High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

A scalable and high-throughput method to identify precise subcellular localization of endogenous proteins is essential for integrative understanding of a cell at the molecular level. Here, we developed a simple and generalizable technique to image endogenous proteins with high specificity, resolution, and contrast in single cells in mammalian brain tissue. The technique, single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (SLENDR), uses in vivo genome editing to insert a sequence encoding an epitope tag or a fluorescent protein to a gene of interest by CRISPR-Cas9-mediated homology-directed repair (HDR). Single-cell, HDR-mediated genome editing was achieved by delivering the editing machinery to dividing neuronal progenitors through in utero electroporation. We demonstrate that SLENDR allows rapid determination of the localization and dynamics of many endogenous proteins in various cell types, regions, and ages in the brain. Thus, SLENDR provides a high-throughput platform to map the subcellular localization of endogenous proteins with the resolution of micro- to nanometers in the brain.

Analysis of key clinical features for achieving complete remission in stage III and IV non-small cell lung cancer patients.

BACKGROUND: Although development of immune checkpoint inhibitors and various molecular target agents has extended overall survival time (OS) in advanced non-small cell lung cancer (NSCLC), a complete cure remains rare. We aimed to identify features and treatment modalities of complete remission (CR) cases in stages III and IV NSCLC by analyzing long-term survivors whose OS exceeded 3 years. METHODS: From our hospital database, 1,699 patients, registered as lung cancer between 1st Mar 2004 and 30th Apr 2011, were retrospectively examined. Stage III or IV histologically or cytologically confirmed NSCLC patients with chemotherapy initiated during this period were enrolled. A Cox proportion hazards regression model was used. Data collection was closed on 13th Feb 2017. RESULTS: There were 164 stage III and 279 stage IV patients, including 37 (22.6%) and 51 (18.3%) long-term survivors and 12 (7.3%) and 5 (1.8%) CR patients, respectively. The long-term survivors were divided into three groups: 3 ≤ OS < 5 years, 5 years ≤ OS with tumor, and 5 years ≤ OS without tumor (CR). The median OS of these groups were 1,405, 2,238, and 2,876 days in stage III and 1,368, 2,503, and 2,643 days in stage IV, respectively. The mean chemotherapy cycle numbers were 16, 20, and 10 in stage III and 24, 25, and 5 in stage IV, respectively. In the stage III CR group, all patients received chemoradiation, all oligometastases were controlled by radiation, and none had brain metastases. Compared with non-CR patients, the stage IV CR patients had smaller primary tumors and fewer metastases, which were independent prognostic factors for OS among long-term survivors. The 80% stage IV CR patients received radiation or surgery for controlling primary tumors, and the surgery rate for oligometastases was high. Pathological findings in the stage IV CR patients revealed that numerous inflammatory cells existed around and inside resected lung and brain tumors, indicating strong immune response. CONCLUSIONS: Multiple line chemotherapies with primary and oligometastatic controls by surgery and/or radiation might achieve cure in certain advanced NSCLC. Cure strategies must be changed according to stage III or IV. This study was retrospectively registered on 16th Jun 2019 in UMIN Clinical Trials Registry (number UMIN000037078).

Plasticity of dendritic spines: Molecular function and dysfunction in neurodevelopmental disorders.

Dendritic spines are tiny postsynaptic protrusions from a dendrite that receive most of the excitatory synaptic input in the brain. Functional and structural changes in dendritic spines are critical for synaptic plasticity, a cellular model of learning and memory. Conversely, altered spine morphology and plasticity are common hallmarks of human neurodevelopmental disorders, such as intellectual disability and autism. The advances in molecular and optical techniques have allowed for exploration of dynamic changes in structure and signal transduction at single-spine resolution, providing significant insights into the molecular regulation underlying spine structural plasticity. Here, I review recent findings on: how synaptic stimulation leads to diverse forms of spine structural plasticity; how the associated biochemical signals are initiated and transmitted into neuronal compartments; and how disruption of single genes associated with neurodevelopmental disorders can lead to abnormal spine structure in human and mouse brains. In particular, I discuss the functions of the Ras superfamily of small GTPases in spatiotemporal regulation of the actin cytoskeleton and protein synthesis in dendritic spines. Multiple lines of evidence implicate disrupted Ras signaling pathways in the spine structural abnormalities observed in neurodevelopmental disorders. Both deficient and excessive Ras activities lead to disrupted spine structure and deficits in learning and memory. Dysregulation of spine Ras signaling, therefore, may play a key role in the pathogenesis of multiple neurodevelopmental disorders with distinct etiologies.

FOXO-regulated DEAF1 controls muscle regeneration through autophagy.

