Reversed Flow Gas Chromatography (R.F.G.C.) was employed to assess the impact of genetic modification on Saccharomyces cerevisiae yeast strains during the process of alcoholic fermentation, utilizing fig syrup. Multiple fermentations were carried out at various temperatures to evaluate the influence of genetic modifications on yeast strain efficiency. The study involved a wild-type yeast strain, W303, as a control, and two genetically modified strains, W_M4_533 and W_M4_558, sharing the same genetic background as the wild type. Notably, the genetic modifications in the Msn4p transcription factor involved the substitution of serine residues with alanine at positions 533 and 558, resulting in the development of psychrophilic or ethanol-resistant strains. Utilizing the R.F.G.C. method enabled the differentiation of the duration of alcoholic fermentation phases, providing insights correlated to the yeast cell life cycle. The values of rate constants (k) for each phase, conducted with both wild-type and genetically modified cells using RFGC, aligned with existing literature. Additionally, the calculation of activation energies for distinct phases revealed lower values for genetically modified strains compared to wild-type strains. This decrease in activation energies suggests enhanced efficiency in the alcoholic fermentation process for the genetically modified strains.

The four solvent extractives obtained from aerial parts of Leucophyllum frutescens were evaluated for their Total Antioxidant Activity (TAA) by ammonium molybdate method, scavenging potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox-Equivalent Antioxidant Capacity (TEAC) assays, metal-reducing potential by Cupric Reducing Antioxidant Capacity (CUPRAC) and Ferric Reducing Antioxidant Power (FRAP) assays, Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and their biological activities. The study concluded that BULE exhibited total antioxidant activity (226.235±1.222 mg AA.Eq.gm-1 DE±S.D) by molybdate method, CHLE exhibited more scavenging potential (DPPH 209.589±8.500 mg trolox Eq.gm-1 DE±S.D and TEAC 210.166±7.954 mg trolox Eq.gm-1 DE±S.D) and reducing potential (CUPRAC 646.889±16.889 mg trolox Eq.gm-1 DE±S.D & FRAP 472.981±15.625 mg trolox Eq.gm-1 DE±S.D). Phytochemical quantification concluded high TPC by BULE (189.369±1.393 mg GA.Eq.gm-1 DE±S.D) and high TFC by CHLE (232.458±1.589 mg Qu.Eq.gm-1 DE±S.D). Strong inhibition of α-glucosidase and urease enzymes was observed by HELE (IC50 0.3321±0.007 mg.ml-1±SD) and BULE (IC50 4.09±0.357 mg.ml-1±SD) extractives, respectively. The hemolytic effect shown by hexane extract (HELE) was higher with HA50 25.545±0.927 ug.ml-1±SD whereas methanol (MELE), chloroform (CHLE), and butanol (BULE) exhibited hemolytic effects at higher concentration with HA50 400.067±1.364, 321.394±1.332, and 332.957±0.465 µg.ml-1±SD, respectively. GC-MS profiling of HELE of L. frutescens was performed for qualitative analysis. The principal phytochemicals tentatively identified by GC-MS analysis of HELE accounts for fatty acids (60.221%), lignans (17.687%), ketones (3.358%), phenols (2.584%), sesquiterpenes (1.265%), and aldehydes (0.345%).

(R)-1-(4-Amino-3,5-dichlorophenyl)-2-bromoethan-1-ol has been synthesised in 93% enantiomeric excess (ee) by asymmetric reduction of the corresponding ketone catalysed by a ketoreductase and NADPH as the co-factor in DMSO. (S)-N-(2,6-Dichloro-4-(1-hydroxyethyl)phenyl)acetamide has been synthesised in >98% ee by the same system. Both synthons are potential precursors for clenbuterol enantiomers. Clenbuterol is a β₂-agonist used in veterinary treatment of asthma in several countries. The drug is listed on the World Anti-doping Agency’s Prohibited list due to its effect on increased protein synthesis in the body. However, racemic clenbuterol has recently been shown to reduce the risk of Parkinson’s disease. In order to reveal which one (or both) of the enantiomers that cause this effect, the pure enantiomers need to be studied separately. Our biocatalytic approach in order to obtain enantiopure clenbuterol should be applicable to industrial scale.

Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth as they account for a considerable fraction of the total lipids in plants and fungi which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, strawberry) were analyzed and revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0 – h26:0). Interestingly, six GIPCs containing t18:2 were observed in raspberry, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment potentially leading to the identification of new GIPCs. For the first time, automated high‑throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain makes it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Abstract：Objective: To compare the effects of fermentation on the chemical composition of Gastrodia Tuder Halimasch Powder, and to provide a basis for the processing, processing and clinical application of this medicinal material. Methods: UHPLC-Q-Orbitrap HRMS was used to identify compound structure by comparing excimer ions, characteristic fragment ions and reference substances, and referring to relevant literature and database search. The main differential components were screened by orthogonal partial least squares-discriminant analysis (OPLS-DA), and the differential components were quantitatively studied by high performance liquid chromatography, and the differences in the types and contents of chemical components before and after fermentation were compared. Results: A total of 77 compounds were identified or preliminarily derived after fermentation, including 21 terpenes, 18 organic acids, 9 flavonoids, 7 nucleosides, 6 amides, 4 amino acids, 2 pyrrolidone derivatives, 2 sterols, 2 alkaloids and 6 others.Through high performance liquid chromatography, it was found that the content of daidzein genistein and ergosterol was significantly increased after fermentation, and a new component ergosterol was produced. Compared with Gastrodia Tuder Halimasch Powder before fermentation, the content of genistin and other components was significantly reduced, and there may be a process of genistin conversion to genistein.Conclusion: The fermentation process will have a certain influence on the types and contents of the chemical components of Gastrodia Tuder Halimasch Powder, and ergosterol, genistein and .daidzein may be the main pharmacodynamic components, which can provide a useful reference for the clinical rational application, quality control and mechanism of action of Gastrodia Tuder Halimasch Powder.

Cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses is a key step. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by an affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. It was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.

Microsamples (collections usually less than 50 µL) have been introduced in pre-clinical, clinical, and research settings to overcome obstacles in sampling via traditional venipuncture. However, venipuncture remains the sampling gold standard for metabolic phenotyping of blood. This pre-sents several challenges in metabolic phenotyping workflows: accessibility for individuals in ru-ral and remote underserved areas (due to the need for trained personnel), the unamenable nature to frequent sampling protocols in longitudinal research (for its invasive nature), and sample col-lection difficulty in the young and elderly. Furthermore, venous sample stability may be compro-mised when temperate conditions necessary for cold-chain transport are beyond control. Alter-natively, research utilising microsamples extends phenotyping possibilities to inborn errors of metabolism, therapeutic drug monitoring, nutrition, as well as sport and anti-doping. Although the application of microsamples in metabolic phenotyping exists, it is still in its infancy, with whole blood being overwhelmingly the primary biofluid collected through the collection method of dried blood spots. Research into metabolic phenotyping of microsamples is limited; however, with advances in commercially available microsampling devices, common barriers such as volumetric inaccuracies and the ‘haematocrit effect’ in dried blood spot microsampling can be overcome. In this review, we provide an overview of the common uses and workflows for mi-crosampling in metabolic phenotyping research. We discuss the advancements in technologies, highlighting key considerations and remaining knowledge gaps for employment of microsamples in metabolic phenotyping research. Supporting the translation of research from the ‘bench to the community’.

Tea (Camellia sinensis) is one of the most popular beverages worldwide. Many types of tea prod-ucts continuously emerge in an endless stream; so, the classification or verification of tea be-comes more difficult. Aroma is a vital indicator of tea quality. The present study deals with the identification of aroma compounds in 18 different kinds of tea belonging to three typical tea va-rieties, including green tea, oolong tea, and black tea, using GC-IMS and GC×GC-O-MS. More-over, the clustering of all 18 tea samples and the in-depth correlation analysis between sensory evaluation and instrumental data were performed using the PCA and OPLS-DA. The results re-vealed that in all 18 kinds of tea, a total of 85 aroma compounds were detected by GC-IMS, whereas 318 were detected by GC×GC-O-MS. The PCA result revealed that green tea, oolong tea, and black tea could be clearly separated based on their peak areas. The OPLS-DA result showed that a total of 49 aroma compounds with VIP value > 1.0 could be considered as the potential in-dicators to quickly classify or verify tea types. This study not only compared the aroma differ-ences across different types of teas, but also provided ideas for the rapid monitoring of tea qual-ity and variety.

Downwind odor characteristics can be very different depending upon the size of the upwind point-source, interim topography, and wind conditions. At one extreme, the downwind odor plume from a relatively large, confined animal feeding operation (CAFO), located on a flat open plain and under stable, near-straight-line wind conditions can be rather broad, sustained and predictable relative to a fixed receptor site downwind. In contrast, the plume from a small point-source (e.g., a roof vent stack) located on irregular topography and under rapidly shifting wind conditions can be intermittent and fleeting. These transient odor events can be surprisingly intense and offensive, in spite of their fleeting occurrence and perception. This work reports on efforts to optimize an environmental odor sampling strategy, which is optimized for the challenges of (1) sampling of such transient odor 'spikes' and (2) the prioritization of odors/odorants from multiple, closely co-located point-sources, under such transient event conditions. Protocol refinement has emerged by way of 2 environmental odor assessment projects which have been undertaken on behalf of the Missouri Department of Natural Resources. The challenge of transient odor events has been mitigated utilizing rapid, odor cued whole-air grab capture sampling into metalized-FEP gas sampling bags, followed by immediate adsorption transfer onto SPME fibers or sorbent tubes for stabilization during the shipment and storage interval between collection and final analysis. Initial results demonstrated approximately 11 fold increases in target odorant yields for 900 mL sorbent tube transfers from 2-3 second 'burst' odor event bag-captures, as compared to equivalent direct collections at the same downwind receptor location but during perceived (stable) odor 'lull' periods. Results-to-date targeting refinement and field trials of this integrated environmental odor assessment strategy are presented. Preliminary application targeting general odor sampling and point-source differentiation utilizing tracer gases is also presented.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well-known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum. Cytoplasm obtained from different E. coli strains was examined as an example of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.