Age-related hearing loss is associated with a decrease in hearing abilities for high frequencies. This increases not only the difficulty to understand speech but also the experienced listening effort. Task based neuroimaging studies in normal-hearing and hearing-impaired participants show an increased frontal activation during effortful speech perception in the hearing-impaired. Whether the increased effort in everyday listening in hearing-impaired even impacts functional brain connectivity at rest is unknown. Nineteen normal-hearing and nineteen hearing-impaired participants with mild to moderate hearing loss participated in the study. Hearing abilities, listening effort and resting state functional connectivity were assessed. Our results indicate no differences in functional connectivity between hearing-impaired and normal-hearing participants. Increased listening effort, however, was related to significantly decreased functional connectivity between the dorsal attention network and the precuneus and superior parietal lobule as well as between the auditory and the inferior frontal cortex. We conclude that already mild to moderate age-related hearing loss can impact resting state functional connectivity. It is however not the hearing loss itself but the individually perceived listening effort that relates to functional connectivity changes. The degraded auditory input leads to an increased effort to process and understand speech. The term listening effort refers to the process of hearing with intention and attention and therefore involves the allocation of attentional as well as cognitive resources to meet the required cognitive demands5. Hearing loss hence decreases available resources in charge of cognitive control, because the neural capacities needed for understanding speech in adverse listening conditions decrease the resources available for other cognitive operations8. As several cognitive abilities such as working memory, episodic memory, attention switching and interference control decline with increasing age, these become even more affected during effortful listening13. Moreover, there is evidence that hearing loss impairs long-term memory14.Age-related hearing loss–also known as presbyacusis–is one of the most common disorders affecting older adults. Presbyacusis affects primarily high frequencies making speech understanding difficult25. Further, effortful speech perception is associated with an increase in neural activation in areas including the cinguloopercular network28, the premotor cortex29, the anterior cingulate cortex30, left inferior frontal cortex31, middle frontal gyrus17, as well as the insula26. However, the influence of the increased listening effort that hearing-impaired people experience in daily life on long-term changes in resting state functional connectivity is still unknown.Previous task-based neuroimaging studies show that recruitment of frontal areas is related to increases in listening effort in younger and elderly volunteers to compensate for the decreased auditory input32. Several resting state networks, such as the default mode, the dorsal attention and the salience network, have been identified and changes have been investigated in a variety of disorders. The default mode network includes brain regions in frontal, parietal and cingulate cortices that are typically active during rest but inactivated during tasks requiring attention35. Previous research shows that the activity within the default mode network is decreased in older participants possibly reflecting a deficit in modulating brain activity and resource allocation39. The dorsal attention network involves the frontal eye fields and the intraparietal sulcus and displays an opposite pattern to the default mode network: inactivated during rest but activated during task35. The salience network comprises areas in the cingulate cortex, insula, prefrontal and supramarginal cortex and has been related to the detection of salient events, the control of behavior and switching of attention40. Previous studies show that the salience network is particularly influenced by aging and hearing impairment41.Resting state functional connectivity is described as spontaneous activity that is organized into coherent networks32. In contrast, another study showed decreased default mode network but increased dorsal attention network connectivity in hearing-impaired compared to normal-hearing subjects35. A recent study by Chen and colleagues demonstrated decreased spontaneous resting state activity in superior temporal gyrus, parahippocampal gyrus and inferior parietal lobule as well as increased activity in middle frontal gyrus, cuneus and postcentral gyrus in hearing-impaired individuals42. These alterations were associated with cognitive and language processing deficits. Puschmann and Thiel43 investigated whether cross-modal reorganization, which is often observed in deaf44 but also mild to moderate hearing-impaired participants45, impacts functional connectivity and found a changed functional connectivity between visual and auditory cortex during audio-visual processing, as well as during rest.Few studies investigated resting state connectivity changes in patients with hearing loss and findings are heterogeneous. In mild to moderately hearing-impaired listeners decreased connectivity was found between the dorsal attention network, the insula and postcentral gyrus, as well as increased connectivity between the default mode network and middle frontal gyrus38. How hearing loss and the increased effort that hearing-impaired individuals are exposed to in daily listening situations adds to this complex mechanism is still an unresolved issue. We here aimed to address how uncompensated age-associated hearing loss and the increased listening effort impact whole brain functional connectivity. For that aim, we conducted resting state connectivity MRI in elderly normal-hearing and hearing-impaired participants. We hypothesized that hearing-impaired participants show increased functional connectivity between auditory and visual cortex and altered connectivity in the default mode network, dorsal attention and salience network43. We further expected a relation between increased listening effort and altered functional connectivity in brain regions previously linked to effortful listening in task based fMRI studies such as frontal and premotor cortices, as well as insula and cingulate cortex30.Differences in brain structure and function in the aging brain can influence sensory and cognitive processing, leading to the assumption of a complex interplay between neuronal structure, function and behavior46). None of the participants reported current or previous use of a hearing aid. Average pure tone audiograms over both ears for the two groups are depicted in Fig. Thirty-nine subjects participated in the study; one subject was excluded due to movement during the resting state measurement. Nineteen participants were classified as normal-hearing (mean age of 63.2 ± 5 years) and served as control group. The other nineteen participants (mean age of 63.5 ± 5.3 years) were classified as hearing-impaired. Hearing abilities for all participants were 30 dB HL or better for frequencies between 125–1000 Hz. High-frequency hearing loss was defined by more than 30 dB HL for the frequencies 2 to 8 kHz. Further, the hearing loss should be symmetrical. People with values less than 30 dB HL in these frequencies were classified as normal-hearing . The second run consisted of a resting state MRI, anatomical MRI and a diffusion MRI measurement. Only the results of the resting state MRI are reported here. Prior to the second run, the participants filled out a listening effort questionnaire (“Höranstrengungsbogen”)47 and a handedness inventory48. The listening effort questionnaire includes seventeen questions about listening situations in everyday life, e.g. ‘You meet with some friends in a café and are able to see them during the conversation. How effortful is it to follow the conversation?’