Inhibitory nitric oxide production and cytotoxic activities of phenolic compounds from Gnetum montanum Markgr.

One new compound, styrene dimer-type listeanol-4-O-α-ʟ-rhamnopyranosyl-(1→4)-ß-ᴅ-glucopyranoside (1), and four known compounds namely listeanol (2), isorhapotigenin (3), genetifolin E (4), gnetifolin K (5) were isolated from the methanolic extract from the aerial part of the Gnetum montanum Markgr. in Viet Nam. Their chemical structures were determined by modern spectroscopic methods (NMR and HR-ESI-MS) and comparison with those of published data. These compounds were evaluated for their anti-inflammatory and cytotoxic activities. Among them, compound 3 exhibited the NO inhibitory production on the RAW264.7 cell line, and inhibited the HepG2 cell line with respective IC50 values of 79.88 ± 5.51 (µg/mL) (L-NMMA 7.90 ± 0.63 µg/mL), and 63.48 ± 3.63 (µg/mL) (Ellipticine 0.40 ± 0.01 µg/mL).

Development of a self-contained microfluidic chip and an internet-of-things-based point-of-care device for automated identification of respiratory viruses.

The COVID-19 pandemic greatly impacted the in vitro diagnostic market, leading to the development of new technologies such as point-of-care testing (POCT), multiplex testing, and digital health platforms. In this study, we present a self-contained microfluidic chip integrated with an internet-of-things (IoT)-based point-of-care (POC) device for rapid and sensitive diagnosis of respiratory viruses. Our platform enables sample-to-answer diagnostics within 70 min by automating RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescence detection. The microfluidic chip is designed to store all the necessary reagents for the entire diagnostic assay, including a lysis buffer, a washing buffer, an elution buffer, and a lyophilized RT-LAMP cocktail. It can perform nucleic acid extraction, aliquoting, and gene amplification in multiple reaction chambers without cross-contamination. The IoT-based POC device consists of a Raspberry Pi 4 for device control and data processing, a CMOS sensor for measuring fluorescence signals, a resistive heater panel for temperature control, and solenoid valves for controlling the movement of on-chip reagent solutions. The proposed device is portable and features a touchscreen for user control and result display. We evaluated the performance of the platform using 11 clinical respiratory virus samples, including 5 SARS-CoV-2 samples, 2 influenza A samples, and 4 influenza B samples. All tested clinical samples were accurately identified with high specificity and fidelity, demonstrating the ability to simultaneously detect multiple respiratory viruses. The combination of the integrated microfluidic chip with the POC device offers a simple, cost-effective, and scalable solution for rapid molecular diagnosis of respiratory viruses in resource-limited settings.

Stigmastane-type steroid saponins from the leaves of Vernonia amygdalina and their α-glucosidase and xanthine oxidase inhibitory activities.

Two new vernonioside K (1) and vernonioside L (2) and four known Δ7,9(11) stigmastane-type steroidal saponins-vernonioside B2 (3), vernoniacum B (4), vernonioside B1 (5), and vernoamyoside A (6)-were isolated from the leaves of Vernonia amygdalina. Their structures were determined by comprehensive spectroscopic analysis with one-dimensional nuclear magnetic resonance, two-dimensional nuclear magnetic resonance, and high-resolution mass spectrometry. All isolated compounds (1-6) were evaluated to determine their inhibitory effects on α-glucosidase and xanthine oxidase. Among them, two new compounds 1 and 2 showed significant inhibition of α-glucosidase with IC50 values of 78.56 ± 7.28 and 14.74 ± 1.57 (µM), respectively, comparable with acarbose as a positive control (127.53 ± 1.73 µM); none of these compounds inhibited xanthine oxidase activity. Compounds 1 and 2 are promising candidates for the development of antidiabetic agents from natural sources.

Effect on Satisfactory Seizure Control and Heart Rate Variability of Thread-Embedding Acupuncture for Drug-Resistant Epilepsy: A Patient-Assessor Blinded, Randomized Controlled Trial.

