A novel liquid chromatography with quadrupole time-of-flight-tandem mass spectroscopy method for ultra-trace level identification and quantification of the genotoxic impurity 2,6-diamino-5-nitropyrimidin-4(3H)-one in valganciclovir hydrochloride.

In the present study, the main objective is to develop an analytical method for ultra-trace level measurement of 2,6-diamino-5-nitropyrimidin-4(3H)-one (DMNP) in valganciclovir hydrochloride (VAL) using liquid chromatography-quadrupole time-of-flight-tandem mass spectroscopy (LC-QTOF-MS/MS). In the early stages of guanine synthesis, DMNP is formed, and guanine is known to be the key starting material for the synthesis of VAL. Taking into consideration DMNP potential genotoxicity, this analytical method has been developed. This method is time saving and suitable for confirming the masses of parent and fragment ions by MS and MS/MS further fragmentation. An isocratic program and Acquity UPLC HSS cyano column (100 × 2.1 mm × 1.8 µm) were used to achieve optimal separation between VAL and the DMNP impurity. A 0.1% ammonia solution in Milli-Q water was used as mobile phase A, and methanol was used as mobile phase B in the ratio 90:10 v/v in isocratic mode. In accordance with the International Conference on Harmonization's requirements, the developed method was validated. The detection and quantification levels were found to be 0.028 and 0.083 ppm respectively. The DMNP impurity is linear from 0.083 to 1.245 ppm levels with correlation coefficient (R2 ) of 0.9960. The recoveries were found to be 97.0-107.9%.

LDL apheresis as an alternate method for plasma LPS purification in healthy volunteers and dyslipidemic and septic patients.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

A comprehensive multiplex PCR based exome-sequencing assay for rapid bloodspot confirmation of inborn errors of metabolism.

BACKGROUND: Tandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). The aim of the study was to evaluate the clinical utility of exome sequencing as a second tier screening method to assist clinical diagnosis of the newborn. METHODS: A novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn bloodspots positive by biochemical testing were analysed by PEARS assay to detect pathogenic mutations relevant to the IEM. RESULTS: In initial validation studies of genomic DNA samples, PEARS assay correctly detected 25 known mutations associated with 17 different IEM. Retrospective gene analysis of newborns with clinical phenylketonuria (PKU), identified compound heterozygote phenylalanine hydroxylase (PAH) gene mutations in eight of nine samples (89%). Prospective analysis of 211 bloodspots correctly identified the two true PKU samples, yielding positive and negative predictive values of 100%. Testing of 8 true positive MS MS samples correctly identified potentially pathogenic compound heterozygote genotypes in 2 cases of citrullinemia type 1 and one case each of methylmalonic acidemia, isobutyryl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency and glutaric acid type II and heterozygous genotypes in 2 cases of autosomal dominant methioninemia. Analysis of 11 of 12 false positive MS MS samples for other IEM identified heterozygous carriers in 8 cases for the relevant genes associated with the suspected IEM. In the remaining 3 cases, the test revealed compound heterozygote mutations in other metabolic genes not associated with the suspected IEM, indicating a misinterpretation of the original MS MS data. CONCLUSIONS: The PEARS assay has clinical utility as a rapid and cost effective second-tier test to assist the clinician to accurately diagnose newborns with a suspected IEM.

Use of serum citrulline concentrations from routine newborn screen as a biomarker for necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC), a leading cause of mortality and morbidity in preterm neonates, lacks a reliable biomarker. Citrulline is primarily produced by enterocytes and correlates with intestinal function. Serum citrulline concentration (CIT) is routinely measured in routine newborn screening (NBS). The purpose of the study is to test if CIT from NBS may predict the occurrence of NEC and whether it correlates with the time to full feeds (TTFF) and length of stay (LOS), serving as a biomarker of NEC and intestinal health. METHODS: In a retrospective case control study conducted on neonates with gestational age of 26-32 weeks, we compared CIT levels between cases (neonates with NEC) and controls (next-born neonate). NBS was collected within first 24 h, at day 5 and when the neonates achieved full feeds and were compared using non-parametric tests. RESULTS: There was no difference in CIT between the controls and cases on day 1 [11.42 (7.42-14.84 vs. 11.93 (6.85-18.8) µmol/L, p = 0.55], on day 5 [11.99 (7.99-16.55) vs. 13.70 (7.42-26.83) µmol/L, p = 0.05], or at full feeds [14.86 (6.85-25.69) vs. 15.7 (7.42-26.26) µmol/L, p = 0.87]. CIT on day 1 did not correlate with TTFF (r = 0.08, p = 0.53) or LOS (r = 0.23, p = 0.06), respectively). CONCLUSIONS: CIT from routine NBS does not serve as a biomarker to predict NEC in preterm neonates.

