The alternative lengthening of telomeres mechanism jeopardizes telomere integrity if not properly restricted

Copyright © 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).A substantial number of human cancers are telomerase-negative and elongate physiologically damaged telomeres through a break-induced replication (BIR)-based mechanism known as alternative lengthening of telomeres (ALT). We recently demonstrated that inhibiting the transcription of the telomeric long noncoding RNA TERRA suppresses telomere damage and ALT features, indicating that telomere transcription is a main trigger of ALT activity. Here we show that experimentally increased TERRA transcription not only increases ALT features, as expected, but also causes rapid loss of telomeric DNA through a pathway that requires the endonuclease Mus81. Our data indicate that the ALT mechanism can endanger telomere integrity if not properly controlled and point to TERRA transcription as a uniquely versatile target for therapy.This work was supported by the Fundação para a Ciência e a Tecnologia (grants PTDC/BIA-MOL/6624/2020, PTDC/MED-ONC/7864/2020 to C.M.A. and B.S., and award 2021.00143.CEECIND to C.M.A.), European Molecular Biology Organization (grant IG3576 to C.M.A.), and LaCaixa Foundation (grant LCF/PR/HP21/52310016 to C.M.A.).info:eu-repo/semantics/publishedVersio

A PSYCHOBIOLOGICAL APPROACH TO IMPROVE EXERCISE ADHERENCE

Studio I La percezione dello sforzo (RPE) \ue8 considerato un determinante negaitvo dell\u2019attivit\ue0 fisica. Questa sensazione \ue8 correlata all\u2019intensit\ue0 dell\u2019esercizio eseguito in un determinato momento. E\u2019 noto che l\u2019esercizio ad elevata intensit\ue0 \ue8 associato ad un tasso di aderenza minore se confrontato con uno ad intensit\ue0 pi\uf9 bassa. La caffeina \ue8 una delle sostanze pi\uf9 utilizzate in tutto il mondo. Inoltre, \ue8 stato ben definito che essa pu\uf2 migliorare le prestazioni in atleti anche grazie a un aumento della potenza prodotta. L\u2019effetto della caffeina sull\u2019 RPE in soggetti sedentari non \ue8 stato molto studiato e risulta controverso. E\u2019 stato indicato che RPE potrebbe non essere modificato a seguito di assunzione di caffeina in questa popolazione durante un esercizio sottomassimale aerobico. Lo scopo dello studio \ue8 stato quello di determinare gli effetti della caffeina in soggetti sedentari a livello della riduzione di RPE durante un esercizio sottomassimale aerobico. Inoltre, uno scopo secondario, \ue8 stato quello di determinare gli effetti della caffeina a livello cardiovascolare e fisiologico. 16 soggetti hanno partecipato allo studio effettuando quattro visite al laboratorio. In questo studio randomizzato incrociato, i partecipanti hanno effettuato una simulazione aerobica sottomassimale, assumendo in un ordine randomizzato 400,200 mg e placebo. E\u2019 stata trovata un\u2019 interazione significativa della caffeina su RPE (p0.05). Concludendo, questo studio ha dimostrato che l\u2019assunzione di 200mg di caffeina 15 minuti prima di un esercizio aerobico sottomassimale hanno prodotto una riduzione di RPE in soggetti sedentari. STUDIO II: E\u2019 ben noto che un\u2019attivit\ue0 fisica effettuata regolarmente, alla giusta intensit\ue0, durata e frequenza produca un miglioramento del fitness e dello stato generale di salute. Come revisionato da Dishman, l\u2019abitudine all\u2019esercizio fisico \ue8 determinata da diversi fattori. Studi precedenti effettuati per indagare l\u2019aderenza all\u2019attivit\ue0 fisica, sono stati focalizzati su interventi psicologici e comportamentali per cercare di migliorarne il livello. Ad oggi, solo pochi studi hanno focalizzato l\u2019attenzione su strategie nutrizionali per poter migliorare l\u2019aderenza all\u2019attivit\ue0 fisica. Lo scopo di questo studio \ue8 stato di implementare un protocollo