The commonality between various muscle diseases is the loss of muscle mass, function, and regeneration, which severely restricts mobility and impairs the quality of life. With muscle stem cells (MuSCs) playing a key role in facilitating muscle repair, targeting regulators of muscle regeneration has been shown to be a promising therapeutic approach to repair muscles. However, the underlying molecular mechanisms driving muscle regeneration are complex and poorly understood. Here, we identified a new regulator of muscle regeneration, Deaf1 (Deformed epidermal autoregulatory factor-1) - a transcriptional factor downstream of foxo signaling. We showed that Deaf1 is transcriptionally repressed by FOXOs and that DEAF1 targets to Pik3c3 and Atg16l1 promoter regions and suppresses their expression. Deaf1 depletion therefore induces macroautophagy/autophagy, which in turn blocks MuSC survival and differentiation. In contrast, Deaf1 overexpression inactivates autophagy in MuSCs, leading to increased protein aggregation and cell death. The fact that Deaf1 depletion and its overexpression both lead to defects in muscle regeneration highlights the importance of fine tuning DEAF1-regulated autophagy during muscle regeneration. We further showed that Deaf1 expression is altered in aging and cachectic MuSCs. Manipulation of Deaf1 expression can attenuate muscle atrophy and restore muscle regeneration in aged mice or mice with cachectic cancers. Together, our findings unveil an evolutionarily conserved role for DEAF1 in muscle regeneration, providing insights into the development of new therapeutic strategies against muscle atrophy.

Proof-of-concept study on a water phantom-based neutron spectrometer: Experimental test with 252Cf and 241Am-Be sources.

Boron neutron capture therapy (BNCT) is a promising cancer treatment that uses energetic ions released from 10B(n, α)7Li reactions. Accurate assessment of neutron energy spectra is important for simulation-based evaluation of neutron doses during BNCT. In this study, a proof-of-concept study was conducted for a neutron spectrometry technique that involves the use of a water phantom, which is commonly used for quality assurance in BNCT, as a moderator. The technique involves applying unfolding to the count rate distribution of the thermal neutron counter measured within the phantom to derive the energy spectrum. We performed experiments using a spherical 3He proportional counter in neutron fields generated by 252Cf and 241Am-Be sources. The results demonstrated that the spectrometer reasonably reproduced neutron spectra and showed the potential of using a water phantom as a moderator for such a technique.

Selective and regulated gene expression in murine Purkinje cells by in utero electroporation.

Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells.

Cerebellar long-term depression requires dephosphorylation of TARP in Purkinje cells.

Cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity-dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K-glu) or of a metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto-inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin-9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K-glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin-9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin.

Intraoperative MR Imaging during Glioma Resection.

One of the major issues in the surgical treatment of gliomas is the concern about maximizing the extent of resection while minimizing neurological impairment. Thus, surgical planning by carefully observing the relationship between the glioma infiltration area and eloquent area of the connecting fibers is crucial. Neurosurgeons usually detect an eloquent area by functional MRI and identify a connecting fiber by diffusion tensor imaging. However, during surgery, the accuracy of neuronavigation can be decreased due to brain shift, but the positional information may be updated by intraoperative MRI and the next steps can be planned accordingly. In addition, various intraoperative modalities may be used to guide surgery, including neurophysiological monitoring that provides real-time information (e.g., awake surgery, motor-evoked potentials, and sensory evoked potential); photodynamic diagnosis, which can identify high-grade glioma cells; and other imaging techniques that provide anatomical information during the surgery. In this review, we present the historical and current context of the intraoperative MRI and some related approaches for an audience active in the technical, clinical, and research areas of radiology, as well as mention important aspects regarding safety and types of devices.

Reevaluation of neurodegeneration in lurcher mice: constitutive ion fluxes cause cell death with, not by, autophagy.

The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding delta2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2(Lc)) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the "autophagic cell death" hypothesis. Although autophagy and cell death were induced by the expression of GluD2(Lc) in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2(Lc) or by removal of extracellular Na(+). In addition, although GluD2(Lc) is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)-Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2(Lc) showed decreased ATP levels and increased AMP-activated protein kinase (AMPK) activities in a manner dependent on extracellular Na(+). Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST-Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2(Lc)-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro.

Multimodality imaging suite: neo-futuristic diagnostic imaging operating suite marks a significant milestone for innovation in medical technology.

In February 2006, Tokai University Hospital officially opened the imaging operation suite, which is the first hybrid neurosurgical procedure suite to combine magnetic resonance imaging, computed tomography and angiography with a neurosurgical operating room. Here, we describe the concept of the imaging operation suite and the first 4 years' experience using this suite.

Improving patient safety in the intra-operative MRI suite using an on-duty safety nurse, safety manual and checklist.

This paper describes the use of an on-duty safety nurse, a surgical safety manual and a checklist as an essential precursor to evaluating how these approaches affect surgical quality, communication in surgery crews and contribute to the safety of surgical care in the intra-operative magnetic resonance imaging (MRI) suite.

