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The novel HLA allele HLA-A*31:215 differs from HLA-A*31:01:02:01 by one nucleotide substitution c.G832A in exon 4. This article is protected by copyright. All rights reserved.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A with no available treatment. Restoring UBE3A levels via downregulation of the paternally cis-acting long non-coding antisense transcript (UBE3A-ATS) is a potential disease modifying. Developing molecules targeting human UBE3A-ATS is challenging because it is expressed only in neurons and lacks animal species sequence conservation. To overcome this, we performed a library screen of locked-nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) on AS patient-derived neurons, identifying initial sequences. Further optimization led to the identification of the ASO, RO7248824, which selectively and potently reduces UBE3A-ATS, while concomitantly upregulating the UBE3A mRNA and protein. These properties held true in both human AS patient- and neurotypical-, as well as cynomolgus monkey-derived neurons. In vivo use of tool molecules in wild-type (WT) and AS Ube3am-/p+ mice, revealed a steep relationship between UBE3A-ATS knock-down and UBE3A mRNA/protein upregulation, whereby an almost 90% downregulation was needed to achieve a 50% upregulation, respectively. This relationship was confirmed in cynomolgus monkeys. Whereby, repeated lumbar intrathecal administrations of RO7248824 was well tolerated without adverse in-life effects or tissue pathology and produced a robust, long lasting (up to 3 months) paternal reactivation of UBE3A mRNA/protein across key monkey brain regions. Our results demonstrate that AS human pluripotent stem cell neurons serve as an excellent translational tool and furthermore LNA-modified ASOs exhibit excellent drug-like properties. Sustained efficacy translated to infrequent, intrathecal dosing and serves as the basis for the ongoing clinical development of RO7248824 for AS.Graphical AbstractGraphical abstract.From AS patient blood to a neuronal screen, identifies clinical ASO with excellent in vivo properties.(1) Patients were recruited. (2) Whereby blood was reprogrammed into hIPSC and subsequently differentiated into neurons. (3) ASOs were designed and screened on human neurons to downregulate the UBE3A-ATS likely via directed RNase H Cleavage of Nascent Transcripts. (4a) RO7248824 was identified that potently and selective reduces UBE3A-ATS, concomitantly with upregulating the UBE3A sense transcript and protein which was used for in vitro pk/pd. (4b) In parallel tool murine ASO were used demonstrate in vivo POC.(5) Pivotal nonhuman primate studies to monitor safety and predict the human dose. (6) RO7248824 is in AS clinical trial.One Sentence SummaryFrom angelman syndrome human neuron screen to cynomolgus monkey proof of concept identifies the clinical molecule RO7248824

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

Well-validated strategies for discovering potent and efficacious antisense oligonucleotides are central to realize the full therapeutic potential of RNA therapy. In this study, we focus on RNA targets where the same sequence of 16-20 nt is found in several regions across the RNA, and not in any other RNA. Targeting such unique repeated regions with oligonucleotides designed as gapmers and capable of recruiting RNase H has previously been proposed as a strategy for identifying potent gapmers. By sequence analysis of the human and monkey transcriptomes, we find that such unique repeated regions in RNA are often conserved between humans and monkeys, which allow pharmacodynamic effects to be evaluated in non-human primates before testing in humans. For eight potential RNA targets chosen in an unbiased fashion, we targeted their unique repeated regions with locked nucleic acid (LNA)-modified gapmers, and for six of them we identified gapmers that were significantly more potent and efficacious in vitro than non-repeat-targeting gapmer controls. We suggest a stochastic model for repeat-targeting gapmers that explains all effects observed so far and can help guide future work. Our results support the targeting of repeated regions as an effective strategy for discovering gapmer antisense oligonucleotides suitable for therapeutic development.

