Photo‐affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids

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Photo-affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids

Leon Duque, Vallavoju, and colleagues report the design and development of photo-affinity and metabolic labeling probes based on the opioid alkaloids. These chemical probes are used to measure localization and protein interactions in a cellular context by confocal microscopy and chemical proteomics. These insights will inform the development of future probes inspired by the opioid alkaloids.


Abstract

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA

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A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA

Tandem Oligonucleotide Synthesis (TOS) of (chemically modified) DNA and RNA using a commercially available cleavable linker. We show that the 2,2′-sulfonyldiethylene linker is effectively incorporated in DNA/RNA sequences, and efficiently cleaved during standard nucleic acid deprotection protocols. Moreover, we utilize our TOS approach to perform ligation experiments to incorporate N3′→P5′ phosphoramidate, and pyrophosphate linkages in DNA strands


Abstract

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2′-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.

Systematic Analysis of the MIO‐forming Residues of Aromatic Ammonia Lyases

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Systematic Analysis of the MIO-forming Residues of Aromatic Ammonia Lyases

Through a systematic analysis of protein sequences, several protein clusters of AAL-like enzymes with unusual MIO-forming motifs (ACG, TSG, SSG, and CSG) were identified, including two novel HALs with CSG motifs, one PAM with a TSG motif, and three novel ETLs without MIO motif. The systematic study and mutagenesis of MIO-groups in this work enable thus functional prediction and mutagenesis research of AALs.


Abstract

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

Endogenous Production and Vibrational Analysis of Heavy‐Isotope‐Labeled Peptides from Cyanobacteria

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Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria

Microcystis aeruginosa cultures were grown in 15N- and 13C-enriched medium to produce heavy-labeled cyanopeptides, which were characterized with vibrational spectroscopy and mass spectrometry. Up to 98 % heavy isotope incorporation was observed for the peptides, which displayed strong vibrational bands associated with diene, thiazole, and phenyl functional groups.


Abstract

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15N/13C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC–MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Numerical Investigation of Different Combustion Models for Dual‐Fuel Engine Combustion Processes

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Numerical Investigation of Different Combustion Models for Dual-Fuel Engine Combustion Processes

The combustion process of a dual-fuel engine was studied by three different models. The peak pressure calculated by CFM is closest to the experimental values. EBM is the most accurate for CO2 emissions. NO x emissions calculated by CTM and EBM are in good agreement with experimental values at low and high speed, respectively. Hydrocarbon emissions calculated by CFM are the most accurate.


Abstract

The combustion process of a dual-fuel engine was calculated by characteristic timescale model (CTM), eddy breakup model (EBM), and coherent flamelet model (CFM) to verify the accuracy of the combustion models. The results show that the peak pressure calculated by EBM is 2.78 % higher than the experimental value, and the peak pressure calculated by CFM is closest to the experimental value. The EBM is the most accurate for CO2 emissions. The deviation of CO emissions from the experimental values calculated by CTM is the smallest (< 1 %). The NO x emission calculated by CTM is good agreement with the experimental value at low speed, and EBM is in best agreement with the experimental value at high speed. The hydrocarbon emission calculated by CFM is the most accurate, with a deviation of less than 0.6 %.

Simulation and Optimization of Biohydrogen Production from Biomass Feed via Anaerobic Digestion

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Simulation and Optimization of Biohydrogen Production from Biomass Feed via Anaerobic Digestion

A simulation model of anaerobic digestion was developed, and sugarcane bagasse, rice straw, and sawdust were examined as feeds for the production of biohydrogen and methane. The operating parameters included in the study were pretreatment temperature, hydraulic retention time, and feed-to-water ratio. These parameters were optimized for better biohydrogen and methane yields.