. Participants are asked to rate the effort they need in the different situations from zero  to ten (extremely effortful). The mean values over all questions was used as a marker of listening effort. After the MRI, a working memory task (Size Comparison Span)49, a pure tone audiometry of the frequencies 125, 250, 500, 1000, 2000, 4000, 6000 and 8000 Hz and a task measuring audiovisual integration (results reported in23) were performed.The study consisted of two MRI measurements separated by a short break outside the MRI. The first run was a task based fMRI measurement (results reported in2*-weighted gradient echo planar imaging (EPI) volumes, TR = 1500 ms, TE = 30 ms, voxelsize = 2.2 × 2.2 × 3.0 mm, 25 slices). Structural images were acquired with a 3-D T1-weighted sequence .MRI data were acquired by a 3 T whole-body Siemens Magnetom Prisma MRI machine with a 20-channel head coil. Resting state data were recorded from all participants while fixating a white dot presented centrally on a black screen  stereotactic space using normalization parameters obtained from a segmentation of the anatomical T1-weighted image and spatial smoothing . The first five images of the resting state scan were skipped. In the CONN toolbox data processing first included linear regression to remove motion and the BOLD signal from white matter and cerebrospinal fluid. Next linear detrending and bandpass-filtering (0.008–0.09 Hz) was applied. Each subject’s seed-to-voxel connectivity maps of a specific seed to the whole brain (Fisher-transformed correlation coefficients) were entered into a second-level analysis. On the group level, between-subject comparisons and a multiple regression analysis with listening effort values across both groups were performed. In the regression analysis, mean high-frequency hearing thresholds (2 to 8 kHz) were used as covariates to identify brain regions related to subjective listening effort independent of the severity of hearing loss. Regions of interest included the auditory cortex as well as the default mode network, salience network and dorsal attention network (see seed regions including MNI coordinates in Table 23). For all data, effects were determined to be significant when passing a threshold of p < 0.05 (FWE cluster size inference). Bonferroni-correction was applied to correct for multiple comparisons (i.e. three networks). Peak coordinates are reported in MNI space. All measures throughout the text and in Fig. We analyzed resting state data with the Statistical Parametric Mapping software package  based on Matlab 2016b and the CONN toolbox for SPM51 with mean values of 25.8 for hearing-impaired and 26.9 for normal-hearing participants. All measures refer to normal cognitive functioning. Significant differences between both groups were not obtained.Working memory scores were 11.47 (±12.8) for the hearing-impaired group and 12.47 (±9) for the normal-hearing group. From participation in other studies we gathered data for 11 hearing-impaired and 13 normal-hearing participants measuring cognitive declineMean values of hearing loss for the high frequencies (2 to 8 kHz) were 39.16 ± 6.1 dB for the hearing-impaired group and 18.92 ± 6.52 dB for the normal-hearing group. Mean values for the low frequencies (125 to 1000 Hz) were 9.8 ± 6.82 dB for the hearing-impaired group and 5.63 ± 5.16 dB for the normal-hearing group. Listening effort was significantly higher in the hearing-impaired group (3.27 ± 1.65) than in the normal-hearing participants (2.26 ± 1.19) (T(36) = 2.128; p = 0.02). High-frequency hearing loss and listening effort correlated significantly .Average connectivity patterns of seed regions for each resting state network can be seen in Fig. Resting state connectivity varied as a function of listening effort. There was a significant negative correlation between connectivity in the dorsal attention network to the precuneus and to superior parietal lobule Table . FurtherWe investigated functional resting state connectivity in normal-hearing and mild to moderate hearing-impaired elderly listeners and its relation to listening effort. We did not find any differences in functional connectivity between normal-hearing and hearing-impaired listeners, neither for any of the assessed networks nor for the auditory cortex. Listening effort, however, was related to reduced resting state connectivity between the dorsal attention network and the precuneus as well as the superior parietal lobule. Further, increased listening effort was associated with a decreased connectivity between auditory and inferior frontal cortex.32 showed decreased connectivity between seed regions in the dorsal attention network with the insula and postcentral gyrus, and increased connectivity between seeds in the default mode network and middle frontal gyrus in mild to moderate hearing-impaired participants. In a prior study, Schmidt and colleagues35 compared tinnitus patients with hearing-impaired participants and normal-hearing participants. Although not focus of the study, they found an increased connectivity of the dorsal attention network to the insula in hearing-impaired participants as compared to normal-hearing subjects35. Recently, Chen et al.42 showed a decreased connectivity in superior temporal gyrus (BA 38), precuneus and inferior parietal lobule as well as increased connectivity in middle frontal gyrus, cuneus and postcentral gyrus in hearing-impaired compared to normal-hearing participants. One explanation for the lack of significant differences between hearing-impaired and normal-hearing participants in our study could be the relatively mild to moderate hearing impairment of the participants. However, the impairment in two of the other studies cited above35 was also mild to moderate with slightly younger subjects. Their normal-hearing participants had a mean age of 50 years while ours had a mean age of 63 years (matched to the hearing-impaired group) with age appropriate hearing abilities. Hence, it may also be the case that differences in resting state connectivity that are present in middle-aged hearing-impaired subjects may be covered by larger age-associated changes in connectivity with increasing age. Other research showed that age has a significant effect on resting state networks41.Differences in resting state connectivity between hearing-impaired and normal-hearing participants were not obtained. In contrast, Husain and colleagues32 did also not find any connectivity differences with seeds in the auditory cortex between hearing-impaired and normal-hearing participants and relate these findings to the mild to moderate severity of hearing loss in their study. Chen et al.42 did also not find any significant effects in primary auditory cortex. On the other hand, a prior study in our lab43 did find an increased connectivity between auditory and visual cortex as a function of hearing loss. Since all of these studies–including ours–employed an eyes-open and fixation cross paradigm for the resting state measurement, the paradigm itself could not be the underlying cause of different results. In contrast to the prior study where the resting state measurement directly followed an audiovisual task, participants in the current study had a short break outside the MRI directly before the resting state measurement. Hence, it is not clear whether take-over effects of previous tasks may have contributed to the different results52.Concerning the direct coupling of visual and auditory cortex, Husain and colleaguesApart from hearing abilities determined by the audiogram we assessed subjective listening effort in everyday situations. Our results suggest that an increase in individually perceived everyday listening effort is associated with a decrease in resting state coupling between the dorsal attention network and the precuneus and superior parietal lobule as well as between the auditory and the inferior frontal cortex.32. We here suggest that listening effort experienced in daily life may be one important factor for changed functional connectivity between the dorsal attention network and the precuneus and superior parietal lobule. The precuneus is part of the default mode network and plays a role in several diseases, including Alzheimer’s55 but also tinnitus. Tinnitus patients exhibit decreased connectivity between the precuneus and other regions of the default mode network, speaking for a less coherent network due to the phantom sound35. Chen and colleagues42 demonstrated neuronal changes in superior temporal gyrus, prefrontal cortex, precuneus and inferior parietal lobule as part of the default mode network in age-related hearing loss. They suggest that decreased connectivity values in the precuneus are responsible for disrupting the default mode network in age-related hearing loss. We here show a disruption of coupling between dorsal attention and default mode network (via precuneus). Dorsal attention and default mode network are often anti-correlated, the dorsal attention network being active during tasks and the default mode network being active at rest. Disruptions in this coupling can be associated with decreased cognitive functions56 or aging39. In long-term tinnitus patients, there was also a decreased connectivity between the dorsal attention network and precuneus57. The decreased connectivity is explained as a suppression and habituation mechanism in response to the phantom sound. Although we excluded participants experiencing tinnitus in our study, the connectivity between the dorsal attention network and precuneus was decreased with high listening effort. In age-related untreated hearing loss, the increased listening effort experienced in daily life may therefore result in a similar disruption between dorsal attention and default mode network as in tinnitus patients. Decreases in functional connectivity were also found within the dorsal attention network, i.e. to the superior parietal lobule. Hence, our data suggest a disruption of between and within network connectivity of the dorsal attention network with increased listening effort.There is some evidence of resting state connectivity changes in the dorsal attention and default mode network in age-related hearing loss45. An increase in frontal activity was previously also associated with effortful listening in healthy participants31. Moreover, effortful speech perception was related to an increase in neural activation in lateral temporal cortex, inferior frontal cortex and premotor cortex18. The inferior frontal gyrus belongs to the core speech network involved in processing acoustic, phonological and lexico-semantic and basic syntactic material58. Additonally, the inferior frontal cortex is connected to the auditory cortex and serves as top-down modulatory control to faciliatate speech understanding. Connections between auditory regions to prefrontal areas, including inferior frontal cortex, are thought to function as a modulatory control and compensatory mechanism during effortful listening18. Previous research showed that speech understanding in adverse listening situations is facilitated by top-down influences accomplished via inferior frontal and premotor cortex, as well as superior temporal sulcus and parietal cortex31. Hence, there is compelling evidence that the inferior frontal cortex has an important role in speech understanding and is particularly involved in processing speech under difficult listening situations. Our results add further evidence, since we show a connection of everyday listening effort and a disruption in resting state coupling between auditory and inferior frontal cortex. Participants experiencing high everyday life listening effort showed a decreased connectivity between auditory cortex and inferior frontal cortex.Moreover, listening effort was related to a decreased connectivity between auditory and inferior frontal cortex. These results are in line with previous work in hearing-impaired participants showing increased frontal recruitment as a compensatory mechanism to control for the decreased auditory input23. Future studies examining both functional connectivity during speech understanding and during resting state are needed to entangle the underlying processes related to the increased listening effort, changes in functional connectivity and the resulting impact on speech understanding.There are two possible explanations for this decreased coupling. First, it may be possible that people experiencing high listening effort have a general coupling deficiency between auditory and frontal cortex which may impact both, the coupling during effortful speech perception and during rest. This may also lead to decreased performance in speech understanding. The second suggestion to explain our results might be that individuals experiencing high listening effort present a higher connectivity between auditory and frontal cortex during effortful listening tasks which may be followed by reduced coupling in situations where the effort is no longer needed. The latter suggestion of increased connectivity during task and decreased connectivity during rest can be better reconciled with the many fMRI studies demonstrating that increased involvement of the frontal lobe during effortful speech perception can facilitate speech understanding. Additional evidence comes from our own previous study in the same sample of subjects, which showed an increased frontal recruitment and preserved performance in speech understanding in hearing-impaired participantsThe variety of resting state connectivity changes related to listening effort seem to reflect more widespread changes in underlying network dynamics. Listening effort experienced in everyday life rather than the hearing loss itself has a significant influence on resting state connectivity. The underlying nature for these changes seems to be a compensatory mechanism to control for decreased auditory input, increased listening effort and decreased neural resources. With regard to the scarce research dealing with functional connectivity changes in mild to moderate age-related hearing loss, we provide new evidence for plastic changes not only in auditory cortex but also in coupling between dorsal attention network, precuneus and superior parietal lobule affecting different resting state networks. We conclude that mainly the individually perceived listening effort in elderly participants leads to altered coupling between different brain areas independent of the hearing impairment itself.


Artefacts are a common and unwanted aspect of any measurement process, especially in a clinical environment, with multiple causes such as environmental changes or motion. In near-infrared spectroscopy (NIRS), there are several existing methods that can be used to identify and remove artefacts to improve the quality of collected data.We have developed a novel Automatic Broadband Artefact Detection (ABroAD) process, using machine learning methods alongside broadband NIRS data to detect common measurement artefacts using the broadband intensity spectrum. Data were collected from eight subjects, using a broadband NIRS monitoring over the frontal lobe with two sensors. Six different artificial artefacts – vertical head movement, horizontal head movement, frowning, pressure, ambient light, torch light – were simulated using movement and light changes on eight subjects in a block test design. It was possible to identify both light artefacts to a good degree, as well as pressure artefacts. This is promising and, by expanding this work to larger datasets, it may be possible to create and train a machine learning pipeline to automate the detection of various artefacts, making the analysis of collected data more reliable. Near infrared spectroscopy (NIRSNear infrared spectroscopy (NIRS)Broadband NIRS)Near infrared spectroscopy (NIRS) instruments use light in the near infrared spectrum  to measure the changes in haemoglobin concentrations. It is also possible, when using a Broadband NIRS system (more than 100 wavelengths), to observe in addition changes in tissue metabolism via quantification of the oxidative state of cytochrome-c-oxidase  or the iAutomatic Broadband Artefact Detection (ABroAD) is the process of identifying patterns within data to try and understand it, preferably in a way that will allow this understanding to be used with new data . Data arThis work aims to develop and use a Automatic Broadband Artefact Detection (ABroAD) platform to identify Broadband NIRS. This is done using the broadband spectra of light rather than the calculated chromophore concentrations. The platform was tested with data generated in a series of experiments wherein subjects simulated artefacts. These data were then used to engineer features that describe each broadband spectrum, before being classified using a random forest classifier.Broadband NIRSAutomatic Broadband Artefact