Migration allows animals to exploit spatially separated and seasonally available resources at a continental to global scale. However, responding to global climatic changes might prove challenging, especially for long-distance intercontinental migrants. During glacial periods, when conditions became too harsh for breeding in the north, avian migrants have been hypothesized to retract their distribution to reside within small refugial areas. Here, we present data showing that an Afro-Palearctic migrant continued seasonal migration, largely within Africa, during previous glacial–interglacial cycles with no obvious impact on population size. Using individual migratory track data to hindcast monthly bioclimatic habitat availability maps through the last 120,000 y, we show altered seasonal use of suitable areas through time. Independently derived effective population sizes indicate a growing population through the last 40,000 y. We conclude that the migratory lifestyle enabled adaptation to shifting climate conditions. This indicates that populations of resource-tracking, long-distance migratory species could expand successfully during warming periods in the past, which could also be the case under future climate scenarios.

RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.

The regular fluctuation of resources across the Globe guides movements of migratory animals. To ensure sufficient reproductive output and maintain viable population sizes, migratory animals should match arrival at breeding areas with local peaks in resource availability. It is generally assumed that breeding phenology dictates the timing of the annual cycle, but this is poorly studied. Here, we use light-level geolocator tracking data to compare the annual spatiotemporal migration patterns of a long-distance migratory songbird, the red-backed shrike, Lanius collurio, breeding at widely different latitudes within Europe. We find that populations use remarkably similar migration routes and are highly synchronized in time. Additional tracks from populations breeding at the edges of the European range support these similar migration patterns. When comparing timing of breeding and vegetation phenology, as a measure of resource availability across populations, we find that arrival and timing of breeding corresponds to the peak in vegetation greenness at northern latitudes. At lower latitudes birds arrive simultaneously with the more northerly breeding populations, but after the local greenness peak, suggesting that breeding area phenology does not determine the migratory schedule. Rather, timing of migration in red-backed shrikes may be constrained by events in other parts of the annual cycle.

Recent technological development has made it possible to pinpoint precise locations of small migratory songbirds throughout their annual cycle, providing the opportunity for improving our understanding of year-round habitat use. Here, we use GPS loggers to map the exact location and habitat use at stationary sites throughout the annual cycle of a long-distance migratory songbird, the Red-backed Shrike Lanius collurio. Although the main staging sites confirmed previous findings from light-level geolocation studies, one individual wintered in south-western Chad, an area with only a few historical records of this species. This study highlights opportunities for answering new questions and gaining more knowledge using fine-scale tracking of migratory songbirds.

We used light-level-based geolocation to study the spatio-temporal behaviour of Siberian Rubythroats Calliope calliope breeding in the Amur region of the Russian Far East. Three retrieved devices revealed long-distance migrations, with southwestward movement from Amur through Northeast China in autumn, with the tracked individuals reaching their wintering grounds in southern China and Indochina without major detours and apparently on a route slightly further west than that of the return migration in spring. A single stopover occurred in two of the three birds in both spring and autumn in China. Migration was faster in spring compared to autumn. The birds spent most of their time in seasonal habitats on their temperate breeding sites, and in less seasonal habitats on their tropical wintering grounds. Departure from and arrival at their breeding site coincided with decreasing and increasing vegetation greenness, respectively. This is the first study presenting year-round tracking data for a songbird migrating from mainland Eurasia to Southeast Asia along the East Asian flyway.

The use of accelerometers has become an important part of biologging techniques for large-sized birds with accelerometer data providing information about flight mode, wing-beat pattern, behaviour and energy expenditure. Such data show that birds using much energy-saving soaring/gliding flight like frigatebirds and swifts can stay airborne without landing for several months. Successful accelerometer studies have recently been conducted also for free-flying small songbirds during their entire annual cycle. Here we review the principles and possibilities for accelerometer studies in bird migration. We use the first annual actograms (for red-backed shrike Lanius collurio) to explore new analyses and insights that become possible with accelerometer data. Actogram data allow precise estimates of numbers of flights, flight durations as well as departure/landing times during the annual cycle. Annual and diurnal rhythms of migratory flights, as well as prolonged nocturnal flights across desert barriers are illustrated. The shifting balance between flight, rest and different intensities of activity throughout the year as revealed by actogram data can be used to analyse exertion levels during different phases of the life cycle. Accelerometer recording of the annual activity patterns of individual birds will open up a new dimension in bird migration research.