[Case report: removing the press through package (PTP) containing a tablet from the subglottic region].

A 58-year-old female patient was prescribed different drugs in both tablet and powder form and inadvertently ingested the tablet along with its press through package (PTP), which thus became lodged in the subglottic region. An emergency operation was then performed to remove this foreign material, under neuroleptic anesthesia, and the foreign material was removed successfully. Furthermore, no abnormal findings, such as enlarged vocal cords, were noted. Vocal cord granuloma developed three months after the operation but it improved after conservative treatment.

Imaging dendritic spines: molecular organization and signaling for plasticity.

The structural plasticity of dendritic spines is considered to be essential for various forms of synaptic plasticity and, ultimately, learning and memory. The process is mediated by signaling pathways that promote the reorganization of the actin cytoskeleton and subsynaptic structures, which in turn cause structural and functional changes in dendritic spines. Recent advances in optical technologies have started to reveal the fine molecular structures and dynamic signaling occurring inside spines, providing significant insights into the molecular regulation of spines. Here, we highlight recent studies to resolve the molecular mechanisms underlying the spine actin cytoskeleton and plasticity with high spatiotemporal resolution. Moreover, we discuss new genome editing-based approaches in imaging the molecular structure and plasticity of dendritic spines.

Simple, Quick, and Safe Dural Incision Technique for Patients with Expected Brain Bulging during Decompressive Craniectomy: "Crank-shaped Dural Incisions".

Brain bulging is an unfavorable outcome in patients with brain swelling who require decompressive craniectomy (DC) to control elevated intracranial pressure (ICP). Although several previous studies have described methods for reducing the operation time during DC in these patients, few have proposed a technique for controlling brain protrusion. Here we describe an effective and simple method for external reduction of ICP and discuss its suitability for patients at risk of brain bulging during DC. After craniectomy, crank-shaped lines extending from a central square dural canopy are all marked on the dura. As the incisions are made, pressure from the swelling brain opens the lines and the protruding cortical surface forms dural windows. The square canopy gradually rotates as it stretches, and along with the remaining dura, functions to gently support and compress the cortex. In the case of insufficient decompression, the incision lines can be extended to further reduce ICP. As the parenchyma is accessible to the surgeon, hematoma removal can be performed through the dural windows. In initial experience of four patients who underwent this technique, ICP was controlled in all cases after surgery and no adverse events occurred. The crank-shaped dural incision method is a simple, quick, and effective technique for external reduction of ICP in patients at risk of brain bulging that is intuitive in the emergency situation and thus can be performed even by relatively inexperienced neurosurgeons.

[Obtaining informed consent in the training of endotracheal intubation by emergency medical technicians].

BACKGROUND: There are some problems in the training of endotracheal intubation enforced for the emergency medical technicians. It is important to obtain the informed consent from preoperative patients which is difficult, and time is spent. In addition, patients sometimes refuse in spite of full explanation about this training. METHODS: We examined the situation where we can obtain the informed consent and what were the reasons the patient refuse this training. RESULTS: The refusal rate was 22.5% and women of their 50's to 60's of ages tended to decline more. And there seemed to be difference in each department slightly. The reasons of the patients who refused the informed consent were an anxiety to the training of endotracheal intubation and unwilling to be sacrificed for that. These reasons comprised about 66%. CONCLUSIONS: We could not find the important point to obtain more informed consent. But it was thought that we should know these results to obtain consent more effectively from now.

A case report of Gorham-Stout disease diagnosed during the course of recurrent meningitis and cholesteatoma.

BACKGROUND: Gorham-Stout disease is a rare bone disorder. Here, we present a case of Gorham-Stout disease diagnosed during follow-up of a patient with cholesteatoma; the disease affected the temporal bone and other sites of the skull. To the best of our knowledge, this is the first report of Gorham-Stout disease diagnosed with recurrent cerebrospinal leakage after surgery to treat cholesteatoma. CASE PRESENTATION: A 25-year-old male patient re-presented to our department for the first time in 7 years with otorrhea in the right ear and recurrent meningitis. The patient had a history of multiple surgeries for cholesteatoma and suffered from recurrent cerebrospinal fluid leakage, which initially was thought to be caused by recurrence of cholesteatoma. Therefore, skull base reconstruction was planned. However, the underlying cause was identified eventually as defects in the temporal bone caused by massive osteolysis due to Gorham-Stout disease. Skull base reconstruction was abandoned because the osteolysis was considered to be progressive. Conservative treatment with infectious control was implemented as an alternative. CONCLUSION: This case describes unusual temporal bone osteolysis after cholesteatoma surgery and the importance of considering the possibility of multiple concurrent diseases in such individuals. The distinguishing features of this case are the fact that the temporal bone had disappeared, and deconstruction was complicated by infection and inflammation caused by cholesteatoma, surgical invasion, and Gorham-Stout disease. Appropriate diagnosis saved the patient from ineffective multiple surgeries for cerebrospinal fluid leakage or cholesteatoma, and improved his quality of life.

