Dual add-on therapy of gemigliptin and dapagliflozin in patients with type 2 diabetes inadequately controlled with metformin alone: The SOLUTION 2 study.

AIM: To evaluate the efficacy and safety of gemigliptin and dapagliflozin dual add-on therapy (GEMI + DAPA) to metformin in type 2 diabetes (T2D) patients who had inadequate glycaemic control on metformin alone, compared with a single add-on of either gemigliptin (GEMI) or dapagliflozin (DAPA) to metformin. MATERIALS AND METHODS: In this randomized, double-blind, double-dummy, active-controlled, parallel-group, phase 3 study, 469 T2D patients treated with a stable dose of metformin for 8 weeks or longer were randomized to receive GEMI + DAPA (n = 157) and either GEMI (n = 156) or DAPA (n = 156). The primary endpoint was change in HbA1c levels from baseline at week 24. RESULTS: Baseline characteristics including body mass index and T2D duration were similar among groups. At week 24, the least square mean changes in HbA1c from baseline were -1.34% with GEMI + DAPA, -0.90% with GEMI (difference between GEMI + DAPA vs. GEMI -0.44% [95% confidence interval {CI}: -0.58% to -0.31%], P < .01) and -0.78% with DAPA (difference between GEMI + DAPA vs. DAPA -0.56% [95% CI: -0.69% to -0.42%], P < .01). Both upper CIs were less than 0, demonstrating the superiority of GEMI + DAPA for lowering HbA1c. The rates of responders achieving HbA1c less than 7% and less than 6.5% were greater with GEMI + DAPA (84.9%, 56.6%) than with GEMI (55.3%, 32.2%) and DAPA (49.3%, 15.3%). The incidence rate of adverse events was similar across groups, with low incidence rates of hypoglycaemia, urinary tract infection and genital infection. CONCLUSIONS: These results suggest that the addition of GEMI + DAPA to metformin as triple combination therapy was effective, safe and well-tolerated, especially for T2D patients who experienced poor glycaemic control on metformin alone.

Integrated high-throughput drug screening microfluidic system for comprehensive ocular toxicity assessment.

Traditional experimental methodologies suffer from a few limitations in the toxicological evaluation of the preservatives added to eye drops. In this study, we overcame these limitations by using a microfluidic device. We developed a microfluidic system featuring a gradient concentration generator for preservative dosage control with microvalves and micropumps, automatically regulated by a programmable Arduino board. This system facilitated the simultaneous toxicological evaluation of human corneal epithelial cells against eight different concentrations of preservatives, allowing for quadruplicate experiments in a single run. In our study, the IC50 values for healthy eyes and those affected with dry eyes syndrome showed an approximately twofold difference. This variation is likely attributable to the duration for which the preservative remained in contact with corneal cells before being washed off by the medium, suggesting the significance of exposure time in the cytotoxic effect of preservatives. Our microfluidic system, automated by Arduino, simulated healthy and dry eye environments to study benzalkonium chloride toxicity and revealed significant differences in cell viability, with IC50 values of 0.0033% for healthy eyes and 0.0017% for dry eyes. In summary, we implemented the pinch-to-zoom feature of an electronic tablet in our microfluidic system, offering innovative alternatives for eye research.

Development of the standard mouse model for human bacterial vaginosis induced by Gardnerella vaginalis.

Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a diminished number of protective bacteria in the vaginal flora. Instead, it is accompanied by a significant increase in facultative and strict anaerobes, including Gardnerella vaginalis (G. vaginalis). BV is one of the most common gynecological problems experienced by reproductive age-women. Because an ideal and standard animal model for human BV induced by G. vaginalis is still underdeveloped, the main objective of this study was to develop a mouse model for human BV induced by G. vaginalis to demonstrate the clinical attributes observed in BV patients. A total of 80 female ICR mice were randomly assigned to 4 groups and intravaginally inoculated with different doses of G. vaginalis: NC (uninfected negative control), PC1 (inoculated with 1 × 105 CFU of G. vaginalis), PC2 (inoculated with 1 × 106 CFU of G. vaginalis) and PC3 (inoculated with 1 × 107 CFU of G. vaginalis). The myeloperoxidase (MPO) activity and serum concentrations of cytokines (IL-1ß, IL-10) in mice administered with G. vaginalis were significantly higher than those of the control group. Gross lesion and histopathological analysis of reproductive tract of mice inoculated with G. vaginalis showed inflammation and higher epithelial cell exfoliation compared to the control group. In addition, vaginal swabs from the mice inoculated with G. vaginalis showed the presence of clue cells, which are a characteristic feature of human BV. Altogether, our results suggested that G. vaginalis is sufficient to generate comparable clinical attributes seen in patients with BV.

Regeneration of taste through sonic hedgehog upregulation by photobiomodulation.

This study investigated photobiomodulation (PBM) effects in Sonic hedgehog (Shh) signaling as a potential approach to taste preservation and regeneration. Primary taste cell (TC) cultures were treated with Shh antagonist vismodegib and irradiated using a continuous wave type 630 nm light-emitting diode (10 mW/cm2 ) array, with single or multiple doses of 30 J/cm2 to determine dose inducing significant upregulation effect. Shh, Ptch, Smo, and Gli1 were significantly upregulated at 120 J/cm2 , used as the minimum dose in vivo. Vismodegib was administered via daily oral gavage for 21 days (30 mg/kg) to induce Shh inhibition in the tongue of rat animal models resulting in taste bud damage and taste dysfunction. PBM treatment using a 630 nm laser (3 W/cm2 ) at a radiant exposure of 120 J/cm2 (24 J/cm2 × 5) successfully upregulated the Shh protein expression, regenerated taste buds, and recovered taste function.

A Modified Length-Based Grading Method for Assessing Coronary Artery Calcium Severity on Non-Electrocardiogram-Gated Chest Computed Tomography: A Multiple-Observer Study.

OBJECTIVE: To validate a simplified ordinal scoring method, referred to as modified length-based grading, for assessing coronary artery calcium (CAC) severity on non-electrocardiogram (ECG)-gated chest computed tomography (CT). MATERIALS AND METHODS: This retrospective study enrolled 120 patients (mean age ± standard deviation [SD], 63.1 ± 14.5 years; male, 64) who underwent both non-ECG-gated chest CT and ECG-gated cardiac CT between January 2011 and December 2021. Six radiologists independently assessed CAC severity on chest CT using two scoring methods (visual assessment and modified length-based grading) and categorized the results as none, mild, moderate, or severe. The CAC category on cardiac CT assessed using the Agatston score was used as the reference standard. Agreement among the six observers for CAC category classification was assessed using Fleiss kappa statistics. Agreement between CAC categories on chest CT obtained using either method and the Agatston score categories on cardiac CT was assessed using Cohen's kappa. The time taken to evaluate CAC grading was compared between the observers and two grading methods. RESULTS: For differentiation of the four CAC categories, interobserver agreement was moderate for visual assessment (Fleiss kappa, 0.553 [95% confidence interval {CI}: 0.496-0.610]) and good for modified length-based grading (Fleiss kappa, 0.695 [95% CI: 0.636-0.754]). The modified length-based grading demonstrated better agreement with the reference standard categorization with cardiac CT than visual assessment (Cohen's kappa, 0.565 [95% CI: 0.511-0.619 for visual assessment vs. 0.695 [95% CI: 0.638-0.752] for modified length-based grading). The overall time for evaluating CAC grading was slightly shorter in visual assessment (mean ± SD, 41.8 ± 38.9 s) than in modified length-based grading (43.5 ± 33.2 s) (P < 0.001). CONCLUSION: The modified length-based grading worked well for evaluating CAC on non-ECG-gated chest CT with better interobserver agreement and agreement with cardiac CT than visual assessment.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Introduction: Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. Methods: We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Results: Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Conclusions: Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00738-8.

