Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.     

Evaluation of the ordering of membranes in multilayer stacks built on an ATR-FTIR germanium crystal with atomic force microscopy: the case of the H(+),K(+)-ATPase-containing gastric tubulovesicle membranes

Polarized attenuated total reflection Fourier transform infrared spectra were recorded on multilayer stacks of native gastric tubulovesicle membranes. The spectral intensity and linear dichroism were measured for average thicknesses ranging between 0 and 100 bilayers. Atomic force microscopy was used to investigate the orientation of the membranes at the top of the stack. Height profiles were obtained along randomly drawn lines and slopes were computed over various distances. Orientation distribution functions were obtained from the slopes and decomposed into Legendre polynomials. It was found that the second Legendre polynomials coefficient characterizing the membrane orientation was always larger than 0.9. It could therefore be concluded that the membrane tilt does not significantly contribute to the infrared dichroism, even for the largest thicknesses tested.     

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.     

Understanding living clathrin-coated pits

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrin-coated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.     

Impacts of Seawalls on Saltmarsh Plant Communities in the Great Bay Estuary,New Hampshire USA

Seawalls are often built along naturally dynamic coastlines, including the upland edge of salt marshes, in order to prevent erosion or to extend properties seaward. The impacts of seawalls on fringing salt marshes were studied at five pairs of walled and natural marshes in the Great Bay Estuary of New Hampshire, USA. Marsh plant species and communities showed no difference in front of walls when compared with similar elevations at paired controls. However, seawalls eliminated the vegetative transition zone at the upper border. Not only did the plant community of the transition zone have high plant diversity relative to the low marsh, but it varied greatly from site to site in the estuary. The effects of seawall presence on other marsh processes, including sediment movement, wrack accumulation, groundwater flow, and vegetation distribution and growth, were examined. Although no statistically significant effects of seawalls were found, variation in the indicators of these processes were largely controlled by wave exposure, site-specific geomorphology and land use, and distance of the sampling station from the upland. Trends indicated there was more sediment movement close to seawalls at high energy sites and less fine grain sediment near seawalls. Both trends are consistent with an increase in energy from wave reflection. The distribution of seawalls bordering salt marshes was mapped for Great and Little Bays and their rivers. Throughout the study area, 3.54% of the marshes were bounded by shoreline armoring (5876 m of seawalls along 165.8 km of marsh shoreline). Localized areas with high population densities had up to 43% of marshes bounded by seawalls. Coastal managers should consider limiting seawall construction to preserve plant diversity at the upper borders of salt marshes and prevent marsh habitat loss due to transgression associated with sea level rise.     

Hydration of phospholipid bilayers in the presence and absence of cholesterol

The number of water molecules bound (unfreezable) by a molecule of dipalmitoyl phosphatidylserine (DPPS) or by a molecule of dipalmitoyl phosphatidylcholine (DPPC) alone or in mixtures with cholesterol was determined by differential scanning calorimetry (DSC). When the phospholipids are in the gel state and in the absence of cholesterol, molecule of DPPS binds about 3.5 molecules of water and molecule of DPPC binds about 6 molecules of water. Number of water molecules bound increases when cholesterol crystallites are formed in the bilayer. For DPPS-cholesterol mixture at X(chol) -0.5, as well as for DPPC-cholesterol mixture at X(chol) -0.5 about 7 water molecules are bound.     

Functional cartography of the ectodomain of the type I interferon receptor subunit ifnar1

Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.     

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied “footprint” region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site. Received: 10 August 1998 / Revised version: 24 September 1998 / Accepted: 24 September 1998     

Xylem and cell turgor pressure probe measurements in intact roots of glycophytes: transpiration induces a change in the radial and cellular reflection coefficients

