Evolutionary conservation of centriole rotational asymmetry in the human centrosome.

Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the 'acorn.' Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly, and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

A multicentre, randomized, double-blinded, placebo-controlled Phase III study to investigate EXtending the time for Thrombolysis in Emergency Neurological Deficits (EXTEND).

BACKGROUND AND HYPOTHESIS: Thrombolytic therapy with tissue plasminogen activator is effective for acute ischaemic stroke within 4·5 h of onset. Patients who wake up with stroke are generally ineligible for stroke thrombolysis. We hypothesized that ischaemic stroke patients with significant penumbral mismatch on either magnetic resonance imaging or computer tomography at three- (or 4·5 depending on local guidelines) to nine-hours from stroke onset, or patients with wake-up stroke within nine-hours from midpoint of sleep duration, would have improved clinical outcomes when given tissue plasminogen activator compared to placebo. STUDY DESIGN: EXtending the time for Thrombolysis in Emergency Neurological Deficits is an investigator-driven, Phase III, randomized, multicentre, double-blind, placebo-controlled study. Ischaemic stroke patients presenting after the three- or 4·5-h treatment window for tissue plasminogen activator and within nine-hours of stroke onset or with wake-up stroke within nine-hours from the midpoint of sleep duration, who fulfil clinical (National Institutes of Health Stroke Score ≥4-26 and prestroke modified Rankin Scale <2) will undergo magnetic resonance imaging or computer tomography. Patients who also meet imaging criteria (infarct core volume <70 ml, perfusion lesion : infarct core mismatch ratio >1·2, and absolute mismatch >10 ml) will be randomized to either tissue plasminogen activator or placebo. STUDY OUTCOME: The primary outcome measure will be modified Rankin Scale 0-1 at day 90. Clinical secondary outcomes include categorical shift in modified Rankin Scale at 90 days, reduction in the National Institutes of Health Stroke Score by 8 or more points or reaching 0-1 at day 90, recurrent stroke, or death. Imaging secondary outcomes will include symptomatic intracranial haemorrhage, reperfusion and or recanalization at 24 h and infarct growth at day 90.

Human skin keloid fibroblasts display bioenergetics of cancer cells.

Cultured human skin keloid fibroblasts (KFs) showed bioenergetics similar to cancer cells in generating ATP mainly from glycolysis as demonstrated by increased lactate production. Activities of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase were also significantly higher compared with normal fibroblasts (NFs). Inhibitors of glycolysis decreased the rate of ATP biosynthesis more significantly in KFs suggesting their reliance on glycolysis. In contrast, ATP generation in NFs was derived mainly from oxidative phosphorylation (OXPHOS), which was more compromised by mitochondrial/respiratory inhibitors. However, when fortified with excess exogenous respiratory substrates, ATP production was increased to a similar maximal level in both types of fibroblasts. In spite of this seemingly equal total capacity, ATP biosynthesis and intracellular ATP concentration were significantly higher in KFs, which further increased their ATP production when exposed to hypoxia and hypoxia-mimetics: desferrioxamine and cobalt chloride. This upregulation was again significantly compromised by glycolytic inhibitors. The rate of generation of reactive oxygen species was lower in KFs possibly due to their switch to aerobic glycolysis from mitochondrial OXPHOS. Thus, cultured skin KFs could provide a human cell model to study the de-regulation of bioenergetics of proliferative cells and their response to the HIF (hypoxia-inducible factor) signaling.

Retroperitoneal fibrosis: a rare cause of both ureteral and small bowel obstruction.

Retroperitoneal fibrosis (RPF) is a rare condition of unclear etiology. It can cause ureteral obstruction. We present the unique case of a 54 years old female, who initially presented with spontaneous perforation of the cecum. Upon exploring the abdomen, the classical glistening white, unyielding retroperitoneal fibrosis was encountered. A right hemicolectomy was performed. Subsequently, the patient presented with bilateral ureteral obstruction, and later on with small bowel obstruction. Ureteral obstruction was treated with stents, and small bowel obstruction was treated with bypass. To our knowledge no case of idiopathic RPF presenting with features of both bilateral ureteral and small bowel obstruction has been reported in the literature.

Basement membrane formation by malignant mouse keratinocyte cell lines in organotypic culture and transplants: correlation with degree of morphologic differentiation.

Six malignant C3H mouse epidermal cell lines (HEL-30, HEL-37, HELP I, HELP IV, HD II, H3L), with different capacities for epidermal differentiation, were analyzed for their organized growth behavior and basement membrane (BM) formation in organotypical cultures in vitro and after transplantation into syngeneic mice. Expression and deposition of five BM components (type IV collagen, laminin, bullous pemphigoid antigen, fibronectin, heparan sulfate proteoglycan) were determined on frozen sections by indirect immunofluorescence. Additionally, synthesis and secretion of BM components by the line HEL-30 (in submersed cultures) were identified by metabolic labeling and immunoprecipitation. Morphologic differentiation features and formation of a structured BM were studied by electron microscopy. All cell lines were tumorigenic and invasive but nevertheless able to synthesize BM constituents in vitro and in vivo, although pronounced variations in the expression and the polarity and continuity of deposition were found. Irrespective of the amount of BM components synthesized, none of the cell lines formed a structured BM in organotypical cultures in vitro. After transplantation the production of BM components was improved and the newly formed epithelia were separated from the mesenchyme by a structured BM. The formation of BM occurred whether the epithelial cells were in immediate contact with the mesenchyme or separated by a 1 to 2 mm thick native collagen gel. Deposition of BM constituents and formation of BM structures occurred both at the superficial epithelium and around invading cell cords. The studies clearly demonstrated that malignant epidermal cells maintain their capacity to synthesize BM components. The extent of production and the polarity of deposition of the constituents and the quality of BM formation were usually higher in well differentiated cell lines and obviously correlated well with their preserved degree of differentiation. Comparable to normal keratinocytes, formation of structured BM requires interaction with living mesenchyme but occurs independently of direct epidermal-mesenchymal contact.