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Medical computed tomography (CT), nuclear magnetic resonance imaging (MRI), and other imaging facility produce a large amount of medical image data which has great diagnosis value. Traditional three-dimensional reconstruction of medical images has high requirements for graphics acceleration hardware and the processing speed. In this study, VxWorks embedded real-time process feature is used for CT or MRI DICOM data to real restoration and establishment of virtual three-dimensional model for realizing volume reconstruction, maximum density projection, multiplane reconstruction, dynamic interactive cutting of any surface, dynamic display of three-dimensional model and two-dimensional sectional image, surgical path planning and interactive surgical simulation, and determine the best surgical scheme. The practical application shows that the virtual simulation environment supports the seamless transplantation of code, function debugging, and interaction and solves the issue of high requirements for hardware. It can meet the needs of scientific research and teaching for clinicians and medical imaging workers and can be widely used in the training of virtual surgical anatomy for medical students.

Exosomes have shown great potential in disease diagnostics and therapeutics. However, current isolation approaches are burdensome and suffer from low speed, yield and purity, limiting basic research and clinical applications. Here, we describe an efficient exosome detection method via the ultrafast-isolation system (EXODUS) that allows automated label-free purification of exosomes from varied biofluids. We obtained the ultra-efficient purification of exosomes by negative pressure oscillation and double coupled harmonic oscillator–enabled membrane vibration. Our two coupled oscillators generate dual-frequency transverse waves on the membranes, enabling EXODUS to outperform other isolation techniques in speed, purity and yield. We demonstrated EXODUS by purifying exosomes from urine samples of 113 patients and validated the practical relevance in exosomal RNA profiling with the high-resolution capability and high-throughput analysis. EXODUS is a high-speed isolation method for the enrichment of exosome from biological fluids with high purity and yield.

Esophageal squamous cell carcinoma (ESCC) is a worldwide malignancy with high mortality rates and poor prognosis due to the lack of effective biomarkers for early detection. Exosomes have been extensively explored as attractive biomarkers for cancer diagnosis and treatment. However, little is known about exosome metabolomics and their roles in ESCC. Here, we performed a targeted metabolomic analysis of plasma exosomes and identified 196 metabolites, mainly including lipid fatty acids, benzene, amino acids, organic acids, carbohydrates and fatty acyls. We systematically compared metabolome patterns of exosomes via machine learning from patients with recrudescence and patients without recrudescence and demonstrated a marker set consisting of 3'-UMP, palmitoleic acid, palmitaldehyde, and isobutyl decanoate for predicting ESCC recurrence with an AUC of 98%. These metabolome signatures of exosomes retained a high absolute fold change value at all ESCC stages and were very likely associated with cancer metabolism, which could be potentially applied as novel biomarkers for diagnosis and prognosis of ESCC.

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

The genetic origins of nanoscale extracellular vesicles in our body fluids remains unclear. Here, we perform a tracking analysis of urinary exosomes via RNA sequencing, revealing that urine exosomes mostly express tissue-specific genes for the bladder and have close cell-genetic relationships to the endothelial cell, basal cell, monocyte, and dendritic cell. Tracking the differentially expressed genes of cancers and corresponding enrichment analysis show urine exosomes are intensively involved in immune activities, indicating that they may be harnessed as reliable biomarkers of noninvasive liquid biopsy in cancer genomic diagnostics and precision medicine.

Electro-transfection is an essential workhorse tool for regulating cellular responses and engineering cellular materials in tissue engineering. However, most of the existing approaches are only focused on cell suspensions in vitro, which fails to mimic an in vivo tissue microenvironment regarding the 3D electric field distribution and mass transport in a biological matrix. However, building a 3D electro-transfection system that is compatible with 3D cell culture for mimicking the in vivo tissue microenvironment is challenging, due to the substantial difficulties in control of the 3D electric field distribution as well as the cellular growth. To address such challenges, we introduce a novel 3D micro-assembly strategy assisted by 3D printing, which enables the molding of 3D microstructures as LEGO® parts from 3D-printed molds. The molded PDMS LEGO® bricks are then assembled into a 3D-cell culture chamber interconnected with vertical and horizontal perfusion microchannels as a 3D channel network. Such a 3D perfusion microchannel network is unattainable by direct 3D printing or other microfabrication approaches, which can facilitate the highly-efficient exchange of nutrition and waste for 3D cell growth. Four flat electrodes are mounted into the 3D culture chamber via a 3D-printed holder and controlled by a programmable power sequencer for multi-directional electric frequency scanning (3D μ-electro-transfection). This multi-directional scanning not only can create transient pores all over the cell membrane, but also can generate local oscillation for enhancing mass transport and improving cell transfection efficiency. As a proof-of-concept, we electro-delivered the pAcGFP1-C1 vector to 3D cultured HeLa cells within peptide hydrogel scaffolding. The expressed GFP level from transfected HeLa cells reflects the transfection efficiency. We found two key parameters including electric field strength and plasmid concentration playing more important roles than the pulse duration and duty cycles. The results showed an effective transfection efficiency of ∼15% with ∼85% cell viability, which is 3-fold higher compared to that of the conventional benchtop 3D cell electro-transfection. This 3D μ-electrotransfection system was further used for genetically editing 3D-cultured Hek-293 cells via direct delivery of CRISPR/Cas9 plasmid which showed successful transfection with GFP expressed in the cytoplasm as the reporter. The 3D-printing enabled micro-assembly allows facile creation of a novel 3D culture system for electro-transfection, which can be employed for versatile gene delivery and cellular engineering, as well as building in vivo like tissue models for fundamentally studying cellular regulation mechanisms at the molecular level.

