Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.

Single-cell transcriptomics of the early developing mouse cerebral cortex disentangle the spatial and temporal components of neuronal fate acquisition.

In the developing cerebral cortex, how progenitors that seemingly display limited diversity end up producing a vast array of neurons remains a puzzling question. The prevailing model suggests that temporal maturation of progenitors is a key driver in the diversification of the neuronal output. However, temporal constraints are unlikely to account for all diversity, especially in the ventral and lateral pallium where neuronal types significantly differ from their dorsal neocortical counterparts born at the same time. In this study, we implemented single-cell RNAseq to sample the diversity of progenitors and neurons along the dorso-ventral axis of the early developing pallium. We first identified neuronal types, mapped them on the tissue and determined their origin through genetic tracing. We characterised progenitor diversity and disentangled the gene modules underlying temporal versus spatial regulations of neuronal specification. Finally, we reconstructed the developmental trajectories followed by ventral and dorsal pallial neurons to identify lineage-specific gene waves. Our data suggest a model by which discrete neuronal fate acquisition from a continuous gradient of progenitors results from the superimposition of spatial information and temporal maturation.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

A Localized Scaffold for cGMP Increase Is Required for Apical Dendrite Development.

Studies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-ß1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development. Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.

Freedom from pulmonary vein stenosis after multiple applications of epicardial ablation energy.

OBJECTIVES: In patients undergoing cardiac surgical procedures, pulmonary vein isolation may be easily accomplished, and it is important to achieve bidirectional conduction block across created lesions. The primary aim of this study was to assess the risk of pulmonary vein stenosis (PVS) after multiple applications of epicardial bipolar radiofrequency energy. METHODS: Thirty-five consecutive patients who were referred for off-pump coronary revascularization with concomitant pulmonary vein isolation and left atrial appendage occlusion were prospectively included in the study. The ablation protocol provided 8 standard epicardial applications of bipolar energy with additional applications until the acute bidirectional conduction block was achieved. Three to 6 months after surgery, patients underwent computed tomography to assess PVS. RESULTS: In all patients, bidirectional conduction block was achieved across the created lesions. In 31 (89%) patients, conduction block was accomplished after the standard 8 energy applications on each side. In 4 (11%) patients, additional applications of energy were needed. All patients had computed tomography (128 total pulmonary veins) scans, which showed no evidence of PVS. CONCLUSIONS: Multiple applications of bipolar radiofrequency energy during off-pump epicardial pulmonary vein isolation did not lead to PVS. Creating bidirectional conduction block using multiple energy applications through created lesions is feasible in all patients using the ablation protocol described.

Intraoperative epicardial focal pulmonary venous electrocardiography in patients with atrial fibrillation.

OBJECTIVES: In patients referred to off-pump coronary artery bypass grafting, pulmonary vein isolation (PVI) may be used for those with persistent atrial fibrillation (AF), an alternative to the Maze procedure. However, the success rate of PVI in persistent AF is limited. The study assesses the prognostic value of focal epicardial electrocardiography of the pulmonary veins (PVs) for surgical ablation results. METHODS: We mapped 140 PV in 35 cases undergoing off-pump coronary artery bypass grafting. Data obtained using a sensing-pacing probe before ablation were analysed. The composite study end-point consisted of the need for electrical cardioversion for in-hospital recurrence of AF and the presence of AF at hospital discharge and after 6 months follow-up confirmed by 24-h Holter electrocardiographic monitoring. RESULTS: In patients with epicardial far-field (FF) signals recorded over at least 1 PV, the composite end-point occurred in 61% (14) vs 25% (3) of patients with no FF signal recorded over any PV (P = 0.04). The presence of FF signals in at least 1 PV significantly increased the risk of composite end-point occurrence (odds ratio 3; P = 0.04). The composite end-point occurred in 86% (6) of patients with FF signals recorded over all PVs and in 39% (11) in the remainder of the study population (P = 0.03). CONCLUSIONS: Intraoperative epicardial focal electrocardiography of PVs revealed more than 40% of PVs had only FF atrial signals. The presence of FF signals in PVs is related to a lower early effectiveness of PVI on ablating AF. Epicardial focal electrocardiography of PVs may be a clinically effective intraoperative tool in the decision-making process between less invasive PVI and the standard Maze procedure.

