Developing BIOTEL: A Semi-Automated Spreadsheet for Estimating Telomere Length and Biological Age

Introduction: Telomere length (TL) is causally related to aging and several age-related diseases. Specifically, the abundance of short telomeres and the rate of telomere shortening are strong determinants of cell homeostasis. Thus, tools for analyzing and manipulating TL data can vastly improve research focused on aging. Aim: In this study, we developed a semi-automated worksheet, BIOTEL, to generate individual and group TL statistics and provide a crude estimation of biological age.Results: Data from the Telomere Length Database Project (TLDP) were implemented to the spreadsheet to produce TL statistics. 150 participants were included, and their age was from 21 to 82 years, and the sex distribution ratio was 52.3%: 47.7% (male: female). Initially, we analyzed the fluorescence intensities of telomeres that were measured on metaphase spread leukocytes using three-dimensional (3D) quantitative-fluorescent in situ hybridization (Q-FISH) procedures (3D DNA FISH) with a (C3TA2)3 peptide nucleic acid (PNA) probe. Raw data of fluorescence intensities, demographic data and medical records from the participants were imported into the worksheet. Basic statistical analyses of TL data were provided through BIOTEL, including TL percentiles, specialized charts for TL distribution including the percentage of critically short telomeres (< 3,000 kilobases), individual telomere profiles, and graphs of biological age vs. chronological age.Conclusion: BIOTEL ver. 2.4 is a functional semi-automated worksheet that calculates a wide range of TL statistics, thus a useful tool with applications in research of telomeres and biological age estimation

Notch signaling pathway in preeclamptic complicated placentas

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p=0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p=0.029), of NICD3 protein levels (p=0.028) and DLL3 mRNA levels (p=0.041) with birth weight centile, and of HEY2 transcript levels with parity (p=0.034) and mode of delivery (p=0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE.Η προεκλαμψία αποτελεί μια από τις κυριότερες αιτίες μητρικής και εμβρυικής/νεογνικής νοσηρότητας και θνητότητας και επιπλέκει το 3-5% των κυήσεων. Προηγούμενες μελέτες έδειξαν ότι μέλη του σηματοδοτικού μονοπατιού Notch (Notch signaling pathway) εκφράζονται στον πλακούντα και παίζουν σημαντικό ρόλο στην ομαλή ανάπτυξη του ενεργοποιώντας μεταγραφικούς παράγοντες οι οποίοι αλλάζουν την έκφραση γονιδίων στόχων. Διαταραχές στη λειτουργία του μονοπατίου Notch σχετίζονται με παθολογική πλακουντοποίηση. Σκοπός της μελέτης αυτής είναι να εξετάσει την έκφραση των υποδοχέων (receptors) NOTCH1,-2,-3,-4, των συνδετών (ligands) DLL1,-3,-4, JAG1,-2 και των γονιδίων στόχων (target genes) HEY1,-2 σε πλακούντες κυήσεων με προεκλαμψία. Εξετάστηκαν δείγματα πλακουντιακού ιστού από 20 προεκλαμπτικές και από 20 φυσιολογικές κυήσεις. Τα επίπεδα mRNA των υπό εξέταση μορίων μετρήθηκαν με ποσοτική (quantitative) Real-Time PCR (qRT-PCR) ενώ η πρωτεϊνική έκφραση της ενδοκυττάριας περιοχής (intracellular domain) των NOTCH2 (NICD2) και NOTCH3 (NICD3) εκτιμήθηκαν με Western Blot (WB). Τα αποτελέσματα μας έδειξαν ότι οι υποδοχείς Notch-1 και Notch-4 αλλά και ο συνδέτης Dll-1 δεν εκφράζονται στους πλακούντες γυναικών με προεκλαμψία ή με φυσιολογική εγκυμοσύνη. Ωστόσο, τα επίπεδα έκφρασης mRNA των NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 ήταν μειωμένα στα παθολογικά δείγματα σε σχέση με τα φυσιολογικά (p<0.01). Η ανάλυση με WB επιβεβαίωσε ότι τα επίπεδα πρωτεϊνικής έκφρασης των NICD2 και NICD3 ήταν επίσης ελαττωμένα στα δείγματα της προεκλαμψίας (p=0.014 και p<0.001, αντίστοιχα). Περαιτέρω στατιστική ανάλυση έδειξε α) μη έκφραση σε γονιδιακό επίπεδο του υποδοχέα NOTCH3 στις εγκύους εκείνες που κάπνιζαν και στη διάρκεια της εγκυμοσύνης σε σχέση με εκείνες που το έκοψαν (p=0.029) και β) στις περιπτώσεις με προεκλαμψία και βάρος νεογνού μικρότερο από την 5η εκατοστιαία θέση παρατηρήσαμε υψηλότερα επίπεδα πρωτεΐνης του NICD-3 (p=0.028) και υψηλότερα επίπεδα mRNA του Dll-3 (p=0.041). Τα αποτελέσματα της μελέτης μας δείχνουν ότι το σηματοδοτικό μονοπάτι Notch αποτελεί σημαντικό μέρος της παθογένειας αυτής της επιπλοκής της εγκυμοσύνης. Περαιτέρω μελέτες απαιτούνται με σκοπό να διερευνηθεί βαθύτερα και να καθοριστεί ο ρόλος των εξετασθέντων μορίων στην προεκλαμψία και να μελετηθεί η δυνητική τους χρήση στην πρόβλεψη και τη διαγνωστική και θεραπευτική προσέγγιση της νόσου

Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans

Summary: C. elegans is a bacteria-eating soil-dwelling nematode. Typical cultivation of laboratory-reared populations occurs on bacteria-covered solid media, where they move along with sinusoidal undulations. Nematodes decelerate when they encounter food. Dopaminergic and serotonergic neurotransmission regulate this behavior. Here, we describe the procedure for determining food-dependent locomotion rate of fed and fasting nematodes. We detail steps for assay plate preparation, C. elegans synchronization, and assessment of locomotion. The behaviors we describe provide information regarding the animal’s physiological neuronal and muscular function.For complete details on the use and execution of this protocol, please refer to Petratou et al. (2023)1 and Sawin et al. (2000).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Downregulation of notch signaling pathway in late preterm and term placentas from pregnancies complicated by preeclampsia.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE

Модулирование процесса старения в организме человека: последние достижения в области биомаркеров для диагностики и лечения

Aging is a complex biological process. Main factors that interplay in the aging process include free radicals and oxidation, insulin and insulin growth factors, sirtuins, mTOR, microbiome, lack of micronutrients, and declining proteasome activity which lead to cellular damage. Damaged cells are being replaced by somatic stem cells which proliferate to generate new cells. For each cell replication the telomeres of the related stem cells become shorter and this is the basic factor that modulates aging. Telomere length shortens with age and leads to senescence. Shorter telomeres are associated with increased incidence of aging related diseases and shorter lifespan. The percentage of short telomeres and rate of telomere shortening predicts longevity in mammals. Measurement of single telomeres through Q-FISH is the only reliable method to evaluate single telomere length and percentage of short telomeres. Repeated measurements at a distance of 6 months or a year can reveal the rate of change of the short telomeres, and response of patients to treatments, lifestyle, diet, supplementation and exercise modifications. A natural product telomerase activator TA-65, an astragalus extract, has been found to lengthen telomere in humans. By experimenting with different combinations of cycloastragenol (astragalus extract active molecule) we’ve able to increase telomerase activation in relation to the control cellsСтарение – это сложный биологический процесс. Основные факторы, участвующие в процессе старения и приводящие к повреждению клеток, включают свободные радикалы, процессы окисления инсулина, инсулиноподобные факторы роста, сиртуины, mTOR, микробиом, отсутствие микроэлементов и снижение активности протеасом. Поврежденные клетки заменяются соматическими стволовыми клетками, которые размножаются, генерируя новые клетки. При каждой клеточной репликации теломеры родственных стволовых клеток становятся короче, и это основной фактор, который модулирует старение. Длина теломер сокращается с возрастом и приводит к старению. Более короткие теломеры ассоциируются с увеличением заболеваний, связанных со старением и сокращением продолжительности жизни. Данные о процентном содержании коротких теломер и скорости сокращения теломер позволяют предсказать продолжительность жизни млекопитающих. Измерение отдельных теломер с помощью Q-FISH является единственным надежным методом оценки длины теломеры и доли коротких теломер. Повторные измерения через 6 месяцев или год могут выявить скорость изменения длины коротких теломер и реакцию пациентов на лечение, изменение образа жизни, диеты, пищевых добавок и физической активности. Было обнаружено, что натуральный продукт – активатор теломеразы TA-65, экстракт астрагала, удлиняет теломеры людей. Экспериментируя с различными комбинациями циклоастрагенола (активная молекула экстракта астрагала), мы смогли достичь активации теломеразы по сравнению с контрольными клеткам

Модулирование процесса старения в организме человека: последние достижения в области биомаркеров для диагностики и лечения

