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Endothelial progenitor cells (EPCs), which are precursors of endothelial cells (ECs), have the capacity to circulate, proliferate and differentiate into mature ECs. EPCs are primarily identified by the uptake of 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein (Dil‐acLDL) and the binding of fluorescein‐isothiocyanate (FITC)‐conjugated Ulex europaeus agglutinin lectin (FITC‐UEA‐I). However, the cytoplasm and nucleus are usually stained by FITC‐UEA‐I via a typical method to double‐stain late EPCs. It is necessary to explore a new method to improve the quality of fluorescence photomicrographs of late EPCs stained with FITC‐UEA‐I. Here, we described an updated protocol for double‐staining late EPCs with Dil‐acLDL and FITC‐UEA‐I, with the cells more optimally stained with FITC‐UEA‐I.

With the rapid development of medical technology, 3D printing technology with realistic representation can perfectly display static human anatomy, while 3D visualisation technology based on Web Graphics Library (WebGL) can promote the rigid replication characteristics of traditional teaching models and express the dynamic spatial relationship between different anatomical structures. Medical students traditionally have less cognition of ankle ligament sprains. In this study, computed tomography (CT) and magnetic resonance imaging (MRI) data of the ankle joints of volunteers were used to print models of the ankle bone, tendon, and ligament using 3D printing technology, and a real-time interactive 3D digital model of the functional ankle joint was designed using 3D visualisation based on WebGL and 2D image real-time rendering technology for interactive teaching. The utility of the 3D printing model combined with the WebGL-based 3D digital teaching model was evaluated in comparison with traditional teaching methods in 24 medical students. The results showed that the total score of students in the experimental group (mean ± SD, 79.48 ± 12.93) was significantly better than that of the control group (61.00 ± 14.94) with P < 0.05. The practical test scores of the experimental group (18.00 ± 2.70) were significantly higher than those of the control group (13.67 ± 4.96) with P < 0.05. In the satisfaction survey, the feedback questionnaire showed that the interactive teaching model of 3D printing technology combined with WebGL-based 3D visualisation technology was recognised by students in terms of quality and overall satisfaction. In addition, female students who used 3D printing combined with WebGL-based 3D visualisation technology as learning aids had a greater difference in practical test scores from the control group than male students. This study has demonstrated that the interactive teaching mode of 3D printing combined with WebGL-based 3D visualisation technology is beneficial to the teaching of medical imaging, enriching the learning experience of students, and increasing the interaction between teachers and students.

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. In β-cells from overweight humans without diabetes, and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+influx. β-cell RNA-seq suggests altered metabolic pathways early following HFD, where we find increased basal oxygen consumption, proton leak, but a more reduced cytosolic redox state. Increased β-cell exocytosis after 2-day HFD is dependent on this reduced intracellular redox and requires thesentrin-specific SUMO-protease-1(SENP1). Mice with either pancreas- or β-cell-specific SENP1 deletion fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day HFD. Mechanistically, redox-sensing by SENP1 requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained β-cell exocytosis and increases SENP1 sensitivity to regulation by redox signals.

Coagulation tests are affected by many factors, such as age, race, and gestation. Although coagulation test results vary by ABO blood type, reference intervals of different ABO blood groups remain to be determined. This study aims to investigate the reference ranges of coagulation tests for different ABO blood groups in the Han population in South China.A retrospective study was conducted in the First Affiliated Hospital of Shantou University Medical College. In all, 9600 individuals aged between 20 and 79 years were included. Coagulation tests, including prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time, and fibrinogen, were performed.There was a significant difference in PT, INR, and aPTT among ABO blood groups. PT and INR varied slightly between ABO blood groups. There was a higher aPTT value in individuals in the O blood group than in those in non-O blood groups, in both males and females across the included age range. No differences were found in thrombin time and fibrinogen between the ABO blood groups.The study provides reference data on coagulation tests from ABO blood groups in South China. The established reference intervals specific to ABO blood type, sex, and age may improve clinical decisions based on coagulation tests.

