Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.

Czech Preschool Children’s Conceptions about Nature

This study focused on young children’s understanding of nature, an issue observed to be a research gap in the scientific community. The question “What nature?” is central to this research. Answers to this question were obtained from 342 children from 21 Czech kindergartens, and results showed 302 preschool and children (aged from 3 to 6.5 years) from the sample displayed a conceptual understanding of nature and expressed their ideas verbally. Qualitative content analysis and comparative analysis (nouns and verbs separately) were performed on the results. Most children interpreted nature through lists of objects or as a space or a concrete place, and most of the objects mentioned related to living nature. Children used verbs describing natural events more often than verbs describing their own or human activities in nature. The comparison between children’s, adults’, and pupils’ concepts of nature shows that children expressed their concepts in a similar, albeit not identical, manner to adults. They expressed the utilitarian and aesthetic value of nature, showed a scientific interest and an emotional connection to nature, and showed their joy in interacting with nature. Children understood nature more positively (no fear, aversion, or efforts to control nature) than adults. Czech children noticed more plants and mushrooms than Norwegian children. A similar percentage of Czech and Australian children included people on their lists of nature. Although certain similarities in children’s answers were noted, each child understands nature individually. Teachers should respect this fact and consider this in environmental and global education.

Extended of TEA: A 256 bits block cipher algorithm for image encryption

<p>This paper introduces an effective image encryption approach that merges a chaotic map and polynomial with a block cipher. According to this scheme, there are three levels of encryption. In the first level, pixel positions of the image are scuffled into blocks randomly based on a chaotic map. In the second level, the polynomials are constructed by taking N unused pixels from the permuted blocks as polynomial coefficients. Finally, the third level a proposed secret-key block cipher called extended of tiny encryption algorithm (ETEA) is used. The proposed ETEA algorithm increased the block size from 64-bit to 256-bit by using F-function in type three Feistel network design. The key schedule generation is very straightforward through admixture the entire major subjects in the identical manner for every round. The proposed ETEA algorithm is word-oriented, where wholly internal operations are executed on words of 32 bits. So, it is possible to efficiently implement the proposed algorithm on smart cards. The results of the experimental demonstration that the proposed encryption algorithm for all methods are efficient and have high security features through statistical analysis using histograms, correlation, entropy, randomness tests, and the avalanche effect.</p>

Regulation of suberin biosynthesis and Casparian strip development in the root endodermis by two plant auxins

The biological function of the auxin phenylacetic acid (PAA) is not well characterized in plants. Although some aspects of its biology; transport, signaling and metabolism have recently been described. Previous work on this phytohormone has suggested that PAA behaves in an identical manner to IAA (indole-3-acetic acid) in promoting plant growth, yet plants require greater concentrations of PAA to elicit the same physiological responses. Here we show that normalized PAA treatment results in the differential expression of a unique list of genes, suggesting that plants can respond differently to the two auxins. This is further explored in endodermal barrier regulation where the two auxins invoke striking differences in the deposition patterns of suberin. We further show that auxin acts antagonistically on Casparian strip (CS) formation as it can circumvent the CS transcriptional machinery to repress CS related genes. Additionally, altered suberin biosynthesis reduces endogenous levels of PAA and CS deficiency represses the biosynthesis of IAA and the levels of both auxins. These findings implicate auxin as a regulator of endodermal barrier formation and highlight a novel role for PAA in root development and differentiation.

Multiscale Entropy Analysis of Gravitational Waves

The first gravitational-wave (GW) signal was detected in the year 2015 indicating tiny distortions of spacetime caused by accelerated masses. We focused on the GW signals consisting of a peak GW strain of 1.0 × 1 0 − 21 that shows merging pairs of large masses. We applied the generalized entropy known as multiscale entropy to the GW interval time series recorded by different observatories (H1, L1, and V1). This enables us to investigate the behavior of entropies on different scales as a method of studying complexity and organization. We found that the entropies of GW interval data with similar physical properties make the identical manner in different scales. Moreover, the results reveal that the signals collected by each observatory have approximately a similar trend in the multiscale analysis results. According to our findings, although different signals have different values for short-range correlations, the long-range correlations are not noticeable in most of them.

