Gene-expression memory-based prediction of cell lineages from scRNA-seq datasets.

Assigning single cell transcriptomes to cellular lineage trees by lineage tracing has transformed our understanding of differentiation during development, regeneration, and disease. However, lineage tracing is technically demanding, often restricted in time-resolution, and most scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational tool allowing to robustly identify small to medium-sized cell lineages solely from scRNA-seq datasets. GEMLI allows to study heritable gene expression, to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. In human breast cancer biopsies, GEMLI reveals previously unknown gene expression changes at the onset of cancer invasiveness. The universal applicability of GEMLI allows studying the role of small cell lineages in a wide range of physiological and pathological contexts, notably in vivo. GEMLI is available as an R package on GitHub ( https://github.com/UPSUTER/GEMLI ).

Optimal control pulses for subspectral editing in low field NMR.

Low field NMR is an inexpensive and low footprint technique to obtain physical, chemical, electronic and structural information on small molecules, but suffers from poor spectral dispersion, especially when applied to the analysis of mixtures. Subspectral editing employing optimal control pulses is a suitable approach to cope with the severe signal superpositions in complex mixture spectra at low field. In this contribution, the use of optimal control pulses is demonstrated to be feasible at a field strength as low as 0.5 T. The optimal control pulse shapes were calculated using the Krotov algorithm. Downsizing the complexity of the algorithm from exponential to polynomial is shown to be possible by using a system approach with each system corresponding to a (small) molecule. In this way compound selective excitation pulses can be calculated. The signals of substructures of the cyclopentenone molecule were excited using optimal control pulses calculated by the Krotov algorithm demonstrating the feasibility of subspectral editing. Likewise, for a mixture of benzoic acid and alanine, editing of the signals of either benzoic acid or alanine employing optimal control pulses was shown to be possible. The obtained results are very promising and can be extended to the targeted analysis of complex mixtures such as biofluids or metabolic samples at low field strengths opening access for benchtop NMR to point of care settings.

Room temperature "optical nanodiamond hyperpolarizer": Physics, design, and operation.

Dynamic Nuclear Polarization (DNP) is a powerful suite of techniques that deliver multifold signal enhancements in nuclear magnetic resonance (NMR) and MRI. The generated athermal spin states can also be exploited for quantum sensing and as probes for many-body physics. Typical DNP methods require the use of cryogens, large magnetic fields, and high power microwave excitation, which are expensive and unwieldy. Nanodiamond particles, rich in Nitrogen-Vacancy (NV) centers, have attracted attention as alternative DNP agents because they can potentially be optically hyperpolarized at room temperature. Here, unraveling new physics underlying an optical DNP mechanism first introduced by Ajoy et al. [Sci. Adv. 4, eaar5492 (2018)], we report the realization of a miniature "optical nanodiamond hyperpolarizer," where 13C nuclei within the diamond particles are hyperpolarized via the NV centers. The device occupies a compact footprint and operates at room temperature. Instrumental requirements are very modest: low polarizing fields, low optical and microwave irradiation powers, and convenient frequency ranges that enable miniaturization. We obtain the best reported optical 13C hyperpolarization in diamond particles exceeding 720 times of the thermal 7 T value (0.86% bulk polarization), corresponding to a ten-million-fold gain in averaging time to detect them by NMR. In addition, the hyperpolarization signal can be background-suppressed by over two-orders of magnitude, retained for multiple-minute long periods at low fields, and deployed efficiently even to 13C enriched particles. Besides applications in quantum sensing and bright-contrast MRI imaging, this work opens possibilities for low-cost room-temperature DNP platforms that relay the 13C polarization to liquids in contact with the high surface-area particles.

Hyperpolarized relaxometry based nuclear T1 noise spectroscopy in diamond.

The origins of spin lifetimes in quantum systems is a matter of importance in several areas of quantum information. Spectrally mapping spin relaxation processes provides insight into their origin and motivates methods to mitigate them. In this paper, we map nuclear relaxation in a prototypical system of [Formula: see text] nuclei in diamond coupled to Nitrogen Vacancy (NV) centers over a wide field range (1 mT-7 T). Nuclear hyperpolarization through optically pumped NV electrons allows signal measurement savings exceeding million-fold over conventional methods. Through a systematic study with varying substitutional electron (P1 center) and [Formula: see text] concentrations, we identify the operational relaxation channels for the nuclei at different fields as well as the dominant role played by [Formula: see text] coupling to the interacting P1 electronic spin bath. These results motivate quantum control techniques for dissipation engineering to boost spin lifetimes in diamond, with applications including engineered quantum memories and hyperpolarized [Formula: see text] imaging.

Enhanced dynamic nuclear polarization via swept microwave frequency combs.