The DEPTOR-mTORC1/2 axis has been shown to play an important, but a context dependent role in the regulation of proliferation and the survival of various cancer cells in cell culture settings. The in vivo role of DEPTOR in tumorigenesis remains elusive. Here we showed that the levels of both DEPTOR protein and mRNA were substantially decreased in human prostate cancer tissues, which positively correlated with disease progression. DEPTOR depletion accelerated proliferation and survival, migration, and invasion in human prostate cancer cells. Mechanistically, DEPTOR depletion not only activated both mTORC1 and mTORC2 signals to promote cell proliferation and survival, but also induced an AKT-dependent epithelial–mesenchymal transition (EMT) and β-catenin nuclear translocation to promote cell migration and invasion. Abrogation of mTOR or AKT activation rescued the biological consequences of DEPTOR depletion. Importantly, in a Deptor-KO mouse model, Deptor knockout accelerated prostate tumorigenesis triggered by Pten loss via the activation of mTOR signaling. Collectively, our study demonstrates that DEPTOR is a tumor suppressor in the prostate, and its depletion promotes tumorigenesis via the activation of mTORC1 and mTORC2 signals. Thus, DEPTOR reactivation via a variety of means would have therapeutic potential for the treatment of prostate cancer.

Enterovirus 71 can cause severe hand, foot, and mouth disease (HFMD) in children. However, little is known about the mechanism of inflammatory disorders caused by EV71 infection and why severe cases are mainly children aged under-three. In current study, using mRNA microarray assay, the differential expression of Placenta-specific 8 (PLAC8) was identified in mice brain. In addition, we found that PLAC8 expression was down-regulated with age in mice lung tissues and human peripheral blood. Then, we further proved that PLAC8 could promote inflammation progress and disturb Th1/Th2/Th17/Treg related cytokines release after EV71 infection using PLAC8 plasmid over-expressed neonatal mouse model. Our data suggest that PLAC8 might play a crucial role in Th cell differentiation and inflammatory damage caused by EV71 infection in infants. Thus, our findings would help understand the causes of severe inflammatory injury in infants during EV71 infection, and provide new insights into the prevention and control of severe HFMD.

Cells use exocytosis to secrete a wide variety of molecules, including proteins, hormones, and neurotransmitters. Exocytosis can be monitored at the single-cell level by using patch-clamp electrophysiology to measure changes in membrane capacitance as vesicles fuse with the cell membrane and release their content. Dispersion of pancreatic islets into single cells allows for individual characterization of electrophysiological characteristics and allows for collection of cellular content for recovery of full-length transcriptomes by use of Smart-seq2. Described in this protocol is the dispersion of pancreatic islets into single cells followed by whole-cell patch clamp electrophysiology which includes parameters representing cell size, exocytosis, sodium channel currents, and calcium channel currents. Cells are then collected individually after recording to be processed for single-cell RNA sequencing.

β-transducin repeat-containing protein (β-TrCP), one of the best-characterized substrate recognition components of the SKP1-CUL1-F-box (SCF) E3 ligase, has two distinct paralogs, β-TrCP1 and β-TrCP2, expressed in mammals. Through governing the ubiquitination and degradation of numerous key regulators, β-TrCP1/2 regulates various cellular physiological and pathological processes. However, whether and how these two proteins cross talk and whether they regulate cell autophagy and proliferation in different manners is completely unknown. Herein, we report that β-TrCP1 and β-TrCP2 are the physiological substrates of SCF E3 ligase and target each other for degradation that is dependent on their β-TrCP degron sequences. Furthermore, glucose deprivation activates AMPK kinase to phosphorylate β-TrCP1 and promotes the subsequent ubiquitination and degradation of β-TrCP1 by β-TrCP2, but does not promote β-TrCP2 degradation by β-TrCP1. Finally, we found that β-TrCP2, not β-TrCP1, preferentially degrades DEPTOR and REDD1, the inhibitors of mTORC1, to activate mTORC1, leading to autophagy inhibition and cell growth. Thus, our study demonstrates that β-TrCP1 and β-TrCP2 mutually target each other for degradation and that β-TrCP2 acts as a dominant paralog in the regulation of cell autophagy and growth, which might be a promising anticancer target.

