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22 pages, 3153 KiB  
Article
Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry Analysis of Δ9-Tetrahydrocannabinol and Cannabidiol in Commercial Suk-Saiyasna Herbal Remedy: Applying Hansen Solubility Parameters for Sample Extraction to Ensure Regulatory Compliance
by Suwimon Sumontri, Wanna Eiamart, Sarin Tadtong and Weerasak Samee
Pharmaceuticals 2024, 17(11), 1502; https://doi.org/10.3390/ph17111502 - 8 Nov 2024
Viewed by 381
Abstract
Background: Suk-Saiyasna is a traditional Thai herbal remedy that comprises 12 distinct herbs. Among these, cannabis leaves constitute 12 of the total 78 components in this formulation. This study specifically examines the portion of the cannabis plant, which accounts for approximately 15.8% of [...] Read more.
Background: Suk-Saiyasna is a traditional Thai herbal remedy that comprises 12 distinct herbs. Among these, cannabis leaves constitute 12 of the total 78 components in this formulation. This study specifically examines the portion of the cannabis plant, which accounts for approximately 15.8% of the overall herbal composition used in the entire remedy. According to the Thailand Narcotics Act of 2022, the Δ9-tetrahydrocannabinol (Δ9-THC) concentration in herbal extracts must not exceed 0.2% by weight. This study aims to quantify the levels of Δ9-THC and cannabidiol (CBD) in commercial Suk-Saiyasna products. Methodology: This research utilizes Hansen Solubility Parameters (HSPs) to identify the optimal solvent for ultrasonic extraction, thereby maximizing cannabinoid yield. An advanced method was developed employing ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS), compliant with AOAC standards to meet regulatory guidelines. The method validation emphasized specificity, linearity, sensitivity, accuracy, and precision. Results: Dichloromethane was chosen due to its favorable HSP values, enabling highly efficient extraction of Δ9-THC and CBD, achieving recovery rates of over 99.9% after the second extraction. This investigation benefits from the accuracy of the UHPLC-MS/MS technique in quantifying cannabinoids in commercial products, with Δ9-THC concentrations observed between 0.00231% and 0.14218%, and CBD concentrations ranging from 0.00002% to 0.01541%, all remaining below the legal limit. Conclusions: The variability in cannabinoid concentrations among various commercial products highlights the need for standardization in the herbal industry. This finding underscores the critical role of rigorous quality control measures in ensuring the safety and efficacy of cannabis-derived products. Full article
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<p>Chromatograms of (<b>a</b>) overlaid standard CBD (orange) and Δ9-THC (blue), (<b>b</b>) dichloromethane extract, (<b>c</b>) ethanol extract, and (<b>d</b>) ethyl acetate extract, detected at 208 nm UV.</p>
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<p>Chromatograms of (<b>a</b>) overlaid standard CBD (orange) and Δ9-THC (blue), (<b>b</b>) dichloromethane extract, (<b>c</b>) ethanol extract, and (<b>d</b>) ethyl acetate extract, detected at 208 nm UV.</p>
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<p>Chromatograms of the initial standard solution and the solution after sonication for 30 min for (<b>a</b>) CBD and (<b>b</b>) Δ9-THC.</p>
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<p>Chromatogram of the extract from the Suk-Saiyasna herbal remedy in commercial brand A, detected at 208 nm UV.</p>
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<p>Mass fragmentation spectra of Δ9-THC in ESI positive mode.</p>
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<p>Mass fragmentation spectra of CBD in ESI positive mode.</p>
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<p>Proposed fragments of Δ9-THC and CBD in ESI positive mode.</p>
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<p>Proposed fragments of Δ9-THC and CBD in ESI positive mode.</p>
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<p>Comparison of overlaid chromatograms: Suk-Saiyasna formulation extract versus spiked standard solutions of CBD and Δ9-THC.</p>
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<p>LC-MS/MS chromatograms of the first, second, and third extractions: (<b>a</b>–<b>c</b>) in-house Suk-Saiyasna remedy, (<b>d</b>–<b>f</b>) expanded chromatogram of the in-house Suk-Saiyasna remedy.</p>
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<p>LC-MS/MS chromatograms of leaf, in-house, and commercial crude extracts.</p>
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28 pages, 12879 KiB  
Review
Classification, Chemical, and Toxicological Properties of Carbamate Nerve Agents
by Georgios Pampalakis
J. Xenobiot. 2024, 14(4), 1729-1756; https://doi.org/10.3390/jox14040092 - 7 Nov 2024
Viewed by 377
Abstract
Nerve agents are usually identified as exceedingly toxic organophosphate compounds like VX and sarin. Nevertheless, although carbamate nerve agents (CNAs) have been developed they constitute the least studied class of nerve agents outside military literature. Recently, CNAs gained popularity after the inclusion of [...] Read more.
Nerve agents are usually identified as exceedingly toxic organophosphate compounds like VX and sarin. Nevertheless, although carbamate nerve agents (CNAs) have been developed they constitute the least studied class of nerve agents outside military literature. Recently, CNAs gained popularity after the inclusion of a small subset of carbamate agents in the Chemical Weapons Convention (CWC) list of Schedule 1 chemicals. Here, a holistic approach was used to identify and categorize the developed CNAs. It is demonstrated that CNAs encompass a highly diverse class of compounds. Their main characteristics include an aromatic group that carries the carbamate moiety. Based on their chemical structure, CNAs were categorized into two generations that are further divided into various subclasses. The second generation of CNAs includes some members that exhibit higher toxicity than VX. CNAs have not been used in any conflict, which may be related to their solid nature that requires sophisticated delivery systems. Since, however, CNAs can be applied as poisons in individualized cases, understanding their chemistry and toxicological properties is important for the development of effective countermeasures. Full article
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<p>Representative examples of the most toxic carbamate pesticides [aldicarb LD<sub>50</sub> rat oral: 650 μg∙kg<sup>−1</sup>; aldoxycarb LD<sub>50</sub> rat oral: 20,000 μg∙kg<sup>−1</sup>, rat iv 14,900 μg∙kg<sup>−1</sup>; bendiocarb LD<sub>50</sub> rat oral: 40,000 μg∙kg<sup>−1</sup>, mouse oral: 45,000 μg∙kg<sup>−1</sup>; carbofuran LD<sub>50</sub> rat oral: 5000 μg∙kg<sup>−1</sup>, mouse iv: 450 μg∙kg<sup>−1</sup>; dog oral: 19,000 μg∙kg<sup>−1</sup>; mouse oral: 2000 μg∙kg<sup>−1</sup>] (data were retrieved from Pubchem, <a href="http://pubchem.ncbi.nlm.nih.gov" target="_blank">http://pubchem.ncbi.nlm.nih.gov</a>, accessed on 21 September 2024).</p>
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<p>Toxicities of representative compounds from subclasses VII, VIII, X, and XI. The selected compounds exhibit LD<sub>50</sub> (sc mice) lower than 500 μg∙kg<sup>−1</sup>. Thus, they may be considered potential CWAs. When a range of toxicity values is given for a compound, e.g., TL-1238 (125–175 μg∙kg<sup>−1</sup>) (in <a href="#jox-14-00092-t002" class="html-table">Table 2</a>), the mean value has been added to generate the graph. As shown, the highest toxicity is exhibited by TL-1345.</p>
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<p>Correlation of structure with toxicity for first-generation CNAs. The LD<sub>50</sub>s are shown as μg∙kg<sup>−1</sup> when administered as a water solution in mice (sc). TL-1309 is a control compound for class VII (lacks the carbamate moiety). (<b>A</b>–<b>G</b>) indicates various examples that compare carbamates and were used to extract the “toxicity rules”.</p>
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<p>Reaction of phenols with dimethylcarbonyl chloride, methyl isocyanate, or phosgene followed by methylamine to generate the first generation of CNAs.</p>
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<p>General synthesis scheme for p-amino-m-alkylphenols. These compounds are precursors of the first generation of CNAs.</p>
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<p>Examples of synthesis schemes for the first generation of CNA TL-1217 (<b>A</b>), TL-1071 (<b>B</b>), and TL-599 (<b>C</b>).</p>
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<p>The phosphonate analogs of the subclasses 19 and 21 of CNAs. The toxicities are given for iv route.</p>
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<p>A synthesis scheme of selected compounds of the second generation of CNAs.</p>
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<p>The structure of KB-16.</p>
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19 pages, 4826 KiB  
Article
Efficient Simultaneous Isolation of Pinostrobin and Panduratin A from Boesenbergia rotunda Using Centrifugal Partition Chromatography
by Wanna Eiamart, Supeecha Wittayalertpanya, Sarin Tadtong and Weerasak Samee
Molecules 2024, 29(21), 5186; https://doi.org/10.3390/molecules29215186 - 2 Nov 2024
Viewed by 493
Abstract
The bioactive flavonoids pinostrobin (PN) and panduratin A (PA) from Boesenbergia rotunda are essential for research and therapeutic applications. This study introduces an innovative method utilizing ultrasound-assisted extraction with n-hexane pre-treatment, followed by one-step centrifugal partition chromatography (CPC) purification. Extraction efficiency was [...] Read more.
