A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

The Upstream 1350~1250 Nucleotide Sequences of the Human ENDOU-1 Gene Contain Critical Cis-Elements Responsible for Upregulating Its Transcription during ER Stress.

ENDOU-1 encodes an endoribonuclease that overcomes the inhibitory upstream open reading frame (uORF)-trap at 5'-untranslated region (UTR) of the CHOP transcript, allowing the downstream coding sequence of CHOP be translated during endoplasmic reticulum (ER) stress. However, transcriptional control of ENDOU-1 remains enigmatic. To address this, we cloned an upstream 2.1 kb (-2055~+77 bp) of human ENDOU-1 (pE2.1p) fused with reporter luciferase (luc) cDNA. The promoter strength driven by pE2.1p was significantly upregulated in both pE2.1p-transfected cells and pE2.1p-injected zebrafish embryos treated with stress inducers. Comparing the luc activities driven by pE2.1p and -1125~+77 (pE1.2p) segments, we revealed that cis-elements located at the -2055~-1125 segment might play a critical role in ENDOU-1 upregulation during ER stress. Since bioinformatics analysis predicted many cis-elements clustered at the -1850~-1250, we further deconstructed this segment to generate pE2.1p-based derivatives lacking -1850~-1750, -1749~-1650, -1649~-1486, -1485~-1350 or -1350~-1250 segments. Quantification of promoter activities driven by these five internal deletion plasmids suggested a repressor binding element within the -1649~-1486 and an activator binding element within the -1350~-1250. Since luc activities driven by the -1649~-1486 were not significantly different between normal and stress conditions, we herein propose that the stress-inducible activator bound at the -1350~-1250 segment makes a major contribution to the increased expression of human ENDOU-1 upon ER stresses.

Donor leukocyte trafficking during human ex vivo lung perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to assess and preserve lungs prior to transplantation. However, its inherent immunomodulatory effects are not completely understood. We examine perfusate and tissue compartments to determine the change in immune cell composition in human lungs maintained on EVLP. METHODS: Six human lungs unsuitable for transplantation underwent EVLP. Tissue and perfusate samples were obtained during cold storage and at 1-, 3- and 6-h during perfusion. Flow cytometry, immunohistochemistry, and bead-based immunoassays were used to measure leukocyte composition and cytokines. Mean values between baseline and time points were compared by Student's t test. RESULTS: During the 1st hour of perfusion, perfusate neutrophils increased (+22.2 ± 13.5%, p < 0.05), monocytes decreased (-77.5 ± 8.6%, p < 0.01) and NK cells decreased (-61.5 ± 22.6%, p < 0.01) compared to cold storage. In contrast, tissue neutrophils decreased (-22.1 ± 12.2%, p < 0.05) with no change in monocytes and NK cells. By 6 h, perfusate neutrophils, NK cells, and tissue neutrophils were similar to baseline. Perfusate monocytes remained decreased, while tissue monocytes remained unchanged. There was no significant change in B cells or T cell subsets. Pro-inflammatory cytokines (IL-1b, G-CSF, IFN-gamma, CXCL2, CXCL1 granzyme A, and granzyme B) and lymphocyte activating cytokines (IL-2, IL-4, IL-6, IL-8) increased during perfusion. CONCLUSIONS: Early mobilization of innate immune cells occurs in both perfusate and tissue compartments during EVLP, with neutrophils and NK cells returning to baseline and monocytes remaining depleted after 6 h. The immunomodulatory effect of EVLP may provide a therapeutic window to decrease the immunogenicity of lungs prior to transplantation.

Attention modulates the contextual similarity effect in negative priming: evidence from task demand and attentional capture.

Negative priming refers to the delayed response to a probe target that was previously a prime distractor. Memory retrieval has been proposed as one critical mechanism for the manifestation of negative priming. This perspective perpetuates that the contextual similarity between prime and probe trials should modulate memory retrieval, and therefore, affect negative priming. However, evidence for the contextual similarity effect in negative priming is mixed. The present study tested the hypothesis that attended contextual cues are more likely to be encoded into a distractor representation, and thus, are more likely to modulate the negative priming effect. By manipulating whether the contextual cues were relevant to the task demand in Section 1, and by manipulating whether cues had an abrupt or simultaneous onset, and by analysing reaction time (RT) distributions of the data in Section 2, our results demonstrated that attended cues produced the contextual similarity effect in negative priming, especially when RTs were long.