Phase and morphology of calcium carbonate precipitated by rapid mixing in the absence of additives.

Calcium carbonate is one of the most common minerals, and its polymorphic formation and transformation pathways from the amorphous to crystalline phases are well documented. However, the effects of locally created pH changes on the preferential formation of amorphous calcium carbonate (ACC) or its crystalline phase remain poorly understood. In this study, the influence of the initial solution pH on the precipitated polymorphs of calcium carbonate was investigated by the rapid mixing of each solution containing calcium or carbonate ions in the absence of additives. The results showed that the amount of recovered ACC particles was associated with the availability of fully deprotonated carbonate ions. A secondary crystalline phase was identified as the vaterite phase, but no polymorphic change to produce the more stable calcite was detected during 5 h of stirring. Interestingly, during the early stage of pouring, the vaterite morphology was dependent on the generated pH range, over which ACC particles were stabilized (pH > 10.3), followed by the hydration-condensation processes. When the pH was sufficiently low (pH < 10.3) for bicarbonate ions to participate in the carbonation reaction, croissant- or cauliflower-like aggregates with layered structures were obtained. In contrast, typical spherical vaterite particles were obtained at a high initial pH when the carbonate ions were dominant. Meanwhile, vaterite particles that were formed in the presence of an excess of carbonate ions were irregular and separate agglomerates. These results elucidate the formation of ACC and the morphologies of the vaterite products.

In silico design and fabrication of an SFI chip-based microspheroid culture system.

The emergence of microfluidic devices and computational fluid dynamics (CFD) has propelled the need for next-generation biomimetic cell culture platforms that are flexible for monitoring and regulation. Therefore, this study evaluated a CFD application in an in silico-designed and spheroid-based flow integration 3D cell culture chip (SFI chip) to illustrate cell culture, drug screening, cytokine delivery, and differentiation of cells in a platform that partially recapitulates the natural environment. Our results show that a flow rate of 0.05 mL h-1 or less induced no physical stress in the SFI chip (15 mm), and uniform cell spheroids (approximately 200 µm) were formed across the platform. The cultured cells were tested in several experimental contexts (co-culture, drug screening, cytokine delivery, and differentiation), demonstrating the usefulness of computational simulation in expediting discovery and simple and effective means to scale the production of standardized cell spheroids cultured under dynamic and natural conditions. Advanced cell culture technologies can be used to accelerate research and discovery and the preclinical and clinical development of cell and cell-free therapies for urgent medical needs.

Sub-picomolar lateral flow antigen detection with two-wavelength imaging of composite nanoparticles.

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0-5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Microfluidic system with light intensity filters facilitating the application of photodynamic therapy for high-throughput drug screening.

BACKGROUND: Photodynamic therapy utilizes light energy with a photosensitizer (a light-sensitive drug) to kill cancer cells through creation of singlet oxygen via light activation. When a photosensitizer is injected into the bloodstream and exposed to a specific wavelength of light, it generates oxygen to destroy or damage nearby cancer cells, while minimizing side effects on normal cells. Although photodynamic therapy is effective for treating cancer, various parameters, such as the optimum light intensity and photosensitizer dose, are currently poorly understood due to the complexity of conventional experimental schemes. METHODS: To effectively perform a simultaneous single parallel test for several different light irradiation conditions on each cell, a microfluidic device was developed to generate eight different intensities from a single light-emitting diode source, through eight different color dye concentrations functioning as light intensity filters. To show that this novel high-throughput microfluidic system can analyze the effects of various light intensities during photodynamic therapy, the optimum light intensities and photosensitizer doses were determined for two different cancer cell lines. RESULTS: Optimum light intensities and photosensitizer were determined for all cell lines. The photodynamic therapy effects in response to different irradiated light intensities were characterized by analyzing cell viability after photosensitizer treatment CONCLUSIONS: : The developed platform is capable of being used as a photodynamic therapy screening tool. The proposed platform provides a simple and robust way to optimize the combined parameters of light intensity and dosage for diverse types of cancer cells.

