Can we accurately measure the concentration of clinically relevant vitamin D metabolites in the circulation? The problems and their consequences

Wzrost zainteresowania oznaczeniami stężenia witaminy D przyczynił się do opracowania w ostatnich latach nowych testów immunochemicznych (manualnych oraz do automatycznych analizatorów). Do laboratoriów diagnostycznych wprowadza się również techniki dotychczas niestosowane w rutynowych oznaczeniach witaminy D, takie jak HPLC (wysokosprawna chromatografia cieczowa) oraz LC-MS/MS (tandemowa spektrometria mas sprzężona z wysokosprawną chromatografią cieczową). W związku z różnorodnością testów i metod pojawia się pytanie o ich wiarygodność. W niniejszej pracy przeglądowej opisano występujące w krwiobiegu metabolity witaminy D, przedstawiamy zalety i wady stosowanych metod oznaczania ich stężenia oraz omawiamy czynniki mające wpływ na wiarygodność wyników tych oznaczeń. (Endokrynol Pol 2013; 64 (3): 238–245)Increased interest in vitamin D measurements in clinical studies has contributed to the development in recent years of several new immunochemical assays (manual and for automatic analyzers). New methods, including HPLC (high performance liquid chromatography), and LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) have also been introduced into routine diagnostic laboratories. Because of the variety of assays and methods used, the question arises which one is the most accurate for the measurement of vitamin D metabolites concentration. In this review, we summarise the advantages and disadvantages of these methods, describe the complexity of vitamin D metabolites pattern in the circulation, and discuss the problem of accurate measuring its concentration. (Endokrynol Pol 2013; 64 (3): 238–245

Czy umiemy wiarygodnie mierzyć stężenia klinicznie ważnych metabolitów witaminy D? Problemy i ich konsekwencje

Wzrost zainteresowania oznaczeniami stężenia witaminy D przyczynił się do opracowania w ostatnich latach nowych testów immunochemicznych (manualnych oraz do automatycznych analizatorów). Do laboratoriów diagnostycznych wprowadza się również techniki dotychczas niestosowane w rutynowych oznaczeniach witaminy D, takie jak HPLC (wysokosprawna chromatografia cieczowa) oraz LC-MS/MS (tandemowa spektrometria mas sprzężona z wysokosprawną chromatografią cieczową). W związku z różnorodnością testów i metod pojawia się pytanie o ich wiarygodność. W niniejszej pracy przeglądowej opisano występujące w krwiobiegu metabolity witaminy D, przedstawiono zalety i wady stosowanych metod oznaczania ich stężenia oraz omówiono czynniki mające wpływ na wiarygodność wyników tych oznaczeń

Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study

Histone pre-mRNAs are cleaved at the 3′ end by a complex that contains U7 snRNP, the FLICE-Associated Huge protein (FLASH) and Histone pre-mRNA Cleavage Complex (HCC) consisting of several polyadenylation factors. Within the complex, the N-terminus of FLASH interacts with the N-terminus of the U7 snRNP protein Lsm11 and together they recruit the HCC. FLASH through its distant C-terminus independently interacts with the C-terminal SANT/Myb-like domain of Nuclear Protein, Ataxia-Telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S-phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium (H/D) exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both H/D exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo

A Strong Neutrophil Elastase Proteolytic Fingerprint Marks the Carcinoma Tumor Proteome

Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic data sets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint com- posed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly over-represented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent can- cer-specific proteolytic fingerprint detected. This se- quence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large frac- tion of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific pep- tides that are possible markers of tumor-infiltrating neu- trophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Data set 1) and PXD004249 (Data set 2). Our results indicate the value of peptide-wise anal- ysis of large global proteomic analysis data sets as op- posed to protein-wise analysis, in which outlier differen- tial peptides are usually neglected

Total testosterone to dihydrotestosterone ratio assessed by LC-MS/MS predicts a worse metabolic profile not only in PCOS patients

Objectives: Total testosterone/dihydrotestosterone ratio (TT/DHT) was found to determine metabolic risk in polycystic ovary syndrome (PCOS). The aim of this study was to analyze whether (TT/DHT) may be helpful in predicting metabolic risk not only in PCOS patients but also in healthy women. Material and methods: Total testosterone (TT), dihydrotestosterone (DHT), androstendione and dehydroepiandrosterone sulphate (DHEA-S) were measured by LC-MS/MS in 36 women with PCOS and in 29 age-matched controls without clinical hyperandrogenism. In all participants, anthropometric data, lipids, adipose tissue percent (%fat), HOMA-IR were also assessed. Results: The studied groups were not different in terms of age, BMI, waist circumference, %fat and HOMA-IR. In the patients group, mean TT and androstendione levels were significantly higher as compared to controls (1.4 nmol/L vs. 1.0 nmol/L, P < 0.001) and (6.6 nmol/L vs. 4.9 nmol/L, P < 0.01), respectively. In the patients group, mean TT/DHT ratio was significantly higher compared to controls (3.6 vs. 2.7, P < 0.01) and correlated with BMI (r = 0.37, P < 0.05), waist circumference (r = 0.44, P < 0.01), %fat (r = 0.30, P < 0.05), as well as with insulin levels (r = 0.38, P < 0.05) and HOMA-IR (r = 0.44, P < 0.05). The association between TT/DHT ratio and unfavorable metabolic parameters was also seen in controls. Conclusion: Total testosterone/dihydrotestosterone ratio assessed by LC-MS/MS correlates with a worse metabolic profile not only in PCOS patients, but also in healthy women

Molecular signatures associated with the treatment of triple-negative MDA-MB231 breast cancer cells with the histone deacetylase inhibitors JAHA and SAHA

Jay Amin Hydroxamic Acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA’s cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 M. JAHA’s lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction and autophagy mechanisms. In order to glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in anti-oxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs

Intrinsically disordered N-terminal domain of the Helicoverpa armigera Ultraspiracle stabilizes the dimeric form via a scorpion-like structure

Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NR