Monitoring the state of charge of vanadium redox flow batteries with an EPR-on-a-Chip dipstick sensor.

The vanadium redox flow battery (VRFB) is considered a promising candidate for large-scale energy storage in the transition from fossil fuels to renewable energy sources. VRFBs store energy by electrochemical reactions of different electroactive species dissolved in electrolyte solutions. The redox couples of VRFBs are VO2+/VO2+ and V2+/V3+, the ratio of which to the total vanadium content determines the state of charge (SOC). V(IV) and V(II) are paramagnetic half-integer spin species detectable and quantifiable with electron paramagnetic resonance spectroscopy (EPR). Common commercial EPR spectrometers, however, employ microwave cavity resonators which necessitate the use of large electromagnets, limiting their application to dedicated laboratories. For an SOC monitoring device for VRFBs, a small, cost-effective submersible EPR spectrometer, preferably with a permanent magnet, is desirable. The EPR-on-a-Chip (EPRoC) spectrometer miniaturises the complete EPR spectrometer onto a single microchip by utilising the coil of a voltage-controlled oscillator as both microwave source and detector. It is capable of sweeping the frequency while the magnetic field is held constant enabling the use of small permanent magnets. This drastically reduces the experimental complexity of EPR. Hence, the EPRoC fulfils the requirements for an SOC sensor. We, therefore, evaluate the potential for utilisation of an EPRoC dipstick spectrometer as an operando and continuously online monitor for the SOC of VRFBs. Herein, we present quantitative proof-of-principle submersible EPRoC experiments on variably charged vanadium electrolyte solutions. EPR data obtained with a commercial EPR spectrometer are in good agreement with the EPRoC data.

The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa.

Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1-a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.

Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association.

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.

The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption.

This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.

The Mushroom Albatrellus confluens: A Minireview on Phytochemistry, Biosynthesis, Synthesis and Pharmacological Activities.

BACKGROUND: Albatrellus confluens is one of the representative species in the Polyporaceae family. Its major mero terpenoid grifolin and related compounds have the potential for drug applications. OBJECTIVE: The current study aims to briefly provide an insightful view of the phytochemistry, biosynthesis, synthesis, and pharmacology of A. confluens metabolites. METHODS: Data collection was performed using electronic resources, e.g., Google Scholar, PubMed, and Sci-Finder from the 1990s to the present, while Albatrellus confluens is the most meaningful keyword in the search for publications. The Latin name Albatrellus confluens (Alb. & Schwein.) Kotl. & Pouzar is in accordance with the name listing on www.mycobank.org. RESULTS: By chromatography column procedures, it indicated that A. confluens species was associated with the presence of 57 secondary metabolites, in which nitrogenous compounds, meroterpenoids, polyene pyrones, and polyesters can be seen as the main phytochemical classes. L-isoleucine was the parent molecule in biosynthetic and synthetic steps of A. confluens nitrogenous compounds. Numerous experiments revealed that A. confluens isolated compounds have a variety of pharmacological activities, such as anticancer, anti-inflammatory, vasorelaxant, and neuroprotective and skin whitening activities. Some isolates become potential cancer inhibitors. Grifolin induced apoptosis and promoted cell cycle arrest in A2780 ovarian cancer cells via the inactivation of the ERK1/2/Akt signaling pathway. Grifolic acid caused osteosarcoma cancer cell deaths by inhibiting NADH generation and ATP production without obvious toxicity. Neoalbaconol caused apoptosis and necroptosis in mice bearing nasopharyngeal C666-1 cancer cells via PDK1-PI3K/Akt signaling inhibition. CONCLUSION: The continuation of chromatographic separation and biomedical research is expected. Modern biological assays for explaining the pharmacological values of A. confluens constituents are warranted. Toxicological and pharmacokinetic assessments are urgently needed.

A chip-based C-band ODNP platform.

In this paper, we present a chip-based C-band ODNP platform centered around an NMR-on-a-chip transceiver and a printed microwave (MW) Alderman-Grant (AG) coil with a broadband tunable frequency range of 528MHz. The printable ODNP probe is optimized for a high input-power-to-magnetic-field conversion-efficiency, achieving a measured ODNP enhancement factor of -151 at microwave power levels of 33.3dBm corresponding to 2.1W. NMR measurements with and without microwave irradiation verify the functionality and the state-of-the-art performance of the proposed ODNP platform. The wide tuning range of the system allows for indirect measurements of the EPR signal of the DNP agent by sweeping the microwave excitation frequency and recording the resulting NMR signal. This feature can, e.g., be used to detect line broadening of the DNP agent. Moreover, we demonstrate experimentally that the wide tuning range of the new ODNP platform can be used to perform multi-tone microwave excitation for further signal enhancement: Using a 10mM TEMPOL solution, we improved the enhancement by a factor of two.

Microwave field mapping for EPR-on-a-chip experiments.