Light‐level geolocator tags use ambient light recordings to estimate the whereabouts of an individual over the time it carried the device. Over the past decade, these tags have emerged as an important tool and have been used extensively for tracking animal migrations, most commonly small birds. Analysing geolocator data can be daunting to new and experienced scientists alike. Over the past decades, several methods with fundamental differences in the analytical approach have been developed to cope with the various caveats and the often complicated data. Here, we explain the concepts behind the analyses of geolocator data and provide a practical guide for the common steps encompassing most analyses – annotation of twilights, calibration, estimating and refining locations, and extraction of movement patterns – describing good practices and common pitfalls for each step. We discuss criteria for deciding whether or not geolocators can answer proposed research questions, provide guidance in choosing an appropriate analysis method and introduce key features of the newest open‐source analysis tools. We provide advice for how to interpret and report results, highlighting parameters that should be reported in publications and included in data archiving. Finally, we introduce a comprehensive supplementary online manual that applies the concepts to several datasets, demonstrates the use of open‐source analysis tools with step‐by‐step instructions and code and details our recommendations for interpreting, reporting and archiving.

Antisense oligonucleotides (AONs) that promote degradation of complementary RNA are being developed as therapeutics. Here, we describe a simple computational workflow for identification of the regions on an RNA that are suitable for targeting with such AONs. The workflow is based on the statistical programming language R, and the calculations and data processing can be carried out on a desktop computer. Our workflow integrates well-established data resources and RNA structure-prediction tools and can be modified easily and expanded as new resources become available.

The current Northern Hemisphere migration systems are believed to have arisen since the last glaciation. In many cases, birds do not migrate strait from breeding to non-breeding areas but fly via a detour. All western European populations of red-backed shrikes Lanius collurio are assumed to reach their southern African wintering grounds detouring via southeast Europe. Based on theoretical considerations under an optimality framework this detour is apparently optimal. Here, we use individual geolocator data on red-backed shrikes breeding in Spain to show that these birds do indeed detour via southeast Europe en route to southern Africa where they join other European populations of red-backed shrikes and return via a similar route in spring. Disregarding potential wind assistance, the routes taken for the tracked birds in autumn were not optimal compared to crossing the barrier directly. For spring migration the situation was quite different with the detour apparently being optimal. However, when considering potential wind assistance estimated total air distances during autumn migration were overall similar and the barrier crossing shorter along the observed routes. We conclude that considering the potential benefit of wind assistance makes the route via southeast Europe likely to be less risky in autumn. However, it cannot be ruled out that other factors, such as following a historical colonisation route could still be important.

Understanding how events throughout the annual cycle interact to influence individual fitness and hence population dynamics is crucial to optimize conservation strategies for migratory birds. Despite major advancements in technology, direct tracking devices for passerine songbirds are still limited by the need for recapturing tagged individuals. Stable-isotope analysis of bird tissue has the opportunity to supply information on a larger number of individuals, although it is limited by the indirect knowledge of the geographical position of the birds. In this study, we provide a first attempt to combine annual spatio-temporal data achieved from direct tracking with information on local environmental conditions by using stable isotopes (δ 13C and δ 15N). We use this approach to investigate if environmental conditions at the wintering area in southern Africa influence timing of spring migration and carry-over to affect breeding performance in a long-distance migratory passerine bird, the Red-backed Shrike (Lanius collurio). We found that individuals wintering in relatively moist conditions (depleted in δ 13C) and at lower latitudes (closer to the breeding grounds) departed later on spring migration than individuals in more xeric habitats. However, the effect of non-breeding area conditions and latitude were not found at subsequent migration stages and late departing individuals spent fewer days on migration towards the breeding grounds. Although conditions in the non-breeding range have previously been suggested to influence population fluctuations in Red-backed Shrikes, we found no evidence of a carry-over effect on breeding performance. This study highlights the potential of combining different approaches when investigating seasonal interactions in migratory animals.

