The impact of death attitudes on death education needs among medical and nursing students.

BACKGROUND: Medical and nursing students will play an essential role in delivering palliative care in the future. Death education is important in preparing them for future palliative care, however, little is known about students education needs and how death attitudes affect such needs in Mainland China. OBJECTIVES: The purpose of this survey was to investigate the death education needs of medical and nursing students and to evaluate the impact of death attitudes on death education needs. DESIGN: Multi-center, cross-sectional survey. SETTINGS: Fourteen medical and nursing colleges & universities in Hunan, Sichuan, Liaoning, Guangdong, Shandong, and Shanxi provinces in China. PARTICIPANTS: The sample included 1044 medical and nursing students from six provinces. METHODS: In this multi-center cross-sectional study, all data were collected through an online questionnaire that included demographic information and questions on death-related experiences. In addition, the Death Attitude Profile-Revised and the Death Education Needs Scale were used to evaluate students' death attitudes and death education needs , respectively. RESULTS: The students had a mean death education needs score of 38.85 ± 7.25 (range: 10-50), yet only 20.9 % of them had received palliative care-related training. Being female (B:3.869, 95 % CI:2.849-4.889), fear of death (B:0.119, 95 % CI:0.005-0.232), and neutral acceptance (B:0.787, 95 % CI:0.638-0.936) were associated with higher death education needs, while death avoidance (B: -0.226, 95 % CI: -0.368 ~ -0.083), approach acceptance (B: -0.126, 95 % CI: -0.215 ~ -0.036), and escape acceptance (B: -0.198, 95 % CI: -0.322 ~ -0.073) were associated with lower death education needs. CONCLUSIONS: The high level of death education needs and low training rate in palliative care among medical and nursing students in mainland China indicates a gap that needs to be addressed. Students' death education needs were affected by gender and death attitudes, which provides implications for the future development of palliative care training models.

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Enhanced Stability and Function of Probiotic Streptococcus thermophilus with Self-Encapsulation by Increasing the Biosynthesis of Hyaluronan.

The harsh conditions of the gastrointestinal tract limit the potential health benefits of oral probiotics. It is promising that oral bioavailability is improved by strengthening the self-protection of probiotics. Here, we report the encapsulation of a probiotic strain by endogenous production of hyaluronan to enhance the effects of oral administration of the strain. The traditional probiotic Streptococcus thermophilus was engineered to produce hyaluronan shells by using traceless genetic modifications and clustered regularly interspaced short palindromic repeat interference. After oral delivery to mice in the form of fermented milk, hyaluronan-coated S. thermophilus (204.45 mg/L hyaluronan in the milk) exhibited greater survival and longer colonization time in the gut than the wild-type strain. In particular, the engineered probiotic strain could also produce hyaluronan after intestinal colonization. Importantly, S. thermophilus self-encapsulated with hyaluronan increased the number of goblet cells, mucus production, and abundance of the microorganisms related to the biosynthesis of short-chain fatty acids, resulting in the enhancement of the intestinal barrier. The coating formed by endogenous hyaluronan provides an ideal reference for the effective oral administration of probiotics.

Heparan sulfate from porcine mucosa promotes amyloid-beta clearance in APP/PS1 mice and alleviates Alzheimer's pathology.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive impairments. Amyloid-ß (Aß) deposition and neurotoxicity play important roles in AD. It has been widely reported that heparan sulfate (HS) proteoglycans play a nonnegligible role in the release, uptake and misfolding of Aß, resulting in the discovery of HS as a therapeutic drug for AD. In this manuscript, HS from porcine mucosa could promote Aß fibrosis and improve the cognitive defects of APPswe/PS1ΔE9 mice. Furthermore, HS enhanced the phagocytosis of neutrophils to clear Aß1-42 from peripheral circulation, reduced peripheral Aß1-42 flow to the brain and increased Aß efflux from the brain. Therefore, the deposition of Aß plaques in the brain was decreased. In addition, HS alleviated neutrophil infiltration and reduced neuroinflammation. Conclusively, HS is a promising neuroprotective candidate for AD treatment.

