Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors

Sensitivity and specificity of blood-fluid levels for oral anticoagulant-associated intracerebral haemorrhage

Intracerebral haemorrhage (ICH) is a life-threatening emergency, the incidence of which has increased in part due to an increase in the use of oral anticoagulants. A blood-fluid level within the haematoma, as revealed by computed tomography (CT), has been suggested as a marker for oral anticoagulant-associated ICH (OAC-ICH), but the diagnostic specificity and prognostic value of this finding remains unclear. In 855 patients with CT-confirmed acute ICH scanned within 48 h of symptom onset, we investigated the sensitivity and specificity of the presence of a CT-defined blood-fluid level (rated blinded to anticoagulant status) for identifying concomitant anticoagulant use. We also investigated the association of the presence of a blood-fluid level with six-month case fatality. Eighteen patients (2.1%) had a blood-fluid level identified on CT; of those with a blood-fluid level, 15 (83.3%) were taking anticoagulants. The specificity of blood-fluid level for OAC-ICH was 99.4%; the sensitivity was 4.2%. We could not detect an association between the presence of a blood-fluid level and an increased risk of death at six months (OR = 1.21, 95% CI 0.28–3.88, p = 0.769). The presence of a blood-fluid level should alert clinicians to the possibility of OAC-ICH, but absence of a blood-fluid level is not useful in excluding OAC-ICH

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Absence of Ca2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory

Ca2+-stimulated adenylyl cyclase (AC) 1 and 8 are two genes that have been shown to play critical roles in fear memory. AC1 and AC8 couple neuronal activity and intracellular Ca2+ increases to the production of cyclic adenosine monophosphate and are localized synaptically, suggesting that Ca2+-stimulated ACs may modulate synaptic plasticity. Here, we first established that Ca2+-stimulated ACs modulate protein markers of synaptic activity at baseline and after learning. Primary hippocampal cell cultures showed that AC1/AC8 double-knockout (DKO) mice have reduced SV2, a synaptic vesicle protein, abundance along their dendritic processes, and this reduction can be rescued through lentivirus delivery of AC8 to the DKO cells. Additionally, phospho-synapsin, a protein implicated in the regulation of neurotransmitter release at the synapse, is decreased in vivo 1 h after conditioned fear (CF) training in DKO mice. Importantly, additional experiments showed that long-term potentiation deficits present in DKO mice are rescued by acutely replacing AC8 in the forebrain, further supporting the idea that Ca2+-stimulated AC activity is a crucial modulator of synaptic plasticity. Previous studies have demonstrated that memory is continually modulated by gene–environment interactions. The last set of experiments evaluated the effects of knocking out AC1 and AC8 genes on experience-dependent changes in CF memory. We showed that the strength of CF memory in wild-type mice is determined by previous environment, minimal or enriched, whereas memory in DKO mice is unaffected. Thus, overall these results show that AC1 and AC8 modulate markers of synaptic activity and help integrate environmental information to modulate fear memory

The Probable Cell of Origin of NF1- and PDGF-Driven Glioblastomas

Primary glioblastomas are subdivided into several molecular subtypes. There is an ongoing debate over the cell of origin for these tumor types where some suggest a progenitor while others argue for a stem cell origin. Even within the same molecular subgroup, and using lineage tracing in mouse models, different groups have reached different conclusions. We addressed this problem from a combined mathematical modeling and experimental standpoint. We designed a novel mathematical framework to identify the most likely cells of origin of two glioma subtypes. Our mathematical model of the unperturbed in vivo system predicts that if a genetic event contributing to tumor initiation imparts symmetric self-renewing cell division (such as PDGF overexpression), then the cell of origin is a transit amplifier. Otherwise, the initiating mutations arise in stem cells. The mathematical framework was validated with the RCAS/tv-a system of somatic gene transfer in mice. We demonstrated that PDGF-induced gliomas can be derived from GFAP-expressing cells of the subventricular zone or the cortex (reactive astrocytes), thus validating the predictions of our mathematical model. This interdisciplinary approach allowed us to determine the likelihood that individual cell types serve as the cells of origin of gliomas in an unperturbed system

Factors associated with paradoxical immune response to antiretroviral therapy in HIV infected patients: a case control study

