Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance phase could also be an adaptive feature to prepare for efficient urea excretion when water becomes available.The African lungfish,  Protopterus aethiopicus, P. amphibicus, P. annectens, and P. dolloi. During drought, African lungfishes can aestivate in subterranean mud cocoons for as long as 4 years  and argininosuccinate lyase  from the skin of P. annectens, which was also expressed in the gills, kidney, liver and skeletal muscle. As several gene duplication events of ut have occurred during the evolution of lower vertebrates, LeMoine and Walsh  are members of the solute transporter family SLC14 that facilitate the passive diffusion of urea down its concentration gradient  was conducted to determine the branchial mRNA expression levels of ut isoforms in P. annectens kept in fresh water (control) or aestivated for 6 days (the induction phase of aestivation) or 6 months (the maintenance phase of aestivation), or aroused for 1 day or 3 days from 6 months of aestivation . Western blotting was conducted to determine the branchial protein abundance of Ut isoforms, using custom-made antibodies developed based on the deduced Ut sequences. It was expected that results obtained would provide insights on whether the mRNA and protein expression of ut/Ut in the gills of P. annectens would be down-regulated during the induction and maintenance phases of aestivation to impede urea excretion and/or to shut down branchial functions, and whether they would be up-regulated during the initial arousal phase to facilitate the excretion of the urea accumulated in the body.Therefore, this study was undertaken to clone and sequence the cDNA coding region of P. annectens (80–150 g body mass) were imported from Central Africa through a fish farm in Singapore. In the laboratory, they were kept in plastic tanks filled with dechlorinated tap water at 25°C. There was no attempt to separate the sexes. Water was changed daily. Fish were acclimated to laboratory conditions for at least 2 weeks and fed with frozen fish meat during the acclimatization period. The procedures used in this study have been approved by the Institutional Animal Care and Use Committee of the National University of Singapore (IACUC 035/09).Specimens of P. annectens were induced to aestivate individually in plastic tanks (85–90% humidity and 27–29°C) containing 15 ml dechlorinated tap water . It took around 6 days for the lungfish to be encased in a brown dried mucus cocoon. In this study, these 6 days were counted as part of the aestivation period and regarded as the induction phase of aestivation. The lungfish were allowed to aestivate for 6 months. After 6 months of aestivation, some lungfish were aroused by adding 200 ml of water into the tank. Subsequently, the aroused lungfish was covered with an additional 800 ml of water.Following the procedure of Chew et al. , P. anneN = 4) kept in fresh water acted as the control and they were killed with an overdose of neutralized 0.05% MS222 after withholding food for 96 h. After 6 days or 6 months of aestivation, some lungfish (N = 4 for each group) were pithed after a blow to the head. After 1 day or 3 days of arousal from 6 months of aestivation, some lungfish (N = 4 for each group) were killed with an overdose of neutralized 0.05% MS222. The gills, eye, brain, heart, liver, spleen, pancreas, gut, kidney, lung, muscle and skin were quickly excised and freeze-clamped with aluminum tongs pre-cooled in liquid nitrogen.Lungfish . RNA was purified by Qiagen RNeasy Mini Kit  and quantified by BioSpec-nano . The integrity of RNA was assessed electrophoretically and gauged by the ratio of 28S/18S rRNA. The purity of RNA was accessed by the absorbance ratio of A260/A280. First strand cDNA synthesis was performed using 4 μg of total RNA, oligo(dT)18 primer and the RevertAid™ first strand cDNA synthesis kit .Total RNA extraction was performed on samples of ut-a2a and ut-a2b from the gills of P. annectens, gene-specific PCR primers  obtained from the skin by Hung et al. /isopropyl β-D-1-thiogalactopyranoside (IPTG) plates. Colony-PCR was then performed on all the selected white colonies. The colonies with insert of expected sizes were selected and grown overnight in LB/ampicillin broth in a shaking incubator . The plasmids were extracted using AxyPrep Plasmid Miniprep Kit  and sequenced. The partial sequences obtained were verified to be ut-a2a and ut-a2b.Ligation of the purified PCR products was performed using pGEM®-T Easy vector . Subsequently, the ligation mixtures were transformed into JM109 P. annectens gills was reverse transcribed into 3′-RACE-Ready cDNA and 5′-RACE-Ready cDNA. Using gene-specific RACE primers (Table ut-a2a and ut-a2b. The cycling conditions involved 30 cycles of 94°C (30 s), 65°C (30 s), and 72°C (4 min). RACE-PCR products were separated by gel electrophoresis, purified and sequenced. To obtain the full coding sequences of ut-a2a and ut-a2b, multiple sequencing was conducted in both directions. Bioedit v7.1.3 , 1 μg of total RNA from .3 Hall,  was usedut-a2a and ut-a2b using ExPASy Proteomic server (http://web.expasy.org/translate/). The alignment of the deduced amino acid sequences with selected Ut/UT from several animal species was performed using Bioedit. Potential N-glycosylation sites were verified using NetNGlyc 1.0 and potential phosphorylation sites were verified using NetPhos 2.0. The transmembrane domains of Ut-a2a and Ut-a2b were identified using MEMSAT3 and MEMSAT-SVM in PSIPRED protein structure prediction server , accompanied by 32 cycles of 95°C (30 s), 55°C (30 s), 72°C (30 s) and a final extension of 72°C (10 min). The PCR products were separated by gel electrophoresis.Using gene-specific qPCR primers of 2b Table , PCR wasut-a2a and ut-a2b in P. annectens gills. The method of relative quantitation does not allow interpretation of the predominant gene expressed in a certain condition and provides only fold-change data.In this study, the method of absolute quantification with reference to a standard curve was used because it was crucial to compare the transcript levels of P. annectens. First strand cDNA synthesis was performed using 4 μg of total RNA, random hexamer primers and RevertAid™ first strand cDNA synthesis kit (Thermo Fisher Scientific). Following the method of Gerwick et al. (6–102 specific copies per 2 μl). The standard was used as a reference to determine the absolute quantities of ut-a2a or ut-a2b transcripts in the qPCR reaction.The Qiagen RNeasy Plus Mini Kit (Qiagen GmbH), which contains gDNA Eliminator spin column (Qiagen GmbH) to remove genomic DNA, was used to purify the RNA from the gills of k et al. , a pure k et al.  was obta−1 of forward and reverse gene-specific qPCR primers each (Table t) were collected. A melt curve analysis was conducted to verify the presence of a single product. Furthermore, the PCR product was separated by gel electrophoresis to confirm the presence of a single band. A standard curve was produced by plotting the natural log of concentration (copies μl−1) on the X-axis and Ct on the Y-axis. The default setting of StepOne™ Software v2.1 (Life Technologies Corporation) was used for the calculation of the Y-intercept, Ct slope, correlation coefficient (r2), and PCR efficiency. The PCR efficiencies for the gene-specific primers of ut-a2a, and ut-a2b were 91.5 and 89.3%, respectively. The linear regression line derived from the standard curve was used to determine the quantity of transcript in an unknown sample, which was expressed as copies of transcripts per ng total RNA.qPCR was carried out in duplicates using the StepOnePlus™ Real-Time PCR System (Life Technologies Corporation). Each qPCR reaction contained 0.3 μmol lP. annectens kept in fresh water (control), or aestivated for 6 days or 6 months, or aroused for 1 day or 3 days from 6 months of aestivation. Each sample was homogenized twice in five volumes (w/v) of ice cold buffer containing 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific), 150 mmol l−1 NaCl, 1 mmol l−1 Na3VO4, 1 mmol l−1 NaF, 50 mmol l−1 Tris HCl, (pH 7.4), 1 mmol l−1 EDTA, 1 mmol l−1 phenylmethylsulfonyl fluoride, 1% sodium deoxycholate, and 1% NP-40, using pre-cooled TissueLyser LT (Qiagen GmbH) at 50 Hz for 2.5 min. Subsequently, the homogenate was centrifuged at 10,000 × g for 20 min at 4°C. The protein concentration in the supernatant was determined . The concentration of the anti-Ut-a2a and anti-Ut-a2b antibodies used for Western blotting was 1.7 μg ml−1.Two rabbit polyclonal antibodies against aa 23–36 (HNTEKQNDKKESVKC) of Ut-a2a and aa 40–53 (LSENTETSVPEYHPC) of Ut-a2b from Following the method of Laemmli , proteinS.E.M.). SPSS version 18  was used for statistical analyses. Levene's Test was used to verify the homogeneity of variance. Differences between means were verified using one-way analysis of variance, followed by multiple comparisons of means using either Dunnett T3 or Tukey post-hoc test, depending on the homogeneity of variance of the data set. Differences were reported as statistically significant at P < 0.05.Results were presented as means ± standard errors of the mean . In comparison, the complete coding cDNA sequence of ut-a2b comprised 1392 bp, coding for 463 amino acids with an estimated molecular mass of 51.2 kDa (accession: KX610112). The similarity between ut-a2a and lfut (accession: EU716115.1) was 99.7%, while the similarity between ut-a2b and lfut was 71.3%.The complete coding cDNA sequence of P. annectens comprised 10 transmembrane regions. Efforts were made to compare the Ut isoforms of P. annectens with H. sapiens UT-B and Desulfovibrio vulgaris dvUT, as their protein structure had been used to formulate the theories of urea transport  and bacteria revealed highly conserved LPXXTXPF motifs and the lack of UT-B ALE domain  in Ut-a2a and Ut-a2b of P. annectens, respectively  of aestivation or after 1 day  of arousal from 6 