Protective Effects of Jujubosides on 6-OHDA-Induced Neurotoxicity in SH-SY5Y and SK-N-SH Cells.

6-hydroxydopamine (6-OHDA) is used to induce oxidative damage in neuronal cells, which can serve as an experimental model of Parkinson's disease (PD). Jujuboside A and B confer free radical scavenging effects but have never been examined for their neuroprotective effects, especially in PD; therefore, in this study, we aimed to investigate the feasibility of jujubosides as protectors of neurons against 6-OHDA and the underlying mechanisms. 6-OHDA-induced neurotoxicity in the human neuronal cell lines SH-SY5Y and SK-N-SH, was used to evaluate the protective effects of jujubosides. These findings indicated that jujuboside A and B were both capable of rescuing the 6-OHDA-induced loss of cell viability, activation of apoptosis, elevation of reactive oxygen species, and downregulation of the expression levels of superoxide dismutase, catalase, and glutathione peroxidase. In addition, jujuboside A and B can reverse a 6-OHDA-elevated Bax/Bcl-2 ratio, downregulate phosphorylated PI3K and AKT, and activate caspase-3, -7, and -9. These findings showed that jujubosides were capable of protecting both SH-SY5Y and SK-N-SH neuronal cells from 6-OHDA-induced toxicity via the rebalancing of the redox system, together with the resetting of the PI3K/AKT apoptotic signaling cascade. In conclusion, jujuboside may be a potential drug for PD prevention.

Rhesus Blood Typing within a Few Seconds by Packing-Enhanced Nanoscattering on Individual Erythrocytes.

A method for the ABO and Rhesus (Rh) blood group typing from individual erythrocytes is proposed in this study. Blood-group-specific antibodies immobilized to gold nanoparticles (BG-AuNP) were utilized for the identification of blood groups from individual erythrocytes by objective-type dark-field microscopy (OTDFM). The scattering of free BG-AuNP and their Brownian motion as well as BG-AuNP attached on erythrocytes is easily observed by OTDFM. The strong scattering intensity caused by BG-AuNP packing-enhanced nanoscattering (PENS) on erythrocytes is first demonstrated. PENS combined with OTDFM allows us to identify blood groups within 5 s for all blood group antigens including A, B, D, C, c, E, and e. This was immediately identified by mixing with BG-AuNP without any washing step or waiting for hemoagglutination. Therefore, PENS combined with OTDFM demonstrates feasibility and advantages for use in emergency transfusions where the blood group of patients is unknown. Moreover, matching RhD+ in the case of emergency transfusions may also be beneficial in reducing the shortage of RhD- red blood cell concentrate in the case of a population with a high frequency in RhD-.

The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2-HO-1 Axis.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. METHODS: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. RESULTS: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. CONCLUSIONS: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.

Cortical Bone Trajectory Instrumentation with Vertebroplasty for Osteoporotic Thoracolumbar Compression Fracture.

BACKGROUND: Osteoporotic spinal fractures commonly occur in elderly patients with low bone mineral density. In these cases, percutaneous vertebroplasty or percutaneous kyphoplasty can provide significant pain relief and improve mobility. However, studies have reported both the recurrence of vertebral compression fractures at the index level after vertebroplasty and the development of new vertebral fractures at the adjacent level that occur without any additional trauma. Pedicle screw fixation combined with percutaneous vertebroplasty has been proposed as an effective procedure for addressing osteoporotic thoracolumbar fractures. However, in osteoporotic populations, pedicle screws can loosen, pullout, or migrate. Currently, the efficacy of cortical bone trajectory screw fixation for osteoporotic fractures remains unclear. Thus, we assessed the effects of using cortical bone trajectory instrumentation with vertebroplasty on patient outcomes. METHOD: We retrospectively reviewed data from 12 consecutively sampled osteoporotic thoracolumbar fracture patients who underwent cortical bone trajectory instrumentation with vertebroplasty. Patients were enrolled beginning in October 2015 and were followed for >24 months. RESULT: The average age was 74 years, and the average dual-energy x-ray absorptiometry T-score was -3.6. The average visual analog scale pain scores improved from 8 to 2.5 after surgery. The average blood loss was 36.25 mL. All patients regained ambulation and experienced reduced pain post-surgery. No recurrent fractures or instrument failures were recorded during follow-up. CONCLUSIONS: Our findings suggest that cortical bone trajectory instrumentation combined with percutaneous vertebroplasty may be a good option for treating osteoporotic thoracolumbar fractures, as it can prevent recurrent vertebral fractures or related kyphosis in sagittal alignment.