This randomized controlled trial investigates the efficacy of thread-embedding acupuncture (TEA) compared to sham TEA in treating drug-resistant epilepsy (DRE). Fifty-four DRE outpatients were randomly divided into two groups: TEA (27 patients) and sham TEA (27 patients). Both groups received four sessions of TEA or sham TEA, spaced four weeks apart, targeting GV20, GV14, BL15, BL18, ST40, and GB34 acupoints. Antiseizure medications were maintained at consistent doses throughout the study. Outcome measures included satisfactory seizure control, seizure freedom, and heart rate (HR) and heart rate variability (HRV) measurements. TEA demonstrated a significantly higher rate of satisfactory seizure control at follow-up compared to the sham TEA group (37% vs. 3.7%, p = 0.003). While no significant intergroup differences were observed in HR, HRV, and HRV components at each stage, the TEA group experienced a significant decrease in HR and a significant increase in HRV posttreatment. This study demonstrates TEA's effectiveness in managing DRE and suggests its impact may relate to heightened parasympathetic nerve activity. Further research with extended follow-up periods is necessary to validate these findings.

Function of the Speech Recognition of the Smartphone to Automatically Operate a Portable Sample Pretreatment Microfluidic System.

We proposed a portable sample pretreatment microsystem, which can be automatically operated through speech recognition in a smartphone app. The proposed sample pretreatment microsystem consists of a microfluidic chip, an air router, pressure and vacuum lines with air pump motors, six 3-way solenoid valves, and a microcontroller with a Bluetooth module. The command of a human voice conducted the whole process of DNA extraction from pathogenic bacterial samples. Thus, manual interference during the DNA extraction is eliminated, preventing any potential infection from human touch. The palm-sized sample pretreatment microsystem can be run by a portable battery or a conventional smartphone charger. Genomic DNA ofSalmonella typhimuriumwas purified on a chip in less than 1 min with an extraction efficiency of 70 ± 5%.

Auricular Acupressure Effect on Autonomic Responses Evoked by a Cold Pressor Test in Healthy Volunteers: A Pilot Study.

Objective: This pilot study was conducted to investigate changes in the pulse rate and blood pressure in healthy volunteers after applying auricular acupressure at the "heart acupoint." Methods: A total of 120 healthy volunteers with hemodynamic indexes within normal limits were randomly allocated into 4 groups to receive auricular acupressure treatment either at the heart acupoint of the left or the right, or in both ears, and one control group without applying auricular acupressure. Results: Before the application of auricular acupressure, there were no statistical differences in pulse rate and blood pressure increments among the four groups during the first cold pressor test. In groups in which auricular pressure was applied, the pulse rate was significantly reduced after the application of auricular acupressure in three groups; however, no statistically significant difference was detected among the groups. Changes in blood pressure were not statistically significant in or among the different groups after applying auricular acupressure. The average recorded pulse rate values during the second cold pressor test (after auricular acupressure) were significantly lower compared to the corresponding values taken during the first cold pressor test (before auricular acupressure) (p < 0.05); however, pulse rate increments during the two cold pressor tests (with and without auricular acupressure) were similar (p > 0.05). Conclusions: These findings suggest that auricular acupressure could be used as an adjunctive nonpharmacological method for reducing the pulse rate.

Heart Rate Variability during Auricular Acupressure at Heart Point in Healthy Volunteers: A Pilot Study.

Heart rate variability (HRV) is the variation in time between each heartbeat. Increasing HRV may contribute to improving autonomic nervous system dysfunctions. Acupuncture stimulation through the vagus plexus in the ear is considered as a method that can improve HRV. In this pilot study, we examined 114 healthy volunteers at the Faculty of Traditional Medicine, University of Medicine and Pharmacy at Ho Chi Minh City, from January to May 2020. During a 20-minute interval, participants were stimulated two times at the acupoint in the left ear with Semen seed. The heart rate and HRV values were monitored before, during, and after acupressure every 5 minutes. When we compared the experimental group with the control group, HRV significantly increased in the stage of ear-stimulated acupressure compared with the stage before and after the auricular acupressure (p=0.01, p=0.04, p=0.04 and p=0.02) and the difference was not statistically significant compared with the phase of nonstimulated (p=0.15, p=0.28). The changes in other values including SDNN (standard deviation of the average NN), RMSSD (root mean square of successive RR interval differences), LF (low-frequency power), and HF (high-frequency power) in all stages were not statistically significant (p=>0.05) between groups. Based on the results, we can determine the increase in HRV when conducting auricular acupressure with stimulation at the heart acupoint on the left ear. This leads to a direction in further studies for clinical application for patients with autonomic nervous disorder.