The bioactive flavonoids pinostrobin (PN) and panduratin A (PA) from Boesenbergia rotunda are essential for research and therapeutic applications. This study introduces an innovative method utilizing ultrasound-assisted extraction with n-hexane pre-treatment, followed by one-step centrifugal partition chromatography (CPC) purification. Extraction efficiency was evaluated using ultra high-performance liquid chromatography (UHPLC), and the isolated compounds were characterized through 1H-NMR and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), adhering to AOAC validation guidelines. Optimal extraction conditions comprised a particle size of 125 μm, a solid-to-liquid ratio of 1:30 g/mL, and a 10 min extraction time, yielding a crude extract of 6.96 ± 0.07%. Using an n-hexane/MeOH/water (5/3.4/1.6, v/v) solvent system in ascending mode, PN (2.16 mg, 98.78% purity) and PA (0.4 mg, 99.69% purity) were isolated from 67 mg of crude extract within 30 min. This streamlined approach enhances purification efficiency, allowing for faster extraction and higher purity, making it a suitable method for commercial applications. Full article
(This article belongs to the Special Issue Chromatography and Extraction Techniques for Chemical Applications)
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<p>RP-HPLC chromatogram of (<b>a</b>) overlaid chromatogram of blank (DMSO in MeOH) compared with standard solution at 10 µg/mL and (<b>b</b>) crude <span class="html-italic">n</span>-hexane extract of <span class="html-italic">B. rotunda</span> at 12.5 µg/mL was spiked standard solution at 2.5 µg/mL.</p>
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<p>The multiple reaction monitoring (MRM) chromatogram of (<b>a</b>) standard compounds and (<b>b</b>) blank (MeOH).</p>
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<p>(<b>a</b>) Chromatographic profiles of extractives from <span class="html-italic">B. rotunda</span> using five different solvents with UAE, and (<b>b</b>) the concentrations of PN and PA in the raw material with different solvents and particle sizes. Data are the mean ± SD (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 compared with the mean of every other parameter.</p>
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<p>Effects of various conditions on extraction of PN and PA from <span class="html-italic">B. rotunda</span> (powder size 125 µm) by UAE with <span class="html-italic">n</span>-hexane as solvent and quantification by UHPLC system. The concentrations of PN and PA at different (<b>a</b>) extraction temperatures in range 30–40 °C and 50–60 °C, (<b>b</b>) ratios of powder/<span class="html-italic">n</span>-hexane in range 1:10 to 1:50 g/mL, (<b>c</b>) extraction times in range 10–120 min, and (<b>d</b>) extraction efficiencies of PN and PA at a single step compared with two and three repetitions. Data are the mean ± SD (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 compared with the mean of every other parameter.</p>
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<p>Effects of various conditions on extraction of PN and PA from <span class="html-italic">B. rotunda</span> (powder size 125 µm) by UAE with <span class="html-italic">n</span>-hexane as solvent and quantification by UHPLC system. The concentrations of PN and PA at different (<b>a</b>) extraction temperatures in range 30–40 °C and 50–60 °C, (<b>b</b>) ratios of powder/<span class="html-italic">n</span>-hexane in range 1:10 to 1:50 g/mL, (<b>c</b>) extraction times in range 10–120 min, and (<b>d</b>) extraction efficiencies of PN and PA at a single step compared with two and three repetitions. Data are the mean ± SD (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 compared with the mean of every other parameter.</p>
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<p>The extraction of PN and PA from <span class="html-italic">B. rotunda</span> using UAE with <span class="html-italic">n</span>-hexane solvent and quantified by UHPLC system. (<b>a</b>) Dried root, (<b>b</b>) dried rhizome, (<b>c</b>) crude <span class="html-italic">n</span>-hexane extract, (<b>d</b>) crude <span class="html-italic">n</span>-hexane extract dissolves in ethyl acetate for injection to CPC system, and (<b>e</b>) HPLC chromatogram of PN and PA from 25 µg/mL of crude <span class="html-italic">n</span>-hexane extract.</p>
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<p>The HPLC chromatogram of PN and PA from crude <span class="html-italic">n</span>-hexane extract in (<b>a</b>) upper and (<b>b</b>) lower phases of <span class="html-italic">n</span>-hexane/MeOH/H<sub>2</sub>O (5/3.4/1.6, <span class="html-italic">v</span>/<span class="html-italic">v</span>) system.</p>
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<p>Preparative CPC (<span class="html-italic">n</span>-hexane: MeOH: H<sub>2</sub>O (5/3.4/1.6, <span class="html-italic">v</span>/<span class="html-italic">v</span>) chromatogram from crude n-hexane extract of the <span class="html-italic">B. rotunda</span>. The PN (Fraction A: 001–004) and PA (Fraction B: 005−008) were detected using UV detection at 220 nm (green line), 230 nm (orange line), 280 nm (pink line),285 nm (black line) and 200-600 nm (blue line).</p>
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<p>The pure crystals of isolated (<b>a</b>) PN and (<b>b</b>) PA from crude <span class="html-italic">n</span>-hexane extract of the <span class="html-italic">B. rotunda</span>.</p>
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<p>The MRM chromatogram of (<b>a</b>) PN and (<b>b</b>) PA derived from fraction A (500 ng/mL of PN) and fraction B (PN 250 of ng/mL) of separation using CPC and zoomed chromatogram. (<b>c</b>) The structures of PA and PN were further confirmed by <sup>1</sup>H NMR (see <a href="#app1-molecules-29-05186" class="html-app">Supplementary Materials</a>).</p>
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19 pages, 693 KiB  
Review
A Review of Wastewater-Based Epidemiology for the SARS-CoV-2 Virus in Rural, Remote, and Resource-Constrained Settings Internationally: Insights for Implementation, Research, and Policy for First Nations in Canada
by Jessica Annan, Rita Henderson, Mandi Gray, Rhonda Gail Clark, Chris Sarin and Kerry Black
Int. J. Environ. Res. Public Health 2024, 21(11), 1429; https://doi.org/10.3390/ijerph21111429 - 28 Oct 2024
Viewed by 815
Abstract
Wastewater-based epidemiology (WBE) is regarded as a support tool for detecting and assessing the prevalence of infectious diseases at a population level. For rural, remote, and resource-constrained communities with little access to other public health monitoring tools, WBE can be a low-cost approach [...] Read more.
Wastewater-based epidemiology (WBE) is regarded as a support tool for detecting and assessing the prevalence of infectious diseases at a population level. For rural, remote, and resource-constrained communities with little access to other public health monitoring tools, WBE can be a low-cost approach to filling gaps in population health knowledge to inform public health risk assessment and decision-making. This rapid review explores and discusses unique considerations of WBE in key settings, with a focus on the detection of the SARS-CoV-2 virus, which has rapidly expanded WBE infrastructure globally. To frame our understanding of possibilities for WBE with First Nations in Alberta, we address the following questions: What are the unique considerations and challenges for WBE under similar contexts in rural, remote, or resource-constrained settings? What are the resources and expertise required to support WBE? This review identifies several unique considerations for WBE in rural, remote, and resource-constrained communities, including costs, accessibility, operator capacity, wastewater infrastructure, and data mobilization—highlighting the need for equity in WBE. In summary, most resource-constrained communities require additional support from external research and/or governmental bodies to undertake WBE. Full article
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<p>PRISMA Chart.</p>
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26 pages, 6242 KiB  
Article
Wireless Sensor Node for Chemical Agent Detection
by Zabdiel Brito-Brito, Jesús Salvador Velázquez-González, Fermín Mira, Antonio Román-Villarroel, Xavier Artiga, Satyendra Kumar Mishra, Francisco Vázquez-Gallego, Jung-Mu Kim, Eduardo Fontana, Marcos Tavares de Melo and Ignacio Llamas-Garro
Chemosensors 2024, 12(9), 185; https://doi.org/10.3390/chemosensors12090185 - 11 Sep 2024
Viewed by 806
Abstract
In this manuscript, we present in detail the design and implementation of the hardware and software to produce a standalone wireless sensor node, called SensorQ system, for the detection of a toxic chemical agent. The proposed wireless sensor node prototype is composed of [...] Read more.