[Comparative Analysis of the Performance of Mascot and IdentiPy Algorithms on a Benchmark Dataset Obtained by Tandem Mass Spectrometry Analysis of Testicular Biopsies].

Proteome profiling of human testicular biopsies was performed using tandem mass spectrometry with electrospray ionization. Protein identification results were compared for the Mascot commercial search engine, the SearchGUI noncommercial package, and their analog IdentiProt based on the open-source IdentiPy algorithm (http://hg.theorchromo.ru/identipy). A feature of IdentiPy is an automatic optimization of MS/MS search parameters. A set of protein identifications obtained with IdentiPy was consequently greater by one third than the sets with the other search engines. For the first time, an IdentiPy/IdentiProt search was conducted within the Progenesis LC-MS framework, which allows spectrum alignment, and the proteome profile obtained with alignment was compared with that obtained using the ProteoWizard converter. A total of 16 human chromosome 18 proteins were identified, including the myelin basic protein, which is not characteristic of testicular tissue.

Dose- and time-dependent effects of triethylene glycol dimethacrylate on the proteome of human THP-1 monocytes.

Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin-based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time- and dose-dependent effects on the proteome of human THP-1 monocytes. The effects of different concentrations (0.07-5 mM) and exposure times (0-72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real-time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time-dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response.

Co-culture of human bone marrow mesenchymal stem cells and macrophages attenuates lipopolysaccharide-induced inflammation in human corneal epithelial cells.

Dry eye syndrome (DES) is considered as an ocular surface inflammatory disease. Previous studies have shown inflammation plays an important role in the progression and onset of DES. Co-culture of human bone marrow mesenchymal stem cells (HBMSCs) and macrophages showed immunomodulatory effects via regulation of cytokine regulation. Thus, the aim of this study was to investigate the effect of the interaction of these cells on in vitro DES model. The conditioned media (CM) from macrophages, HBMSCs, and HBMSCs + macrophages were treated to human corneal epithelial cells, which showed significant reduction in IL-1α and IL-1ß expression levels in HBMSCs + macrophages group. Moreover, the IL-1 Receptor Antagonist (IL-1RA) was highly expressed in the CM from the HBMSCs + macrophages group. Wounded eyes of mice were treated with IL-1RA at 0-100 ng/mL for 16 h, the wound size was reduced. The results of this study might lead to the identification of new therapeutic targets for DES.

An LC-MS/MS method to quantify acylcarnitine species including isomeric and odd-numbered forms in plasma and tissues.

Acylcarnitines are intermediates of fatty acid and amino acid oxidation found in tissues and body fluids. They are important diagnostic markers for inherited diseases of peroxisomal and mitochondrial oxidation processes and were recently described as biomarkers of complex diseases like the metabolic syndrome. Quantification of acylcarnitine species can become challenging because various species occur as isomers and/or have very low concentrations. Here we describe a new LC-MS/MS method for quantification of 56 acylcarnitine species with acyl-chain lengths from C2 to C18. Our method includes amino acid-derived positional isomers, like methacrylyl-carnitine (2-M-C3:1-CN) and crotonyl-carnitine (C4:1-CN), and odd-numbered carbon species, like pentadecanoyl-carnitine (C15:0-CN) and heptadecanoyl-carnitine (C17:0-CN), occurring at very low concentrations in plasma and tissues. Method validation in plasma and liver samples showed high sensitivity and excellent accuracy and precision. In an application to samples from streptozotocin-treated diabetic mice, we identified significantly increased concentrations of acylcarnitines derived from branched-chain amino acid degradation and of odd-numbered straight-chain species, recently proposed as potential biomarkers for the metabolic syndrome. In conclusion, the LC-MS/MS method presented here allows robust quantification of isomeric acylcarnitine species and extends the palette of acylcarnitines with diagnostic potential derived from fatty acid and amino acid metabolism.

