Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation.

The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect human pluripotent stem cell-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide chromatin immunoprecipitation enrichment and mechanistic studies show that p38 MAPK-mediated CCAAT enhancer binding protein delta (CEBPD) upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in mesenchymal stem cells exerts a negative regulatory effect on osteogenesis through a similar mechanism. Chromatin immunoprecipitation-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.

Perfluorooctanesulfonic Acid Detection Using Molecularly Imprinted Polyaniline on a Paper Substrate.

Perfluorinated compounds like perfluorooctanesulfonic acid (PFOS) are synthetic water pollutants and have accumulated in environments for decades, causing a serious global health issue. Conventional assays rely on liquid chromatography and mass spectroscopy that are very expensive and complicated and thus limit the large-scale monitoring of PFOS in wastewater. To achieve low-cost and accurate detection of PFOS, we designed a paper-based sensor with molecularly imprinted polyaniline electrodes that have recognition sites specific to PFOS. The calibration curve of resistivity ratios as a function of PFOS concentrations has a linear range from 1 to 100 ppt with a coefficient of determination of 0.995. The estimated limit of detection is 1.02 ppt. We also investigated attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) spectra of the surface of the polyaniline (PANI) electrodes to propose the potential recognition sites in polyaniline matrix and the detection mechanism. This electrical paper sensor with low cost and excellent sensitivity and selectivity provides the potential for large-scale monitoring of wastewater.

A Low-Cost Paper Glucose Sensor with Molecularly Imprinted Polyaniline Electrode.

For the hundreds of millions of worldwide diabetic patients, glucose test strips are the most important and commonly used tool for monitoring blood glucose levels. Commercial test strips use glucose oxidases as recognition agents, which increases the cost and reduces the durability of test strips. To lower the cost of glucose sensors, we developed a paper-based electrical sensor with molecularly imprinted glucose recognition sites and demonstrated the determination of various glucose concentrations in bovine blood solutions. The sensing electrode is integrated with molecular recognition sites in the conductive polymer. A calibration graph as a function of glucose concentration in aqueous solution was acquired and matched with a correlation coefficient of 0.989. We also demonstrated the determination of the added glucose concentrations ranging from 2.2 to 11.1 mM in bovine blood samples with a linear correlation coefficient of 0.984. This non-enzymatic glucose sensor has the potential to reduce the health care cost of test strips as well as make glucose sensor test strips more accessible to underserved communities.

Nurse-Administered Auricular Point Acupressure for Cancer-Related Pain.

PURPOSE: The study aimed to (1) examine the feasibility of providing a training course on auricular point acupressure (APA) for clinical oncology nurses to integrate APA into real-world nursing care settings, and (2) examine the effectiveness of APA on cancer-related pain (CRP) under usual inpatient oncology ward conditions. METHODS: This was a 2-phase feasibility study. Phase 1, an in-person, 8 hour training program was provided to oncology nurses. Phase 2, a prospective and feasibility study was conducted to evaluate the integration of APA into nursing care activities to manage CRP. Oncology patients were included if their pain was rated at ≥4 on a 0 to 10 numeric rating scale in the past 24 hours. Patients received 1 APA treatment administered by the nurses and were instructed to stimulate the points for 3 days. Study outcomes (pain intensity, fatigue, and sleep disturbance), pain medication use, and APA practice were measured by a phone survey daily. RESULTS: Ten oncology nurses received APA training in phase 1. APA had been added to the hospital's electronic health records (EHRs) as a pain treatment. In phase 2, 33 oncology patients received APA treatment with a 100% adherence rate (pressing the seeds 3 times per day, 3 minutes per time based on the suggestion). The side effects of APA were minimal (~8%-12% felt tenderness on the ear). After 3 days of APA, patients reported 38% pain relief, 39% less fatigue, and 45% improvement in sleep disturbance; 24% reduced any type of pain medication use and 19% reduced opioid use (10 mg opioids using milligram morphine equivalent). The major barrier to integrating APA into routine nursing practice was time management (how to include APA in a daily workflow). CONCLUSION: It is feasible to provide 8-hour training to oncology nurses for mastering APA skill and then integrating APA into their daily nursing care for patients with CRP. Based on the promising findings (decreased pain, improved fatigue and sleep disturbance, and less opioid use), the next step is to conduct a randomized clinical trial with a larger sample to confirm the efficacy of APA for oncology nurses to treat CRP in real-world practice.ClinicalTrial.gov identifier number: NCT04040140.

