C-terminally truncated α- and β-tubulin variants, both ending with an -EEEG sequence, are identified in vivo: αΔ3-tubulin, which has a specific neuronal distribution pattern (distinct from that of αΔ2-tubulin) and seems to be related to dynamic microtubules, and βΔ4-tubulin, corresponding to β2A/B-tubulin modified by truncation of four C-terminal residues, which is ubiquitously present in cells and tissues. Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development

2020-05-22 DAILY UNM GLOBAL HEALTH COVID-19 BRIEFING

Executive Summary: NM Highlights: 12 positive cases at NMSU. Short-term rentals restrictions. Dine-in reopening. US Highlights: Models to forecast COVID-19 deaths. Places of worship reopening. Contact tracing at University of Alabama. International Highlights: More deaths among poor. Mortality data in Italy. High death toll in Brazil. Global vaccines fall behind. Italy cases drop. Vaccines to go through India. Epidemiology Highlights: Children not pandemic drivers. Healthcare Policy Recommendations: Burnout of HCWs. NYC surgeons’ response. Foggy glasses tips. Practice Guidelines: Recommendations are provided on surgical strategies during COVID-19, management of hypertension, anesthesia practice, avoiding drug-induced cardiovascular impairments, arrhythmia management, elective surgery reboot, nutritional management, stroke care, neurointerventional surgery, blood management. Testing: Unregulated tests identified. Interpreting test results. Drugs, Vaccines, Therapies, Clinical Trials: Vaccine trials results. Vaccine moves to trials. Monoclonal antibody identified. Hydroxychloroquine no benefit. Adjuvant corticosteroid therapy. Old drugs may benefit. Vaccines inducing antibodies. Other Science: Mortality and CKD/liver disease. Transmission in pregnancy. Pathophysiology insights. Readmission characteristics. Sodium and severity. Simulated sunlight disinfects

Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology