Absolute quantification
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Placental insufficiency-related complications are one of the leading causes of maternal and perinatal morbidity and mortality. This study investigated the quantification of placenta-specific microRNAs (miRNAs) in the maternal circulation during gestation in a cohort of women with normally progressing pregnancies, the differentiation between placental insufficiency-related complications and normally progressing pregnancies, and the differentiation between placental insufficiency and normally progressing pregnancies during the early stages of gestation. Both absolute and relative quantification of placenta-specific miRNAs (ie, miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525, and miR-526a) was determined in 50 women with normally progressing pregnancies, 32 with complicated pregnancies [21 with preeclampsia with or without intrauterine growth retardation (IUGR) and 11 with IUGR], and 7 women with pregnancies at various gestational stages who later developed preeclampsia and/or IUGR using real-time PCR and a comparative C(T) method relative to normalization factor (ie, geometric mean of ubiquitous miR-16 and let-7d). Both quantification approaches revealed significant increases in extracellular placenta-specific miRNA levels over time in women with normally progressing pregnancies; however, they were not able to differentiate between normally progressing and complicated pregnancies at the time of preeclampsia and/or IUGR onset. Nevertheless, significant elevation of extracellular miRNA levels was observed during early gestation (ie, within the 12th to 16th weeks) in pregnancies with later onset of preeclampsia and/or IUGR. Early gestation extracellular miRNA screening can differentiate between women with normally progressing pregnancies and those who may later develop placental insufficiency-related complications.
- MeSH
- komplikace těhotenství etiologie patologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- mikro RNA krev genetika MeSH
- placenta metabolismus MeSH
- placentární insuficience genetika MeSH
- preeklampsie etiologie patologie MeSH
- retrospektivní studie MeSH
- růstová retardace plodu etiologie patologie MeSH
- studie případů a kontrol MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AbstractA proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.
- Publikační typ
- časopisecké články MeSH
A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.
- MeSH
- biotest metody normy MeSH
- izotopové značení MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- peptidy chemická syntéza chemie MeSH
- proteiny analýza chemie MeSH
- proteolýza MeSH
- proteomika metody MeSH
- referenční standardy MeSH
- rozpouštědla MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
2 sv. + 1 CD-ROM
- MeSH
- chudoba MeSH
- hodnocení rizik MeSH
- přenos infekční nemoci MeSH
- rizikové faktory MeSH
- snížení rizika poškození MeSH
- socioekonomické faktory MeSH
- zdravotní stav MeSH
- znečištění životního prostředí MeSH
- Konspekt
- Lékařské vědy. Lékařství
- NLK Obory
- management, organizace a řízení zdravotnictví
- lékařství
- NLK Publikační typ
- publikace WHO
BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.
- MeSH
- hmotnostní spektrometrie * metody MeSH
- lidé MeSH
- metabolomika * metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Úvod: Unilaterální idiopatická kondylární hyperplazie mandibuly (UCH) je stav vedoucí k poruchám skusu či k obličejové asymetrii. Důležitou roli při plánování terapie hraje scintigrafie skeletu, pomocí níž hodnotíme růstovou aktivitu kondylu. Dosavadní metodiky stanovují relativní aktivitu vzhledem k protilehlému kondylu nebo k jiné kostní struktuře. Ověřovali jsme možnosti absolutní kvantifikace aktivity novou technologií xSPECT-Quant firmy Siemens. Metodika: SPECT/CT vyšetření hlavy po aplikaci 99mTc-HDP bylo provedeno u 21 pacientů (8 mužů, 13 žen, průměrný věk 31,7 ± 11,9 let) s kondylární hyperplazií. Modifikovanou iterativní rekonstrukcí OSCGM (Ordered Subset Conjugate Gradient Minimization) jsme získali hodnoty SUV (průměrné – SUVavg a maximální – SUVmax). Srovnávali jsme podíly hodnot SUV v oblastech zájmu nad oběma kondyly s dosud standardním hodnocením na našem pracovišti, tj. s poměry aktivit ze sumovaného transaxiálního řezu prostého tomogramu. Za signifikantní asymetrii byl považován jednostranný podíl na celkové aktivitě vyšší než 55 %. Výsledky: Rozdíl mezi poměrem z SUVavg a poměrem impulzů z konvenčního tomogramu nebyl signifikantní (p = 0,1907), což neplatilo pro SUVmax (p = 0,01417), kde byla patrná signifikantní proporcionální i systematická chyba. Patrná byla klesající tendence SUVmax s rostoucím věkem. Hodnoty SUVmax se při prokazatelné asymetrii nelišily signifikantně od těch ze zbytku souboru (p = 0,9654 pro relativněji aktivnější kondyly, p = 0,1654 pro relativně méně aktivní kondyly). Závěr: Hodnocení růstové aktivity při UCH má význam při plánování chirurgického řešení. Poměr hodnot SUVavg z nového systému xSPECT-Quant odpovídá konvenční metodice semikvantitativního porovnávání osteoblastické aktivity na SPECT, na rozdíl od poměru založeném na hodnotách SUVmax.
