AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation.

Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical applications. Here, we develop a muscone-induced transgene system packaged into adeno-associated virus (AAV) vectors (AAVMUSE) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (PCRE). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAVMUSE enables remote, muscone dose- and exposure-time-dependent control of luciferase expression in the livers or lungs of mice for at least 20 weeks. Moreover, we apply this AAVMUSE to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone-after only one injection of AAVMUSE-can achieve long-term controllable expression of therapeutic proteins (ΔhFGF21 or ΔmIL-4). Our odorant-molecule-controlled system can advance gene-based precision therapies for human diseases.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae.

Cycloalkanes have broad applications as specialty fuels, lubricants, and pharmaceuticals but are not currently available from renewable sources, whereas, production of microbial cycloalkanes such as cyclopropane fatty acids (CFA) has bottlenecks. Here, a systematic investigation was undertaken into the biosynthesis of CFA in Saccharomyces cerevisiae heterologously expressing bacterial CFA synthase. The enzyme catalyzes formation of a 3-membered ring in unsaturated fatty acids. Monounsaturated fatty acids in phospholipids (PL) are the site of CFA synthesis; precursor cis-Δ9 C16 and C18 fatty acids were enhanced through OLE1 and SAM2 overexpression which enhanced CFA in PL. CFA turnover from PL to storage in triacylglycerols (TAG) was achieved by phospholipase PBL2 overexpression and acyl-CoA synthase to increase flux to TAG. Consequently, CFA storage as TAG reached 12 mg g-1 DCW, improved 3-fold over the base strain and >22% of TAG was CFA. Our research improves understanding of cycloalkane biosynthesis in yeast and offers insights into processing of other exotic fatty acids.

Muscone inhibits the progression of atherosclerotic plaques in mice aorta by inhibiting the NF-κB/p65 pathway.

Atherosclerosis (AS) is considered to be one of the main pathogenic factors of coronary heart disease, cerebral infarction and peripheral vascular disease. Oxidative stress and inflammation run through the occurrence and development of atherosclerosis and related cardiovascular events. Muscone is a natural extract of deer musk and also the main physiological active substance of musk. This study investigated the impact of muscone on atherosclerosis. ApoE-/- mice were used to establised AS model and injected with low-dose (4 mg/kg/day) or high-dose (8 mg/kg/day) of muscone intraperitoneally for 4 weeks. Then aortic tissues were collected, and pathological sections of the aorta were prepared for oil red staining, HE and masson staining. The changes of MDA, SOD, VCAM-1, NF-κB, and TNF-α were observed by Western blotting or immunofluorescence staining. The results showed that high-dose muscone could effectively reduce the plaque area/aortic root area and relative atherosclerotic area, reduce the collagen composition in plaque tissue. In addition, we also found that high-dose muscone can effectively increase MDA level, reduce the level of SOD, and inhibit the expression of VCAM-1, NF-κB/p65, TNF-α in arterial plaques. Our results indicate that the administration of muscone has the benefit of inhibiting atherosclerosis. The potential mechanisms may be associated with antioxidant effect and inhibition of inflammatory reaction in arterial plaques. With the increasing understanding of the relationship between muscone and atherosclerosis, muscone has high potential value as a new drug to treat atherosclerosis.

Cycloalkane degradation by an uncultivated novel genus of Gammaproteobacteria derived from China's marginal seas.

The consumption of cycloalkanes is prevalent in low-temperature marine environments, likely influenced by psychrophilic microorganisms. Despite their significance, the primary active species responsible for marine cycloalkane degradation remain largely unidentified due to cultivation challenges. In this study, we provide compelling evidence indicating that the uncultured genus C1-B045 of Gammaproteobacteria is a pivotal participant in cycloalkane decomposition within China's marginal seas. Notably, the relative abundance of C1-B045 surged from 15.9% in the methylcyclohexane (MCH)-consuming starter culture to as high as 97.5% in MCH-utilizing extinction cultures following successive dilution-to-extinction and incubation cycles. We used stable isotope probing, Raman-activated gravity-driven encapsulation, and 16 S rRNA gene sequencing to link cycloalkane-metabolizing phenotype to genotype at the single-cell level. By annotating key enzymes (e.g., alkane monooxygenase, cyclohexanone monooxygenase, and 6-hexanolactone hydrolase) involved in MCH metabolism within C1-B045's representative metagenome-assembled genome, we developed a putative MCH degradation pathway.

Muscone alleviates neuronal injury via increasing stress granules formation and reducing apoptosis in acute ischemic stroke.

As the main bioactive component of musk, muscone has been reported to have marked protective effects in treating acute ischemic stroke (AIS). However, the specific anti-stroke mechanism of muscone still needs further research. In the current investigation, the PC12 cells OGD/R and the rat transient MCAO/R models were utilized as the AIS models. Serum hepatic and renal functional indexes (ALT, AST, BUN, and Cr) and cell viability were determined to select the appropriate muscone concentrations for in vitro and in vivo experiments. TTC, Hematoxylin and eosin (H&E), and Live/Dead staining were utilized to evaluate the protective effects of muscone in injured tissues and cells. Western blotting analysis, TUNEL staining, propidium iodide, and annexin V staining were applied to detect the anti-apoptotic effect of muscone. Double-label immunofluorescence staining of T-cell intracellular antigen-1 (TIA1) and Ras-GAP SH3 domain-binding protein 1 (G3BP1) was performed to observe whether muscone regulated the SG formation level. Molecular docking, TIA1 silencing and TIA1 overexpression experiments were employed to investigate the molecular mechanism underlying the regulation of SG formation by muscone. The 2, 3, 5-Triphenyl-tetrazolium chloride (TTC) staining and live/dead staining showed the AIS injury level of MCAO/R rat and the OGD/R PC12 cells were attenuated by muscone administration. The muscone significantly minimized the apoptosis rate in MCAO/R rats and OGD/R PC12 cells following flow cytometry analysis, western blotting analysis, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The double-label immunofluorescence staining data revealed that muscone promoted the SG formation level in OGD/R PC12 cells and the cortex MCAO/R rats. The results of molecular docking, TIA1 silencing and TIA1 overexpression experiments revealed that muscone could bind to TIA1 protein and regulate its expression level, thereby promoting the formation of stress granules and exerting a protective effect against AIS injury. This study indicated that the significant protective effect of muscone in reducing apoptosis levels might be via promoting SG formation under AIS conditions. This study further explores the therapeutic effect and anti-apoptosis mechanism of muscone in AIS, which may provide a potential candidate drug for the clinical treatment of AIS injury.

Comparative Study of Sorption Phenomena Between Three Medications and Syringes Made of Cyclic Olefin Copolymer or Polypropylene.

INTRODUCTION: Medical syringes are widely used in hospitals to store and administer drugs, and the contact time between the drugs and these syringes can vary from a few minutes to several weeks like for pharmaceutical preparations. The aim of this comparative study was to evaluate the potential sorption phenomena occurring between three drugs (paracetamol, diazepam and insulin aspart) and polypropylene syringes (PP) or syringes made of Cyclic Olefin Copolymer (COC). MATERIALS AND METHODS: 50 mL 3-part syringes made of either COC with crosslinked silicone on the barrel inner surface (COC-CLS) and a bromobutyl plunger seal, or PP lubricated with silicone oil (PP-SOL) with a polyisoprene plunger seal were used. RESULTS: COC-CLS syringes induced less sorption of diazepam and insulin than PP-SOL syringes and the plunger seal material seemed to be the main cause of these interactions. An alkalinization of the medications in contact with the PP-SOL syringes was observed. It could be caused by leachable compounds and should be investigated further. CONCLUSION: This work shows once again that it is essential to consider content-container interactions to help improve the safe use of parenteral drugs.

Aerobic biodegradation of cycloalkanes in non-aqueous extracted oil sands tailings.

Management of growing volumes of fluid fine tailings (FFT) is a significant challenge for oil sands industry. A potential alternative non-aqueous solvent extraction (NAE) process uses cycloalkane solvent such as cyclohexane or cyclopentane with very little water and generates smaller volumes of 'dry' solids (NAES) with residual solvent. Here we investigate remediation of NAES in a simulated bench-scale upland reclamation scenario. In the first study, microcosms with nutrient medium plus FFT as inoculum were amended with cyclohexane and incubated for ∼1 year, monitoring for cyclohexane biodegradation under aerobic conditions. Biodegradation of cyclohexane occurred under aerobic conditions with no metabolic intermediates detected. A second study using NAES mixed with FFT spiked with cyclohexane and cyclopentane, with or without additional nutrients (nitrogen and phosphorus), showed complete and rapid aerobic biodegradation of both cycloalkanes in NAES inoculated with FFT and supplemented with nutrients. 16S rRNA gene sequencing revealed dominance of Rhodoferax and members of Burkholderiaceae during aerobic cyclohexane biodegradation in FFT, and Hydrogenophaga, Acidovorax, Defluviimonas and members of Porticoccaceae during aerobic biodegradation of cyclohexane and cyclopentane in NAES inoculated with FFT and supplemented with nutrients. The findings indicate that biodegradation of cycloalkanes from NAES is possible under aerobic condition, which will contribute to the successful reclamation of oil sands tailings for land closure.

Fabrication of Implantable Microelectrode Array using Cyclic Olefin Copolymer and SU-8 via Photocrosslinking Lamination.

In this work, a fabrication process for implantable electrodes using a Cyclic Olefin Copolymer (COC) substrate and a SU-8 passivation layer was presented. COC and SU-8 were shown to be suitable for implantable neural electrodes due to their biocompatibility, chemical resistance, and thermal stability. The electrodes were successfully patterned on the COC film, and the SU-8 passivation layer was coated while maintaining site-opened via photolithography. The photocrosslinking lamination of the substrate and passivation layer was used to produce electrodes with fine line widths of 20um without applying heat.

Low dead space encapsulation and integration of circuits for retinal prosthesis based on cyclic olefin copolymer.

A retinal prosthesis is a device that can provide artificial vision to people who have lost their sight from certain retinal disorder. Because the device needs to be inserted into the body, high flexibility and reliability is required. Recently, devices using thermoplastic polymers such as LCP and COC as substrates have been studied. Being a highly functional integrated device, retinal prosthesis poses many design challenges. Among them, the stimulation chip embedding can be a particularly important task. Although it is common to use a wire bonding method for chip embedding, there are several limitations that are difficult to apply to implantable device. In this investigation, a novel approach is developed for high spaceefficient electrical connections and perform reliable encapsulation of integrated circuits to replace wire bonding. Since designing and manufacturing the stimulator chip used in retinal prosthesis requires non-negligible cost, a silicon die with the identical shape was selected as a substitute for testing purposes.

Non-target screening of volatile organic compounds in spray-type consumer products and their potential health risks.

Widespread use of spray-type consumer products can raise significant concerns regarding their effects on indoor air quality and human health. In this study, we conducted non-target screening using gas chromatography-mass spectrometry (GC-MS) to analyze VOCs in 48 different spray-type consumer products. Using this approach, we tentatively identified a total of 254 VOCs from the spray-type products. Notably, more VOCs were detected in propellant-type products which are mostly solvent-based than in trigger-type ones which are mostly water-based. The VOCs identified encompass various chemical classes including alkanes, cycloalkanes, monoterpenoids, carboxylic acid derivatives, and carbonyl compounds, some of which arouse concerns due to their potential health effects. Alkanes and cycloalkanes are frequently detected in propellant-type products, whereas perfumed monoterpenoids are ubiquitous across all product categories. Among the identified VOCs, 12 compounds were classified into high-risk groups according to detection frequency and signal-to-noise (S/N) ratio, and their concentrations were confirmed using reference standards. Among the identified VOCs, D-limonene was the most frequently detected compound (freq. 21/48), with the highest concentration of 1.80 mg/g. The risk assessment was performed to evaluate the potential health risks associated with exposure to these VOCs. The non-carcinogenic and carcinogenic risks associated with the assessed VOC compounds were relatively low. However, it is important not to overlook the risk faced by occupational exposure to these VOCs, and the risk from simultaneous exposure to various VOCs contained in the products. This study serves as a valuable resource for the identification of unknown compounds in the consumer products, facilitating the evaluation of potential health risks to consumers.

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

Methylated cycloalkanes fuel a novel genus in the Porticoccaceae family (Ca. Reddybacter gen. nov).

Cycloalkanes are abundant and toxic compounds in subsurface petroleum reservoirs and their fate is important to ecosystems impacted by natural oil seeps and spills. This study focuses on the microbial metabolism of methylcyclohexane (MCH) and methylcyclopentane (MCP) in the deep Gulf of Mexico. MCH and MCP are often abundant cycloalkanes observed in petroleum and will dissolve into the water column when introduced at the seafloor via a spill or natural seep. We conducted incubations with deep Gulf of Mexico (GOM) seawater amended with MCH and MCP at four stations. Within incubations with active respiration of MCH and MCP, we found that a novel genus of bacteria belonging to the Porticoccaceae family (Candidatus Reddybacter) dominated the microbial community. Using metagenome-assembled genomes, we reconstructed the central metabolism of Candidatus Reddybacter, identifying a novel clade of the particulate hydrocarbon monooxygenase (pmo) that may play a central role in MCH and MCP metabolism. Through comparative analysis of 174 genomes, we parsed the taxonomy of the Porticoccaceae family and found evidence suggesting the acquisition of pmo and other genes related to the degradation of cyclic and branched hydrophobic compounds were likely key events in the ecology and evolution of this group of organisms.