Characterization and Quantification by LC-MS/MS of the Chemical Components of the Heating Products of the Flavonoids Extract in Pollen Typhae for Transformation Rule Exploration.

The Traditional Chinese Medicine herbs Pollen Typhae and Pollen Typhae Carbonisatus have been used as a hemostatic medicine promoting blood clotting for thousands of years. In this study, a reliable, highly sensitive method based on LC-MS/MS has been developed for differentiation of the heating products of total flavonoids in Pollen Typhae (FPT-N). Twenty three peaks were detected and 18 peaks have been structurally identified by comparing retention times, high resolution mass spectrometry data, and fragment ions with those of the reference substances and/or literature data. Additionally, 15 compounds have been quantified by multiple reaction monitoring in the negative ionization mode. It was found that the contents of the characterized compounds differed greatly from each other in FPT-N samples. Among them, the content of huaicarbon B significantly increased at first, while it decreased after heating for 25 min, which could be considered as the characteristic component for distinguishing FPT-N. The present study provided an approach to rapidly distinguish the differences of FPT-N samples. In addition, the actively summarized characteristic fragmentation might help deducing the structure of unknown flavonols compounds. Furthermore, transformation rules of flavonoids during the heating process in carbonisatus development could contribute to hemostatic therapeutic component exploration.

Analysis of benzo[c]phenanthridine alkaloids in Eschscholtzia californica cell culture using HPLC-DAD and HPLC-ESI-MS/MS.

Effective HPLC-DAD and HPLC-ESI-MS/MS methods have been developed for the analysis of eight benzo[c]phenanthridine alkaloids (sanguinarine, chelirubine, macarpine, chelerythrine, dihydrosanguinarine, dihydrochelirubine, dihydromacarpine and dihydrochelerythrine), which are important metabolites in Eschscholtzia californica cell culture. By adopting a ternary gradient pump system, the dihydro-form alkaloids hardly separable from each other could be successfully separated, and all the target alkaloids could be simultaneously quantified with the LOD values of 0.01-0.79 µg/mL and the LOQ values of 0.03-3.59 µg/mL. This HPLC-DAD method was further confirmed by HPLC-ESI-MS/MS system in multiple reaction monitoring mode. Each separated HPLC peak was identified as the target alkaloid, showing its relevant ionized molecule and selected fragment ion. By applying the established method, alkaloid production during the E. californica cell culture could be successfully monitored and some valuable information on its metabolism could be deduced.

Evaluation of Bacillus sp. MZS10 for decolorizing Azure B dye and its decolorization mechanism.

To evaluate decolorization and detoxification of Azure B dye by a newly isolated Bacillus sp. MZS10 strain, the cultivation medium and decolorization mechanism of the isolate were investigated. The decolorization was discovered to be dependent on cell density of the isolate and reached 93.55% (0.04 g/L) after 14 hr of cultivation in a 5 L stirred-tank fermenter at 2.0 g/L yeast extract and 6.0 g/L soluble starch and a small amount of mineral salts. The decolorization metabolites were identified with ultra performance liquid chromatography-tandem mass spectroscopy (UPLC-MS). A mechanism for decolorization of Azure B was proposed as follows: the C=N in Azure B was initially reduced to -NH by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent quinone dehydrogenase, and then the -NH further combined with -OH derived from glucose to form a stable and colorless compound through a dehydration reaction. The phytotoxicity was evaluated for both Azure B and its related derivatives produced by Bacillus sp. MZS10 decolorization, indicating that the decolorization metabolites were less toxic than original dye. The decolorization efficiency and mechanism shown by Bacillus sp. MZS10 provided insight on its potential application for the bioremediation of the dye Azure B.

Plasma niacin is inversely associated with hyperlipidemia in participants with diabetes among Chinese adults.