Aging is a complex biological process. Main factors that interplay in the aging process include free radicals and oxidation, insulin and insulin growth factors, sirtuins, mTOR, microbiome, lack of micronutrients, and declining proteasome activity which lead to cellular damage. Damaged cells are being replaced by somatic stem cells which proliferate to generate new cells. For each cell replication the telomeres of the related stem cells become shorter and this is the basic factor that modulates aging. Telomere length shortens with age and leads to senescence. Shorter telomeres are associated with increased incidence of aging related diseases and shorter lifespan. The percentage of short telomeres and rate of telomere shortening predicts longevity in mammals. Measurement of single telomeres through Q-FISH is the only reliable method to evaluate single telomere length and percentage of short telomeres. Repeated measurements at a distance of 6 months or a year can reveal the rate of change of the short telomeres, and response of patients to treatments, lifestyle, diet, supplementation and exercise modifications. A natural product telomerase activator TA-65, an astragalus extract, has been found to lengthen telomere in humans. By experimenting with different combinations of cycloastragenol (astragalus extract active molecule) we’ve able to increase telomerase activation in relation to the control cellsСтарение – это сложный биологический процесс. Основные факторы, участвующие в процессе старения и приводящие к повреждению клеток, включают свободные радикалы, процессы окисления инсулина, инсулиноподобные факторы роста, сиртуины, mTOR, микробиом, отсутствие микроэлементов и снижение активности протеасом. Поврежденные клетки заменяются соматическими стволовыми клетками, которые размножаются, генерируя новые клетки. При каждой клеточной репликации теломеры родственных стволовых клеток становятся короче, и это основной фактор, который модулирует старение. Длина теломер сокращается с возрастом и приводит к старению. Более короткие теломеры ассоциируются с увеличением заболеваний, связанных со старением и сокращением продолжительности жизни. Данные о процентном содержании коротких теломер и скорости сокращения теломер позволяют предсказать продолжительность жизни млекопитающих. Измерение отдельных теломер с помощью Q-FISH является единственным надежным методом оценки длины теломеры и доли коротких теломер. Повторные измерения через 6 месяцев или год могут выявить скорость изменения длины коротких теломер и реакцию пациентов на лечение, изменение образа жизни, диеты, пищевых добавок и физической активности. Было обнаружено, что натуральный продукт – активатор теломеразы TA-65, экстракт астрагала, удлиняет теломеры людей. Экспериментируя с различными комбинациями циклоастрагенола (активная молекула экстракта астрагала), мы смогли достичь активации теломеразы по сравнению с контрольными клеткам

Determination of DNA damage and telomerase activity in stanozolol-treated rats

Anabolic androgenic steroids (AAS) are performance -enhancing drugs commonly abused by atheletes. Stanozolol is a synthetic testosterone-derived anabolic steroid. Although it is well known that AAS have several side-effects, there are only few toxicological studies available on the toxic effects and mechanisms of action of stanozolol. The aim of this study was to investigate the genotoxic effects of stanozolol and to determine its effects on telomerase activity in Sprague-Dawley male rats. For this purpose, 34 male rats were divided into 5 groups as follows: i) the control group (n=5); ii) the propylene glycol (PG)-treated group (n=5); iii) the stanozolol-treated group (n=8); iv) the PG-treated group subjected to exercise (n=8); and v) the stanozolol-treated group subjected to exercise (n=8). PG is used as a solvent control in our study. Stanozolol (5 mg/kg) and PG (1 ml/kg) were injected subcutaneously 5 days/week for 28 days. After 28 days, the animals were sacrificed, and DNA damage evaluation (comet assay) and telomerase activity assays were then performed using peripheral blood mononuclear cells (PBMCs). Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS kit. The results of this study revealed that stanozolol treatment induced DNA damage, while exercise exerted a protective effect. Stanozolol treatment without exercise stimulation was associated with a significant increase in telomerase activity in the PBMCs

Telomerase inhibitors and activators in aging and cancer : A systematic review

The main aim of the present systematic review was to summarize the most frequently used telomerase regulators with an impact on aging and cancer that are referred to in in vitro and in vivo studies. For this purpose, a systematic review of the available literature on telomerase regulators referred to in articles from PubMed and Scopus libraries published from 2002 to 2021 and in accordance with PRISMA 2020 criteria, was conducted. Articles were included if they met the following criteria: They referred to telomerase modulators in aging and in cancer and were in vitro and/or in vivo studies, while studies that did not provide sufficient data or studies not written in English were excluded. In the present systematic review, 54 publications were included, of which 29 were full-text published studies, 11 were full-text reviews, 10 structure-based design studies and 4 abstracts are reported in this review. Telomerase regulators were then categorized as synthetic direct telomerase inhibitors, synthetic indirect telomerase inhibitors, synthetic telomerase activators, natural direct telomerase activators, natural telomerase inhibitors and natural indirect telomerase activators, according to their origin and their activity. On the whole, as demonstrated herein, telomerase regulators appear to be promising treatment agents in various age-related diseases. However, further in vivo and in vitro studies need to be performed in order to clarify the potentiality of telomerase as a therapeutic target.Title in WoS: tau elomerase inhibitors and activators in aging and cancer: A systematic review</p

Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.

<p>Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.</p

Schematic representation of Notch pathway molecules mRNA expression profile in our series of preeclampsia-complicated late preterm and term placentas.

<p>Red: Downregulation; White: Normal expression; Sky Blue: Overexpression.</p