Successful Radiofrequency Ablation of the Right Lateral Accessory Pathway with Modified Carpentier Reconstruction Surgery in a Patient with Ebstein Anomaly Concomitant of Secundum Atrial Septal Defect, Atrial Fibrillation, and Wolff-Parkinson-White Syndrome.

Ebstein anomaly (EA) results from the failure of proper delamination of the tricuspid valve leaflets from the right ventricle (RV) myocardium. The severity of EA occurs on a spectrum that results in varying degrees of tricuspid regurgitation, atrial dilation, RV dilation, and dysfunction. These effects have the potential to create substrates that can give rise to atrial arrhythmia, ventricular arrhythmia, and a greater incidence of Wolff-Parkinson-White (WPW) syndrome Wackel et al. (2018) accounting for 0.5% of all congenital heart diseases (Oh et al. 1985). In the case of atrial fibrillation and WPW, it is very dangerous for the patient because of hemodynamic compromise, syncope, and sudden death. In this case report, we share our experience in using radiofrequency ablation to ablate right lateral accessory pathway, with modified Carpentier technique in operation to treat an adult patient diagnosed with Ebstein anomaly, atrial septal defect, atrial fibrillation, and WPW syndrome.

A handheld-type total integrated capillary electrophoresis system for SARS-CoV-2 diagnostics: Power, fluorescence detection, and data analysis by smartphone.

A micro-capillary electrophoresis (µCE) system is one of the widely adopted techniques in the molecular diagnostics and DNA sequencing due to the benefits of high resolution, rapid analysis, and low reagent consumption, but due to the requirements of bulky high-power suppliers and an expensive laser-induced fluorescence detector module, the conventional set-up of µCE system is not adequate for point-of-care (POC) molecular diagnostics. In this study, we constructed a miniaturized and integrated µCE system which can be manipulated by a smartphone. The smartphone not only powers two boost converters and an excited laser, but also controls the relay for the power switch. Moreover, the complementary metal-oxide-semiconductor (CMOS) camera of the smartphone was used for detecting the fluorescence signal of amplicons amplified with reverse transcription-polymerase chain reaction (RT-PCR). We also developed a web-based application so that the raw data of the recorded fluorescence intensity versus the running time can display typical capillary electropherograms on the smartphone. The total size of the hand-held µCE system was 9.6 cm [Width] × 22 cm [Length] × 15.5 cm [Height], and the weight was ∼1 kg, which is suitable for POC DNA testing. In the integrated smartphone-associated µCE system, we could accurately analyze two genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely N gene and S gene along with two bracket ladders in 6 min to identify SARS-CoV-2. Such an advanced µCE platform can be applied for a variety of on-site molecular diagnostics fields with user-friendliness.

Prediction of acute rejection in kidney transplanted patients based on the point-of-care isothermal molecular diagnostics platform.

In this study, we proposed an advanced point-of-care molecular diagnostic technology to evaluate the acute rejection (AR) in kidney transplanted patients. On the contrary to the conventional PCR method, we developed a colorimetric loop mediated isothermal amplification (LAMP) for quantitative analysis of the six biomarkers related to AR (CD3ϵ, IP-10, Tim-3-HAVCR2, CXCL9, PSMB9, C1QB) with a reference gene (18S rRNA). Using urinary cDNA samples of transplanted patients, it turned out that three biomarkers among six, namely IP-10, Tim-3-HAVCR2 and C1QB, have significant discrepancy in quantity between the stable graft (STA) patient and the AR patient. The AR prediction model using these three biomarkers was established, which could estimate the immune-rejection in the patients with 93.3% of accuracy. For the point-of-care (POC) molecular diagnostics for the AR evaluation, we constructed a centrifugal microfluidic platform, in which the RNA extraction from the clinical urinary samples, the quantitative reverse-transcription (RT)-LAMP reaction, and the data analysis based on the AR prediction model could be performed in a serial order. Ten blind clinical samples were analyzed on the POC genetic analyzer, showing 100% match with the validated qPCR data. Thus, the proposed advanced molecular diagnostic platform enables us to perform the timely treatment for the transplanted patients who are suffering from the allograft failure and side effects such as infection and malignancy.

