Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

A temperature-independent model of dual calibration standards for onsite and point-of-care quantification analyses via electrophoresis titration chip.

Electrophoresis titration chip (ETC) is a versatile tool for onsite and point-of-care quantification analyses because it affords naked-eye detection and a straightforward quantification format. However, it is vulnerable to changes in environmental temperature, which regulates the electrophoretic migration by affecting the ion mobility and the target recognition by influencing the enzyme activity. Therefore, the quantification accuracy of the ETC tests was severely compromised. Rather than using the dry bath or heating/cooling units, we proposed a facile model of dual calibration standards (DCS) to mathematically eliminate the effects of temperature on quantification accuracy. To verify our model, we deployed the ETC device at different temperatures ranging from 5 to 40 °C. We further utilized the DCS-ETC to determine the protein content and uric acid concentration in real samples outside the laboratory. All the experimental results showed that our model significantly stabilized the quantification recovery from 35.31-153.44 % to 99.38-103.44 % for protein titration; the recovery of uric acid titration is also stable at 96.25-106.42 %, suggesting the enhanced robustness of the ETC tests. Therefore, DCS-ETC is a field-deployable test that can offer reliable quantification performance without extra equipment for temperature control. We envision that it is promising to be used for onsite applications, including food safety control and disease diagnostics.

High-resolution nucleic acid detection using online polyacrylamide gel electrophoresis platform.

Polyacrylamide gel electrophoresis (PAGE) is one of the most popular techniques for the separation and detection of nucleic acids. However, it requires a complicated detection procedure and offline detection format, which inevitably leads to band broadening and thus compromises the separation resolution. To overcome this problem, we developed an online PAGE (OPAGE) platform by integrating the gel electrophoresis apparatus with the gel imaging system, so as to obviate the need for the complicated detection procedure. Notably, OPAGE enabled the real-time monitoring of the separation process and the immediate imaging of the separation results once the electrophoresis ended. Using a series of synthetic DNAs with different lengths as samples, we demonstrated that the OPAGE platform enhanced 32-64 % of the number of theoretical plates, showed a robust dynamic range of 0.1-12.5 ng/µL, and realized a limit of detection as low as 0.08 ng/µL DNA. Based on our results, we anticipate that the OPAGE platform is a promising alternative to traditional nucleic acid gel electrophoresis for simple and high-resolution detection and quantification and nucleic acid.

Marker-Free Isoelectric Focusing Patterns for Identification of Meat Samples via Deep Learning.

Isoelectric focusing (IEF) is a powerful tool for resolving complex protein samples, which generates IEF patterns consisting of multiplex analyte bands. However, the interpretation of IEF patterns requires the careful selection of isoelectric point (pI) markers for profiling the pH gradient and a trivial process of pI labeling, resulting in low IEF efficiency. Here, we for the first time proposed a marker-free IEF method for the efficient and accurate classification of IEF patterns by using a convolutional neural network (CNN) model. To verify our method, we identified 21 meat samples whose IEF patterns comprised different bands of meat hemoglobin, myoglobin, and their oxygen-binding variants but no pI marker. Thanks to the high throughput and short assay time of the microstrip IEF, we efficiently collected 1449 IEF patterns to construct the data set for model training. Despite the absence of pI markers, we experimentally introduced the severe pH gradient drift into 189 IEF patterns in the data set, thereby omitting the need for profiling the pH gradient. To enhance the model robustness, we further employed data augmentation during the model training to mimic pH gradient drift. With the advantages of simple preprocessing, a rapid inference of 50 ms, and a high accuracy of 97.1%, the CNN model outperformed the traditional algorithm for simultaneously identifying meat species and cuts of meat of 105 IEF patterns, suggesting its great potential of being combined with microstrip IEF for large-scale IEF analyses of complicated protein samples.

[Detection and analysis of moving reaction boundary-based electrophoresis distance using smartphone images].

Electrophoresis titration (ET) based on the moving reaction boundary (MRB) theory can detect the analyte contents in different samples by converting content signals into distance signals. However, this technique is only suitable for on-site qualitative testing, and accurate quantification relies on complex optical equipment and computers. Hence, applying this method to real-time point-of-care testing (POCT) is challenging. In this study, we developed a smartphone-based ET system based on a visual technique to achieve real-time quantitative detection. First, we developed a portable quantitative ET device that can connect to a smartphone; this device consisted of five components, namely, an ET chip, a power module, a microcontroller, a liquid crystal display screen, and a Bluetooth module. The device measured 10 cm×15 cm×2.5 cm, weighed 300 g, and was easy to hold. Thus, it is suitable for on-site testing with a run time of only 2-4 min. An assistant mobile software program was also developed to control the device and perform ET. The colored electrophoresis boundary can be captured using the smartphone camera, and quantitative detection results can be obtained in real time. Second, we proposed a quantitative algorithm based on ET channels. The software was used to recognize the boundary migration distance of three channels, a standard curve based on two given contents of the standards was established using the two-point method, and the content of the test sample was calculated. Human serum albumin (HSA) and uric acid (UA) were used as a model protein and biosample, respectively, to test the performance of the detection system. For HSA detection, different HSA solutions were mixed with a polyacrylamide gel (PAG) stock solution, phenolphthalein was added as an indicator, and sodium persulfate and tetramethyl ethylenediamine (TEMED) were used to promote polymerization to form a gel. For UA detection, agarose gel was filled into the ET channel, the UA sample, urate oxidase, and leucomalachite green were added into the anode cell and incubated for 20 min. ET was then performed. The fitting goodness (R2) values of HSA and UA were 0.9959 and 0.9935, respectively, with a linear range of 0.5-35.0 g/L and a log-linear range of 100-4000 µmol/L. The limits of detection for HSA and UA were 0.05 g/L and 50 µmol/L, respectively, and the corresponding relative standard deviations (RSDs) were not greater than 2.87% and 3.21%, respectively. These results demonstrate that the detection system has good accuracy and sensitivity. Clinical samples collected from healthy volunteers were used as target blood samples, and the developed system was used to measure serum total protein and UA levels. Serum samples from five volunteers were selected, standard curves of total serum protein and UA were established, and the test results were compared with hospital standard testing results. The relative errors for serum total protein and UA were less than 6.03% and 6.21%, respectively, and the corresponding RSDs were less than 3.72% and 5.84%, respectively. These findings verify the accuracy and reliability of the proposed detection system. The smartphone-based ET detection system introduced in this paper presents several advantages. First, it enables the portable real-time detection of total serum protein and UA. Second, compared with traditional ET strategies based on colored boundaries, it does not rely on optical detection equipment or computers to obtain quantitative detection results; as such, it can reduce the complexity of the operation and provide portability and real-time metrics. Third, the detection of two biomarkers, serum total protein and UA, is achieved on the same device, thereby improving the multitarget detection potential of the ET method. These advantages render the developed method a promising detection platform for clinical applications and real-time POCT.

Nucleo-cytoplasmic shuttling of 14-3-3 epsilon carrying hnRNP C promotes autophagy.

Translocation of 14-3-3 protein epsilon (14-3-3ε) was found to be involved in Triptolide (Tp)-induced inhibition of colorectal cancer (CRC) cell proliferation. However, the form of cell death induced by 14-3-3ε translocation and mechanisms underlying this effect remain unclear. This study employed label-free LC-MS/MS to identify 14-3-3ε-associated proteins in CRC cells treated with or without Tp. Our results confirmed that heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C) were exported out of the nucleus by 14-3-3ε and degraded by ubiquitination. The nucleo-cytoplasmic shuttling of 14-3-3ε carrying hnRNP C mediated Tp-induced proliferation inhibition, cell cycle arrest and autophagic processes. These findings have broad implications for our understanding of 14-3-3ε function, provide an explanation for the mechanism of nucleo-cytoplasmic shuttling of hnRNP C and provide new insights into the complex regulation of autophagy.

[Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection].