Effects of heparan sulfate from porcine mucosa on Aß1-42-induced neurotoxicity in vitro and in vivo.

Amyloid-ß (Aß) deposition and neurotoxicity play an important role in Alzheimer's disease (AD). Notably, the nonnegligible role of endogenous heparan sulfate (HS) in the release, uptake and misfolding of Aß sheds light on the discovery of HS as an effective drug for AD. In this work, the effects of HS from porcine mucosa (PMHS) on Aß1-42-induced neurotoxicity were investigated both in vitro and in vivo. The in vitro AD model was established in SH-SY5Y via treatment with oligomeric Aß1-42, and the in vivo AD model was established by intracerebroventricular injection of Aß1-42 to KM mice. The results showed that in vitro, PMHS could ameliorate the inflammation and apoptosis response of SH-SY5Y cells induced by Aß1-42; in vivo, PMHS could not only improve the cognitive impairment induced by Aß1-42 but also inhibit neuroinflammation and apoptosis in the brain. Furthermore, PMHS lowered the levels of Aß1-42 in the peripheral circulation and brain by improving the phagocytosis function of neutrophils. This is the first report that PMHS enhances the phagocytosis function of neutrophils to alleviate Aß-induced neurotoxicity. Moreover, our work verified the feasibility of peripheral Aß clearance for improving neurotoxicity. Conclusively, we believe that PMHS could be developed into neuroprotective drugs for AD.

Low molecular weight chondroitin sulfate ameliorates pathological changes in 5XFAD mice by improving various functions in the brain.

Our previous study found that low molecular weight chondroitin sulfate (LMWCS) had neuroprotective effects against the toxicity of amyloid-ß (Aß) peptides both in vitro and in vivo, and we speculated that the effects might be related with its anti-oxidative activities. In this study, the anti-Alzheimer's disease (AD) activity of LMWCS was further studied in 5XFAD transgenic mice. After 4-month gavage, the levels of Aß1-42 level, amyloid precursor protein (APP) and presenilin 1 (PS1) were significantly decreased in the brains of 5XFAD mice, indicating the alteration of APP metabolism by LMWCS. Besides, LMWCS inhibited the secretions of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6. Furthermore, the suppression of neuroinflammation by LMWCS was supported by the decreased expressions of glial fibrillary acidic protein (GFAP) and toll-like receptor 2 (TLR2) in the brains. LMWCS also reduced the production of reactive oxygen species (ROS) and the level of phospho-tau (Ser404) in the brains. Nevertheless, the changes in the behavior tests were moderate. In conclusion, LMWCS administration ameliorated APP metabolism, neuroinflammation, ROS production and tau protein abnormality in the brains of 5XFAD mice, displaying the potential to improve the pathological changes of AD mouse brain. LMWCS could be considered as a promising anti-AD drug candidate, nonetheless, the therapy regimen need to be optimized to improve its pharmacotherapy efficacy.

Simultaneous detection of genotype and phenotype enables rapid and accurate antibiotic susceptibility determination.

Multidrug resistant organisms are a serious threat to human health1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug resistant organisms increase mortality3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution5, require several days before informing key clinical decisions. Rapid AST would transform the care of patients with infection while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here we describe a rapid assay for combined genotypic and phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 h faster than standard workflows, with <4 h assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.

Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

Continuous and High-Throughput Electromechanical Lysis of Bacterial Pathogens Using Ion Concentration Polarization.

Electrical lysis of mammalian cells has been a preferred method in microfluidic platforms because of its simple implementation and rapid recovery of lysates without additional reagents. However, bacterial lysis typically requires at least a 10-fold higher electric field (∼10 kV/cm), resulting in various technical difficulties. Here, we present a novel, low-field-enabled electromechanical lysis mechanism of bacterial cells using electroconvective vortices near ion selective materials. The vortex-assisted lysis only requires a field strength of ∼100 V/cm, yet it efficiently recovers proteins and nucleic acids from a variety of pathogenic bacteria and operates in a continuous and ultrahigh-throughput (>1 mL/min) manner. Therefore, we believe that the electromechanical lysis will not only facilitate microfluidic bacterial sensing and analysis but also various high-volume applications such as the energy-efficient recovery of valuable metabolites in biorefinery pharmaceutical industries and the disinfection of large-volume fluid for the water and food industries.