Detection (ABroAD) data were collected from eight different subjects in a block test design using a Custom-built broadband NIRS systemBroadband NIRS, based on a system previously described by Bale et al. , at a saThe start and end of each artefact, as well as the start and end of the experiment, was marked in the output data as an event using the LabVIEW software which collects data from the NIRS system. All artefacts were simulated in the order listed above for all subjects.At each time point a spectrum of light was collected for 1340 wavelengths between 610 and 920 nm, as seen in Fig. power density fraction, sample entropy, autocorrelation and area under the curve.Feature engineering describes the process by which new featuresNear infrared spectroscopy (NIRS)Artefacts are generated from existing data. The features were chosen by looking at different spectra for each artefact and attempting to identify differences that could then be summarised in a single number or measureAutomatic Broadband Artefact Detection (ABroAD). These were: The ambient light artefact is one of the most noticeable, as the fluorescent lights used in the room led to spikes in intensity at specific wavelengths, as seen in Fig. Automatic Broadband Artefact Detection (ABroAD)Sample entropy is a modification of approximate entropy, chosen due to it being more computationally efficient, and is a measure of the complexity level within a signal . Figure Automatic Broadband Artefact Detection (ABroAD)Autocorrelation is the correlation of a signal with a time delayed copy of itself as a function of delay. Figure Many artefacts, particularly those due to changes in light, showed an increase in intensity for many wavelengths, increasing the area under the curve. This could be calculated by Automatic Broadband Artefact Detection (ABroAD)Integrating under the light spectrum using the trapezoidal rule. Good separation can be seen in Fig. i, the spectrum at each time point, tj is converted into a four-dimensional feature vector, xi,j of the form {xi, j, 1, xi, j, 2, xi, j, 3, xi, j, 4} and assigned a true classification yi,j according to the artefact simulated at that time point. This dataset is split into test and training sets, based on the subject number, i. This ensures that the algorithm is tested on data from an unseen subject.Figure The training data are then fed into a machine learning pipeline consisting of two main steps: Automatic Broadband Artefact Detection (ABroAD). Scaling is done using the ‘RobustScaler’ from the Scikit-learn library  and is dThis is fitted using a Automatic Broadband Artefact Detection (ABroAD). The training data are split by subject into training and test sets “M times”, with M = 10, allowing retesting of the Automatic Broadband Artefact Detection (ABroAD) of the overall training set. The classifier is run for different parameter combinations, and the set that provides the best final score is chosen as the best estimator. This is then trained on the total training set and tested on the initial test setNear infrared spectroscopy (NIRS)Artefacts. This final score allows the effectiveness of the chosen method to be evaluated.j out of the total number of predictions of that class, whilst recall is the fraction of correct classifications for a class j out of the total number of actual occurrences of that class.The Automatic Broadband Artefact Detection (ABroAD) chosen here is the `weighted F1-score’ – where a perfect classification has a score of 1 and no correct classifications would have a score of 0 – which accounts for both precision (p) and recall (r) and is able to deal with the class imbalance inherent in the data. It is defined as F1 = 2*p*r/(p+r). Precision is the fraction of correct classifications for a class Initially, all Automatic Broadband Artefact Detection (ABroAD) were considered together. A distinct difference can be seen between the F1-scores for light artefacts and for motion artefacts. Therefore, it was decided to additionally consider datasets that contained just motion artefacts and just light artefacts to determine if classification could be improved by doing so.ambient: 0.89 vs 0.08, torch: 0.96 vs 0.01). Across both sensors there is a clear inability to detect motion artefacts, but this is improved by omitting light artefacts when classifying. Additionally, for both sensors the algorithm is able to detect the non-presence of an artefact when only considering light artefacts.Automatic Broadband Artefact Detection (ABroAD) were selected randomly, splitting by subject. The same test and training splits were used for all results. The algorithm was trained using data from subjects 1, 2, 3, 4, 6 and 8 and tested against data from subjects 5 and 7. Figure We have developed a Broadband NIRS. We have shown that it can detect the non-presence of light artefacts across both long and short sensors, as well as the ability to determine the presence of specific light artefact types in the long-distance sensor.The algorithm shows difficulty Detecting motion artefacts, particularly those due to horizontal and vertical movement. This may be because the choice of engineered features does not adequately capture information that can distinguish these artefacts, or it may be that these artefacts were not adequately simulated during data collection. Double cross-validationNear infrared spectroscopy (NIRS)Artefacts  will be New features can be easily added into the process and, with further data collection, the platform can be improved to detect these artefacts. Additionally, whilst the data used here are from a broadband NIRS system, there is the potential for data from accelerometers and external light sensors to be used with the platform where broadband data are not available.


Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced  DNA double strand DNA breaks (DSBs) are a dangerous type of DNA damage that if left unrepaired lead to cell death (reviewed in ). To avoSimilar to other pathways of homologous recombination, BIR is initiated by 5′ to 3′ DSB end resection , resultiThis unusual mode of DNA synthesis likely underlies BIR’s 1000-fold elevation in frameshift mutations compared to S-phase replication ,7. This kataegis , which are cytidine deaminases that convert cytidines to deoxyuridines in the context of ssDNA (reviewed in (It is believed that iewed in ). The maiewed in ). Howeveiewed in ,25–28. Aiewed in ,29,30. Ciewed in . While Aiewed in . In addiiewed in , althougkataegis. Here, using yeast Saccharomyces cerevisiae as a model, we determine whether mutagenic ssDNA is produced along different positions of the BIR track and we also characterize its potential to promote APOBEC-induced mutagenesis. Using a ura3-29 reporter we demonstrate similarly high levels of damage-induced mutations at MAT locus (near the site of BIR invasion), at 16 kb, and at 90 kb from the site of BIR invasion suggesting that mutagenic ssDNA is formed over the entire track of BIR. Also, we demonstrate that translesion synthesis (TLS) polymerase Pol ζ mediates spontaneous and damage-induced base substitutions resulting from accumulation of ssDNA during BIR. Finally, we report that BIR-associated ssDNA is a potent substrate for the APOBEC3A (A3A) enzyme in yeast, and that this enzyme promotes formation of mutation clusters along the track of BIR. We used ung1Δ mutants lacking uracil glycosylase as a tool allowing us to determine the full mutagenic potential of A3A during BIR. Under these conditions, A3A/BIR–induced mutation clusters contained up to 222 mutations in the BIR track. In the presence of uracil glycosylase, the number of mutations in the clusters was reduced due to error-free bypass, which did not rely on Mph1, Ubc13 or on BER enzymes, but proceeded through homologous recombination (HR). However, despite the reduction in mutations, this HR pathway instead stimulated GCRs. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced mutation clusters observed previously in human cancers (i.e. high mutation density and association with GCRs) which suggests that BIR might represent an important initiating factor for kataegis formation.Since BIR promotes accumulation of ssDNA and is implicated in cancer development, we hypothesized that ssDNA generated during BIR could serve as a substrate for APOBEC to produce mutations and All yeast strains used in these experiments are isogenic to and are derivatives of AM1003  which ishmlΔ::ADE1/ hmlΔ::ADE3 MATa-LEU2-tel/MATα-inc hmrΔ::HPH FS2Δ::NAT/FS2leu2/leu2-3,112 thr4 ura3-52 ade3::GAL::HO ade1 met13.URA3 on Chr. V and LYS2 on Chr. II were deleted using the delitto perfetto approach : TCTTTCTGCAATTATTGCACGCCTCCTCGTGAGTAGTGACCGTGCGAACAAAAGAGTCATTACAACGAGGAAATAGAAGAagtcagtgagcgaggaagc and ATATTTGCTGCTATACTACCAAATGGAAAAATATAAGATACACAATATAGATAGTATTAAAAAAACGTGTATACGTTATTattgtactgagagtgcacc (see in (ura3-29 reporter was inserted at position 90 kb by amplifying the reporter from Ori1 and Ori2 plasmids using the following primers (5′-3′):TATGTCTAGTATGCTGGATTTAAACTCATCTGTGATTTGTGGATTTAAAAGGTCTTTAATGGGTATTTTATTCATTTTTTagtcagtgagcgaggaagc and TGCTGCATTTTGTCCGCGTGCCATTCTTCAGCGAGCAGAGAAGACAAGACATTTTGTTTTACACCGGAGCCAAACTGTGAGattgtactgagagtgcacc. The ACAACGTTTCCAAAAGTTAGTTAAATACATACGTCTATTTACTAAGCAAGAAATATATCATGACAAGCCCAAATATTATAagtcagtgagcgaggaagc and GCAAGACTACCAGGATCTTTTATCTGATAAGCTCAAATTACCATATTGCTTAATTTCTTACTACTTGTTATAGTGAAAAGattgtactgagagtgcaccung1::r, rad30::BleorKanMX, rev3::BSD, mph1:: KanMX, ubc13::KanMX, ntg1::KanMX, ntg2::KanMX, rad59::KanMX, mus81::KanMX, KanMX::Bleo, or rad1::BSD disruptions were constructed by transformation with a PCR-derived blasticidin (BSD) marker (TEF/BSD from Invitrogen), KanMX marker  strain with CRISPR-Cas9 expression vector bRA89 modified to express sgRNAs targeting the sequences (5′-3′): TCCTTTGATATCGAGTGTGC  and synthetic complete medium were prepared as described . The YEPADE3-specific probe and a 345 kb chromosome that hybridized to an ADE1-specific probe. BIR outcomes with rearrangements were defined as having two copies of Chr. III where at least one of the copies deviated from its expected size.To induce DSBs, galactose was added ) to a yeast culture that was grown to log phase in YEP-Lac medium. MMS was added to cultures at concentrations of 1.5 mM 30 min after the addition of galactose. Cultures were further incubated at 30°C with agitation for 7 h  as described and reported in rev3Δrad30Δ strains with four individual experiments performed, the range of the median was calculated and reported in U test. The rate of Ura+ mutagenesis in the presence of MMS was determined similarly to :The rate of Urasome III ,7. RatesPrimer 1: GTGTGCTTCATTGGATGTTCGTAC and Primer 2:AAAAGGCCTCTAGGTTCCTTTGTT. The resulting 600 bp-long PCR fragment was Sanger sequenced using the following primer (5′-3′): CTGGAGTTAGTTGAAGCATTAGG 26). WholI Figure . Since aI Figure ,26. CoveI Figure .MATa locus of the truncated copy of chromosome III (Chr. III) , close to the beginning of BIR synthesis, at 16 kb centromere distal to MAT, and at 90kb (close to the very end of the chromosome)  into the ssDNA accumulated during leading strand synthesis of BIR  Figure  6). The. TheMATae Figure . Distal this DSB . We havethis DSB ,6,15. Ththis DSB . To teste Figure . This reitutions ,36. We i) Figure . At eachsome III hr. III   for different positions in both Ori1 and Ori2 yeast strains  compared to the spontaneous level of base substitutions , regardless of the position of ) Figure  and C. Ms Figure . We therura3-29 positions. , which creates a variety of DNA lesions, including mutagenic N3-methyl cytosine lesions that are specifically formed in ssDNA regions ,45,46. W. Figure . In Ori2s Figure . The simura3-29 by transforming them with a centromeric plasmid expressing A3A under the control of the TetO7 promoter  on BIR-induced mutagenesis. In ssDNA, A3A deaminates cytidine into deoxyuridine (dU), which in turn can be excised by uracil glycosylase to produce a mutagenic abasic (AP) site, ultimately resulting in C to T and C to G substitutions . We exprpromoter ,43. The A Figure . In contA Figure , the A3As Figure . This bis Figure ,47. ThusIn our earlier study , we demoREV3 gene, which encodes the catalytic subunit of Pol ζ  in rev3Δ strains did not result in further decrease of spontaneous or MMS-induced mutagenesis (pol3-01 mutation that eliminates the proofreading activity of Pol δ (pol3-01 affects the spectra of A3A/BIR-induced mutagenesis and observed that the fraction of C to G mutations was significantly reduced while the fraction of C to T mutations was significantly increased in pol3-01 mutants as compared to POL3 (wild type) strains  and asss Figure . An even3 Figure . Finallys Figure . Furtheragenesis . Finallyof Pol δ  led to dof Pol δ . We furts Figure . Togetheung1Δ derivatives  of our strains that contained the Ori2 ura3-29 reporter at the 90 kb position. We observed that the level of A3A-induced mutagenesis in the absence of UNG1 was 19-fold higher than in UNG1 A3A strains    obtained in the UNG1 A3A strain contained gross chromosomal rearrangements . The frequency of GCRs was significantly lower among Ura+ outcomes in the ung1Δ A3A strain where only 2 of 28 outcomes (7%) were rearranged  in UNG1s Figure  and D. TMPH1, which encodes a helicase involved in the error-free template switching pathway (NTG1 with NTG2 and APN1 with APN2) significantly affected the level of A3A induced mutagenesis associated with BIR  frequently associated with complex chromosomal rearrangements. To determine whether A3A stimulates the formation of mutation clusters during BIR, we sequenced the genomes of 50 independent Ura+ revertants formed during BIR in the presence of A3A. All these Ura+ revertants were obtained in strains containing the ura3-29 reporter at the 90kb position. Selecting Ura+ revertants for whole genome sequencing (WGS) ensured that A3A was expressed in these cells during BIR and also that BIR synthesis reached at least the 90 kb position. Additionally, these 50 sequenced Ura+ strains were a subset of 86 Ura+ repair events that were previously analyzed by CHEF gel electrophoresis  were from ung1Δ A3A, while the remaining 25 Ura+ (14 non-rearranged and 11 rearranged) were obtained from the UNG1 A3A strain.We previously suggested that APOBEC activity on BIR intermediates could be a potent source of s Figure –C and seung1Δ A3A isolates, we identified groups of mutations where each mutation was no more than 100 kb from the next one and subsequently statistically evaluated whether such groups could be generated by the random distribution of mutations in the genome by comparison to a negative binomial distribution . In addition, the median density of mutations in the Chr III (BIR) clusters formed in A3A-expressing ung1Δ yeast, ∼1 mutation/5 kb  than in the non-BIR (non-Chr. III) clusters, where it was 1 mutation/11 kb . Groups3 Figure , while t3 Figure . In addi genomes . The occclusters ,43. Howeb Figure , was sigon/11 kb .ung1Δ A3A strains. A3A-promoted BIR mutation clusters occupied large areas of Chr. III, located on both the 5′ and 3′ ends of the DSB  , while nsitions .ung1Δ isolates that were rearranged, one of them, RE_34 (ung1Δ) , followed by initiation of BIR synthesis (primary BIR)    containeary BIR) . This symilar to ) followe+ isolates formed during BIR in the UNG1 A3A strain by WGS. Our mutation analysis demonstrated that expression of A3A in all of these isolates led to the formation of BIR-associated mutation clusters (located in the right arm of Chr. III) (UNG1 A3A clusters was significantly lower compared to BIR clusters in ung1Δ A3A (P < 0.0001)  with those in Figure ung1Δ A3A). In particular, the median number of mutations in the UNG1A3A clusters is 7 with an average of 9 and the median number of mutations in the ung1Δ A3A clusters is 23 with the average being 65  (P < 0.005)  . However) Figure ; also co5 Figure . In addi) Figure . The lowUNG1 A3A background demonstrates, that 149 out of 184 heterozygous mutations analyzed are in TC motifs targeted by A3A (75% are in the TCW A3A consensus motif) . This is consistent with their occurrence via a mutagenic bypass of AP sites by translesion polymerase Pol ζ  suggested that they resulted from a single round of BIR repair  . The majse Pol ζ ,50. In Uas RE_20  where BIA strain . The difUNG1-proficient A3A-expressing strains suggested that A3A-induction of abasic sites in the nascent BIR synthesis tract may also promote more complex rearrangement structures frequently observed in association with kataegis events in cancer. We therefore investigated the mechanisms of formation for 11 rearranged BIR events