Evidence is limited regarding the association of plasma niacin with the risk of hyperlipidemia in participants with diabetes. We aimed to determine the relationship between plasma niacinamide/nicotinic acid and hyperlipidemia in participants with/without diabetes. Plasma niacinamide/nicotinic acid concentrations were measured using high-performance liquid chromatography-tandem mass spectroscopy. Multivariable logistic regression analyses were performed to evaluate the association between plasma niacin and hyperlipidemia in participants with diabetes and nondiabetes in a cross-sectional study. Compared to the first quartile, plasma nicotinamide, nicotinic acid, and niacin (nicotinamide plus nicotinic acid) were associated with a 54%, 50%, and 52% lower risk of hyperlipidemia in diabetic participants, respectively, but no significant association was observed in nondiabetic participants. These inverse associations persisted across subgroups stratified by sex, age, body mass index, smoking status, alcohol consumption, and physical activity. In addition, the fully adjusted odds ratios (95% confidence intervals) for hypercholesterolemia and hypertriglyceridemia among diabetic participants were 0.54 (0.38, 0.77) and 0.61 (0.44, 0.85), respectively, when comparing to the first quartile of plasma niacin concentrations (all Ptrend < .001). This study of 2647 participants observed that plasma niacin was inversely associated with hyperlipidemia in those with diabetes.

Development of dispersive solid phase extraction based on magnetic metal organic framework for the extraction of sunitinib in biological samples and its determination by high performance liquid chromatography-tandem mass spectrometry.

Herein, a simple, sensitive, and reliable dispersive solid phase extraction was reported for the efficient extraction of sunitinib from biological samples. To facilitate the extraction of the desired analyte from urine and plasma samples, magnetic MIL-101Cr (NH2) @SiO2 @ NiFe2O4 was synthesized by a hydrothermal method and applied as an effective sorbent during the extraction process. After adsorption of the drug using 10 mg of MIL-101Cr (NH2) @ SiO2 @ NiFe2O4 nanoparticles through vortexing (1 min), the sorbent was separatedfrom the sample solution using a magnet. To eluate the drug, the sorbent containing the sunitinib was contacted with 100 µL dimethylformamide. The eluent was analyzed by high performance liquid chromatography-tandem mass spectrometry. Reasonable validation data consisting of low limits of detection (0.14, 0.35, and 0.70 ng mL-1 in deionized water, plasma, and urine) and quantification (0.48, 1.2, and 2.4 ng mL-1 in deionized water, plasma, and urine, respectively), a wide linear range of the calibration curve (0.48-200, 1.2-200, and 2.4-100 ng mL-1 in deionized water, plasma, and urine, respectively) good extraction recovery (76 %), and low relative standard deviations for inter- and intra-day precisions (6.9 %) were obtained by the method. Eventually, the proposed procedure was effectively implemented on both plasma and urine samples, yielding successful outcomes.

Isolation of Momordica charantia seed lectin and glycosidases from the protein bodies: Lectin-glycosidase (ß-hexosaminidase) protein body membrane interaction reveals possible physiological function of the lectin.

Momordica charantia seeds are known to contain a galactose specific lectin that has been well characterized. Seed extracts also contain glycosidases such as the ß-hexosaminidase, α-mannosidase and α-galactosidase. In the present study, lectin was affinity purified from the seed extracts and protein bodies isolated by sucrose density gradient centrifugation. From the protein bodies, lectin was identified and ß-hexosaminidase was isolated by lectin affinity chromatography and subsequently separated from other glycosidases by gel filtration. In the native PAGE, the purified ß-hexosaminidase migrated as a single band with a molecular weight of ∼235 kDa and by zymogram analysis using 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide substrate it was confirmed as ß-hexosaminidase. Under reducing conditions in SDS-PAGE, the purified enzyme dissociated into three bands (Mr 33, 20 and 15 kDa). The prominent bands (20 and 15 kDa) showed immunological cross-reactivity with the human Hexosaminidase B antibody in a western blot experiment. In gel digestion of the purified enzyme, followed by proteomic analysis using tandom MS/MS revealed sequence identity as compared to the genomic sequence of the Momordica charantia with a score of 57 (24% sequence coverage). Additionally, by CD analysis the purified ß-hexosaminidase showed 39.1% of α-helix. Furthermore, secondary structure variations were observed in presence of substrate, lectin and at different pH values. Protein body membrane prepared from the isolated protein bodies showed a pH dependent interaction with the purified lectin and mixture of glycosidases.

