Genotyping of the Galactosemia GALT 591 A>G Mutation by Pyrosequencing

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S128-S129
Author(s):  
J B Bamford ◽  
J M Ramirez ◽  
W A Prewit ◽  
W J Robinson ◽  
K M Bennett
Keyword(s):  
Standard Deviation ◽  
Limit Of Detection ◽  
Pcr Amplification ◽  
The Body ◽  
Clinical Environment ◽  
Wild Type ◽  
High Resolution Melt ◽  
Clinical Sensitivity ◽  
Pyrosequencing Assay ◽  
Validation Parameters

Abstract Introduction/Objective This project aimed to design a pyrosequencing assay capable of genetically analyzing the presence of GALT 591 A>G mutation for the diagnosis of galactosemia. Galactosemia is an autosomal recessive disorder that affects enzyme activity of galactose-1-phosphate uridylytransferase (GALT). This enzyme is responsible for proper processing of galactose to glucose; if not broken down the accumulation of galactose in the body results in developmental delays, clouding of the eyes, speech difficulties, and intellectual disabilities. Pyrosequencing offers a valuable platform in assessing whether patients are wild type unaffected (A:A), heterozygous carrier (A:G), or homozygous affected (G:G) at the alleles within the GALT 591 locus by measuring percentage of the wild type A allele and the mutant G allele. Methods PCR primers were designed for the mutation locus, and PCR amplification was optimized for the target 77 bp product, with verification by gel electrophoresis. The pyrosequencing assay was designed and validated on the Qiagen PyroMark Q24 instrument. Control samples included commercially purchased DNA from the Coriell Cell Repository. Results One hundred and thirty-three reactions were utilized to establish the following assay validation parameters: accuracy (100% for G:G, 95% for A:G and A:A), precision (determined by mean and standard deviation with a standard deviation of 0.6 for G:G, 4.55 for A:G and 4.85 for A:A), and limit of detection (10 ng DNA pre-PCR and 2 µL of PCR product loaded). Twenty-six blinded samples were utilized to test assay clinical performance, as compared against a high resolution melt curve assay. Conclusion It was determined that detection of GALT 591 A>G via pyrosequencing is highly sensitive and specific with a clinical sensitivity of 100% and a clinical specificity of 95.83%. It was concluded that this assay could be applied in a clinical environment for GALT 591 A>G genotyping to aid in galactosemia diagnosis.

Erythroferrone Regulates Hepcidin Expression Independently of Matriptase 2

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3616-3616 ◽  
Author(s):  
Leon Kautz ◽  
Sharraya Aschemeyer ◽  
Victoria Gabayan ◽  
Tomas Ganz ◽  
Elizabeta Nemeth
Keyword(s):  
Limit Of Detection ◽  
Hematological Parameters ◽  
Isolated Hepatocytes ◽  
Bmp Signaling ◽  
The Body ◽  
Deficiency Anemia ◽  
Wild Type ◽  
Hepcidin Expression ◽  
Undetectable Plasma ◽  
Transmembrane Serine Protease

Abstract Introduction: The iron-regulatory hormone hepcidin regulates the body iron stores and its expression is repressed when erythropoietic activity intensifies to meet the iron requirements for erythropoiesis (e.g. during anemia). Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and the spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with mutations in transmembrane serine protease 6 (TMPRSS6) have iron-refractory iron deficiency anemia (IRIDA) but increased hepcidin production despite a severe anemia and elevated EPO levels. Recently, it has been suggested that matriptase-2 activity facilitates ERFE-mediated suppression of hepcidin. We therefore investigated the potential crosstalk between ERFE and Matriptase 2. Methods: We first measured serum ERFE concentration in Tmprss6-/- mice. To assess the contribution of ERFE to the phenotype of Tmprss6-/-mice, we next generated Tmprss6-/-mice with disrupted Erfe (Erfe+/- Tmprss6-/-; Erfe-/- Tmprss6-/- and Erfe+/+ Tmprss6-/-). To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from wild-type (WT) or Tmprss6-/- mice with conditioned medium from cells expressing recombinant ERFE or not. Results: While wild-type mice have undetectable plasma ERFE (below the 500 pg/ml limit of detection), plasma ERFE concentration was elevated in Tmprss6-/- to levels comparable to those of WT animals 24 hours after phlebotomy (~3 ng/ml) but was lower than ERFE levels in thalassemic mice (~10 ng/ml). Ablation of Erfe in Tmprss6-/- mice did not result in any change in hematological parameters, hepcidin expression and iron levels compared to Tmprss6-/- animals at 6 weeks of age. However, treatment of WT and Tmprss6-/-hepatocytes with ERFE resulted in a comparable suppression of hepcidin mRNA expression. Conclusion: Although matriptase-2 may dampen the BMP signaling under the influence of EPO, it is not part of the ERFE signaling pathway. Disclosures Ganz: Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor; Keryx Biopharmaceuticals: Consultancy. Nemeth:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor.

scholarly journals Fatal N-Ethylhexedrone Intoxication

2020 ◽  
Author(s):  
Ewa Domagalska ◽  
Laura Banaszkiewicz ◽  
Mateusz Kacper Woźniak ◽  
Marzena Kata ◽  
Beata Szpiech ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Data Interpretation ◽  
The Body ◽  
Limited Data ◽  
Limit Of Quantification ◽  
Low Concentrations ◽  
Validation Parameters ◽  
Plastic Bag ◽  
Cocaine Hydrochloride

Abstract N-Ethylhexedrone [2-(ethyloamino)-1-phenylhexan-1-one; α-ethylaminohexanophenone (NEH)] is one of the most recent synthetic cathinones that appeared on the illegal market in late 2015. The majority of information concerning the model of consumption of NEH and its impact on the body originates only from self-reports from gray literature websites and drug forums. There are only limited data associated with the concentrations of NEH in blood samples available in the literature. This article presents a case of fatal NEH intoxication and a method for the determination of this substance in whole blood. A 21-year-old man without any diagnosed diseases was admitted to the hospital due to disorientation, aggression and finally loss of consciousness. Hyperthermia (>41°C), tachycardia (>160 beats per minute), tachypnea (20 breaths per minute), blood pressure (110/60 mmHg) and acute kidney failure were diagnosed. After a few hours of hospitalization, the patient died. A plastic bag with a white powder was found in his underwear. Analysis of the powder by another laboratory revealed cocaine hydrochloride; however, no cocaine or its metabolites were found in the biological material upon testing in our laboratory. Therefore, re-analysis of the powder was performed, and NEH was identified. Liquid–liquid extraction followed by liquid chromatography-triple quadrupole-mass spectrometry (LC-MS/MS) analysis were used for the determination of NEH in blood. The validation parameters were as follows: calibration range 1–250 ng/mL, accuracy 106.5–109.9%, precision 3.5–6.3%, recovery 90.1–96.9%, limit of detection 0.07 ng/mL and limit of quantification 1 ng/mL. NEH was quantified in the blood at a concentration of 145 ng/mL. Additionally, amphetamine at low concentrations and 11-nor-9-karboksy-Δ9-tetrahydrokannabinol (THC-COOH) were detected. Our study provided information on the possible lethal concentration and toxidrome that clinicians can observe for NEH-intoxicated patients and can be helpful during the preparation of toxicology analysis reports for a court of law for proper data interpretation.

scholarly journals Validation of the spectrophotometric method for the dosing of some combined capsule

2021 ◽  
Vol 64 (4) ◽  
pp. 10-16
Author(s):  
Livia Uncu ◽  
◽  
Vladilena Evtodienco ◽  
Ecaterina Mazur ◽  
Elena Donici ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Spectrophotometric Method ◽  
Limit Of Detection ◽  
Limit Values ◽  
Minimum Error ◽  
Quantification Limit ◽  
Relative Standard ◽  
Validation Parameters ◽  
High Degree

Background: UV-Vis spectrophotometry remains the most accessible spectral method with a high degree of sensitivity and information. The advantage of the method consists in its universality, the ability to combine with other methods, the minimum error, as well as its economic efficiency. The objective of this study was the determination of some validation parameters for the spectrophotometric method of dosing piracetam and nicergoline in combined capsules. Material and methods: Agilent 8453 UV-Vis spectrophotometer, reference standards of piracetam and nicergoline, 0.1 M HCl methanolic solution. Validation of the spectrophotometric method according to the requirements of the ICH guide “Q2R1: For analytical procedures and validation”. Results: Linearity was investigated on concentration ranges 5-40 µg / mL. The regression (R2 ) values were 0.9998 for nicergoline and 0.998 for piracetam, respectively. The limit of detection was 1.737 µg / mL for nicergoline and 0.369 µg / mL for piracetam. Quantification limit values were also calculated as 5.265 and 1.118 µg / mL for nicergoline and piracetam, accordingly. The results obtained showed that the developed spectrophotometric method is accurate, precise and robust, because the value of the relative standard deviation was less than 1.0%. Conclusions: The developed spectrophotometric method showed specificity, linearity, accuracy, precision and robustness, and can be applied on the concentration range between 80-120% of the nominal value of the content of nicergoline and piracetam in the preparation.

Fast Method for the Determination of Residual Solvents in Radiopharmaceutical Products

2017 ◽  
Vol 68 (4) ◽  
pp. 666-670 ◽  
Author(s):  
Mirela Mihon ◽  
Catalin Stelian Tuta ◽  
Alina Catrinel Ion ◽  
Dana Niculae ◽  
Vasile Lavric
Keyword(s):  
Gas Chromatography ◽  
Control Method ◽  
Limit Of Detection ◽  
The Body ◽  
Biologically Active ◽  
Injection Technique ◽  
Fast Method ◽  
Residual Solvent ◽  
Residual Solvents

The aim of this work was the development and validation of a fast analytical method to determine the residual solvents content in radiopharmaceuticals such as: 18F-Fluorodeoxyglucose (18F-FDG), 18F-Fluoroestradiol (18F-FES), 18F-Fluorothymidine (18F-FLT),18F-Fluoromisonidazole (18F-FMISO). Radiopharmaceuticals are radioactive preparations for medical purposes used in nuclear medicine as tracers in diagnostic imaging and treatment of certain diseases. Positron Emission Tomography (PET) is a medical imaging technique that consists in introducing into the body of a small amount of a biologically active chemical compound labelled with a short lived positron-emitting radioisotope (18F, 11C, 68Ga). Residual solvents are critical impurities in radiopharmaceuticals that can affect labelling, stability and physicochemical properties of drugs. Therefore, the determination of these solvents is essential for quality control of radiopharmaceuticals. Validation of the control method for residual solvents by gas chromatography is referred by the European Pharmacopoeia using a special injection technique (head space). The parameters of the method, which comply with International Conference on Harmonization guidelines, are: accuracy, precision, linearity, limit of detection, limit of quantification and robustness. The proposed method (direct gas chromatography injection) proved to be linear, precise, accurate and robust. Good linearity was achieved for all the solvents and correlation coefficients (R2) for each residual solvent were found more than 0.99.

Roux-en-Y Gastrointestinal Bypass Promotes Activation of TGR5 and Peptide YY

2020 ◽  
Vol 20 (8) ◽  
pp. 1262-1267
Author(s):  
Haojun Yang ◽  
Hanyang Liu ◽  
YuWen Jiao ◽  
Jun Qian
Keyword(s):  
Body Weight ◽  
Food Intake ◽  
Metabolic Diseases ◽  
Peptide Yy ◽  
The Body ◽  
Wild Type ◽  
L Cells ◽  
Body Weights ◽  
Gastrointestinal Bypass ◽  
G Protein Coupled

Background: G protein-coupled bile acid receptor (TGR5) is involved in a number of metabolic diseases. The aim of this study was to identify the role of TGR5 after Roux-en-Y gastric bypass (GBP). Methods: Wild type and TGR5 knockout mice (tgr5-/-) were fed a high-fat diet (HFD) to establish the obesity model. GBP was performed. The changes in body weight and food intake were measured. The levels of TGR5 and peptide YY (PYY) were evaluated by RT-PCR, Western blot, and ELISA. Moreover, the L-cells were separated from wild type and tgr5-/- mice. The levels of PYY in L-cells were evaluated by ELISA. Results: The body weights were significantly decreased after GBP in wild type mice (p<0.05), but not tgr5-/- mice (p>0.05). Food intake was reduced after GBP in wild type mice, but also not significantly affected in tgr5-/- mice (p>0.05). The levels of PYY were significantly increased after GBP compared with the sham group (p<0.05); however, in tgr5-/- mice the expression of PYY was not significantly affected (p>0.05). After INT-777 stimulation in L-cells obtained from murine intestines, the levels of PYY were significantly increased in L-cells tgr5+/+ (p<0.05). Conclusion: Our study suggests that GBP up-regulated the expression of TGR5 in murine intestines, and increased the levels of PYY, which further reduced food intake and decreased the body weight.

scholarly journals Validation method for body-mounted sensor attachments

2020 ◽  
Vol 6 (2) ◽  
Author(s):  
Katharina Schmidt ◽  
David Hochmann
Keyword(s):  
Evaluation Method ◽  
Tracking System ◽  
The Body ◽  
Exploratory Research ◽  
Body Segment ◽  
Validation Method ◽  
Sensor Position ◽  
Validation Parameters ◽  
Measurement Units ◽  
Soft Tissue Artifact

AbstractSmall sensor devices like inertial measurement units enable mobile movement and gait analysis, whereby existing systems differ in data acquisition, data processing, and gait parameter calculation. Concerning the validation, recent studies focus on the captured motion and the influence of sensor positioning with respect to the accuracy of the computed biomechanical parameters in comparison to a reference system. Although soft tissue artifact is a major source of error for skin-mounted sensors, there are no investigations regarding the relative movement between the body segment and sensor attachment itself. The aim of this study is to find an evaluation method and to determine parameters that allow the validation of various sensor attachment types and different sensor positionings. The analysis includes the comparison between an adhesive and strap attachment variant as well as the frontal and lateral sensor placement. To validate different attachments, an optical marker-based tracking system was used to measure the body segment and sensor position during movement. The distance between these two positions was calculated and analyzed to determine suitable validation parameters. Despite the exploratory research, the results suggest a feasible validation method to detect differences between the attachments, independent of the sensor type. To have representative and statistically validated results, further studies that involve more participants are necessary.

scholarly journals Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution

2021 ◽  
Vol 22 (15) ◽  
pp. 8261
Author(s):  
Juraj Piestansky ◽  
Michaela Matuskova ◽  
Ivana Cizmarova ◽  
Dominika Olesova ◽  
Peter Mikus
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Background Electrolyte ◽  
Branched Chain Amino Acids ◽  
Fused Silica ◽  
Suitable Alternative ◽  
Enhanced Resolution ◽  
Validation Parameters ◽  
Branched Chain

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids—isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68–359.24 µM for valine, 91.76–95.67 µM for isoleucine, and 196.78–251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.

scholarly journals Development of a Methodology for Estimating the Ergosterol in Meat Product-Borne Toxigenic Moulds to Evaluate Antifungal Agents

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 438
Author(s):  
Micaela Álvarez ◽  
Alicia Rodríguez ◽  
Elena Bermúdez ◽  
Elia Roncero ◽  
María J. Andrade
Keyword(s):  
Antifungal Agents ◽  
Limit Of Detection ◽  
Meat Product ◽  
Meat Products ◽  
Ergosterol Biosynthesis ◽  
Array Detector ◽  
Meat Industry ◽  
Ergosterol Content ◽  
Validation Parameters ◽  
Toxigenic Moulds

Antifungal agents are commonly used in the meat industry to prevent the growth of unwanted moulds, such as toxigenic ones, on dry-cured meat products. For enhancing the application of antifungals, their mode of action must be evaluated. Their effect on the mould ergosterol content is one of the most studied ones, since it is the target site of some commercialised antifungals or of those that are in development. The aim of this study was to develop a methodology for determining how the antifungal agents used in the meat industry work. A method for analysing ergosterol was firstly developed using high-performance liquid chromatography with fluorescence detection coupled to a diode array detector (HPLC-FLD/DAD). The chromatographically optimised conditions (gradient and mobile phases) allowed us to reduce the time per analysis with respect to previously published methods up to 22 min. Withing the six checked extraction methods, method 5, showing the best mean recovery values (99.51%), the shortest retention time (15.8 min), and the lowest standard deviation values (9.92) and working temperature (60 °C), was selected. The limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. All the validation parameters corroborated the method’s suitability. Finally, its feasibility for evaluating the effect of a commercial antifungal preparation (AP) and different herbs that are frequently added to meat products on the ergosterol content of several toxigenic moulds was studied. Differences at the strain level were obtained in the presence of AP. Moreover, the addition of herbs significantly reduced the ergosterol content in Penicillium nordicum up to 83.91%. The developed methodology is thus suitable for screening the antifungals’ role in altering mould ergosterol biosynthesis before their application in real meat products.

scholarly journals Involvement of Huntingtin in Development and Ciliary Beating Regulation of Larvae of the Sea Urchin, Hemicentrotus pulcherrimus

2021 ◽  
Vol 22 (10) ◽  
pp. 5116
Author(s):  
Hideki Katow ◽  
Tomoko Katow ◽  
Hiromi Yoshida ◽  
Masato Kiyomoto
Keyword(s):  
Sea Urchin ◽  
The Body ◽  
Brdu Incorporation ◽  
Wild Type ◽  
Blastula Stage ◽  
Ciliary Beating ◽  
Marker Proteins ◽  
Disease Protein ◽  
Pattern Regulation ◽  
Hemicentrotus Pulcherrimus

The multiple functions of the wild type Huntington’s disease protein of the sea urchin Hemicentrotus pulcherrimus (Hp-Htt) have been examined using the anti-Hp-Htt antibody (Ab) raised against synthetic oligopeptides. According to immunoblotting, Hp-Htt was detected as a single band at around the 350 kDa region at the swimming blastula stage to the prism larva stage. From the 2-arm pluteus stage (2aPL), however, an additional smaller band at the 165 kDa region appeared. Immunohistochemically, Hp-Htt was detected in the nuclei and the nearby cytoplasm of the ectodermal cells from the swimming blastula stage, and the blastocoelar cells from the mid-gastrula stage. The Ab-positive signal was converged to the ciliary band-associated strand (CBAS). There, it was accompanied by several CBAS-marker proteins in the cytoplasm, such as glutamate decarboxylase. Application of Hp-Htt morpholino (Hp-Htt-MO) has resulted in shortened larval arms, accompanied by decreased 5-bromo-2-deoxyuridin (BrdU) incorporation by the ectodermal cells of the larval arms. Hp-Htt-MO also resulted in lowered ciliary beating activity, accompanied by a disordered swirling pattern formation around the body. These Hp-Htt-MO-induced deficiencies took place after the onset of CBAS system formation at the larval arms. Thus, Hp-Htt is involved in cell proliferation and the ciliary beating pattern regulation signaling system in pluteus larvae.

scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kyoko Yoshizaki ◽  
Akihiro Hirata ◽  
Hiroyuki Matsushita ◽  
Naohito Nishii ◽  
Mifumi Kawabe ◽  
...  
Keyword(s):  
Mutant Allele ◽  
False Negative ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Buccal Swab ◽  
Wild Type ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
Rflp Assay

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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