analyzed by WGS in UNG1 A3A strains by determining their chromosomal structure by CHEF gel analysis in combination with the analysis of coverage maps and the location of clustered mutations obtained by WGS.The elevated frequency of rearrangements in TEF sequences of NAT and rBleo, which resulted in deletion of a large chromosomal region  DSB resection and invasion of the recipient into the donor chromosome initiating BIR synthesis (primary BIR), (ii) A3A-promoted deamination of cytosines in ssDNA (resulting from resection or synthesis) leading to accumulation of dU in the recipient, (iii) Ung1-promoted conversion of dU into AP sites, (iv) Stalling of the lagging strand synthesis and/or breakage at the position of the AP site behind the BIR bubble promoting GCR. The exact details of how these steps were completed varied among different repair outcomes, which can explain the variety of phenotypes observed in the outcomes see the  details.kataegis events observed in cancer.We demonstrate in this work that BIR promotes accumulation of long regions of ssDNA throughout the synthesis tract of this DSB repair pathway. This ssDNA can be targeted by DNA damaging agents, like APOBEC cytidine deaminases, which can lead to high levels of base substitutions and the formation of long and dense mutation clusters. In addition, we determined that DNA polymerase Pol ζ mediates formation of mutations and mutation clusters induced by MMS and APOBEC during BIR. Finally, we propose that an error-free pathway must exist that allows the bypass of DNA damage induced by APOBEC in the context of BIR without formation of mutations. However, this pathway can frequently lead to GCRs, resulting in complex events similar to + reversions (which can possibly result from the increased level of dNTPs during BIR (rev3Δ could result from Pol δ errors that escaped correction by proofreading and mismatch repair (the latter being very inefficient during BIR).Interestingly, in the context of BIR proceeding without additional damaging agents, a proofreading defect of Pol δ led to dramatic increase of Uraring BIR ), but itring BIR ,50) that+ revertants was lower at the beginning of BIR as compared to the increase that we observed at later steps of BIR (at 16kb and 90kb positions). We propose this difference could be explained by a higher efficiency of error-free bypass of AP sites at the beginning of BIR as compared to later steps of BIR progression.Based on the increase in base substitution frequency at MAT, 16kb, and 90kb positions, we propose that each part of the BIR track (until the end of the chromosome) is present as ssDNA for a considerable period of time. Therefore, we propose that BIR proceeds via migration bubble (promoting accumulation of ssDNA) from its initiation until the end of the chromosome. This would argue against the idea that the migrating bubble driving BIR at the beginning, is later converted into a normal replication fork, which was proposed based on the observation of frequent template switching at the beginning, but not at the end of BIR . We propung1Δ cells lacking uracil glycosylase, deamination of cytidines by A3A in ssDNA formed during BIR leads to accumulation of uracils in DNA that are not processed further ). During lagging strand BIR synthesis, a replicative polymerase (likely Pol δ), incorporates adenines across from these uracils, which leads to the formation of C to T transitions through the entire track of BIR. This is similar to the mechanism of C to T mutations induced by APOBECs described in other yeast systems in the absence of uracil glycosylase  base substitutions among mutations. This is similar to what was reported for mutagenesis induced by APOBEC3B at eroded telomeres in baker's yeast  did not affect the frequency of mutations in UNG1 strains even though it affected their spectrum. Using yeast as a model, Poltoratsky and Pavlov  can be er cells ,66 wouldRaw sequencing reads can be accessed from the NCBI Sequence Read Archive database under bioproject accession number PRJNA517571.gkz651_Supplemental_FilesClick here for additional data file.


Diagnosis of hereditary hearing loss (HHL) as a heterogeneous disorder is very important especially in countries with high rates of consanguinity where the autosomal recessive pattern of inheritance is prevalent. Techniques such as next-generation sequencing, a comprehensive genetic test using targeted genomic enrichment and massively parallel sequencing (TGE + MPS), have made the diagnosis more cost-effective. The aim of this study was to determine HHL variants with comprehensive genetic testing in our country.GJB2 negative individuals with HHL were referred to the Kariminejad-Najmabadi Pathology and Genetics Center, Tehran, one of the reference diagnostic genetic laboratories in Iran, during a 3-year period between 2014 and 2017. They were screened with the OtoSCOPE test, the targeted genomic enrichment and massively parallel sequencing (TGE + MPS) platform after a detailed history had been taken along with clinical evaluation.Fifty GJB2 negative patients (64%), 34 known pathogenic and novel variants were detected of which 16 (47%) were novel, identified in 10 genes of which the most prevalent were CDH23, MYO7A and MYO15A.Among 32 out of 50 These results provide a foundation from which to make appropriate recommendations for the use of comprehensive genetic testing in the evaluation of Iranian patients with hereditary hearing loss. To dateGJB2 mutations are the most prevalent cause of HL among several genes related to autosomal recessive nonsyndromic hearing loss (ARNSHL)  –6. OtherGJB2 negative HL cases using the OtoSCOPE test, which can screen all the genes involved in hearing loss at once.The fact that early diagnosis in HHL cases may be helpful in prevention and treatment elucidates the need to find the most efficient and cost-effective way to investigate the genetic causes of HL in every population. Hence, this report represents our experience of investigating 50 GJB2 negative individuals with HHL were referred to the Kariminejad-Najmabadi Pathology and Genetics Center, Tehran, one of the reference diagnostic genetic laboratories in Iran, during a 3-year period between 2014 and 2017. Their detailed history was taken along with a clinical evaluation.Fifty All patients completed consent forms and their family pedigrees were drawn to determine the pattern of inheritance.deafnessvariationdatabase.org).Hearing thresholds were measured by pure-tone audiometry following standard protocols . The tarAll results were discussed at a multidisciplinary meeting. The variants in each patient were discussed individually and, in the context of unique clinical information, the most comprehensive diagnosis was provided. Positive results were confirmed via Sanger sequencing before reporting.MYO7A, CDH23 and USH2A, with novel and known variants. Auditory neuropathy was associated with one novel variant in the OTOF gene  to study conservation of missense variants and functional significance.MYO7A, CDH23 and USH2A genes.In the present study, 50 individuals with HHL were studied. Because of the heterogeneity and the role of different loci and genes in HHL, an affordable technique was required to minimize the cost and time needed for diagnosis. OtoSCOPE was chosen for detection of causative variants related to hearing loss as it can screen all the genes involved in hearing loss at once. After evaluating with this test, 16 known variants were detected in 16 individuals in whom four showed retinitis pigmentosa (RP) and hearing loss with homozygous and compound heterozygous variants in MYO7A, CDH23 and USH2A with novel and known variants contributed to both RP and hearing loss. Two of these genes, MYO7A and CDH23, are among five genes involved in neuro-sensory hearing loss  in Iran. It may account for 1%–4% of severe-to-profound HL . This rate ranged from 10% to 83% in several small cohort studies . Other pPlatforms such as OtoSCOPE, providing comprehensive genetic screening for deafness, will allow clinicians to improve patient care by providing prognostic information, and in cases with both RP and hearing loss, offer families preventative strategies to minimize the rate of progression of retinitis pigmentosa.Ethical issues  have been completely observed by the authors.


Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution . Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse ( 2E(X)4E, required for its endonuclease function, and this activity is postulated to represent a “resolvase”activity for class I COs. We generated a point mutant (Mlh3DN) in the endonuclease domain without altering the overall structure of MutLγ. Mlh3DN/DN males have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and chromosome pairing. The MLH3DN mutation permits normal loading of MutLγ, but key DSB repair factors persist in Mlh3DN/DN males, indicating a temporal delay in repair and suggesting a mechanism by which alternative DSB repair pathways may be selected. Thus, the endonuclease domain of MLH3 is important for normal processing of DSB repair intermediates.Meiosis is a specialized cell division whereby a diploid cell undergoes one round of DNA replication followed by two rounds of division, yielding up to four haploid gametes. This process depends on tethering of maternal and paternal homologous chromosomes, and by the formation of crossovers (COs) between homologs during prophase I. COs arise from programmed double-strand breaks (DSBs), and can form via one of at least two mechanisms . In mouse, class I represents the major CO pathway, with the MLH1-MLH3 (MutLγ) complex being critical. MLH3 contains a conserved metal binding motif, DQHA(X) Meiosis is a specialized cell division process in which a diploid parental cell undergoes one round of DNA replication followed by two rounds of division, resulting in up to four haploid gametes. Successful halving of the genome during meiosis I depends on the tethering of maternal and paternal homologous chromosomes during meiotic prophase I, and their subsequent release at the first meiotic division. This tethering is ensured by homologous recombination, leading to the formation of crossovers; by synapsis, the formation of a tripartite proteinaceous structure, the synaptonemal complex, or SC between homologous chromosomes; and by cohesion between replicated sister chromatids that ensures appropriate tension on the metaphase I spindle ,2. Thus,S. cerevisiae, where it was shown that the former occur earlier in meiotic prophase I, and subsequent work suggested that they appeared primarily through synthesis-dependent strand annealing (SDSA)[M. musculus, only 10% of DSBs are repaired as COs, while the majority are mostly repaired as NCOs, presumably via SDSA or other pathways, [Meiotic recombination begins with the introduction of a large number of programmed double-strand breaks (DSBs), which are repaired as non-crossovers (NCOs) or crossovers (COs). Evidence for distinct NCO versus CO pathways was obtained in ng (SDSA),5. In M.athways, –9.S. cerevisiae) [COs can form via one of at least two distinct mechanisms (referred to as class I and class II), each of which is used in varying degrees in different eukaryotic organisms ,10,11. Tevisiae) ,10,11. Cevisiae) .Sgs1 , Top3 and Rmi1 [In the class I CO pathway, DSBs are processed and resected to form single-end invasion (SEI) intermediates. This is followed by displacement of single strand DNA (ssDNA) from the recipient homolog to produce a double Holliday junction (dHJ). The yeast ZMM proteins Msh4 and Msh5 form a complex known as MutSγ that associates with a subset of these intermediate structures –22. At land Rmi1 ,24. STR and Rmi1 . MutSγ iand Rmi1 ,26. The and Rmi1 , recruitand Rmi1 ,29.M. musculus MLH1-MLH3 heterodimer localizes to sites that are destined to become class I COs and the absence of either subunit in male spermatocytes leads to a dramatic decrease, but not complete absence, of chiasmata  [S. cerevisiae MutLγ can bind to single and double-stranded DNA , as well as a variety of branched DNA structures [in vivo functions of MutLγ remains unclear.Though not formally considered to be ZMM proteins, MLH1 and MLH3 are critical for most, if not all, class I CO events in numerous organisms ,29–36. Iof a CO) ,29,37–40ructures ,41–43. HM. musculus is dependent on MLH3, and on its heterodimeric interaction with MLH1 [2E(X)4E, originally discovered in the human MutL homolog, PMS2, and found to be required for human MutLα (hMLH1/hPMS2) endonuclease function [S. cerevisiae have shown that a single point mutation in the endonuclease motif of yeast Mlh3 (mlh3-D523N) disrupts its endonucleolytic activity and results in meiotic crossover defects similar to full mlh3 (mlh3Δ) null mutants, yet does not affect the protein stability of Mlh3 or its interaction with Mlh1 [S. cerevisiae revealed that mutation of any conserved residue results in a null or near-null phenotype with respect to crossing over [S. cerevisiae suggest that MutLγ plays a direct role in resolving dHJs to generate COs through its endonuclease activity.Class I CO formation in ith MLH1 ,29,37. Iith MLH1 . Furtherfunction . This puith Mlh1 . Furthering over . Biocheming over ,43. CollMlh3DN) in which the endonuclease domain was disrupted at the orthologous residue to the D523N mutation in yeast, allowing the overall structure of MLH3 to remain intact, as determined by the ability to form a stable complex with MLH1. By mutating the catalytic domain of MLH3, we hypothesized that the mutant MutLγ complex would remain structurally intact and thus might reveal a functional interplay with other meiotic CO functions. We demonstrate that normal function of the MLH3 endonuclease domain is required for resolution of DSB repair intermediates towards CO formation and thus for late meiotic recombination events. Mlh3DN/DN spermatocytes exhibit grossly normal DSB formation and early processing events, and normal timing of synapsis through early prophase I. Mlh3DN/DN spermatocytes exhibit appropriate localization of MLH3 and MLH1 to the synaptonemal complex during pachynema, along with pro-crossover factors HEI10 and CDK2, phenotypes that are clearly different from that observed in Mlh3-/- males. However, Mlh3DN/DN diakinesis-staged spermatocytes show significantly fewer chiasmata compared to wild-type mice (WT), but significantly more when compared to Mlh3-/- males, suggesting either that the MLH3DN protein retains partial endonuclease activity, or that the presence of the MutLγ complex, albeit altered in its endonuclease capacity, can invoke MLH3-independent repair pathways to become active by interfering with normal resolution of recombination intermediates. In line with these suggestions, we find that the RecQ helicase, BLM, is upregulated throughout