months of aestivation, as compared to control fish  and 3.4-fold (P < 0.05), respectively, as compared to the control  or 6 months  of aestivation, or after 1 day  of arousal from 6 months of aestivation  have been identified in dvUT of P. annectens contain several potential phosphorylation sites and at least one N-glycosylation site, which are in agreement with those of UT-A and UT-B of H. sapiens  and Ut-a2b (with an estimated molecular mass of ~51.2 kDa) would exhibit a higher molecular mass of ~50 and ~55 kDa, respectively, through Western blotting. Similarly, it has been reported that the bands of Ut/UT of Rhinella marina  from the skin of P. annectens. As for the gills, the expression levels of ut-a2a and ut-a2b appeared to be comparable, although quantitative conclusions cannot be drawn based on PCR results alone. Using qPCR, we confirmed that the gills of P. annectens kept in fresh water had similar transcript levels of ut-a2a and ut-a2b. Therefore, it is logical to deduce that both Ut-a2a and Ut-a2b would contribute to urea excretion through the gills of P. annectens, particularly during the arousal phase. More importantly, the transcript levels and protein abundance of ut-a2a/Ut-a2a and ut-a2b/Ut-a2b remained either unchanged or increased significantly in the gills during the maintenance phase, which were unexpected as no urea excretion could have occurred.The expression of g et al.  reportedut-a2a/Ut-a2a in the gills of P. annectens after 6 days (the induction phase) of aestivation. However, the transcript level of ut-a2a increased significantly while the protein abundance of Ut-a2a remained unchanged as compared with the control after 6 months of aestivation (the maintenance phase). The significant increase in ut-a2a transcripts cannot be explained by decreased degradation of ut-a2a transcripts alone. There must be continuous transcription of ut-a2a in the gills of P. annectens during the maintenance phase of aestivation. It is apparent that the transcriptional ut-a2a and translation of Ut-a2a is momentarily disengaged resulting in the unchanged protein abundance of Ut-a2a in the gills. This can be interpreted as an adaptive feature to prepare for arousal, as the increased transcript level of ut-a2a would prepare for a subsequent increase in the production of Ut-a2a during the arousal phase. As expected, the ut-a2a transcripts in the gills continued to increase upon arousal (1 day) from 6 months of aestivation and the Ut-a2a protein abundance increased concurrently by ~4-fold. These results confirm that the increase in translation of Ut-a2a is activated when water becomes available upon arousal. In addition, they imply that, besides the skin , the maintenance phase (15.5-fold), and the arousal phase (7.8-fold) as compared with the control. While a decrease in the degradation of Ut-a2b might have contributed in part to the continuous increase of Ut-a2b protein abundance during the 6 months of aestivation, there must be an up-regulation in the translation of Ut-a2b between the induction phase and the maintenance phase of aestivation. Of note, during the induction and maintenance phases, P. annectens gills are covered with a thick layer of mucus (Sturla et al., In contrast with Scaphiopus couchii, accumulate urea during aestivation (Jones, Rana sylvatica (Muir et al., P. annectens to eliminate it from the body with high efficiency during the initial phase of arousal. Similarly, marsupials (Hume, Urea can act as an osmolyte to maintain cell volume in conditions of osmotic stress Yancey, . Some frn Jones, . The hign Jones, . It is pn Jones, , reversin Jones, , and conn Jones,  like thels Hume,  and Coluls Hume,  accumulaut-a2a and the protein abundance of Ut-a2b in the gills of P. annectens after 6 months of aestivation. Similarly, the mRNA expression levels of cps III (Loong et al., ass, asl (Chng et al., bhmt1 (Ong et al., asl in the brain (Chng et al., P. annectens during the maintenance phase of aestivation. In addition, increases in protein abundance during the maintenance phase have been reported for Bhmt1 in the liver and muscle (Ong et al., P. annectens. Taken together, these results indicate that the supposition of expression and syntheses of all genes/proteins being suppressed in every organ in an African lungfish during the maintenance phase of aestivation may not be valid (Ip and Chew, As conservation of metabolic fuels is essential during extended periods of aestivation without food, metabolic depression has long been regarded as an important feature of aestivation Storey, . MoreoveYI designed the experiment. JO, BC, XC, KH, WW, and SC were involved in the subjection of animal and the collection of samples. YC performed all experiments and analyzed the data. SL and YI were involved in the analysis of data and the approval of manuscript. YI and YC wrote the manuscript. All authors were involved in the revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tensor tympani contraction may have a "signature" audiogram. This study demonstrates audiometric findings during voluntary tensor tympani contraction.Five volunteers possessing the ability to voluntarily contract their tensor tympani muscles were identified and enrolled. Tensor tympani contraction was confirmed with characteristic tympanometry findings. Study subjects underwent conventional audiometry. Air conduction and bone conduction threshold testing was performed with and without voluntary tensor tympani contraction.Changes in air conduction and bone conduction thresholds during voluntary tensor tympani contraction.Audiometric results demonstrate a low frequency mixed hearing loss resulting from tensor tympani contraction. Specifically, at 250 Hz, air conduction thresholds increased by 22 dB and bone conduction thresholds increased by 10 dB.Previous research has demonstrated a low frequency conductive hearing loss in the setting of tensor tympanic contraction. This is the first study to demonstrate a low frequency mixed hearing loss associated with tensor tympani contraction. This finding may aid in the diagnosis of disorders resulting from abnormal tensor tympani function. Tensor tympani contraction should be included on the differential for low frequency mixed hearing loss. The contribution of the tensor tympani muscle to middle ear function and dysfunction remains a topic of controversy. EMG studies have demonstrated TT activity during vocalization, yawning, swallowing, laughing, coughing, and both face and head movements, but not to auditory stimuli , 2. It hIn order to better understand the role of the TT in otologic symptoms and conditions, it is important to establish objective indicators of TT function. Recently, our group  identifDetermining the audiometric effects of TT contraction may provide additional objective evidence in the form of characteristic audiograms thereby implicating its possible contribution to otologic conditions. If these audiometric patterns are seen, then the tympanometric markers could also be sought. Additionally, an accurate audiometric characterization may allow the implication of TT dysfunction in cases of hearing loss, or other otologic symptoms. Politzer articulated the phenomenon of TT contraction and its association with hearing loss in his 1909 textbook, where he described “a deafness that occurs during yawning brought on by simultaneous contraction of the tensor tympani” . AlthougOur group  recentlThis study was approved by our institutional research ethics board.Five subjects capable of voluntary TT contraction were recruited for audiometric testing. TT contraction was confirmed with modified reflex decay testing Fig. , accordiPure tone audiometry in five subjects with the TT in the relaxed and contracted state was collected. All subjects had normal pre-test air conduction thresholds of less than 25 dB with no pre-existing air bone gap at the tested frequencies. TT contraction produced a mixed hearing loss, with a reduction in air conduction and bone conduction thresholds at 250 Hz, 500 H, 1000 Hz and 2000 Hz during TT contraction. These changes were temporary and reversed with cessation of tensor tympanic contraction. Fig. The TT muscle measures 20 to 25 mm in length, arises from the cartilaginous Eustachian tube, adjacent sphenoid bone and its bony semi canal; turning on the cochleariform process, it terminates as a tendon inserting on the medial aspect of the malleus . ActivatThe audiometric effect of voluntary TT contraction has been documented previously. In 1943, the case of a lieutenant capable of voluntary TT contraction was reviewed. A low frequency hearing loss was identified during voluntary TT contraction. However, bone conduction thresholds were not tested . In 1960Our group  used laIn this study, all patients showed varying degrees of reversible low frequency mixed hearing loss during voluntary tensor tympani contraction – a finding that has not been shown previously. The mechanism for this loss is proposed to be due to a combination of effects. TT contraction results in stiffening of the ossicular mechanism, which could result in a conductive hearing loss. The low frequency sensorineural component of hearing loss induced by TT contraction may be theoretically explained by a number of mechanisms. One is the loss of the osteo-tympanal contribution to bone conduction thresholds . In thisAaron et al. found that the reversal of the positive deflection on modified reflex decay testing is specific to tensor tympanic contraction . HoweverOur study is the first to demonstrate a low frequency mixed hearing loss resulting from voluntary contraction of the tensor tympani muscle in five ears. This audiometric finding serves as an additional objective marker of tensor tympani contraction. TT contraction should be included in the differential for cases of low frequency mixed hearing loss, particularly if there is decreased compliance on tympanometric testing, as noted by us previously .