Wearable Biosensor with Molecularly Imprinted Conductive Polymer Structure to Detect Lentivirus in Aerosol.

The coronavirus disease (COVID-19) pandemic has increased pressure to develop low-cost, compact, user-friendly, and ubiquitous virus sensors for monitoring infection outbreaks in communities and preventing economic damage resulting from city lockdowns. As proof of concept, we developed a wearable paper-based virus sensor based on a molecular imprinting technique, using a conductive polyaniline (PANI) polymer to detect the lentivirus as a test sample. This sensor detected the lentivirus with a 4181 TU/mL detection limit in liquid and 0.33% to 2.90% detection efficiency in aerosols at distances ranging from 30 cm to 60 cm. For fabrication, a mixture of a PANI monomer solution and virus were polymerized together to form a conductive PANI sensing element on a polyethylene terephthalate (PET) paper substrate. The sensing element exhibited formation of virus recognition sites after the removal of the virus via ultrasound sonication. A dry measurement technique was established that showed aerosol virus detection by the molecularly imprinted sensors within 1.5 h of virus spraying. This was based on the mechanism via which dispensing virus droplets on the PANI sensing element induced hybridization of the virus and molecularly imprinted virus recognition templates in PANI, influencing the conductivity of the PANI film upon drying. Interestingly, the paper-based virus sensor was easily integrated with a wearable face mask for the detection of viruses in aerosols. Since the paper sensor with molecular imprinting of virus recognition sites showed excellent stability in dry conditions for long periods of time, unlike biological reagents, this wearable biosensor will offer an alternative approach to monitoring virus infections in communities.

A hydraulic soft microgripper for biological studies.

We have developed a microscale hydraulic soft gripper and demonstrated the handling of an insect without damage. This gripper is built on Polydimethylsiloxane (PDMS) with the soft material casting technique to form three finger-like columns, which are placed on a circular membrane. The fingers have a length of 1.5 mm and a diameter of 300 µm each; the distance between the two fingers is 600 µm of center-to-center distance. A membrane as a 150 µm soft film is built on top of a cylindrical hollow space. Applying pressure to the interior space can bend the membrane. Bending the membrane causes the motion of opening/closing of the gripper, and as a result, the three fingers can grip an object or release it. The PDMS was characterized, and the experimental results were used later in Abaqus software to simulate the gripping motion. The range of deformation of the gripper was investigated by simulation and experiment. The result of the simulation agrees with the experiments. The maximum 543 µN force was measured for this microfluidic-compatible microgripper and it could lift a ball that weighs 168.4 mg and has a 0.5 mm diameter. Using this microgripper, an ant was manipulated successfully without any damage. Results showed fabricated device has great a potential as micro/bio manipulator.

A Hybrid Structure of Piezoelectric Fibers and Soft Materials as a Smart Floatable Open-Water Wave Energy Converter.