Left Atrial Ganglionated Plexi Detection is Related to Heart Rate and Early Recurrence of Atrial Fibrillation after Surgical Ablation.

INTRODUCTION:: Left atrial ganglionated plexi ablation is an adjuvant technique used to increase the success rate of surgical ablation of atrial fibrillation. Ganglionated plexi ablation requires previous detection. We aimed to assess determinants of successful ganglionated plexi detection and to correlate range of ganglionated plexi ablation with risk of early atrial fibrillation recurrence. METHODS:: The study involved 34 consecutive patients referred for surgical coronary revascularization with concomitant atrial fibrillation ablation. Ganglionated plexi detection was done by inducing vagal reflexes in the area of the pulmonary veins and left atrial fat pads. RESULTS:: Detection of GP was successful in 85% of the patients. There was no difference in preoperative characteristics nor in atrial fibrillation type between patients in whom ganglionated plexi detection was successful and others. The number of detected ganglionated plexi correlated significantly only with preoperative resting heart rate. Significant negative correlation was found in patients with preoperative heart rate>75 beat/min in terms of total number of detected ganglionated plexi (P=0.04). Average number of detected ganglionated plexi was significantly higher in patients with in-hospital atrial fibrillation recurrence requiring electrical cardioversion (3.8±3) in comparison to rest of the study population (2±1.3; P=0.02). In patients in whom 4 or more ganglionated plexi were detected, significantly increased risk of in-hospital atrial fibrillation recurrence was observed (OR 15; 95% CI 1.5-164; P=0.003). CONCLUSION:: Left atrial ganglionated plexi detection was unsuccessful in a considerable percentage of patients. Preoperative heart rate significantly influenced positive ganglionated plexi detection and number of ablated ganglia. Higher number of detected ganglionated plexi was related with early recurrence of atrial fibrillation.

Left atrial ganglionated plexi detection is related to heart rate and early recurrence of atrial fibrillation after surgical ablation

Abstract INTRODUCTION: Left atrial ganglionated plexi ablation is an adjuvant technique used to increase the success rate of surgical ablation of atrial fibrillation. Ganglionated plexi ablation requires previous detection. We aimed to assess determinants of successful ganglionated plexi detection and to correlate range of ganglionated plexi ablation with risk of early atrial fibrillation recurrence. METHODS: The study involved 34 consecutive patients referred for surgical coronary revascularization with concomitant atrial fibrillation ablation. Ganglionated plexi detection was done by inducing vagal reflexes in the area of the pulmonary veins and left atrial fat pads. RESULTS: Detection of GP was successful in 85% of the patients. There was no difference in preoperative characteristics nor in atrial fibrillation type between patients in whom ganglionated plexi detection was successful and others. The number of detected ganglionated plexi correlated significantly only with preoperative resting heart rate. Significant negative correlation was found in patients with preoperative heart rate>75 beat/min in terms of total number of detected ganglionated plexi (P=0.04). Average number of detected ganglionated plexi was significantly higher in patients with in-hospital atrial fibrillation recurrence requiring electrical cardioversion (3.8±3) in comparison to rest of the study population (2±1.3; P=0.02). In patients in whom 4 or more ganglionated plexi were detected, significantly increased risk of in-hospital atrial fibrillation recurrence was observed (OR 15; 95% CI 1.5-164; P=0.003). CONCLUSION: Left atrial ganglionated plexi detection was unsuccessful in a considerable percentage of patients. Preoperative heart rate significantly influenced positive ganglionated plexi detection and number of ablated ganglia. Higher number of detected ganglionated plexi was related with early recurrence of atrial fibrillation.