Blood Plasma Separation Using a Fidget-Spinner.

In this study, we present a simple, hand-powered, and electricity-free centrifuge platform based on a commercially available "fidget-spinner." The centrifugal force provided by this inexpensive and easy-to-use toy is sufficient to separate whole blood, producing a plasma yield rate and purity of 30% and 99%, respectively, separated in as little as 4-7 min. We verified the separated plasma by performing a paper-based HIV-1 p24 capsid protein enzyme-linked immunosorbent assay, which achieved a recovery rate of up to 98%, indicating the plasma features extremely low matrix interference effects. These results demonstrate the reliability of the platform for practical use, in addition to greatly reducing the overall cost and time of analysis while retaining detection precision, making it suitable for medical applications in resource-limited regions of the world.

A collective tracking method for preliminary sperm analysis.

BACKGROUND: Total motile sperm count (TMSC) and curvilinear velocity (VCL) are two important parameters in preliminary semen analysis for male infertility. Traditionally, both parameters are evaluated manually by embryologists or automatically using an expensive computer-assisted sperm analysis (CASA) instrument. The latter applies a point-tracking method using an image processing technique to detect, recognize and classify each of the target objects, individually, which is complicated. However, as semen is dense, manual counting is exhausting while CASA suffers from severe overlapping and heavy computation. METHODS: We proposed a simple frame-differencing method that tracks motile sperms collectively and treats their overlapping with a statistical occupation probability without heavy computation. The proposed method leads to an overall image of all of the differential footprint trajectories (DFTs) of all motile sperms and thus the overall area of the DFTs in a real-time manner. Accordingly, a theoretical DFT model was also developed to formulate the overall DFT area of a group of moving beads as a function of time as well as the total number and average speed of the beads. Then, using the least square fitting method, we obtained the optimal values of the TMSC and the average VCL that yielded the best fit for the theoretical DFT area to the measured DFT area. RESULTS: The proposed method was used to evaluate the TMSC and the VCL of 20 semen samples. The maximum TMSC evaluated using the method is more than 980 sperms per video frame. The Pearson correlation coefficient (PCC) between the two series of TMSC obtained using the method and the CASA instrument is 0.946. The PCC between the two series of VCL obtained using the method and CASA is 0.771. As a consequence, the proposed method is as accurate as the CASA method in TMSC and VCL evaluations. CONCLUSION: In comparison with the individual point-tracking techniques, the collective DFT tracking method is relatively simple in computation without complicated image processing. Therefore, incorporating the proposed method into a cell phone equipped with a microscopic lens can facilitate the design of a simple sperm analyzer for clinical or household use without advance dilution.

Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations.

Here we present the realization of a novel fluorescence detection method based on the electromigration of fluorescent molecules within a nanocapillary combined with the laser excitation through a platinum (Pt)-coated nanocapillary. By using the Pt nanocapillary assisted focusing of a laser beam, we completely remove the background scattering on the tip of the electrophoretic nanocapillary. In this excitation geometry, we demonstrate a 1000-fold sensitivity enhancement (1.0 nM to 1.0 pM) compared to the detection in microcapillaries with epifluorescence illumination and fluorescence spectrophotometry. Due to a significant electroosmotic flow, we observe a decelerating migration of DNA molecules close to the tip of the electrophoretic nanocapillary. The reduced DNA translocation velocity causes a two-step stacking process of molecules in the tip of the nanocapillary and can be used as a way to locally concentrate molecules. The sensitivity of our method is further improved by a continuous electrokinetic injection of DNA molecules followed by sample zone stacking on the tip of the nanocapillary. Concentrations ranging from 0.1 pM to 1.0 fM can be directly observed on the orifice of the electrophoretic nanocapillary. This is a 1000-fold improvement compared to traditional capillary electrophoresis with laser-induced fluorescence.