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Antisense oligonucleotides (AONs) that promote degradation of complementary RNA are being developed as therapeutics. Here, we describe a simple computational workflow for identification of the regions on an RNA that are suitable for targeting with such AONs. The workflow is based on the statistical programming language R, and the calculations and data processing can be carried out on a desktop computer. Our workflow integrates well-established data resources and RNA structure-prediction tools and can be modified easily and expanded as new resources become available.

Protandry, the earlier arrival of males at the breeding grounds relative to females, is common in migratory birds. However, due to difficulties in following individual birds on migration, we still lack knowledge about the spatiotemporal origin of protandry during the annual cycle, impeding our understanding of the proximate drivers of this phenomenon. Here, we use full annual cycle tracking data of red-backed shrikes Lanius collurio to investigate the occurrence of sex-related differences in migratory pattern, which could be viewed as precursors (proximate causes) to protandry. We find protandry with males arriving an estimated 8.3 days (SE = 4.1) earlier at the breeding area than females. Furthermore, we find that, averaged across all departure and arrival events throughout the annual cycle, males migrate an estimated 5.3 days earlier than females during spring compared to 0.01 days in autumn. Event-wise estimates suggest that a divergence between male and female migratory schedules is initiated at departure from the main non-breeding area, thousands of kilometres from-, and several months prior to arrival at the breeding area. Duration of migration, flight speed during migration and spatial locations of stationary sites were similar between sexes. Our results reveal that protandry might arise from sex-differential migratory schedules emerging at the departure from the main non-breeding area in southern Africa and retained throughout spring migration, supporting the view that sex-differential selection pressure operates during spring migration rather than autumn migration.

Following ongoing technological advances, an increasing amount of full-year tracking data on individual migratory movements is becoming available. This opens up the opportunity to study how migration develops within individuals in consecutive years and the extent to which the migratory program is constrained. Such knowledge is essential for understanding the degree of individual flexibility during the annual cycle, which may help identifying potential bottlenecks, where the range of individual decisions is restricted. In this study, we investigate repeatability in time of a long-distance migratory songbird, the red-backed shrike Lanius collurio, tracked across consecutive years (n = 7). Furthermore, we explore the population variability and dependencies between consecutive events of departure and arrival throughout the annual cycle in this species (n = 15). We find that individuals show high repeatability in timing of departure from their two main non-breeding areas in sub-Saharan Africa. In contrast, low repeatability is found in timing of arrivals to stationary sites throughout the annual cycle. Population variation in timing of departure and arrival was similar across all events, ranging from 30 to 41 days, and was highly dependent on timing of preceding events. We conclude that timing of departures is the key event potentially controlled by the individual innate migration program, while arrivals are more flexible, likely dependent on the environmental conditions experienced en route in red-backed shrikes. Still, apparent flexibility in the individual schedule may be hampered by overall constraints of the annual cycle. The annual migration schedule of migratory animals is controlled by a combination of endogenous and exogenous factors. Understanding the temporal dynamics within and between individuals across the annual cycle is important to assess to which extent the migratory schedule is constrained in time. By using full-annual cycle tracking data of individual red-backed shrikes tracked across consecutive years, we find that individuals are highly consistent in their decision to depart from their main non-breeding areas in sub-Saharan Africa, whereas arrivals are less consistent throughout the annual cycle. Overall, the migration schedule is highly constrained across the annual cycle, with each arrival and departure event being dependent on the previous event. Our results suggest that departure decision is underlying endogenous control and that little flexibility is available throughout this complex migration system.

Covalent or noncovalent surface functionalization of soft-matter structures is an important tool for tailoring their function and stability. Functionalized surfaces and nanoparticles have found numerous applications in drug delivery and diagnostics, and new functionalization chemistry is continuously being developed in the discipline of bottom-up systems chemistry. The association of polar functional molecules, e.g., molecular recognition agents, with soft-matter structures can be achieved by derivatization with alkyl chains, allowing noncovalent anchoring into amphiphilic membranes. We report the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayer's hydrophilic surface can significantly increase the degree of structure functionalization.

