Myb-binding protein 1A (MYBBP1A) is essential for early embryonic development, controls cell cycle and mitosis, and acts as a tumor suppressor

MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.NHMRC: This work was supported by Associazione Italiana Ricerche sul Cancro (AIRC) grant 8929 and European Community FP7 201681 ‘‘Prepobedia’’ to FB, the Australian National Health and Medical Research Council to RK and TJG (project ID000115). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

High-throughput analytical methods employing microextraction techniques: Towards fast analyses lasting a few seconds

Green Analytical Chemistry (GAC) principles have influenced the development of analytical methods with minimal sample handling and operations, in contrast to conventional techniques, which are often laborious and time-consuming. Since the introduction of microextraction techniques in the 1990s, various approaches, configurations, and sorptive phases have been proposed to replace Solid Phase Extraction (SPE) and Liquid-Liquid Extraction (LLE), covering a wide range of matrices and analytes, focused on chromatography and mass spectrometry instrumentation. The main features of microextraction techniques are simplicity, low solvent consumption, and minimum residue generation. The demand for fast results and the large number of samples, along with advances in analytical instrumentation, have led to the coupling of microextraction techniques with automation and/or the development of technologies for multiple extractions at the same time, known as parallel extractions. As a result of its popularity in medical and pharmaceutical sciences, the 96-well plate has been successfully adapted for Solid Phase Microextraction (SPME) and Liquid Phase Microextraction (LPME), significantly reducing the time required to process a large number of samples. This review presents some of the basic principles of microextraction techniques, and it contextualizes and compares analytical methods published in the period of 2018 to early 2023 in the microextraction context for high-throughput analyses

Disposable Pipette Extraction (DPX) Coupled to HPLC-DAD as an Alternative for the Determination of Phthalic Monoesters in Urine Samples

Phthalates are widely used in industry, but adverse effects on human health have been reported due to exposure to these chemicals. In the human body, they are metabolized into phthalic monoesters, which are used to monitor human exposure and assess risk. Urine is one of the main biological samples used, due to its easy access and collection, and also being the main elimination pathway for phthalates. Urine samples are complex; therefore, sample preparation is a critical step. Disposable pipette extraction (DPX) has not previously been reported for quantifying phthalates in urine and is here presented as a fast and low sample consumption method. A fully optimized RP-DPX method was developed for determination of free monomethyl phthalate, monobutyl phthalate, monobenzyl phthalate, and monoethylhexyl phthalate from urine samples. Analytical parameters of merit were obtained. The values of R2 were ≥0.9832, and the LOD and LOQ varied from 3.0 to 7.6 μg L−1 and 10 to 25 μg L−1, respectively. Intraday (n = 3) and interday (n = 9) precision were ≤13.6 and 15.6%. The accuracy, as relative recovery, presented a range from 83 to 120%. The method was robust after performing the Youden test. Compared to other methods, this work stands out due to its short extraction time and sample consumption

Disposable Pipette Extraction (DPX) Coupled to HPLC-DAD as an Alternative for the Determination of Phthalic Monoesters in Urine Samples

Phthalates are widely used in industry, but adverse effects on human health have been reported due to exposure to these chemicals. In the human body, they are metabolized into phthalic monoesters, which are used to monitor human exposure and assess risk. Urine is one of the main biological samples used, due to its easy access and collection, and also being the main elimination pathway for phthalates. Urine samples are complex; therefore, sample preparation is a critical step. Disposable pipette extraction (DPX) has not previously been reported for quantifying phthalates in urine and is here presented as a fast and low sample consumption method. A fully optimized RP-DPX method was developed for determination of free monomethyl phthalate, monobutyl phthalate, monobenzyl phthalate, and monoethylhexyl phthalate from urine samples. Analytical parameters of merit were obtained. The values of R2 were ≥0.9832, and the LOD and LOQ varied from 3.0 to 7.6 μg L−1 and 10 to 25 μg L−1, respectively. Intraday (n = 3) and interday (n = 9) precision were ≤13.6 and 15.6%. The accuracy, as relative recovery, presented a range from 83 to 120%. The method was robust after performing the Youden test. Compared to other methods, this work stands out due to its short extraction time and sample consumption

Alternative Green Extraction Phases Applied to Microextraction Techniques for Organic Compound Determination

The use of green extraction phases has gained much attention in different fields of study, including in sample preparation for the determination of organic compounds by chromatography techniques. Green extraction phases are considered as an alternative to conventional phases due to several advantages such as non-toxicity, biodegradability, low cost and ease of preparation. In addition, the use of greener extraction phases reinforces the environmentally-friendly features of microextraction techniques. Thus, this work presents a review about new materials that have been used in extraction phases applied to liquid and sorbent-based microextractions of organic compounds in different matrices

