Concurrent stable and unstable cortical correlates of human wrist movements

Cortical activity has been shown to correlate with different parameters of movement. However, the dynamic properties of cortico‐motor mappings still remain unexplored in humans. Here, we show that during the repetition of simple stereotyped wrist movements both stable and unstable correlates simultaneously emerge in human sensorimotor cortex. Using visual feedback of wrist movement target inferred online from MEG, we assessed the dynamics of the tuning properties of two neuronal signals: the MEG signal below 1.6 Hz and within the 4 to 6 Hz range. We found that both components are modulated by wrist movement allowing for closed‐loop inference of movement targets. Interestingly, while tuning of 4 to 6 Hz signals remained stable over time leading to stable inference of movement target using a static classifier, the tuning of cortical signals below 1.6 Hz significantly changed resulting in steadily decreasing inference accuracy. Our findings demonstrate that non‐invasive neuronal population signals in human sensorimotor cortex can reflect a stable correlate of voluntary movements. Hence, we provide first evidence for a stable control signal in non‐invasive human brain‐machine interface research. However, as not all neuronal signals initially tuned to movement were stable across days, a careful selection of features for real‐life applications seems to be mandatory. Hum Brain Mapp 35:3867–3879, 2014. © 2014 Wiley Periodicals, Inc .     

The “speed” of acuity in scotopic vs. photopic vision

Graefe's Archive for Clinical and Experimental Ophthalmology - The effect of duration of optotype presentation on visual acuity measures has been extensively studied under photopic conditions....     

Comparison of various surgical approaches for extensive bilateral colorectal liver metastases

Purpose

Tailored operative strategies have been proposed for patients with bilobar colorectal liver metastases (CLM). The aim of the study was to evaluate the long-term outcome, safety and efficacy, including cancer-specific survival, morbidity, and mortality, of three different surgical strategies for extensive bilateral CLM.

Methods

This is a retrospective study of a prospective database of 356 consecutive patients, who underwent hepatic resection due to CLM between January 2003 and January 2009. Fifty-nine patients underwent three different therapeutic approaches: 22 patients with portal vein embolization (PVE) + staged resections, 11 patients with staged resections solely, and 26 patients with an extensive liver resection and simultaneous or subsequent radiofrequency ablation (RFA).

Results

The three groups were comparable regarding their general patient characteristics. The overall morbidity and mortality rates were 27.1 and 1.7 %, respectively. There were no significant differences in morbidity, mortality, or survival between the three groups. The median survival of all patients was 48 months, with a recurrence-free survival of 30 months.

Conclusions

The clearance of bilobar CLM can be achieved by various strategies, all of them providing an acceptable mortality rate and survival for the patients. Therefore, patients with bilobar liver metastases should receive a procedure tailored for their individual extent of disease.     

Expanding the spectrum of PTH1R mutations in patients with primary failure of tooth eruption

Objectives

Primary failure of tooth eruption (PFE) is a rare autosomal-dominant disease characterized by severe lateral open bite as a consequence of incomplete eruption of posterior teeth. Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been shown to cause PFE likely due to protein haploinsufficiency. To further expand on the mutational spectrum of PFE-associated mutations, we report here on the sequencing results of the PTH1R gene in 70 index PFE cases.

Materials and methods

Sanger sequencing of the PTH1R coding exons and their immediate flanking intronic sequences was performed with DNA samples from 70 index PFE cases.

Results

We identified a total of 30 unique variants, of which 12 were classified as pathogenic based on their deleterious consequences on PTH1R protein while 16 changes were characterized as unclassified variants with as yet unknown effects on disease pathology. The remaining two variants represent common polymorphisms.

Conclusions

Our data significantly increase the number of presently known unique PFE-causing PTH1R mutations and provide a series of variants with unclear pathogenicity which will require further in vitro assaying to determine their effects on protein structure and function.

Clinical relevance

Management of PTH1R-associated PFE is problematic, in particular when teeth are exposed to orthodontic force. Therefore, upon clinical suspicion of PFE, molecular DNA testing is indicated to support decision making for further treatment options.     

Assessment of smoking behaviour in a dental setting: a 1-year follow-up study using self-reported questionnaire data and exhaled carbon monoxide levels

Objectives

This study analyses the changes in smoking habits over the course of 1 year in a group of patients referred to an oral medicine unit.

Materials and methods

Smoking history and behaviour were analysed at baseline and after 1 year based on a self-reported questionnaire and on exhaled carbon monoxide levels [in parts per million (ppm)]. During the initial examination, all smokers underwent tobacco use prevention and cessation counselling.

Results

Of the initial group of 121 patients, 98 were examined at the follow-up visit. At the baseline examination, 33 patients (33.67 %) indicated that they were current smokers. One year later, 14 patients (42.24 % out of the 33 smokers of the initial examination) indicated that they had attempted to stop smoking at least once over the follow-up period and 15.15 % (5 patients) had quit smoking. The mean number of cigarettes smoked per day by current smokers decreased from 13.10 to 12.18 (p?=?0.04). The exhaled CO level measurements showed very good correlation with a Spearman's coefficient 0.9880 for the initial visit, and 0.9909 for the follow-up examination. For current smokers, the consumption of one additional cigarette per day elevated the CO measurements by 0.77 ppm (p?<?0.0001) at the baseline examination and by 0.84 ppm (p?<?0.0001) at the 1-year follow-up.

Conclusions

In oral health care, where smoking cessation is an important aspect of the treatment strategy, the measurement of exhaled carbon monoxide shows a very good correlation with a self-reported smoking habit.

Clinical relevance

Measurement of exhaled carbon monoxide is a non-invasive, simple and objective measurement technique for documenting and monitoring smoking cessation and reduction.     

Clinical efficacy and patients' acceptance of a rubber interdental bristle. A randomized controlled trial

Objectives

Interdental cleaning is an essential component of home plaque control to prevent periodontitis and caries. There is limited data on the efficacy of commonly used metal-core interdental brushes in comparison to metal-free interdental brushes. The aim of this study was to compare a new rubber interdental bristle (Fuchs®) with a standard metal-core interdental brush (TePe®) for their impact upon gingival bleeding, plaque removal, and patient experience.

Materials and methods

A single-blind, prospective, randomized, and controlled clinical trial with a crossover design was used to measure plaque index (Turesky-Modified Quigley & Hein Index), bleeding index (Eastman Interdental Bleeding Index by Caton & Polson), and patient satisfaction by means of questionnaires in 39 patients. Each patient was randomly assigned with regard to the sequence of interdental product used and recalled.

Results

Both groups showed statistically significant decreases of plaque after a single usage, respectively. Bleeding was statistically significantly reduced after 4 weeks, with no statistically significant differences concerning between the two tested interdental brushes. Rubber interdental bristles reached significantly higher scores with regard to patient acceptance in overall assessment and in sub-items for less pain during usage, comfort of brushing, and willingness to buy the product.

Conclusion

Rubber interdental bristles were similarly effective compared to the interdental brushes. In addition, rubber interdental bristles were significantly more comfortable for participants than metal-core brushes.

Clinical relevance

Rubber interdental bristles can be used as an alternative interdental cleaning product which may be more accepted by patients.     

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.