Dynamic nuclear polarization (DNP) has enabled enormous gains in magnetic resonance signals and led to vastly accelerated NMR/MRI imaging and spectroscopy. Unlike conventional cw-techniques, DNP methods that exploit the full electron spectrum are appealing since they allow direct participation of all electrons in the hyperpolarization process. Such methods typically entail sweeps of microwave radiation over the broad electron linewidth to excite DNP but are often inefficient because the sweeps, constrained by adiabaticity requirements, are slow. In this paper, we develop a technique to overcome the DNP bottlenecks set by the slow sweeps, using a swept microwave frequency comb that increases the effective number of polarization transfer events while respecting adiabaticity constraints. This allows a multiplicative gain in DNP enhancement, scaling with the number of comb frequencies and limited only by the hyperfine-mediated electron linewidth. We demonstrate the technique for the optical hyperpolarization of 13C nuclei in powdered microdiamonds at low fields, increasing the DNP enhancement from 30 to 100 measured with respect to the thermal signal at 7T. For low concentrations of broad linewidth electron radicals [e.g., TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl)], these multiplicative gains could exceed an order of magnitude.

A novel method for quantitative measurements of gene expression in single living cells.

Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.

An Outbreak of Porcine Reproductive and Respiratory Syndrome Virus in Switzerland Following Import of Boar Semen.

An outbreak of porcine reproductive and respiratory syndrome virus (PRRSV) occurred in November 2012 in Switzerland (CH), traditionally PRRSV-free. It was detected after a German boar stud informed a semen importer about the detection of PRRSV during routine monitoring. Tracing of semen deliveries revealed 26 Swiss sow herds that had used semen from this stud after its last negative routine monitoring and 62 further contact herds. All herds were put under movement restrictions and examined serologically and virologically. As a first measure, 59 sows from five herds that had previously been inseminated with suspicious semen were slaughtered and tested immediately. Investigations in the stud resulted in 8 positive boars with recent semen deliveries to CH (Seven with antibodies and virus, one with antibodies only). In one boar out of six tested, virus was detected in semen. Of the 59 slaughtered sows, five from three herds were virus-positive. In one herd, the virus had spread, and all pigs were slaughtered or non-marketable animals euthanized. In the remaining herds, no further infections were detected. After confirmatory testings in all herds 3 weeks after the first examination gave negative results, restrictions were lifted in January 2013, and Switzerland regained its PRRSV-free status. The events demonstrate that import of semen from non-PRRS-free countries--even from negative studs--poses a risk, because monitoring protocols in boar studs are often insufficient to timely detect an infection, and infections of sows/herds occur even with low numbers of semen doses. The outbreak was eradicated successfully mainly due to the high disease awareness of the importer and because immediate actions were taken before clinical or laboratory diagnosis of a single case in the country was made. To minimize the risk of an introduction of PRRSV in the future, stricter import guidelines for boar semen have been implemented.

A cryogenic receiver for EPR.

Cryogenic probes have significantly increased the sensitivity of NMR. Here, we present a compact EPR receiver design capable of cryogenic operation. Compared to room temperature operation, it reduces the noise by a factor of ≈2.5. We discuss in detail the design and analyze the resulting noise performance. At low microwave power, the input noise density closely follows the emission of a cooled 50Ω resistor over the whole measurement range from 20K up to room temperature. To minimize the influence of the microwave source noise, we use high microwave efficiency (≈1.1-1.7mTW(-1/2)) planar microresonators. Their efficient conversion of microwave power to magnetic field permits EPR measurements with very low power levels, typically ranging from a few µW down to fractions of nW.

Room temperature strong coupling between a microwave oscillator and an ensemble of electron spins.

We demonstrate theoretically and experimentally the possibility to achieve the strong coupling regime at room temperature with a microwave electronic oscillator coupled with an ensemble of electron spins. The coupled system shows bistable behaviour, with a broad hysteresis and sharp transitions. The coupling strength and the hysteresis width can be adjusted through the number of spins in the ensemble, the temperature, and the microwave field strength.

Embryonic stem cell-based screen for small molecules: cluster analysis reveals four response patterns in developing neural cells.