Application of Parallel-DPX for the determination of potential cancer biomarkers in urine samples using cork as natural extraction phase followed by GC–MS

Early-stage diagnosis is important to discover possible cancers in the human body. Bioanalytical methods are tools that allow for these studies. Biogenic amines are markers of several cancers in the organism, such as colorectal, lung and pancreatic. In this study, the determination of three important amines, putrescine (Put), spermidine (Spd) and cadaverine (Cad), was proposed. The use of Parallel-DPX-Cork is demonstrated for the first time in this type of application, permitting an effective methodology based on Green Analytical Chemistry (GAC) principles, with separation/quantification by GC–MS in human urine. Multivariate and univariate designs were applied, and the sample was prepared as follows: 10 min for alkaline hydrolysis under agitation with NaOH (2 mol L−1), added in a proportion of 1:8 (v/v) with the sample, at 60 °C, followed by 10 min of centrifugation at 3500 rpm, 5 min for derivatization with chloroformate isobutyl (180 µL), sample pH adjusted at 12; the extraction step was performed using 6 cycles, with 2 mL for each cycle (totalizing 12 mL of sample per experiment) and 25 mg of cork; the desorption step was carried out using 2 cycles with ethyl acetate (300 µL). LODs and LOQs were 15.2 and 50 ng mL−1 for all analytes. Intraday and interday precisions ranged from 2 to 22% and 9 to 28%, respectively. For the relative recoveries, the range was 62% to 130%. Five urine samples, collected from male and female volunteers, were analyzed using the methodology proposed, and the analytes concentrations obtained lower than LOQ and LOD

Natural sparkling guava wine: volatile and physicochemical characterization

ABSTRACT: Although different tropical fruit species have been used in the development of fermented beverages, there are only few references in the literature to the production of natural sparkling wines from fruits other than grapes. In this sense, the objective of the present research was the development and physicochemical and volatile characterization of a natural sparkling guava wine produced by the champenoise method. Volatile compounds were identified by gas chromatography coupled to mass spectrometry using the headspace solid-phase microextraction (HS-SPME) technique on samples. Eighty-nine volatile compounds were detected, of which 51 were identified. Esters were the predominant class of volatile compounds (a total of 26), followed by alcohols (10), terpenes (9), ketones (3), and acids (3). Volatile compounds with possible odoriferous activity were reported in the beverage, including ethyl octanoate, ethyl 5-hexenoate, phenethyl acetate, (E)-β-damascenone, (E)-ethyl cinnamate, 2-methyl butyl acetate, 3-methylbutanol, ethyl 3-(E)-hexenoate, and methyl 5-hexenoate. Natural sparkling guava wine produced showed a complex composition of fruity and floral aromas. Furthermore, the use of the champenoise method, traditionally applied to grapes, enabled the manufacture of a natural sparkling guava wine with physicochemical characteristics equivalent to those of sparkling wines made from grapes

Specific down-regulation of <i>Mybbp1a</i> in wild type ES cells blocks proliferation and induces activation of caspase-3.

<p>(<b>A</b>) ES cells were infected with the <i>Mybbp1a</i>-specific shRNA3 or with an empty non-target (NT) lentivirus vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039723#s2" target="_blank">Materials and Methods</a>), and the proliferation rate was measured starting two days after infection (0 h) every 24 h, using a cell counter. (<b>B</b>). At the end of the experiment (72 h) the cells were lysed and the extract immunoblotted with Mybbp1a-specific antibodies (anti-p160C) using Tubulin as loading control. (<b>C, D</b>). The extracts were also immunoblotted against Oct4 (for specificity of the down-regulation) and cleaved-Caspase 3 for apoptosis using Vinculin as loading control.</p

<i>MYBBP1A</i> down-regulation induces apoptosis.

<p>(<b>A</b>) HeLa cells were transiently transfected with siRNA1, 2 or 3, or with control High-GC or Medium-GC oligonucleotides, or with Lipofectamine only, for 48 h. The figure shows the determination of early apoptotic cells by flow cytometry (i.e. Annexin V positive and 7AAD-negative) (left panel, circled gate) 48 h post transfection. The histogram on the right shows the quantification in the various samples at the different times after transfection. In untreated cells only 1% of the cells were in apoptosis. (<b>B</b>) Western-blot analysis of HeLa cells transiently transfected for 48 h with CTL (untreated cells), LIPO (treated only with lipofectamine), HIGH GC (transfected with High-GC control), siRNA1 (transfected with MYBBP1A-specific siRNA1). The immunoblot was performed against MYBBP1A, active Caspase 3 and Caspase 9; tubulin is shown as loading control.</p