In this paper, we studied the effect of mobility-on-demand (MoD) services' pricing and matching decisions on mitigating the negative impacts of curbside stopping activities. We first developed a set of macroscopic models of MoD services, curbside stopping systems, road networks, and their interactions. In particular, we generalized an existing queueing model of curbside stopping systems to the multiple-pickup-locations context, and formulated MoD services' demand guidance decision problem as an integer program. We also proposed a novel and efficient numerical method to find the equilibria of the system. Then, we apply simulation optimization methods over the proposed model to search for optimal MoD operations. Our numerical experiments showed that, compared with optimal pricing decisions, optimal matching decisions could better balance the welfare of the MoD service and its negative impact on road network capacities. This observation implied that matching mechanisms could be competitive alternatives to congestion pricing in mitigating the curbside stopping problem.

DEP-domain containing mTOR-interacting protein (DEPTOR), a natural mTOR inhibitor, has essential roles in several processes, including cell growth, metabolism, apoptosis, and immunity. DEPTOR expression has been shown to be diversely controlled at transcriptional levels in cell- and context-specific manners. However, whether there is a general mechanism for the regulation of DEPTOR expression remains largely unknown. Here, we report that DEPTOR is a downstream target of the tumor suppressor, p53, whose activity is positively correlated with DEPTOR expression both in vitro in cell cultures and in vivo in mouse tissues. Mechanistically, p53 directly binds to the DEPTOR promoter and transactivates its expression. Depletion of the p53-binding site on the DEPTOR promoter by CRISPR-Cas9 technology decreases DEPTOR expression and promotes cell proliferation and survival by activating AKT signaling. Importantly, inhibition of AKT by small molecular inhibitors or genetic knockdown abrogates the induction of cell growth and survival induced by deletion of the p53-binding region on the DEPTOR promoter. Furthermore, p53, upon activation by the genotoxic agent doxorubicin, induces DEPTOR expression, leading to cancer cell resistance to doxorubicin. Together, DEPTOR is a direct p53 downstream target and contributes to p53-mediated inhibition of cell proliferation, survival, and chemosensitivity.

We study the curbside stopping activities of on-demand mobility services from a macroscopic perspective. We develop a queueing network model of the curbside stopping system and integrate this model into existing macroscopic analyzing frameworks of on-demand mobility services. Theoretical analysis and numerical experiments both show that service operators can provide services at a level beyond the social optimum and introduce negative externalities to the system. Moreover, improvements in services’ operational efficiencies are not sufficient to resolve this problem. Therefore, it is necessary to introduce system-level solutions such as introduction of additional curbside stopping spaces.

Reliable prediction of short-term passenger flow could greatly support metro authorities’ decision processes, help passengers to adjust their travel schedule, or, in extreme cases, assist emergency management. The inflow and outflow of the metro station are strongly associated with the travel demand within metro networks. The purpose of this paper is to obtain such prediction. We first collect the origin-destination information from the smart-card data and explore the passenger flow patterns in a metro system. We then propose a data driven framework for short-term metro passenger flow prediction with the ability to utilize both spatial and temporal related information. The approach adopts two forecasts as basic models and then uses a probabilistic model selection method, random forest classification, to combine the two outputs to achieve a better forecast. In the experiments, we compare the proposed model with four other prediction models, i.e., autoregressive-moving-average, neural networks, support vector regression, and averaging ensemble model, as well as the basic models. The results indicate that the proposed approach outperforms the others in most cases. The origin-destination flows extracted from smart-card data can be successfully exploited to describe different metro travel patterns. And the framework proposed here, especially the probabilistic combination method, can improve the performance of short-term transportation prediction.