Electron paramagnetic resonance-on-a-chip (EPRoC) devices use small voltage-controlled oscillators (VCOs) for both the excitation and detection of the EPR signal, allowing access to unique sample environments by lifting the restrictions imposed by resonator-based EPR techniques. EPRoC devices have been successfully used at multiple frequencies (7 to 360 gigahertz) and have demonstrated their utility in producing high-resolution spectra in a variety of spin centers. To enable quantitative measurements using EPRoC devices, the spatial distribution of the B1 field produced by the VCOs must be known. As an example, the field distribution of a 12-coil VCO array EPRoC operating at 14 gigahertz is described in this study. The frequency modulation-recorded EPR spectra of a "point"-like and a thin-film sample were investigated while varying the position of both samples in three directions. The results were compared to COMSOL simulations of the B1-field intensity. The EPRoC array sensitive volume was determined to be ~19 nanoliters. Implications for possible EPR applications are discussed.

A conformal multi-port MIMO patch antenna for 5G wireless devices.

This paper presents a multiple-input-multiple-output (MIMO) antenna array with low-profile and flexible characteristics. Multiple microstrip patches are arranged in the E-plane configuration and decoupled by shorted quarter-wavelength stubs. The antenna has a small element spacing of 0.032 λ, where λ is a free-space wavelength at the center frequency. To demonstrate the feasibility of the proposed concept, a 1 × 4 MIMO array prototype is fabricated. The measured results on the fabricated prototype demonstrate that the MIMO antenna has good operation features at 4.8 GHz with a reflection coefficient of less than -10 dB and an isolation of better than 20 dB. Besides, good radiation patterns and broadside gain of around 4.5 dBi are also attained. The antenna also works in the bending mode and has the capability of extending to large-scale MIMO arrays. Such attractive features prove the utility of the proposed antenna in various modern electronic devices.

High Seroprevalence of Anti-SARS-CoV-2 Antibodies in Children in Vietnam: An Observational, Hospital-Based Study.

Background: The robustness of sero-surveillance has delineated the high burden of SARS-CoV-2 infection in children; however, these existing data showed wide variation. This study aimed to identify the serostatus of antibodies against SARS-CoV-2 and associated factors among children following the fourth pandemic wave in Vietnam. Methods: A cross-sectional study was conducted at Vietnam National Children's Hospital (VNCH) between March 13 and April 3, 2022. Thus, 4032 eligible children seeking medical care for any medical condition not related to acute COVID-19 infection were tested for IgG SARS-CoV-2 antibodies by ADVIA Centaur® SARS-CoV-2 IgG (sCOVG) assay using the residuals of routine blood samples. Results: The median age of enrolled children was 39 (IQR = 14−82) months. The overall seropositive prevalence was 59.2% (95%CI = 57.6−60.7) and the median antibody titer was 4.78 (IQR 2.38−9.57) UI/mL. The risk of seropositivity and the median antibody titer were not related to gender (58.6% versus 60.1%, 4.9 versus 4.6 UI/mL, all p > 0.05). Children aged ≤12 months were likely to be seropositive compared to children aged 36 to <60 months (59.2% versus 57.5%, p = 0.49) and those aged ≥144 months (59.2% versus 65.5%, p = 0.16). Children aged ≥144 months exhibited a significantly higher titer of protective COVID-19 antibodies than other age groups (p < 0.001). In multivariate logistic regression, we observed independent factors associated with SARS-CoV-2 seropositivity, including the age 13 to <36 months (OR = 1.29, 95%CI = 1.06−1.56, p = 0.01), 60 to <144 months (OR = 0.79, 95%CI = 0.67−0.95, p = 0.01), ≥144 months (OR = 1.84, 95%CI = 1.21−2.8, p = 0.005), the presence of infected household members (OR = 2.36, 95%CI = 2.06−2.70, p < 0.001), participants from Hanoi (OR = 1.54, 95%CI = 1.34−1.77, p < 0.001), underlying conditions (OR = 0.71, 95%CI = 0.60−0.85, p ≤ 0.001), and using corticosteroids or immunosuppressants (OR = 0.64, 95%CI = 0.48−0.86, p = 0.003). Conclusions: This study highlights a high seroprevalence of antibodies against SARS-CoV-2 among children seeking medical care for non-acute COVID-19-related conditions in a tertiary children's hospital in Hanoi, Vietnam. In the context of reopening in-person schools and future emerging COVID-19 variants, this point will also be a key message about the necessity of "rush-out" immunization coverage for children, especially those under the age of five years.

On the modeling of amplitude-sensitive electron spin resonance (ESR) detection using voltage-controlled oscillator (VCO)-based ESR-on-a-chip detectors.