di lavoro aerobico di 12 settimane, con una frequenza di 3 sedute di allenamento a settimana per verificare l\u2019efficacia dell\u2019assunzione di 200 mg di caffeina 60 minuti prima di ogni seduta di allenamento nell\u2019aumentare l\u2019aderenza ad un programma di allenamento ad elevata intensit\ue0. A seguito della randomizzazione, i soggetti hanno effettuato un test di VO2MAX per definire il proprio picco di Potenza [W] e le relative intensit\ue0 di allenamento per le prime 6 settimane. La stessa procedura \ue8 stata osservata dopo le prime sei settimane ed al termine dello studio. E stato inoltre chiesto ai soggetti di compilare un questionario iniziale relativo a IPAQ, EMI-2 e BRUMS, per poter definire qualsiasi differenza di attivit\ue0 fisica quitidiana oltre allo stato d\u2019animo e la motivazione. I parametri relativi all\u2019aderenza sono stati misurati a 6 e 12 settimane di allenamento per poter identificare differenze tra i due gruppi (CAF-PLAC). Risultati: attendance al programma di allenamento \ue8 risultata significativamente differente nelle settimane da 1 a 6 [CAF(12.92\ub13.75)PLAC(15.92\ub1 2.54)t(23)=-2.99,p=0.030],6 a 12 [CAF(4.00\ub15.34) PLAC(8.67 \ub1 5.34) t(23) =-4.67,p= 0.040] e nelle 12 settimane di allenamento [CAF(16.92 \ub1 7.98)PLAC(24.58\ub16.49)t(23)=-7.66,p=0.040]. La percentuale media di tempo completato \ue8 risultata significativamente differente nelle settimane da 1 a 6 CAF(68.01\ub119.46)PLAC(85.50\ub114.19) t(23) =-17.49,p=0. 018] e da 1 a 12 [CAF(47.01 \ub1 22.18)PLAC(68.29 \ub1 18.02) t(23) = -21.28, p = 0.015]. Il confronto del tempo medio di allenamento speso in ogni singola seduta di allenamento \ue8 stato trovato significativamente differente nelle sessioni 12,15,17,21,22,33,35(P0.05). I risultati relativi al questionari EMI-2 non hanno mostrato alcuna interazione nei confronti pre e post dei gruppi caff e plac in relazione ai parametri AFFILIATION(p=0.370),APPEARANCE(p =0.685),ENJOYMENT(p = 0.643),HEALTHPRESSURE(p = 0.855),HILLHEALTH AVOIDANCE(p=0.394),NIMBLENESS(p=0.597),POSITIVEHEALTH(p=0.227), REVITALISATION(p=0.324),SOCIALRECOGNITION(p=0.364),STRENGTHENDURANCE(p=0.696)and WEIGHTMANAGEMENT(p=0.929). Inoltre, solo per quanto riguarda I parametri AFFILIATION,POSITIVEHEALTH , \ue8 stato trovato un main effect del tempo(P0.05). I risultati relativi alla scala PACES non hanno mostrato alcuna interazione significativa tra caff e plac in riferimento al parametro PACESSCALESCORING(p=0.794). Nessun main effect del tempo \ue8 stato trovato (P>0.05). I risultati relativi al questionario IPAQ hanno mostrato un\u2019interazione significativa nel confronto pre-post tra i gruppi caff e plac in relazione ai parametri IPAQHIGHINTENSITYDAYS(p0.05). E\u2019 stato trovato un main effect significativo del tempo per i parametri IPAQHIGHINTENSITYDAYS, IPAQHIGHINTENSITYMINUTES (P<0.05). I risultati relativi ai parametri fisiologici e percettivi hanno mostrato la mancanza di qualsiasi interazione nel confronto pre-post tra I gruppi caff e plac in merito al PPO (p = 0.683). Lo stesso ha subito una significativa variazione nel tempo (P<0.05)(p = 0.003). L\u2019ANOVA effettuata per i parametri VO2 PEAK e HR pre-post non ha rilevato alcun cambiamento statisticamente significativo (VO2 PEAK (p = 0.462), HR (p = 0.856)). E\u2019 stato di fatto identificato un main effect del tempo per la variabile HR (p< 0.05, p = 0.001). L\u2019analisi dei dati relativi a RPE pre-post confrontando i due gruppi caff e plac non ha mostrato alcuna differenza significativa in termini di sforzo percepito durante ogni singola seduta (p = 0.487). E\u2019 stato identificato un main effect del tempo (p< 0.05,p=0.001). Tutti i dati sono presentati come media\ub1SD. Conclusioni: Questo studio ha dimostrato che non \ue8 presente alcun effetto della caffeina nella riduzione della percezione dello