A contribution to the debate about social recognition of marriage-like and family-like social phenomena

The paper elaborates on the relation between marriage and the family, on one hand, and marriage-like and family-like homosexual unions such as so-called homosexual marriage and same-sex union (or same-sex life partnership). With a brief theoretical introduction and a comparative overview of the manner in which this matter is regulated in other countries, certain solutions are analyzed from the Proposal Draft Law on Same-Sex Unions of Serbia (2021). It is indicated that it is no longer possible to equalize marriage and the family with the same-sex union because the content of these concepts is substantially different. The society is interested in protecting marriage and the family and to take special care of them since they perform extremely important social functions, whereas without some of them the survival of the society itself would be brought to question (the reproduction function). On the other hand, homosexual marriage and/or same-sex unions do not perform any social function, but are formed exclusively for the purpose of relatively longer-standing satisfaction of sexual needs of same-sex partners. Since the society and the state do not protect marriage only because sexuality is regulated within it, but because it legitimizes sexual relationships aimed at having children, in the event of homosexual marriage-like unions it should also be proceeded in an identical manner: solely those rights and obligations of partners deriving from a relatively longer-standing homosexual partnership should be legally regulated. Moreover, having in mind the principle that every man's right is limited by other people's rights, it is impermissible to give priority to the protection of the rights of same-sex partners for the purpose of longer-standing homosexual relationships over the protection of children's rights. Children need both a father and a mother (an ideal condition), and the family in which they will be socialized. In line with those homosexual marriage-like unions, neither adoption nor assisted insemination can be allowed.

Healthcare clinic and pharmacy chains in Kenya and Nigeria: How are they regulated, and what opportunities and risks do they present to national regulatory systems?

Introduction Regulating highly fragmented healthcare markets is a major challenge in low- and middle-income countries, with compliance often very poor. We investigated market consolidation through the emergence of clinic and pharmacy chains in Kenya and Nigeria and explored the regulatory opportunities and risks that chains present. Methods The study was conducted in Nairobi, Kenya and the Federal Capital Territory, Nigeria, with chains defined as businesses with four or more establishments. Data were collected through document reviews and 26 interviews with chain operators, professional associations and regulators between September and December 2018. A thematic analysis was conducted. Results While chain clinics and pharmacies still accounted for a relatively small share of these markets, their importance was growing. We identified both organic-growth chains which had started as single business locations and gradually expanded, and investor-driven chains that used external investment capital to rapidly buy out businesses or establish new ones. In both countries, chains were regulated in an identical manner to independents, with all branches treated as independent units. Regulators neither capitalized on opportunities presented nor guarded against potential risks. The chains’ brand visibility and centralized management systems were seen as having the potential to encourage self-regulation, and improve regulatory efficiency, but regulators were not exploiting this. Potential risks identified were chains gaining excessive market power, and encouraging greater commercialization of professional healthcare, putting public health at risk. Chains tended to have regular interaction with regulators, which some feared could lead to excessive influence over the regulatory process. Conclusion As the market shares of clinic and pharmacy chains continues to rise, regulatory bodies should modify regulatory requirements and process to take advantage of chains’ brand visibility and centralized management and quality assurance systems, as well as guarding against the risks of monopolization, commercialization and regulatory capture.

Strain-Specific Adaptations of Streptococcus mitis-oralis to Serial In Vitro Passage in Daptomycin (DAP): Genotypic and Phenotypic Characteristics

Viridans group streptococci (VGS), especially the Streptococcus mitis-oralis subgroup, are pivotal pathogens in a variety of invasive endovascular infections, including “toxic shock” in neutropenic cancer patients and infective endocarditis (IE). Previously, we showed that the serial in vitro passage of S. mitis-oralis strains in sublethal daptomycin (DAP) resulted in rapid, high-level and stable DAP-resistance (DAP-R), which is accompanied by distinct changes in several genotypic and phenotypic signatures: (1) the disappearance of two key membrane phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL); (2) increased membrane fluidity; (3) increased positive surface charge; (4) single nucleotide polymorphisms (SNPs) in two loci involved in CL biosynthesis (pgsA; cdsA); and (5) DAP hyperaccumulation. The current study examined these same metrics following in vitro serial DAP passages of a separate well-characterized S. mitis-oralis bloodstream isolate (SF100). Although some metrics seen in prior DAP post-passage strains were recapitulated with SF100 (e.g., pgsA SNPs, enhanced membrane fluidity), we observed the following major differences (comparing the parental versus post-passage variant): (1) no change in PG content; (2) reduced, but not absent, CL, with enhancement in phosphatidic acid (PA) content; (3) an unusual pattern of CL localization; (4) significantly decreased positive surface charge; (5) no difference in DAP accumulation; and (6) no cdsA SNPs. Thus, S. mitis-oralis strains are not “pre-programmed” phenotypically and/or genotypically to adapt in an identical manner during the evolution of the DAP-R.

