Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria's activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

Development of Integrated Systems for On-Site Infection Detection.

The modern healthcare system faces an unrelenting threat from microorganisms, as evidenced by global outbreaks of new viral diseases, emerging antimicrobial resistance, and the rising incidence of healthcare-associated infections (HAIs). An effective response to these threats requires rapid and accurate diagnostic tests that can identify causative pathogens at the point of care (POC). Such tests could eliminate diagnostic uncertainties, facilitating patient triaging, minimizing the empiric use of antimicrobial drugs, and enabling targeted treatments. Current standard methods, however, often fail to meet the needs of rapid diagnosis in POC settings. Culture-based assays entail long processing times and require specialized laboratory infrastructure; nucleic acid (NA) tests are often limited to centralized hospitals due to assay complexity and high costs. Here we discuss two new POC tests developed in our groups to enable the rapid diagnosis of infection. The first is nanoPCR that takes advantages of core-shell magnetoplasmonic nanoparticles (MPNs): (i) Au shell significantly accelerates thermocycling via volumetric, plasmonic light-to-heat conversion and (ii) a magnetic core enables sensitive in situ fluorescent detection via magnetic clearing. By adopting a Ferris wheel module, the system expedites multisamples in parallel with a minimal setup. When applied to COVID-19 diagnosis, nanoPCR detected SARS-CoV-2 RNA down to 3.2 copy/µL within 17 min. In particular, nanoPCR diagnostics accurately identified COVID-19 cases in clinical samples (n = 150), validating its clinical applicability. The second is a polarization anisotropy diagnostic (PAD) system that exploits the principle of fluorescence polarization (FP) as a detection modality. Fluorescent probes were designed to alter their molecular weight upon recognizing target NAs. This event modulates the probes' tumbling rate (Brownian motion), which leads to changes in FP. The approach is robust against environmental noise and benefits from the ratiometric nature of the signal readout. We applied PAD to detect clinically relevant HAI bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus). The PAD assay demonstrated detection sensitivity down to the single bacterium level and determined both drug resistance and virulence status. In summary, these new tests have the potential to become powerful tools for rapid diagnosis in the infectious disease space. They do not require highly skilled personnel or labor-intensive analyses, and the assays are quick and cost-effective. These attributes will make nanoPCR and PAD well-aligned with a POC workflow to aid physicians to initiate prompt and informed patient treatment.

Portable glucose meter-utilized label-free and washing-free telomerase assay.

We herein describe a portable glucose meter (PGM)-utilized label-free and washing-free method for the facile determination of telomerase activity that relies on the kinase-catalyzed cascade enzymatic reaction (KCER) that transduces the telomerase activity to the glucose level. In the sensor, the telomerase that elongates telomere sequences ((TTAGGG)n) from the 3'-terminus of telomerase substrate primer (TSP) consumes deoxynucleoside triphosphate (dNTP), which serves as a phosphate source for KCER promoted by hexokinase and pyruvate kinase. Thus, the presence of telomerase protects KCER from working effectively, resulting in the maintenance of an initial, high glucose level that is readily determined using hand-held PGM. With this strategy, the telomerase activities in various types of cell lines were successfully determined with high sensitivity. Furthermore, the ability of this method to screen candidate inhibitors for telomerase activity was also verified.

Quantitative Trait Locus Mapping of Clubroot Resistance and Plasmodiophora brassicae Pathotype Banglim-Specific Marker Development in Brassica rapa.

Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines "09CR500" and "09CR501", respectively. The resistance of "09CR500" to Plasmodiophora brassicae pathotype "Banglim" was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. PbBrA08Banglim was identified as having a logarithm of odds value of 7.9-74.8, and a phenotypic variance of 26.0-97.1% with flanking marker "09CR.11390652" in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. PbBrA08Banglim was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the "09CR.11390652" marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.

Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color.

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.

Fluorescent S1 nuclease assay utilizing exponential strand displacement amplification.

We herein devise a simple and label-free strategy to determine S1 nuclease activity by exploiting the target-induced inhibition of exponential strand displacement amplification (eSDA). In principle, a DNA probe that is designed to produce a large amount of duplexes through a process of eSDA, is degraded by the catalytic activity of S1 nuclease. This reaction blocks the initiation of eSDA, leading to the quite-reduced fluorescence of a double-stranded DNA specific fluorescent dye, SYBR Green I compared to the one in the absence of S1 nuclease. With this simple but novel approach, the S1 nuclease activity was selectively assayed with the high sensitivity. In addition, this system was successfully demonstrated to possess the capability to screen potential inhibitors against S1 nuclease.

