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To keep in (active) reserve some technical entities equates with the action of stocking them; specifying that such an action should be interpreted as being a case of "waiting", therefore a retention "stock" for a limited period. Referring to such equipment, identical to those in working conditions, the retention may become, at any time, "by rotation", operating, according to a schedule established by the managerial forum. It is the case of a "mobile" retention, defined by a quantity, usually a constant of "the stock". Obviously, exceptions are those entities upon which the decider factor decides the intervention of maintenance. [ABSTRACT FROM AUTHOR]

Introducere: Neoplasmele mieloproliferative cronice cromozom Philadelphia negative (Ph-MPN) -policitemia vera (PV), trombocitemia esenţială (TE) şi mielofibroza primară (MP) - sunt caracterizate de o rată mare de complicaţii trombotice, parţial date de activarea trombocitară. Trombocitele mari sunt mai active, cu o funcţie procoagulantă crescută şi au rol patogenic demonstrat în tromboza arterială şi venoasă. În studiul nostru am încercat să stabilim dacă indicii trombocitari (MPV, PDW, P-LCR), furnizaţi de analizoarele de hematologie în hemoleucograma completă, sunt modificaţi la pacienţii diagnosticaţi cu Ph-MPN comparativ cu subiecţii sănătoşi. Material şi metodă: Hemoleucograma, ce a inclus şi indicii trombocitari, a fost determinată la 102 pacienţi Ph- MPN şi la 102 subiecţi sănătoşi cu ajutorul analizorului de hematologie Sysmex XS 1000i (metoda impedanţei), iar testele biochimice (glucoza şi parametrii metabolismului lipidic), pe analizorul Architect c8000. Prezenţa mutaţiei JAK2 V617F, la pacienţii cu Ph-MPN, a fost evidenţiată prin tehnica ARMS-PCR. Rezultate: Indicii de volum plachetari (IVP) măsuraţi prin metoda impedanţei, nu au fost semnificativ modificaţi la pacienţii cu Ph-MPN comparativ cu cei ai subiecţilor sănătoşi. Totuşi, am observat o moderată corelaţie între valoarea acestor indici şi prezenţa mutaţiei JAK2V617F, fără însă a se atinge o diferenţă semnificativă din punct de vedere statistic. IVP au fost crescuţi la micul subgrup de pacienţi trataţi cu anagrelide şi scăzuţi la cei trataţi cu simvastatin, comparativ cu pacienţii care nu au primit aceste terapii. Concluzii: Folosind metoda impedanţei în determinarea indicilor trombocitari, în studiul nostru nu am demonstrat o diferenţă semnificativă între valorile indicilor trombocitari la pacienţii cu Ph- MPN comparativ cu subiecţii sănătoşi. Sunt necesare studii ulterioare pentru a demonstra o posibilă corelaţie între indicii trombocitari şi mutaţia JAK2V617F, respectiv cu tratamentul cu anagrelide şi statine.

The impact of the COVID-19 pandemic demands effective prognostic tools for precise risk evaluation and timely intervention. This study utilized the APTASHAPE technology to profile plasma proteins in COVID-19 patient samples. Employing a highly diverse 2'-fluoro-protected RNA aptamer pool enriched toward proteins in the plasma samples from COVID-19 patients, we performed a single round of parallel selection on the derivation cohort and identified 93 discriminatory aptamers capable of distinguishing COVID-19 and healthy plasma samples. A subset of these aptamers was then used to predict 30-day mortality with high sensitivity and specificity in a validation cohort of 165 patients. We predicted 30-day mortality with areas under the curve (AUCs) of 0.91 in females and 0.68 in males. Affinity purification coupled with mass spectrometry analysis of the aptamer-targeted proteins identified potential biomarkers associated with disease severity, including complement system components. The study demonstrates the APTASHAPE technology as an unbiased approach that not only aids in predicting disease outcomes but also offers insights into gender-specific differences, shedding light on the nuanced aspects of COVID-19 pathophysiology. In conclusion, the findings highlight the promise of APTASHAPE as a valuable tool for estimating risk factors in COVID-19 patients and enabling stratification for personalized treatment management., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy ( 1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism., (© 2022 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