Neural differentiation of embryonic stem cells (ESC) is considered a promising model to perform in vitro testing for neuroactive and neurotoxic compounds. We studied the potential of a dual reporter murine ESC line to identify bioactive and/or toxic compounds. This line expressed firefly luciferase under the control of the neural cell-specific tubulin alpha promoter (TUBA1A), and renilla luciferase under the control of the ubiquitous translation elongation factor 1-alpha-1 (EEF1A1) promoter. During neural differentiation, TUBA1A activity increased, while EEF1A1 activity decreased. We first validated our test system using the known neurotoxin methyl mercury. This compound altered expression of both reporter genes, with ESC-derived neural precursors being affected at markedly lower concentrations than undifferentiated ESCs. Analysis of a library of 1040 bioactive compounds picked up 127 compounds with altered EEF1A1 and/or TUBA1A promoter activity, which were classified in 4 clusters. Cluster 1 (low EEF1A1 and TUBA1A) was the largest cluster, containing many cytostatic drugs, as well as known neurodevelopmental toxicants, psychotropic drugs and endocrine disruptors. Cluster 2 (high EEF1A1, stable TUBA1A) was limited to three sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) was small, but markedly enriched in neuroactive and neurotoxic compounds. Cluster 4 (stable EEF1A1, high TUBA1A) was heterogeneous, containing endocrine disruptors, neurotoxic and cytostatic drugs. The dual reporter gene assay described here might be a useful addition to in vitro drug testing panels. Our two-dimensional testing strategy provides information on complex response patterns, which could not be achieved by a single marker approach.

Single-molecule force spectroscopy of the Aplysia cell adhesion molecule reveals two homophilic bonds.

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.

Fermentation of six different forages in the semi-continuous fermentation technique Caesitec.

The aim of the present study was to compare carbohydrate degradation of forages which store carbohydrates either predominantly as fructan or starch, in horses' hindgut. The effects of an abrupt change from hay-based feeding to green fodder-based feeding on the caecal flora were tested with the in vitro hindgut simulation technique 'Caesitec'. Six trials with different forages (English ryegrass, tall fescue, grass mixture-horses, grass mixture-cows, lucerne, white clover) were conducted. During a 4-day stabilisation period, samples were taken once a day before loading the fermenters with hay. After diet-change to forage-based feeding, samples were taken four times a day. Ammonia and pH-value were measured before and 1, 2 and 6 h after loading the 'Caesitec'. Gas formation was measured daily. Bacterial numbers, lactate and short chain fatty acids were detected at four time-points of each trial. The grass mixtures contained the highest amounts of fructan. The pH-values were in the physiological range from pH 6 up to 7 (6.58-6.83) by feeding all forages. Gas formation, anaerobic and aerobic bacterial numbers increased after diet change from hay to any forage. The maximum amount of fructan (3.75 g/kg) in swiss pasture did not cause a permanent pathological change in the hindgut-flora.

The mammalian circadian timing system: synchronization of peripheral clocks.

Mammalian physiology has to adapt to daily alternating periods during which animals either forage and feed or sleep and fast. The adaptation of physiology to these oscillations is controlled by a circadian timekeeping system, in which a master pacemaker in the suprachiasmatic nucleus (SCN) synchronizes slave clocks in peripheral organs. Because the temporal coordination of metabolism is a major purpose of clocks in many tissues, it is important that metabolic and circadian cycles are tightly coordinated. Recent studies have revealed a multitude of signaling components that possibly link metabolism to circadian gene expression. Owing to this redundancy, the implication of any single signaling pathway in the synchronization of peripheral oscillators cannot be assessed by determining the steady-state phase, but instead requires the monitoring of phase-shifting kinetics at a high temporal resolution.

Electron paramagnetic resonance investigation of the electron-doped manganite La(1-x)Te(x)MnO3 (0.1 ≤ x ≤ 0.2).

The electron paramagnetic resonance (EPR) properties of the electron-doped manganite La(1-x)Te(x)MnO(3) (0.1 ≤ x ≤ 0.2) are investigated based on the data of EPR spectra, resistivity, and magnetic susceptibility. With decreasing temperature from 400 K, the EPR linewidth ΔH(PP) decreases and passes through a minimum at T(min), then substantially increases with further decreasing temperature. The broadening of the EPR linewidth above T(min) can be understood in terms of the increase in the relaxation rate of spin of e(g) polarons to the lattice with increasing temperature due to the similarity between the temperature dependence of the linewidth ΔH(pp)(T) and the conductivity σ(T). For the samples with x = 0.1 and 0.15, the conductivity activation energy E(σ) is comparable with the activation energy E(a) deduced from the linewidth. Whereas for the x = 0.2 sample, there is a large difference between E(σ) (0.2206 eV) and E(a) (0.0874 eV).

Visualization of spin dynamics in single nanosized magnetic elements.

The design of future spintronic devices requires a quantitative understanding of the microscopic linear and nonlinear spin relaxation processes governing the magnetization reversal in nanometer-scale ferromagnetic systems. Ferromagnetic resonance is the method of choice for a quantitative analysis of relaxation rates, magnetic anisotropy and susceptibility in a single experiment. The approach offers the possibility of coherent control and manipulation of nanoscaled structures by microwave irradiation. Here, we analyze the different excitation modes in a single nanometer-sized ferromagnetic stripe. Measurements are performed using a microresonator set-up which offers a sensitivity to quantitatively analyze the dynamic and static magnetic properties of single nanomagnets with volumes of (100 nm)(3). Uniform as well as non-uniform volume modes of the spin wave excitation spectrum are identified and found to be in excellent agreement with the results of micromagnetic simulations which allow the visualization of the spatial distribution of these modes in the nanostructures.

