Oscillatory Behavior of Neutrophils under Opposing Chemoattractant Gradients Supports a Winner-Take-All Mechanism

Neutrophils constitute the largest class of white blood cells and are the first responders in the innate immune response. They are able to sense and migrate up concentration gradients of chemoattractants in search of primary sites of infection and inflammation through a process known as chemotaxis. These chemoattractants include formylated peptides and various chemokines. While much is known about chemotaxis to individual chemoattractants, far less is known about chemotaxis towards many. Previous studies have shown that in opposing gradients of intermediate chemoattractants (interleukin-8 and leukotriene B₄), neutrophils preferentially migrate toward the more distant source. In this work, we investigated neutrophil chemotaxis in opposing gradients of chemoattractants using a microfluidic platform. We found that primary neutrophils exhibit oscillatory motion in opposing gradients of intermediate chemoattractants. To understand this behavior, we constructed a mathematical model of neutrophil chemotaxis. Our results suggest that sensory adaptation alone cannot explain the observed oscillatory motion. Rather, our model suggests that neutrophils employ a winner-take-all mechanism that enables them to transiently lock onto sensed targets and continuously switch between the intermediate attractant sources as they are encountered. These findings uncover a previously unseen behavior of neutrophils in opposing gradients of chemoattractants that will further aid in our understanding of neutrophil chemotaxis and the innate immune response. In addition, we propose a winner-take-all mechanism allows the cells to avoid stagnation near local chemical maxima when migrating through a network of chemoattractant sources.     

STDP Installs in Winner-Take-All Circuits an Online Approximation to Hidden Markov Model Learning

In order to cross a street without being run over, we need to be able to extract very fast hidden causes of dynamically changing multi-modal sensory stimuli, and to predict their future evolution. We show here that a generic cortical microcircuit motif, pyramidal cells with lateral excitation and inhibition, provides the basis for this difficult but all-important information processing capability. This capability emerges in the presence of noise automatically through effects of STDP on connections between pyramidal cells in Winner-Take-All circuits with lateral excitation. In fact, one can show that these motifs endow cortical microcircuits with functional properties of a hidden Markov model, a generic model for solving such tasks through probabilistic inference. Whereas in engineering applications this model is adapted to specific tasks through offline learning, we show here that a major portion of the functionality of hidden Markov models arises already from online applications of STDP, without any supervision or rewards. We demonstrate the emergent computing capabilities of the model through several computer simulations. The full power of hidden Markov model learning can be attained through reward-gated STDP. This is due to the fact that these mechanisms enable a rejection sampling approximation to theoretically optimal learning. We investigate the possible performance gain that can be achieved with this more accurate learning method for an artificial grammar task.     

GFP示踪观察移植瘤原代培养细胞的生长规律

采用GFP稳定表达的细胞系(293-BAC)接种裸鼠形成移植瘤.取肿瘤组织进行原代培养,通过GFP示踪和细胞形态学变化,观察了肿瘤组织中细胞的生长规律.肿瘤细胞释放并生长在组织块附近,向周围空间延伸.种植时散落的薄层组织细胞则直接贴壁生长.生长的鼠源间质细胞占总细胞量的1%～3%,散在或在组织新生肿瘤细胞外围集中生长.原代培养的细胞在传代5代以后,其中的鼠源细胞消失.经过成瘤和传代过程的肿瘤细胞生长性能稳定、来源纯净,是相关研究的好材料.观察到的细胞生长规律可为移植瘤的相关研究提供参考.     

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.     

Automated Turbidimetric Bioassay Readout Instrument Using a Multiple Flow-Cell System

An instrument has been developed which permits the automatic quantitation of the turbidities of microbiological assay samples. Assay tubes were fed to the instrument at the end of the incubation period. The turbidity readings were automatically converted to digital data which were printed on International Business Machines (IBM) cards and from which potencies were calculated by an IBM computer. The instrument operated at a speed of over 240 tube readings per hour and was totally automatic in sample-mixing, readout, and data recording. The instrument is being used routinely at The Upjohn Co. for the turbidimetric bioassay of vitamins, with a coefficient of variation among repeated turbidity readings of 0.12 to 0.23%.     

The Temporal Structure of Vertical Arm Movements

The present study investigates how the CNS deals with the omnipresent force of gravity during arm motor planning. Previous studies have reported direction-dependent kinematic differences in the vertical plane; notably, acceleration duration was greater during a downward than an upward arm movement. Although the analysis of acceleration and deceleration phases has permitted to explore the integration of gravity force, further investigation is necessary to conclude whether feedforward or feedback control processes are at the origin of this incorporation. We considered that a more detailed analysis of the temporal features of vertical arm movements could provide additional information about gravity force integration into the motor planning. Eight subjects performed single joint vertical arm movements (45° rotation around the shoulder joint) in two opposite directions (upwards and downwards) and at three different speeds (slow, natural and fast). We calculated different parameters of hand acceleration profiles: movement duration (MD), duration to peak acceleration (D PA), duration from peak acceleration to peak velocity (D PA-PV), duration from peak velocity to peak deceleration (D PV-PD), duration from peak deceleration to the movement end (D PD-End), acceleration duration (AD), deceleration duration (DD), peak acceleration (PA), peak velocity (PV), and peak deceleration (PD). While movement durations and amplitudes were similar for upward and downward movements, the temporal structure of acceleration profiles differed between the two directions. More specifically, subjects performed upward movements faster than downward movements; these direction-dependent asymmetries appeared early in the movement (i.e., before PA) and lasted until the moment of PD. Additionally, PA and PV were greater for upward than downward movements. Movement speed also changed the temporal structure of acceleration profiles. The effect of speed and direction on the form of acceleration profiles is consistent with the premise that the CNS optimises motor commands with respect to both gravitational and inertial constraints.     