P0127ROUTINE USE OF COMBINATION CD3/PAS STAIN IN EVALUATING RENAL ALLOGRAFT BIOPSIES FOR REJECTION RESULTS IN MORE ACCURATE BANFF SCORES FOR TUBULITIS

Background and Aims The BANFF Classification of renal allograft rejection consists of scores for cellular rejection which rely primarily on the evaluation of lymphocytic infiltration into tubules (t), non-atrophic interstitial inflammation (i), total inflammation (ti), and endotheliatis (v.) CD3/PAS is a combination stain that highlights T lymphocytes (CD3) and basement membrane (PAS). The original BANFF scoring was developed using a hematoxylin and eosin (H&E) stain only. This study compares scores for (t) using H&E versus CD3/PAS. Method 50 consecutive renal allograft biopsies were retrieved and on each case H&E and CD3/PAS combination stains (Ventana/Roche tissue Diagnostics Clone 2GV6 for CD3) were performed. One blinded group of matching CD3/PAS and H&E slides were evaluated and reviewed independently by two renal pathologists (Reviewer 1 and 2). Each case was graded in an identical manner following criteria designated for (t) as previously described in the Banff Classification for Renal Allograft Biopsies. Briefly (t)=0 no lymphocytes in 10 tubule cross section (tcx), (t)=1 between 1-4 lymphocytes per tcx, (t)=2 5-10 lymphocytes per tcx and (t)=3 greater than 10 lymphocytes per tcx. Cases were not graded for (v) as there were too few cases with endotheliatis to allow for statistical evaluation. The difference between CD3/PAS versus H&E scores were tested for significance using the Wilcoxon Signed Rank test. All p values were two-tailed and values of &lt;0.05 were considered statistically significant. Results Mean age was 44 years with a range of 9 -75 years. Standard deviation (SD) for age was 18 years. Gender distribution was 20% female and 80% male. 28 cases were graded as (t) = 0 in H&E versus 18 in the CD3/PAS group (59% versus 36%). 17 cases were graded as (t)=1 in H&E group versus 19 in the CD3/PAS group (36% versus 38% respectively). 2 cases were graded (t)=&gt;2 in H&E versus 12 cases in CD3/PAS groups (4% versus 24% respectively). In all categories of (t) there was a higher (t) score when using a CD3/PAS with 42% showing a one digit score increase and 8% showing a 2 digit increase. The difference in scoring for (t) between the H&E set versus the CD3/PAS set for both Reviewers 1 and 2 were statistically significant (p&lt;0.0001). Conclusion This study showed statistically significant higher tubulitis scores when using a CD3/PAS combination stain to grade allograft biopsies. Since (t) scores directly affect the diagnosis of cellular rejection, we propose routine use of CD3/PAS in addition to H&E when reviewing renal allograft biopsies. Compared with H&E, CD3/PAS makes recognition and quantification of lymphocytes crossing the tubular basement membrane easier to detect and therefore more accurate.

4447 Leptin supplementation prevents the loss of hypoglycemia-induced glucagon release following exposure to six days of severe caloric restriction in mice

OBJECTIVES/GOALS: We have recently shown that mice exposed to six days of 60% caloric restriction acutely display reduced hypoglycemia-induced glucagon release following refeeding, and that this effect is concurrent with low leptin levels. The current study was conducted to ascertain if leptin treatment during caloric restriction would reverse this effect. METHODS/STUDY POPULATION: Three groups of mice were used, an ad libitum (Ad-lib) fed group and two caloric restriction (CR) groups, one of which received twice daily leptin injection (0.5-1μg/g; IP) and the other vehicle (saline) during their caloric restriction. CR mice were placed on 60% caloric restriction for 6 consecutive days. Ad lib mice were housed in an identical manner but fed ad libitum during this same period. Following 6 days of restriction, CR mice were given ad lib access to food for 16 h. After the 16 h period of refeeding, both CR and ad lib mice began a 6 h fast which was immediately followed by a hypoglycemic insulin tolerance test (ITT). ITTs consisted of a variable dose of insulin intended to achieve a blood glucose of ~45 mg/dL within 60 minutes, at which time blood was collected for glucagon and corticosterone assays. RESULTS/ANTICIPATED RESULTS: The mean blood glucose levels during the ITT at 45 and 60 minutes post injection across all three groups were 46.8 + 3.1 and 37.0 + 2.4, respectively. There were no significant differences in glucose levels between the three groups at these two time points. As expected, saline treated CR mice displayed significantly reduced serum glucagon levels in response to the ITT relative to Ad-lib mice (23.5 + 10.9 vs. 91.7 + 20.8 pg/mL, p = 0.009). In contrast, leptin-treated CR mice maintained their hypoglycemia-induced glucagon response to the ITT (78.0 + 16.8 pg/mL, p>0.99 vs. Ad-lib group). In addition, although corticosterone levels in saline treated CR mice were numerically lower than in Ad-lib mice, this difference was not statistically significance (3928 + 277 vs. 4571 + 178 pg/mL, p = 0.179). DISCUSSION/SIGNIFICANCE OF IMPACT: Diabetes patients on insulin therapy often develop impaired hypoglycemic counter-regulation which can lead to life-threatening hypoglycemic complications. Our results suggest that leptin may hold promise as a therapeutic intervention for the prevention of impaired hypoglycemic counter-regulation in persons with diabetes.