Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

HP1 proteins regulate nucleolar structure and function by secluding pericentromeric constitutive heterochromatin.

Nucleoli are nuclear compartments regulating ribosome biogenesis and cell growth. In embryonic stem cells (ESCs), nucleoli containing transcriptionally active ribosomal genes are spatially separated from pericentromeric satellite repeat sequences packaged in largely repressed constitutive heterochromatin (PCH). To date, mechanisms underlying such nuclear partitioning and the physiological relevance thereof are unknown. Here we show that repressive chromatin at PCH ensures structural integrity and function of nucleoli during cell cycle progression. Loss of heterochromatin proteins HP1α and HP1ß causes deformation of PCH, with reduced H3K9 trimethylation (H3K9me3) and HP1γ levels, absence of H4K20me3 and upregulated major satellites expression. Spatially, derepressed PCH aberrantly associates with nucleoli accumulating severe morphological defects during S/G2 cell cycle progression. Hp1α/ß deficiency reduces cell proliferation, ribosomal RNA biosynthesis and mobility of Nucleophosmin, a major nucleolar component. Nucleolar integrity and function require HP1α/ß proteins to be recruited to H3K9me3-marked PCH and their ability to dimerize. Correspondingly, ESCs deficient for both Suv39h1/2 H3K9 HMTs display similar nucleolar defects. In contrast, Suv4-20h1/2 mutant ESCs lacking H4K20me3 at PCH do not. Suv39h1/2 and Hp1α/ß deficiency-induced nucleolar defects are reminiscent of those defining human ribosomopathy disorders. Our results reveal a novel role for SUV39H/HP1-marked repressive constitutive heterochromatin in regulating integrity, function and physiology of nucleoli.

Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease.

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/ß-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/ß-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/ß-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

Adults remember their birth weight, an important anamnestic item for cardiovascular and renal disease risk stratification.

To assess how many adults remember their own birth weight, an important anamnestic item for cardiovascular and renal disease risk stratification, we conducted an inquiry among all employees of public hospitals of Ente Ospedaliero Cantonale (EOC) in Ticino region (Southern Switzerland). The results show that the vast majority of adults remember their own birth weight. Hence, it is reasonable to include this information in the stratification of risk for cardiovascular and renal diseases.

[Artificial ulcer with perforating bone defect on the scalp due to repeated - scratching attacks in dementia syndrome]. / Artifizielles Ulkus mit perforierendem Knochendefekt an der Schädelkalotte durch wiederholte ­ Kratzattacken bei demenziellem Syndrom.

HISTORY: We report on a 72-year-old demented patient with a palm-sized skin defect on the scalp, who has been manipulating her head several times a day with her fingers but also with sharp objects for many months. CLINICAL FINDINGS: A 4 × 6 cm circular, cleanly granulated ulcer was visible on the high parietal surface. On general examination, the patient was little affected: no fever, no signs of inflammation around the ulcer, no evidence of meningitis. EXAMINATIONS: There was no histological evidence of the presence of a malignant or benign tumor. A CT of the skull showed a bony defect measuring 2.6 × 3.3 cm without involvement of the dura. The whole body CT without pathological findings. THERAPY AND COURSE: First, a protective helmet was made for the patient. Subsequently, a titanium plate was surgically inserted as a bone substitute and the defect was closed by means of transpositionoplasty. In the two-year follow-up, no tumor disease was detected. CONCLUSION: Due to the lack of evidence of tumorigenesis in a follow-up period of two years, we evaluate the lesion as an artificial ulcer with perforating bone defect caused by repeated scratching attacks in the context of dementia syndrome.

The Effect of Silicon Grade and Electrode Architecture on the Performance of Advanced Anodes for Next Generation Lithium-Ion Cells.

To increase the specific capacity of anodes for lithium-ion cells, advanced active materials, such as silicon, can be utilized. Silicon has an order of magnitude higher specific capacity compared to the state-of-the-art anode material graphite; therefore, it is a promising candidate to achieve this target. In this study, different types of silicon nanopowders were introduced as active material for the manufacturing of composite silicon/graphite electrodes. The materials were selected from different suppliers providing different grades of purity and different grain sizes. The slurry preparation, including binder, additives, and active material, was established using a ball milling device and coating was performed via tape casting on a thin copper current collector foil. Composite electrodes with an areal capacity of approximately 1.70 mAh/cm² were deposited. Reference electrodes without silicon were prepared in the same manner, and they showed slightly lower areal capacities. High repetition rate, ultrafast laser ablation was applied to these high-power electrodes in order to introduce line structures with a periodicity of 200 µm. The electrochemical performance of the anodes was evaluated as rate capability and operational lifetime measurements including pouch cells with NMC 622 as counter electrodes. For the silicon/graphite composite electrodes with the best performance, up to 200 full cycles at a C-rate of 1C were achieved until end of life was reached at 80% relative capacity. Additionally, electrochemical impedance spectroscopies were conducted as a function of state of health to correlate the used silicon grade with solid electrolyte interface (SEI) formation and charge transfer resistance values.

Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

[Smoking Behaviour after Partial Resection of the Larynx: The Role of Causal Attribution]. / Rauchverhalten von Krebspatienten nach Kehlkopfteilresektion: Welche Bedeutung hat die Kausalattribution?