Xylem probe measurements in the roots of intact plants of wheat and barley revealed that the xylem pressure decreased rapidly when the roots were subjected to osmotic stress (NaCl or sucrose). The magnitude of the xylem pressure response and, in turn, that of the radial reflection coefficients (σ_r) depended on the transpiration rate. Under very low transpiration conditions (darkness and high relative humidity), σ_r assumed values of the order of about 0·2–0·4. The σ_r values of excised roots were also found to be rather low, in agreement with data obtained using the root pressure probe of Steudle. For transpiring plants (light intensities at least 10 μmol m^?2 s^?1; relative humidity 20–40%) the response was nearly 1:1, corresponding to radial reflection coefficients of σ_r= 1. Further increase of the light intensity to about 400 μmol m^?2 s^?1 resulted in a slight but significant decrease of the σ_r values to about 0·8. Similar measurements on maize roots confirmed our previous results (Zhu et al. 1995, Plant, Cell and Environment 18, 906–912) that, in intact transpiring plants at low light intensities of about 10 μmol m^?2 s^?1 and at relative humidities of 20–40% as well as in excised roots, the xylem pressure response was much less than expected from the external osmotic pressure (σ_r values 0·3–0·5). In contrast to wheat and barley, very high light intensities (about 700 μmol m^?2 s^?1) were needed to shift the radial reflection coefficients of maize roots to values of about 0·9. Osmotically induced xylem pressure changes were apparently linked to changes in turgor pressure in the root cortical parenchyma cells, as shown by simultaneous measurements of xylem and cell turgor pressure. In analogy to the σ_r values of the respective glycophytes, the σ_c values of the root cortical cells of wheat and barley were close to unity, whereas σ_c for maize was significantly smaller (about 0·7) under laboratory conditions. When the light intensity was increased up to about 700 μmol m^?2 s^?1 the cellular reflection coefficient of maize roots increased to about 0·95. In contrast to the σ_r values, the σ_c values of the three species investigated remained almost unchanged when the leaves were exposed to darkness and humidified air or when the roots were cut. The transpiration-dependent (species-specific) pattern of the cellular and radial reflection coefficients of the root compartment of the three glycophytes apparently resulted from (flow-dependent) concentration-polarization and sweep-away effects in the roots of intact plants. The data could be explained straightforwardly terms of theoretical considerations outlined previously by Dainty (1985, Acta Horticulturae 171, 21–31). The far-reaching consequences of this finding for root pressure probe measurements on excised roots, for the occurrence of pressure gradients under transpiring conditions, and for the non-linear flow-force relationships in roots found by other investigators are discussed.     

肌电图多参数检测对2型糖尿病患者无症状性周围神经病变的诊断价值分析

摘要 目的：探讨肌电图对无症状糖尿病周围神经病变（DPN）的诊断价值。方法：选取2020年1月~2022年12月就诊于本院的2型糖尿病（T2DM）患者142例,根据是否伴随DPN将患者分为DPN组（n=68）与单纯T2DM组（n=74）。患者均进行神经肌电图检测,包括神经传导（NCS）、F波、H反射的检测;比较两组各项肌电图参数和NCS、F波、H反射异常率,并应用受试者工作特征（ROC）曲线评价NCS异常、F波异常、H反射异常及其联合对DPN的诊断效能。结果：相比单纯T2DM组,DPN组正中神经、尺神经等神经的远端运动潜伏期（DML）显著延长（P＜0.05）,复合肌肉动作电位显著降低（P＜0.05）;同时DPN组感觉传导参数感觉传导速度（SNCV）、感觉神经动作电位（SNAP）低于单纯T2DM组（P＜0.05）。相比单纯T2DM组,DPN组尺神经和胫后神经的平均潜伏期（Fmean）和F波离散度（Fchd）均显著延长（P＜0.05）。DPN组胫后神经的最短潜伏期（Hmin）相比单纯T2DM组显著延长（P＜0.05）。DPN组NCS异常率、F波异常率以及H反射异常率均显著高于单纯T2DM组（P＜0.05）。 ROC曲线分析显示,NCS、F波、H反射诊断DPN的曲线下面积分别为0.659、0.614、0.671,三者联合的AUC为0.753,相比各单一指标均显著提高（P＜0.05）。结论：肌电图对于无症状DPN有着重要诊断作用,NCS、F波、H反射三项联合检测有助于提高DPN的早期诊断效能。