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The performance of a mixture of CH3F/He (1/9) as a reaction gas for the determination of As in biological fluids using a quadrupole ICP-MS instrument has been explored. A simple (dilute-and-shoot) interference-free method has been developed to quantify As concentrations at trace and ultra-trace levels in matrices with a high Cl content. As+ reacts with CH3F (through CH3F addition, followed by HF elimination) with high efficiency forming AsCH2+ as the primary reaction product, which can be monitored at a mass-to-charge ratio of 89, free from the Cl-based interferents (e.g., 40Ar35Cl+ and 40Ca35Cl+) that hamper the monitoring of 75As+. Matrix effects are overcome by the use of Te as an internal standard and the addition of 3% v/v ethanol to all samples and calibration standard solutions. The method presented was validated by analysing a set of reference materials (blood, serum and urine) and by assessing As recovery from a set of real blood samples. With this method, the limit of detection was calculated to be 0.8 ng L−1 As, favourably comparable to the vast majority of values reported in the literature, even with those obtained using more sophisticated sector-field instrumentation. [ABSTRACT FROM AUTHOR]

Researchers have dedicated efforts to refining genetic part assembly techniques, responding to the demand for complex DNA constructs. The optimization efforts, targeting enhanced efficiency, fidelity, and modularity, have yielded streamlined protocols. Among these, Golden Gate cloning has gained prominence, offering a modular and hierarchical approach for constructing complex DNA fragments. This method is instrumental in establishing a repository of reusable parts, effectively reducing the costs and proving highly valuable for high-throughput DNA assembly projects. In this review, we delve into the main protocol of Golden Gate cloning, providing refined insights to enhance protocols and address potential challenges. Additionally, we perform a thorough evaluation of the primary modular cloning toolkits adopted by the scientific community. The discussion includes an exploration of recent advances and challenges in the field, providing a comprehensive overview of the current state of Golden Gate cloning., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

(1) Background: Cold exposure induces metabolic adaptations that can promote health benefits, including increased energy disposal due to lipid mobilization in adipose tissue (AT). This study aims to identify easily measurable biomarkers mirroring the effect of cold exposure on AT. (2) Methods: Transcriptomic analysis was performed in peripheral blood mononuclear cells (PBMCs) and distinct AT depots of two animal models (ferrets and rats) exposed to cold, and in PBMCs of cold-exposed humans. (3) Results: One week of cold exposure (at 4 °C) affected different metabolic pathways and gene expression in the AT of ferrets, an animal model with an AT more similar to humans than that of rodents. However, only one gene, Tlcd4 , was affected in the same way (overexpressed) in aortic perivascular and inguinal AT depots and in PBMCs, making it a potential biomarker of interest. Subsequent targeted analysis in rats showed that 1 week at 4 °C also induced Tlcd4 expression in brown AT and PBMCs, while 1 h at 4 °C resulted in reduced Tlcd4 mRNA levels in retroperitoneal white AT. In humans, no clear effects were observed. Nevertheless, decreased PBMC TLCD4 expression was observed after acute cold exposure in women with normal weight, although this effect could be attributed to short-term fasting during the procedure. No effect was evident in women with overweight or in normal-weight men. (4) Conclusions: Our results obtained for different species point toward TLCD4 gene expression as a potential biomarker of cold exposure/fat mobilization that could tentatively be used to address the effectiveness of cold exposure-mimicking therapies.

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells., Competing Interests: The authors confirm that there are no conflicts of interest.

The impact of the lockdown, during the period from March to June in 2020, upon the air quality of the Basque Country in northern Spain is analyzed. The evaluation accounts for the meteorology of the period. Daily and sub-daily analysis of aerosol and ozone records show that the territory was repeatedly affected by episodes of pollutants from outer regions. Three episodes of PM 10 and ten of PM 2.5 were caused by transported anthropogenic European sulfates, African dust, and wildland fires. The region, with a varied orographic climatology, shows high and diverse industrial activity. Urban and interurban road traffic of the region decreased by 49% and 53%, respectively, whereas industrial activity showed a lower reduction of 20%. Consequently, the average concentrations of NO 2 in the cities during the period fell to 12.4 µg·m -3 (-45%). Ozone showed up to five exceedances of the WHOAQG for the daily maximum 8-h average in both rural and urban sites, associated with transport through France and the Bay of Biscay, under periods of European blocking anticyclones. However, averages showed a moderate decrease (-11%) in rural environments, in line with the precursor reductions, and disparate changes in the cities, which reproduced the weekend effect of their historical records. The PM 10 decreased less than expected (-10% and -21%, in the urban and rural environments, respectively), probably caused by the modest decrease of industrial activity around urban sites and favorable meteorology for secondary aerosol formation, which could also influence the lower changes observed in the PM 2.5 (-1% and +3% at the urban and rural sites, respectively). Consequently, in a future low NOx traffic emission scenario, the inter-regional PM and ozone control will require actions across various sectors, including the industry and common pollution control strategies.