<p>Abstract</p> <p>Background</p> <p>A paradoxical immunologic response (PIR) to Highly Active Antiretroviral Therapy (HAART), defined as viral suppression without CD4 cell-count improvement, has been reported in the literature as 8 to 42%, around 15% in most instances. The present study aims to determine, in a cohort of HIV infected patients in Brazil, what factors were independently associated with such a discordant response to HAART.</p> <p>Methods</p> <p>A case-control study (1:4) matched by gender was conducted among 934 HIV infected patients on HAART in Brazil. Cases: patients with PIR, defined as CD4 < 350 cells/mm³(hazard ratio for AIDS or death of at least 8.5) and undetectable HIV viral load on HAART for at least one year. Controls: similar to cases, but with CD4 counts ≥ 350 cells/mm³. Eligibility criteria were applied. Data were collected from medical records using a standardized form. Variables were introduced in a hierarchical logistic regression model if a p-value < 0.1 was determined in a bivariate analysis.</p> <p>Results</p> <p>Among 934 patients, 39 cases and 160 controls were consecutively selected. Factors associated with PIR in the logistic regression model were: total time in use of HAART (OR 0.981; CI 95%: 0.96-0.99), nadir CD4-count (OR 0.985; CI 95%: 0.97-0.99), and time of undetectable HIV viral load (OR 0.969; CI 95%: 0.94-0.99).</p> <p>Conclusions</p> <p>PIR seems to be related to a delay in the management of immunodeficient patients, as shown by its negative association with nadir CD4-count. Strategies should be implemented to avoid such a delay and improve the adherence to HAART as a way to implement concordant responses.</p

Incident Tuberculosis during Antiretroviral Therapy Contributes to Suboptimal Immune Reconstitution in a Large Urban HIV Clinic in Sub-Saharan Africa

Antiretroviral therapy (ART) effectively decreases tuberculosis (TB) incidence long-term, but is associated with high TB incidence rates in the first 6 months. We sought to determine the incidence and the long-term effects of TB during ART on HIV treatment outcome, and the risk factors for incident TB during ART in a large urban HIV clinic in Uganda.Routinely collected longitudinal clinical data from all patients initiated on first-line ART was retrospectively analysed. 5,982 patients were included with a median baseline CD4+ T cell count (CD4 count) of 117 cells/mm(3) (interquartile range [IQR]; 42, 182). In the first 2 years, there were 336 (5.6%) incident TB events in 10,710 person-years (py) of follow-up (3.14 cases/100 pyar [95% CI 2.82-3.49]); incidence rates at 0-3, 3-6, 6-12 and 12-24 months were 11.25 (9.58-13.21), 6.27 (4.99-7.87), 2.47 (1.87-3.36) and 1.02 (0.80-1.31), respectively. Incident TB during ART was independently associated with baseline CD4 count of <50 cells/mm(3) (hazard ratio [HR] 1.84 [1.25-2.70], P = 0.002) and male gender (HR 1.68 [1.34-2.11], P<0.001). After two years on ART, the patients who had developed TB in the first 12 months had a significantly lower median CD4 count increase (184 cells/mm(3) [IQR; 107, 258, n = 118] vs 209 cells/mm(3) [124, 309, n = 2166], P = 0.01), a larger proportion of suboptimal immune reconstitution according to two definitions (increase in CD4 count <200 cells/mm(3): 57.4% vs 46.9%, P = 0.03, and absolute CD4 count <200 cells/mm(3): 30.4 vs 19.9%, P = 0.006), and a higher percentage of immunological failure according to the WHO criteria (13.6% vs 6.5%, P = 0.003). Incident TB during ART was independently associated with poor CD4 count recovery and fulfilling WHO immunological failure definitions.Incident TB during ART occurs most often within 3 months and in patients with CD4 counts less than 50 cells/mm(3). Incident TB during ART is associated with long-term impairment in immune recovery

A Novel Function of DELTA-NOTCH Signalling Mediates the Transition from Proliferation to Neurogenesis in Neural Progenitor Cells

A complete account of the whole developmental process of neurogenesis involves understanding a number of complex underlying molecular processes. Among them, those that govern the crucial transition from proliferative (self-replicating) to neurogenic neural progenitor (NP) cells remain largely unknown. Due to its sequential rostro-caudal gradients of proliferation and neurogenesis, the prospective spinal cord of the chick embryo is a good experimental system to study this issue. We report that the NOTCH ligand DELTA-1 is expressed in scattered cycling NP cells in the prospective chick spinal cord preceding the onset of neurogenesis. These Delta-1-expressing progenitors are placed in between the proliferating caudal neural plate (stem zone) and the rostral neurogenic zone (NZ) where neurons are born. Thus, these Delta-1-expressing progenitors define a proliferation to neurogenesis transition zone (PNTZ). Gain and loss of function experiments carried by electroporation demonstrate that the expression of Delta-1 in individual progenitors of the PNTZ is necessary and sufficient to induce neuronal generation. The activation of NOTCH signalling by DELTA-1 in the adjacent progenitors inhibits neurogenesis and is required to maintain proliferation. However, rather than inducing cell cycle exit and neuronal differentiation by a typical lateral inhibition mechanism as in the NZ, DELTA-1/NOTCH signalling functions in a distinct manner in the PNTZ. Thus, the inhibition of NOTCH signalling arrests proliferation but it is not sufficient to elicit neuronal differentiation. Moreover, after the expression of Delta-1 PNTZ NP continue cycling and induce the expression of Tis21, a gene that is upregulated in neurogenic progenitors, before generating neurons. Together, these experiments unravel a novel function of DELTA–NOTCH signalling that regulates the transition from proliferation to neurogenesis in NP cells. We hypothesize that this novel function is evolutionary conserved