Selection of O‐negative induced pluripotent stem cell clones for high‐density red blood cell production in a scalable perfusion bioreactor system

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Human pluripotent stem cell‐derived red blood cells could help alleviate worldwide blood shortages and provide a secure year‐round supply. However, current generation methods lack the necessary scalability. Here, we present the selection of O‐negative hiPSC clones and demonstrate the small‐scale generation of functional erythroblasts in a high‐density perfusion bioreactor system.     

基于农户感知的黄河下游人-水关系结构分析

人-水关系研究是社会-生态系统研究的重要领域。识别黄河下游农户对人-水关系的感知及其影响因素,对黄河下游因人施策开展生态保护与高质量发展政策调控具有重要作用。但已有研究多从区域水资源、水环境、水文化等某一种或几种关系开展居民感知调查及归因,忽视了人-水关系之间结构化的相互影响机制。基于对黄河下游山东省10个县的农户发放的1651份问卷,经过验证性因子分析后,最终引入水资源与生态关系、水文化与旅游关系、居住地生态功能、河口湿地功能、文化旅游功能五个潜变量,建立结构方程模型发现：农户对水资源与生态关系的认识正向影响其对居住地生态功能和河口湿地功能的评估,农户对水文化与旅游关系的认识正向影响其对居住地生态功能、河口湿地功能和文化旅游功能的评估。研究结果通过基于农户感知刻画人-水关系结构,揭示人-水系统功能认知的影响机制,有助于为黄河下游地区生态保护和乡村振兴提供决策参考。     

Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis

Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl₄ in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE₂. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.     

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.     

水分和氮磷水平对小麦碳同位素分辨率和水分利用效率的影响

 依据盆栽试验数据,利用植物稳定性碳同位素分辨率的理论模型,研究了水分和氮磷营养对小麦叶片碳同位素分辨率(Carbon-isotope discrimination △)的影响。结果表明：水分差异引起碳同位素分辨率较大变异,碳同位素分辨率随土壤相对含水量(Soil relative water content)的提高而提高,在土壤相对含水量为60％～70％条件下碳同位素分辨率最高。缺水时磷水平提高,碳同位素分辨率提高。水分利用效率(Water use efficiency)与碳同位素分辨率关系受土壤水分和养分水平的影响。缺水条件下水分利用效率与碳同位素分辨率之间为负相关,充分供水下为正相关;在低氮水平下的关系不明显,施氮150kg·hm-2时相关性显著。     

Structure-based model for light-harvesting properties of nucleic acid nanostructures

Programmed self-assembly of DNA enables the rational design of megadalton-scale macromolecular assemblies with sub-nanometer scale precision. These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient in silico evaluation of nucleic acid nanostructures for diverse light-harvesting and photonic applications.     

Regulation of Monocarboxylic Acid Transporter-1 by cAMP Dependent Vesicular Trafficking in Brain Microvascular Endothelial Cells

In this study, a detailed characterization of Monocarboxylic Acid Transporter-1 (Mct1) in cytoplasmic vesicles of cultured rat brain microvascular endothelial cells shows them to be a diverse population of endosomes intrinsic to the regulation of the transporter by a brief 25 to 30 minute exposure to the membrane permeant cAMP analog, 8Br-cAMP. The vesicles are heterogeneous in size, mobility, internal pH, and co-localize with discreet markers of particular types of endosomes including early endosomes, clathrin coated vesicles, caveolar vesicles, trans-golgi, and lysosomes. The vesicular localization of Mct1 was not dependent on its N or C termini, however, the size and pH of Mct1 vesicles was increased by deletion of either terminus demonstrating a role for the termini in vesicular trafficking of Mct1. Using a novel BCECF-AM based assay developed in this study, 8Br-cAMP was shown to decrease the pH of Mct1 vesicles after 25 minutes. This result and method were confirmed in experiments with a ratiometric pH-sensitive EGFP-mCherry dual tagged Mct1 construct. Overall, the results indicate that cAMP signaling reduces the functionality of Mct1 in cerebrovascular endothelial cells by facilitating its entry into a highly dynamic vesicular trafficking pathway that appears to lead to the transporter''s trafficking to autophagosomes and lysosomes.     

Prevalence and co-infection of Toxoplasma gondii and Neospora caninum in Apodemus sylvaticus in an area relatively free of cats

The protozoan parasite Toxoplasma gondii is prevalent worldwide and can infect a remarkably wide range of hosts despite felids being the only definitive host. As cats play a major role in transmission to secondary mammalian hosts, the interaction between cats and these hosts should be a major factor determining final prevalence in the secondary host. This study investigates the prevalence of T. gondii in a natural population of Apodemus sylvaticus collected from an area with low cat density (<2·5 cats/km2). A surprisingly high prevalence of 40·78% (95% CI: 34·07%-47·79%) was observed despite this. A comparable level of prevalence was observed in a previously published study using the same approaches where a prevalence of 59% (95% CI: 50·13%-67·87%) was observed in a natural population of Mus domesticus from an area with high cat density (>500 cats/km2). Detection of infected foetuses from pregnant dams in both populations suggests that congenital transmission may enable persistence of infection in the absence of cats. The prevalences of the related parasite, Neospora caninum were found to be low in both populations (A. sylvaticus: 3·39% (95% CI: 0·12%-6·66%); M. domesticus: 3·08% (95% CI: 0·11%-6·05%)). These results suggest that cat density may have a lower than expected effect on final prevalence in these ecosystems.     

Subunit interface dynamics in hexadecameric rubisco

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L₂ dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.     

土壤干旱条件下磷素营养对春小麦水分状况和光合作用的影响

研究了土壤干旱条件下磷素营养对春小麦水分状况和光合作用的影响。结果表明：土壤干旱下,磷素营养明显改善了春小麦的水分状况,维持了较高的ψｗ和ＲＷＣ,并因而导致了较高的净光合速率和较大的气孔开度。不同干旱程度下,限制春小麦光合的因子并不相同,轻度干旱下,光合降低主要是气孔的作用,而严重干旱下则主要是非气孔因子的作用。无论何种水分水平下,施磷处理的净光合速率皆高于不施磷处理,与其较大的气孔导度和叶肉光合