Underpinning beneficial maize response to application of minimally processed homogenates of red and brown seaweeds.

Sap from the fresh seaweed Kappaphycus alvarezii (KA) has been reported to improve crop growth, quality, and stress alleviation. However, limited studies are reported for the minimally processed aqueous homogenates (MPHs) derived from dry seaweeds. The present investigation was envisaged to characterize the MPHs from the red seaweed KA and a brown seaweed Sargassum wightii (SW) and also assess the effect of foliar application on maize (Zea mays) crop performance when applied alone or in proportions ranging from 0% to 100%. Two doses (0.35% and 0.7%) were compared with control. Both the MPHs contained several compounds like retronecine, tyrosyl-glycine, hexyl 2-furoate, 1-phosphatidyl-1D-myo-inositol, 12-(2,3-dihydroxycyclopentyl)-2-dodecanone, and trihomomethionine and many others that have known bioactivity for enhancing plant growth and providing stress tolerance. Both doses of MPHs enhanced crop growth and yield; however, the best response was in general observed at a lower dose. The MPH of SW at 100% gave the highest seed yield at a lower dose, which was also on par with that obtained under a lower dose of 100% KA. Other combinations, 80:20 and 40:60 KA : SW, were also found to give comparable yields. The highest dose of 100% MPH of SW was found on par with control, a phenomenon that was investigated in detail with respect to metabolites and antioxidant profile in leaves as well as membrane modeling. Higher ROS and certain sugar and organic acids were observed in 100% MPH of SW at a higher dose, although none of the antioxidant enzymes were significantly affected, nor was there any change in membrane characteristics of the leaf with respect to control as well as lower dose. Improvements in the seed yield were attributed to improved photosynthate production on account of higher dry matter accumulation in the MPH-treated plants, which may also be attributed to the presence of bioactive compounds in the biostimulants. In the future, it is imperative to direct scientific investigations towards the quantification and identification of the most effective concentrations of these compounds within MPHs to optimize plant responses. The study indicated the beneficial use of the MPHs towards increasing crop production by employing optimum dose as foliar spray to crops.

Arginine-induced metabolomic perturbation in Streptococcus mutans.

BACKGROUND: Streptococcus mutans is a major pathogen responsible for dental caries. Arginine is a promising potential caries preventive agent which can inhibit the growth of S. mutans. However, the mechanism whereby arginine inhibits S. mutans growth remains unclear. AIM: To assess the impact of arginine-induced metabolomic perturbations on S. mutans under biofilm conditions. METHODS: We identified 5,933 and 7,413 ions in positive (ESI+) and negative (ESI-) electrospray ion modes, respectively, with a total of 11.05% and 11.58% differential ions subsequently detected in two respective modes. Further analyses of these metabolites led to identification of 8 and 22 metabolic pathways that were affected by arginine treatment in ESI+ and ESI- modes. RESULTS: Once or twice daily treatments of S. mutans biofilms with arginine resulted in reductions in biofilm biomass. Significant reductions in EPS production were observed following twice daily arginine treatments. Identified metabolites that were significantly differentially abundant following arginine treatment were associated with glycolysis metabolism, amino sugar and nucleotide sugar metabolism, and peptidoglycan synthesis. CONCLUSIONS: Arginine can reduce S. mutans biofilm growth and acid production by inhibiting glycolysis, amino sugar and nucleotide sugar metabolism, and peptidoglycan synthesis.

Proteomic dataset of Listeria monocytogenes exposed to sublethal concentrations of free and nanoencapsulated nisin.