In this manuscript, we present in detail the design and implementation of the hardware and software to produce a standalone wireless sensor node, called SensorQ system, for the detection of a toxic chemical agent. The proposed wireless sensor node prototype is composed of a micro-controller unit (MCU), a radio frequency (RF) transceiver, a dual-band antenna, a rechargeable battery, a voltage regulator, and four integrated sensing devices, all of them integrated in a package with final dimensions and weight of 200 × 80 × 60 mm and 0.422 kg, respectively. The proposed SensorQ prototype operates using the Long-Range (LoRa) wireless communication protocol at 2.4 GHz, with a sensor head implemented on a hetero-core fiber optic structure supporting the surface plasmon resonance (SPR) phenomenon with a sensing section (L = 10 mm) coated with titanium/gold/titanium and a chemically sensitive material (zinc oxide) for the detection of Di-Methyl Methyl Phosphonate (DMMP) vapor in the air, a simulant of the toxic nerve agent Sarin. The transmitted spectra with respect to different concentrations of DMMP vapor in the air were recorded, and then the transmitted power for these concentrations was calculated at a wavelength of 750 nm. The experimental results indicate the feasibility of detecting DMMP vapor in air using the proposed optical sensor head, with DMMP concentrations in the air of 10, 150, and 150 ppm in this proof of concept. We expect that the sensor and wireless sensor node presented herein are promising candidates for integration into a wireless sensor network (WSN) for chemical warfare agent (CWA) detection and contaminated site monitoring without exposure of armed forces. Full article
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<p>Hardware architecture of the proposed wireless sensor node.</p>
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<p>Wireless sensor node: (<b>a</b>) 3D model isometric (top/front/left) view, (<b>b</b>) 3D model lateral view, and (<b>c</b>) integrated and packaged wireless sensor node prototype.</p>
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<p>Architecture of the SensorQ system. Showing deployed wireless sensor nodes at the bottom of the figure connected to the communications gateway mounted on UAVs. The communications gateway makes data available to the end user through the MQTT protocol and 4G/5G wireless communications links.</p>
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<p>Wireless sensor node electronics: (<b>a</b>) communications side view and (<b>b</b>) sensors side view. A description of each part according to enclosed numbers is provided in <a href="#chemosensors-12-00185-t003" class="html-table">Table 3</a>.</p>
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<p>Wireless sensor node antenna: (<b>a</b>) top view, showing the stacked dual band antenna setup and (<b>b</b>) bottom view showing interconnections and power divider network.</p>
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<p>Gateway electronics. A description of each part according to enclosed numbers is provided in <a href="#chemosensors-12-00185-t005" class="html-table">Table 5</a>.</p>
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<p>Graphical representation of the proposed sensor probe supporting the SPR effect with stacked material layers deposited on the SMF: longitudinal optical fiber section (left) and optical fiber cross sections (right).</p>
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<p>Representation of data collection by the WSN composed of one gateway and three sensor nodes operating under low-power listening mode.</p>
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<p>Representation of the frame-slotted ALOHA’s (FSA) time organization whilst the gateway is collecting data from each sensor node into a defined sequence of frames (top), slot representation (bottom).</p>
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<p>Wireless sensor node software architecture based on four inter-related layers (L1–L4): L1 is for the Hardware Abstraction Layer, L2 is for the Real-Time Operating System, L3 is for the drivers to access other devices, and L4 is for the Application Layer.</p>
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<p>Wireless software architecture of the gateway based on four inter-related layers (L1–L4): L1 is for the interface with different peripherals, L2 is for the Raspbian operating system of the Raspberry Pi, L3 is for the MQT client, a GNSS receiver, and a Lora radio transceiver driver, and L4 is for the parallel running tasks.</p>
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<p>Screenshot of the configuration dashboard, which allows for the manipulation of several parameters regarding the experiment, the MAC layer, the PHY layer, and the commands sections.</p>
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<p>Deployment of different data collected in the measurements dashboard (screenshot), such as environmental conditions (gas concentration and temperature), the status (RSSI, acceleration, and battery level), and the location (GPS position and altitude) from two sensor node prototypes.</p>
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<p>Average data collection time depending on (<b>a</b>) the number of slots per frame for a given number of sensor nodes (each node sends 1 data packet of 22 bytes), (<b>b</b>) the number of sensor nodes for a given number of slots per frame (each node sends 1 data packet of 22 bytes), and (<b>c</b>) the number of slots per frame for a given number of sensor nodes (each node sends 10 data packets of 22 bytes or 1 data packet of 220 bytes). All results are presented for SF-6. (<b>a</b>) Data collection time over number of slots (single packet of 22 bytes), (<b>b</b>) data collection time over number of sensor nodes (single packet of 22 bytes), and (<b>c</b>) data collection time over number of slots (10 packets of 22 bytes or 1 packet of 220 bytes).</p>
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<p>Sensor head experimental setup based on the optical fiber hetero-core structure coated with Ti/Au/Ti/ZnO.</p>
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<p>Normalized transmitted intensity for different concentrations of DMMP mixed in the air and interaction with our proposed sensing probe (dots: measured data; dashed line: trend).</p>
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30 pages, 9351 KiB  
Article
Achieving Financial Feasibility and Carbon Emission Reduction: Retrofit of a Bangkok Shopping Mall Using Calibrated Simulation
by Kongkun Charoenvisal, Atch Sreshthaputra and Sarin Pinich
Buildings 2024, 14(8), 2512; https://doi.org/10.3390/buildings14082512 - 15 Aug 2024
Viewed by 1365
Abstract
This study investigated the building energy retrofit potential of a shopping mall in Bangkok, Thailand, using a combined building energy modeling and economic analysis approach to achieve a balance between carbon emission reduction and financial feasibility. The study adopted ASHRAE Guideline 14, a [...] Read more.
This study investigated the building energy retrofit potential of a shopping mall in Bangkok, Thailand, using a combined building energy modeling and economic analysis approach to achieve a balance between carbon emission reduction and financial feasibility. The study adopted ASHRAE Guideline 14, a standard for energy modeling accuracy, using whole-building calibrated simulation to evaluate the energy, energy cost, and operational carbon emission reduction achievable through the proposed energy conservation measures. The calibrated model demonstrated high accuracy, achieving an NMBE of 1.10% and CVRMSE of 3.77% for energy consumption, and NMBE of 0.15% and CVRMSE of 5.44% for peak energy demand compared to the monthly data. The economic analysis employed indicators such as NS, AIRR, and DPB, along with MACC analysis, to assess the financial viability of the ECMs and examine the impact of carbon credit cost savings on the analysis results. This case study highlights the critical role of energy modeling and economic analysis in evaluating building retrofits. The findings demonstrate the potential for carbon emission reduction and financial benefits with the case study building achieving up to 12.5% energy cost savings and carbon emission reduction based on a prospective building lifespan of 40 years without compromising financial sustainability. Full article
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<p>Simplified building configuration of the case study shopping mall.</p>
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<p>Actual building energy consumption by end use.</p>
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<p>A comparison of actual and simulated monthly electricity consumption data with an NMBE of 0.15% and CVRMSE 0f 5.44%.</p>
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<p>A comparison of actual and simulated monthly peak electricity demand data with an NMBE of 0.15% and CVRMSE of 5.44%.</p>
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<p>Reference model energy consumption by end use.</p>
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<p>Net savings (NS) results.</p>
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<p>Adjusted internal rate of return (AIRR) results.</p>
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<p>Discounted payback (DPB) results.</p>
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<p>MACC analysis results based on a 1% escalation rate.</p>
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<p>MACC analysis results based on a 2% escalation rate.</p>
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<p>MACC analysis results based on a 3% escalation rate.</p>
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<p>The results of a sensitivity analysis of economic indicators for ECM09 under different carbon credit cost scenarios: (<b>a</b>) NS results, (<b>b</b>) AIRR results, and (<b>c</b>) DPB results.</p>
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28 pages, 11202 KiB  
Article
Anti-Inflammatory Activities of Yataprasen Thai Traditional Formulary and Its Active Compounds, Beta-Amyrin and Stigmasterol, in RAW264.7 and THP-1 Cells
by Jaenjira Angsusing, Sudarshan Singh, Weerasak Samee, Sarin Tadtong, Leanne Stokes, Maria O’Connell, Hanna Bielecka, Nopparut Toolmal, Supachoke Mangmool and Chuda Chittasupho
Pharmaceuticals 2024, 17(8), 1018; https://doi.org/10.3390/ph17081018 - 1 Aug 2024
Viewed by 2050
Abstract
Yataprasen (YTPS) remedy formulary, a national Thai traditional medicine formulary, comprises 13 herbal plants. It has been extensively prescribed to relieve osteoarthritis and musculoskeletal pain in the Thai traditional medicine healthcare system. The aim of this study was to investigate the antioxidant and [...] Read more.