We describe a method and device (< 1.2 g) for recording, processing and storing data about activity and location of individuals of free-living songbirds throughout the annual cycle. Activity level was determined every five minutes from five 100 ms samples of accelerometer data with 5 s between the sampling events. Activity levels were stored on an hourly basis throughout the annual cycle, allowing periods of resting/sleep, continuous flight and intermediate activity (foraging, breeding) to be distinguished. Measurements from a light sensor were stored from preprogrammed key stationary periods during the year to provide control information about geographic location. Successful results, including annual actogram, were obtained for a red-backed shrike Lanius collurio carrying out its annual loop migration between northern Europe and southern Africa. The shrike completed its annual migration by performing > 66 (max. 73) nocturnal migratory flights (29 flights in autumn and > 37, max. 44, in spring) adding up to a total of > 434 (max. 495) flight hours. Migratory flights lasted on average 6.6 h with maximum 15.9 h. These flights were aggregated into eight travel episodes (periods of 4-11 nights when flights took place on the majority of nights). Daytime resting levels were much higher during the winter period compared to breeding and final part of spring migration. Daytime resting showed peaks during days between successive nocturnal flights across Sahara, continental Africa and the Arabian Peninsula, indicating that the bird was mostly sleeping between these long migratory flights. Annual activity and flight data for free-living songbirds will open up many new research possibilities. Main topics that can be addressed are e.g. migratory flight performance (total flight investment, numbers and characteristics of flights), timing of stationary periods, activity patterns (resting/sleep, activity level) in different phases of the annual cycle and variability in the annual activity patterns between and within individuals.

Oscillations are commonly observed in cellular behavior and span a wide range of timescales, from seconds in calcium signaling to 24 hours in circadian rhythms. In between lie oscillations with time periods of 1-5 hours seen in NF-κB, p53 and Wnt signaling, which play key roles in the immune system, cell growth/death and embryo development, respectively. In the first part of this article, we provide a brief overview of simple deterministic models of oscillations. In particular, we explain the mechanism of saturated degradation that has been used to model oscillations in the NF-κB, p53 and Wnt systems. The second part deals with the potential physiological role of oscillations. We use the simple models described earlier to explore whether oscillatory signals can encode more information than steady-state signals. We then discuss a few simple genetic circuits that could decode information stored in the average, amplitude or frequency of oscillations. The presence of frequency-detector circuit downstream of NF-κB or p53 would be a strong clue that oscillations are important for the physiological response of these signaling systems.

Recent advances in tracking technology are based on the use of miniature sensors for recording new aspects of individual migratory behaviour. In this study, we have used activity data loggers with barometric and temperature sensors to record the flight altitudes as well as ground elevations during stationary periods of migratory songbirds. We tracked one individual of red-backed shrike and one great reed warbler along their autumn migration from Europe to Africa. Both individuals performed their migration stepwise in travel segments and climbed most metres during the passage across the Mediterranean Sea and the Sahara Desert and least metres during the first flight segment in Europe. The great reed warbler reached its highest flight altitude of 3950 m a.s.l. during the travel segment from Europe to west Africa, while the red-backed shrike reached 3650 m a.s.l as maximum flight altitude during its travel segment from Sahel to southern Africa. Both individuals used both lowlands and highlands for resting periods along their migrations. Furthermore, temperature decreased with increasing altitude during migratory flights for both individuals, highlighting the potential to determine flight duration from temperature measurements. Finally, we discuss how barometric data could be used to investigate birds’ responses to changes in air pressure as a cue for departures on migratory flights. This new technique, i.e. using a miniature data logger with barometric pressure sensor to estimate flight altitudes and ground elevations, will open up new avenues for research and importantly advance our understanding on how small birds behave during migratory flights. (Less)

The historical co-evolution of biological motility models with models of Brownian motion is outlined. Recent results for how to derive cell-type-specific motility models from experimental cell trajectories are reviewed. Experimental work in progress, which tests the generality of this phenomenological model building is reported. So is theoretical work in progress, which explains the characteristic time scales and correlations of phenomenological models in terms of the dynamics of cytoskeleton, lamellipodia, and pseudopodia.