The use of genetically modified varieties tolerant to herbicides (HT varieties) and resistant to insects (Bt varieties) in combination with application of a broad-spectrum herbicide such as glyphosate could be an effective option for the simultaneous control of weeds and pests in maize. Nevertheless, the possible impact of these tools on nontarget arthropods still needs to be evaluated. In a field study in central Spain, potential changes in populations of canopy-dwelling arthropods in Bt maize under different weed management options, including glyphosate application, were investigated. Canopy-dwelling arthropods were sampled by visual inspection and yellow sticky traps. The Bt variety had no effect on any group of studied arthropods, except for the expected case of corn borers-the target pests of Bt maize. Regarding the effects of herbicide regimes, the only observed difference was a lower abundance of Cicadellidae and Mymaridae on yellow sticky traps in plots not treated with pre-emergence herbicides. This effect was especially pronounced in a treatment involving two glyphosate applications. The decrease in Cicadellidae and Mymaridae populations was associated with a higher density of weeds in plots, which may have hindered colonization of the crop by leafhoppers. These differences, however, were only significant in the last year of the study. The low likelihood of the use of glyphosate- and herbicide-tolerant varieties for weed control triggering important effects on the nontarget arthropod fauna of the maize canopy is discussed., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.

Safety assessments guard against unintended effects for human health and the environment. When new products are compared with accepted reference products by broad arrays of measurements, statistical analyses are usually summarised by significance tests or confidence intervals per endpoint. The traditional approach is to test for statistical significance of differences. However, absence or presence of significant differences is not a statement about safety. Equivalence limits are essential for safety assessment. We propose graphs to present the results of equivalence tests over the array of endpoints. It is argued that plots of the equivalence limit scaled difference (ELSD) are preferable over plots of the standardised effect size (SES) used previously for similar assessments. The ELSD method can be used either with externally specified equivalence limits or with equivalence limits estimated from (historical) data. The method is illustrated with two examples: first, environmental safety of MON810 Bt maize was assessed using field trial count data of arthropods; second, human safety of herbicide tolerant NK603 maize was assessed using haematological, biochemical and organ weight data from a 90-day rat feeding study. All assessed endpoints were classified in EFSA equivalence categories I or II, implying full equivalence or equivalence more likely than not., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae , so that neutral mutations were simply added and recombined in vivo With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivo IMPORTANCE Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae ., (Copyright © 2018 Martin-Diaz et al.)

The use of glyphosate, as a post-emergence broad-spectrum herbicide in genetically modified glyphosate-tolerant (GT) cotton, supposes a big change in weed management programs with respect to a conventional regime. Thus, alterations in arable flora and arthropod fauna must be considered when evaluating their potential impacts. A 3-year farm-scale study was conducted in a 2-ha GT cotton crop, in southern Spain, to compare the effects of conventional and glyphosate herbicide regimes on weed abundance and diversity and their consequences for ground-dwelling predators. Surveys reveal that weed density was relatively low within all treatments with a few dominant species, with significantly higher weed densities and modifications of the floristic composition in glyphosate-treated plots that led to an increase in the abundance of Portulaca oleracea and to a reduction in plant diversity. The activity-density of the main predatory arthropod taxa (spiders, ground beetles, rove beetles and earwigs) varied among years, but no significant differences were obtained between conventional and glyphosate herbicide regimes. However, significant differences between treatments were obtained for ground beetles species richness and diversity, being higher under the glyphosate herbicide regime, and a positive correlation with weed density could be established for both parameters. The implications of these findings to weed control in GT cotton are discussed.

Objectives: Cold exposure induces hyperphagia to counteract fat loss related to lipid mobilization and thermogenic activation. The aim of this study was investigate on the molecular mechanisms involved in cold-induced compensatory hyperphagia. Methods: We analyzed the effect of cold exposure on gene expression of orexigenic and anorexigenic peptides, and of leptin signaling-related genes in the hypothalamus of rats at different ages (1, 2, 4, and 6 months), as well as in ferrets. We also evaluated the potential of peripheral blood mononuclear cells to reflect hypothalamic molecular responses. Results: As expected, cold exposure induced hypoleptinemia in rats, which could be responsible for the increased ratio of orexigenic/anorexigenic peptides gene expression in the hypothalamus, mainly due to decreased anorexigenic gene expression, especially in young animals. In ferrets, which resemble humans more closely, cold exposure induced greater changes in hypothalamic mRNA levels of orexigenic genes. Despite the key role of leptin in food intake control, the effect of cold exposure on the expression of key hypothalamic leptin signaling cascade genes is not clear. In our study, cold exposure seemed to affect leptin signaling in 4-month-old rats (increased Socs3 and Lepr expression), likely associated with the smaller-increase in food intake and decreased body weight observed at this particular age. Similarly, cold exposed ferrets showed greater hypothalamic Socs3 and Stat3 gene expression. Interestingly, peripheral blood mononuclear cells (PBMC) mimicked the hypothalamic increase in Lepr and Socs3 observed in 4-month-old rats, and the increased Socs3 mRNA expression observed in ferrets in response to cold exposure. Conclusions: The most outstanding result of our study is that PBMC reflected the specific modulation of leptin signaling observed in both animal models, rats and ferrets, which points forwards PBMC as easily obtainable biological material to be considered as a potential surrogate tissue to perform further studies on the regulation of hypothalamic leptin signaling in response to cold exposure.