Serum total protein refers to the sum of all proteins in the serum, and its content determination is relevant to human health monitoring and disease diagnosis. However, existing detection techniques present a number of limitations; for example, the Kjeldahl method suffers from the negative effects of interfering substances such as non-protein nitrogen (NPN). Although the electrophoresis titration (ET) method has solved interference problems to some extent, the current ET technique relies on optical detection methods, which increases the tediousness of the operation. This study addresses the challenge of accurate serum total protein detection by combining the traditional ET technique with capacitively coupled contactless conductivity detection (C4D). The research contributions of this work are multifold. First, it presents the first development of an ET-C4D detection system, which consists of six components: an ET power module, an ET chip, a C4D sensing module, a detection module, a data acquisition card, and software. The developed system can capture the conductivity of substances in the channel using the software developed by our laboratory during ET. The detection system can be used to quantify the total protein content in human serum without the addition of specific labeling reagents or using optical detection equipment, and its running time is approximately 300 s. Second, this research proposes the corresponding principle of the system. Under an electric field, ion migration results in different pH levels before and after the boundary, leading to a protein surface charge difference. The maintenance of the electrical neutrality of the substances in the detection channel is related to the protein surface charge; therefore, the ion concentration distribution of the substances in the detection channel changes as the protein surface charge varies. A plot of conductivity as a function of running time showed an "inverted clock shape", first falling and then rising. Owing to the addition of different types and concentrations of proteins, the microenvironment of the entire system changes, resulting in different changes in conductivity. Third, the performance of the detection system was tested using human serum albumin (HSA) standard protein, which was mixed with polyacrylamide gel (PAG) mother liquor, riboflavin, etc., and irradiated under ultraviolet light for 10 min to form a gel. The ET experiments were then carried out. The shape of the conductivity curve was consistent with the proposed principle, and the higher the HSA concentration, the lower the conductivity curve trough, followed by a lagged time of the trough. Quantitative analysis of the conductivity signals showed that the linear range was 0.25-3.00 g/L, with a linearity of up to 0.98. The limit of detection (LOD) was 0.01 g/L, the relative standard deviation (RSD) was 1.90%, and the relative error of the test values was <7.20%, indicating the good detection stability and sensitivity of the system. Clinical samples collected from healthy volunteers were used as target blood samples for serum total protein content measurement using our detection system. Blood samples from a volunteer were used to obtain a standard curve, and the serum samples of other four volunteers were selected for ET-C4D and biuret detection. The results showed that the relative errors between the two methods were within 4.43%, indicating the accuracy and reliability of the detection system. The advantages of the ET-C4D detection system proposed in this paper are as follows: (i) ET-C4D realizes the rapid detection of total serum protein content based on the ET technique; (ii) compared with the traditional protein ET technique, the ET-C4D method does not rely on specific labeling components or optical detection equipment, thereby reducing the complexity of the operation; and (iii) the output signal of ET-C4D can be used for quantitative analysis with excellent analytical performance and high accuracy. These merits highlight the potential of the developed system for clinical application and biochemical analysis.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

A handheld contactless conductivity detector for monitoring the desalting of low-volume virus and cell samples.

Desalting of biosamples is crucial for analytical techniques intolerant to abundant salts. However, there is no simple tool to monitor the desalting of low-volume biosamples so far. Here we developed a handheld capacitively coupled contactless conductivity detector (hC4D) as a miniaturized device to measure the conductivity of 75 µL biosamples. Polyether-ether-ketone (PEEK) tubing was selected as the sample reservoir for sample loading via a pipette. Another pipetting of air pushed the sample solution out of the tubing to recollect the sample. Owing to the low sample consumption and easy sample recollection, hC4D is advantageous for testing expensive biosamples, such as viruses and cells. In addition, the whole process of sample injection, conductivity measurement, recollection, and calibration of conductivity can be completed within 1 min. To verify the feasibility of hC4D, we monitored the desalting progress of gel filtration (GF) of 200 µL blood samples, ultrafiltration (UF) of 300 µL virus samples, and dialysis of 7 mL cell samples. Three rounds of GF and UF completely removed the salts but led to poor sample recovery. In contrast, low concentrations of residual salts remained and better recovery was achieved after two rounds of GF and UF. We further utilized the hC4D to monitor the dialysis and tuned the salt concentration in the cell sample, such that we maintained the viability of cells in a low conductivity environment. These results indicated that hC4D is a promising tool for optimizing the desalting procedure of low-volume biosamples.