Yataprasen (YTPS) remedy formulary, a national Thai traditional medicine formulary, comprises 13 herbal plants. It has been extensively prescribed to relieve osteoarthritis and musculoskeletal pain in the Thai traditional medicine healthcare system. The aim of this study was to investigate the antioxidant and anti-inflammatory properties of the bioactive compounds (β-amyrin and stigmasterol) of YTPS remedy formulary ethanolic extract, along with its composition. The YTPS formulary extract contains 70.30 nM of β-amyrin and 605.76 nM of stigmasterol. The YTPS formulary extract exhibited ABTS and DPPH free radical scavenging activity, with IC50 values of 144.50 ± 2.82 and 31.85 ± 0.18 µg/mL, respectively. The ethanolic extract of YTPS at a concentration of 1000 µg/mL showed a significant (p < 0.01) anti-inflammatory effect, mainly by reducing IL-6 and TNF-α release in response to LPS. NO production was prominently lowered by 50% at 24.76 ± 1.48 µg/mL, 55.52 ± 24.40 µM, and more than 570 µM of YTPS formulary extract, β-amyrin, and stigmasterol, respectively. Major components of YTPS, β-amyrin, and stigmasterol exerted significant anti-inflammatory effects by inhibiting LPS-induced IL-1β, IL-6, TNF-α secretion in THP-1 cells. Our findings suggest that the ethanolic extract from YTPS holds promise as an alternative topical treatment for osteoarthritis and inflammatory disorders, potentially with fewer side effects than non-steroidal anti-inflammatory medications (NSAIDs). Full article
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<p>(<b>A</b>) ABTS radical scavenging activity; (<b>B</b>) DPPH radical scavenging activity; (<b>C</b>) FRAP value of ascorbic acid (3.91–2000 µg/mL), gallic acid (3.91–2000 µg/mL), quercetin (3.91–2000 µg/mL), Trolox<sup>®</sup> (3.91–2000 µg/mL), YTPS formulary extract (9.77–5000 µg/mL), individual extract in the formulary (9.77–5000 µg/mL), β-amyrin (4.57–234 µM), and stigmasterol (4.73–242 µM).</p>
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<p>Effect of (<b>A</b>) YTPS formulary extract (1–300 µg/mL), (<b>B</b>) β-amyrin (1.8–230 µM), and (<b>C</b>) stigmasterol (1.88–240 µM) on the cell viability of RAW264.7 cells using MTT assay. Cells were seeded in 96-well plates at a density of 1 × 10<sup>4</sup> cells/well. The cells were then treated with YTPS formulary extract and incubated for 24 h before conducting the MTT assay. Data are expressed as a percentage of control (media only). Data were gathered from three separate experiments; each was conducted with triplicate wells (n = 3). The descriptive statistics were analyzed by GraphPad Prism version 9.5.0.</p>
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<p>Effect of (<b>A</b>) β-amyrin (1.8–230 µM), (<b>B</b>) stigmasterol (1.88–240 µM), (<b>C</b>) YTPS formulary extract (11.72–1500 µg/mL), and (<b>D</b>–<b>O</b>) individual extract (10–100 µg/mL) on the cell viability of THP-1 cells. Cell viability was assessed using an Alamar blue metabolic assay by treating the cells with Resazurin dye solution for 4 h after 24 h of incubation of the samples. Alamar blue fluorescence was measured, and data are expressed as a percentage of control (media only). Supernatants were collected and measured with FlexStation<sup>®</sup> 3 at 570 nm excitation and 600 nm emission using SoftMax Pro 5.4.6 software for data recording and analysis. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The descriptive statistics were analyzed by GraphPad Prism version 9.5.0.</p>
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<p>Effect of (<b>A</b>) β-amyrin (1.8–230 µM), (<b>B</b>) stigmasterol (1.88–240 µM), (<b>C</b>) YTPS formulary extract (11.72–1500 µg/mL), and (<b>D</b>–<b>O</b>) individual extract (10–100 µg/mL) on the cell viability of THP-1 cells. Cell viability was assessed using an Alamar blue metabolic assay by treating the cells with Resazurin dye solution for 4 h after 24 h of incubation of the samples. Alamar blue fluorescence was measured, and data are expressed as a percentage of control (media only). Supernatants were collected and measured with FlexStation<sup>®</sup> 3 at 570 nm excitation and 600 nm emission using SoftMax Pro 5.4.6 software for data recording and analysis. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The descriptive statistics were analyzed by GraphPad Prism version 9.5.0.</p>
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<p>Effects of (<b>A</b>) YTPS formulary extract (1.95–250 µg/mL), (<b>B</b>) β-amyrin (1.8–230 µM), and (<b>C</b>) stigmasterol (1.88–240 µM) on NO release from LPS-treated RAW264.7 cells. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The data were analyzed by GraphPad Prism version 9.5.0 with one-way ANOVA and Dunnett’s multiple comparisons test (statistical significance denoted by <span class="html-italic">p</span> &lt; 0.05 compared with cells treated with LPS).</p>
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<p>Effects of (<b>A</b>) β-amyrin (20 µM), (<b>B</b>) stigmasterol (20 µM), (<b>C</b>) YTPS formulary extract (1000 µg/mL), and (<b>D</b>–<b>O</b>) individual extract (50 or 100 µg/mL) tested at non-toxic concentrations on IL-1β release in THP-1 cells. Cells were treated at different concentrations of treatments for 3 h and stimulated with LPS for 24 h. The supernatants were collected and ELISA was performed according to the manufacturer’s protocol. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The data were analyzed by GraphPad Prism version 9.5.0 with one-way ANOVA and Dunnett’s multiple comparisons test (statistical significance denoted **** <span class="html-italic">p</span> &lt; 0.0001 compared with cells treated with LPS).</p>
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<p>Effects of (<b>A</b>) β-amyrin (20 µM), (<b>B</b>) stigmasterol (20 µM), (<b>C</b>) YTPS formulary extract (1000 µg/mL), and (<b>D</b>–<b>O</b>) individual extracts (50 or 100 µg/mL) tested at non-toxic concentrations on the IL-6 release in THP-1 cells. Cells were treated at different concentrations of treatments for 3 h and stimulated with LPS for 24 h. The supernatants were collected and ELISA was performed according to the manufacturer’s protocol. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The data were analyzed by GraphPad Prism version 9.5.0 with one-way ANOVA and Dunnett’s multiple comparisons test (statistical significance denoted by **** <span class="html-italic">p</span> &lt; 0.0001, compared with cells treated with LPS).</p>
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<p>Effects of (<b>A</b>) β-amyrin (20 µM), (<b>B</b>) stigmasterol (20 µM), (<b>C</b>) YTPS formulary extract (1000 µg/mL), and (<b>D</b>–<b>O</b>) individual extracts (50 or 100 µg/mL) tested at non-toxic concentrations on the TNF-α release in THP-1 cells. Cells were treated at different concentrations of treatments for 3 h and stimulated with LPS for 24 h. The supernatants were collected and ELISA was performed according to the manufacturer’s protocol. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The data were analyzed by GraphPad Prism version 9.5.0 with one-way ANOVA and Dunnett’s multiple comparisons test (statistical significance denoted by **** <span class="html-italic">p</span> &lt; 0.0001 compared with cells treated with LPS).</p>
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<p>Effect of the YTPS formulary extract (3.91–500 µg/mL) on the cell viability of HaCaT cells. Cells were treated at different concentrations for 24 h and treated with MTT reagent for 2 h, and data are expressed as a percentage of control (media only). Data were gathered from three separate experiments; each conducted with triplicate wells (n = 3). The descriptive statistics were analyzed by GraphPad Prism version 9.5.0.</p>
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<p>Effects of (<b>A</b>) β-amyrin (1–20 µM), (<b>B</b>) stigmasterol (1–20 µM), (<b>C</b>) YTPS formulary extract (1–300 µg/mL), and (<b>D</b>–<b>N</b>) individual extracts (10–100 µg/mL) on the cell viability of HEK293 cells. Cell viability was assessed using an Alamar blue metabolic assay by treating the cells with Resazurin dye solution for 4 h after 24 h of incubation of samples. Alamar blue fluorescence was measured, and data are expressed as a percentage of control (media only). Supernatants were collected and measured with FlexStation<sup>®</sup> 3 at 570 nm excitation and 600 nm emission using SoftMax Pro 5.4.6 software for data recording and analysis. Data were gathered from three separate experiments, each conducted in triplicate (n = 3). The descriptive statistics were analyzed by GraphPad Prism version 9.5.0.</p>
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<p>The extraction process of YTPS formulary and individual plant ingredients. The dried powder (300 g) was macerated with 95% ethanol (3000 mL), filtered, and evaporated until dry.</p>
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14 pages, 3283 KiB  
Article
Preclinical Evaluation of a Novel Series of Polyfluorinated Thalidomide Analogs in Drug-Resistant Multiple Myeloma
by Blaire E. Barton, Matthew K. Collins, Cindy H. Chau, Hyoyoung Choo-Wosoba, David J. Venzon, Christian Steinebach, Kathleen M. Garchitorena, Bhruga Shah, Eric L. Sarin, Michael Gütschow and William D. Figg
Biomolecules 2024, 14(6), 725; https://doi.org/10.3390/biom14060725 - 19 Jun 2024
Viewed by 1210
Abstract
Immunomodulatory imide drugs (IMiDs) play a crucial role in the treatment landscape across various stages of multiple myeloma. Despite their evident efficacy, some patients may exhibit primary resistance to IMiD therapy, and acquired resistance commonly arises over time leading to inevitable relapse. It [...] Read more.