Right atrium positioning for exposure of right pulmonary veins during off-pump atrial fibrillation ablation.

OBJECTIVES: Concomitant surgical ablation of atrial fibrillation (AF) is recommended for patients undergoing off-pump coronary revascularization in the presence of this arrhythmia. Achievement of optimal visualization of pulmonary veins while maintaining stable haemodynamic conditions is crucial for proper completion of the ablation procedure. This study evaluates the safety and feasibility of right atrial positioning using a suction-based cardiac positioner as opposed to compressive manoeuvres for exposure during off-pump surgical ablation for AF. METHODS: Thirty-four consecutive patients underwent pulmonary vein isolation, ganglionated plexi ablation and left atrial appendage occlusion during off-pump coronary artery bypass grafting. Right atrial suction positioning was used to visualize right pulmonary veins. Safety and feasibility end points were analysed intraoperatively and in the early postoperative course. RESULTS: In all patients, right atrial positioning created optimal conditions to complete transverse and oblique sinus blunt dissection, correct placement of a bipolar ablation probe, detection and ablation of ganglionated plexi and conduction block assessment. In all patients, this entire right-sided ablation procedure was completed with a single exposure manoeuvre. Feasibility end points were achieved in all study patients. CONCLUSIONS: This report documents the safety and feasibility of right atrial exposure using a suction-based cardiac positioner to complete ablation for AF concomitant with off-pump coronary revascularization. This technique may be widely adopted to create stable haemodynamic conditions and optimal visualization of the right pulmonary veins.

Comparison of safety of radial and femoral approaches for coronary catheterization in interventional cardiology.

BACKGROUND: The femoral approach has been the preferably used access in interventional cardiology as well for coronary diagnostics as for percutaneous coronary intervention, being perceived as easy and facilitating quick access with relatively low risk. Due to the results of the latest studies, however, the radial approach has become increasingly popular. The aim of this study was a safety analysis of cardiological interventional procedures (i.e., coronarography and PCI) according to the vessel approach. MATERIAL/METHODS: The 204 coronary interventions done in our Department of Interventional Cardiology were retrospectively analyzed. All the procedures were classified according to femoral or radial access. The incidence of local complications (e.g., major bleedings and hematomas) was assessed as well as the volume of contrast agent administered during the procedure and the radiation dose. RESULTS: It has been shown that radial approach, which is obviously more comfortable for patients, reduces the risk of local complications (0 vs. 2.97% and 0 vs. 3.96%) and does not lead to increased radiation exposure (p=0.88). However, there could be a larger volume of contrast agent administered (p=0.029), which in some cases could increase the risk of contrast-induced nephropathy. CONCLUSIONS: The radial approach should be recommended as a first choice because it is safer than the classical femoral approach, but one must be cautious in choosing radial approach patients with renal insufficiency.

Pulmonary artery aneurysm in an adult patient with idiopathic dilatation of the pulmonary artery.

Idiopathic dilatation of the pulmonary artery (IDPA) is a rare congenital heart disease. It has been described for almost one hundred years, and numerous definitions have been proposed. The IDPA diagnostic criteria have not been updated for years. Secondary to primary disease, pulmonary artery aneurism was recognised as a lethal defect; however, long-term follow-up of patients with IDPA has not been well researched. Thus, indications to medical or surgical treatment are not evidence based. Here, we present a rare case of a 54-year-old patient with IDPA, who remained under observation for 36 years without surgical intervention.

[New directions in the therapy of pulmonary arterial hypertension]. / Nowe kierunki w leczeniu tetniczego nadcisnienia plucnego.