Perivascular adipose tissue (PVAT) surrounds blood vessels and releases paracrine factors, such as cytokines, which regulate local inflammation. The inflammatory state of PVAT has an important role in vascular disease; a pro-inflammatory state has been related with atherosclerosis development, whereas an anti-inflammatory one is protective. Cold exposure beneficially affects immune responses and, could thus impact the pathogenesis of cardiovascular diseases. In this study, we investigated the effects of one-week of cold exposure at 4°C of ferrets on aortic PVAT (aPVAT) versus subcutaneous adipose tissue. Ferrets were used because of the similarity of their adipose tissues to those of humans. A ferret-specific Agilent microarray was designed to cover the complete ferret genome and global gene expression analysis was performed. The data showed that cold exposure altered gene expression mainly in aPVAT. Most of the regulated genes were associated with cell cycle, immune response and gene expression regulation, and were mainly down-regulated. Regarding the effects on immune response, cold acclimation decreased the expression of genes involved in antigen recognition and presentation, cytokine signalling and immune system maturation and activation. This immunosuppressive gene expression pattern was depot-specific, as it was not observed in the inguinal subcutaneous depot. Interestingly, this depression in immune response related genes was also evident in peripheral blood mononuclear cells (PBMC). In conclusion, these results reveal that cold acclimation produces an inhibition of immune response-related pathways in aPVAT, reflected in PBMC, indicative of an anti-inflammatory response, which can potentially be exploited for the enhancement or maintenance of cardiovascular health.

Odour complaints are frequent nowadays, particularly nearby industrial facilities emitting odorous compounds. Among all compounds susceptible of causing odour annoyance, reduced sulphur compounds (RSC) were studied, due to their unpleasant odour and low odour threshold. RSC ambient air mixing ratios, meteorological conditions and population complaints were analysed in an area of complex topography in order to identify their potential sources. Mixing ratios of three compounds, dimethyl sulphide (DMS), carbon disulphide (CDS) and dimethyl disulphide (DMDS), were continuously monitored using an online gas chromatograph coupled with a mass spectrometer detector (GC-MSD), which was placed in a mobile air quality monitoring unit. Measurement campaigns were performed during 2012 and 2013 for periods of 7-25 days in an urban area (four campaigns, N = 1368) and an urban area surrounded by industrial activities (three campaigns, N = 564). During such campaigns, RSC mixing ratios were frequently above their odour thresholds, which did not always involve citizen complaints. Average RSC ambient air mixing ratios tended to be lower in the urban area (DMS 0.06-0.33, CDS 0.05-0.10, DMDS 0.07-0.30 μg m -3 ) than in the industry surrounded one (DMS 0.30-2.39, 0.05-0.18, DMDS 0.09-0.62 μg m -3 ). The DMS/DMDS mixing ratio was frequently above 1, being a paper mill one of the main sources of RSC in the area. DMS/DMDS ratios below 1 were also recorded, suggesting a waste treatment plant as the RSC source or older air masses coming from the paper mill.