In this paper, we present an in-depth analysis of a voltage-controlled oscillator (VCO)-based sensing method for electron spin resonance (ESR) spectroscopy, which greatly simplifies the experimental setup compared to conventional detection schemes. In contrast to our previous oscillator-based ESR detectors, where the ESR signal was encoded in the oscillation frequency, in the amplitude-sensitive method, the ESR signal is sensed as a change of the oscillation amplitude of the VCO. Therefore, using VCO architecture with a built-in amplitude demodulation scheme, the experimental setup reduces to a single permanent magnet in combination with a few inexpensive electronic components. We present a theoretical analysis of the achievable limit of detection, which uses perturbation-theory-based VCO modeling for the signal and applies a stochastic averaging approach to obtain a closed-form expression for the noise floor. Additionally, the paper also introduces a numerical model suitable for simulating oscillator-based ESR experiments in a conventional circuit simulator environment. This model can be used to optimize sensor performance early on in the design phase. Finally, all presented models are verified against measured results from a prototype VCO operating at 14 GHz inside a 0.5 T magnetic field.

Rapid-scan electron paramagnetic resonance using an EPR-on-a-Chip sensor.

Electron paramagnetic resonance (EPR) spectroscopy is the method of choice to investigate and quantify paramagnetic species in many scientific fields, including materials science and the life sciences. Common EPR spectrometers use electromagnets and microwave (MW) resonators, limiting their application to dedicated lab environments. Here, novel aspects of voltage-controlled oscillator (VCO)-based EPR-on-a-Chip (EPRoC) detectors are discussed, which have recently gained interest in the EPR community. More specifically, it is demonstrated that with a VCO-based EPRoC detector, the amplitude-sensitive mode of detection can be used to perform very fast rapid-scan EPR experiments with a comparatively simple experimental setup to improve sensitivity compared to the continuous-wave regime. In place of a MW resonator, VCO-based EPRoC detectors use an array of injection-locked VCOs, each incorporating a miniaturized planar coil as a combined microwave source and detector. A striking advantage of the VCO-based approach is the possibility of replacing the conventionally used magnetic field sweeps with frequency sweeps with very high agility and near-constant sensitivity. Here, proof-of-concept rapid-scan EPR (RS-EPRoC) experiments are performed by sweeping the frequency of the EPRoC VCO array with up to 400 THz s-1, corresponding to a field sweep rate of 14 kT s-1. The resulting time-domain RS-EPRoC signals of a micrometer-scale BDPA sample can be transformed into the corresponding absorption EPR signals with high precision. Considering currently available technology, the frequency sweep range may be extended to 320 MHz, indicating that RS-EPRoC shows great promise for future sensitivity enhancements in the rapid-scan regime.

Comparing a standard and tailored approach to scaling up an evidence-based intervention for antiretroviral therapy for people who inject drugs in Vietnam: study protocol for a cluster randomized hybrid type III trial.

BACKGROUND: People who inject drugs (PWID) bear a disproportionate burden of HIV infection and experience poor outcomes. A randomized trial demonstrated the efficacy of an integrated System Navigation and Psychosocial Counseling (SNaP) intervention in improving HIV outcomes, including antiretroviral therapy (ART) and medications for opioid use disorder (MOUD) uptake, viral suppression, and mortality. There is limited evidence about how to effectively scale such intervention. This protocol presents a hybrid type III effectiveness-implementation trial comparing two approaches for scaling-up SNaP. We will evaluate the effectiveness of SNaP implementation approaches as well as cost and the characteristics of HIV testing sites achieving successful or unsuccessful implementation of SNaP in Vietnam. METHODS: Design: In this cluster randomized controlled trial, two approaches to scaling-up SNaP for PWID in Vietnam will be compared. HIV testing sites (n = 42) were randomized 1:1 to the standard approach or the tailored approach. Intervention mapping was used to develop implementation strategies for both arms. The standard arm will receive a uniform package of these strategies, while implementation strategies for the tailored arm will be designed to address site-specific needs. PARTICIPANTS: HIV-positive PWID participants (n = 6200) will be recruited for medical record assessment at baseline; of those, 1500 will be enrolled for detailed assessments at baseline, 12, and 24 months. Site directors and staff at each of the 42 HIV testing sites will complete surveys at baseline, 12, and 24 months. OUTCOMES: Implementation outcomes (fidelity, penetration, acceptability) and effectiveness outcomes (ART, MOUD uptake, viral suppression) will be compared between the arms. To measure incremental costs, we will conduct an empirical costing study of each arm and the actual process of implementation from a societal perspective. Qualitative and quantitative site-level data will be used to explore key characteristics of HIV testing sites that successfully or unsuccessfully implement the intervention for each arm. DISCUSSION: Scaling up evidence-based interventions poses substantial challenges. The proposed trial contributes to the field of implementation science by applying a systematic approach to designing and tailoring implementation strategies, conducting a rigorous comparison of two promising implementation approaches, and assessing their incremental costs. Our study will provide critical guidance to Ministries of Health worldwide regarding the most effective, cost-efficient approach to SNaP implementation. TRIAL REGISTRATION: NCT03952520 on Clinialtrials.gov. Registered 16 May 2019.

Structural Insights into KCTD Protein Assembly and Cullin3 Recognition.

Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2-ubiquitin, while the N-terminal region interacts with various BTB (bric-à-brac, tramtrack, broad complex) domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain-containing proteins, the KCTD proteins, is also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9 and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by cryo-electron microscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family.