Immediate early genes (IEGs) were traditionally used as markers of neuronal activity in striatum in response to stimuli including drugs of abuse such as psychostimulants. Early studies using these neuronal activity markers led to important insights in striatal neuron subtype responsiveness to psychostimulants. Such studies have helped identify striatum as a critical brain center for motivational, reinforcement and habitual behaviors in psychostimulant addiction. While the use of IEGs as neuronal activity markers in response to psychostimulants and other stimuli persists today, the functional role and implications of these IEGs has often been neglected. Nonetheless, there is a subset of research that investigates the functional role of IEGs in molecular, cellular and behavioral alterations by psychostimulants through striatal medium spiny neuron (MSN) subtypes, the two projection neuron subtypes in striatum. This review article will address and highlight the studies that provide a functional mechanism by which IEGs mediate psychostimulant molecular, cellular and behavioral plasticity through MSN subtypes. Insight into the functional role of IEGs in striatal MSN subtypes could provide improved understanding into addiction and neuropsychiatric diseases affecting striatum, such as affective disorders and compulsive disorders characterized by dysfunctional motivation and habitual behavior. Immediate early genes (IEGs) are activated transiently and rapidly throughout the brain by many cellular stimuli including psychostimulants. Traditionally, IEGs are used as markers of neuronal activity, in striatum and other brain regions, in response to psychostimulants. The striatum consists of the dorsal striatum, which regulates actions and habits vs. ventral striatum (a.k.a.- nucleus accumbens- NAc), which is involved in motivation and reinforcement -∆FosB line crossed to a NSE-tetracycline transactivator (ttA) line resulted in expression of ∆FosB in striatal D1-MSNs  induction in striatum with acute psychostimulants is D1 receptor dependent (Daunais and McGinty, While studies examining psychostimulant-mediated IEG function in D1-MSN vs. D2-MSN subtypes are sparse, they have provided some insight into mechanisms by which IEGs act in these neuron subtypes. This includes actions primarily through D1-MSNs in psychostimulant-mediated molecular, cellular and behavioral plasticity. Overall we focus on three select IEGs that have been examined in MSN subtypes in psychostimulant action. However, examination of other psychostimulant relevant IEGs, such as Arc and CREB (Carlezon et al., MKL and RC both contributed to the writing of the manuscript, as well as the preparations of the figure and table.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Substitutional donor atoms in silicon are promising qubits for quantum computation with extremely long relaxation and dephasing times demonstrated. One of the critical challenges of scaling these systems is determining inter-donor distances to achieve controllable wavefunction overlap while at the same time performing high fidelity spin readout on each qubit. Here we achieve such a device by means of scanning tunnelling microscopy lithography. We measure anti-correlated spin states between two donor-based spin qubits in silicon separated by 16 ± 1 nm. By utilising an asymmetric system with two phosphorus donors at one qubit site and one on the other (2P−1P), we demonstrate that the exchange interaction can be turned on and off via electrical control of two in-plane phosphorus doped detuning gates. We determine the tunnel coupling between the 2P−1P system to be 200 MHz and provide a roadmap for the observation of two-electron coherent exchange oscillations. Donor impurities in silicon are promising candidates as qubits but in order to create a large-scale quantum computer inter-qubit coupling must be introduced by precise positioning of the donors. Here the authors demonstrate the fabrication, manipulation and readout of a two qubit phosphorous donor device. Physical systems ranging from trapped ions2, single photons3, superconducting circuits4 and semiconductor quantum dots5 have demonstrated this exquisite level of control. In 1998, Loss and Divincenzo6 proposed the use of a controllable exchange interaction in semiconductor quantum dots to perform a two-qubit logic gate. In the same year, Kane7 proposed how this could be achieved in donor-based devices. Here, the wavefunction overlap between two electrons on neighbouring donor atoms placed ~20 nm apart is controlled using an exchange gate between them. Harnessing this exchange interaction to perform a universal two-qubit quantum logic gate is the next step for donor-based architectures.Controlling the interaction strength between two quantum particles lies at the heart of quantum information processing. One must have access to classical control fields that, while tuning the environment of quantum particles, are sufficiently decoupled from them as to not disturb their quantum states8; the controlled-rotation (C-ROT) gate9 and a direct two-electron SWAP operation10. While the first two protocols require the use of high frequency microwave fields for electron spin resonance11, a direct two-electron SWAP necessitates the ability to turn on and off the exchange interaction between the electrons over orders of magnitude for high fidelity two-qubit operations. Notably, while the extent of a single donor wavefunction is well understood14, modelling the exchange coupling between two donor electrons is more complex17 due to multi-valley interference effects18. To this end, a critical challenge for donor-based architectures is to know the distance required between the donors in order to turn the exchange interaction on and off with external gates16.Three approaches exist for donor qubits: a controlled-phase (CZ) gate19 and atomic manipulation via scanning-tunnelling-microscopy (STM) hydrogen lithography20. Despite much success in accessing randomly placed donor spins, ion implantation has yet to demonstrate donor placement precision below ~6 nm, while STM lithography has demonstrated donor placement down at the atomic scale21.To date, two main methods for donor placement in silicon exist: ion implantationIn this paper we use STM lithography that allows both the precision placement of donor atoms for direct and independent spin-measurement of electrons near a readout structure and, most importantly, the control of the exchange interaction between them. We measure the anti-correlated spin states that arise due to the formation of two-electron singlet-triplet states as a function of their wavefunction overlap, which is controlled by in-plane detuning gates. By observing the onset of these anti-correlated spin states as a function of detuning pulse voltage and time, we estimate the magnitude of tunnel coupling between the two donor qubits, and provide a roadmap towards coherent exchange gates for future devices.7. Recent tight binding simulations have shown that it is difficult to tune the exchange energy in a 1P−1P donor configuration using such a gate22. Instead, it has been proposed that the exchange energy could be tuned over five orders of magnitude22 by confining electrons in an asymmetric 2P−1P configuration and by utilising ‘tilt’ control using two opposing detuning gates rather than a central J-gate; see Fig. In the original Kane proposal an exchange gate between the donors was suggested to directly tune the exchange coupling between the qubits24  charge sensor. The SET quantum dot is operated with a source-drain bias of 2.5 mV, has a charging energy of ~6 meV and is controlled predominantly via gate GS. All data in this paper was taken in a dilution refrigerator with a base temperature of ~100 mK (electron temperature ~200 mK).The device, shown in Fig. Figure 25. To measure the spin of R we employ an energy-selective tunnelling technique23 where the electrochemical potential of the single-electron transition from the 1 → 0 charge state is split by the Zeeman energy in a static magnetic field Bz; see blue arrows in Fig. The direct measurement of anti-correlated electrons hinges upon the ability to independently measure their spin states26. The charge transition for this qubit borders the 1 → 2 charge states, but because the chemical potential from the one-electron spin-up and -down states to the two-electron singlet state are also split by the Zeeman energy, a similar readout method is allowed . In this case, we utilise a spin-dependent loading mechanism from the SET to L. The combination of these two distinct readout techniques avoids the need to pulse over large voltages in order to reach the – charge transition. Both readout methods are equivalent and give rise to a current ‘blip’ through the SET which is used to discriminate between spin-up and -down electrons. The average readout fidelity of spin-up and -down are estimated to be 96.2 ± 1.1% and 97.6 ± 2.1% for qubit-L and -R respectively  charge region midway between the two readout positions  = tc). The detuning axis VGL = −0.9 VGR  and is shown by the white arrow in Fig. VGL via the lever arm VGL.The realisation of a two-qubit logic gate hinges on the ability to controllably turn on and off an interaction between quantum particles. We show this here by pulsing towards the – charge transition where an exchange interaction between the two electrons arises as a consequence of the Pauli-exclusion principle5, shown by the open black circles in Fig. Pij for ij ∈ {↑↑, ↑↓, ↓↑,↑↑}, as shown in Fig. We start by initialising either state from Eq.  by loadiρ↑↓ is initialised, after pulsing to P↑↓ ~ 0.5 and P↓↑ ~ 0, indicating no spin flip-flops have occurred during the 50 ms pulse duration. However, anti-correlated spins can be seen in Fig. P↑↓ and P↓↑ → 0.25. Furthermore, we see that both P↑↑ and P↓↓ remain constant at approximately 0 and 0.5 respectively as they represent populations of the triplet states ϕ = −0.243 ± 0.028 with a p-value In the case where tc, we repeat the same pulsing scheme as above while modifying the detuning pulse duration from 0.1 to 2 ms and compare our results to a spin-level theoretical model; see Fig. Hze and Hzn are the electron and nuclear Zeeman energies, with γe ≈ 28.024 GHz/T and γn ≈ 17.235 MHz/T gyromagnetic ratios respectively29. The hyperfine term, Hhf is separated into two components as it has been predicted that the