Here we present a protocol for isolation and purification of exosomes using exosome detection via the ultrafast-isolation system (EXODUS). This protocol is associated with our Nature Methods paper: Exosome Detection via the Ultrafast-isolation System: EXODUS.

Generating open-domain conversational responses in the desired style usually suffers from the lack of parallel data in the style. Meanwhile, using monolingual stylistic data to increase style intensity often leads to the expense of decreasing content relevance. In this paper, we propose to disentangle the content and style in latent space by diluting sentence-level information in style representations. Combining the desired style representation and a response content representation will then obtain a stylistic response. Our approach achieves a higher BERT-based style intensity score and comparable BLEU scores, compared with baselines. Human evaluation results show that our approach significantly improves style intensity and maintains content relevance.

Microfluidic technology has been attractive and widely employed for solving problems in biomedical research field. However, sophisticated microfabrication process limited its utility and access from biologists and physicians who are unfamiliar with microfabrication, in turn, significantly hindered the biomedical discovery. Since past 5 years, 3D printing technology has been introduced for fabricating microfluidic devices and serves as the game-changing role in the field. 3D printing converts computer-assisted design (CAD) into a physical object in a single process, and the process is easy, fast, and versatile with low-cost. This chapter will focus on the 3D printing technology and development, as well as the applications for enabling microfluidic fabrication of both monolithic, 3D-printed microfluidic device and mold-based polydimethylsiloxane (PDMS) microfluidic device casting. The direct 3D printing of microfluidic automixer has been demonstrated as proof-of-concept to be employed in point-of-care diagnosis of anemia. The 3D molding of PDMS microfluidic devices was also discussed, which overcomes the lengthy and complicated protocols via soft lithography, offering quick 3D microstructure prototyping. The application of 3D printing in microfluidic technology has shown tremendous potential in tissue engineering, sample preparation techniques, cell processing, and biochemical detection, which can facilitate a paradigm shift in methodology used in studying complex biological systems.

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

Isolation and purification of extracellular vesicles (EVs) from plasma is essential to understand the EV circulation mechanism and discover biomarkers for the early detection of diseases. However, the size range of lipoprotein particles such as high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) overlap that of EVs, making it difficult to remove lipoproteins from EVs. Here, we propose a method for the high efficiency separation of EVs in plasma using agarose gel electrophoresis based on their differences in size and zeta potential properties. Electrophoresis track assays revealed that EVs propagate more slowly than HDL but more quickly than LDL and VLDL in 1% agarose gel with pH 7.4 Tris-Acetate-EDTA (TAE) buffer. The size and morphology of the electrophoresis-recovered products were characterized to be consistent with typical EVs. In addition, the biological function of recovered EVs was investigated with cell uptake tests. The feasibility of this method was further verified with human plasma samples. In summary, this technique has the potential to become a convenient and efficient approach for high-purity EV separation.

One of the most crucial problem on training personalized response generation models for conversational robots is the lack of large scale personal conversation data. To address the problem, we propose a two-phase approach, namely initialization then adaptation, to first pre-train an optimized RNN encoder-decoder model (LTS model) in a large scale conversational data for general response generation and then fine-tune the model in a small scale personal conversation data to generate personalized responses. For evaluation, we propose a novel human aided method, which can be seen as a quasi-Turing test, to evaluate the performance of the personalized response generation models. Experimental results show that the proposed personalized response generation model outperforms the state-of-the-art approaches to language model personalization and persona-based neural conversation generation on the automatic evaluation, offline human judgment and the quasi-Turing test.

