Separation and detection of D-/L-serine by conventional HPLC.

D-serine has a role as an endogenous allosteric agonist of N-methyl-D-aspartate (NMDA) receptor in the mammalian brain. In this study, we present a detailed description of our method that measures D-/L-serine by using conventional high performance liquid chromatography (HPLC). • We reacted D-serine and L-serine with ortho-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) to form diastereomeric isoindole derivatives, then we separated and detected them by conventional reversed phase HPLC with electrochemical detector (ECD). • We present typical measurement data of rat brain homogenate as an example of a convenient, appropriate method for measuring brain concentrations of D-serine. • Since many peaks appear in biological samples, we confirmed that the peaks were derived from serine by treating the sample with D-amino oxidase and catalase to decompose D-serine. As a results, one peak disappeared, suggesting that it is derived from D-serine.

Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

Fetal blockade of nicotinic acetylcholine transmission causes autism-like impairment of biological motion preference in the neonatal chick.

Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects.

Combined predisposed preferences for colour and biological motion make robust development of social attachment through imprinting.

To study how predisposed preferences shape the formation of social attachment through imprinting, newly hatched domestic chicks (Gallus gallus domesticus) were simultaneously exposed to two animations composed of comparable light points in different colours (red and yellow), one for a walking motion and another for a linear motion. When a walking animation in red was combined with a linear one in yellow, chicks formed a learned preference for the former that represented biological motion (BM). When the motion-colour association was swapped, chicks failed to form a preference for a walking in yellow, indicating a bias to a specific association of motion and colour. Accordingly, experiments using realistic walking chicken videos revealed a preference for a red video over a yellow one, when the whole body or the head was coloured. On the other hand, when the BM preference had been pre-induced using an artefact moving rigidly (non-BM), a clear preference for a yellow walking animation emerged after training by the swapped association. Even if the first-seen moving object was a nonbiological artefact such as the toy, the visual experience would induce a predisposed BM preference, making chicks selectively memorize the object with natural features. Imprinting causes a rapid inflow of thyroid hormone in the telencephalon leading to the induction of the BM preference, which would make the robust formation of social attachment selectively to the BM-associated object such as the mother hen.

Gene expression of Dio2 (thyroid hormone converting enzyme) in telencephalon is linked with predisposed biological motion preference in domestic chicks.

Filial imprinting leads to the formation of social attachment if training is performed during a brief sensitive period after hatching. We found that thyroid hormone (3,5,3'-triiodothyronine, T3) acts as a critical determining factor of the sensitive period in domestic chicks. Imprinting upregulates gene expression of the converting enzyme (Dio2, type 2 iodothyronine deiodinase) in the telencephalon, leading to increased brain T3 content. If systemically applied, T3 facilitates imprinting in aged chicks even after the sensitive period is over. Imprinting is also associated with the rapid development of visual perception. Exposure to motion pictures induces a predisposed preference to Johansson's biological motion (BM), and those individuals with higher BM preference are more easily imprinted. Here, we examined whether Dio2 expression is also linked with BM predisposition. Chicks were trained by a rotating red block, and tested for imprinting (experiment 1) and BM preference (experiment 2). To examine the time courses of behavioural and physiological processes, Dio2 expression in telencephalon was compared among three groups: naïve control chicks, and chicks trained for a short (0.5 h) or long period (2 h). In experiment 1, higher Dio2 expression appeared in the 2-h group than in the 0.5-h/control groups, but it was not correlated with the individual imprinting score. In experiment 2, a significant positive correlation appeared between Dio2 expression and BM preference in 2-h-trained chicks. Memory priming by T3 is therefore functionally linked to BM preference induction, leading to successful imprinting to natural objects even when they are initially exposed to artificial objects.

Thyroid Hormone Sensitizes the Imprinting-Associated Induction of Biological Motion Preference in Domestic Chicks.

Filial imprinting is associated with induction of predisposed preference to animations that bear visual features of Johansson's biological motion (BM), and the induction is limited to a few days after hatching. As thyroid hormone (3,5,3'-triiodothyronine, T3) plays a critical role in determining the sensitive period of imprinting, we examined if exogenously applied T3 (or iopanoic acid, IOP; a selective inhibitor for converting enzymes) could also sensitize (or desensitize) the BM induction. Chicks were trained by using a non-BM stimulus (rotating red toy) according to a conventional imprinting procedure. Trained chicks were tested for preference to a point-light BM animation (walking chick) over a non-BM animation (linear motion), and for the preference for the familiarized stimulus (red toy) over an unfamiliar one (yellow toy). In 1-day chicks, those injected with IOP showed significantly lower scores than controls on both BM and imprinting tests. In 4-days chicks, those injected with T3 showed higher scores than control, but the difference in BM score was not significant. Imprinting and the accompanying T3 surge may be necessary for the predisposed BM preference to appear in 1-day chicks. Even after the conventional sensitive period is over, exogenous T3 can partly re-sensitize the BM preference as it does imprinting.

Preference for biological motion in domestic chicks: sex-dependent effect of early visual experience.

To examine the effects of early visual experience on preference for biological motion (BM), newly hatched chicks were exposed to a point-light animation (a visual stimulus composed of identical light points) depicting the following features of a hen: a walking hen (a BM stimulus), a rotating hen (a non-BM stimulus), a pendulum stimulus, a random motion stimulus and a stationary pattern. Chicks were then tested in a binary choice task, choosing between walking-hen and rotating-hen stimuli. Males exhibited a preference for BM if they had been trained with any animation except the stationary pattern stimulus, suggesting that the BM preference was not learned, but induced by motion stimuli. We found a significant positive correlation between the number of approaches in training and the preference in the test, but locomotion alone did not cause preference for BM. In contrast, females exhibited a particularly strong preference for walking-hen stimuli, but only when they had been trained with it. Furthermore, females (but not males) trained with random motion showed a preference for walking hen over walking cat (a biological motion animation depicting a cat), possibly suggesting that females are choosier than males. Chicks trained with a stationary pattern and untrained controls did not show a significant preference. The induction of BM preference is discussed in terms of possible ecological background of the sex differences.