A fluorescent polarization-based assay for the identification of disruptors of the RCAN1/calcineurin A protein complex

5 pages, 4 figures, a table. 19891949 [PubMed]Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identifiedThis work was supported by grants from Fundació La Marató de TV3 (Ref. 030830), the Spanish Ministry of Education and Science (SAF2006-04815, BIO2004-00998, BIO2007-60066, CTQ2005-00995/BQU), the Fundación Mutua Madrileña 2007 and from the Generalitat de Catalunya (Ref. 2006 BE 00051)Peer reviewe

Natural history of carotid artery free-floating thrombus-A single center, consecutive cohort analysis.

Introduction Carotid free-floating thrombus (CFFT) is a rare cause of stroke and is thought to be associated with a high risk of recurrent cerebrovascular ischaemic events. The existing data on the natural history and optimal treatment modalities of CFFT is scanty and no clear recommendations exist. Objective A retrospective analysis, single-center cohort of consecutive patients diagnosed with CFFT was conducted, investigating the risk for recurrent cerebrovascular ischaemic events. Methods We performed a single-center retrospective analysis including all patients presenting at our tertiary center between January 2005 and December 2020 with symptoms consistent with ischaemic stroke and/or transient ischaemic attack. Digital subtraction angiography (DSA), computed tomography angiography (CTA) or magnetic resonance angiography (MRA) were used to diagnose CFFT. In all included patients, CFFT was confirmed with a second imaging modality. CFFT was defined on imaging as a defect in contrast filling extending into the carotid lumen. We gathered information on vascular risk factors, diagnosis and follow-up methods, modality of treatment and neurological outcome. A survival analysis was performed, assessing the risk for recurrent cerebrovascular events. Results In total, N = 62 patients presenting with symptomatic CFFT were included. Mean age was 68 years, 69% (43/62) of patients were male, 52% (32/62) current or previous smokers, 76% (47/62) suffered from arterial hypertension, 68% (42/62) from dyslipidaemia, and 31% (19/62) from diabetes mellitus. Overall, 71% (44/62) of patients received any kind of intervention [endovascular or surgical carotid thrombo-endartectomy (CEA)] at any time point during follow-up. Sixteen percent of patients (10/62) received intervention within 48 h after diagnosis of CFFT. The survival analysis and Kaplan-Meier model censoring patients at the time of intervention or last follow-up showed that the risk for any recurrent ischaemic stroke was 19.7% within the first 7 days and 27.4% within 3 months after diagnosis. No patients experienced a new ischaemic stroke beyond 11 days after diagnosis of CFTT (n = 17). Conclusion The risk of recurrent ischaemic events in patients with CFFT is high, especially in the first week after diagnosis. Prospective studies are needed to further investigate the optimal management of these patients

Effects of formalin fixation on polarimetric properties of brain tissue: fresh or fixed?

Imaging Mueller polarimetry (IMP) appears as a promising technique for real-time delineation of healthy and neoplastic tissue during neurosurgery. The training of machine learning algorithms used for the image post-processing requires large data sets typically derived from the measurements of formalin-fixed brain sections. However, the success of the transfer of such algorithms from fixed to fresh brain tissue depends on the degree of alterations of polarimetric properties induced by formalin fixation (FF). Comprehensive studies were performed on the FF induced changes in fresh pig brain tissue polarimetric properties. Polarimetric properties of pig brain were assessed in 30 coronal thick sections before and after FF using a wide-field IMP system. The width of the uncertainty region between gray and white matter was also estimated. The depolarization increased by 5% in gray matter and remained constant in white matter following FF, whereas the linear retardance decreased by 27% in gray matter and by 28% in white matter after FF. The visual contrast between gray and white matter and fiber tracking remained preserved after FF. Tissue shrinkage induced by FF did not have a significant effect on the uncertainty region width. Similar polarimetric properties were observed in both fresh and fixed brain tissues, indicating a high potential for transfer learning

The Development of Cephalic Armor in The Tokay Gecko (Squamata: Gekkonidae: \u3cem\u3eGekko gecko\u3c/em\u3e)