This work investigates the possibilities of high-resolution continuum source graphite furnace molecular absorption spectrometry for the direct determination of Cl in solid samples via the CaCl molecule and measurement of its molecular absorption. The method proposed is based on addition of 400µg Ca as molecule-forming reagent and of 20µgPd as chemical modifier, which helps to stabilize the analyte and enhances sensitivity. The molecular spectrum for CaCl offers different lines with different limits of detection and linear ranges, which permitted to analyze solid samples with different Cl contents. The lowest limit of detection (0.75ng Cl, corresponding to 0.75µgg -1 for a typical sample mass of 1mg) could be achieved by combination of three of the most sensitive lines in the vicinity of 620.862nm, while the amplest linear range (up to 860ng Cl) was achieved by selection of the less sensitive line at 377.501nm. The method developed enabled the direct determination of Cl in solid samples using simple external calibration with aqueous standards. Good precision (5-9% RSD) and accuracy was attained in a series of certified samples of very different nature (i.e. coal, iron oxide, polyethylene, human hair, pine needles, rice flour and milk) and with very different Cl contents, ranging from about 50µgg -1 to 1% (w/w) Cl. The method appears as particularly useful for Cl determination in samples with elevated Ca contents, for which biased results with other diatomic molecules, such as AlCl or SrCl, may be obtained., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Peripheral blood mononuclear cells (PBMC) are increasingly used for nutrigenomic studies. In this study, we aimed to identify whether these cells could reflect the development of an obesogenic profile associated with the intake of high-fat (HF) diets. We analysed, by real-time RT-PCR, the dietary response of key genes related to lipid metabolism, obesity and inflammation in PBMC of control rats, rats fed a cafeteria or a commercial HF diet and rats fed a control diet after the intake of a cafeteria diet (post-cafeteria model). Cafeteria diet intake, which resulted in important overweight and related complications, altered the expressions of most of the studied genes in PBMC, evidencing the development of an obesogenic profile. Commercial HF diet, which produced metabolic alterations but in the absence of noticeably increased body weight, also altered PBMC gene expression, inducing a similar regulatory pattern as that observed for the cafeteria diet. Regulation of carnitine palmitoyltransferase I (Cpt1a) mRNA expression was of special interest; its expression reflected metabolic alterations related to the intake of both obesogenic diets (independently of increased body weight) even at an early stage as well as metabolic recovery in post-cafeteria animals. Thus, PBMC constitute an important source of biomarkers that reflect the increased adiposity and metabolic deregulation associated with the intake of HF diets. In particular, we propose an analysis of Cpt1a expression as a good biomarker to detect the early metabolic alterations caused by the consumption of hyperlipidic diets, and also as a marker of metabolic recovery associated to weight loss.

Because of the discovery of brown adipose tissue (BAT) in humans, there is increased interest in the study of induction of this thermogenic tissue as a basis to combat obesity and related complications. Cold exposure is one of the strongest stimuli able to activate BAT and to induce the appearance of brown-like (brite) adipocytes in white fat depots (browning process). We analyzed the potential of peripheral blood mononuclear cells (PBMCs) to reflect BAT and retroperitoneal white adipose tissue (rWAT) response to 1-wk cold acclimation (4°C) at different ages of rat development (1, 2, 4, and 6 mo). As expected, cold exposure increased fatty acid β-oxidation capacity in BAT and rWAT (increased Cpt1a expression), explaining increased circulating nonesterified free fatty acids and decreased adiposity. Cold exposure increased expression of the key thermogenic gene, Ucp1, in BAT and rWAT, but only in 1-mo-old animals. Additionally, other brown/brite markers were affected by cold during the whole developmental period studied in BAT. However, in rWAT, cold exposure increased studied markers mainly at early age. PBMCs did not express Ucp1, but expressed other brown/brite markers, which were cold regulated. Of particular interest, PBMCs reflected adipose tissue-increased Cpt1a mRNA expression in response to cold (in older animals) and browning induction occurring in rWAT of young animals (1 mo) characterized by increased Cidea expression and by the appearance of a high number of multilocular CIDE-A positive adipocytes. These results provide evidence pointing to PBMCs as an easily obtainable biological material to be considered to perform browning studies with minimum invasiveness., (Copyright © 2015 the American Physiological Society.)

Aim: To compare the ability of model for end-stage liver disease (MELD)-Na and Maddrey discrimination function index (DFI) to predict mortality at 30 and 90 d in patients with alcoholic hepatitis (AH)., Methods: We prospectively assessed 52 patients with AH. Demographic, clinical and laboratory parameters were obtained. MELD-Na and Maddrey DFI were calculated on admission. Short-term mortality was assessed at 30 and 90 d. Receiver operating characteristic curve analysis was performed., Results: Thirty-day and 90-d mortality was 44% and 58%, respectively. In the univariate analysis, sodium levels was associated with mortality at 30 and 90 d (P = 0.001 and P = 0.03). Child stage, encephalopathy, ascites, or types of treatment were not associated with mortality. MELD-Na was the only predictive factor for mortality at 90 d. For 30-d mortality area under the curve (AUC) was 0.763 (95%CI: 0.63-0.89) for Maddrey DFI and 0.784 for MELD-Na (95%CI: 0.65-0.91, P = 0.82). For 90-d mortality AUC was 0.685 (95%CI: 0.54-0.83) for Maddrey DFI and 0.8710 for MELD-Na (95%CI: 0.76-0.97, P = 0.041)., Conclusion: AH is associated with high short-term mortality. Our results show that MELD-Na is a more valuable model than DFI to predict short-term mortality.