hyperfine constants will be different for varying donor cluster configurations30. Here for simplicity we assume the bulk-like value of AL = AR = A = 117.53 MHz29 and define the static field to be To ascertain the value of the inter-dot tunnel coupling, ρ↑↓ from Eq.  charge region – inter-dot transition. From an electrostatic model of our device we estimate tc = 0.2 GHz  this makes the operation of coherent oscillations challenging31. Indeed, in this particular device we measured gate RMS voltage noise of 50 μV from shot to shot, equivalent to detuning noise of δIt can be seen from Fig. tc = 2.5 GHz after pulsing to a detuning 29Si Overhauser field expected in natural silicon32 as well as randomised P donor nuclear spins of the donor atoms themselves.Figure ν as well as the dephasing time τd, allowing us to determine the figure of merit ντd as a function of tunnel coupling and detuning pulse position; see Fig. ντd only becomes significant beyond the boundary ΔBz = 3A/2 for values of tc > 2 GHz, providing a lower bound on the required tunnel coupling for coherent control. Figure ντd as a function of tunnel coupling for a detuning pulse to the boundary ΔBz = 3A/2. These results indicate that at high tunnel coupling, the observation of exchange oscillations will ultimately be hindered by the presence of the fluctuating 29Si Overhauser field. In the case where qubits exist in a spin vacuum, as in 28Si, only charge noise is relevant and ντd can be seen to increase monotonically as a function of tunnel coupling – charge transition and represent the first direct measurement of correlated electron spins in donor-based devices. While the small tunnel coupling (0.2 GHz) in the present device prohibited measurement of coherent oscillations, we show our results agree with recent studies34. While extensive studies have been conducted for deterministic single P donor incorporation35, similar studies will need to be developed to determine the optimal lithographic patch for deterministic 2P incorporation. Crucially, recent theory predicts that the 2P−1P configuration we present in this paper both increases the tunability of the tunnel coupling and at the same time suppresses the ‘exchange fluctuations’ known for two single donors, and may therefore be less sensitive to the exact atomistic donor positions than two coupled single donors22. Furthermore, our ability to directly place the donor with <1 nm accuracy along with the reproducible demonstration of high fidelity single-shot spin-readout in multiple devices25, bode well for the future scalability of donor qubit quantum computers.With the atomic precision placement of donors using STM lithography it will be possible to further optimise the inter-donor distance to control the coherent coupling between two donor qubits with order-of-magnitude accuracy3 and annealed (320 °C) to incorporate P atoms into the silicon substrate36 with ~1/4 monolayer density (2 × 1014/cm2) allowing for quasi-metallic conduction in all electrodes37.The device, shown in Fig. L and GR by an arbitrary waveform generator (Agilent 81180A), connected via a bias tee to the gate along with a constant-voltage source. The SET current, ISET, was amplified and converted into a voltage signal at room temperature, low-pass filtered to 1 kHz bandwidth, and acquired with a fast digitising oscilloscope.For all electrical measurements, the device was mounted on a high-frequency printed circuit board within a copper enclosure, thermally anchored to the cold finger of a dilution refrigerator with a base temperature of 50 mK. Voltage pulses were applied to gates GThe data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


What is the relationship between children's thoughts and feelings about their donor and their security of attachment to their solo mothers in middle childhood?Children with higher levels of secure–autonomous attachment to their mothers were more likely to have positive perceptions of the donor, and those with higher levels of insecure–disorganized attachment to their mothers were more likely to perceive him negatively.There is limited understanding of the factors that contribute to children's thoughts and feelings about their donor in solo mother families. In adolescence, an association was found between adolescents’ curiosity about donor conception and their security of attachment to their mothers.19 children were administered the Friends and Family Interview and Donor Conception Interview between December 2015 and March 2016 as part of the second phase of a longitudinal, multi-method, multi-informant study of solo mother families.All children were aged between 7 and 13 years and had been conceived by donor insemination to solo mothers. Interviews were conducted in participants’ homes. The Friends and Family Interview was rated according to a standardized coding scheme designed to measure security of attachment in terms of secure–autonomous, insecure–dismissing, insecure–preoccupied and insecure–disorganized attachment patterns. Quantitative analyses of the Donor Conception Interview yielded two factors: interest in the donor and perceptions of the donor. Qualitative analyses of the Donor Conception Interview were conducted using qualitative content analysis and thematic analysis.r = 0.549, P = 0.015), and those with higher levels of insecure–disorganized attachment to their mothers were more likely to perceive him negatively . Children's narratives about the donor depicted him as a stranger (n = 8), a biological father (n = 4), a social parent (n = 3), or in ambivalent terms (n = 4).Statistically significant associations were found between the perception of the donor scale and the secure–autonomous and insecure–disorganized attachment ratings. Children with higher levels of secure–autonomous attachment to their mothers were more likely to have positive perceptions of the donor (Findings are limited by the wide age range of children within a small overall sample size. Participants were those willing and able to take part in research on donor conception families. The statistical significance of correlation coefficients was not corrected for multiple comparisons.Findings highlight the importance of situating children's ideas about the donor within family contexts. It is recommended that those working with donor conception families consider this when advising parents about whether, what and how to tell children about donor conception.This study was funded by the Wellcome Trust [097857/Z/11/Z]. The authors have no conflicts of interest to declare. There has been a worldwide increase in the number of single women accessing fertility treatment with donated gametes , or did not consent to participation (n = 1). All participants had been told about their donor conception, and the majority  were told this information before the age of 3; the remainder were told at ages 4 , 8  and 12 . Most children  were conceived using an anonymous donor; the remainder  were conceived using an identifiable donor. Ten children (53%) were male and 9 (47%) were female. Twelve children (63%) were singletons, five (26%) had one sibling also conceived through donor insemination. Two children (11%) had two siblings, one conceived naturally and one conceived through donor insemination. Four mothers (21%) were now in relationships, three (16%) of which were cohabiting, although all stated that they remained single parents.Interviews were conducted with 19 children aged between 7 and 13 years . Four children were not interviewed because they had not yet been told about the donor's role in their conception  interviewed children on their own at home. Each interview lasted ~1 h in duration. Interviews were transcribed and anonymised, and imported into the qualitative software program Atlas.ti.Children were administered a modified version of a semi-structured interview (Four variables were created from the interview schedule: (i) ‘description of donor’, (ii) ‘feelings about donor’, (iii) ‘thoughts about donor’ and (iv) ‘questions for donor’, and rated according to a standardized coding scheme. Description of donor and feelings about donor were each rated on a three-point scale ranging from ‘1’ negative through ‘2’ neutral to ‘3’ positive. Thoughts about donor was rated on a three-point scale from ‘1’ no through ‘2’ sometimes to ‘3’ yes. Questions for donor was rated on a four-point scale ranging from ‘1’ no questions through ‘2’ questions about the conception process and ‘3’ questions about the donor to ‘4’ questions about a relationship with the donor. Rating reliability for the ‘description of the donor’ and ‘feelings about the donor’ scales was ensured by the qualitative content analyses described below.r = 0.72, P < 0.001) and between thoughts about donor and questions for donor . Based on these findings, two scales were created. The first, perception of the donor, comprised description of donor and feelings about donor. The second, interest in the donor, comprised thoughts about donor and questions for donor. Higher scores on these scales indicated more positive perceptions of, and greater interest in, the donor, respectively.Pearson correlation coefficients were calculated to examine relationships between the four variables. A significant positive association was found between description of donor and feelings about donor (n = 5) were discussed until total agreement was reached.Qualitative content analyses  conducteChildren were administered a modified version of the FFI , a semi-Interviews were coded using the FFI Rating and Classification System  were unable or unwilling to do so. Explanations ranged from describing the fertility clinic or hospital, to mentioning the donor, to details about the conception process:There's a special part in the hospital where you can go if you don't have a boyfriend … so you can just go ask … if people volunteer to help make a baby. (11-year-old boy)I was made by a donor, that's about all I know. (9-year-old girl)To make a baby you need sperm from a man and an egg from a woman and some very kind men helped my Mum make me and they donated their sperms. (9-year-old girl)Overall, 14 children (74%) were able to explain at least some aspect of donor conception; the remainder , confused , surprised , neutral  or positive :I didn't really know what my Mum was talking about. I thought she was talking gibberish. (10-year-old boy)I just remember accepting it and moving