An open-water wave energy converter (OWEC) made of a new soft platform has been developed by combining piezoelectric macro-fiber composites (MFCs) and a low-cost elastomer. In the past decades, numerous types of water wave energy conversion platform have been developed and investigated, from buoys to overtopping devices. These harvesters mainly use electromagnetic-based generators, and they have faced challenges such as their enormous size, high deployment and maintenance costs, and negative effects on the environment. These problems hinder their practicality and competitiveness. In this paper, a soft open-water wave energy converter is introduced which integrates piezoelectric MFCs and bubble wrap into an elastomer sheet. The performance of the OWEC was investigated in a wave flume as a floatable structure. The maximum 29.7 µW energy harvested from the small OWEC represents a promising energy conversion performance at low frequencies (<2 Hz). The elastomer was able to protect the MFCs and internal electrical connections without any degradation during the experiment. In addition, the OWEC is a foldable structure, which can reduce the deployment costs in real-world applications. The combination of no maintenance, low fabrication cost, low deployment cost, and moderate energy harvesting capability may advance the OWEC platform to its real-world applications.

High speed digital protein interaction analysis using microfluidic single molecule detection system.

The understanding of protein interaction dynamics is important for signal transduction research but current available techniques prove difficult in addressing this issue. Thus, using the microfluidic approach, we developed a digital protein analytical platform and methodology named MAPS (Microfluidic system Analyzing Protein in Single complex) that can measure the amount of target proteins and protein complexes at the digitally single molecule resolution. By counting protein events individually, this system can provide rough protein interaction ratios which will be critical for understanding signal transduction dynamics. In addition, this system only requires less than an hour to characterize the target protein sample, which is much quicker than conventional approaches. As a proof of concept, we have determined the interaction ratios of oncogenic signaling protein complexes EGFR/Src and EGFR/STAT3 before and after EGF ligand stimulation. To the best of our knowledge, this is the first time that the interaction ratio between EGFR and its downstream proteins has been characterized. The information from MAPS will be critical for the study of protein signal transduction quantitation and dynamics.

Rapid prototyping of nanofluidic systems using size-reduced electrospun nanofibers for biomolecular analysis.

Biomolecular transport in nanofluidic confinement offers various means to investigate the behavior of biomolecules in their native aqueous environments, and to develop tools for diverse single-molecule manipulations. Recently, a number of simple nanofluidic fabrication techniques has been demonstrated that utilize electrospun nanofibers as a backbone structure. These techniques are limited by the arbitrary dimension of the resulting nanochannels due to the random nature of electrospinning. Here, a new method for fabricating nanofluidic systems from size-reduced electrospun nanofibers is reported and demonstrated. As it is demonstrated, this method uses the scanned electrospinning technique for generation of oriented sacrificial nanofibers and exposes these nanofibers to harsh, but isotropic etching/heating environments to reduce their cross-sectional dimension. The creation of various nanofluidic systems as small as 20 nm is demonstrated, and practical examples of single biomolecular handling, such as DNA elongation in nanochannels and fluorescence correlation spectroscopic analysis of biomolecules passing through nanochannels, are provided.

Rapid detection of two-protein interaction with a single fluorophore by using a microfluidic device.

We have developed a microfluidics based platform and methodology named MAPS (microfluidic system for analyzing proteins in single complex) for detecting two protein interactions rapidly using a single fluorophore. Target proteins were labelled with Quantum dot 525 (QD525) via specific polyclonal antibodies, and were transported through the microfluidic channel subsequently, where the 375 nm excitation laser light was focused to form a detection volume. Photon bursts from target proteins passing through the detection volume were recorded and their photon burst histograms were plotted which demonstrated roughly the specific protein interaction ratio based on their population and statistical behavior. As a proof of concept, Src/STAT3 protein complex interaction ratios with and without EGF stimulation were obtained by MAPS within 1 h and the results were well matched with the one obtained by the conventional immunoprecipitation/Western blot (IP/WB).

Isolation of cancer-derived extracellular vesicle subpopulations by a size-selective microfluidic platform.