Response to growth hormone according to provocation test results in idiopathic short stature and idiopathic growth hormone deficiency.

PURPOSE: To investigate growth response in children with either idiopathic short stature (ISS) or growth hormone (GH) deficiency (GHD). METHODS: The data of prepubertal GHD or ISS children treated using recombinant human GH were obtained from the LG Growth Study database. GHD children were further divided into partial and complete GHD groups. Growth response and factors predicting growth response after 1 and 2 years of GH treatment were investigated. RESULTS: This study included 692 children (98 with ISS, 443 partial GHD, and 151 complete GHD). After 1 year, changes in height standard deviation score (ΔHt-SDS) were 0.78, 0.83, and 0.96 in ISS, partial GHD, and complete GHD, respectively. Height velocity (HV) was 8.72, 9.04, and 9.52 cm/yr in ISS, partial GHD, and complete GHD, respectively. ΔHt-SDS and HV did not differ among the 3 groups. Higher initial body mass index standard deviation score (BMI-SDS) and midparental height standard deviation score (MPH-SDS) were predictors for better growth response after 1 year in ISS and the partial GHD group, respectively. In the complete GHD group, higher Ht-SDS and BMI-SDS predicted better growth response after 1 year. After 2 years of GH treatment, higher BMI-SDS and MPH-SDS predicted a better growth outcome in the partial GHD group, and higher MPH-SDS was a predictor of good growth response in complete GHD. CONCLUSION: Clinical characteristics and growth response did not differ among groups. Predictors of growth response differed among the 3 groups, and even in the same group, a higher GH dose would be required when poor response is predicted.

Particulate Matter Exposure Aggravates IL-17-Induced Eye and Nose Inflammation in an OVA/Poly(I:C) Mouse Model.

PURPOSE: Data on the effects of direct particulate matter (PM) exposure on the eyes and the nose are limited. Here, an interleukin (IL)-17/neutrophil-dominant ovalbumin (OVA)/polyinosinic-polycytidylic acid (Poly(I:C)) mouse model was used to evaluate the effect of different-sized titanium dioxide (TiO2) particles on the eyes and the nose. We also examined whether IL-17-neutralizing antibody (IL-17Ab) treatment could reverse TiO2 effects. METHODS: The nasal cavities and conjunctival sacs of each mouse were challenged with OVA and Poly(I:C) to induce neutrophil-dominant inflammation and then exposed to micro- and nano-TiO2. Subsequently, IL-17Ab was administered to investigate the role of IL-17 and inflammatory parameters. RESULTS: Micro- and nano-TiO2 resulted in significant decreases in tear-break-up time and increases in corneal damage. Airborne micro-TiO2 also increased nasal rubbing and sneezing counts compared with the OVA/Poly(I:C). Micro-TiO2 exposure increased infiltration of neutrophils and IL-17A+ cells in the conjunctival tissues and the nasal mucosae. In addition, these increased symptoms and inflammation in the eyes and the nose by micro-TiO2 exposure were inhibited by the IL-17Ab, suggesting IL-17 dependency. CONCLUSIONS: TiO2 aggravated IL-17-induced eye and nose inflammation and the IL-17Ab alleviated inflammation in the OVA/Poly(I:C) mouse model. These results may help develop a therapeutic modality for PM exposure and provide evidence for PM-associated diseases.

Comparison of Sensitivity Encoding (SENSE) and Compressed Sensing-SENSE for Contrast-Enhanced T1-Weighted Imaging in Patients With Crohn Disease Undergoing MR Enterography.