Sensitive detection of DNA from Chlamydia trachomatis by using flap endonuclease-assisted amplification and graphene oxide-based fluorescence signaling.

A simple and sensitive method is described for the determination of DNA. It relies on the use of (a) an invasive reaction that is catalyzed by flap endonuclease 1 (FEN 1), and (b) graphene oxide (GO)-based fluorescence signalling. The presence of target DNA mediates the formation of the invasive structure, and this induces FEN 1 to catalyze multiple cycles of cleavage reaction at the junction, thereby liberating numerous fluorophore-labeled flaps. The released flaps are intentionally designed too short to be adsorbed onto GO. Hence, intense green fluorescence whose maximum emission is observed at 520 nm after excitation at 480 nm is restored even in the presence of GO. The method can be applied to the determination of target DNA from Chlamydia trachomatis, one of the major pathogenic bacteria causing sexually transmitted diseases. The assay is sensitive and specific with the limit of detection of 6.7 pM, and was applied to reliable determination of Chlamydia trachomatis DNA in human serum. Graphical abstract Flap endonuclease 1 (FEN 1)-catalyzed invasive reaction and graphene oxide (GO)-based fluorescence signalling are integrated to develop a novel and sensitive target DNA detection method.

A simple and sensitive detection of small molecule-protein interactions based on terminal protection-mediated exponential strand displacement amplification.

We herein describe a simple and sensitive strategy to detect a small molecule-protein interaction based on terminal protection-mediated exponential strand displacement amplification (eSDA). In principle, the small molecule linked to a DNA probe protects the DNA probe against the exonuclease I-catalyzed degradation after its binding to the corresponding target protein. The protected DNA probe then serves as a template to promote eSDA. Consequently, a large number of duplexes are produced, which leads to a high fluorescence from a double-stranded DNA specific fluorescent dye, SYBR Green I. As a model system to prove this sensing strategy, the interaction between biotin and streptavidin (SA), which is known to be the strongest among the non-covalent biological interactions, was selected and its analytical performance was thoroughly investigated. As a result, SA was sensitively detected with the limit of detection of 16 pM. In addition, the practical applicability of this method was successfully demonstrated by reliably determining the SA in human serum.

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction.

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Enzyme-Free Colorimetric Detection of Cu2+ by Utilizing Target-Triggered DNAzymes and Toehold-Mediated DNA Strand Displacement Events.

A new enzyme-free system for colorimetric Cu2+ detection, which relies on target-triggered DNAzymes and toehold-mediated DNA strand-displacement circuits, is described. The system employs a DNAzyme designed to undergo self-cleavage in the presence of Cu2+ and release a catalyst strand that triggers a sequential toehold-mediated strand displacement reaction. This event leads to the release of a split G-quadruplex DNAzyme strand that is initially caged and inactivated by a blocker strand. A fuel strand is further incorporated for the recycling of the catalyst strand to promote another toehold-mediated strand displacement event, which consequently produces a large number of active split G-quadruplex DNAzymes. By employing this design principle, target Cu2+ was very successfully identified with a detection limit of 1.31 nm based on the distinct colorimetric signal developed by the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Finally, the practical capability of this sensing system was very successfully demonstrated by its use to reliably determine Cu2+ in tap water.

Quality assurance for a multicenter Phase II study of stereotactic ablative radiotherapy for hepatocellular carcinoma ≤5 cm: a planning dummy run.