Extracellular vesicles (EVs) play an important role in intercellular communication. Recently, there has been increasing interest in EVs as potential diagnostic biomarkers and therapeutic vehicles. However, the molecular properties and cargo information of EV subpopulations have not yet been fully investigated due to lack of reliable and reproducible EV separation technology. Current approaches have faced difficulties with efficiently isolating EVs from biofluids, especially subpopulations of small EVs. Here, we report an EV isolation method based on a size-selective microfluidic platform (ExoSMP) via nanomembrane filtration and electrophoretic force. This unique platform offers an enhanced approach to sorting a heterogeneous population of EVs based on size, with the additional advantages of being label-free and low-cost, and featuring a short processing time (<1 h), and convenient integration with downstream analysis. In this research, we used ExoSMP to demonstrate the isolation of cancer-derived small EVs (30-120 nm) with high recovery (94.2%) and reproducibility at an optimum sample flow rate. Furthermore, we investigated isolation of EV subpopulations by altering nanomembrane combinations with different pore size combinations (50 and 100 nm, 30 and 100 nm, 30 and 200 nm, and 30 and 50 nm). This ExoSMP technique can serve as a standardized EV isolation/separation tool, facilitating the clinical prospects of EVs and opening up a new avenue for future point-of-care applications in liquid biopsies.

A Low-cost and Enzyme-free Glucose Paper Sensor.

A low-cost and enzyme-free glucose paper sensor is presented as a promising alternative to glucose test strips. This paper-based glucose sensor is prepared with molecularly imprinted (MIP) polyaniline (PANI) electrode. The determination of glucose concentrations was studied by the impedance change of the paper sensor before and after the blood samples dispensing at a low frequency. A comparison of the linear and polynomial regression was applied to analyze the impedance ratio as a function of glucose concentrations. The proposed glucose paper sensor showed a limit of detection (LoD) of 1.135 mM. This novel and non-enzymatic paper sensor suggests a low-cost glucose test assay and can improve the quality of routine testing for diabetic patients.

Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant (Kd) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

Development of size-selective microfluidic platform.

Exosomes are nanosized extracellular vesicles that play a significant role in cell-cell communication. Recently, there is significant interest in exosome-related fundamental research, especially subgroups of exosomes as potential biomarkers for cancer diagnosis and prognosis. In this paper, we report a new size selective isolation method via elastic lift force and nanomembrane filtration and demonstrated the liposome recovery rate of 92.5% from a mixture solution of 1 µm polystyrene beads, 100 nm liposomes and proteins as a proof of concept for exosome isolation. This single microfluidic platform offers an improved approach with short processing time (<; 2 hours) and low cost, and shows potential broad applicability to cancer biomarker studies.

Autologous hematopoietic stem cell transplantation in extranodal natural killer/T cell lymphoma: a multinational, multicenter, matched controlled study.

Extranodal natural killer (NK)/T cell lymphoma, nasal type, is a recently recognized distinct entity and the most common type of non-B cell extranodal lymphoma in Asia. This retrospective analysis studied the potential survival benefits of hematopoeitic stem cell transplantation (HSCT) compared with a historical control group. A total of 47 patients from 3 previously published series of HSCT were matched according to NK/T cell lymphoma International Prognostic Index (NKIPI) risk groups and disease status at transplantation with 107 patients from a historical control group for analysis. After a median follow-up of 116.5 months, the median survival time was not determined for the HSCT group, but it was 43.5 months for the control group (95% confidence interval [CI] = 6.7 to 80.3 months; P = .127, log-rank test). In patients who were in complete remission (CR) at the time of HSCT or at surveillance after remission, disease-specific survival rates were significantly higher in the HSCT group compared with the control group (disease-specific 5-year survival rate, 87.3% for HSCT vs 67.8% for non-HSCT; P = .027). In contrast, in subgroup analysis on non-CR patients at the time of HSCT or non-HSCT treatment, disease-specific survival rates were not significantly prolonged in the HSCT group compared with the control group (1-year survival rate, 66.7% for HSCT vs 28.6% for non-HSCT; P = .141). The impact of HSCT on the survival of all patients was significantly retained at the multivariate level with a 2.1-fold (95% CI =1.2- to 3.7-fold) reduced risk of death (P = .006). HSCT seems to confer a survival benefit in patients who attained CR on postremission consolidation therapy. These findings suggest that, in particular, patients in CR with high NKIPI risk scores at diagnosis should receive full consideration for HSCT.

