抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

/pdf/improved-patch-clamp-techniques-for-high-resolution-current-tmno572o60.pdf

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

RELION: implementation of a Bayesian approach to cryo-EM structure determination.

In cultures of dissociated rat hippocampal neurons, persistent potentiation and depression of glutamatergic synapses were induced by correlated spiking of presynaptic and postsynaptic neurons. The relative timing between the presynaptic and postsynaptic spiking determined the direction and the extent of synaptic changes. Repetitive postsynaptic spiking within a time window of 20 msec after presynaptic activation resulted in long-term potentiation (LTP), whereas postsynaptic spiking within a window of 20 msec before the repetitive presynaptic activation led to long-term depression (LTD). Significant LTP occurred only at synapses with relatively low initial strength, whereas the extent of LTD did not show obvious dependence on the initial synaptic strength. Both LTP and LTD depended on the activation of NMDA receptors and were absent in cases in which the postsynaptic neurons were GABAergic in nature. Blockade of L-type calcium channels with nimodipine abolished the induction of LTD and reduced the extent of LTP. These results underscore the importance of precise spike timing, synaptic strength, and postsynaptic cell type in the activity-induced modification of central synapses and suggest that Hebb’s rule may need to incorporate a quantitative consideration of spike timing that reflects the narrow and asymmetric window for the induction of synaptic modification.

https://www.jneurosci.org/content/jneuro/18/24/10464.full.pdf

Synaptic Modifications in Cultured Hippocampal Neurons: Dependence on Spike Timing, Synaptic Strength, and Postsynaptic Cell Type

Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated analysis and discovery of unexpected structures without bias from a reference map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

We propose a definition of local resolution for three-dimensional electron cryo-microscopy (cryo-EM) density maps that uses local sinusoidal features. Our algorithm has no free parameters and is applicable to other imaging modalities, including tomography. By evaluating the local resolution of single-particle reconstructions and subtomogram averages for four example data sets, we report variable resolution across a 4- to 40-A range.

/pdf/quantifying-the-local-resolution-of-cryo-em-density-maps-194ktl42w0.pdf

Quantifying the local resolution of cryo-EM density maps.

The aims of analysis of single channel records can be considered in two categories. The first is to allow one to observe results at leisure in order to determine their qualitative features. It may, for example, be found that the single-channel currents were not all of the same amplitude or that they showed obvious grouping into bursts or that artifacts appeared on the record that might be misleading. These effects are often not easy to see on the oscilloscope screen as an experiment proceeds (especially since it is common practice for the signal to be rather heavily filtered for observation during the experiment). For this aim to be fulfilled, highly automated methods of analysis are not desirable.

Fitting and Statistical Analysis of Single-Channel Records

Data transformations for improved display and fitting of single-channel dwell time histograms

1. Single myelinated nerve fibres 12-17 mum in diameter from Rana temporaria and Rana pipiens were voltage clamped at 2-5 degrees C. Potassium currents were blocked by internal Cs(+) and external tetraethylammonium ion. Series resistance compensation was employed.2. Sets of 80-512 identical, 20 ms depolarizations were applied, with the pulses repeated at intervals of 300-600 ms. The resulting membrane current records, filtered at 5 kHz, showed record-to-record variations of the current on the order of 1%. From each set of records the time course of the mean current and the time course of the variance were calculated.3. The variance was assumed to arise primarily from two independent sources of current fluctuations: the stochastic gating of sodium channels and the thermal noise background in the voltage clamp. Measurement of the passive properties of the nerve preparation allowed the thermal noise variance to be estimated, and these estimates accounted for the variance observed in the presence of tetrodotoxin and at the reversal potential.4. After the variance sigma(2) was corrected for the contribution from the background, its relationship to the mean current I could be fitted by the function sigma(2) = iI-I(2)/N expected for N independent channels having one non-zero conductance level. The single channel currents i corresponded to a single-channel chord conductance gamma = 6.4 +/- 0.9 pS (S.D.; n = 14). No significant difference in gamma was observed between the two species of frogs. The size of the total population of channels ranged from 20,000 to 46,000.5. The voltage dependence of i corresponded closely to the form of the instantaneous current-voltage relationship of the sodium conductance, except at the smallest depolarizations. The small values of i at small depolarizations may have resulted from the filtering of high-frequency components of the fluctuations.6. It is concluded that sodium channels have only two primary levels of conductance, corresponding to ;open' and ;closed' states of the channel.7. The fraction p(max) of channels open at the time of the peak conductance was found to be 0.59 +/- 0.08 (S.D.; n = 5) and 0.9 +/- 0.1 (S.D.; n = 3) for depolarizations to -5 and +125 mV, respectively. (50 ms hyperpolarizations to -105 mV preceded the depolarizations in each case.) These values are similar to those predicted by Hodgkin-Huxley kinetics.8. Fluctuations in the firing threshold of neurones are expected from the stochastic gating of sodium channels. A prediction of the size of these fluctuations based on the measured properties of the channels gives a value of about 1% for the relative spread, which agrees with experimental values in the literature.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1980.sp013426

Fred J. Sigworth

Papers

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

Quantifying the local resolution of cryo-EM density maps.

Fitting and Statistical Analysis of Single-Channel Records

Data transformations for improved display and fitting of single-channel dwell time histograms

The variance of sodium current fluctuations at the node of Ranvier