OBJECTIVE: The Korean Radiation Oncology Group (12-02) investigated the outcome of stereotactic ablative radiotherapy for hepatocellular carcinoma ≤5 cm using 60 Gy in three fractions. To evaluate dosimetric differences and compliance in a multicenter trial, a planning dummy run procedure was performed. METHODS: All six participating institutions were provided the contours of two dummy run cases. Plans were performed following the study protocol to cover the planning target volume with a minimum of 90% of the prescription dose and to satisfy the constraints for organs at risk. We assessed the institutional variations in plans using dose-volume histograms. RESULTS: Different planning techniques were applied: static intensity-modulated radiotherapy in two institutions, CyberKnife in two institutions and RapidArc in two institutions. The conformity index of all 12 plans was ≤1.2. In terms of the planning target volume coverage, all participants followed our study protocol. For the second dummy run case, located in Segment 8 near the heart, the minimum dose of the planning target volume (D99%: dose covering 99% of the planning target volume) was variable because there was no mention of constraints of D99% of the planning target volume in the study protocol. As an important organ at risk, the normal liver volumes receiving <17 Gy in all 12 plans were >700 ml. CONCLUSIONS: Dosimetric parameters showed acceptable compliance with the study protocol. However, we found the possibility of underdose to the planning target volume if the hepatocellular carcinoma lesion was located near organs at risk such as the heart. Based on this dummy run, we will conduct individual case reviews to minimize the effects of study protocol deviation.

Determination of RNase H activity via real-time monitoring of target-triggered rolling circle amplification.

The authors describe a method for real-time monitoring of the activity of ribonuclease H (RNase H). It is based on target-triggered rolling circle amplification (RCA). It utilizes a specially designed primer that contains a RNA sequence in the center and an amino group at the 3'-end. In the absence of RNase H, the primer when hybridized to a circular DNA template is not extended by DNA polymerase due to the amino group at the 3'-end. In contrast, the presence of RNase H specifically degrades the RNA sequence of the primer hybridized to the circular DNA template. This results in the conversion of the 3'-amino group to a 3'-hydroxy group and thereby enables the extension reaction promoted by DNA polymerase. This, consequently, leads to efficient RCA producing a long concatenated DNA strand. Its generation can be monitored in real-time by using the fluorescent dye SYBR green II which is specific for single-stranded DNA. Based on this RNase H-triggered RCA, RNase H activity can be selectively determined at levels as low as 0.019 U·mL-1 with a total assay time of <5 min. The diagnostic capability of this assay was demonstrated by monitoring the activity of RNase H in tumor cells. Graphical abstract Schematic of real-time monitoring of ribonuclease H (RNase H) activity based on target-triggered rolling circle amplification (RCA). RNase H that degrades RNA in primer, converts 3'-amino group to 3'-hydroxy group, which promotes RCA with fluorescence enhancement of the probe SYBR green II.

Change in Posterior Pharyngeal Space After Counterclockwise Rotational Orthognathic Surgery for Class II Dentofacial Deformity Diagnosed With Obstructive Sleep Apnea Based on Cephalometric Analysis.

Although maxillomandibular advancement (MMA) is an orthognathic surgical procedure used to manage obstructive sleep apnea (OSA) in individuals who are noncompliant with continuous positive airway pressure therapy, simple MMA encounters problems in terms of aesthetic outcomes in Asian populations with preexisting dentoalveolar protrusion. Our current prospective investigation describes changes in posterior pharyngeal space and aesthetic outcomes after counterclockwise rotational orthognathic surgery, which is known to be quite difficult in terms of the maintenance of the skeletal stability in skeletal class II patients with OSA. This prospective study investigated the surgical outcome of patients who suffered from OSA following counterclockwise rotational orthognathic surgery. The patients were skeletal class II patients who underwent orthognathic surgery between March 2013 and December 2014. Cephalometric posterior airway analysis and a questionnaire for facial perception were used to assess pharyngeal airway and patient perception of facial appearance. A total of 14 patients were included. Satisfactory results were achieved without complications in all OSA patients. The airway parameters for anteroposterior length significantly increased. Thirteen patients answered a questionnaire on their facial appearance, and the visual analog scale averaged 7.31 points, indicating a favorable facial appearance. Counterclockwise rotational orthognathic surgery without maxilla advancement for the correction of OSA can effectively increase the posterior pharyngeal space, with favorable aesthetic results. With thoughtful application, this novel approach may be an alternative to standard approaches for the correction of OSA using orthognathic surgery.

A sensitive dual colorimetric and fluorescence system for assaying the activity of alkaline phosphatase that relies on pyrophosphate inhibition of the peroxidase activity of copper ions.