sforzo e miglioramento dell\u2019aderenza all\u2019attivit\ue0 fisica in soggetti sedentari. Inoltre, da un punto di vista fisiologico, a causa di una riduzione relativa al tempo totale di allenamento ed alla percentuale di tempo di allenamento completato, non abbiamo dimostrato alcun miglioramento del fitness aerobico. Dall\u2019altro lato, l\u2019aderenza all\u2019esercizio relativa al gruppo placebo \ue8 risultata essere significativamente differente rispetto al gruppo caffeina.Study I Rating of perceived exertion (RPE) is a negative determinant of physical activity. This conscious sensation is related to the intensity of the performed task. It is well known that high-intensity exercise is associated to a lower rate of adherence when compared to a moderate intensity regimen. Caffeine is one of the most widely used drugs in the world. Furthermore, it has been well established that it can enhances performance in athletes either by a higher power production. The effect of caffeine on RPE in unfit people is poorly understood and controversial. It has been suggested that RPE could be not affected by caffeine in this population during a sub-maximal aerobic exercise. The aim of this study was to establish whether caffeine affects RPE in sedentary healthy subjects during a sub-maximal aerobic exercise. A secondary aim was to determine the effects of caffeine on cardiovascular and physiological variables. 16 healthy subjects were involved in the study. In this randomised cross-over study participants visited the laboratory four times. After a VO2MAX determination each subject was asked to perform a sub-maximal aerobic simulation taking in a randomised order 400,200mg and placebo. There was a significant treatment x workload interaction of caffeine on RPE(p<0.05,p=0.006). Pairwises comparisons indicated that a dosage of 200 mg(M=15.66,SD=1.69)(p<0.05,p=0.014) and 400mg (M=15.47,SD=1.26)(p<0.05,p=0.000) of caffeine were significantly different in reducing RPE compared to placebo (M=16.94,SD=1.94). No significant differences were found between the 200,400mg of caffeine(p=1.000). No significant treatment x workload interaction was found on VO2(p=0.480). There was a significant main effect of workload on VO2 on the three different conditions(p<0.05,p=0.000). No treatment x workload interaction on VCO2(p=0.408). Significant main effect of workload(p<0.05,p=0.000) was found. No treatment x workload interaction on VE(p=0.388). Significant main effect of workload(p<0.05,p=0.000) was found. No treatment x workload interaction on HR(p=0.548). Significant main effect of workload (p<0.05,p=0.000) was found. No treatment x workload interaction on La(p=0.390). Significant main effect of workload (p<0.05,p=0.045). No treatment x workload interaction on RPM(p=0.659). A significant main effect of workload(p<0.05,p=0.004) was found. No significant treatment x workload interaction on SV(p=0.284). A significant main effect of workload(p<0.05,p=0.049) was found. No significant treatment x workload interaction on CO(p=0.285). A significant main effect of workload (p<0.05,p=0.001) was found. No significant treatment x workload interaction on SBP (p=0.538) A significant main effect of workload(p<0.05,p=0.001) was found. No significant treatment x workload interaction on DBP(p=0.972). There was not any significant main effect of workload(p= .194). In conclusion, this study demonstrated that 200 mg of caffeine taken on a chewing-gum based pill 15 minutes before a sub-maximal aerobic exercise were able to reduce RPE in sedentary individuals. STUDY II: It is well known that physical activity improves physical fitness and several health outcomes whether performed on a regular basis, at the right intensity, duration and frequency. As reviewed by Dishman physical activity behaviour is determined by several factors. Previous studies on