Predicting evacuation demand, including its generation and dissipation process, for urban rail transit systems under disruptions, such as line and station closure, often requires comprehensive historical data recorded under homogeneous situations. However, data under disruptions are hard to collect due to various reasons, which makes traditional methods impractical in evacuation demand prediction. To address this problem from the modeling perspective, we develop a data-efficient approach to predict evacuation demand for urban rail transit systems under disruptions. Our model-based approach mainly uses historical data obtained from the natural state, when no shocks take place. We first formulate the mathematical representation of the evacuation demand for every type of urban rail transit station. Input variables in this step are location features related to the station under the disruption, as well as an origin–destination matrix under the natural state. Then, based on these mathematical expressions, we develop a simulation system to imitate the spatio-temporal evolution of evacuation demand within the whole network under disruptions. The transport capacity drop under disruptions is used to describe the disruption situation. Several typical scenarios from the Shanghai metro network are used as examples to implement the proposed method. The results show that our method is able to predict the generation and dissipation processes of evacuation demand, as well model how severely stations will be affected by given disruptions. One general observation we draw from the results is that the most vulnerable stations under disruption, where the locations peak evacuation demand occurs, are mainly turn-back stations, closed stations, and the transfer stations near closed stations. This paper provides new insight into evacuation demand prediction under disruptions. It could be used by transport authorities to better respond to the urban rail transit system disruption.

Summary: The tumor suppressor p53 plays a critical role in integrating a wide variety of stress responses. Therefore, p53 levels are precisely regulated by multiple ubiquitin ligases. In this study, we report that FBXW7, a substrate recognition component of the SKP1-CUL1-F-box (SCF) E3 ligase, interacts with and targets p53 for polyubiquitination and proteasomal degradation after exposure to ionizing radiation or etoposide. Mechanistically, DNA damage activates ATM to phosphorylate p53 on Ser33 and Ser37, which facilitates the FBXW7 binding and subsequent p53 degradation by SCFFBXW7. Inactivation of ATM or SCFFBXW7 by small molecular inhibitors or genetic knockdown/knockout approaches extends the p53 protein half-life upon DNA damage in an MDM2-independent manner. Biologically, FBXW7 inactivation sensitizes cancer cells to radiation or etoposide by stabilizing p53 to induce cell-cycle arrest and apoptosis. Taken together, our study elucidates a mechanism by which FBXW7 confers cancer cell survival during radiotherapy or chemotherapy via p53 targeting. : In this study, Cui et al. demonstrate that upon DNA damage, ATM is activated to phosphorylate p53 to facilitate its binding with FBXW7, leading to p53 ubiquitination and proteasomal degradation. Biologically, FBXW7 inactivation sensitizes cancer cells to radiation or etoposide by stabilizing p53 to induce cell-cycle arrest and apoptosis. Keywords: FBXW7, p53, E3 ligase, DNA damage, radiosensitivity, ATM, SCF, CRL, ubiquitination, degradation

Pancreatic islet-cells regulate glucose levels in the body through insulin and glucagon secretion; dysfunction of these cells leads to severe disease such as diabetes. Several single-cell transcriptome studies have shown heterogeneous gene expression in major islet cell-types such as beta and alpha cells. However, it has remained an open challenge to reconcile the heterogeneity in transcriptomic signatures with physiological observations of functional heterogeneity. Here we combined electrophysiological profiling and single-cell RNA sequencing of the same cells (pancreas patch-seq) to link transcriptomic phenotypes of islet cells to their physiologic properties. We collected 1,369 pancreas patch-seq cells from fresh and cryopreserved tissue and assessed function-gene expression networks in nondiabetic, type 1 diabetic (T1D) and type 2 diabetic (T2D) humans. We discovered that ‘hub’ genes and pathways drive functional heterogeneity in nondiabetic beta-cells and show that specific transcriptional programs correlate with physiologic dysfunction early in T2D. We have also found evidence that alpha-cell transcriptional plasticity acts as a driver of dysfunction in T1D. Disclosure J. Camunas Soler: None. X. Dai: None. Y. Hang: None. S. Kim: None. P. MacDonald: None. Funding Chan Zuckerberg Biohub; California Institute for Regenerative Medicine; National Institutes of Health