The role of awareness in the cognitive control of single-prime negative priming.

Single-prime negative priming refers to a phenomenon whereby repeating a single prime as a probe target slows responses to that target. This phenomenon is modulated by cognitive control when the contingency between the prime and probe target is higher than chance. The present study investigated the role of prime awareness and awareness of the contingency within the control mechanism during single-prime negative priming. Results showed that while single-prime negative priming occurred regardless of participant awareness, the control mechanism was modulated by prime awareness and perceived contingency.

Development of an Internal Real-Time Wireless Diagnostic Tool for a Proton Exchange Membrane Fuel Cell.

To prolong the operating time of unmanned aerial vehicles which use proton exchange membrane fuel cells (PEMFC), the performance of PEMFC is the key. However, a long-term operation can make the Pt particles of the catalyst layer and the pollutants in the feedstock gas bond together (e.g., CO), so that the catalyst loses reaction activity. The performance decay and aging of PEMFC will be influenced by operating conditions, temperature, flow and CO concentration. Therefore, this study proposes the development of an internal real-time wireless diagnostic tool for PEMFC, and uses micro-electro-mechanical systems (MEMS) technology to develop a wireless and thin (<50 µm) flexible integrated (temperature, flow and CO) microsensor. The technical advantages are (1) compactness and three wireless measurement functions; (2) elastic measurement position and accurate embedding; (3) high accuracy and sensitivity and quick response; (4) real-time wireless monitoring of dynamic performance of PEMFC; (5) customized design and development. The flexible integrated microsensor is embedded in the PEMFC, three important physical quantities in the PEMFC, which are the temperature, flow and CO, can be measured simultaneously and instantly, so as to obtain the authentic and complete reaction in the PEMFC to enhance the performance of PEMFC and to prolong the service life.

In-Situ Measurement of High-Temperature Proton Exchange Membrane Fuel Cell Stack Using Flexible Five-in-One Micro-Sensor.

In the chemical reaction that proceeds in a high-temperature proton exchange membrane fuel cell stack (HT-PEMFC stack), the internal local temperature, voltage, pressure, flow and current nonuniformity may cause poor membrane material durability and nonuniform fuel distribution, thus influencing the performance and lifetime of the fuel cell stack. In this paper micro-electro-mechanical systems (MEMS) are utilized to develop a high-temperature electrochemical environment-resistant five-in-one micro-sensor embedded in the cathode channel plate of an HT-PEMFC stack, and materials and process parameters are appropriately selected to protect the micro-sensor against failure or destruction during long-term operation. In-situ measurement of the local temperature, voltage, pressure, flow and current distributions in the HT-PEMFC stack is carried out. This integrated micro-sensor has five functions, and is favorably characterized by small size, good acid resistance and temperature resistance, quick response, real-time measurement, and the goal is being able to be put in any place for measurement without affecting the performance of the battery.

A magnetic induction heating system with multi-cascaded coils and adjustable magnetic circuit for hyperthermia.

Based on the characteristics of cancer cells that cannot survive in an environment with temperature over 42 °C, a magnetic induction heating system for cancer treatment is developed in this work. First, the methods and analyses for designing the multi-cascaded coils magnetic induction hyperthermia system are proposed, such as internal impedance measurement of power generator, impedance matching of coils, and analysis of the system. Besides, characteristics of the system are simulated by a full-wave package for engineering optimization. Furthermore, by considering the safety factor of patients, a two-sectional needle is designed for hyperthermia. Finally, this system is employed to test the liver of swine in ex-vivo experiments, and through Hematoxylin and Eosin (H&E) stain and NADPH oxidase activity assay, the feasibility of this system is verified.

Cold pretreatment amplifies the responses of in vitro Eucalyptus grandis shoots to cryopreparative drying.