The molecular composition of blood is a signature of human health, reflected in the thousands of blood biomarkers known for human diseases. However, establishing robust disease markers is challenging due to the diversity of individual samples. New sequencing methods have simplified biomarker discovery for circulating DNA and RNA while protein profiling is still laborious and costly. To harness the power of high-throughput sequencing to profile the protein content of a biological sample, we developed a method termed APTASHAPE that uses oligonucleotide aptamers to recognize proteins in complex biofluids. We selected a large pool of 2'Fluoro protected RNA sequences to recognize proteins in human plasma and identified a set of 33 cancer-specific aptamers. Differential enrichment of these aptamers after selection against 1 μl of plasma from individual patients allowed us to differentiate between healthy controls and bladder cancer-diagnosed patients (91% accuracy) and between early non-invasive tumors and late stage tumors (83% accuracy). Affinity purification and mass spectrometry of proteins bound to the predictive aptamers showed the main target proteins to be C4b-binding protein, Complement C3, Fibrinogen, Complement factor H and IgG. The APTASHAPE method thus provides a general, automated and highly sensitive platform for discovering potential new disease biomarkers., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Cancer.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants., Competing Interests: Competing interest statement: Aarhus University has filed a patent on the sequence of RBD-PB6. J.V., L.C., D.M.D., and J.K. appear as inventors in the patent (pending)., (Copyright © 2021 the Author(s). Published by PNAS.)

PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated. Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme m Aconitase, accumulation of NAD + , and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches. We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors.)

Human thermogenic adipose tissue mitigates metabolic disease, raising much interest in understanding its development and function. Here, we show that human thermogenic adipocytes specifically express a primate-specific long non-coding RNA, LINC00473 which is highly correlated with UCP1 expression and decreased in obesity and type-2 diabetes. LINC00473 is detected in progenitor cells, and increases upon differentiation and in response to cAMP. In contrast to other known adipocyte LincRNAs, LINC00473 shuttles out of the nucleus, colocalizes and can be crosslinked to mitochondrial and lipid droplet proteins. Up- or down- regulation of LINC00473 results in reciprocal alterations in lipolysis, respiration and transcription of genes associated with mitochondrial oxidative metabolism. Depletion of PLIN1 results in impaired cAMP-responsive LINC00473 expression and lipolysis, indicating bidirectional interactions between PLIN1, LINC00473 and mitochondrial oxidative functions. Thus, we suggest that LINC00473 is a key regulator of human thermogenic adipocyte function, and reveals a role for a LincRNA in inter-organelle communication and human energy metabolism., Competing Interests: Competing Interests Statement The authors declare no competing interests.

Induced pluripotent stem cells (iPSCs) have recapitulated several aspects of Parkinson's disease (PD), but most iPSCs are derived from familial cases, which account for only about 15% of patients. Thus, while the emphasis has justifiably been on using iPSCs to model rare familial cases, models for the most common forms of PD are critically lacking. Here, we report the generation of an iPSC-based model of idiopathic PD (iPD) with or without RS1491923, which is a common risk variant in the LRRK2 locus. Consistent with GWA studies, we found large variability in our datasets. However, iPSC-derived neurons carrying the risk allele emerged for displaying subtle disturbances of cellular degradative systems, in line with familial PD models. We also observed that treatment with the LRRK2 inhibitor CZC-25146 slightly reduced a marker of aSYN pathology in all iPD lines. Future iPSC-based studies may need to be structured similarly to large GWA studies in order to obtain relevant statistical power. However, results from this pilot study suggest that iPSC-based modeling represents an attractive way to investigate idiopathic diseases.

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Induced pluripotent stem cells (iPSCs) hold great promise to model diseases, where the disease affected cell type is difficult to access. A major obstacle for the development of disease models is the lack of well characterized control iPSCs from old people not affected by such a disease. Furthermore, gene-editing approaches often require iPSCs from healthy donors, where pathogenic mutations can be inserted if patient material is not available. Here, we report the generation of an iPSC line (16423 #6) from a 77-year-old woman, who did not display any disease symptoms at the time, when the skin biopsy was taken., (Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.)