SummaryPancreatic islet cells regulate glucose homeostasis through insulin and glucagon secretion; dysfunction of these cells leads to severe diseases like diabetes. Prior single-cell transcriptome studies have shown heterogeneous gene expression in major islet cell-types; however it remains challenging to reconcile this transcriptomic heterogeneity with observed islet cell functional variation. Here we achieved electrophysiological profiling and single-cell RNA sequencing in the same islet cell (pancreas patch-seq) thereby linking transcriptomic phenotypes to physiologic properties. We collected 1,369 cells from the pancreas of donors with or without diabetes and assessed function-gene expression networks. We identified a set of genes and pathways that drive functional heterogeneity in β-cells and used these to predict β-cell electrophysiology. We also report specific transcriptional programs that correlate with dysfunction in type 2 diabetes (T2D) and extend this approach to cryopreserved cells from donors with type 1 diabetes (T1D), generating a valuable resource for understanding islet cell heterogeneity in health and disease.Key findingsPancreas patch-seq provides a single-cell survey of function-transcriptome pairing in 1,369 islet cells from donors with and without diabetesExpression of a specific subset of genes predicts β-cell electrophysiology in transcriptome-function networks.Compromised β-cell function in T2D correlates with altered ETV1 expression and inflammatory pathwaysFunctional heterogeneity in α-cells maps to ER stress and islet lineage markersApplication of patch-seq to cells from rare cryopreserved islets from donors with T1D

Sideflash is a kind of lightning striking on the side wall of tall buildings. This unexpected thunder stroke often leads to big property losses and endangers lives of people. However, the origin and development mechanism of this lightning stroke are still not clear. To simulate the sideflash, a 3-D numerical model of negative cloud-to-ground lightning is built which is composed of two parts: the thundercloud model and the stepped leader model. The thundercloud model is based on the 3-D dynamics and electrification coupled model. And the stepped leader model is based on the Dielectric Breakdown Model (DBM). The Poisson's Equation solved by the Successive Over-Relaxation method is employed to refresh the spatial electric potential field. Finally, some buildings with different heights are set on the ground. In the numerical simulation, some sideflashes are observed. Based on the comparison experiments, the origin of these phenomena is discussed.

An efficient Pt-free counter electrode (CE) based on poly(3,4-ethylenedioxythiophene) (PEDOT) film for dye-sensitized solar cells (DSSCs) has been developed via solid-state polymerization (SSP) approach. The polymerization was simply initiated by sintering the monomer, 2,5-diiodo-3,4-ethylenedioxythiophene (DIEDOT), at the temperature of 135 °C. Furthermore, DIEDOT is not only quite suitable and economic for large scale synthesis, but also very stable in air. Under optimized conditions, the power conversion efficiency of a DSSC with a N719-sensitized TiO2 photo-anode and the SSP-PEDOT CE is 6.38% measured under standard 1 sun illumination (100 mW cm−2, AM 1.5).

Electromagnetism such as gravity can be considered an omnipresent force, which plays an important role in the functional activities of organisms. However, electromagnetic waves with different frequencies have aroused various harmful factors in human beings and wildlife. With the tunable dielectric property, the material can get a controllable electromagnetic absorption range against different types of electromagnetic pollution. In this study, we reported that nano γ-Fe2O3 can regularly tune the dielectric properties of the three dimensional (3D) polypyrrole (PPy) aerogel. On the one hand, it has advantages such as a rapid synthesis strategy (only several minutes), facile purification process (with only water), and on the other hand, this aerogel based absorber can reach effective EA bandwidths of 6.76 GHz (≤−10 dB, 90% electromagnetic energy was attenuated) and 1.88 GHz (≤−20 dB, 99% electromagnetic energy was attenuated) simultaneously at a thickness of 2.5 mm.