Purpose and experimental design: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease and improved biomarkers would help identify high-risk individuals. The aim of this study was to discover candidate biomarkers for DN in the plasma peptidome in an in-house cross-sectional cohort (n=122) of type 1 diabetic patients diagnosed with normo-, micro-, and macroalbuminuria. Results: Automated, high-throughput, and reproducible (interassay median CV: 13–14%) plasma peptide profiling protocols involving RPC18 and weak cation exchange magnetic beads on a liquid handling workstation with a MALDI-TOF-MS readout were successfully established. Using these protocols and a combined univariate (Kruskal–Wallis) and multivariate (independent component analysis) statistical analysis approach, ten single peptides and three multi-peptide candidate biomarkers were found. Employment of RPC18 and weak cation exchange magnetic beads proved to be complementary. Conclusions and clinical relevance: The proteins found in this study, including C3f and apolipoprotein C-I, represent new candidate biomarkers for DN from the plasma peptidome. The automated procedures and implementation of independent components analysis provide a fast and informative system for analyzing individual patient samples in protein biomarker discovery.

Background As part of a clinical proteomics program focused on diabetes and its complications we are looking for new and better protein biomarkers for diabetic nephropathy. The search for new and better biomarkers for diabetic nephropathy has, with a few exceptions, previously focused on either hypothesis-driven studies or urinary based investigations. To date only two studies have investigated the proteome of blood in search for new biomarkers, and these studies were conducted in sera from patients with type 2 diabetes. This is the first reported in depth proteomic study where plasma from type 1 diabetic patients was investigated with the goal of finding improved candidate biomarkers to predict diabetic nephropathy. In order to reach lower concentration proteins in plasma a pre-fractionation step, either hexapeptide bead-based libraries or anion exchange chromatography, was performed prior to surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis. Results Proteomic analysis of plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric, gave rise to 290 peaks clusters of which 16 were selected as the most promising biomarker candidates based on statistical performance, including independent component analysis. Four of the peaks that were discovered have been identified as transthyretin, apolipoprotein A1, apolipoprotein C1 and cystatin C. Several yet unidentified proteins discovered by this novel approach appear to have more potential as biomarkers for diabetic nephropathy. Conclusion These results demonstrate the capacity of proteomic analysis of plasma, by confirming the presence of known biomarkers as well as revealing new biomarkers for diabetic nephropathy in plasma in type 1 diabetic patients.

The Wnt signaling pathway transducing the stabilization of β-catenin is essential for metazoan embryo development and is misregulated in many diseases such as cancers. In recent years models have been proposed for the Wnt signaling pathway during the segmentation process in developing embryos. Many of these include negative feedback loops where Axin2 plays a key role. However, Axin2 null mice show no segmentation phenotype. We therefore propose a new model where the negative feedback involves Dkk1 rather than Axin2. We show that this model can exhibit the same type of oscillations as the previous models with Axin2 and as observed in experiments. We show that a spatial Wnt gradient can consistently convert this temporal periodicity into the spatial periodicity of somites, provided the oscillations in new cells arising in the presomitic mesoderm are synchronized with the oscillations of older cells. We further investigate the hypothesis that a change in the Wnt level in the tail bud during the later stages of somitogenesis can lengthen the time period of the oscillations and hence the size and separation of the later somites.