Abstract

Biomass energy is a renewable energy source that is carbon-neutral, versatile, and can never go extinct. In the current study, a simulation model of an anaerobic digester with parameters such as pretreatment temperature (PTT), hydraulic retention time (HRT), and feed-to-water (F/W) ratio was developed and a design of experiment was created. The results showed that the higher hemicellulose content in sugarcane bagasse (SB) gave a decent H2 yield (13–23 %). The significance sequence was PTT > F/W > HRT. The optimal values were HRT of 15–16 d; F/W of 1.5 (SB), 1.26 [rice straw (RS)], and 0.5 [sawdust (SD)]; and PTT of 63.3 (SB), 68.8 (RS), and 75 °C (SD). The optimal H2 yield was 19.80 (SB), 20.94 (RS), and 21.41 % (SD). Therefore, the present simulation and optimization showed concrete results to raise H2 yield.

Advancements, Challenges, and Pathways to Sustainable Biofuel Production

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Advancements, Challenges, and Pathways to Sustainable Biofuel Production

A comprehensive review of all available physical, physiochemical, thermochemical, and biochemical techniques is given that are effectively applied for the production of biofuels (bioethanol, biodiesel, bio-oil, biochar, biogas, and other valuable chemicals) from biomass. The concept of waste-to-wealth in biomass conversion is emphasized whereby low-value biomass is transformed into high-value products.


Abstract

The discovery of new energy sources plays a vital role to fulfil the needs of energy and depletion of fossil fuel sources. An inclusive review of all available technologies for the synthesis of biofuels from biomass is presented. Amongst the innovative biomass conversion technologies, thermochemical technology encompasses significant potential practices and is required for expansion. Universally, biofuels are gaining importance due to the depletion of non-renewable fuel sources in transport and industries by equivalent work proficiencies. The reaction mechanism thoroughly explores all aspects associated with reaction conditions, types of reactor, and products. Biomass thermochemical conversion technologies will be influential in producing sustainable biofuels.

Hormones Concentration in an Aqueous Two‐Phase System: Experimental and Computational Analysis

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Hormones Concentration in an Aqueous Two-Phase System: Experimental and Computational Analysis

Aqueous two-phase systems based on protic ionic liquids (PILs) allowed 250-fold concentration of hormones in both phases, and thus 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), and progesterone (PROG) were identified and quantified in water samples previously diagnosed as free of these micropollutants. The COSMO-RS method was used to understand hormone migration between the two phases.


Abstract

Estrogens are emergent pollutants found in low levels in water bodies, and for this reason they require concentration for quantification. In this study, aqueous two-phase systems (ATPSs) were used as an alternative strategy to improve hormone detection and quantification from real water samples. In addition, a predictive computational method (COSMO-RS) was used to understand better the migration of hormones between the two phases at the molecular level. Water samples previously analyzed as hormone-free were added to the 250-fold concentrated ATPS, where it was possible to detect and quantify concentrations of these micropollutants in the range of 9.3–29.19 ng L−1.

Numerical Performance Investigation of a Side‐outlet Cyclone for Particulate Matter and Polymer Powder Separation

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Numerical Performance Investigation of a Side-outlet Cyclone for Particulate Matter and Polymer Powder Separation

A cyclone with a side-outlet configuration was analyzed by computational fluid dynamics simulation and validated with experimental results. The renormalization group k-ε model indicated a pressure drop of 80.8412 Pa and separation efficiency of 45.48 %. The operating pressure drop was one-third of the mathematical model result, while separation efficiency was 15 % lower than the experimental value.


Abstract

The side-outlet cyclone, featuring a novel geometric design with a lateral gas outlet, proves advantageous in space-constrained environments compared to traditional cyclones with top gas outlets. Herein, a cyclone separator with a side-outlet configuration was numerically investigated using the renormalization group k-ε model and pressure drop and separation efficiency as the performance parameters. Simulation results revealed that the side-outlet cyclone operates at a low to moderate pressure drop of 80.8412 Pa, with a removal efficiency of 45.48 %. The operating pressure drop was found to be only one-third of the computed pressure drop from mathematical modeling, with a removal efficiency 15 % lower than the experimental value. With varying cyclone design modifications, suitable experimental procedures and measuring approaches are desirable to further accurately corroborate these results.