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders. MDS is frequently associated with deletions on chromosome 5q as well as aberrant DNA methylation patterns including hypermethylation of key tumor suppressors. We have previously shown that hypermethylation and silencing of the non-coding RNA VTRNA2-1 are correlated with poor outcomes in acute myeloid leukemia patients. In this study, we find that VTRNA1-2 and VTRNA1-3, both located on chromosome 5q, can be regulated and silenced by promoter DNA methylation, and that the hypomethylating agent 5-aza-2-deoxycytidine causes reactivation these genes. In normal hematopoiesis, we find that vault RNAs (vtRNAs) show differential methylation between various hematopoietic cell populations, indicating that allele-specific methylation events may occur during hematopoiesis. In addition, we show that VTRNA1-3 promoter hypermethylation is frequent in lower risk MDS patients and is associated with a decreased overall survival.

Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML) suggesting the presence of tumor suppressor(s) at the locus. However, definitive identification of putative tumor suppressor genes remains controversial. Here we show that a 106-nucleotide noncoding RNA vault RNA2-1 (vtRNA2-1), previously misannotated as miR886, could potentially play a role in the biology and prognosis of AML. vtRNA2-1 is transcribed by polymerase III and is monoallelically methylated in 75% of healthy individuals whereas the remaining 25% of the population have biallelic hypomethylation. AML patients without methylation of VTRNA2-1 have a considerably better outcome than those with monoallelic or biallelic methylation (n = 101, P = .001). We show that methylation is inversely correlated with vtRNA2-1 expression, and that 5-azanucleosides induce vtRNA2-1 and down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), whose activity has been shown to be modulated by vtRNA2-1. Because pPKR promotes cell survival in AML, the data are consistent with vtRNA2-1 being a tumor suppressor in AML. This is the first study to show that vtRNA2-1 might play a significant role in AML, that it is either mono- or biallelically expressed in the blood cells of healthy individuals, and that its methylation state predicts outcome in AML.

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

RNA-interference has been recognized as a powerful tool to control gene function and has been used for gene silencing by knocking down mRNA. Chemically modified RNAs, especially 2'-O-modification, successfully improved their physicochemical and pharmaceutical properties such as stability, nuclease resistance and delivery. Here, we report the synthesis of adenosine building blocks with different 2'-tethered modifications like aminoethyl and guanidinoethyl and show that they are compatible with RNAi function. They enhance the half life of the siRNA in serum suggesting that these modifications can enhance the pharmacokinetic properties and knock down activity of siRNAs in vivo.

The presenilin-1 (PS-1)/S 182 gene at chromosome 14q24.3 is, when mutated, the most common disease gene in autosomal dominant early-onset Alzheimer's disease. Substitution of methionine 146 of the gene product for either valine or leucine co-segregates with Alzheimer's disease with the age of onset in the late thirties or early forties. Here we describe a new substitution of methionine 146 for isoleucine that co-segregates with Alzheimer's disease with age of the onset in the early forties. All identified missense mutations in methionine codon 146 replace one hydrophobic amino acid (Met) with another (Val, Leu, Ile) and correspond to any nucleotide change at the first or third position of the codon. Second position mutations invariably lead to replacement of the hydrophobic methionine with a hydrophilic amino acid that may severely affect the function of the protein. The fact that no second position mutations have been identified so far may support the hypothesis that the protein product of PS-1 plays a crucial role during development.

A high-performance liquid chromatographic method for the analysis of coenzyme A thioesters which employs continuous-flow fast atom bombardment mass spectrometric detection is presented. The chromatographic system utilizes gradient elution with reversed-phase conditions using ammonium acetate-acetonitrile from both standard analytical (3.9 mm I.D.) and microbore (1 mm I.D.) columns. Applications to coenzyme A thioesters of various acyl group chain length (C2-C18) and functionality (-COOH, -OH, -C = C-) are described. The system is also applied to an in vitro enzyme reaction (crotonase) to directly follow the disappearance of substrate and appearance of product. The mass spectrometry of coenzyme A thioesters, their chromatographic behavior, system stability, and sensitivity of detection are discussed.