Immunomodulatory imide drugs (IMiDs) play a crucial role in the treatment landscape across various stages of multiple myeloma. Despite their evident efficacy, some patients may exhibit primary resistance to IMiD therapy, and acquired resistance commonly arises over time leading to inevitable relapse. It is critical to develop novel therapeutic options to add to the treatment arsenal to overcome IMiD resistance. We designed, synthesized, and screened a new class of polyfluorinated thalidomide analogs and investigated their anti-cancer, anti-angiogenic, and anti-inflammatory activity using in vitro and ex vivo biological assays. We identified four lead compounds that exhibit potent anti-myeloma, anti-angiogenic, anti-inflammatory properties using three-dimensional tumor spheroid models, in vitro tube formation, and ex vivo human saphenous vein angiogenesis assays, as well as the THP-1 inflammatory assay. Western blot analyses investigating the expression of proteins downstream of cereblon (CRBN) reveal that Gu1215, our primary lead candidate, exerts its activity through a CRBN-independent mechanism. Our findings demonstrate that the lead compound Gu1215 is a promising candidate for further preclinical development to overcome intrinsic and acquired IMiD resistance in multiple myeloma. Full article
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<p>Structures of thalidomide, the first-generation thalidomide analog CPS49, and the thalidomide analogs Gu1210, Gu1213, Gu1214, and Gu1215.</p>
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<p>Treatment with Gu compounds inhibited multiple myeloma cell proliferation in vitro. (<b>A</b>) The RPMI-8226 cell proliferation during 72-h treatment was most potently inhibited by Gu1210, Gu1213, Gu1214, and Gu1215 at a concentration of 10 μM compared to that of the vehicle control (0.5% DMSO). (<b>B</b>) The JJN3 cell proliferation during 24-h treatment was most potently inhibited by Gu1210, Gu1213, Gu1214, and Gu1215 at a concentration of 10 μM compared to that of the vehicle control (0.5% DMSO). (<b>C</b>) The 72-h treatment of RPMI-8226 cells with four lead analogs at a range of concentrations (0.1 μM–20 μM) showed a dose-dependent response, with Gu1215 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>D</b>) The 24-h treatment of JJN3 cells with the four lead analogs at a range of concentrations (0.1 μM–20 μM) showed a dose-dependent response, with Gu1215 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001). Note: a 5 µM dose represents two independent replicates. (<b>E</b>) The 72-h treatment of MOLP-8 cells with the four lead analogs at a range of concentrations (0.1 μM–10 μM) showed dose-dependent responses, with Gu1214 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001). These data are representative of at least three independent experiments with at least three replicates per experiment. In all the figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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<p>Treatment with Gu compounds inhibited multiple myeloma cell proliferation in vitro. (<b>A</b>) The 72-h treatment of MM1/R10R cells with the four lead analogs at a range of concentrations (0.1 μM–10 μM) showed a dose-dependent response, with Gu1215 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>B</b>) The 72-h treatment of MM1.S cells with the four lead analogs at a range of concentrations (0.1 μM–10 μM) showed a dose-dependent response, with Gu1210 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>C</b>) The 72-h treatment of U266/R10R cells with the four lead analogs at a range of concentrations (0.05 μM–10 μM) showed a dose-dependent response, with Gu1210 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001). (<b>D</b>) The 72-h treatment of U266 cells with the four lead analogs at a range of concentrations (0.05 μM–10 μM) showed a dose-dependent response, with Gu1215 exhibiting the most potent inhibition (**** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001). These data are representative of at least three independent experiments with at least three replicates per experiment. In all the figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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<p>Treatment with Gu compounds reduced spheroid growth in vitro. (<b>A</b>) The 72-h treatment of MOLP-8 spheroids at a range of concentrations (0.1–10 µM) produced a dose-dependent response, with Gu1213 exhibiting the most potent inhibition of spheroid growth (**** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001). (<b>B</b>) The 72-h treatment of RPMI-8226 spheroids with the lead compounds at a range of concentrations (0.1–10 µM) also produced a dose-dependent response, with Gu1215 exhibiting the most potent inhibition of spheroid growth (**** <span class="html-italic">p</span> &lt; 0.0001). (<b>C</b>,<b>D</b>) Representative images of the spheroids are shown. These data represent at least three independent experiments performed in triplicates. In all figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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<p>In vitro testing was conducted to assess Gu compounds’ effects on the inflammatory response in THP-1 cells, using a TNF-α ELISA. The vehicle control was 0.5% DMSO, while thalidomide (200 μM) was used as a comparator. THP-1 cells were exposed to the vehicle alone or with the compound for 1 h prior to the 3-h LPS challenge (1 ng/mL). Treatment of THP-1 cells with the four lead analogs (Gu1210, Gu1213, Gu1214, and Gu1215) at a range of concentrations (250 nM–750 nM) demonstrated a dose-dependent inhibition of TNF-α expression, with Gu1215 being the most potent inhibitor (**** <span class="html-italic">p</span> &lt; 0.0001). The results shown are representative of at least three independent experiments with at least three replicates per experiment. In all the figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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<p>In vitro screening was conducted in an endothelial tube formation angiogenesis assay. The vehicle control was 0.5% DMSO, CPS49 (30 μM) was used as a positive control, and thalidomide (100 μM) was used as a comparator. (<b>A</b>) The graph represents the mean area of lattice formation relative to the vehicle control. The treatment of HUVEC cells with four lead analogs (Gu1210, Gu1213, Gu1214, and Gu1215) at a range of concentrations (1 µM–10 µM) showed a dose-dependent inhibition of tube formation (**** <span class="html-italic">p</span> &lt; 0.0001, * <span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) Representative images of the tube formation assay. Results shown are representative of at least three independent experiments with at least three replicates per experiment. In all figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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<p>Ex vivo testing of thalidomide analogs in the human saphenous vein model of angiogenesis. The vehicle control was 0.5% DMSO, TNP-470 (50 µM) was used as a positive control, and thalidomide (100 µM) was included as a comparator. (<b>A</b>) The graph represents the mean area of microvessel outgrowth relative to the vehicle control. Fourteen-day incubation of human saphenous vein rings with Gu1213 and Gu1215 at 10 µM and 25 µM demonstrated a dose-dependent response, with Gu1215 exhibiting the most potent angiogenesis inhibition (*** <span class="html-italic">p</span> &lt; 0.005, ** <span class="html-italic">p</span> &lt; 0.01). (<b>B</b>) Representative images of human saphenous vein rings treated with the vehicle control, 50 µM TNP-470, 100 µM Thalidomide, or the indicated thalidomide analog at 10 M for 14 days. The results shown are representative of at least two independent experiments with at least two rings per experiment. In all figures, any cases in which the concentrations are grouped in a bracket indicate that all of the values within the bracket are individually statistically different from the vehicle control.</p>
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37 pages, 9291 KiB  
Article
New Heterostilbene and Triazole Oximes as Potential CNS-Active and Cholinesterase-Targeted Therapeutics
by Milena Mlakić, Tena Čadež, Goran Šinko, Irena Škorić and Zrinka Kovarik
Biomolecules 2024, 14(6), 679; https://doi.org/10.3390/biom14060679 - 11 Jun 2024
Viewed by 1167
Abstract
New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite [...] Read more.