Genome editing in the mammalian brain using the CRISPR-Cas system.

Recent advances in genome editing technologies such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 have enabled the rapid and efficient modification of endogenous genomes in a variety of cell types, accelerating biomedical research. In particular, precise genome editing in somatic cells in vivo allows for the rapid generation of genetically modified cells in living animals and holds great promise for the possibility of directly correcting genetic defects associated with human diseases. However, because of the limited efficiency and suitability of these technologies in the brain, especially in postmitotic neurons, the practical application of genome editing technologies has been largely limited in the field of neuroscience. Recent technological advances overcome significant challenges facing genome editing in the brain and have enabled us to precisely edit the genome in both mitotic cells and mature postmitotic neurons in vitro and in vivo, providing powerful means for studying gene function and dysfunction in the brain. This review highlights the development of genome editing technologies for the brain and discusses their applications, limitations, and future challenges.

Retrospective review of previous minor leak before major subarachnoid hemorrhage diagnosed by MRI as a predictor of occurrence of symptomatic delayed cerebral ischemia.

OBJECTIVE This study attempted to determine whether a previous minor leak correlated with the occurrence of symptomatic delayed cerebral ischemia (sDCI). METHODS The authors retrospectively evaluated sDCI-related clinical features and findings from MRI, including T1-weighted imaging (T1WI)-FLAIR mismatch at the time of admission, in 151 patients admitted with subarachnoid hemorrhage (SAH) within 48 hours of ictus. RESULTS The overall incidence of sDCI was 23% (35 of 151 patients). In all subjects, multivariate analysis revealed that World Federation of Neurosurgical Societies Grades II-V, age 70 years or older, presence of rebleeding after admission, a previous minor leak before the major SAH attack as diagnosed by T1WI-FLAIR mismatch, acute infarction on diffusion-weighted imaging, and CT SAH score were significantly associated with occurrence of sDCI. In patients with no previous minor leak before major SAH as diagnosed by T1WI-FLAIR mismatch, the incidence of sDCI was only 7% (7 of 97 patients). CONCLUSIONS A previous minor leak before major SAH as diagnosed by T1WI-FLAIR mismatch represents an important sDCI-related factor. When the analysis was restricted to patients with true acute SAH without a previous minor leak diagnosed by T1WI-FLAIR mismatch, the incidence of sDCI was extremely low.

Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain.

Precise genome editing via homology-directed repair (HDR) in targeted cells, particularly in vivo, provides an invaluable tool for biomedical research. However, HDR has been considered to be largely restricted to dividing cells, making it challenging to apply the technique in postmitotic neurons. Here we show that precise genome editing via HDR is possible in mature postmitotic neurons as well as mitotic cells in mice brain by combining CRISPR-Cas9-mediated DNA cleavage and the efficient delivery of donor template with adeno-associated virus (AAV). Using this strategy, we achieved efficient tagging of endogenous proteins in primary and organotypic cultures in vitro and developing, adult, aged, and pathological brains in vivo. Thus, AAV- and CRISPR-Cas9-mediated HDR will be broadly useful for precise genome editing in basic and translational neuroscience.

Occurrence of spot signs from hypodensity areas on precontrast CT in intracerebral hemorrhage.

OBJECTIVE: Both the spot signs, which is a bright spot on computed tomography angiography (CTA) source images, and hypodensity areas within a hematoma on precontrast CT scans, which presumably represent uncoagulated blood, have been reported to be predictive of hematoma enlargement in acute spontaneous intracerebral hematoma (ICH). The aim was to investigate densities on precontrast CT scans in an area within a hematoma that matched the locations of spot signs on CTA source images. METHODS: In consecutive cases of spontaneous ICH admitted within 6 h after onset, early spot signs on CTA source images and delayed spot signs on delayed-phase CT scans 90 s after CTA were evaluated. RESULTS: Of 177 patients undergoing CTA, 41 (23.2%) showed early spot signs. Among 146 patients who underwent delayed-phase CT scans, 23 (15.8%) demonstrated delayed spot signs but not early spot signs. Spot signs originated from hypodensity areas, including densities <50 HU, in 30 of 35 patients (85.7%) with early spot signs and in 8 of 23 (34.8%) with delayed spot signs. Early spot signs arose from hypodensity areas more frequently than delayed spot signs (p < 0.05). Hematoma enlargement was observed in 10 of 24 patients (41.7%) with early spot signs, but in none with delayed spot signs. DISCUSSION: Some hypodensity areas within ICHs may indicate uncoagulated blood related to ongoing leakage, which are seen as spot signs. Minimum densities in hypodensity areas might correlate with the speed and volume of bleeding.