Integrative structure and function of the yeast exocyst complex

Exocyst is an evolutionarily conserved hetero‐octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative‐stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride cross‐links from chemical‐crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo‐EM structure, cross‐links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo‐EM structure, except for Sec5. While not observed in the cryo‐EM map, the integrative model localizes the N‐terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low‐resolution structural data can inform biologically relevant hypotheses, even in the absence of high‐resolution data.     

基于不同可加性方法的黑龙江省红松人工林林分生物量模型

大尺度估算森林生物量一直是人们关注的焦点,而构建林分水平的生物量模型是一种估算森林乔木层生物量的方法。本研究基于聚合法1、聚合法2、平差法、分解法构建红松人工林林分生物量模型,并对比分析4种可加性方法的预测精度,为黑龙江省红松人工林的生物量预测提供科学依据。各模型均使用权函数来消除各模型的异方差,并以留一交叉验证法(LOOCV)作为各模型的检验方法。结果表明: 平差法的整体预测能力略优于聚合法1、聚合法2和分解法,预测精度排序为平差法>聚合法1>聚合法2>分解法;分别对比不同林分断面积的预测能力时,4种可加性方法的预测精度不一致。当红松人工林的林分断面积分布于0～10或50～60 m²·hm^-2区间时,建议采用分解法的参数估计值,而林分断面积分布于其他区间时,建议采用平差法的参数估计值。     

Using Integrative Modeling Platform to compute,validate, and archive a model of a protein complex structure

Biology is advanced by producing structural models of biological systems, such as protein complexes. Some systems are recalcitrant to traditional structure determination methods. In such cases, it may still be possible to produce useful models by integrative structure determination that depends on simultaneous use of multiple types of data. An ensemble of models that are sufficiently consistent with the data is produced by a structural sampling method guided by a data‐dependent scoring function. The variation in the ensemble of models quantified the uncertainty of the structure, generally resulting from the uncertainty in the input information and actual structural heterogeneity in the samples used to produce the data. Here, we describe how to generate, assess, and interpret ensembles of integrative structural models using our open source Integrative Modeling Platform program ( https://integrativemodeling.org ).     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.     

Geometrical optimisation of a biochip microchannel fluidic separator

This article reports on the geometric optimisation of a T-shaped biochip microchannel fluidic separator aiming to maximise the separation efficiency of plasma from blood through the improvement of the unbalanced separation performance among different channel bifurcations. For this purpose, an algebraic analysis is firstly implemented to identify the key parameters affecting fluid separation. A numerical optimisation is then carried out to search the key parameters for improved separation performance of the biochip. Three parameters, the interval length between bifurcations, the main channel length from the outlet to the bifurcation region and the side channel geometry, are identified as the key characteristic sizes and defined as optimisation variables. A balanced flow rate ratio between the main and side channels, which is an indication of separation effectiveness, is defined as the objective. It is found that the degradation of the separation performance is caused by the unbalanced channel resistance ratio between the main and side channel routes from bifurcations to outlets. The effects of the three key parameters can be summarised as follows: (a) shortening the interval length between bifurcations moderately reduces the differences in the flow rate ratios; (b) extending the length of the main channel from the main outlet is effective for achieving a uniformity of flow rate ratio but ineffective in changing the velocity difference of the side channels and (c) decreasing the lengths of side channels from upstream to downstream is effective for both obtaining a uniform flow rate ratio and reducing the differences in the flow velocities between the side branch channels. An optimisation process combining the three parameters is suggested as this integration approach leads to fast convergent process and also offers flexible design options for satisfying different requirements.     

Night,sight, and feeling safe: An exploration of aspects of Warlpiri and Western sleep

Sleeping leaves those asleep ‘blind’ and hence oblivious to potential or real danger. Such dangers are heightened further and more feared at night, the main time for sleep. In this article, I link ideas about sleep and nighttime social practices with questions about vision. My aim is to tease out some of the meanings implied in cross‐culturally distinct solutions to the protection of sleepers at night. I proceed by contrasting ethnographic data from the remote Aboriginal settlement of Yuendumu, Northern Territory, with select elements of the cultural history of Euro‐American sleep. Through ethnographic vignettes, I illuminate how people at Yuendumu commonly arrange themselves in yunta, or rows of sleepers, at night, and how some sleepers awake regularly during the night to ensure the others’ safety. I contrast this with Euro‐American ways of providing a sense of safety to the sleeper through practices of domestic fortification. My comparison revolves around the notion of sight, which in the Euro‐American West is clearly linked to ideas of knowledge, and at Yuendumu, as I demonstrate, imbued with a sense of care. I conclude by relating the gained insights to participant observation as anthropological method.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-α-mediated apoptosis

Tumour necrosis factor-α(TNF-α) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-α increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-α induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-α (50ng/ml), a slow rise in intracellular free Ca²⁺ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3h after the addition of TNF-α) were observed in fluo-3 and furared or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-α in the presence of BAPTA/AM, an intracellular Ca²⁺ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-α-mediated Ca²⁺ rise. Moreover, when cells were exposed to TNF-α with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca²⁺ are two cross-talk messengers important in TNF-α-mediated apoptosis.     

TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR,blocks tumor angiogenesis

Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.