High Cycle-Life Twistocaloric Cooling of Poly-p-Phenylene Benzodioxole Fibers.

It is an urgent need to develop efficient solid state cooling technologies and materials with high cycle life. Poly-p-phenylene benzodioxole (PBO) is a high performance fiber with excellent mechanical properties. In this work, for the first time, elasto- and twistocaloric cooling of PBO fibers by stretching and twisting of the PBO fiber bundles is reported. The cooling temperature reaches -0.4 and -1.3 K, for fiber stretching and twisting, respectively. A self-coiled PBO fiber achieves maximum cooling of -3.7 K upon stretching by 35% strain, with an exceptionally high cycle life of 200 000 times. During the twisting of the PBO fibers, reversible changes in the intensity of the diffraction peaks in X-ray diffraction patterns are observed. A strain-sensitive color change application is realized by coating a self-coiled PBO fiber with liquid crystallite dyes. This work provides new perspectives for PBO fibers as a high cycle-life solid-state refrigeration material.

Catalytic Asymmetric Intermolecular [3 + 2] Photocycloaddition of Cyclopropylamines with Electron-Deficient Olefins.

An enantioselective intermolecular [3 + 2] cycloaddition of N-arylcyclopropylamines with 2-aryl acrylates/ketones and cyclic ketone-derived terminal olefins via asymmetric photoredox catalysis is reported. A dual catalyst system involving DPZ and a chiral phosphoric acid is effective for the transformations, leading to a wide array of valuable cyclopentylamines with high yields, ee's, and drs. Among them, elaborate modulation of the ester group of 2-aryl acrylates was shown to be effective in improving reactivity, thereby enabling the success of the transformations.

Detection and Identification of an Unknown Impurity in Ephedrine HCl 5 mg/mL Cyclic Olefin Syringes: Formulation Development.

An unknown impurity was detected in in-house prepared ephedrine hydrochloride (HCl) 5 mg/mL prefilled sterilized syringes when applying a stability-indicating British Pharmacopoeia 2018 impurity method for ephedrine injection. Ultraviolet, chromatographic, mass spectral, and physicochemical methods were combined to identify the unknown impurity. The unknown impurity was identified as methcathinone, which is generated from ephedrine drug substance through an oxidation reaction. A formulation study, in which different process adjustments were tested, was carried out to reduce the amount of unknown impurity. Nitrogen gassing in combination with 0.05 M citrate buffer addition proved to be the most potent process adjustment in reducing methcathinone formation in ephedrine HCl 5 mg/mL prefilled sterilized syringes after 4 months of storage in the dark at room temperature (20 °C ± 5 °C). More detailed research on the long-term stability of the reformulated ephedrine HCl drug product is currently underway, with promising results for up to 9 months gathered already.

Transannular C-H functionalization of cycloalkane carboxylic acids.

Cyclic organic molecules are common among natural products and pharmaceuticals1,2. In fact, the overwhelming majority of small-molecule pharmaceuticals contain at least one ring system, as they provide control over molecular shape, often increasing oral bioavailability while providing enhanced control over the activity, specificity and physical properties of drug candidates3-5. Consequently, new methods for the direct site and diastereoselective synthesis of functionalized carbocycles are highly desirable. In principle, molecular editing by C-H activation offers an ideal route to these compounds. However, the site-selective C-H functionalization of cycloalkanes remains challenging because of the strain encountered in transannular C-H palladation. Here we report that two classes of ligands-quinuclidine-pyridones (L1, L2) and sulfonamide-pyridones (L3)-enable transannular γ-methylene C-H arylation of small- to medium-sized cycloalkane carboxylic acids, with ring sizes ranging from cyclobutane to cyclooctane. Excellent γ-regioselectivity was observed in the presence of multiple ß-C-H bonds. This advance marks a major step towards achieving molecular editing of saturated carbocycles: a class of scaffolds that are important in synthetic and medicinal chemistry3-5. The utility of this protocol is demonstrated by two-step formal syntheses of a series of patented biologically active small molecules, prior syntheses of which required up to 11 steps6.