New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite the development of a large number of oxime compounds that should have the capacity to reactivate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The activity of these two enzymes, crucial for neurotransmission, is blocked by OP, which has the consequence of disturbing normal cholinergic nerve signal transduction in the peripheral and CNS, leading to a cholinergic crisis. The oximes in use have one or two pyridinium rings and cross the brain–blood barrier poorly due to the quaternary nitrogen. Following our recent study on 2-thienostilbene oximes, in this paper, we described the synthesis of 63 heterostilbene derivatives, of which 26 oximes were tested as inhibitors and reactivators of AChE and BChE inhibited by OP nerve agents–sarin and cyclosarin. While the majority of oximes were potent inhibitors of both enzymes in the micromolar range, we identified several oximes as BChE or AChE selective inhibitors with the potential for drug development. Furthermore, the oximes were poor reactivators of AChE; four heterocyclic derivatives reactivated cyclosarin-inhibited BChE up to 70%, and cis,trans-5 [2-((Z)-2-(5-((E)-(hydroxyimino)methyl)thiophen-2-yl)vinyl)benzonitrile] had a reactivation efficacy comparable to the standard oxime HI-6. In silico analysis and molecular docking studies, including molecular dynamics simulation, connected kinetic data to the structural features of these oximes and confirmed their productive interactions with the active site of cyclosarin-inhibited BChE. Based on inhibition and reactivation and their ADMET properties regarding lipophilicity, CNS activity, and hepatotoxicity, these compounds could be considered for further development of CNS-active reactivators in OP poisoning as well as cholinesterase-targeted therapeutics in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Full article
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<p>The general structures of the new oxime derivatives: triazole oximes (<b>A</b>), heterostilbene oximes with the oxime group directly attached to the furan or thiophene ring (<b>B</b>,<b>C</b>), diheterostilbene oximes (<b>D</b>) and diheterostilbene oximes with one triazole ring (<b>E</b>).</p>
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<p>New triazole oximes <b>1</b>–<b>3</b> are the only non-stilbene type oximes with the oxime group directly attached to the triazole ring.</p>
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<p>New oxime derivatives <b>4</b>–<b>21</b> with a heterostilbene skeleton.</p>
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<p>(<b>A</b>) Physico-chemical properties—molecular weight (MW), number of hydrogen bond donors and acceptors (HBDs and HBAs), number of rotating bonds (RBs), lipophilicity (AlogP), and polar surface area (PSA), of uncharged oximes in relation to the recommended values (red line). (<b>B</b>) Correlation plot for prediction of BBB penetration. The recommended values of CNS-active drugs are given by Pajouhesh and Lenz [<a href="#B23-biomolecules-14-00679" class="html-bibr">23</a>].</p>
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<p>Conformation of the complex between native human BChE and <span class="html-italic">trans,anti</span>-<b>4</b> (<b>A</b>) and <span class="html-italic">cis,syn</span>-<b>5</b> (<b>B</b>). Interactions with amino acid residues are represented as dashed lines: hydrophobic (purple), hydrogen bonds (green), and electrostatic (orange). Crystal structure of human BChE was used (PDB code 2PM8) [<a href="#B44-biomolecules-14-00679" class="html-bibr">44</a>].</p>
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<p>Reactivation of cyclosarin-inhibited BChE by 100 µM oximes in terms of the reactivation rate constants, <span class="html-italic">k</span><sub>obs</sub> (grey columns), maximal reactivation in 240 min (purple columns), and inhibition of BChE by oximes (green columns). The presented values are means of at least two experiments measured at 25 °C.</p>
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<p>Minimized conformation of a complex in near-attack conformation between cyclosarin-inhibited BChE and <span class="html-italic">cis,syn</span>-<b>5</b> (<b>A</b>) and <span class="html-italic">cis,syn</span>-<b>16</b> (<b>B</b>). The Van der Waals distance of the oxime group from the oxygen (Oγ) of catalytic serine (Ser198) is red. Oxime interactions with active site residues are represented as dashed lines: hydrophobic (purple and yellow) and hydrogen bonds (green). The crystal structure of human BChE inhibited by tabun (PDB code 3DJY) [<a href="#B60-biomolecules-14-00679" class="html-bibr">60</a>] was used for the modeling of cyclosarin-inhibited BChE [<a href="#B58-biomolecules-14-00679" class="html-bibr">58</a>].</p>
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<p>New uncharged heterocyclic oximes are potent human AChE (grey) and human BChE (blue) inhibitors and reactivators of human BChE that are inhibited by cyclosarin (yellow).</p>
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<p>First synthetic steps towards uncharged oximes <b>4</b>–<b>21</b>.</p>
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<p>Vilsmeier formylation of new heterostilbenes and their conversion to uncharged oximes <b>4</b>–<b>9</b>.</p>
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<p>Synthetic pathway of new uncharged oximes <b>10</b>–<b>13</b> (R = CH<sub>3</sub>, OCH<sub>3</sub>, Cl, CN, N(CH<sub>3</sub>)<sub>2</sub>).</p>
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<p>Synthetic route of new uncharged oximes <b>14</b>–<b>21</b> (R = CH<sub>3</sub>, OCH<sub>3</sub>, Cl, CN; X = O, S).</p>
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11 pages, 1959 KiB  
Article
Cholesterol Oxime Olesoxime Assessed as a Potential Ligand of Human Cholinesterases
by Dora Kolić, Goran Šinko, Ludovic Jean, Mourad Chioua, José Dias, José Marco-Contelles and Zrinka Kovarik
Biomolecules 2024, 14(5), 588; https://doi.org/10.3390/biom14050588 - 15 May 2024
Viewed by 1044
Abstract
Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood–brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase [...] Read more.
Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood–brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10–30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes. Full article
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<p>Chemical structures of olesoxime, standard oxime reactivators of AChE—pralidoxime (2-PAM) and asoxime (HI-6), and organophosphate compounds (tabun, sarin, cyclosarin, VX, and paraoxon).</p>
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<p>The inhibition profile and estimated binding affinities of AChE and BChE for olesoxime in terms of IC<sub>50</sub> (±SEM) values measured at 25 °C. Total inhibition was not achieved due to olesoxime’s low solubility in buffer solution at concentrations higher than 100 µM.</p>
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<p>Complex between native human AChE (<b>A</b>) and BChE (<b>B</b>) with olesoxime. Interactions with amino acid residues are represented as dashed lines: hydrophobic (purple) and hydrogen bond (green). Crystal structure of human AChE (PDB code 4PQE) [<a href="#B25-biomolecules-14-00588" class="html-bibr">25</a>] and human BChE was used (PDB code 2PM8) [<a href="#B26-biomolecules-14-00588" class="html-bibr">26</a>].</p>
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<p>Reactivation screening of nerve agent-inhibited BChE by 0.1 mM olesoxime (±SEM) expressed in terms of the observed reactivation rate constant (<span class="html-italic">k</span><sub>obs</sub>) and maximum reactivation percentage (React<sub>max</sub>) determined at 25 °C within 24 h (n = 2).</p>
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<p>Kinetics of olesoxime-assisted reactivation of cyclosarin-inhibited BChE (±SEM) determined at 25 °C within 4 h (n = 2).</p>
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<p>Combined reactivation capability of 0.1 or 1.0 mM oxime 2-PAM with or without 50 µM olesoxime (OLE) on sarin- and cyclosarin-BChE conjugates (±SEM). The observed reactivation rate constant (<span class="html-italic">k</span><sub>obs</sub>) and maximum reactivation percentage (React<sub>max</sub>) were determined at 25 °C within 24 h (n = 2).</p>
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14 pages, 6774 KiB  
Article
Effects of the ZrO2 Crystalline Phase and Morphology on the Thermocatalytic Decomposition of Dimethyl Methylphosphonate
by Xuwei Wang, Peng Sun, Ziwang Zhao, Yimeng Liu, Shuyuan Zhou, Piaoping Yang and Yanchun Dong
Nanomaterials 2024, 14(7), 611; https://doi.org/10.3390/nano14070611 - 30 Mar 2024
Viewed by 1096
Abstract
Thermocatalytic decomposition is an efficient purification technology that is potentially applicable to degrading chemical warfare agents and industrial toxic gases. In particular, ZrO2 has attracted attention as a catalyst for the thermocatalytic decomposition of dimethyl methylphosphonate (DMMP), which is a simulant of [...] Read more.