Quantification of colorimetric isothermal amplification on the smartphone and its open-source app for point-of-care pathogen detection.

The increasing risk of infectious pathogens, especially in the under-developed countries, is demanding the development of point-of-care (POC) nucleic acid testing in the low-resource setting conditions. Here, we describe a methodology for colorimetric quantitative analysis of nucleic acid using an easy-to-build smartphone-based platform, offering low-cost, portability, simplicity in operation, and user-friendliness. The whole system consists of a hand-held box equipped with a smartphone, a film heater, a white LED, a loop-mediated isothermal amplification (LAMP) chip, and a DC converter, and all the processes were powered by a portable battery of 5 V. Upon the amplification of the target gene by an Eriochrome Black T-mediated LAMP reaction, the color of the LAMP reaction was changed from violet to blue that was real-time recorded by a smartphone camera. To keep track of the progress of the color change, we developed a novel mobile app in which a hue value was accepted as an indicator for color transition and for determining the threshold time of the amplification reaction. A calibration curve could be generated by plotting the logarithm of the known concentration of the DNA templates versus the threshold time, and it can be used to predict the copy number of nucleic acids in the test samples. Thus, the proposed mobile platform can inform us of not only qualitative but also quantitative results of the pathogens. We believe that this advanced colorimetric approach and the mobile app can expand the potentials of the smartphone for the future POCT system in the bio-diagnostic fields.

An Integrated Smartphone-Based Genetic Analyzer for Qualitative and Quantitative Pathogen Detection.

The use of the smartphone is an ideal platform to realize the future point-of-care (POC) diagnostic system. Herein, we propose an integrated smartphone-based genetic analyzer. It consists of a smartphone and an integrated genetic analysis unit (i-Gene), in which the power of the smartphone was utilized for heating the gene amplification reaction, and the camera function was used for imaging the colorimetric change of the reaction for quantitative and multiplex foodborne pathogens. The housing of i-Gene was fabricated by using a 3D printer, which was equipped with a macro lens, white LEDs, a disposable microfluidic chip for loop-mediated isothermal amplification (LAMP), a thin-film heater, and a power booster. The i-Gene was installed on the iPhone in alignment with a camera. The LAMP mixture for Eriochrome Black T (EBT) colorimetric detection was injected into the LAMP chip to identify Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus. The proportional-integral-derivative controller-embedded film heater was powered by a 5.0 V power bank to maintain 63 °C for the LAMP reaction. When the LAMP proceeded, the color was changed from violet to blue, which was real-time monitored by the smartphone complementary metal oxide semiconductor camera. The images were transported to the desktop computer via Wi-Fi. The quantitative LAMP profiles were obtained by plotting the ratio of green/red intensity versus the reaction time. We could identify E. coli O157:H7 with a limit of detection of 101 copies/µL within 60 min. Our proposed smartphone-based genetic analyzer offers a portable, simple, rapid, and cost-effective POC platform for future diagnostic markets.

Nucleic acid diagnostics on the total integrated lab-on-a-disc for point-of-care testing.

Since the emergence of the lab-on-a-chip technology in 1979, a variety of microfluidic devices have been developed and utilized for chemical and biological applications. Among the microfluidic devices, the centrifugal microfluidic device or lab-on-a-disc (LOAD) has advanced remarkably due to simple operation by the rotation, total integration, and high-throughput capability. Moreover, the centrifugal microdevices do not need complex tubing and pumping systems, which render them ideal for point-of-care testing (POCT) system. Owing to these characteristics, the centrifugal microdevices have been extensively used for bio-diagnostics. In particular, molecular diagnostics, which are regarded as an essential method for definite determination of the targets related with diseases, have been widely applied on the LOAD. In this review paper, we focus on the molecular diagnostics on the LOAD. The steps for the molecular diagnostics such as cell lysis, genome purification, gene amplification, amplicon detection, and data analysis can be performed individually or totally on the LOAD. Future directions of the LOAD in the fields of bio-diagnostics is to realize POCT for U-healthcare monitoring. In this context, the latest LOAD strategies for molecular diagnostics are summarized in this review paper, which would provide an insight for future POCT platform.

Point-of-care genetic analysis for multiplex pathogenic bacteria on a fully integrated centrifugal microdevice with a large-volume sample.