on. (12-year-old girl)Twelve children (63%) reported remembering being told about their donor conception, and 9 (48%) recalled initial responses to this information: feeling different (Of the seven children (37%) who could not remember being told, three (16%) reported that they had always known.n = 10, 52%) reported that they did not think about the donor. Six children (32%) said they thought about him only occasionally, and three (16%) reported that they did think about him:Yeah, before I heard the description of him, I always imagined him to have black hair, a moustache, blue eyes, that's how I always imagined him. (8-year-old boy)Approximately half of the children I don't really know [what sort of person he is]. I don't really care. Probably not a trustworthy one … because of his job. (12-year-old girl)Negative descriptions included:I really don't think he'd be the best Dad because if he doesn't even want to see his own child then I don't think he'd be the best. (9-year-old girl)I just feel like I want to be with him, want him to live with us. (8-year-old boy)Negative feelings included:He's probably quite a kind person … the sort of person who wants to help people. (11-year-old girl)I think he would be kind, he would be caring, he would be nice, maybe loving. (8-year-old boy)Positive descriptions included:I kind of admire him I suppose. (11-year-old girl)I feel that he helped me a lot to be alive. (9-year-old girl)Positive feelings included:n = 15, 79%) had at least one question for the donor  of children's donor narratives drew upon notions of biological and social parenthood. Some children  distinguished between the donor's role as a biological contributor and the role of a social parent, while others appeared to either confuse  or entirely conflate  these roles. Conversely, several children  described the donor in minimal terms, as neither a biological contributor nor social parent. Narratives thus corresponded to one of four subthemes on a continuum: (i) donor as stranger, (ii) donor as biological father, (iii) ambivalence towards donor and (iv) donor as social parent.More than half These narratives were generally brief. They did not include a description of the donor as either a biological or social parent, and did not identify him as family:I've no idea who they are … I actually wonder who he is. (13-year-old boy)I don't know what he's like because I've never met him. (8-year-old girl)Some narratives were positive, describing the donor as ‘kind’ and ‘helpful,’ while others were either neutral or negative. For some children, answering questions about what the donor might be like seemed challenging:It's just a guy's characteristics and parts apparently … Whenever [other people] ask I just say ‘don't have a Dad’. (12-year-old boy)Other children stated that they did not have a father:He has dark features, dark eyebrows, eyes … Mum says you have his eyes or hair. And we wouldn't have these kind of features if our Dad was blonde. (12-year-old boy)Apparently it was a man that was tall, that's probably why we're tall. (12-year-old girl)Narratives about the donor as a biological father often emphasized his physical characteristics:I think he's quite a lively and musical person who enjoys going outside and running around and being all crazy and whatever because that's a bit like me. (12-year-old girl)One child also described the donor's personality on the basis of her own:If people ask something about my Dad … They ask ‘Do you know him?’ and I probably say ‘no’ or something like, ‘but I have a sheet that says about him.’ (10-year-old boy)These narratives generally described the donor as ‘technically my Dad in a way’, ‘sort of’ or ‘kind of like family’. One child mentioned using information provided by the fertility clinic when discussing the donor:I felt fine [when first told] because I was used to not having a Dad and I didn't really care … I wish I could just meet him … because he's my Dad after all. (13-year-old girl)It's normal for me because I'm just used to not having a Dad … I don't really want to meet him because if I do I'll probably just miss him, because I guess he is kind of my Dad. (11-year-old boy)Ambivalent narratives often included multiple interpretations of the donor, and mixed feelings about him and his status as a father:I'd describe him as a half Dad like he's half not my Dad but he half is my Dad. (11-year-old boy)My Dad … He's not really family, maybe genetically or biologically whatever you say, not really my family. (10-year-old boy)Children were also ambivalent as to whether or not the donor was family:Mum said that one day when I'm older I could find my biological Dad … I feel quite happy because I kind of like not having a Dad. (10-year-old girl)In one narrative, mixed feelings about the donor were discussed in relation to making contact with him in the future:I think there are eight others so far that are the same as me, same Dad … He was quite kind because … he wanted to make life even though he didn't want to see us for some reason. I can't think of him really … I don't know if I would like him or hate him if I did see him … he would have to be shared out between about 13 other kids, so. (10-year-old boy)In another, ambivalence was highlighted in relation to donor siblings:I don't really think he'd be the best Dad because he doesn't want to be … I'd still call him Dad. (9-year-old girl)I know what I feel like but I also don't know because I really want to meet him someday but I also don't … because I'm not sure what he'll be like and yeah, I'm not sure if he will want to see me. (9-year-old girl)Narratives in this category were likely to mention the possibility of a social relationship with the donor, or the current lack thereof, with multiple feelings expressed:Now you have to be 18 to see him, but when I'm 18 I want to see him and I want to meet some of my half-brothers and sisters. (9-year-old girl)[I'd ask him] ‘Where do my other brothers and sisters live? And are you going to live here?’ (8-year-old boy)These narratives were also likely to describe the donor as family. Two referred to donor siblings:r = 0.549, P = 0.015), showing that children with higher levels of secure-autonomous attachment had more positive perceptions of the donor. In addition, a significant negative association was found between the insecure–disorganized scale of the FFI and the perception of the donor scale , reflecting more negative perceptions among children with higher levels of insecure–disorganized attachment.Pearson correlation coefficients were calculated between children's attachment ratings on each of the four dimensions of the FFI coding scheme and children's scores on the perception of the donor and interest in the donor scales to examine the relationship between attachment security and children's perceptions of, and interest in, the donor Table . A signiFindings suggest that in middle childhood, children in solo mother families are diverse in their thoughts, feelings and narratives about the donor; while some children feel positively, others are neutral or negative in their evaluations. Additionally, although some children do not appear to have thoughts about or questions for the donor, others seem curious about him. Children's questions for the donor ranged from those focussed on the possibility of a relationship, to wanting identifying, or non-identifying, information. These findings—both about the diversity of children's perceptions of the donor, and their differing levels of curiosity about him—are in keeping with what is known about the perspectives of donor-conceived children at a similar developmental stage in both same-sex and opposite-sex two-parent families (This study also investigated the relationship between children's ideas about the donor and their patterns of attachment to their mothers. In middle childhood, children with secure-autonomous attachments continue to perceive their caregivers as both reliable and responsive to their needs, while those with insecure–disorganized attachments exhibit behaviours that indicate that a consistent strategy towards their caregivers is lacking . ChildreChildren's interviews were also analysed qualitatively, illustrating that for some children, the donor remains a stranger about whom little is known or thought, while for others, he is a biological relative, who may be responsible for some of their traits. Some children seem to find positioning the donor difficult, and thus describe him in ambivalent terms, as at once a biological and social parent. For a minority, the donor is primarily understood in a social role, and these children are likely to report wanting to meet him, and/or other children conceived using his gametes. Notions of biological and social parenthood are also found in the donor narratives of children in same-sex two-parent families (Understanding the factors that contribute to particular narratives about the donor among the present sample is challenging. While previous research has established that age at disclosure is important for perceptions of the donor and donor conception (It seems clear that for some children, ideas about the donor are shaped by information shared by their mothers. Findings may thus be said to reflect the power asymmetry of parent–child relationships at this developmental stage . FurtherAlthough middle childhood remains an understudied developmental stage in research on attachment , finding