Thermocatalytic decomposition is an efficient purification technology that is potentially applicable to degrading chemical warfare agents and industrial toxic gases. In particular, ZrO2 has attracted attention as a catalyst for the thermocatalytic decomposition of dimethyl methylphosphonate (DMMP), which is a simulant of the nerve gas sarin. However, the influence of the crystal phase and morphology on the catalytic performance of ZrO2 requires further exploration. In this study, monoclinic- and tetragonal-phase ZrO2 (m- and t-ZrO2, respectively) with nanoparticle, flower-like shape and hollow microsphere morphologies were prepared via hydrothermal and solvothermal methods, and their thermocatalytic decomposition of DMMP was systematically investigated. For a given morphology, m-ZrO2 performed better than t-ZrO2. For a given crystalline phase, the morphology of hollow microspheres resulted in the longest protection time. The exhaust gases generated by the thermocatalytic decomposition of DMMP mainly comprised H2, CO2, H2O and CH3OH, and the by-products were phosphorus oxide species. Thus, the deactivation of ZrO2 was attributed to the deposition of these phosphorous oxide species on the catalyst surface. These results are expected to help guide the development of catalysts for the safe disposal of chemical warfare agents. Full article
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<p>Schematic of the custom-built apparatus for evaluation of the thermocatalytic decomposition performance.</p>
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<p>XRD spectra of ZrO<sub>2</sub> catalysts: m-ZrO<sub>2</sub> (<b>a</b>) nanoparticles, (<b>c</b>) flower-like shapes and (<b>e</b>) hollow microspheres; t-ZrO<sub>2</sub> (<b>b</b>) nanoparticles, (<b>d</b>) flower-like shapes and (<b>f</b>) hollow microspheres.</p>
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<p>TEM and HRTEM images of ZrO<sub>2</sub> catalysts: (<b>a</b>) m-ZrO<sub>2</sub> nanoparticles and (<b>b</b>) t-ZrO<sub>2</sub> nanoparticles; SEM and HRTEM images of ZrO<sub>2</sub> catalysts: (<b>c</b>) m-ZrO<sub>2</sub> with flower-like shapes, (<b>d</b>) t-ZrO<sub>2</sub> with flower-like shapes, (<b>e</b>) m-ZrO<sub>2</sub> with hollow microspheres, and (<b>f</b>) t-ZrO<sub>2</sub> with hollow microspheres.</p>
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<p>DMMP conversion rates over time of the six ZrO<sub>2</sub> catalysts.</p>
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<p>H<sub>2</sub>-TPR curves of the m-ZrO<sub>2</sub> catalysts with different morphologies.</p>
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<p>Mass spectrometry results for the exhaust gases from the thermocatalytic decomposition of DMMP by the m-ZrO<sub>2</sub> catalysts: (<b>a</b>) nanoparticles, (<b>b</b>) flower-like shapes, and (<b>c</b>) hollow microspheres (The pink line is the curve of the heating program).</p>
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<p>XPS spectra (P2p) of m-ZrO<sub>2</sub> (<b>a</b>) nanoparticles, (<b>b</b>) flower-like shapes, and (<b>c</b>) hollow microspheres.</p>
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<p>Proposed reaction mechanism for DMMP decomposition.</p>
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17 pages, 592 KiB  
Review
Metabolic Dysfunction-Associated Steatohepatitis and Progression to Hepatocellular Carcinoma: A Literature Review
by Haider Ghazanfar, Nismat Javed, Abeer Qasim, George Sarin Zacharia, Ali Ghazanfar, Abhilasha Jyala, Elona Shehi and Harish Patel
Cancers 2024, 16(6), 1214; https://doi.org/10.3390/cancers16061214 - 20 Mar 2024
Cited by 5 | Viewed by 2698
Abstract
The prevalence of metabolic-associated fatty liver disease (MAFLD) is increasing globally due to factors such as urbanization, obesity, poor nutrition, sedentary lifestyles, healthcare accessibility, diagnostic advancements, and genetic influences. Research on MAFLD and HCC risk factors, pathogenesis, and biomarkers has been conducted through [...] Read more.
The prevalence of metabolic-associated fatty liver disease (MAFLD) is increasing globally due to factors such as urbanization, obesity, poor nutrition, sedentary lifestyles, healthcare accessibility, diagnostic advancements, and genetic influences. Research on MAFLD and HCC risk factors, pathogenesis, and biomarkers has been conducted through a narrative review of relevant studies, with a focus on PubMed and Web of Science databases and exclusion criteria based on article availability and language. Steatosis marks the early stage of MASH advancement, commonly associated with factors of metabolic syndrome such as obesity and type 2 diabetes. Various mechanisms, including heightened lipolysis, hepatic lipogenesis, and consumption of high-calorie diets, contribute to the accumulation of lipids in the liver. Insulin resistance is pivotal in the development of steatosis, as it leads to the release of free fatty acids from adipose tissue. Natural compounds hold promise in regulating lipid metabolism and inflammation to combat these conditions. Liver fibrosis serves as a significant predictor of MASH progression and HCC development, underscoring the need to target fibrosis in treatment approaches. Risk factors for MASH-associated HCC encompass advanced liver fibrosis, older age, male gender, metabolic syndrome, genetic predispositions, and dietary habits, emphasizing the requirement for efficient surveillance and diagnostic measures. Considering these factors, it is important for further studies to determine the biochemical impact of these risk factors in order to establish targeted therapies that can prevent the development of HCC or reduce progression of MASH, indirectly decreasing the risk of HCC. Full article
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<p>Hypothetical model of a hepatocyte showing possible pathogenic mechanisms.</p>
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14 pages, 5693 KiB  
Article
Synthesis and Application of Polymer SXFA in the Detection of Organophosphine Agents with a SAW Sensor
by Cancan Yan, Molin Qin, Tengxiao Guo, Lin Zhang, Junchao Yang and Yong Pan
Polymers 2024, 16(6), 784; https://doi.org/10.3390/polym16060784 - 12 Mar 2024
Cited by 1 | Viewed by 1026
Abstract
The effective detection of isopropyl methylfluorophosphonate (GB, sarin), a type of organophosphine poisoning agent, is an urgent issue to address to maintain public safety. In this research, a gas-sensitive film material, poly (4-hydroxy-4,4-bis trifluoromethyl)-butyl-1-enyl)-siloxane (SXFA), with a structure of hexafluoroisopropyl (HFIP) functional group [...] Read more.
The effective detection of isopropyl methylfluorophosphonate (GB, sarin), a type of organophosphine poisoning agent, is an urgent issue to address to maintain public safety. In this research, a gas-sensitive film material, poly (4-hydroxy-4,4-bis trifluoromethyl)-butyl-1-enyl)-siloxane (SXFA), with a structure of hexafluoroisopropyl (HFIP) functional group was synthesized by using methyl vinylpropyl dichlorosilane and hexafluoroacetone trihydrate as initial materials. The synthesis process products were characterized using FTIR. SXFA was prepared on a 200 MHz shear surface wave delay line using the spin-coating method for GB detection. A detection limit of <0.1 mg/m3 was achieved through conditional experiments. Meanwhile, we also obtained a maximum response of 2.168 mV at a 0.1 mg/m3 concentration, indicating the much lower detection limit of the SAW-SXFA sensor. Additionally, a maximum response standard deviation of 0.11 mV with a coefficient of variation of 0.01 and a maximum recovery standard deviation of 0.22 mV with a coefficient of variation of 0.02 were also obtained through five repeated experiments. The results show that the SAW-SXFA sensor has strong selectivity and reproducibility, good selectivity, positive detection ability, high sensitivity, and fast alarm performance for sarin detection. Full article
(This article belongs to the Special Issue Polymer Materials in Sensors, Actuators and Energy Conversion II)
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<p>Equilibrium partitioning of vapor molecules between gas phase and polymer.</p>
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<p>Schematic of sensing mechanism.</p>
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<p>Synthesis route of SXFA.</p>
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<p>Schematic of SAW-SXFA sensor response mechanism.</p>
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<p>IR spectrum of methylallyldichlorosilane.</p>
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<p>IR spectrum of methylvinylpolysiloxane.</p>
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<p>FTIR spectrum of SXFA.</p>
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<p>SEM image of SAW sensor delay line of SXFA sensitive film.</p>
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<p>Response of SAW-SXFA sensor with concentration changes in GB and DMMP (19.6 °C, RH = 27%).</p>
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<p>Comparison of GB and DMMP at 2.7 mg/m<sup>3</sup> (17.9 °C, RH = 28%).</p>
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<p>Detection of GB (1.6 mg/m<sup>3</sup>) by SAW-SXFA sensor (18.6 °C, RH = 28%).</p>
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<p>SAW-SXFA sensor for minimum detection concentration of GB (18.6 °C, RH = 29%).</p>
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<p>Reproducibility of GB by SAW-SXFA sensor (16.9 °C, RH = 28%).</p>
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<p>Column chart of interference gas response (19.3 °C, RH = 26%).</p>
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18 pages, 4667 KiB  
Article
Hierarchical Nanoheterostructure of HFIP-Grafted α-Fe2O3@Multiwall Carbon Nanotubes as High-Performance Chemiresistive Sensors for Nerve Agents
by Xuechun Wang, Jingyuan Liu, Rumin Li, Jing Yu, Qi Liu, Jiahui Zhu and Peili Liu
Nanomaterials 2024, 14(3), 305; https://doi.org/10.3390/nano14030305 - 2 Feb 2024
Cited by 1 | Viewed by 1204
Abstract
New and efficient sensors of nerve agents are urgently demanded to prevent them from causing mass casualties in war or terrorist attacks. So, in this work, a novel hierarchical nanoheterostructure was synthesized via the direct growth of α-Fe2O3 nanorods onto [...] Read more.