Study of extracellular vesicles (EVs), particularly exosomes, holds significant promise; however, it is technically challenging to define these small and molecularly diverse nanovesicles. With intrinsic molecular payload and biodegradability, molecular engineering of exosomes opens new avenues for mediating cellular responses and developing novel nano-delivery systems in precision therapeutics. Microfluidic lab-on-chip technology is playing pivotal roles in this emerging field. In this review, we have examined scientific advancements of microfluidic technology for engineering exosomes and assessed future applications and perspectives in developing precision therapeutics; this can serve the community via identification of potential new research areas or technologies that are urgently needed in precision therapeutics.

Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids. Graphical abstract Rapid quantitative analysis of prolyl dipeptides in urine using flow-gated capillary electrophoresis coupled with laser-induced fluorescence detection.

Recent years have seen the field of extracellular vesicle (EV) studies burgeoning. This is mainly because EV constituents including nucleic acid, proteins, lipids, and metabolites are promising sources towards disease biomarker discovery. However, EV study remains challenging due to the complexity of biofluids as well as technical limitations during sample preparation. Here, we proposed a simple method combing ultrafiltration (UF) and phospholipid affinity to collect high purity EVs from 30 mL of urine sample for their metabolomic profiling. Ultracentrifugation (UC) for EV isolation was applied as a reference method. Western blot (WB) analysis, nanoparticles tracking analysis (NTA) and electron microscopy (EM) were used to assess EV protein markers and to characterize vesicle size and morphology. The results revealed that more than 1010 EV particles could be isolated from a 30 mL urine sample by the proposed method, and the resulting EVs carry specific protein markers and had a typical “cup shape” morphology. This suggests that our method is suitable for EV isolation and can provide sufficient EV quantity to ensure downstream analysis. Further untargeted metabolomic profiling of isolated EVs by UHPLC-QTOF-MS detected 433 metabolites by our methods and 432 metabolites by UC with a MS/MS similarity score greater than 0.7. We then applied the lipid metabolites-targeted method using UHPLC-QTrap-MS with the MRM mode, which successfully detected 467 compounds from urine EVs. This indicates that UF integrating phospholipid affinity is a reliable method for metabolic analysis of urinary EVs, which holds the potential for EV clinical application towards biomarker investigation from their metabolites.

Amino acid analysis is an important procedure to measure free amino acids (AAs) in various samples including proteins, blood plasma, urine, foods and beverages. Due to the complexity of these samples, chemical separations are required to determine the composition and quantities of AAs. Here, we report a fast and accurate approach to quantify AAs in beers. This method was based on a flow-gated capillary electrophoresis (CE) system coupled with alternate injections enabled by a micro-fabricated switch. AAs in beer samples and standard solutions were fluorogenically derivatized with 2,3-naphthalenedicarboxaldehyde (NDA) in the presence of cyanide. The two derivatized mixtures were then loaded into two separate micro-syringes and were alternately delivered to the flow gate for rapid injections and separations. Here, the standards functioned as side-by-side calibration and quantitation references. The separation of 21 AA derivatives was achieved within 90 seconds under an electric field of 1470 V cm−1 by using optimized background electrolytes consisting of 40 mM sodium tetraborate, 60 mM sodium dodecyl sulfate, and 2 mM 2-hydroxypropyl-β-cyclodextrin, and theoretical plates of >300k for most AAs were obtained. The beer matrix effect was studied, and the quantitation of AAs was conducted by directly comparing the peak heights of analytes with those of their individual self-standards. The method was validated with respect to the linearity, precision, and accuracy, and was applied to the measurements of 21 AAs in eight different beer samples. The results demonstrated the quantitative capability of flow-gated CE coupled with alternate injections for side-by-side calibration that was able to compensate for potential drift of the CE system. The developed method is expected to be valuable for high-throughput amino acid analysis of a variety of samples.

Capillary electrokinetic chromatography is generally recognized as a versatile and robust capillary electromigration technique for the separation of enantiomers. In this mode, one or more chiral selectors are added to the background electrolyte acting as pseudostationary phases. Within the various chiral selectors that have been applied to enantioseparations in capillary electrokinetic chromatography, cyclodextrins are by far the most often used selectors because of their versatility, structural variety and commercial availability. This is reflected in the large number of applications of cyclodextrins to analytical enantioseparations that have been reported between January 2012 and July 2016, the period of time covered by this review. Many of these applications cover aspects of life sciences such as drug analysis, bioanalysis or food analysis. Despite the large number of commercially available cyclodextrins, new derivatives have been developed in order to achieve altered enantioselectivities or to further broaden the application range. Cyclodextrins have also been used to demonstrate the validity of theoretical models of electromigration as well as complex formation equilibria in enantioseparations. Finally, recent studies for an understanding of the molecular basis of the chiral recognition between cyclodextrins and the analytes are discussed.