Thus, the present study explored how groove was felt and defined, as well as how musical factors induced the sensation of wanting to move in cross-cultural context. A listening experiment was conducted using drum breaks as stimuli. Stimuli consisted of various rhythm patterns at six tempi from 60 to 200 BPM. The main findings are that: (1) an optimal tempo for groove existed for drum breaks at around 100–120 BPM, (2) an optimal tempo existed for the sensation of wanting to move the body in specific directions , (3) groove and nori shared a similar concept of wanting to move but differed on several points . Overall, the present study suggests that there is an optimal tempo for body movement related to groove. This finding has implications for the use of music or rhythmic stimuli to induce smooth motion in rehabilitation, therapy, or dance.The tendency for groove-based music to induce body movements has been linked to multiple acoustical factors. However, it is unclear how or whether tempo affects groove, although tempo significantly affects other aspects of music perception. To address this issue, the present study investigated effects of tempo, specific rhythmic organizations of patterns, and syncopation on groove and the induction of the sensation of wanting to move. We focused on the directions of body movement in particular by taking into account  Movements such as body sway, head nodding, and foot tapping frequently emerge while listening to music in our daily lives. The feeling that induces body movement has been defined as groove , a parallel that speaks to the potential generality of the concept groove across different musical and cultural traditions. Nori is frequently described as groove  to Japanese people. In music, nori relates not only to groove, but also to rhythm or beat  and yoko-nori . In Japanese, tate means “vertical” and yoko means “horizontal.” In general, vertical nori, which includes bouncing or a “headbanging”-like back-and-forth movement, is used to embody the rhythm of pieces with fast tempi, such as pop, rock, and punk . This type of movement includes the anteroposterior movement that was measured by Toiviainen et al.  an optimal tempo for groove exists, (2) an optimal tempo for inducing the sensation of wanting to move depends on directions of movement , and (3) groove and nori signify concepts that partially overlap.The present study aimed to investigate the influence of tempo on the groove in musical rhythms. As mentioned earlier, the results of previous studies using commercially available music imply that an optimal tempo for groove, or phenomena related to groove exist. By using simple drum breaks, we isolated effects of tempo while excluding other factors  that could potentially influence groove in commercial recordings. Drum breaks were created based on rhythm patterns and degrees of syncopation that were found to influence groove in previous studies, with the additional manipulation of tempo over a wide range of values. Furthermore, by focusing on associations between groove and the direction of body movement, the present study also aimed to shed light on the relationship between the Western concept of groove and the Japanese concept of All participants were informed as follows: their participation was voluntary; they were free to leave if they felt uncomfortable; absence or withdrawal of participation provided no disadvantage for participants. The data were anonymized. Following a presentation of oral and written instructions, all participants agreed and signed a letter of consent.Thirty-eight participants  aged 18 to 40 years , all majoring in music at Tokyo University of the Arts, were recruited for the study. Mean musical experience was 15.00 years (SD = 5.77). The groove ratings from three participants were discarded because these individuals had missing responses or responded in a way that suggested that they did not understand the concept of groove, when replying to the question “What types of performance do you think are groovy?” (Q. 1 in Appendix).Thirty drum breaks were employed as stimuli. Each drum breaks comprised hi-hat, snare drum, and bass drum sounds, which were synthesized using GarageBand . To create different types of stimuli, five different rhythm patterns were used as bases for the drum breaks Figure . The 30 The number of hi-hat, snare drum, and bass drum sounds used in each drum break was the same across the patterns, since imbalances in the number of different tones in the drum breaks could affect perceived complexity. Each stimulus consisted of eight hi-hat, two snare, and three bass drum attacks in a bar.To minimize potential causes of variance, only the position of the bass drum sounds relative to the patterns' quadruple metrical structure (four quarter notes per bar) was manipulated to create the different rhythm patterns. The position in the pattern of the hi-hat and snare drum was the same for each stimulus.The bass drum sounded on the first beat in all stimuli. This configuration was intended to help the participants perceive the sense of meter as easily as possible.Each drum break had different degrees of syncopation, which were calculated based on the Witek et al.  index thThe differences between 8th note rhythm patterns and 16th note rhythm patterns were created by shifting the note position of the bass drum by a 16th note. The degree of syncopation was similar across patterns . However, due to the controlled stimuli conditions, it was not possible to create 8th note rhythm patterns with a high level of syncopation. Therefore, we compared low and mid-level of syncopation separately for 8 and 16th note rhythm patterns.Each drum break was presented at six different tempi: 60, 75, 100, 120, 150, and 200 BPM. These tempi were selected to fit within natural musical tempo range, in accordance with studies of Moelants  and Parnnori, questions verifying the hypothesis that the groove ratings in Japan are similar to those in studies conducted in Western countries, and a question about back-and-forth and side-to-side movements related to nori. As Table nori , 3.5 m from the closest seat, using iTunes for playback. Before the experiment, two drum breaks that were not used in the experiment were presented as practice. Participants also responded the open-ended questionnaires (Appendix). During the listening test, participants were instructed to listen to the stimulus for at least one bar, and then start rating the stimulus. Since each stimulus contained repeated presentations of the same rhythm pattern, most participants made their rating before the end of each stimulus. Each trial was about 40 s. The whole experiment lasted ~25 min.The experiment was conducted in a soundproof room at Tokyo University of the Arts. Because of the capacity of the room, we conducted the experiment in seven sessions, with three to eight participants tested in each session. Participants were seated in the room and listened to 30 drum breaks (five patterns × six tempi) in random order and rated each stimulus with pencil and paper according to 11 items , we applied linear regression analysis and quadratic regression analysis, and then compared the sum of squared error (SSE) of each model.Secondly, to investigate whether optimal tempo differs among rhythm patterns for groove, and differs between the sensation of wanting to move back-and-forth and side-to-side, we applied Monte Carlo simulation. The procedure is as follows.B2 and B are known , we conducted t-tests (two-sided) to investigate whether the optimal tempo between each rhythm pattern, and between back-and-forth and side-to-side is significantly different.Likewise, the distribution of the difference between the optimal tempo of each rhythm pattern, and between the optimal tempo of wanting to move back-and-forth and side-to-side can be calculated. To this end, after obtaining the distribution of the difference in optimal tempo between each rhythm pattern by using a Monte Carlo simulation  = 11.244, p < 0.01, ηp2 = 0.249; Greenhouse-Geisser correction under the assumption of the violation of sphericity] and tempo  were statistically significant. The interaction was also significant . This result suggests that the influence of tempo on groove rating depended on rhythm patterns. As Figure The mean rating for the item “Performance with high groove” for each stimulus , calculated across participants, is displayed in Figure mpo [F2.81, 96.251p-value was multiplied by the number of comparisons). Generally, the groove ratings for around 100–120 BPM were significantly higher than those for fast or slow tempi. In addition, the groove ratings for 16_mid were higher than for other stimuli. Multiple comparisons revealed that drum breaks performed at 200 BPM showed lower ratings than those at other tempi (p < 0.05). Drum breaks performed at 60 BPM showed lower ratings than those at 100 and 120 BPM (p < 0.01). Drum breaks performed at 120 BPM showed higher ratings than those at 150 BPM . The ratings for 8_low were significantly lower than the ratings for the other four rhythm patterns (p < 0.05). The ratings for 16_mid were significantly higher than the ratings for 16_low and 16_high (p < 0.05).Subsequently, multiple comparisons employing 6 tempi and 5 types of rhythm pattern were conducted using the Bonferroni correction . In addition, the value of B2  was negative and statistically significant for all patterns (ps < 0.01). These results suggest the existence of an optimal tempo for each rhythm pattern. Regarding the quadratic regression, the peak of the regression curve indicates that the predicted optimal tempo is from 107 to 126 BPM, although the adjusted R2 in this analysis was small.To investigate whether an optimal tempo for groove existed, linear and quadratic regression analyses employing groove ratings as dependent variables were conducted for each rhythm pattern using 210 data points i.e., 35 participants' ratings for each rhythm pattern at the 6 tempi Figure . As a reWe applied Monte Carlo simulation to investigate whether the optimal tempi differ among rhythm patterns . However, no significant difference was found.p < 0.05) except 8_low, both in back-and-forth and side-to-side scenarios . In addition, the value of B2 for all patterns except 8_low was negative and significant (p < 0.05). These results suggest that optimal tempi for inducing the sensation of wanting to move for 8_mid, 16_low, 16_mid, and 16_high exist. In all rhythm patterns, the estimated optimal tempi based on the quadratic regression for back-and-forth scenarios were faster than those for side-to-side, with the exception of 8_low.To test hypothesis 2, we investigated whether an optimal tempo that induces the sensation of wanting to move existed. Linear and quadratic regression analyses were conducted for each rhythm pattern, setting tempo as an independent variable and the ratings as the dependent variable. Next, the goodness of fit was compared between the two models. The ratings of “wanting to move my body back-and-forth” and “wanting to move my body side-to-side” were employed Table . The sumAgain, we applied Monte Carlo simulation to investigate whether the optimal tempi differ between back-and-forth and side-to-side . However, again, no significant difference was found.nori, Pearson's correlation coefficients between the ratings for “Performance with high groove” and “This playing has a