New and efficient sensors of nerve agents are urgently demanded to prevent them from causing mass casualties in war or terrorist attacks. So, in this work, a novel hierarchical nanoheterostructure was synthesized via the direct growth of α-Fe2O3 nanorods onto multiwall carbon nanotube (MWCNT) backbones. Then, the composites were functionalized with hexafluoroisopropanol (HFIP) and successfully applied to detect dimethyl methylphosphonate (DMMP)-sarin simulant gas. The observations show that the HFIP-α-Fe2O3@MWCNT hybrids exhibit outstanding DMMP-sensing performance, including low operating temperature (220 °C), high response (6.0 to 0.1 ppm DMMP), short response/recovery time (8.7 s/11.9 s), as well as low detection limit (63.92 ppb). The analysis of the sensing mechanism demonstrates that the perfect sensing performance is mainly due to the synergistic effect of the chemical interaction of DMMP with the heterostructure and the physical adsorption of DMMP by hydrogen bonds with HFIP that are grafted on the α-Fe2O3@MWCNTs composite. The huge specific surface area of HFIP-α-Fe2O3@MWCNTs composite is also one of the reasons for this enhanced performance. This work not only offers a promising and effective method for synthesizing sensitive materials for high-performance gas sensors but also provides insight into the sensing mechanism of DMMP. Full article
(This article belongs to the Special Issue Nanoscale Material-Based Gas Sensors)
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<p>Illustration diagram of the synthesis and HFIP chemical functionalization of prepared HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs.</p>
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<p>(<b>a</b>,<b>b</b>) Photos and schematics of the sensors before and after covering with sensing materials, respectively, and (<b>c</b>) illustration diagram of the sensor circuit.</p>
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<p>Structural characterization of the prepared samples, (<b>a</b>,<b>b</b>) SEM and TEM images of MWCNTs; (<b>c</b>) TEM image of MWCNTs-COOH; (<b>d</b>) SEM images of the α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs composites; (<b>e</b>) TEM image of the α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs composites and corresponding SAED pattern inset on f; (<b>f</b>) HRTEM image of the α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs composites; (<b>g1</b>–<b>g4</b>) TEM and TEM mapping patterns of HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs, respectively.</p>
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<p>(<b>a</b>) XRD patterns of the α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTts nanostructures, α-Fe<sub>2</sub>O<sub>3</sub> and MWCNTs; (<b>b</b>) FT-IR spectra of the representative samples at different preparation stages.</p>
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<p>(<b>a</b>) Survey XPS spectra of different samples; high-resolution XPS spectrum of O 1s of α-Fe<sub>2</sub>O<sub>3</sub> (<b>b</b>), α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>c</b>), and HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNT composites (<b>d</b>); high-resolution XPS spectrum of Fe 2p of α-Fe<sub>2</sub>O<sub>3</sub> (<b>e</b>) and α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>f</b>).</p>
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<p>Nitrogen adsorption–desorption isotherms and Barrett–Joyner–Halenda (BJH) pore size–volume distribution (inset) of MWCNTs (<b>a</b>), α-Fe<sub>2</sub>O<sub>3</sub> (<b>b</b>), α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>c</b>), and HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>d</b>).</p>
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<p>(<b>a</b>) The responses of α-Fe<sub>2</sub>O<sub>3</sub>, α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs, and HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs to 1 ppm DMMP versus the test temperature; (<b>b</b>) the responses of α-Fe<sub>2</sub>O<sub>3</sub>, α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs, and HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs toward various gases; (<b>c</b>) the responses of HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs toward DMMP in the presence of other interfering gases.</p>
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<p>(<b>a</b>) The dynamic response curves of the α-Fe<sub>2</sub>O<sub>3</sub>, α-Fe<sub>2</sub>O<sub>3</sub>@MWCNT, and HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNT sensors in the concentration range 0.1–1 ppm DMMP at their working temperature; (<b>b</b>) the responsivity of the HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNT sensor versus DMMP concentration and the linear fitting results; (<b>c</b>) response–recovery times with 1 ppm DMMP concentrations; (<b>d</b>) multiple-cycle test curves of sensors at their working temperature with 1 ppm DMMP; (<b>e</b>) long-term stability of sensors at their working temperature with 1 ppm DMMP; (<b>f</b>) responses of the sensors at different values of RH.</p>
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<p>Mott–Schottky plots of MWCNTs (<b>a</b>), α-Fe<sub>2</sub>O<sub>3</sub> (<b>b</b>), and α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>c</b>).</p>
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<p>(<b>a</b>) Energy band diagram of α-Fe<sub>2</sub>O<sub>3</sub> and MWCNTs; (<b>b</b>) energy band diagram of the α-Fe<sub>2</sub>O<sub>3</sub>@MWCNT composite; schematic diagram of the DMMP sensing mechanism of sensors based on αFe<sub>2</sub>O<sub>3</sub>@MWCNT composites (<b>c</b>), and hybrid HFIP-α-Fe<sub>2</sub>O<sub>3</sub>@MWCNTs (<b>d</b>). The abbreviation Ec is the conduction band, Ev is the valence band, Ef is the Fermi level, Eg is the energy bandgap, Φw is the work function, and χ is the electron affinity.</p>
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14 pages, 3796 KiB  
Article
Rapid Non-Contact Detection of Chemical Warfare Agents by Laser Photoacoustic Spectroscopy
by Luca Fiorani, Claudio Ciceroni, Isabella Giardina and Fabio Pollastrone
Sensors 2024, 24(1), 201; https://doi.org/10.3390/s24010201 - 29 Dec 2023
Cited by 1 | Viewed by 1676
Abstract
Nerve agents have recently been used in battlefield operations, espionage wars, and terrorist attacks. These compounds, like some pesticides, cause organophosphate poisoning. The rapid, noncontact detection of a sarin simulant in the liquid phase has been demonstrated at the Diagnostics and Metrology Laboratory [...] Read more.
Nerve agents have recently been used in battlefield operations, espionage wars, and terrorist attacks. These compounds, like some pesticides, cause organophosphate poisoning. The rapid, noncontact detection of a sarin simulant in the liquid phase has been demonstrated at the Diagnostics and Metrology Laboratory of the Italian National Agency for New Technologies, Energy and Sustainable Economic Development using laser photoacoustic spectroscopy, an infrared absorption technology. The first measurements, carried out with an experimental system based on a quantum cascade laser and developed for the assessment of food authenticity in the “fingerprint region”, show that a detection limit of one nanolitre is within the reach of the instrument when chemometric analysis is applied. Full article
(This article belongs to the Section Optical Sensors)
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<p>(<b>Left</b>): Block diagram of the LPAS system. BS: beam splitter, C: photoacoustic cell, CH: chopper, F: active low pass filter, M: mirror, MP: microphone, PC: personal computer, PM: power meter, W: window. Grey continuous line: continuous wave laser beam, grey dotted line: modulated laser beam, black continuous line: signal, black dotted line: control. (<b>Right</b>): exploded view of the photoacoustic cell: on the left, the drawer in which the sample holder is hollowed out (when inserted in the cell, the seal is ensured by a double o-ring): on the top, from top to bottom, window flange, window, and o-ring; on the right, microphone flange; on the bottom, three vibration dampening rubber feet.</p>
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<p>Spectral composition of the four QCL modules of which the DRS Daylight Solutions MIRcat-1200 is made. Continuous lines: continuous wave (CW) operation. Dashed lines: pulsed operation. Blue, green, cyan and red lines correspond to module 1, 2, 3 and 4, respectively.</p>
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<p>GUI of the photoacoustic laser system (developed in the ENEA-funded TecHea project [<a href="#B25-sensors-24-00201" class="html-bibr">25</a>]).</p>
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<p>(<b>Top left</b>): handling of DMMP under a chemical hood. (<b>Top right</b>): preparation of a DMMP-soaked filter-paper disc; the LPAS system sample holder is visible on the table. (<b>Bottom left</b>): inserting the disc into the holder. (<b>Bottom right</b>): LPAS system with an aspiration system.</p>
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<p>LPAS spectrum of DMMP (60 replicas).</p>
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<p>Relative error of the LPAS spectrum of DMMP (60 replicas).</p>
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<p>(<b>Top</b>): average spectrum of DMMP (LPAS signal and NIST absorbance). (<b>Middle</b>): average spectrum of ethanol (LPAS signal and NIST absorbance). (<b>Bottom</b>): average spectra of DMMP and ethanol. Note the difference in the signal scales.</p>
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<p>The 2D PCA of the 60 spectra of each sample C: activated carbon; D: DMMP; E: ethanol; P: blank disc).</p>
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<p>(<b>Left</b>): 3D PCA of the 10 spectra of each sample (C.E.: confidence ellipse). (<b>Right</b>): predicted DMMP vs. actual DMMP. The line is the identity function. In fact, the linear fit nearly coincides with it, having intercept: 0.08 ± 0.72; slope: 0.99987 ± 0.00054; R-squared: 1.0. The point corresponding to 0 actual DMMP cannot be plotted in the logarithmic graph but was included in all calculations.</p>
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