Introduction: Unilateral idiopathic condylar hyperplasia (UCH) is a condition that may cause malocclusion or facial asymmetry. Bone scintigraphy is important in planning of surgical treatment. It can assess the growing activity of the condyle. Conventional methodologies use the percentage difference between the activity of the two condyles or ratio of the condyle activity to a different bone reference area. We verified the possibility of absolute quantification of activity with the new Siemens xSPECT Quant technology. Methods: SPECT/CT examination of the head after 99mTc-HDP administration was performed in 21 patients (8 men, 13 women, mean age 31.7 ± 11.9 years) with condyle hyperplasia. SUV values (average – SUVavg and maximum –SUVmax) were obtained by modified iterative reconstruction OSCGM (Ordered Subset Conjugate Gradient Minimization). We compared the SUV values in the regions of interest over the two condyles with conventional assessement represented by counting the ratio of activities from the summated transaxial slices. Significant asymmetry was defined as a 55 % or higher condylar uptake. Results: The difference between the ratio of SUVavg and the ratio of counts from the conventional technique was not significant (p = 0.1907). The differences were found for SUVmax (p = 0.01417), where significant proportional and systematic error were evident. The decreasing tendency of SUVmax with increasing age was apparent. The SUVmax values did not differ condyles with asymetrically higher activity from those of the rest of the population (p = 0.9654, p = 0.1654 for a relatively less active condyle respectively). Conclusions: The evaluation of growth activity in UCH is important in planning the optimal treatment approach. The ratio of SUVavg values from the new xSPECT-Quant system corresponds to the conventional methodology of semi-quantitative comparison of osteoblastic activity on SPECT, unlike the ratio based on SUVmax values.
- Klíčová slova
- unilaterální kondylární hyperplazie,
- MeSH
- dospělí MeSH
- hyperplazie * diagnostické zobrazování MeSH
- klinická studie jako téma MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- nemoci mandibuly * diagnostické zobrazování MeSH
- počítačové zpracování obrazu MeSH
- SPECT/CT * metody využití MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Úvod a cíl studie: Uterinní NK (uNK) buňky jsou specializovanou subpopulací NK (natural killer) lymfocytů nacházejících se v endometriu. Hrají klíčovou roli v regulaci imunitní odpovědi a v procesu implantace embrya. Cílem této studie je retrospektivní analýza výsledků léčby metodou in vitro fertilizace (IVF) v souboru žen, které podstoupily imunofenotypizaci uNK buněk a na základě výsledků tohoto vyšetření byly, nebo nebyly léčeny imunomodulační terapií. Metody: Studie zahrnovala 122 pacientek, které podstoupily imunofenotypizaci uNK buněk v období od dubna do prosince 2023. Imunofenotypizace byla provedena metodou průtokové cytometrie. Pacientky byly roztříděny do čtyř skupin dle fenotypu uNK buněk: normální nálezy, nízké absolutní a relativní počty uNK (LOW-IMMUNE profil), nízké počty uNK v kombinaci s nežádoucím posunem směrem k cytotoxickému uNKdim imunofenotypu (MIXED-IMMUNE profil) a normální počty uNK, ale nežádoucí posun v poměru cytotoxických a regulačních uNK s cytotoxickým fenotypem (OVER-IMMUNE profil). Byly hodnoceny výsledky embryotransferu a výskyt potratů do ukončeného 12. týdne těhotenství v jednotlivých skupinách. Výsledky: Nejvyšší míra dosažení klinické gravidity byla nalezena v léčené skupině OVER- -IMMUNE (70 %), následované skupinou MIXED-IMMUNE (60 %). Skupina LOW-IMMUNE se od neléčené NORMAL skupiny signifikantně nelišila (p = 0,205). Nedostatečná imunitní aktivace (LOW-IMMUNE profil) byla signifikantně nejčastěji sdružena s prvotrimestrální těhotenskou ztrátou (p < 0,0001). Závěr: Tato studie přináší nové poznatky o potenciálu imunofenotypizace uNK buněk a následné imunomodulační terapie v léčbě poruch plodnosti. Ačkoli výsledky naznačují možné klinické přínosy, je zapotřebí dalšího výzkumu k potvrzení těchto zjištění a k objasnění mechanizmů, které vedou ke zlepšení výsledků léčby technikami asistované reprodukce.
Introduction and objective: Uterine NK (uNK) cells, a specialized subpopulation of natural killer (NK) lymphocytes located in the endometrium, play a crucial role in regulating the immune response and in the process of embryo implantation. This study aims to retrospectively analyze the outcomes of in vitro fertilization (IVF) treatment in a cohort of women who underwent uNK cell immunophenotyping with subsequent immunomodulatory therapy applied based on the results. Methods: The study included 122 patients who underwent uNK cell immunophenotyping between April and December 2023. Immunophenotyping was performed using flow cytometry. Patients were categorized into four groups according to their uNK cell phenotypes: normal findings, low absolute and relative numbers of uNK cells (LOW-IMMUNE profile), low numbers of uNK cells combined with the shift towards the cytotoxic uNKc dim immunophenotype (MIXED-IMMUNE profile), and normal numbers of uNK cells, but an undesirable shift in the ratio of cytotoxic to regulatory uNK cells towards the cytotoxic uNK dim phenotype (OVER-IMMUNE profile). Embryo transfer outcomes and the occurrence of miscarriages up to the 12th week of pregnancy were evaluated in each group. Results: The highest clinical pregnancy rate was observed in the treated OVER-IMMUNE group (70%), fol lowed by the MIXED-IMMUNE group (60%). The LOW-IMMUNE group did not differ significantly from the untreated NORMAL group (P = 0.205). Insufficient immune activation (LOW-IMMUNE profile) was significantly associated with first-trimester pregnancy loss (P < 0.0001). Conclusion: This study provides new insights into the potential benefits of uNK cell immunophenotyping and subsequent immunomodulatory therapy in treating fertility disorders. While the results indicate possible clinical advantages, further research is necessary to confirm these findings and elucidate the mechanisms leading to improved outcomes in assisted reproductive techniques.
- Klíčová slova
- uterinní NK buňky, imunofenotypizace lymfocytů, opakované selhání implantace,
- MeSH
- buňky NK MeSH
- endometrium cytologie MeSH
- fertilizace in vitro * MeSH
- habituální potrat MeSH
- imunofenotypizace MeSH
- imunomodulace MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- retrospektivní studie MeSH
- ženská infertilita * MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