The cellular proteins of L. monocytogenes exposed to free and liposome-encapsulated nisin at sublethal concentration were hydrolyzed by trypsin and examined by tandem mass spectrometry (MS/MS) to obtain proteomic data. In the present study, we use the STRING v11.05 database analyze the interactions among the 78 upregulated proteins from L. monocytogenes obtained after treatment with sublethal concentrations of free and nanoliposome-encapsulated nisin. As result, from the upregulated proteins by free nisin was determined a network with 140 edges with two relevant nodes, containing ribosomal proteins and transmembrane transport proteins (SecD and ABC transport system). These two sets of proteins present biological connection as a group, with strong interactions and are related to detoxification and other Listeria response mechanisms. In addition, a high amount of membrane proteins was identified in the free nisin treatment. On the other hand, in the interaction analysis of upregulated proteins by liposome-loaded nisin, was found 156 edges with a single protein network, the same observed in free nisin, related to ribosomal proteins. Therefore, according with this analysis, the encapsulation of nisin into liposomes cause upregulation of ribosomal and decrease of L. monocytogenes response proteins as compared with free nisin.

Twenty Years of Neonatal Screening for Congenital Adrenal Hyperplasia in North-Eastern Italy: Role of Liquid Chromatography-Tandem Mass Spectrometry as a Second-Tier Test.

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) based on 17-hydroxyprogesterone (17-OHP) concentration in dried blood spots has been taking place in North-Eastern Italy since 2001. Since 2017, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been introduced, for the first time in Italy, as a second-tier test. AIMS: Our study aims to evaluate, on the one hand, the effectiveness of the newborn screening for CAH after 20 years of testing and, on the other, the impact that the introduction of the second-tier test had on the diagnostic accuracy of the screening program. METHODS: Since 2001 dried blood spots taken from newborns have been screened with a time-resolved fluoroimmunoassay for 17-OHP determination. Over the years, the cut-off levels of 17-OHP were adjusted according to gestational age. Since 2017, a second-tier test in LC-MS/MS was introduced for samples displaying fluoroimmunoassay 17-OHP exceeding the cut-off. RESULTS: In total, 862,521 newborns have been screened over a period of 20 years. The total incidence of 21-hydroxylase deficiency (21-OHD) was 1:25,368, moreover, a case of 11-ß-hydroxylase deficiency was identified. All these diagnoses were genetically confirmed. The sensitivity and specificity of the screening program were 97% and 99.4%, respectively. The use of LC-MS/MS as a second-tier test significantly reduced the recall rate and increased the positive predictive value. CONCLUSIONS: Screening for CAH is useful in the neonatal diagnosis of a classic form of 21-OHD, allowing a precocious treatment of affected children. The introduction of an LC-MS/MS second-tier reduced the recall rate, avoiding unnecessary blood withdrawal and medical evaluations and preventing stress to families. Furthermore, it helped identify rarer forms of CAH.

Heavy-Metal Contents and the Impact of Roasting on Polyphenols, Caffeine, and Acrylamide in Specialty Coffee Beans.

The aim of this study was to determine the effect of roasting on the contents of polyphenols (PPH), acrylamide (AA), and caffeine (CAF) and to analyze heavy metals in specialty coffee beans from Colombia (COL) and Nicaragua (NIC). Samples of NIC were naturally processed and COL was fermented anaerobically. Green beans from COL (COL-GR) and NIC (NIC-GR) were roasted at two levels, light roasting (COL-LIGHT and NIC-LIGHT) and darker roasting (COL-DARK and NIC-DARK), at final temperatures of 210 °C (10 min) and 215 °C (12 min), respectively. Quantitative analyses of PPH identified caffeoylquinic acids (CQA), feruloylquinic acids, and dicaffeoylquinic acids. Isomer 5-CQA was present at the highest levels and reached 60.8 and 57.7% in COL-GR and NIC-GR, 23.4 and 29.3% in COL-LIGHT and NIC-LIGHT, and 18 and 24.2% in COL-DARK and NIC-DARK, respectively, of the total PPH. The total PPH contents were highest in COL-GR (59.76 mg/g dry matter, DM). Roasting affected the contents of PPH, CAF, and AA (p < 0.001, p < 0.011 and p < 0.001, respectively). Nickel and cadmium contents were significantly higher in the COL-GR than in the NIC-GR beans. Darker roasting decreased AA content, but light roasting maintained similar amounts of CAF and total PPH.

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.