Recent years are the time of dynamic development of pulmonary arterial pressure pharmacotherapy. By introducing the goal oriented therapy the survival in this group of patients has significantly increased. Apart from the pharmacotherapy used according to the ESC guidelines, new attempts of interventional treatment based on denervation of pulmonary artery have also been taken. Constantly, the new clinical trials (tests?) of drugs acting via new metabolic pathways have been conducted. They include for example: soluble guanylate cyclase stimulators, tyrosine kinase inhibitors, serotonin receptors inhibitors, Rhokinase inhibitors, VIP analogues. One of the newmedicines is riociguat, the effectiveness and safety of which have been confirmed in the PATENT and CHEST study. However, the small number and clinical diversity in the group of the PAH patients cause significant difficulties with the extrapolation of the results of trials according to the guidelines of the therapy.

[Reversible pulmonary hypertension as a consequence of dasatinib treatment in a patient with chronic myeloid leukemia and scleroderma]. / Odwracalne nadcisnienie plucne jako powiklanie leczenia dazatinibem pacjentki z przewleklq bialaczka szpikowa i twardzina ukladowa.

Pulmonary hypertension may be associated with many clinical conditions, including connective tissue diseases. We present a case of a patient with sclerodermia and chronic myeloid leukemia, who developed severe symptoms of pulmonary hypertension. Atypical clinical course of pulmonary hypertension, including complete remission of clinical symptoms and hemodynamic improvement provoked critical approach to the etiology of pulmonary hypertension. Taking into account the temporal coincidence with the use of dasatinib (a tyrosine kinase inhibitor) and a few case reports in the literature, it appears that reversible pulmonary hypertension in our patient was associated with the use of dasatinib. Sclerodermia in a previous medical history, an acknowledged risk factor for pulmonary hypertension, initially delayed the correct diagnosis. Reclassification changes the clinical prognosis of pulmonary hypertension in this specific case and allowed to terminate of specific treatment of pulmonary hypertension with good results.

[Selected, biochemical markers of hypoxia]. / Wybrane, biochemiczne markery hipoksji.

Although tissues may exist regardless of reduced oxygen pressure, this requires glycolytic ATP generation, which is very expensive from the energetic viewpoint. Hypoxia is defined as the condition in which oxygen pressure is reduced at the level of bodily tissues. There are many clinical situations during which decreased tissue oxygenation may occur. It may be transient or chronic, as well as systemic or local. An emergent need exists for monitoring and diagnosis with respect to numerous possible clinical circumstances leading to hypoxia and its life-threatening consequences. The assessment of global oxygen homeo-stasis relies on blood gas analysis and lactate concentration, but such an approach does not fully reflect the local oxygenation of tissues. Oxygen needle microelectrode measurements reveal great differences in tissue pO2 levels. Local pO2 levels depend on many factors, among which the most important are: the distance to the nearest capillary, the extracellular and intracellular fluid diffusion rates and intracellular measurements of the number and activity levels of mitochondria. Thus, nowadays, it is impossible to establish an accurate normal value ranges for local tissue pO2. Oxygen deficiency is an important gene regulator. A sequence-specific DNA-binding factor, the hypoxia induced factor (HIF), is the fundamental hypoxia response protein. 70 genes identified so far have been found to be HIF-dependent. They are responsible for increased oxygen delivery, i.e. by boosting angiogensis due to vascular endothelial growth factor (VEGF) release and the enhancement of red blood cell production by erythropoietin (EPO). VEGF-induced angiogenesis is one of several key hypoxia adaptations. An enhanced vascular bed in response to hypoxia affects almost every bodily tissue and organ. This was observed particularly in skeletal muscles as well as in the brain. The expression of a few hypoxia markers does not require HIF activation. An especially interesting member of this group is osteopontin (OPN), whose synthesis increases during hypoxia. OPN was originally linked to bone remodeling, but currently it seems to posses an important role in immunity, inflammation and tumor pathogenesis. Quantification of hypoxia is clinically essential both for therapy and prognosis. Taking account of the fact that the concept of oxygen pressure at the tissue level is not quantitative (norms do not exist, results are incomparable), biochemical markers are preferable. Particularly significant in this context are hypoxia-induced proteins such as HIF, EPO, VEGF or potentially OPN.