Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering.

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a -1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

ABCA3-related interstitial lung disease beyond infancy.

BACKGROUND: The majority of patients with childhood interstitial lung disease (chILD) caused by pathogenic variants in ATP binding cassette subfamily A member 3 (ABCA3) develop severe respiratory insufficiency within their first year of life and succumb to disease if not lung transplanted. This register-based cohort study reviews patients with ABCA3 lung disease who survived beyond the age of 1 year. METHOD: Over a 21-year period, patients diagnosed as chILD due to ABCA3 deficiency were identified from the Kids Lung Register database. 44 patients survived beyond the first year of life and their long-term clinical course, oxygen supplementation and pulmonary function were reviewed. Chest CT and histopathology were scored blindly. RESULTS: At the end of the observation period, median age was 6.3 years (IQR: 2.8-11.7) and 36/44 (82%) were still alive without transplantation. Patients who had never received supplemental oxygen therapy survived longer than those persistently required oxygen supplementation (9.7 (95% CI 6.7 to 27.7) vs 3.0 years (95% CI 1.5 to 5.0), p=0.0126). Interstitial lung disease was clearly progressive over time based on lung function (forced vital capacity % predicted absolute loss -1.1% /year) and on chest CT (increasing cystic lesions in those with repetitive imaging). Lung histology pattern were variable (chronic pneumonitis of infancy, non-specific interstitial pneumonia, and desquamative interstitial pneumonia). In 37/44 subjects, the ABCA3 sequence variants were missense variants, small insertions or deletions with in-silico tools predicting some residual ABCA3 transporter function. CONCLUSION: The natural history of ABCA3-related interstitial lung disease progresses during childhood and adolescence. Disease-modifying treatments are desirable to delay such disease course.

Do Children With Functional Abdominal Pain Benefit More From a Pain-Specific Cognitive-Behavioral Intervention Than From an Unspecific Attention Control Intervention? Results of a Randomized Controlled Trial.

INTRODUCTION: We aimed to compare the efficacy of cognitive-behavioral therapy (CBT) among children with functional abdominal pain with an attention control (AC), hypothesizing the superiority of CBT group intervention regarding pain intensity (primary outcome), pain duration and frequency (further primary outcomes), functional disability, and quality of life and coping strategies (key secondary outcomes). METHODS: We conducted a prospective, multicenter, randomized controlled efficacy trial (RCT) with 4 time points (before intervention, after intervention, 3-month follow-up, and 12-month follow-up). One hundred twenty-seven children aged 7-12 years were randomized to either the CBT (n = 63; 55.6% girls) or the AC (n = 64; 57.8% girls). RESULTS: Primary endpoint analysis of the logarithmized area under the pain intensity curve showed no significant difference between groups (mean reduction = 49.04%, 95% confidence interval [CI] -19.98%-78.36%). Treatment success rates were comparable (adjusted odds ratio = 0.53, 95% CI 0.21-1.34, number needed to treat = 16). However, time trend analyses over the course of 1 year revealed a significantly greater reduction in pain intensity (40.9%, 95% CI 2.7%-64.1%) and pain duration (43.6%, 95% CI 6.2%-66.1%) in the CBT compared with the AC, but not in pain frequency per day (1.2, 95% CI -2.7 to 5.2). In the long term, children in the CBT benefitted slightly more than those in the AC with respect to functional disability, quality of life, and coping strategies. DISCUSSION: Both interventions were effective, which underlines the role of time and attention for treatment efficacy. However, in the longer term, CBT yielded more favorable results.

Reactive Exercises with Interactive Objects: Interim Analysis of a Randomized Trial on Task-Driven NMES Grasp Rehabilitation for Subacute and Early Chronic Stroke Patients.