BACKGROUND. Long acquisition times for breath-hold contrast-enhanced (CE) T1-weighted imaging in MR enterography (MRE) protocols result in reduced image quality. OBJECTIVE. The purpose of this study was to compare CE T1-weighted imaging performed using sensitivity encoding (SENSE) and compressed sensing-SENSE (CS-SENSE) in terms of image quality and diagnostic performance for active inflammation in Crohn disease (CD). METHODS. This retrospective study included 41 patients (31 men, 10 women; mean age, 34 ± 12 [SD] years) who underwent MRE for known or suspected CD between June 2020 and September 2020. MRE was performed in one of two scanning rooms depending on scheduling availability. Per institutional protocol, in one room, the enteric phase was acquired using SENSE (acceleration factor, 3) and the portal phase was acquired using CS-SENSE (acceleration factor, 5); this order was reversed in the other room. Two radiologists independently assessed sequences for subjective image quality measures at the patient level and for active inflammation at the bowel-segment level. Mean image quality scores between readers were computed. Diagnostic performance for active inflammation was compared between SENSE and CS-SENSE using generalized estimating equations; a separate experienced radiologist reviewed the full MRE protocol to establish the reference standard. RESULTS. The mean acquisition time of CE T1-weighted imaging was 17.2 ± 1.1 seconds for SENSE versus 11.5 ± 0.8 seconds for CS-SENSE (p < .001). CS-SENSE scored significantly better than SENSE in overall image quality (4.2 ± 0.7 vs 3.7 ± 1.1; p = .02), motion artifacts (4.0 ± 0.8 vs 3.6 ± 1.2; p = .006), and aliasing artifacts (4.8 ± 0.4 vs 4.2 ± 0.6; p < .001). CS-SENSE scored significantly worse than SENSE in synthetic appearance (4.6 ± 0.5 vs 4.8 ± 0.4; p = .003). Contrast, sharpness, and blurring were not different between sequences (p > .05). For reader 1, CS-SENSE, compared with SENSE, showed a sensitivity of 86% versus 81% (p = .09), specificity of 88% versus 83% (p = .08), and accuracy of 87% versus 82% (p = .56). For reader 2, CS-SENSE, compared with SENSE, showed a sensitivity of 92% versus 79% (p = .006), specificity of 90% versus 98% (p = .16), and accuracy of 91% versus 86% (p = .002). CONCLUSION. Use of CS-SENSE for CE T1-weighted imaging in MRE protocols results in reduced scan times with reduced artifact and improved image quality. CLINICAL IMPACT. The benefits of CS-SENSE in MRE protocols may improve the diagnostic performance for active inflammation in CD.

Repurposing macitentan with nanoparticle modulates tumor microenvironment to potentiate immune checkpoint blockade.

Immune checkpoint therapy (ICT), which reinvigorates cytotoxic T cells, provides clinical benefits as an alternative to conventional cancer therapies. However, its clinical response rate is too low to treat an immune-excluded tumor, owing to the presence of abundant stromal elements impeding the penetration of immune cells. Here, we report that macitentan, a dual endothelin receptor antagonist approved by the FDA to treat pulmonary arterial hypertension, can be repositioned to modulate the desmoplastic tumor microenvironment (TME). In the 4T1 orthotopic tumor model, the polymeric nanoparticles bearing macitentan (M-NPs) prevent fibrotic progression by regulating the function of cancer-associated fibroblasts, attenuate the biogenesis of cancer cell-derived exosomes, and modulate the T cell subsets and distribution in TME. These results demonstrate that the M-NPs effectively reorganize the immunosuppressive TME by targeting the endothelin-1 axis and consequently exhibit synergistic antitumor effects in combination with ICT.

Modular Counter-Fischer-Indole Synthesis through Radical-Enolate Coupling.

A single-electron transfer mediated modular indole formation reaction from a 2-iodoaniline derivative and a ketone has been developed. This transition-metal-free reaction shows a broad substrate scope and unconventional regioselectivity trends. Moreover, important functional groups for further transformation are tolerated under the reaction conditions. Density functional theory studies reveal that the reaction proceeds by metal coordination, which converts a disfavored 5-endo-trig cyclization to an accessible 7-endo-trig process.

Validating a Series of Photonumeric Rating Scales for Use in Facial Aesthetics Using Statistical Analysis of Intra- and Inter-rater Reliability.