Point of care testing (POCT) of cholesterol in blood serum via a moving reaction boundary electrophoresis titration chip.

Cholesterol (CHO) in human blood is one of the most frequently and crucially quantified substances in diagnostic laboratories. However, visual and portable point of care testing (POCT) methods have been rarely developed for the bioassay of CHO in blood samples. Here, we developed an electrophoresis titration (ET) model, a chip device of ∼60 grams, and a quantification method for the POCT of CHO in blood serum based on a moving reaction boundary (MRB). In this model, the selective enzymatic reaction is integrated with an ET chip for visual and portable quantification. At first, CHO reacted with cholesterol oxidase (CHOx) in the anode well, producing H2O2 and cholest-4-en-3-one in the solution. H2O2 further oxidized the colorless and chargeless leucocrystal violet (LCV) dye into violet colored positively charged crystal violet (CV+) and, under the influence of the electric field, the CV+ migrates in the ET channels and is titrated by the alkali of sodium hydroxide immobilized in the ET channels. The length covered by the MRB was measured as a function of the CHO content. The relevant experiments validated the feasibility of the model and method. Furthermore, the experiments revealed the high selectivity, portability, and visuality of the ET-MRB model, device, and method. Finally, the experiments showed a fair sensitivity of LOD of 5 µM, good linearity of 10-1000 µM (r2 = 0.9919), fair stability (intra-day RSD of less than 5.09% and an inter-day RSD of less than 6.36%), and high recovery (99.4-105%). All the data and results indicate the potential of the ET-MRB model, chip device, and method for POCT of CHO in human blood samples.

Isoelectric point barcode and similarity analysis with the earth mover's distance for identification of species origin of raw meat.

In this work, by combining the microcolumn isoelectric focusing (mIEF) and similarity analysis with the earth mover's distance (EMD) metric, we proposed the concept of isoelectric point (pI) barcode for the identification of species origin of raw meat. At first, we used the mIEF to analyze 14 meat species, including 8 species of livestock and 6 species of poultry, to generate 140 electropherograms of myoglobin/hemoglobin (Mb/Hb) markers. Secondly, we binarized the electropherograms and converted them into the pI barcodes that only showed the major Mb/Hb bands for the EMD analysis. Thirdly, we efficiently developed the barcode database of 14 meat species and successfully used the EMD method to identify 9 meat products thanks to the high throughput of mIEF and the simplified format of the barcode for similarity analysis. The developed method had the merits of facility, rapidity and low cost. The developed concept and method had evident potential to the facile identification of meat species.

Quadruple UV LED Array for Facile, Portable, and Online Intrinsic Fluorescent Imaging of Protein in a Whole Gel Electrophoresis Chip.

Intrinsic fluorescence imaging (IFI) has been used for the stain-free detection of proteins in slab gel. However, complicated detection setups and small irradiation area limited the development of facile, online, and portable imaging of the whole slab gel. We here designed a quadruple UV LED array to produce even and powerful area light for direct irradiation of gel electrophoresis chip (GEC) at 275 nm. In addition, we only used a filter of 365 nm, a UV camera lens, and a CCD for IFI detection. We integrated the simple detection setup with the small GEC to construct the IFI-GEC device with a portable size of 15 × 15 × 38 cm. We detected three model proteins to demonstrate the good evenness of the LED array and the online imaging of the whole GEC. Furthermore, the reproducible IFI-GEC detection was completed within 10 min and the LOD was as low as 40 ng for lysozyme detection. All results indicated the potential of the IFI-GEC device for online and portable detection of proteins without staining.

Review on the Effects of Electrochemical Exfoliation Parameters on the Yield of Graphene Oxide.