Reusable Functionalized Hydrogel Sorbents for Removing Long- and Short-Chain Perfluoroalkyl Acids (PFAAs) and GenX from Aqueous Solution.

Per- and poly-fluoroalkyl substances (PFASs) are man-made chemicals that are toxic and widely detected in the environment, including drinking water sources. A cost-effective treatment process for PFASs is currently not available. We developed reusable hydrogel sorbents to remove long- and short-chain perfluoroalkyl acids and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX), which is are emerging PFAS. Through fluoridation and amination of poly(ethylene glycol) diacrylate (PEGDA), the newly synthesized sorbents can sorb the five targeted PFASs (perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorobutanesulfonic acid (PFBS), and perfluorobutanoic acid (PFBA) and GenX) to different degrees from aqueous solution. Aminated PEGDA showed the highest sorption capacity for all five PFASs, particularly for PFBA and PFBS. The bifunctionalized PEGDA showed higher capacities for PFOA and PFOS, suggesting that both hydrophobic interactions and charges contribute to the sorption. Both aminated and bifunctionalized sorbents can remove GenX from water, with the highest sorption capacity of 98.7 µmol g aminated PEGDA-1 within 6 h. The absorbed PFASs on the sorbents were observed and characterized by Fourier-transform infrared spectroscopy. The spent sorbents were reusable after readily regenerated with 70% methanol contained 1% NaCl.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R2], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level.

An optofluidic device for surface enhanced Raman spectroscopy.

We have developed an optofluidic device that improves the sensitivity of surface enhanced Raman spectroscopy (SERS) when compared to other SERS approaches. This device has a pinched and step microchannel-nanochannel junction that can trap and assemble nanoparticles/target molecules into optically enhanced SERS active clusters by using capillary force. These SERS active clusters provide an electromagnetic enhancement factor of approximately 10(8). In addition, due to the continuous capillary flow that can transport nanoparticles/target molecules into the junction sites, the numbers of nanoparticles/target molecules and SERS active sites are increased. As a result, the detection limit of SERS for adenine molecules was better than 10 pM.

A magneto-fluidic nanoparticle trapping platform for surface-enhanced Raman spectroscopy.

A microfluidic device utilizing magnetically activated nickel (Ni) micropads has been developed for controlled localization of plasmonic core-shell magnetic nanoparticles, specifically for surface enhanced Raman spectroscopy (SERS) applications. Magnetic microfluidics allows for automated washing steps, provides a means for easy reagent packaging, allows for chip reusability, and can even be used to facilitate on-chip mixing and filtration towards full automation of biological sample processing and analysis. Milliliter volumes of gold-coated 175-nm silica encapsulated iron oxide nanoparticles were pumped into a microchannel and allowed to magnetically concentrate down into 7.5 nl volumes over nano-thick lithographically defined Ni micropads. This controlled aggregation of core-shell magnetic nanoparticles by an externally applied magnetic field not only enhances the SERS detection limit within the newly defined nanowells but also generates a more uniform (∼92%) distribution of the SERS signal when compared to random mechanical aggregation. The microfluidic flow rate and the direction and strength of the magnetic field determined the overall capture efficiency of the magneto-fluidic nanoparticle trapping platform. It was found that a 5 µl/min flow rate using an attractive magnetic field provided by 1 × 2 cm neodymium permanent magnets could capture over 90% of the magnetic core-shell nanoparticles across five Ni micropads. It was also observed that the intensity of the SERS signal for this setup was 10-fold higher than any other flow rate and magnetic field configurations tested. The magnetic concentration of the ferric core-shell nanoparticles causes the SERS signal to reach the steady state within 30 min can be reversed by simply removing the chip from the magnet housing and sonicating the retained particles from the outlet channel. Additionally, each magneto-fluidic can be reused without noticeable damage to the micropads up to three times.