Enriched environments and tools are believed to promote grasp rehabilitation after stroke. We designed S2, an interactive grasp rehabilitation system consisting of smart objects, custom orthoses for selective grasp constraining, and an electrode array system for forearm NMES. Motor improvements and perceived usability of a new enriched upper limb training system for sub-acute stroke patients was assessed in this interim analysis. INCLUSION CRITERIA: sub-acute stroke patients with MMSE>20, ipsilesional MI>80%, and contralesional MI<80%. Effects of 30-min therapy supplements, conventional vs. S2 prototype, are compared through a parallel two-arms dose-matched open-label trial, lasting 27 sessions. Clinical centres: Asklepios Neurologische Klinik Falkenstein, Königstein im Taunus, Germany, and Clinica Villa Beretta, Costa Masnaga, Italy. Assessment scales: ARAT, System Usability, and Technology Acceptance. METHODOLOGY: 26 participants were block randomized, allocated to the study (control N=12, experimental N=14) and underwent the training protocol. Among them, 11 participants with ARAT score at inclusion below 35, n = 6 in the experimental group, and n = 5 in the control group were analysed. RESULTS: participants in the enriched treatment group displayed a larger improvement in the ARAT scale (+14.9 pts, pval=0.0494). Perceived usability differed between clinics. No adverse effect was observed in relation to the treatments. Trial status: closed. CONCLUSIONS: The S2 system, developed according to shared clinical directives, was tested in a clinical proof of concept. Variations of ARAT scores confirm the feasibility of clinical investigation for hand rehabilitation after stroke.

Mobilization upon Cooling.

Phase transitions between different aggregate states are omnipresent in nature and technology. Conventionally, a crystalline phase melts upon heating as we use ice to cool a drink. Already in 1903, Gustav Tammann speculated about the opposite process, namely melting upon cooling. So far, evidence for such "inverse" transitions in real materials is rare and limited to few systems or extreme conditions. Here, we demonstrate an inverse phase transition for molecules adsorbed on a surface. Molybdenum tetraacetate on copper(111) forms an ordered structure at room temperature, which dissolves upon cooling. This transition is mediated by molecules becoming mobile, i.e., by mobilization upon cooling. This unexpected phenomenon is ascribed to the larger number of internal degrees of freedom in the ordered phase compared to the mobile phase at low temperatures.

Natural variants of ELF3 affect thermomorphogenesis by transcriptionally modulating PIF4-dependent auxin response genes.

BACKGROUND: Perception and transduction of temperature changes result in altered growth enabling plants to adapt to increased ambient temperature. While PHYTOCHROME-INTERACTING FACTOR4 (PIF4) has been identified as a major ambient temperature signaling hub, its upstream regulation seems complex and is poorly understood. Here, we exploited natural variation for thermo-responsive growth in Arabidopsis thaliana using quantitative trait locus (QTL) analysis. RESULTS: We identified GIRAFFE2.1, a major QTL explaining ~18 % of the phenotypic variation for temperature-induced hypocotyl elongation in the Bay-0 x Sha recombinant inbred line population. Transgenic complementation demonstrated that allelic variation in the circadian clock regulator EARLY FLOWERING3 (ELF3) is underlying this QTL. The source of variation could be allocated to a single nucleotide polymorphism in the ELF3 coding region, resulting in differential expression of PIF4 and its target genes, likely causing the observed natural variation in thermo-responsive growth. CONCLUSIONS: In combination with other recent studies, this work establishes the role of ELF3 in the ambient temperature signaling network. Natural variation of ELF3-mediated gating of PIF4 expression during nightly growing periods seems to be affected by a coding sequence quantitative trait nucleotide that confers a selective advantage in certain environments. In addition, natural ELF3 alleles seem to differentially integrate temperature and photoperiod information to induce architectural changes. Thus, ELF3 emerges as an essential coordinator of growth and development in response to diverse environmental cues and implicates ELF3 as an important target of adaptation.

Encounter times in overlapping domains: application to epidemic spread in a population of territorial animals.

We develop an analytical method to calculate encounter times of two random walkers in one dimension when each individual is segregated in its own spatial domain and shares with its neighbor only a fraction of the available space, finding very good agreement with numerically exact calculations. We model a population of susceptible and infected territorial individuals with this spatial arrangement, and which may transmit an epidemic when they meet. We apply the results on encounter times to determine analytically the macroscopic propagation speed of the epidemic as a function of the microscopic characteristics: the confining geometry, the animal diffusion constant, and the infection transmission probability.

Next-to-leading-order results for five, six, and seven jets in electron-positron annihilation.

We present next-to-leading order corrections in the leading color approximation for jet rates in electron-positron annihilation up to seven jets. The results for the two-, three-, and four-jet rates agree with known results. The NLO jet rates have been known previously only up to five jets. The results for the six- and seven-jet rate are new. The results are obtained by a new and efficient method based on subtraction and numerical integration.

Vectorial Electron Spin Filtering by an All-Chiral Metal-Molecule Heterostructure.