Recent years have witnessed many breakthroughs in research on graphene as well as a significant improvement in the electrochemical synthesis methods of graphene oxide (GO). GO is a derivative of graphene which has attracted the focus of worldwide scientists and researchers because of its hydrophilic and easily functionalized properties. The electrochemical approach is popular because it saves time, creates zero explosion risk, releases no hazardous gases, and avoids environmental pollution. Although recent publications show that the green, rapid, and mass electrochemical synthesis of GO has more advantages as compared with the traditional Hummers method, it is crucial to study the effects of reaction parameters. Herein, we review recent various works regarding the influences of various reaction parameters on the synthesis of GO sheets. The advancement, current challenges, and solutions of electrochemical synthesis methods of GO are also outlined. Through this review, we hope to spark some clear ideas for anyone who wants to scale up the yield of GO.

[In-site electrophoretic elution of excessive fluorescein isothiocyanate from fluorescent particles in gel for image analysis].

The sensitivity, accuracy, and efficiency of fluorescent particle detection can be improved by purifying the fluorescent-dye-labeled particles. In this study, an in-site model of electrophoretic elution (EE) was developed for the facile and efficient removal of unconjugated fluorescent dyes after labeling reactions, thereby facilitating the sensitive fluorescent imaging of proteins captured by microbeads. First, bovine serum albumin (BSA) and magnetic beads (MBs) were chosen as the model protein and particles, respectively, and an MBs-BSA complex was synthesized by mixing the beads with the BSA solution. Second, excessive fluorescein isothiocyanate (FITC) was added to the EP tube with MBs-BSA suspension for the fluorescent labeling of BSA, and a labeled compound was obtained after 8-h incubation in the dark at 4 ℃. The unpurified MBs-BSAFITC was obtained by removing the supernatant, leaving 5 µL of the residual solution in the EP tube. The obtained MBs-BSAFITC solution was added to a 50-µL phosphate buffer solution (PBST, containing 0.01% Triton X-100, pH 7.4). Third, gel suspension was prepared by mixing the MBs-BSAFITC solution with the low-gelling-temperature agarose gel (10 g/L) and filled into an electrophoresis channel. To demonstrate the high efficiency of the in-site model of EE for removing excessive FITC, a 10-mm hydrogel segment was prepared using MBs-BSAFITC sandwiched between two blank hydrogels and filled into a 50-mm-long electrophoresis tube (outer diameter: 5 mm; inner diameter: 3 mm) for the EE. Subsequently, the filled channel was set in an electrophoresis device to construct the in-site EE model. The particle size of the MBs was larger than the pore size of the gel, and the fluorescent beads were physically immobilized in the gel while the excessive FITC was removed from the channel by electrophoresis. Before an EE run, the original fluorescence image of the target gel was captured using a CCD camera. After the 30-min EE (50 V, 6 mA, pH 7.4 PBS), the fluorescence image was also recorded by the CCD camera. The fluorescent images were converted to a grayscale intensity map. To simplify the calculation, a simple fluorescent image analysis method was developed. The side view of the grayscale intensity map is a two-dimensional plot of peaks. Each peak indicates a fluorescent spot at a given position along the length of the channel when the distribution density of the particles is low, and the peak value is the grayscale intensity of the fluorescent spot. The statistical peak numbers and values can be used to approximate fluorescent spots on the image. After image processing and calculations, 27.8% of the average grayscale intensity of the fluorescent spot was retained, comparing the average gray value of the bright spot before and after EE, and 97.6% of excessive FITC in the channel was cleared, obtained by calculating the decreased background fluorescence grayscale intensity after EE. The particle-to-background signal ratio (P/B ratio, PBr) increased from 1.08 to 12.2 after EE with an exposure time of 1.35 s. In addition, different exposure times were explored during the fluorescence detection. Increasing the exposure time from 1.35 to 2.35 s enhanced PBr from 12.2 to 15.5, which could effectively increase the signal-to-noise ratio. An appropriate increase in exposure time also allowed the detection of many weak fluorescent particles that were previously undetectable, indicating increased sensitivity of the fluorescence detection. The EE model has the following advantages: (i) increase in specificity by eluting FITC absorbed to the surface of beads; (ii) high efficiency in the removal of free FITC with more than 97% clearance; (iii) rapid decrease in noise in the mass hydrogel (within 30 min). This method can be used in beads/spots-based immunoassay in gel, immuno-electrophoresis, and fluorescent staining of protein/nucleic acid bands in gel electrophoresis.