Armored skin resulting from the presence of bony dermal structures, osteoderms, is an exceptional phenotype in gekkotans (geckos and flap-footed lizards) only known to occur in three genera: Geckolepis, Gekko, and Tarentola. The Tokay gecko (Gekko gecko LINNAEUS 1758) is among the best-studied geckos due to its large size and wide range of occurrence, and although cranial dermal bone development has previously been investigated, details of osteoderm development along a size gradient remain less well-known. Likewise, a comparative survey of additional species within the broader Gekko clade to determine the uniqueness of this trait has not yet been completed. Here, we studied a large sample of gekkotans (38 spp.), including 18 specimens of G. gecko, using X-rays and high-resolution computed tomography for visualizing and quantifying the dermal armor in situ. Results from this survey confirm the presence of osteoderms in a second species within this genus, Gekko reevesii GRAY 1831, which exhibits discordance in timing and pattern of osteoderm development when compared with its sister taxon, G. gecko. We discuss the developmental sequence of osteoderms in these two species and explore in detail the formation and functionality of these enigmatic dermal ossifications. Finally, we conducted a comparative analysis of endolymphatic sacs in a wide array of gekkotans to explore previous ideas regarding the role of osteoderms as calcium reservoirs. We found that G. gecko and other gecko species with osteoderms have highly enlarged endolymphatic sacs relative to their body size, when compared to species without osteoderms, which implies that these membranous structures might fulfill a major role of calcium storage even in species with osteoderms

Expertise in surgical neuro-oncology. Results of a survey by the EANS neuro-oncology section

Introduction: Technical advances and the increasing role of interdisciplinary decision-making may warrant formal definitions of expertise in surgical neuro-oncology. Research question: The EANS Neuro-oncology Section felt that a survey detailing the European neurosurgical perspective on the concept of expertise in surgical neuro-oncology might be helpful. Material and methods: The EANS Neuro-oncology Section panel developed an online survey asking questions regarding criteria for expertise in neuro-oncological surgery and sent it to all individual EANS members. Results: Our questionnaire was completed by 251 respondents (consultants: 80.1%) from 42 countries. 67.7% would accept a lifetime caseload of >200 cases and 86.7% an annual caseload of >50 as evidence of neuro-oncological surgical expertise. A majority felt that surgeons who do not treat children (56.2%), do not have experience with spinal fusion (78.1%) or peripheral nerve tumors (71.7%) may still be considered experts. Majorities believed that expertise requires the use of skull-base approaches (85.8%), intraoperative monitoring (83.4%), awake craniotomies (77.3%), and neuro-endoscopy (75.5%) as well as continuing education of at least 1/year (100.0%), a research background (80.0%) and teaching activities (78.7%), and formal interdisciplinary collaborations (e.g., tumor board: 93.0%). Academic vs. non-academic affiliation, career position, years of neurosurgical experience, country of practice, and primary clinical interest had a minor influence on the respondents’ opinions. Discussion and conclusion: Opinions among neurosurgeons regarding the characteristics and features of expertise in neuro-oncology vary surprisingly little. Large majorities favoring certain thresholds and qualitative criteria suggest a consensus definition might be possible

Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination

The contribution of oligodendrocytes to remyelination in functional recovery after spinal cord injury is not fully understood. Here, the authors show that oligodendrocyte progenitor cell differentiation is not required for functional recovery after spinal cord injury in mice

Expertise in surgical neuro-oncology. Results of a survey by the EANS neuro-oncology section

Introduction: Technical advances and the increasing role of interdisciplinary decision-making may warrant formal definitions of expertise in surgical neuro-oncology. Research question: The EANS Neuro-oncology Section felt that a survey detailing the European neurosurgical perspective on the concept of expertise in surgical neuro-oncology might be helpful. Material and methods: The EANS Neuro-oncology Section panel developed an online survey asking questions regarding criteria for expertise in neuro-oncological surgery and sent it to all individual EANS members. Results: Our questionnaire was completed by 251 respondents (consultants: 80.1%) from 42 countries. 67.7% would accept a lifetime caseload of >200 cases and 86.7% an annual caseload of >50 as evidence of neuro-oncological surgical expertise. A majority felt that surgeons who do not treat children (56.2%), do not have experience with spinal fusion (78.1%) or peripheral nerve tumors (71.7%) may still be considered experts. Majorities believed that expertise requires the use of skull-base approaches (85.8%), intraoperative monitoring (83.4%), awake craniotomies (77.3%), and neuro-endoscopy (75.5%) as well as continuing education of at least 1/year (100.0%), a research background (80.0%) and teaching activities (78.7%), and formal interdisciplinary collaborations (e.g., tumor board: 93.0%). Academic vs. non-academic affiliation, career position, years of neurosurgical experience, country of practice, and primary clinical interest had a minor influence on the respondents’ opinions. Discussion and conclusion: Opinions among neurosurgeons regarding the characteristics and features of expertise in neuro-oncology vary surprisingly little. Large majorities favoring certain thresholds and qualitative criteria suggest a consensus definition might be possible

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system