Degradation potential and pathways of methylcyclohexane by bacteria derived from Antarctic surface water.

Cycloalkanes pose a tremendous environmental risk due to their high concentration in petroleum hydrocarbons and hazardous effects to organisms. Numerous studies have documented the biodegradation of acyclic alkanes and aromatic hydrocarbons. However, insufficient attention has been paid to studies on the microbial degradation of cycloalkanes, which might be closely linked to psychrophilic microbes derived from low-temperature habitats. Here we show that endemic methylcyclohexane (MCH, an abundant cycloalkane species in oil) consumers proliferated in seawater samples derived from the Antarctic surface water (AASW). The MCH-consuming bacterial communities derived from AASW exhibited a distinct species composition compared with their counterparts derived from other cold-water habitats. We also probed Colwellia and Roseovarius as the key active players in cycloalkane degradation by dilution-to-extinction-based incubation with MCH as sole source of carbon and energy. Furthermore, we propose two nearly complete MCH degradation pathways, lactone formation and aromatization, concurrently in the high-quality metagenome-assembled genomes of key MCH consumer Roseovarius. Overall, we revealed that these Antarctic microbes might have strong interactions that enhance the decomposition of more refractory hydrocarbons through complementary degradation pathways.

Transforming Inert Cycloalkanes into α,ω-Diamines by Designed Enzymatic Cascade Catalysis.

Aliphatic α,ω-diamines (DAs) are important monomer precursors that are industrially produced by energy-intensive, multistage chemical reactions that are harmful to the environment. Therefore, the development of sustainable green DA synthetic routes is highly desired. Herein, we report an efficient one-pot in vivo biocatalytic cascade for the transformation of cycloalkanes into DAs with the aid of advanced techniques, including the RetroBioCat tool for biocatalytic route design, enzyme mining for finding appropriate enzymes and microbial consortia construction for efficient pathway assembly. As a result, DAs were successfully produced by the designed microbial consortia-based biocatalytic system. In particular, the highest biosynthesis productivity record of 1,6-hexanediamine was achieved when using either cyclohexanol or cyclohexane as a substrate. Thus, the developed biocatalytic process provides a promising alternative to the dominant industrial process for manufacturing DAs.

Highly selective catalytic transfer hydrodeuteration of cyclic alkenes.

Selective deuterium installation into small molecules is becoming increasingly desirable not only for the elucidation of mechanistic pathways and studying biological processes but also because of deuterium's ability to favorably adjust the pharmacokinetic parameters of bioactive molecules. Fused bicyclic moieties, especially those containing heteroatoms, are prevalent in drug discovery and pharmaceuticals. Herein, we report a copper-catalyzed transfer hydrodeuteration of cyclic and heterocyclic alkenes, which enables the synthesis of chromans, quinolinones, and tetrahydronaphthalenes that are precisely deuterated at the benzylic position. We also demonstrate the ability to place one deuterium atom at the homobenzylic site of these scaffolds with high regioselectivity by swapping transfer reagents for their isotopic analogs. Furthermore, examples of chemoselective transfer hydrogenation and transfer deuteration are disclosed, allowing for the simultaneous incorporation of two vicinal hydrogen or deuterium atoms into a double bond.

Fast and Bioorthogonal Release of Isocyanates in Living Cells from Iminosydnones and Cycloalkynes.

Bioorthogonal click-and-release reactions are powerful tools for chemical biology, allowing, for example, the selective release of drugs in biological media, including inside animals. Here, we developed two new families of iminosydnone mesoionic reactants that allow a bioorthogonal release of electrophilic species under physiological conditions. Their synthesis and reactivities as dipoles in cycloaddition reactions with strained alkynes have been studied in detail. Whereas the impact of the pH on the reaction kinetics was demonstrated experimentally, theoretical calculations suggest that the newly designed dipoles display reduced resonance stabilization energies compared to previously described iminosydnones, explaining their higher reactivity. These mesoionic compounds react smoothly with cycloalkynes under physiological, copper-free reaction conditions to form a click pyrazole product together with a released alkyl- or aryl-isocyanate. With rate constants up to 1000 M-1 s-1, this click-and-release reaction is among the fastest described to date and represents the first bioorthogonal process allowing the release of isocyanate electrophiles inside living cells, offering interesting perspectives in chemical biology.