Cellular Heterogeneity Facilitates the Functional Differences Between Hair Follicle Dermal Sheath Cells and Dermal Papilla Cells: A New Classification System for Mesenchymal Cells within the Hair Follicle Niche.

Mesenchymal stem cells (MSCs) are known for their self-renewal and multi-lineage differentiation potential, with these cells often being evaluated in the regulation and maintenance of specific cellular niches including those of the hair follicle. Most mesenchymal stem cells in the hair follicles are housed in the dermal papilla (DP) and dermal sheath (DS), with both niches characterized by a broad variety of cellular subsets. However, while most previous studies describing the hair follicle mesenchymal niche treated all DP and DS cells as Hair Follicle Mesenchymal Stem Cells (HF-MSCs), the high number of cellular subsets would suggest that these cells are actually too heterogenous for such a broad definition. Given this we designed this study to evaluate the differentiation processes in these cells and used this data to create a new set of classifications for DP and DS cells, dividing them into "hair follicle mesenchymal stem cells (HF-MSCs)", "hair follicle mesenchymal progenitor cells (HF-MPCs)", and "hair follicle mesenchymal functional cells (HF-MFCs)". In addition, those cells that possess self-renewal and differentiation were re-named hair follicle derived mesenchymal multipotent cells (HF-MMCs). This new classification may help to further our understanding of the heterogeneity of hair follicle dermal cells and provide new insights into their evaluation.

Postoperative survival of extrahepatic and intrahepatic cholangiocarcinoma after surgery: a population-based cohort.

OBJECTIVES: The study was designed to clarify the difference between extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) in postoperative cancer-specific death. DESIGN: Patients diagnosed with ECC and ICC after surgery, who are identified from the Surveillance, Epidemiology and End Results programme, are eligible for this retrospective cohort study. SETTING: Survival between groups was compared using the traditional Kaplan-Meier method and the cumulative incidence function (CIF) method. Propensity score-matched (PSM) analysis was conducted to balance the differences in vital variables between groups. The HR and 95% CI for ECC relative to ICC were used to quantify the risk of death. Subgroup analysis was further used to evaluate the stability of the differences between groups. RESULTS: The study included 876 patients with ECC and 1194 patients with ICC. Before PSM, with the Kaplan-Meier method, postoperative overall survival and cancer-specific death for ECC were worse than those for ICC. However, with the CIF method, no difference in postoperative cancer-specific death was found. After PSM, all differences in the considered traits were balanced, and 173 pairs of patients were retained. Survival analysis found that there was no difference in postoperative all-cause death (Kaplan-Meier method, p=0.186) or cancer-specific death (Kaplan-Meier and CIF methods, p=0.500 and p=0.913, respectively), which was consistent with subgroup analysis. CONCLUSIONS: ECC and ICC showed no difference in postoperative cancer-specific death, both in the natural state and in multiple variable-matched conditions. TRIAL REGISTRATION NUMBER: researchregistry4175.

[Multi-channel contactless conductivity detection device for online detection of free-flow electrophoresis separation].