A novel and highly sensitive colorimetric and fluorescence assay for the accurate determination of alkaline phosphatase (ALP) activity has been developed. The assay takes advantage of the inhibition of the peroxidase activity of Cu(2+) ions caused by complexation with pyrophosphate (PPi), a natural substrate for ALP. This inhibition disappears when PPi undergoes ALP catalyzed hydrolysis to generate phosphate, which does not bind to Cu(2+) ions. Thus, ALP causes generation of uncomplexed Cu(2+) ions, which promote multiple oxidation reactions of Amplex UltraRed in the presence of hydrogen peroxide in conjunction with the production of intense fluorescence and colorimetric signals. By employing the fluorescence and colorimetric assay strategies, ALP can be detected at respective concentrations as low as 4.3 pM and 5.4 pM, detection limits that are much lower than those associated with previously described methods. The practical diagnostic capability of the assay system has been demonstrated by its use to detect ALP in human blood serum.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SIGNIFICANCE: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe.

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0-10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing.

Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).

Tower microneedle minimizes vitreal reflux in intravitreal injection.

Intravitreal injection is widely used for easy control of drug levels in posterior segment of the eye by injecting the drug directly with hypodermic needles. Patients, however, often experience complications from intravitreal injection due to repeated injections, increased intraocular pressure, and infection. In addition, injected drug reflux after intravitreal injection makes it challenging to maintain predetermined drug dose due to the drug loss through backward effusions. Here, we described that the Tower Microneedle can reduce initial reflux and bleb formation due to its smaller outer diameter compared to a traditional hypodermic needle. Furthermore, we use phenylephrine hydrochloride for pupil expansion and demonstrated that Tower Microneedle induced similar pupil expansions using only half the drug volume, in the same period of time, compared to the 31 Gauge hypodermic needle. Consequently, Tower Microneedle achieves the same therapeutic effect in the vitreous body using fewer drugs than a traditional hypodermic needle due to the decreased backward drug effusion. Tower Microneedle described herein holds great promise for intravitreal injection with less reflux and lower drug dosage.

An optimized hollow microneedle for minimally invasive blood extraction.

The healthcare system relies widely on biochemical information obtained from blood sample extracted via hypodermic needles, despite the invasiveness and pain associated with this procedure. Therefore, an alternative micro-scale needle for minimally invasive blood sampling is highly desirable. Traditional fabrication techniques to create microneedles do not generate needles with the combined features of a sharp tip, long length, and hollow structure concurrently. Here, we report the fabrication of a microneedle long enough to reach blood vessels and sharp enough to minimize nerve contact for minimally invasive blood extraction. The microneedle structure was precisely controlled using a drawing lithography technique, and a sharp tip angle was introduced using a laser-cutting system. We investigated the characteristics of a microneedle with a length of 1,800 µm length, an inner diameter of 60 µm, a tip diameter of 120 µm, and a 15° bevel angle through in-vitro liquid extraction and mechanical strength analysis. We demonstrated that the proposed structure results in blood extraction at a reasonable rate, and that a microneedle with this geometry can reliably penetrate skin without breaking. We integrated this microneedle into a blood extraction device to extract a 20 µl volume of mouse blood in-vivo. Our optimized, hollow microneedle can potentially be incorporated with other cutting-edge technologies such as microactuators, biosensors, and microfluidic chips to create blood analysis systems for point-of-care diagnostics.

Glycyrrhizin alleviates neuroinflammation and memory deficit induced by systemic lipopolysaccharide treatment in mice.

The present study investigated the effects of glycyrrhizin (GRZ) on neuroinflammation and memory deficit in systemic lipopolysaccharide (LPS)-treated C57BL/6 mice. Varying doses of GRZ was orally administered (10, 30, or 50 mg/kg) once a day for 3 days before the LPS (3 mg/kg) injection. At 24 h after the LPS injection, GRZ significantly reduced TNF-α and IL-1ß mRNA at doses of 30 and 50 mg/kg. COX-2 and iNOS protein expressions were significantly reduced by GRZ at doses of 30 and 50 mg/kg. In the Morris water maze test, GRZ (30 mg/kg) significantly prolonged the swimming time spent in the target and peri-target zones. GRZ also significantly increased the target heading and memory score numbers. In the hippocampal tissue, GRZ significantly reduced the up-regulated Iba1 protein expression and the average cell size of Iba1-expressing microglia induced by LPS. The results indicate that GRZ ameliorated the memory deficit induced by systemic LPS treatment and the effect of GRZ was found to be mediated through the inhibition of pro-inflammatory mediators and microglial activation in the brain tissue. This study supports that GRZ may be a putative therapeutic drug on neurodegenerative diseases associated with cognitive deficits and neuroinflammation such as Alzheimer's disease.