Capturing the radical ion-pair intermediate in DNA guanine oxidation.

Although the radical ion pair has been frequently invoked as a key intermediate in DNA oxidative damage reactions and photoinduced electron transfer processes, the unambiguous detection and characterization of this species remain formidable and unresolved due to its extremely unstable nature and low concentration. We use the strategy that, at cryogenic temperatures, the transient species could be sufficiently stabilized to be detectable spectroscopically. By coupling the two techniques (the cryogenic stabilization and the time-resolved laser flash photolysis spectroscopy) together, we are able to capture the ion-pair transient G+•⋯Cl- in the chlorine radical-initiated DNA guanine (G) oxidation reaction, and provide direct evidence to ascertain the intricate type of addition/charge separation mechanism underlying guanine oxidation. The unique spectral signature of the radical ion-pair G+•⋯Cl- is identified, revealing a markedly intense absorption feature peaking at 570 nm that is distinctive from G+• alone. Moreover, the ion-pair spectrum is found to be highly sensitive to the protonation equilibria within guanine-cytosine base pair (G:C), which splits into two resolved bands at 480 and 610 nm as the acidic proton transfers along the central hydrogen bond from G+• to C. We thus use this exquisite sensitivity to track the intrabase-pair proton transfer dynamics in the double-stranded DNA oligonucleotides, which is of critical importance for the description of the proton-coupled charge transfer mechanisms in DNA.

Protective Effects of Puerarin against Aß 1-42-Induced Learning and Memory Impairments in Mice.

Puerarin is a major isoflavone glycoside from the root of Pueraria lobata. It has been reported that puerarin can protect neurons from oxidative stress-induced apoptosis. Emerging evidence suggests that oxidative damage is associated with Aß-induced neuronal death. In the current study, we evaluated the effect of puerarin on Alzheimer's disease induced by Aß and explored the potential mechanisms underlying this effect. We found that the escape latency of the Morris water maze was decreased in groups treated with puerarin compared to the model group (p < 0.01). In addition, there were significant differences between treated groups and the model group mice in a Y-maze test (p < 0.01). Furthermore, puerarin recovered the levels of brain-derived neurotrophic factor, phosphorylated tau, malondialdehyde, acetylcholine esterase, glycogen synthase kinase-3beta, and the activity of superoxide dismutase to some extent in the hippocampus and cerebral cortex. Shrinkage of nuclei and swollen and eccentrically dispersed neuronal bodies were observed in the hippocampus of Aß-treated mice. These data demonstrate that puerarin might protect against cognitive deficits, oxidative stress, and neurodegeneration induced by Aß1-42.

Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

Schisantherin B ameliorates Aß1-42-induced cognitive decline via restoration of GLT-1 in a mouse model of Alzheimer's disease.

Accumulation of amyloid beta (Aß) peptide and hyperphosphorylated tau protein has been proposed to play roles in neural destruction which induce Alzheimer's disease (AD) progresses, glutamate transporter type 1 (GLT-1) and Glycogen synthase kinase3ß (GSK3ß) may be the pathological links between Aß and tau pathology. Schisantherin B (STB) is one bioactive of lignans isolated from Schisandra chinensis (Turcz.) Baill which has been commonly used as a traditional herbal medicine for thousands of years. This paper was designed to investigate the effects of STB on improving the cognitive function and neurodegeneration in the mouse model of Alzheimer's disease induced by Aß1-42, and its possible mechanism were Glutamate transporter GLT-1, tau and GSK3ß. It was found that successive intracerebroventricular (ICV) administration of STB (0.15mg/kg) for 5days significantly attenuated Aß1-42-induced learning and memory impairment as measured by the Locomotor activity test, Y-maze test and Morris water maze test. Furthermore, STB at a dose of 0.15mg/kg restored the activities of GLT-1 and GSK3ß while decreasing the levels of hyperphosphorylated tau protein in the hippocampus and cerebral cortex. The results suggested that STB might protect against cognitive deficits and neurodegeneration induced by Aß1-42 in mice by regulating the GLT-1 restoration as well as the capacity of GSK3ß.

