Natural Organochlorines as precursors of 3-monochloropropanediol esters in vegetable oils

During high-temperature refining of vegetable oils, 3-monochloropropanediol (3-MCPD) esters, possible carcinogens, are formed from acylglycerol in the presence of a chlorine source. To investigate organochlorine compounds in vegetable oils as possible precursors for 3-MCPD esters, we tested crude palm, soybean, rapeseed, sunflower, corn, coconut, and olive oils for the presence of organochlorine compounds. Having found them in all vegetable oils tested, we focused subsequent study on oil palm products. Analysis of the chlorine isotope mass pattern exhibited in high-resolution mass spectrometry enabled organochlorine compound identification in crude palm oils as constituents of wax esters, fatty acid, diacylglycerols, and sphingolipids, which are produced endogenously in oil palm mesocarp throughout ripening. Analysis of thermal decomposition and changes during refining suggested that these naturally present organochlorine compounds in palm oils and perhaps in other vegetable oils are precursors of 3-MCPD esters. Enrichment and dose-response showed a linear relationship to 3-MCPD ester formation and indicated that the sphingolipid-based organochlorine compounds are the most active precursors of 3-MCPD esters

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Metabolite profiling of Boesenbergia rotunda tissue culture callus related to embryogenesis and plant regeneration / Theresa Ng Lee Mei

Boesenbergia rotunda or fingerroot ginger is commonly found in South East Asia and traditionally used to treat common illnesses. Interest in the medicinal properties of B. rotunda has led to the tissue culture studies of this plant. The exploitation of culture conditions can be expected to affect production of different calli types and cell metabolites. Hence, analysis of primary and secondary metabolites as well as hormones was performed by Ultra Performance Liquid Chromatography Mass Spectrometry to determine the biochemical changes related to embryogenesis and plant regeneration. This was complemented by histological characterization study by microscopy. Primary metabolite profiles showed higher levels of glutamine, arginine and lysine in B. rotunda embryogenic callus compared to non-embryogenic tissues (suspension cells, dry and watery calli). The metabolite markers for embryogenic competency were confirmed in sieved embryogenic cells. Rhizome had the highest flavonoid levels while shoot tips the lowest indicating that flavonoids in shoot tips may result from diffusion from the rhizome. The low endogenous auxin level in embryogenic callus suggests active auxin metabolism to stimulate cell division and elongation for embryogenesis. Histo-morphological study indicated that embryogenic callus can be characterized by the presence of starch granules, fibrils on cell surfaces and bright fluorescent spots after diphenylboric acid 2-aminoethylester staining. Cells in watery callus were non-proliferative, lacking fluorescent spots, nuclei and starch granules, however had apparently higher flavonoid levels, possibly due to higher stain specificity towards selected flavonoids in B. rotunda. Ultimately, the identification of primary metabolite and cell morphology markers in B.rotunda cell cultures together with ongoing genomic studies can improve understanding of molecular processes related to embryogenesis and plant regeneration

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda).

Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue

Relative abundance of metabolites markers in embryogenic callus and sieved embryogenic cells (n = 3 biological replicates).

<p>EC: Embryogenic callus; EC_S: sieved embryogenic cells. Error bars indicate standard deviation, asterisk represents p-value <0.05 by student T-test.</p

Morphology and histology of <i>B</i>. <i>rotunda</i> explant and callus.

<p><b>A-D</b>: morphology of samples; A: cross section of 1 cm x 1 cm shoot base tissue; B: friable pale yellowish callus; C: compact, dense and dry callus, D: spongy and wet callus; <b>E-H</b>: SEM images (100x magnification); E: regular-shaped and -sized cells with arrows showing the presence of starch; F: regular-shaped cells with fibrils; G: rounded, compact cells; H: elongated and irregular-shaped cells; <b>I-L</b>: morphology of each sample viewed under fluorescent microscopy with diphenylboric acid 2-aminoethylester (DPBA) stain (100x magnification); I: fluorescent yellowish-green lining of cell membrane, J: fluorescent greenish-blue spots observed with yellow lining of cell membrane; K: fluorescent greenish blue spots observed with yellow lining of cell membrane; L: yellowish lining of cell membrane; <b>M-P</b>: morphology of each sample viewed under light microscopy with Periodic acid Schiff (PAS) stain (100x magnification); E: organized and compact cells with presence of vascular bundles (VB) and purplish-red starch granules; F: presence of dark blue clusters indicates active cell division and purplish-red starch granules; G: presence of dark blue clusters indicates active cell division and red-purplish starch granules; H: irregular-shaped and -sized cells without starch granules. SB: shoot base; EC: embryogenic callus; DC: dry callus; WC: watery callus.</p

Principal Component Analysis (PCA) plot showing three clusters in callus and explant tissues from <i>B</i>. <i>rotunda</i> (n = 3 biological replicates).

<p>Blue ellipse: embryogenic callus (EC); orange ellipse with green; dry callus (DC) and with purple: watery callus (WC); and red ellipse: shoot base (SB).</p

Concentrations (% dry extract) of secondary metabolites in <i>B</i>. <i>rotunda</i> shoot (n = 3 biological replicates).

<p>Error bars indicate standard deviation; RH: rhizome; SB: shoot base; T1: region of shoot 1–5 cm distal from the shoot base; T2: region of shoot 6–10 cm distal from the shoot base.</p