The discovery of the electrons' chiral induced spin selective transmission (CISS) through chiral molecules has opened the pathway for manipulating spin transport in nonmagnetic structures on the nanoscale. CISS has predominantly been explored in structurally helical molecules on surfaces, where the spin selectivity affects only the spin polarization of the electrons along their direction of propagation. Here, we demonstrate a spin selective electron transmission for the point-chiral molecule 3-methylcyclohexanone (3-MCHO) adsorbed on the chiral Cu(643)R surface. Using spin- and momentum-resolved photoelectron spectroscopy, we detect a spin-dependent electron transmission through a single layer of 3-MCHO molecules that depends on all three components of the electrons' spin. Crucially, exchanging the enantiomers alters the electrons' spin component oriented parallel to the terraces of the Cu(643)R surface. The findings are attributed to the enantiomer-specific adsorption configuration on the surface. This opens the intriguing opportunity to selectively tune CISS by the enantiospecific molecule-surface interaction in all-chiral heterostructures.

A Natural History of Erectile Dysfunction in Elderly Men: A Population-Based, Twelve-Year Prospective Study.

There is a wide variation in the development and course of erectile dysfunction (ED) in men, which confirms the need for prospective studies. We conducted a cross-sectional analysis among the general male population at the baseline (n = 359) and in a follow-up survey (n = 218) 12 years later. The prospective 12-year study included 189 men. ED was assessed using the International Index of Erectile Function questionnaire. The mean age of the participants was 62.0 years at the baseline, while at the 12-year follow-up it was 71.6 years. The crude prevalence of ED was 61.6% at the baseline and 78.9% at the follow-up, and the prevalence tended to increase with age. All of the men aged 75 years or more had at least mild ED. The incidence of ED in every thousand person years was 53.5. A total of 54.5% of the men experienced ED progression, while 39.2% reported no changes in erectile function, and 6.3% experienced ED regression during the 12-year study. The likelihood of ED progression was higher in the older compared with younger age group (odds ratio, OR 5.2 (95% CI: 1.1-26.2)), and the likelihood of ED regression was lower among men with increased depression symptoms (OR 0.3 (95% CI: 0.1-0.6)) and among men with a decreased interest in their sexual life (OR 0.1 (95% CI: 0.0-0.6)). Lifestyle factors such as the consumption of alcohol and smoking were not significantly associated with ED.

Comparison of the Lung Clearance Index in Preschool Children With Primary Ciliary Dyskinesia and Cystic Fibrosis.

BACKGROUND: Previous studies showed that the lung clearance index (LCI) determined by multiple-breath washout (MBW) is sensitive to detecting early lung disease in preschool children with cystic fibrosis (CF). In preschool children with primary ciliary dyskinesia (PCD), data on the onset and severity of lung disease and on the sensitivity of the LCI as a noninvasive quantitative outcome measure remain limited. RESEARCH AND STUDY QUESTION: Is MBW feasible and sensitive to detect ventilation inhomogeneity in preschool children with PCD? STUDY DESIGN AND METHODS: This was a prospective, cross-sectional, multicenter study and included preschoolers with PCD, preschoolers with CF, and healthy control (HC) participants. LCI was determined using nitrogen MBW and was compared among the three groups. RESULTS: LCI was determined in 27 children with PCD, 34 children with CF, and 30 HC participants (mean age, 4.8 years; range, 2.2-6.9 years). The LCI in preschool children with PCD was increased (median, 9.1; 95% CI, 8.6-10.3) compared with HC participants (median, 7.0; 95% CI, 6.7-7.1; P < .0001), but did not differ from preschool children with CF (median, 8.6; 95% CI, 8.4-9.7; P = .71). The feasibility in the PCD group was 93.1% and was similar to that in the CF group (91.9%) and in HC participants (85.7%; P = .55). INTERPRETATION: This study demonstrated early onset of lung disease in preschool children with PCD and indicated that lung disease severity in PCD may be similar to that in CF during preschool years. These data support a need for early diagnostic monitoring and therapy and suggest the LCI as a noninvasive diagnostic tool and as a potential end point in clinical trials testing early interventions in children with PCD.

A benzodiazepine activator locks Kv7.1 channels open by electro-mechanical uncoupling.

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

MOSAiC-ACA and AFLUX - Arctic airborne campaigns characterizing the exit area of MOSAiC.