We present a fully integrated portable centrifugal microsystem for multiplex detection of food poisoning bacteria with a large volume of sample up to 1 mL. The microsystem consists of a portable genetic analyzer and a fully integrated centrifugal microdevice. The centrifugal microdevice is designed with two units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions for loop-mediated isothermal amplification (LAMP) reaction are stored inside the cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device is designed with 20 reaction chambers for simultaneous detection of food-borne bacteria in one test. To increase the amount of a sample to 1 mL, we incorporated the super absorbent polymer (SAP) in the waste chamber to absorb the sample and the washing solution during the device operation. The whole process was automatically conducted including designated solution release, bead-based DNA extraction, isothermal gene amplification by Eriochrome Black (EBT)-mediated LAMP reaction, and colorimetric and UV-visible detection of amplicons. The ratio between Abs640nm and Abs570nm was used as a criterion to confirm the positive result, and the result was positive upon the condition of Abs640/Abs570 ≥ 1.0. To demonstrate the pathogenic bacteria detection on our proposed microsystem, we targeted three kinds of bacteria (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) for monoplex and multiplex detection. The whole process from sample to result was completed within 1 h with a low limit of detection (LOD) of 102 cells/mL.

Anti-inflammatory and wound healing activities of calophyllolide isolated from Calophyllum inophyllum Linn.

Due to the high-cost and limitations of current wound healing treatments, the search for alternative approaches or drugs, particularly from medicinal plants, is of key importance. In this study, we report anti-inflammatory and wound healing activities of the major calophyllolide (CP) compound isolated from Calophyllum inophyllum Linn. The results showed that CP had no effect on HaCaT cell viability over a range of concentrations. CP reduced fibrosis formation and effectively promoted wound closure in mouse model without causing body weight loss. The underlying molecular mechanisms of wound repair by CP was investigated. CP markedly reduced MPO activity, and increased M2 macrophage skewing, as shown by up-regulation of M2-related gene expression, which is beneficial to the wound healing process. CP treatment prevented a prolonged inflammatory process by down-regulation of the pro-inflammatory cytokines-IL-1ß, IL-6, TNF-α, but up-regulation of the anti-inflammatory cytokine, IL-10. This study is the first to indicate a plausible role for CP in accelerating the process of wound healing through anti-inflammatory activity mechanisms, namely, by regulation of inflammatory cytokines, reduction in MPO, and switching of macrophages to an M2 phenotype. These findings may enable the utilization of CP as a potent therapeutic for cutaneous wound healing.

Quantitative trait loci mapping of partial resistance to Diamondback moth in cabbage (Brassica oleracea L).

KEY MESSAGE: The resistance to Diamondback moth insect in cabbage is governed by many minor loci in quantitative nature, and at least four genetic loci should be incorporated in marker-assisted breeding program for developing partially resistant DBM cabbage cultivars. The Diamondback moth (DBM), Plutella xylostella (L.), is the most destructive insect infesting cruciferous plants worldwide. Earlier studies have reported that the glossy leaves of cabbage are associated with resistance to this insect. However, until now, genetics of DBM resistance has not been studied in detail, and no QTL/gene mapping for this trait has been reported. In this paper, we report quantitative trait loci (QTL) mapping of DBM-resistant trait using 188 randomly selected segregating F 3 population derived from crossing a partially DBM-resistant glossy leaf cabbage (748) with a susceptible smooth cabbage line (747). Quantitative trait loci mapping using phenotypic data of four consecutive years (2008, 2009, 2010, and 2011) on DBM insect infestation detected a total of eight QTL on five linkage groups suggesting that DBM resistance is a quantitative in nature. Of these QTL, four QTL, i.e., qDbm 1 on LG1, qDbm5 and qDbm6 on LG7, and qDbm8 on LG9, were detected in different tests and years. The QTL, qDbm6 on LG7, was consecutively detected over 3 years. Tightly linked molecular markers have been developed for qDbm8 QTL on LG9 which could be used in marker-assisted breeding program. Our research demonstrated that for desired DBM resistance cultivar breeding, those four genetic loci have to be taken into consideration. Furthermore, the comparative study revealed that DBM resistance QTL is conserved between close relative model plant Arabidopsis thaliana and Brassica oleracea genome.

Genic microsatellite markers in Brassica rapa: development, characterization, mapping, and their utility in other cultivated and wild Brassica relatives.

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.