exercise adherence have been focused on psychological and behavioural interventions in order to improve this behaviour. To date, there are just few studies that used nutritional strategies in order to improve exercise adherence. The aim of the study was to implement in a randomized controlled trial a 3 times per week for 12 weeks aerobic training in order to test the hypothesis that 200 mg of caffeine assumed before each training session could increase the adherence to a vigorous aerobic training program. After randomization, subjects performed a VO2 MAX in order to define the PPO [W] and training intensities for the first 6 weeks of training. Same procedures for the mid-test and post-test. Was also asked to each subject to perform a baseline mid and post-test assessment of IPAQ, EMI-2 and BRUMS questionnaires in order to detect any difference on the general level of physical activity during daily activities and on mood and motivation. Adherence parameters were measured at 6 and 12 weeks of training in order to detect differences between caffeine and placebo group. Subjects visited the gym 3 times/week for 12 weeks. Results: Attendance to the exercise program were significantly different on the weeks 1 to 6 [CAF(12.92\ub13.75)PLAC(15.92\ub1 2.54)t(23)=-2.99,p=0.030],6 to 12 [CAF(4.00\ub15.34) PLAC(8.67 \ub1 5.34) t(23) =-4.67,p= 0.040] and all the 12 weeks of training [CAF(16.92 \ub1 7.98)PLAC(24.58\ub16.49)t(23)=-7.66,p=0.040].The mean percentage of time completed was significantly different on the weeks 1 to 6 [CAF(68.01\ub119.46)PLAC(85.50\ub114.19) t(23) =-17.49,p=0. 018] and from week 1 to 12 [CAF(47.01 \ub1 22.18)PLAC(68.29 \ub1 18.02) t(23) = -21.28, p = 0.015]. The comparison of mean total time performed for each training was found significantly different at session 12,15,17,21,22,33,35(P0,05). The EMI2 results did not show any interaction between the pre\u2013post test comparisons between caf and plac groups on AFFILIATION(p = 0.370),APPEARANCE(p =0.685),ENJOYMENT(p = 0.643),HEALTHPRESSURE(p = 0.855),HILLHEALTH AVOIDANCE(p=0.394),NIMBLENESS(p=0.597),POSITIVEHEALTH(p=0.227), REVITALISATION(p=0.324),SOCIALRECOGNITION(p=0.364),STRENGTHENDURANCE(p=0.696)and WEIGHTMANAGEMENT(p=0.929). A significant main effect of time was found on AFFILIATION,POSITIVEHEALTH (P0.05). PACES scale results did not show any significant interaction between caf and plac on PACESSCALESCORING(p=0.794). No main effect of time (p= 0.095). The IPAQ results presented a significant treatment x workload interaction on the pre-post comparisons between caf and plac on IPAQHIGHINTENSITYDAYS (p0.05). Main effect of time was found significantly different for IPAQHIGHINTENSITYDAYS,IPAQHIGHINTENSITYMINUTES (P<0.05) PHYSIOLOGICAL PARAMETERS: The pre-post test analysis of the PPO did not show a significant treatment x workload interaction between caf and plac (p = 0.683). Significant main effect of time (P<0.05)(p = 0.003). ANOVAs performed on VO2 PEAK and HR pre-post test did not reveal a significant treatment x workload interaction (VO2 PEAK (p = 0.462), HR (p = 0.856)). Significant main effect of time for HR (p< 0.05, p = 0.001). The data analysis of RPE pre-post test comparing caf and plac groups, did not reveal a significant difference accounted for the effort perceived during each training sessions (p = 0.487). Significant main effect of time(p< 0.05,p=0.001). All the data are presented by mean\ub1SD. Conclusions: This study demonstrated that there is not any effect of caffeine on perception of effort and adherence improvement in sedentary subjects. From a physiological point of view, due to a reduction in total time of training and percentage of total training completed we do not have demonstrated an improvement in aerobic fitness. On the other hand, adherence for the placebo group was significantly different compared to caffeine