Adaptation to metabolic acidosis and its recovery are associated with changes in anion exchanger distribution and expression in the cortical collecting duct.

It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in ß-intercalated cells and the basolateral anion exchanger (AE1) in α-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and α-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6-20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base.

Invasive pneumococcal disease in children prior to implementation of the conjugate vaccine in the Zurich region, Switzerland.

OBJECTIVE: To describe symptoms, disease manifestations and outcome of invasive pneumococcal disease in children prior to implementation of the pneumococcal vaccine. PATIENTS AND METHODS: Analysis of children younger than 16 years of age with invasive pneumococcal disease (IPD; n = 119). Children with culture-confirmed IPD, without underlying illness at risk for invasive disease, were included. RESULTS: IPD in 90 children (age: median 2, mean 3.2 years) included 15 with meningitis, 16 with septicaemia, 14 with bacteraemia, 24 with pneumonia and 21 with skin, bone and joint infections. Symptoms of IPD most often described were fever and gastrointestinal symptoms (abdominal pain, vomiting, or diarrhoea), and coughing. More than 90% of children with pneumonia were coughing. Most importantly, clinical signs significantly predictive for severe IPD included tachycardia for sepsis, tachypnea for pneumonia, and meningeal signs for meningitis. Leukocyte, neutrophil and platelet counts were lower and C-reactive protein concentrations were higher on admission in children with complicated than in children with uncomplicated IPD but, due to wide overlap of these numbers, the difference was not of prognostic help to predict clinical course and outcome. Overall, 40% of children with IPD manifested complications and IPD showed a mortality rate of 6.6%. CONCLUSIONS: IPD is a serious disease with a high complication rate and mortality. The clinical signs tachycardia, tachypnea, and meningism were highly predictive for severe IPD. The initial clinical presentation and laboratory evaluation were mostly unpredictable with respect to complications and outcome in contrast to the clinical signs.

Scaling of sensitivity and efficiency in planar microresonators for electron spin resonance.

Electron spin resonance (ESR) of volume-limited samples or nanostructured materials can be made significantly more efficient by using microresonators whose size matches that of the structures under investigation. We describe a series of planar microresonators that show large improvements over conventional ESR resonators in terms of microwave conversion efficiency (microwave field strength for a given input power) and sensitivity (minimum number of detectable spins). We explore the dependence of these parameters on the size of the resonator and find that both scale almost linearly with the inverse of the resonator size. Scaling down the loops of the planar microresonators from 500 down to 20 mum improves the microwave efficiency and the sensitivity of these structures by more than an order of magnitude and reduces the microwave power requirements by more than two orders of magnitude.

Plant enemy-derived elicitors increase the foliar tannin concentration of Onobrychis viciifolia without a trade-off to growth.

BACKGROUND AND AIMS: Molecular experiments suggest that the regulation of the biosynthesis of condensed tannin (CT) is sensitive to the presence of plant enemies. The enemy-specific response of CT concentrations to simulated attacks by pathogenic fungi, bacteria or herbivores was studied in Onobrychis viciifolia grown at four levels of nutrient availability. It was hypothesized that CT concentrations increase in response to an attack, and that constitutive and induced levels of CT are higher at low than at high nutrient availability. Investment in CT was also predicted to be negatively related to plant growth. METHODS: Recently discovered substances by which plants recognize their opponents (i.e. elicitors) were used to simulate attacks to Onobrychis viciifolia grown at 0.0027, 0.075, 0.67 or 2 mm phosphorus in the nutrient solution. KEY RESULTS: Relative growth rate and final biomass (P < 0.001) were highest at 0.67 mm of phosphorus. CT concentrations decreased with increasing phosphorus availability, from 94.9 to 69.0 mg g(-1) leaf dry weight (P < 0.001). Compared with unscathed plants, sterile mere mechanical wounding reduced tannin concentrations from 83.8 to 69.3 mg g(-1) leaf dry weight (P < 0.01). Local CT concentrations were higher when wounded leaves were additionally treated with fungal (+15.9 %), bacterial (+19.6 %) or insect (+31.0 %) elicitors (each elicitor; P < 0.05); however, only the insect elicitor (saliva of the lepidopteron Spodoptera littoralis) induced CT concentrations higher than those of unscathed leaves. CONCLUSIONS: CT concentrations were inducible in the vicinity of the wound but the level of induction was unrelated to the nutrient status of the plant. There was no evidence of a growth-defence trade-off. The inverse relationship between CT concentrations and nutrient availability appears to reflect passive growth dilution at high nutrient availability, rather than surplus CT production at low nutrient availability.