MBD结构域和SRA结构域识别甲基化DNA的结构机理

DNA胞嘧啶5-甲基化修饰是表观遗传重要的修饰之一,其对基因表达的调控依赖下游的识别蛋白识别和传递甲基化信号.本文围绕两种主要的甲基化DNA识别结构域——MBD结构域和SRA结构域,综述了它们识别不同修饰形式DNA的结构基础以及发挥功能的分子机理.     

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann^1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor³ for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L''Etoile et al.,⁴ uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC⁴. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC⁵. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC^6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.     

Tripled Readout Slices in Multi Time-Point pCASL Using Multiband Look-Locker EPI

Multi time-point pseudo-continuous arterial spin labelling (pCASL) with a Look-Locker EPI readout can sample the signal curve of blood kinetics at multiple time points after the labelling pulse. However, due to signal relaxation of labelled blood, the number of readout slices is limited. The aim of this study is to employ a multiband excitation technique to triple the number of readout slices in multi time-point pCASL. The multiband technique, along with 2-fold in-plane parallel imaging, was incorporated into the Look-Locker EPI for the multi time-point sampling of blood kinetic behaviour following the pCASL labelling scheme. The performance evaluation of the multiband and the single-band techniques were performed on four healthy subjects using a 32-channel head RF coil at 3T. Quantitative perfusion maps were analysed using a combination of labelling with and without flow suppression gradients. The perfusion maps provided by the multiband accelerated multi time-point pCASL were in good agreement with the conventional single-band technique. Multiband acceleration caused SNR loss but offered quantitative perfusion maps in 6.23 min with 18 slices compared with 6 slices within the same time period for the single-band method. As conclusion, the multiband technique can successfully triple the number of readout slices while achieving comparable perfusion data in the same measurement time as the conventional single-band readout.     

The Temporal Structure of Behaviour and Sleep Homeostasis

The amount and architecture of vigilance states are governed by two distinct processes, which occur at different time scales. The first, a slow one, is related to a wake/sleep dependent homeostatic Process S, which occurs on a time scale of hours, and is reflected in the dynamics of NREM sleep EEG slow-wave activity. The second, a fast one, is manifested in a regular alternation of two sleep states – NREM and REM sleep, which occur, in rodents, on a time scale of ∼5–10 minutes. Neither the mechanisms underlying the time constants of these two processes – the slow one and the fast one, nor their functional significance are understood. Notably, both processes are primarily apparent during sleep, while their potential manifestation during wakefulness is obscured by ongoing behaviour. Here, we find, in mice provided with running wheels, that the two sleep processes become clearly apparent also during waking at the level of behavior and brain activity. Specifically, the slow process was manifested in the total duration of waking periods starting from dark onset, while the fast process was apparent in a regular occurrence of running bouts during the waking periods. The dynamics of both processes were stable within individual animals, but showed large interindividual variability. Importantly, the two processes were not independent: the periodic structure of waking behaviour (fast process) appeared to be a strong predictor of the capacity to sustain continuous wakefulness (slow process). The data indicate that the temporal organization of vigilance states on both the fast and the slow time scales may arise from a common neurophysiologic mechanism.     

The Role of Temporal Information in Perisaccadic Mislocalization

In dynamic environments, it is crucial to accurately consider the timing of information. For instance, during saccades the eyes rotate so fast that even small temporal errors in relating retinal stimulation by flashed stimuli to extra-retinal information about the eyes’ orientations will give rise to substantial errors in where the stimuli are judged to be. If spatial localization involves judging the eyes’ orientations at the estimated time of the flash, we should be able to manipulate the pattern of mislocalization by altering the estimated time of the flash. We reasoned that if we presented a relevant flash within a short rapid sequence of irrelevant flashes, participants’ estimates of when the relevant flash was presented might be shifted towards the centre of the sequence. In a first experiment, we presented five bars at different positions around the time of a saccade. Four of the bars were black. Either the second or the fourth bar in the sequence was red. The task was to localize the red bar. We found that when the red bar was presented second in the sequence, it was judged to be further in the direction of the saccade than when it was presented fourth in the sequence. Could this be because the red bar was processed faster when more black bars preceded it? In a second experiment, a red bar was either presented alone or followed by two black bars. When two black bars followed it, it was judged to be further in the direction of the saccade. We conclude that the spatial localization of flashed stimuli involves judging the eye orientation at the estimated time of the flash.     

Sos恢复系统--一种新型的酵母双杂交系统

Sos恢复系统是一种新型的不依赖转录激活机制的酵母双杂交系统,原理是通过蛋白质的相互作用将Sos富集在细胞膜上激活Ras信号通路,使得酵母温度敏感缺陷株可以在限制温度下生长。与传统的酵母双杂交系统相比,Sos恢复系统的主要优点在于被研究的蛋白质的相互作用发生在胞质而不是核中;而且,它更适合研究转录因子以及在胞质中行使生理功能的蛋白质。Sos恢复系统克服了传统的酵母双杂交系统的一些限制,大大拓展了酵母双杂交技术的应用范围。     

Temporal arrest

Two patients are presented in which documented periods of asystole were caused by temporal epilepsy. Pacemaker implantation prevented total collapse but episodes of near collapse continued. After institution of anti-epileptic drug treatment both patients are free of symptoms.