The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organism

ALTernative functions for human FANCM at telomeres

Copyright © 2019 Domingues-Silva, Silva and Azzalin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The human FANCM ATPase/translocase is involved in various cellular pathways including DNA damage repair, replication fork remodeling and R-loop resolution. Recently, reports from three independent laboratories have disclosed a previously unappreciated role for FANCM in telomerase-negative human cancer cells that maintain their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. In ALT cells, FANCM limits telomeric replication stress and damage, and, in turn, ALT activity by suppressing accumulation of telomeric R-loops and by regulating the action of the BLM helicase. As a consequence, FANCM inactivation leads to exaggerated ALT activity and ultimately cell death. The studies reviewed here not only unveil a novel function for human FANCM, but also point to this enzyme as a promising target for anti-ALT cancer therapy.Research in the Azzalin laboratory is supported by the European Molecular Biology Organization (IG3576) and the Fundação para a Ciência e a Tecnologia (IF/01269/2015; PTDC/BIA-MOL/29352/2017; PTDC/MED-ONC/28282/2017). Publication costs were supported by UID/BIM/50005/2019, project funded by the Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado.info:eu-repo/semantics/publishedVersio

TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells

© The Author(s) 2021. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.This work was supported by the European Molecular Biology Organization (IG3576), the Fundação para a Ciência e a Tecnologia (IF/01269/2015; PTDC/MED-ONC/28282/2017; PTDC/BIA-MOL/29352/2017) and the European Union’s Horizon 2020 Research and Innovation Programme (GA No. 857119 – RiboMed).info:eu-repo/semantics/publishedVersio

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cell

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cells

A Maintenance Management Model. Upgrading and Experimentation

The paper deals with the first results of the activities of BIG Building Innovative Governance srl, Academic Spin-Off and Innovative Start Up, which provides smart services for Building Maintenance and Facility Management also launching circular R&amp;D actions in the relative reference areas. The contribution introduces some actions actually in progress geared to upgrading the Maintenance Management Model, M3, specifically aimed at managing the life cycle of buildings. The current stage of development corresponds to a TRL 4. TRL 8 is that expected by the end of 2020. Characterized by interconnectivity and scalability, the Maintenance Management Model is a dynamic, collaborative and implementable system, whose architecture consists of three separated but strongly interconnected devices: an information interface system, a collaborative platform, a remote cloud. An ICT network infrastructure able to activate using BIM models (IFC and COBie standards) circular information workflows between all operators and/or users involved in Maintenance and Facility Management processes. It allows the development and/or implementation of information management and sharing models based on Open Data and Semantic Web. Its use will also promote shared lexicons and the circulation of knowledge within a holistic process of managing information from and for Maintenance. The use of BIM models and the possibility of collecting and managing a large amount of data will be oriented to the structuring of information feedback databases according to ISO 15686 guidelines. In this way, available information can be usefully transferred in life cycle assessments and service life prediction of materials and components

Paradigms

The paper investigates and analyses creation, success and evolution of different Paradigms that have characterised mankind modern history, strongly connecting social, scientific, technological and economic contexts. The paper critically outlines their characteristics, peculiarities and logical structures, and then re-interpret them in the light of the ongoing transition and particularly of the changes that the digital transformation is determining on the important subjects of environmental policies, of technological innovation – and therefore of ‘living’ – and particularly of sustainable building in the digital era. By underlining the success of a new habitat – the digital ecosystem – this reading associates to the mentioned Paradigms, new characteristics outlining attributes, areas, problems and developments of a possible ‘second life’, of which is not quite simple today to foresee results on the architectural languages, the transformations of the implemental procedures, the new characteristic, besides ecological and digital, the ones already known