BACKGROUND: Eucalyptus grandis is an important commercial forestry species in South Africa. Little is known about its response (which is both drought and cold sensitive) to cold exposure and subsequent drying. OBJECTIVE: The study was to investigate the responses of E. grandis in vitro shoots to cold pretreatment. METHODS: E. grandis in vitro shoots were subjected to cold pretreatment (5, 10 and 15 degrees C at different exposure periods of 1 or 3 days), different drying times (20, 40, 60 and 80 min over activated silica gel), and the combination of the selected cold pretreatment regime and subsequent drying. RESULTS: Cold pretreatment alone did not cause detrimental effects but elicited the accumulation of phenolic acids. Shoots exposed to 5 degrees C for 3 days had significantly higher levels of reactive oxygen species as well. The accumulation of ROS and phenolic acids was also noted in unacclimated, dried shoots, especially after 80 min of drying. In addition, these shoots had significantly higher levels of total soluble sugars, lower levels of starch and elevated proline levels. CONCLUSION: This osmotic adjustment strategy was amplified in cold acclimated, dried shoots (10 degrees C, 3 days; 80 min drying), which also had significantly lower levels of ROS, increased levels of phenolic acid, and higher water content and viability.

Attentional control and priming: The distractor set hypothesis.

Repeating a single-prime stimulus as a target to respond to usually facilitates responses. However, sometimes, prime repetition slows the responses and produces the single-prime negative priming effect. In this study, the distractor set hypothesis was proposed as a mechanism of attentional control that can contribute toward single-prime negative priming. In Experiments 1a-1d, an integrated Stroop task was used. The results showed that the prime produced negative priming only when it matched the form of the competing distractors. A separate Stroop task was used in Experiments 2 and 3 and a flanker task was used in Experiments 4a and 4b. In both tasks, the results suggested that a prime produced negative priming when the location of the prime matched that of the distractors. In Experiment 5, alternative explanations including the effects of the prime-to-distractor similarity and the target set were examined. The results revealed that the distractor set, rather than the target set and the similarity between the prime and distractor, could better account for the negative priming effect. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Bio-Inspired Functional Materials for Environmental Applications.

With the global population expected to reach 9.7 billion by 2050, there is an urgent need for advanced materials that can address existing and developing environmental issues. Many current synthesis processes are environmentally unfriendly and often lack control over size, shape, and phase of resulting materials. Based on knowledge from biological synthesis and assembly processes, as well as their resulting functions (e.g., photosynthesis, self-healing, anti-fouling, etc.), researchers are now beginning to leverage these biological blueprints to advance bio-inspired pathways for functional materials for water treatment, air purification and sensing. The result has been the development of novel materials that demonstrate enhanced performance and address sustainability. Here, an overview of the progress and potential of bio-inspired methods toward functional materials for environmental applications is provided. The challenges and opportunities for this rapidly expanding field and aim to provide a valuable resource for researchers and engineers interested in developing sustainable and efficient processes and technologies is discussed.

The risk analysis index is an independent predictor of outcomes after lung cancer resection.

BACKGROUND: The Risk Analysis Index (RAI) is a frailty assessment tool based on an accumulation of deficits model. We mapped RAI to data from the Society of Thoracic Surgeons (STS) Database to determine whether RAI correlates with postoperative outcomes following lung cancer resection. METHODOLOGY/PRINCIPAL FINDINGS: This was a national database retrospective observational study based on data from the STS Database. Study patients underwent surgery 2018 to 2020. RAI was divided into four increasing risk categories. The associations between RAI and each of postoperative complications and administrative outcomes were examined using logistic regression models. We also compared the performance of RAI to established risk indices (American Society of Anesthesiology (ASA) and Charlson Comorbidity Index (CCI)) using areas under the Receiver Operating Characteristic (ROC) curves (AUC). Results: Of 29,420 candidate patients identified in the STS Database, RAI could be calculated for 22,848 (78%). Almost all outcome categories exhibited a progressive increase in marginal probability as RAI increased. On multivariable analyses, RAI was significantly associated with an incremental pattern with almost all outcomes. ROC analyses for RAI demonstrated "good" AUC values for mortality (0.785; 0.748) and discharge location (0.791), but only "fair" values for all other outcome categories (0.618 to 0.690). RAI performed similarly to ASA and CCI in terms of AUC score categories. CONCLUSIONS/SIGNIFICANCE: RAI is associated with clinical and administrative outcomes following lung cancer resection. However, its overall accuracy as a surgical risk predictor is only moderate and similar to ASA and CCI. We do not recommend routine use of RAI for assessment of individual patient risk for major lung resection.