Background: Growing demand for minimally invasive aesthetic procedures to correct age-related facial changes and optimize facial proportions has been met with innovation but has created an unmet need for objective assessment tools to evaluate results empirically. Objectives: The purpose of this study is to establish the intra- and inter-rater reliability of ordinal, photonumeric, 4-, or 5-point rating scales for clinical use to assess facial aesthetics. Methods: Board-certified plastic surgeons and dermatologists (3 raters) performed live validation of jawline contour, temple volume, chin retrusion, nasolabial folds, vertical perioral lip lines, midface volume loss, lip fullness, and crow's feet dynamic- and at rest-rating scales over 2 rounds, 2 weeks apart. Subjects selected for live validation represented the range of scores and included 54 to 83 subjects for each scale. Test-retest reliability was quantitated through intra- and inter-rater reliability, determined from the mean weighted kappa and round 2 intraclass correlation coefficients, respectively. The clinical significance of a 1-grade difference was assessed through rater comparison of 31 pairs of side-by-side photographs of subjects with the same grade or a different grade on the developed scales. Results: The study demonstrated substantial to near-perfect intra- and inter-rater reliability of all scales when utilized by trained raters to assess a diverse group of live subjects. Furthermore, the clinical significance of a 1-point difference on all the developed scales was established. Conclusions: The high test-retest reliability and intuitive layout of these scales provide an objective approach with standardized ratings for clinical assessment of various facial features.

Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range.

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core-shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL-1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

Indirect co-culture of stem cells from human exfoliated deciduous teeth and oral cells in a microfluidic platform.

Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

Inhibition of hardy kiwifruit (Actinidia aruguta) ripening by 1-methylcyclopropene during cold storage and anticancer properties of the fruit extract.

Hardy kiwifruits (Actinidia arguta) were treated with 20 µl/l 1-methylcyclopropene (1-MCP) for 16 h at 10 °C and subsequently stored at 1 ± 0.5 °C. Anticancer properties of the fruit extracts were tested against five different human cancer cells. The hardy kiwifruits, without 1-MCP treatment, showed increases in both respiration and ethylene production rates during fruit storage. The 1-MCP treatment remarkably inhibited fruit ripening by reducing respiration and ethylene production. Fruits with the 1-MCP treatment could be stored for up to 5 weeks by maintaining higher fruit firmness, ascorbic acid and total phenolic contents compared to the control. The hardy kiwifruit extracts showed anti-proliferative effects to Hep3B and HeLa cells but not to HT29, HepG2 and LoVo cells. These results suggest that the application of 1-MCP at harvest effectively delayed the ripening process of the fruits, and the fruit extract had beneficial effects for the prevention of human cancer growth.

High-Throughput Cytotoxicity Testing System of Acetaminophen Using a Microfluidic Device (MFD) in HepG2 Cells.

A lab-on-a-chip (LOC) is a microfluidic device (MFD) that integrates several lab functions into a single chip of only millimeters in size. LOC provides several advantages, such as low fluidic volumes consumption, faster analysis, compactness, and massive parallelization. These properties enable a microfluidic-based high-throughput drug screening (HTDS) system to acquire cell-based abundant cytotoxicity results depending on linear gradient concentration of drug with only few hundreds of microliters of the drug. Therefore, a microfluidic device was developed containing an array of eight separate microchambers for cultivating HepG2 cells to be exposed to eight different concentrations of acetaminophen (APAP) through a diffusive-mixing-based concentration gradient generator. Every chamber array with eight different concentrations (0, 5.7, 11.4, 17.1, 22.8, 28.5, 34.2, or 40 mM) APAP had four replicating cell culture chambers. Consequently, 32 experimental results were acquired with a single microfluidic device experiment. The microfluidic high-throughput cytotoxicity device (µHTCD) and 96-well culture system showed comparable cytotoxicity results with increasing APAP concentration of 0 to 40 mM. The HTDS system yields progressive concentration-dependent cytotoxicity results using minimal reagent and time. Data suggest that the HTDS system may be applicable as alternative method for cytotoxicity screening for new drugs in diverse cell types.