Free-flow electrophoresis (FFE) is an all-liquid-phase electrophoresis technique without any supporting media, which has both analytical and preparative functions. Compared to other electrophoresis techniques, FFE has been used for the separation of peptides, proteins, cells, and microorganisms due to its advantages of mild separation environment, high recovery, and sustainable separation. Both the online detection of the characteristic parameters for each component solution and the real-time control of the progress of the separation experiment are of considerable importance for the study of FFE separation. Since the existing FFE devices do not have the online detection function, there are obvious deficiencies in their practicability. The absence of online detection function not only made it impossible to track the progress of the separation experiment in real time, but also made it difficult to detect the properties of the component solutions, which still require offline testing after separation. In this study, a multi-channel capacitively coupled contactless conductivity detection (MC-C4D) device has been developed to solve this problem, and an automatic measurement software has also been developed. The MC-C4D device used a parallel time-sharing contactless conductivity detection technique. It consisted of several contactless conductivity detection modules arranged in parallel, which in turn consisted of a number of contactless conductivity cells that were switched on/off by analog multiplexers for detecting the conductivity of the solution flowing through the cells in real time. The number of cells was equal to the number of components of the FFE. The components were connected to each of the FFE flow channels, such that the MC-C4D device could be used to measure the conductivity of the solution flowing through each channel in parallel online. To verify the performance of the MC-C4D device, calibration was conducted by using potassium chloride (KCl) standard solutions on MC-C4D device. The experimental data showed that the detection range of MC-C4D was 0.015-2.5 mS/cm, and the limit of detection (LOD) was 0.002 mS/cm. The intra-day relative standard deviation (RSD, n=3) was 2.31%, the measurement relative error (RE) was 3.03%, and the measurement difference between channels was 1.60%. All these data validated that the device had the advantages of wide detection range, low LOD, good repeatability, high accuracy, and low variation between channels. The MC-C 4D device was also applied to reciprocating free-flow isoelectric focusing (RFFIEF) electrophoresis for real-time online detection of the conductivity of each component solution during protein focusing. At the start of isoelectric focusing, when the ions had not reached equilibrium loading in the electric field and the pH gradient had not yet been fully developed, there was little difference in conductivity between the different channels and the channel conductivity curve was relatively flat. As the experiment progressed, the proteins gradually started to enrich the anodic end. As the proteins accumulated towards the isoelectric point, their own net charge gradually decreased, and thus, the conductivity of the solution in the channels near the anodic region also decreased. Under sufficient isoelectric focusing, protein enrichment was evident. In the focusing region, the conductivity of the solution in the corresponding channel decreased further. There was also an increase in the conductivity of the solution in the corresponding channel due to the accumulation of ions near the electrode ends. These results showed that the MC-C4D device not only enabled real-time online detection of the conductivity of each component solution in FFE, but also aided in mastering the progress of separation experiment in RFFIEF, thus improving the practicality of the FFE device. Thus, the MC-C4D device, which had the advantages of good performance, small size, simple circuit system, easy installation and commissioning, and low cost, could play an important role in multi-channel measurement, online inspection, and process monitoring.

Longitudinal shear wave elasticity measurements of millimeter-sized biomaterials using a single-element transducer platform.

Temporal variations of the extracellular matrix (ECM) stiffness profoundly impact cellular behaviors, possibly more significantly than the influence of static stiffness. Three-dimensional (3D) cell cultures with tunable matrix stiffness have been utilized to characterize the mechanobiological interactions of elasticity-mediated cellular behaviors. Conventional studies usually perform static interrogations of elasticity at micro-scale resolution. While such studies are essential for investigations of cellular mechanotransduction, few tools are available for depicting the temporal dynamics of the stiffness of the cellular environment, especially for optically turbid millimeter-sized biomaterials. We present a single-element transducer shear wave (SW) elasticity imaging system that is applied to a millimeter-sized, ECM-based cell-laden hydrogel. The single-element ultrasound transducer is used both to generate SWs and to detect their arrival times after being reflected from the side boundaries of the sample. The sample's shear wave speed (SWS) is calculated by applying a time-of-flight algorithm to the reflected SWs. We use this noninvasive and technically straightforward approach to demonstrate that exposing 3D cancer cell cultures to X-ray irradiation induces a temporal change in the SWS. The proposed platform is appropriate for investigating in vitro how a group of cells remodels their surrounding matrix and how changes to their mechanical properties could affect the embedded cells in optically turbid millimeter-sized biomaterials.

Sonoporation based on repeated vaporization of gold nanodroplets.

BACKGROUND: Gold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase-shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring. PURPOSE: In this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation. METHODS: Perfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5- and 10-MHz focused US receivers. Numbers of calcein-AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells. RESULTS: We demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm2 for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm2 , respectively). CONCLUSION: We demonstrated the feasibility of vaporization-based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.