With the depletion of shallow metal resources in the land, the development of seabed resources has become a new direction. In order to achieve safe and efficient exploitation of seabed metal mineral resources, a similar simulation test platform was established for seabed mining of metal minerals in this paper. Similar simulation materials and the best material ratios were selected to make simulated specimens. A series of mechanical pressurization devices and model sample were prototyped and submarine simulation mining test was conducted. During the simulation mining test, stress and strain changes and microscopic observations of crack evolution were monitored by the static strain test system. These indicators revealed the characteristics of ore-rock mechanical change in the development of submarine mental resources. By exploring the best safe thickness of seabed resource mining, it provides effective data reference for subsequent seabed resource development, so as to realize safe and efficient mining.

Detailed protocol for dispersing human and mouse islets into single cells for patch clamp and imaging experiments

Glucagon is synthesized and released by pancreatic α-cells and, along with insulin, maintains blood glucose levels within the physiological range. We seek to understand the impact of glucose on the biophysical properties of α-cells in T2D. We studied α-cells from human donors (34 nondiabetic, and 14 T2D) and from mice fed a high fat diet (HFD) by whole-cell patch-clamp electrophysiology, with and without combined single-cell RNA sequencing (patch-seq). We found that, opposite to what is observed in β-cells, depolarization-induced exocytosis and Ca2+-channel function is enhanced at low glucose (LG, 1 mM G) and suppressed at high glucose (HG, 5-20 mM G) in nondiabetic (ND) α-cells. This is reversed in α-cells from T2D donors, which display a β-cell-like phenotype where HG amplifies exocytosis and LG inhibits it. Using pharmacological antagonists of Ca2+ channels, we found that LG increases P/Q-type Ca2+ currents in human α-cells, which are specifically linked to glucagon exocytosis. Similar results were obtained from α-cells of HFD mice for 10-14 weeks, where α-cell exocytosis and glucagon secretion were both enhanced, while exocytosis shifted from a dependency on P/Q-type (α-cell-like) to L-type (β-cell-like) Ca2+ channels. Finally, given that the Ca2+ responsiveness of α-cells is variable (suggesting cell- to-cell heterogeneity) and the function of α-cells is dysregulated in diabetes, we applied combined computational approaches and single-cell RNA sequencing with patch-clamp analysis (patch-seq) to link single human α-cell function with transcriptomic profiles. We found distinct α-cell sub-populations in human islets that are either resistant to, or sensitive to, dysfunction in the form of a shift towards a more ’β-cell like’ functional phenotype in T2D. In all, we provide new information to understand normal function of α-cells and important determinants of glucagon secretion in health and diabetes. Disclosure X. Dai: None. J. Camunas Soler: None. L. Briant: None. A.F. Spigelman: None. Y. Hang: None. P. Rorsman: None. S. Kim: None. P. MacDonald: None.

Micro carbonaceous spheres have been successfully synthesized from watermelon via a one-pot hydrothermal reaction. After a quick annealing process to remove the surface oxygen groups, the as-prepared spheres presented an excellent microwave absorption performance at the Ku band (12–18 GHz), with a loading ratio of 25 wt% in a paraffin-based composite at 2.0 mm thickness.

Governments in developing counties often promote intercropping (crops intercropped with fruit trees on cultivated land) schemes in order to improve smallholders’ income. However, the implementation of such schemes is often hindered by inappropriate institutional environments and inefficient project management. It is important to assess the impacts of such intercropping programs, especially since such a cultivation strategy can often align closely with smallholders’ livelihood strategies, particularly in poor and remote rural regions. This paper attempts to assess the impact of an intercropping program on participants’ incomes in rural Xinjiang (China), and to explore the possible shortcomings in the program’s design and implementation. We apply a propensity score matching method, based on a survey dataset of 352 households, supplemented with descriptive analysis based on our anecdotal field observations. The findings demonstrate that the intercropping program had negative effects on the incomes that participants derived from farming, their off-farm income and their gross income. Overall, participants experienced significant losses of income. Anecdotal observations show that land tenure insecurity played a crucial role in negating the anticipated income improvement effect of this program. Farmer’s perceptions that they have limited security of tenure made them reluctant to invest the necessary time and resources to make the new cropping systems a success, while the available subsidies only partially covered the costs involved. In addition, the program led to a significant drop in yields of field crops as the trees were competing for a limiting and fixed supply of irrigation water.