Two airborne field campaigns focusing on observations of Arctic mixed-phase clouds and boundary layer processes and their role with respect to Arctic amplification have been carried out in spring 2019 and late summer 2020 over the Fram Strait northwest of Svalbard. The latter campaign was closely connected to the Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) expedition. Comprehensive datasets of the cloudy Arctic atmosphere have been collected by operating remote sensing instruments, in-situ probes, instruments for the measurement of turbulent fluxes of energy and momentum, and dropsondes on board the AWI research aircraft Polar 5. In total, 24 flights with 111 flight hours have been performed over open ocean, the marginal sea ice zone, and sea ice. The datasets follow documented methods and quality assurance and are suited for studies on Arctic mixed-phase clouds and their transformation processes, for studies with a focus on Arctic boundary layer processes, and for satellite validation applications. All datasets are freely available via the world data center PANGAEA.

Electrochemical insertion of lithium in mechanochemically synthesized Zn2SnO4.

We studied the electrochemical insertion of Li in mechanochemically prepared Zn(2)SnO(4). The mechanism of the electrochemical reaction was investigated by using X-ray diffraction, nuclear magnetic resonance spectroscopy, and Mössbauer spectroscopy. Changes in the morphology of the Zn(2)SnO(4) particles were studied by in situ scanning electron microscopy. The results were compared with mixtures of SnO(2) + ZnO and with Zn(2)SnO(4) prepared by conventional solid-state synthesis and showed that the mechanochemically prepared Zn(2)SnO(4) exhibits the best cyclic stability of these samples.

Predictors of High Resource Consumption in Alcohol Intoxicated Patients in the Emergency Department.

Background: previous studies have reported that the incidence of alcohol-related visits to emergency departments (ED) has increased, but little is known about how the necessary resources per visit have changed, or about the predictors and reasons for resource consumption. Methods: a retrospective analysis was performed of all consultations with a primary or secondary diagnosis of acute alcohol intoxication admitted to the ED of Bern University Hospital, Switzerland, between 1 June 2012, and 31 May 2017. Clinical characteristics and resource consumption were extracted and analysed over time. Results: in all, 196,045 ED consultations included 2586 acute alcohol intoxications, corresponding to 1.3% of the total. The incidences of acute alcohol intoxications have tended to increase over the last five years, and a growing number of visits have consumed high resources (consultations above the 75th percentile for total resource consumption). High resource consumption was associated with greater age and the male gender (p < 0.001). The main predictors of resource consumption were fractures (Odds ratio (OR): 3.9, 95% CI 2.8-5.3, p < 0.001), dislocations (OR 3.7, 95%: 1.5-9.1, p < 0.001), and traumatic brain injury (3.5, 2.5-5.1, p < 0.001). Consultations consuming high resources mostly required radiology resources (45%); consultations consuming low or normal resources mostly required physicians' work (45%) or nurses' work (27%). Conclusions: the number of alcohol intoxications consuming high resources has increased over the last five years. Acute alcohol intoxication associated with trauma is resource intensive, especially with regard to radiology resources. This underlines the need for further efforts to prevent alcohol-related traffic accidents, for examples.

Reconstructing the phylogeny of 21 completely sequenced arthropod species based on their motor proteins.

BACKGROUND: Motor proteins have extensively been studied in the past and consist of large superfamilies. They are involved in diverse processes like cell division, cellular transport, neuronal transport processes, or muscle contraction, to name a few. Vertebrates contain up to 60 myosins and about the same number of kinesins that are spread over more than a dozen distinct classes. RESULTS: Here, we present the comparative genomic analysis of the motor protein repertoire of 21 completely sequenced arthropod species using the owl limpet Lottia gigantea as outgroup. Arthropods contain up to 17 myosins grouped into 13 classes. The myosins are in almost all cases clear paralogs, and thus the evolution of the arthropod myosin inventory is mainly determined by gene losses. Arthropod species contain up to 29 kinesins spread over 13 classes. In contrast to the myosins, the evolution of the arthropod kinesin inventory is not only determined by gene losses but also by many subtaxon-specific and species-specific gene duplications. All arthropods contain each of the subunits of the cytoplasmic dynein/dynactin complex. Except for the dynein light chains and the p150 dynactin subunit they contain single gene copies of the other subunits. Especially the roadblock light chain repertoire is very species-specific. CONCLUSION: All 21 completely sequenced arthropods, including the twelve sequenced Drosophila species, contain a species-specific set of motor proteins. The phylogenetic analysis of all genes as well as the protein repertoire placed Daphnia pulex closest to the root of the Arthropoda. The louse Pediculus humanus corporis is the closest relative to Daphnia followed by the group of the honeybee Apis mellifera and the jewel wasp Nasonia vitripennis. After this group the rust-red flour beetle Tribolium castaneum and the silkworm Bombyx mori diverged very closely from the lineage leading to the Drosophila species.