good “Nori”” . “I feel like my body resonates with the rhythm” showed the highest correlation, also positive, with groove ratings. “I want to move my body” and “pleasure” also showed high positive correlations, and the correlation with nori was statistically significant. The ratings for nori were also highly (positively) correlated with items related to wanting to move. However, the ratings for nori showed low correlations with items related to pleasure and no significant correlation with items related to pulsing. Furthermore, nori was positively correlated to a fast tempo  and dancing . In addition, the results of open-ended questions on groove revealed that nori was mentioned most frequently as an alternative term for groove, although participants explained groove with terms such as body movement, nori, timing deviation, and a sense of unity (Appendix).To investigate the relationship between groove and nori. The main findings are as follows: (1) an optimal tempo for groove induction existed for drum breaks at around 100–120 BPM, (2) an optimal tempo for the sensation of wanting to move in different directions , (3) groove and nori shared a similar concept of wanting to move but differed on several points .The present study tested the hypothesis that there is an optimal tempo for both groove and the induction of the sensation of wanting to move by musical rhythms. To address this issue, the present study differed from previous work in two aspects: by varying both tempi and rhythmic patterns in a controlled fashion, and by focusing on the relationship between tempo and groove over a relatively wide tempo range. A secondary aim was to investigate associations between groove and the sensation of the direction of body movement with regard to the concept of First, the present study provided evidence for the existence of an optimal tempo for groove. The results showed inverted U-shaped relationships between groove rating and tempo. Groove ratings were low for extremely slow or fast tempi, whereas groove ratings were high at around 100–120 BPM. The estimated optimal tempo for groove was 107–126 BPM. This result is in accordance with previous tempo ranges associated with groove, such as 115–144 BPM  and yoko-nori, namely the side-to-side direction of body movement, which is related to slower tempo in Japan  but also for preferred directions of movement. Our findings also suggested that tempo affects preferences for vertical or horizontal directions of movement, while previous studies suggested that changes in pitch and loudness were related to vertical motion   Nori, , n.d.a, m) Nori,  are simim) Nori, . Accordinori are not exactly the same. The correlation between nori and “I feel pleasure” was lower than the correlation between groove and pleasure. In addition, neither the significant positive correlation between nori and “fast tempo” nor the absence of a correlation between nori and pulsing were similar to the correlation between groove and those items. Moreover, this study's results regarding nori accord with the qualitative definition of nori, which involves the factors of fast tempo  to examine what factors determine optimal tempi for groove. It is also necessary to examine associations between optimal tempo and other factors of groove such as micro-timing (Kilchenmann and Senn, SK designed the study. TE and AM collected data. SK and TE analyzed and interpreted data. SK, TE, and PK drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The role of cardiac troponins as diagnostic biomarkers of myocardial injury in the context of acute coronary syndrome (ACS) is well established. Since the initial 1st-generation assays, 5th-generation high-sensitivity cardiac troponin (hs-cTn) assays have been developed, and are now widely used. However, its clinical adoption preceded guidelines and even best practice evidence. This review summarizes the history of cardiac biomarkers with particular emphasis on hs-cTn. We aim to provide insights into using hs-cTn as a quantitative marker of cardiomyocyte injury to help in the differential diagnosis of coronary versus non-coronary cardiac diseases. We also review the recent evidence and guidelines of using hs-cTn in suspected ACS. A rapid and accurate diagnosis is critical in patients with presumed acute coronary syndrome for the initiation of effective evidence-based medical management and revascularization. The third universal definition of myocardial infarction defines an acute myocardial infarction (AMI) as evidence of myocardial necrosis in a patient with the clinical features of acute myocardial ischemia, and defines the 99th percentile of cardiac troponins as the decision value for AMI . ClinicaThe aim of the current article is to review the history and evolution of cardiac biomarkers of acute coronary syndrome, define what troponin is, and aid in the use of the hs-cTn in clinical practice according to current guidelines.Aspartate Transaminase (AST) became the first biomarker used in the diagnosis of AMI  Fig. 1)1). AST wIn 1965, a new protein constituent of the cardiac myofibrillar apparatus was discovered, which subsequently came to be known as troponin . In the troponin T attaches the troponin complex to the actin filament;troponin C acts as the calcium binding site;troponin I inhibits interaction with myosin heads in the absence of sufficient calcium ions.Troponin is a component of the contractile apparatus within skeletal and cardiac myocytes. Along with calcium ions, troponin proteins regulate and facilitate the interaction between actin and myosin filaments as part of the sliding filament mechanism of muscle contraction. Cardiac troponin (cTn) is a complex comprising three subunits:Troponin C is synthesised in skeletal and cardiac muscle. Troponin T and I isoforms are highly specific and sensitive to cardiac myocytes and, therefore, are known as cardiac troponins (cTn). The detection of cTn-T or cTn-I in the blood stream is, therefore, a highly specific marker for cardiac damage . 92–95% The plasma half-life of cTn is around 2 h. Although the precise mechanism by which troponin is eliminated from the body remains unclear, it is hypothesized that troponin is cleared, at least in part, by the renal reticulo-endothelial system .Most hospitals now have replaced conventional cTn tests with the new 5th generation hs-cTn T and I assays which can detect troponin at concentrations 10- to 100-fold lower than conventional assays Fig. . VariousThere is a misconception that troponin elevation is secondary only to myocyte injury and necrosis. There are six mechanisms that have been proposed to explain the release of troponin into the bloodstream: normal cell turnover, myocyte necrosis, apoptosis or programmed cell death, proteolytic fragmentation, increased cell membrane permeability and membranous blebs.Whether or not cTn is detectable, or even elevated, is therefore dependent on the balance of a host of interdependent factors, including the sensitivity of the test. Furthermore, there are potentially other, as yet not described mechanisms involved in the release of cTn. For example, it is still unknown why cTn is elevated in certain extra-cardiac disease processes such as sepsis. Whether ischemia causes elevated cTn in the absence of myocyte necrosis remains controversial . Some anThere is a risk of misinterpretation of elevated troponin results. Almost 13% of patients presenting with raised hs-cTn and chest pain eventually prove not to have ACS . Hs-cTn More sensitive cTn assays also maintain high diagnostic accuracy in patients with renal dysfunction when assay-specific higher optimal cutoff levels are used . CurrentBoth the European Society of Cardiology (ESC) guidelines on the definition of AMI and suspected ACS endorse the use of hs-cTn assays , 40. TheTo maintain a high specificity, it is important to distinguish acute from chronic hs-cTn elevation. Acute causes of hs-cTn elevation are associated with a corresponding significant rise or fall of hs-cTn. Acute cardiomyocyte injury causes a steep release of troponins, such as in AMI, shock, myocarditis, pulmonary embolus, Tako-tsubo (stress-induced) cardiomyopathy. Chronic, stable elevations of hs-cTn at or above the 99th percentile without a significant rise or fall are common in patients with structural heart disease . In thesTo differentiate acute from chronic troponin elevation and to maintain a high specificity, clinical evaluation (pre-test probability) and serial testing of hs-cTn are warranted. Various rule-in and rule-out algorithms have been proposed using different time points and cutoff values, including the question whether absolute or relative hs-cTn changes should be used. The use of any of these change criteria increases specificity (at the price of slightly decreased sensitivity) . DespiteOptimal cutoffs for (absolute and relative) changes and the earliest time points of the second hs-cTn measurement will have to be determined for each assay and clinical background  separately and are the subject of current research.Figure Due to a more atypical presentation, women remain a challenging group with regard to the diagnosis of myocardial infarction. Studies regarding gender-specific lower thresholds for women for the diagnosis of acute myocardial infarction have not shown consistent results: Shah et al. proposed that women-specific lower diagnostic thresholds for hs-cTn may double the diagnosis of myocardial infarction in women, and identify those at high risk of re-infarction and death . On the Accelerated diagnostic protocols using hs-cTn assays have now been widely proposed , endorseMore recently, several studies have tested lower cutoffs for hs-cTn for ruling out AMI , 48, 50.Cardiac biomarkers for diagnosis of AMI have become more and more sensitive in recent decades. The currently used hs-cTn assays are highly valuable for rule-in and rule-out of AMI. International guidelines have been published for appropriate use of hs-cTn. Acute changes in hs-cTn complement the quantitative information provided by hs-cTn, and help in the differential diagnosis of diseases with chronic, stable troponin elevations vs. diseases with acute troponin elevations and acute cardiac damage. Serial testing of hs-cTn does not differentiate Type 1 from Type 2 myocardial infarction and, hence, integrating the results of hs-cTn measurements with robust clinical assessment remains the optimal approach.


Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity of the pathogen in its host environment. However, it is yet largely unknown how the streptococcal metabolism adapts to the different host niches encountered during infection. Our previous isotopologue profiling studies on S. suis grown in porcine blood and cerebrospinal fluid (CSF) revealed conserved activities of central carbon metabolism in both body fluids. On the other hand, they suggested differences in the de novo amino acid biosynthesis. This prompted us to further dissect S. suis adaptation to porcine blood and CSF by RNA deep sequencing (RNA-seq). In blood, the majority of differentially expressed genes were associated with transport of alternative carbohydrate sources and the carbohydrate metabolism . In CSF, predominantly genes involved in the biosynthesis of branched-chain and aromatic amino acids were differentially expressed. Especially, isoleucine biosynthesis seems to be of major importance for S. suis in CSF because several related biosynthetic genes were more highly expressed. In conclusion, our data revealed niche-specific metabolic gene activity which emphasizes a selective adaptation of S. suis to host environments. Streptococcus suis is an important facultative pathogen in pigs and is considered a neglected zoonotic pathogen glucose in isotopologue profiling experiments, we were able to analyze de novo amino acid biosynthesis of S. suis in porcine blood or CSF. We found that the central carbon metabolic activity was almost similar in both body fluids. However, these studies suggested different activities of the amino acid metabolism in CSF. For instance, the branched-chain amino acid (BCAA) isoleucine seemed to be preferentially synthesized de novo by S. suis in CSF but not in blood. BCAA biosynthesis is a tightly transcriptionally regulated metabolic pathway in bacteria [S. suis in more detail. For this, the S. suis serotype 2 strain 10 was incubated in porcine blood or CSF for six hours according to our previous study [In a previous study we analyzed the primary carbohydrate metabolism of bacteria . This prus study  and globOur experiments revealed 434 and 422 differentially expressed genes in porcine blood and CSF, respectively, when compared to Todd-Hewitt broth (THB) media. In blood, 356 and 78 genes were higher-expressed and lower-expressed, respectively; whereas in CSF we found 206 higher-expressed genes and 216 lower-expressed genes . FurtherS. suis, the EMP pathway is preferred for the metabolism of glucose [SSU0181, SSU0182, zwf (SSU1025), rpe (SSU1785), and deoB (SSU1269), showed increased expression  needed for the synthesis of adenine, guanine, and uracil were more highly expressed in porcine blood than in THB medium. In contrast, in CSF only two genes associated with PPP  and purine biosynthesis genes  were more highly expressed. Remarkably, pyrimidine biosynthesis genes (carAB and pyrBCDER) were also more highly expressed when compared to THB media, suggesting an enhanced need for uracil with a prolonged time of incubation in CSF. Overall, these data suggested differences in the sugar and nucleotide metabolism when S. suis was grown in blood or CSF.In order to generate energy, imported carbohydrates need to be catabolized by the Embden–Meyerhof–Parnas (EMP) pathway or the pentose-phosphate pathway (PPP). In  glucose . Our RNApression . This obN-acetylgalactosamine, mannose, fructose, or ascorbate, or ATP-binding-cassette (ABC) transporters such as msmK (SSU1701), which encodes for an ATPase subunit of ABC transporters and thereby contributes to the utilization of multiple carbohydrates and the host colonization of S. suis [Streptococcus agalactiae in which a higher expression of multiple sugar transport systems was also observed when bacteria were grown in human blood [galK, galET) which are involved in the formation of glucose-1-phosphate from galactose. Glucose-1-phosphate may also originate from the degradation of maltodextrins which might be transported by an ABC transporter  after enzymatic digestion of more complex α-glucans, such as glycogen by an amylopullulanase (apuA) [glgABC or into the EMP pathway after conversion to glucose-6-phosphate. Overall, the increased expression of genes involved in carbohydrate uptake and conversions might indicate a lack of glucose as a primary sugar source and trigger the uptake of alternative carbohydrates. Indeed, several of these genes have been found to be part of the regulon of the transcriptional regulator catabolite control protein A (CcpA) which is an important regulator of glucose-mediated carbon catabolite repression and activation of metabolic genes in S. suis [S. agalactiae and Streptococcus pyogenes were incubated in human blood for much shorter time periods after which glucose should not yet be exhausted [Furthermore, we found that most of the differentially expressed genes in blood 17.5%) encode for proteins involved in the carbohydrate metabolism and transport (G)  which po S. suis . These dan blood . In addi.5% encode (apuA) . Most li S. suis ,20. On txhausted ,13. ThesS. suis first has to overcome amino acid starvation. Thus, we found that after growth in CSF, 14.42% of differentially expressed genes were assigned to amino acid metabolism and transport (E) (SSU1360-SSU1364), for a polar amino acid ABC transporter , and for a peptide transport system (SSU1660-SSU1664) were increased in expression, indicating the demand of these sources by S. suis grown in CSF. Determination of the amino acid concentrations in porcine CSF (SSU1192-1195 (glnQ4) and SSU1851-1853 (glnQ5) encoding for two putative glutamine ABC transporter systems was decreased  and threonine  were highly expressed compared to control and blood conditions  of the shikimate pathway, important for aromatic amino acid biosynthesis as shown previously by isotopologue profiling [In contrast to blood, in CSF only a few genes (6.62%) of carbohydrate metabolism and transport (G)  were affport (E) . Respectcine CSF  revealedecreased B. Thus, xpressed ,24. Acconingitis . Notably leucine . This mino acids . Neverthnditions B, indicarofiling , were inS. suis serotype 2 strain after growth in porcine blood or CSF. In blood, S. suis seems to be sufficiently provided with amino acids, whereas a broader spectrum of carbohydrates is used and PPP gene expression is increased. In contrast, in CSF the requirement for amino acids seems to dominate S. suis adaptation as genes associated with amino acid transport and biosynthesis, such as those belonging to branched-chain or aromatic amino acid synthesis, were increased in expression. Overall, our results revealed a transcriptional adaptation of S. suis to porcine blood and CSF including environment-specific changes of metabolic gene expression in these host environments.In conclusion, we observed specific transcriptional profiles of a ® syringe filter), and frozen at −80 °C until use. S. suis strain 10  was subcultured on Columbia blood agar plates containing 6% (v/v) sheep blood (Oxoid). For growth experiments overnight cultures in Todd-Hewitt broth (THB) were adjusted to an OD600 of 0.02 in fresh THB on the next day. Bacteria were grown to early-log phase (OD600 of 0.2), pelleted, washed in PBS, and resuspended to approx. 108 CFU/mL. Ten mL blood or 2 mL CSF were inoculated with S. suis to concentrations of 1 × 107 or 1 × 104 CFU/mL, respectively. Bacteria were incubated under gentle rotation at 37 °C [g for 5 min at 4 °C. The bacteria-containing supernatant was placed in a new collecting tube and centrifuged at 15,550 × g for 5 min at 4 °C to pellet the bacteria. Bacteria grown in CSF were pelleted directly from the fluid by centrifugation at 15,550 × g for 5 min at 4 °C. As a control, S. suis strain 10 was grown in THB medium to an OD600 of 0.2, representing the early-log phase of bacterial growth [Heparinized porcine blood was drawn from healthy pigs, as registered at the Lower Saxonian State Office for Consumer Protection and Food Safety (permit number: 33.9-42502-05-11A137). The collection of blood samples was performed in line with the recommendations of the German Society for Laboratory Animal Science and the German Veterinary Association for Protection of Animals. CSF was taken from clinically healthy pigs and done according to the registered Lower Saxonian State Office for Consumer Protection and Food Safety with the permit number 33.9-42502-04-08/1612. Collected CSF samples were pooled, sterile filtered . The cells were then disrupted three times for 45 s at intensity settings 6.5 by using a FastPrep® instrument  and cooled on ice. Furthermore, RNA was separated from DNA as well as proteins by chloroform. RNA was precipitated by 2-Propanol and purified by using the RNeasy Mini kit (Qiagen) according to the instructions of the manual that includes a treatment with DNase. RNA quality was verified by gel electrophoresis  and then further processed by the MICROBEnrich™ Kit  according to the manufactures recommendation. Sequencing libraries of two independent biological replicates were prepared and sequenced using 50 bp single-ends sequencing on a HiSeq2500 instrument . In brief, libraries of 300 bp were prepared according the manufacturer’s instructions “TrueSeq RNA Sample Prep Guide” (Illumina). Protocol fragments the RNA, synthesizes first- and second-strand cDNA and ligates adapters to the ends of the cDNA fragments. After PCR enrichment of cDNA fragments, purification with AMPure beads XP and QC on Bioanalyzer/Qbit templates go on the Illumina HiSeq2500 Sequencer system. The fluorescent images were processed to sequences and transformed to FastQ format using the Genome Analyzer Pipeline Analysis software 1.8.2 (Illumina). The sequence output was controlled for general quality features using the fastq-mcf tool of ea-utils [S. suis P1/7 using BWA version 0.7.5 [q-value ≤ 0.01 were considered as significantly differentially expressed and genes with raw counts in all replicates were included for further analysis.Total RNA from e et al. . Brieflyea-utils  and was on 0.7.5  and SAMton 0.7.5 . Genes wFor verification of the RNA-seq data RT-qPCR was performed as previously described .For the determination of amino acids in porcine CSF and serum samples, 0.25 mL of sulfosalicylic acid was added to 1 mL of CSF or serum, intensively mixed, chilled for 30 min at 4 °C and centrifuged for 10 min at 3000 rpm. The supernatant was mixed 1:1 with sample diluent buffer. Following, amino acids were determined by ion-exchange chromatography  and results evaluated according to established methods .Raw data sets are available in the European Nucleotide Archive Repository (PRJEB14724).