OBJECTIVE: The aim of our study was to examine how different causal attributions in patients with laryngeal cancer are associated with smoking behaviours (smoking cessation rates and amount of cigarettes per day) after partial resection of the larynx. METHODS: Multicentre prospective cohort study including 4 interviews: between diagnosis and partial resection of larynx (t1), one week (t2), 3 months (t3) and 12 months (t4) after surgery. Presented in this study are t1 and t4. A total of 134 patients (mean age 62 years, 93% male) were interviewed at t1 and t4 between 2007 and 2013. Key items were causal attribution as well as previous and current smoking behaviour. Patients were grouped according to the subjectively stated causal attribution. Results were analysed descriptively and group as well as mean value comparisons were conducted. RESULTS: Smoking was the most commonly stated causal attribution (43.3%). The quantity of cigarettes decreased significantly by about 6 cigarettes from 17 (range 3-40) to 11 (range 2-30) cigarettes per day in this group (p=0.001). 25% of patients did not recognize a reason for their illness. In longitudinal analyses of all groups of different causal attributions, there was a non-significant decrease in the percentage of smokers. CONCLUSION: We show that causal attribution does not affect smoking cessation rate significantly in a positive way. But active smokers after PRL reduce their quantity of cigarettes per day significantly. This impact is more noticeable in patients who were able to define a causal attribution. Psycho-oncological care, information services and smoking cessation programs could contribute to this effect by making the causal attribution a subject of discussion.

Effects of Finding the Speech-Language Pathologist Likeable on Postlaryngectomy Speech Intelligibility Outcomes.

INTRODUCTION: Speech-language pathologists (SLPs) work with patients after total laryngectomy (TL) to regain verbal communication. The influence of the quality of the therapeutic relationship on the success of TL voice rehabilitation in terms of speech intelligibility is not known. Finding each other likeable is an important factor in establishing and maintaining interpersonal relationships in everyday life. The fit of therapist and client is relevant to the therapeutic relationship. The purpose of this study therefore was to assess the association between the degree of SLPs' likeability ratings and postlaryngectomy speech intelligibility. METHODS: In a multicentre prospective cohort study, participants rated their SLPs' likeability after finishing TL rehabilitation. Speech intelligibility was measured objectively with the Post-Laryngectomy Telephone Intelligibility Test and subjectively with the Questionnaire for Adjustment after Laryngectomy. The association of SLPs' likeability with speech intelligibility was analysed using hierarchical logistic regression, expressed with odds ratios (OR) with corresponding 95% confidence intervals (CI). RESULTS: Altogether 124 patients from 13 institutions participated. The degree of finding the SLP likeable was not significantly associated with objective speech intelligibility (OR 1.30; 95% CI 0.78-2.18; p = 0.32) or subjective speech intelligibility (OR 1.01; 95% CI 0.60-1.72; p = 0.96) after controlling for age, sex and education factors. DISCUSSION/CONCLUSION: In this patient cohort, there was no evidence for an association between ratings of SLPs' likeability and speech intelligibility outcomes after rehabilitation. Future studies could consider the use of alternative instruments for measuring likeability.

Dynamic association of human Ebp1 with the ribosome.

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.

Mechanisms of OCT4-SOX2 motif readout on nucleosomes.

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

An RNA Thermometer Activity of the West Nile Virus Genomic 3'-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching.

The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity.

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

The RGG/RG motif of AUF1 isoform p45 is a key modulator of the protein's RNA chaperone and RNA annealing activities.

The RNA-binding protein AUF1 regulates post-transcriptional gene expression by affecting the steady state and translation levels of numerous target RNAs. Remodeling of RNA structures by the largest isoform AUF1 p45 was recently demonstrated in the context of replicating RNA viruses, and involves two RNA remodeling activities, i.e. an RNA chaperone and an RNA annealing activity. AUF1 contains two non-identical RNA recognition motifs (RRM) and one RGG/RG motif located in the C-terminus. In order to determine the functional significance of each motif to AUF1's RNA-binding and remodeling activities we performed a comprehensive mutagenesis study and characterized the wildtype AUF1, and several variants thereof. We demonstrate that each motif contributes to efficient RNA binding and remodeling by AUF1 indicating a tight cooperation of the RRMs and the RGG/RG motif. Interestingly, the data identify two distinct roles for the arginine residues of the RGG/RG motif for each RNA remodeling activity. First, arginine-mediated stacking interactions promote AUF1's helix-destabilizing RNA chaperone activity. Second, the electropositive character of the arginine residues is the major driving force for the RNA annealing activity. Thus, we provide the first evidence that arginine residues of an RGG/RG motif contribute to the mechanism of RNA annealing and RNA chaperoning.

Multicenter prospective study on the use and outcome of rehabilitation after total laryngectomy in Germany.

BACKGROUND: The purpose of this study was to assess the use and outcome of rehabilitation after total laryngectomy in Germany. METHODS: We enrolled patients who were scheduled for total laryngectomy, approached them again after surgery, after 3 months and 1 year. Patients completed questionnaires and were interviewed. RESULTS: Of 309 participants, 14% had not received any rehabilitation. Reasons for nonuse were primarily ongoing treatment and poor health. Users of rehabilitation had 4 times the odds of attaining any ability to speak compared to nonusers (odds ratio 3.8, P = .02). The main aim of rehabilitation from the perspective of the users was speech rehabilitation, mentioned by 71% before starting rehabilitation. This was also what most users (27%) found the most helpful part of rehabilitation. CONCLUSIONS: Patients are interested in attending rehabilitation. They especially want to improve their speech capacity, which indeed is better among users of rehabilitation than in those without.