The construction of a self-reproducing chemical system possessing simplified capabilities of a living cell is a major scientific challenge and a milestone for Artificial Life research. Minimal living physicochemical systems should encompass “information”, “energy transformation” and “co-localization” in a mutually interdependent manner. A fundamental design strategy to achieve such minimal living system was outlined in Rasmussen et al. (2003, 2004), and to reach that goal we have defined a chemical dependency between each of the three component subsystems. The replication of the information molecules must depend on the formation of the container, while the formation of the container must depend on the work of the metabolism, likewise, the work of the metabolism must depend on the replication of the information molecules. This work focuses of the information replication processes, where we have recently developed a template-directed synthesis of oligonucleotides by non-enzymatic ligation (Cape et al., 2012), which is connected to the growth of the container by the action of a Ruthenium catalyst.

Shoulder overuse injuries frequently occur among competitive swimmers (Wanivenhaus et al, 2012), however, the aetiology of shoulder dysfunction remains unclear (Zemek et al., 1995). Increased shoulder mobility as well as Generalised Joint Hypermobility (GJH), are both suggested being predisposing risk factors for shoulder injuries (Pink et al., 2000, Zemek et al., 1995). An association between GJH and shoulder mobility among young, competitive swimmers has not been studied. The aim of this study was firstly to evaluate the association between GJH and horizontal shoulder abduction (HSA) in young, competitive swimmers, and secondly to describe reference values for HSA among this group. Methods In total, 92 pain-free competitive swimmers (age 10-15 years) participated. GJH was evaluated by the Beighton test (BT) a 0-9 scoring system. GJH was classified at cut points ≥5/9, ≥6/9 and ≥7/9. Shoulder mobility was measured as HSA using an inclinometer in a standardized protocol format. A multiple regression analysis was used to reveal associations between GJH and horizontal shoulder abduction, adjusted for sex and age. Results Overall, significant associations between GJH and HSA were found. An increase in the BT score was positively associated with an increase of HSA, seen as an increased HSA of 3.9 degrees at BT cut point ≥5/9, 5.7 degrees at BT cut point ≥6/7 and 7.9 degrees at BT cut point ≥7/9. Normative values for HSA ranged from 40 to 52 degrees, with a trend towards decreasing HSA by increasing age. Conclusion In this study, a significant association was observed between GJH and HSA, as the horizontal shoulder abduction increased with increasing BT scores. The predictive validity of GJH and HSA with respect to shoulder injuries must be assessed in future studies Shoulder overuse injuries frequently occur among competitive swimmers (Wanivenhaus et al, 2012), however, the aetiology of shoulder dysfunction remains unclear (Zemek et al., 1995). Increased shoulder mobility as well as Generalised Joint Hypermobility (GJH), are both suggested being predisposing risk factors for shoulder injuries (Pink et al., 2000, Zemek et al., 1995). An association between GJH and shoulder mobility among young, competitive swimmers has not been studied. The aim of this study was firstly to evaluate the association between GJH and horizontal shoulder abduction (HSA) in young, competitive swimmers, and secondly to describe reference values for HSA among this group. Methods In total, 92 pain-free competitive swimmers (age 10-15 years) participated. GJH was evaluated by the Beighton test (BT) a 0-9 scoring system. GJH was classified at cut points ≥5/9, ≥6/9 and ≥7/9. Shoulder mobility was measured as HSA using an inclinometer in a standardized protocol format. A multiple regression analysis was used to reveal associations between GJH and horizontal shoulder abduction, adjusted for sex and age. Results Overall, significant associations between GJH and HSA were found. An increase in the BT score was positively associated with an increase of HSA, seen as an increased HSA of 3.9 degrees at BT cut point ≥5/9, 5.7 degrees at BT cut point ≥6/7 and 7.9 degrees at BT cut point ≥7/9. Normative values for HSA ranged from 40 to 52 degrees, with a trend towards decreasing HSA by increasing age. Conclusion In this study, a significant association was observed between GJH and HSA, as the horizontal shoulder abduction increased with increasing BT scores. The predictive validity of GJH and HSA with respect to shoulder injuries must be assessed in future studies

Archived version of the supplemental online manual on light-level geolocator analyses. Note, that the manual is constantly updated and released on http://geolocationmanual.vogelwarte.ch