Comparison of Muscular Activity and Movement Performance in Robot-Assisted and Freely Performed Exercises.

End-effector-based robotic systems are, in particular, suitable for extending physical therapy in stroke rehabilitation. An adequate therapy and thus the recovery of movement can only be guaranteed if the physiological muscular activation and movement performance are influenced as little as possible by the robot itself. Yet, this relation has not been investigated in the literature. Therefore, 20 healthy subjects performed free and robot-assisted exercises under different control settings supported by an end-effector-based system. The control settings differed concerning changes in the end-effector velocity and the stiffness of the robot joints. During the exercises, data from inertial measurement unit sensors, robot kinematics, and surface electromyography were collected for the upper limbs. The results showed an increase in muscular activity during robot-assisted movements compared to freely performed movements and also differences in movement performance. The change of the control setting influenced the muscular activation, but not the movement performance. The results of the study revealed that the robot could not be regarded as only a passive element. This should be kept in mind in future robotic rehabilitation systems in order to reduce the influences of the robot itself and thus to optimize the therapy.

Time-frequency coherence of categorized sEMG data during dynamic contractions of biceps, triceps, and brachioradialis as an approach for spasticity detection.

The assessment of muscular interactions between biceps, triceps, and brachioradialis can be used as an approach for the detection of spasticity in the upper limbs. A crucial prerequisite for the aforementioned validation of muscular interactions is the calculation of time frequencies due to the non-stationary characteristics of electromyographic (EMG) signals and thus the estimation of coherences. Adding biomechanical parameters increases the validity of the assessment process and simplifies the comparison of EMG data as a result of categorization. In this numerical-experimental study, a method will be introduced by using the smoothed pseudo Wigner-Ville distribution and a categorization algorithm to estimate and categorize coherences between biceps, triceps, and brachioradialis during dynamic contractions. The categorization will be performed according to the type of contraction, external load, joint angle, and angular velocity and will be used to assess 10 healthy subjects and 6 patients with spasticity. Generally, the introduced method shows the velocity dependence of coherence during spasticity in extension movements as well as much stronger muscular co-activation between triceps, biceps, and brachioradialis in spastic patients in comparison to healthy subjects. Furthermore, the influence of variables e.g. as joint angle, angular velocities, and type of contraction on the coherence is quantified.

Roles of Lactose and Fructose Malabsorption and Dietary Outcomes in Children Presenting with Chronic Abdominal Pain.

Intolerance to lactose or fructose is frequently diagnosed in children with chronic abdominal pain (CAP). However, the causal relationship remains a matter of discussion. A cohort of 253 patients, aged 7-12 years, presenting with unexplained CAP received standardized diagnostics. Additional diagnostic tests were performed based on their medical history and physical and laboratory investigations. Fructose and lactose hydrogen breath tests (H2BT) as well as empiric diagnostic elimination diets were performed in 135 patients reporting abdominal pain related to the consumption of lactose or fructose to evaluate carbohydrate intolerance as a potential cause of CAP. Carbohydrate malabsorption by H2BT was found in 55 (41%) out of 135 patients. An abnormal increase in H2BT was revealed in 30% (35/118) of patients after fructose consumption and in 18% (20/114) of patients after lactose administration. Forty-six percent (25/54) reported pain relief during a diagnostic elimination diet. In total, 17 patients had lactose malabsorption, 29 fructose malabsorption, and nine combined carbohydrate malabsorption. Carbohydrate intolerance as a cause of CAP was diagnosed at follow-up in only 18% (10/55) of patients with malabsorption after the elimination of the respective carbohydrate. Thus, carbohydrate malabsorption appears to be an incidental finding in children with functional abdominal pain disorders, rather than its cause. Therefore, testing of carbohydrate intolerance should only be considered in children with a strong clinical suspicion and with the goal to prevent long-term unnecessary dietary restrictions in children suffering from CAP.