High-efficiency broadband anti-Stokes emission from Yb3+-doped bulk crystals.

We investigate the broadband anti-Stokes emission (BASE) from Yb3+-doped crystals with a laser diode (LD) pumping at 940 nm. Our experiment reveals that Yb3+-doped crystals with random cracks are able to generate bright BASE at room temperature and atmospheric pressure. By examining the various characteristics of the crystals and the emitted light, we supply a theory for interpreting the underlying physics for this variety of BASE. In particular, we take into consideration the effects of energy migration, avalanche process, and charge-transfer luminescence. This represents the first time, to the best of our knowledge, that BASE was obtained from Yb3+-doped bulk crystals with a high optical-optical efficiency.

Broad spectrum immunomodulation using biomimetic blood cell margination for sepsis therapy.

Sepsis represents a systemic inflammatory response caused by microbial infection in blood. Herein, we present a novel comprehensive approach to mitigate inflammatory responses through broad spectrum removal of pathogens, leukocytes and cytokines based on biomimetic cell margination. Using a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), we performed extracorporeal blood filtration with the developed microfluidic blood margination (µBM) device. Circulating bacteremia, leukocytes and cytokines in blood decreased post-filtration and significant attenuation of immune cell and cytokine responses were observed 3-5 days after intervention, indicating successful long-term immunomodulation. A dose-dependent effect on long-term immune cell count was also achieved by varying filtration time. As proof of concept for human therapy, the µBM device was scaled up to achieve ∼100-fold higher throughput (∼150 mL h(-1)). With further multiplexing, the µBM technique could be applied in clinical settings as an adjunctive treatment for sepsis and other inflammatory diseases.

Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications.

Large-Volume Microfluidic Cell Sorting for Biomedical Applications.

Microfluidic cell-separation technologies have been studied for almost two decades, but the limited throughput has restricted their impact and range of application. Recent advances in microfluidics enable high-throughput cell sorting and separation, and this has led to various novel diagnostic and therapeutic applications that previously had been impossible to implement using microfluidics technologies. In this review, we focus on recent progress made in engineering large-volume microfluidic cell-sorting methods and the new applications enabled by them.

High-throughput protease activity cytometry reveals dose-dependent heterogeneity in PMA-mediated ADAM17 activation.

As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases.

Direct observation of guanine radical cation deprotonation in G-quadruplex DNA.

Although numerous studies have been devoted to the charge transfer through double-stranded DNA (dsDNA), one of the major problems that hinder their potential applications in molecular electronics is the fast deprotonation of guanine cation (G(+•)) to form a neutral radical that can cause the termination of hole transfer. It is thus of critical importance to explore other DNA structures, among which G-quadruplexes are an emerging topic. By nanosecond laser flash photolysis, we report here the direct observation and findings of the unusual deprotonation behavior (loss of amino proton N2-H instead of imino proton N1-H) and slower (1-2 orders of magnitude) deprotonation rate of G(+•) within G-quadruplexes, compared to the case in the free base dG or dsDNA. Four G-quadruplexes AG3(T2AG3)3, (G4T4G4)2, (TG4T)4, and G2T2G2TGTG2T2G2 (TBA) are measured systematically to examine the relationship of deprotonation with the hydrogen-bonding surroundings. Combined with in depth kinetic isotope experiments and pKa analysis, mechanistic insights have been further achieved, showing that it should be the non-hydrogen-bonded free proton to be released during deprotonation in G-quadruplexes, which is the N2-H exposed to solvent for G bases in G-quartets or the free N1-H for G base in the loop. The slower N2-H deprotonation rate can thus ensure less interruption of the hole transfer. The unique deprotonation features observed here for G-quadruplexes open possibilities for their interesting applications as molecular electronic devices, while the elucidated mechanisms can provide illuminations for the rational design of G-quadruplex structures toward such applications and enrich the fundamental understandings of DNA radical chemistry.

The expression and characterization of functionally active soluble CD83 by Pichia pastoris using high-density fermentation.

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.