prophase I in Mlh3DN/DN spermatocytes, perhaps aiding the recruitment of other repair proteins. To explore the increase in residual chiasmata observed at diakinesis in Mlh3DN/DN males relative to that of Mlh3-/- males, we demonstrate that co-incident loss of the class II CO pathway in Mlh3DN/DNMus81-/- double mutant males results in altered distribution of MutLγ, with an increased proportion of synapsed autosomes bearing no MutLγ foci. Furthermore, the proportion of chiasmata remaining in these double mutants is between that of Mlh3DN/DN and Mlh3-/- males, suggesting that MUS81-EME1 may account for only a proportion of these additional chiasmata, the mutant MutLγ retains residual resolvase activity, and/or mutant MutLγ can recruit other proteins to perform this resolvase activity at a subset of recombination intermediate sites. Collectively, our data show that the endonuclease activity of MLH3 is important for normal processing of DSB repair intermediates through the Class I pathway.To investigate the function of the putative endonuclease domain of MLH3 in mammalian meiotic recombination, we generated a point mutant mouse ..M. muscuMlh3+/DN mice were phenotypically similar to WT littermates and displayed full fertility. Mlh3DN/DN males are also grossly normal when compared to WT littermates, survive into adulthood, and live normal lifespans. Mlh3DN/DN males also exhibit normal mating behaviors as determined by observing a vaginal plug in WT females the morning after mating. However, breeding between multiple sets of Mlh3DN/DN males and WT females never resulted in offspring over a four-year period.Male Mlh3-/- males [Mlh3DN/DN males show complete infertility, accompanied by significantly reduced testes size when compared to WT  components, SYCP3 and SYCP1, marking the axial/lateral elements and the transverse filaments, respectively. Prophase I progression in WT spreads is characterized by the initial accumulation of SYCP3 signal in discrete dots along chromosomes at leptonema, and these dots gradually coalesce into continuous filaments along the chromosome cores in zygonema . By late zygonema, the RAD51 counts were significantly lower in Mlh3-/- spermatocytes compared to WT and Mlh3DN/DN animals (p<0.001 by unpaired t-test with Welch’s correction). By pachynema, RAD51 foci frequency in spermatocytes from WT mice decreased to very low numbers, as did that of Mlh3-/- males , similar to the increased focus frequency observed for RAD51. However, unlike RAD51, the RPA focus counts were not significantly different between genotypes at pachynema and diplonema  is a mammalian RecQ DNA helicase whose  and COs ,24. Duri and COs –56. ReceMlh3-/-, we stained prophase I chromosome spreads with an antibody against BLM. In zygonema, as previously reported, WT cells show the accumulation of BLM foci on the cores in high numbers, and this frequency is elevated in spermatocytes from both Mlh3DN/DN and Mlh3-/- spermatocytes , and cyclin-dependent kinase-2 (CDK2) ,65,66. I spreads . This isCO sites .Mlh3DN/DN mice, with a frequency of one to two foci per chromosome . CDK2 and HEI10 focus counts for WT or Mlh3DN/DN males were not statistically different from each other. Taken together, these observations demonstrate that loading of HEI10 and CDK2 on class I CO designated sites is affected differently by mutation of Mlh3: complete loss of MLH3 results in failure to load CDK2 and hyper-accumulation of HEI10, while altered endonuclease activity of MLH3 results in normal loading of both CDK2 and HEI10. Thus, the physical accumulation of MutLγ is required for normal loading of associated pro-crossover maturation factors.HEI10 was recently shown to co-localize with MutLγ at sites of class I CO, and its localization is dependent on Cyclin N-terminal Domain-containing-1 (CNTD1),61. HEI1Mlh1 and Mlh3 were amplified from cDNA and cloned into pFastBac1 vectors as described in the Methods. The MLH1-MLH3 and MLH1-MLH3-D1185N complexes were expressed from Sf9 cells infected with baculoviruses containing MBP-Mlh1 and His10-Mlh3 or His10-Mlh3-D1185N constructs .Mlh3DN/DN males compared to Mlh3-/- animals prompted us to ask whether some or all of these crossovers were dependent on the activity of the MUS81-EME1 heterodimer. Previous studies in our lab showed that Mus81-/- animals show increased accumulation of MutLγ, resulting in normal chiasmata counts, suggesting that class I CO events are up-regulated in the absence of the class II machinery [Mlh3 alleles to the Mlh3DN variant on the Mus81-/- mutant background yielded MLH1 focus counts that were significantly reduced compared to those observed in Mlh3+/+Mus81-/- mice  cells showed synapsed chromosomes without any MLH1 foci in pachynema , while in Mlh3+/DNMus81-/- males, this proportion increased to 67% (8/12), with up to 3 E0 chromosomes per cell , with as many as 6 E0 bivalents being observed . Thus, loss of the class II pathway in addition to the mutation of the endonuclease domain of Mlh3 only partially reduces residual chiasmata counts to the levels observed in Mlh3-/- animals. Interestingly, despite reduced chiasmata, the overall number of bivalent structures observed in diakinesis preparations from Mus81-/-Mlh3DN/DN males is the same as that seen in Mlh3DN/DN males, and is significantly elevated above that seen in Mlh3-/- single mutant males , this is not sufficient to drive CO resolution along the class I CO pathway.Importantly, the phenotypic consequences of loss of a functional MutLγ complex occur despite normal accumulation of both MutSγ and MutLγ indicating that the physical presence of these complexes is not sufficient to ensure complete CO resolution. However, CO licensing (defined by MutSγ deposition) and designation (defined by MutSγ and MutLγ deposition) are both normal in Mlh3-/- males [Mlh3-/- and Mlh3DN/DN males, suggesting that the effect of MutLγ loss on DSB repair processing is quite different from the presence of a defective MutLγ complex. Most importantly, while RAD51 is recruited in elevated numbers to chromosome cores of Mlh3DN/DN males, it fails to be cleared effectively in pachynema, perhaps because the defective MutLγ complex blocks subsequent processing of DSB repair intermediates. Intriguingly, the significantly altered RAD51 accumulation in leptonema in both Mlh3 mutants indicates a role for MutLγ prior to pachynema, far earlier than has been defined thus far. Indeed, analogous early pairing roles for MutLγ have been proposed for M. sordaria and S. cerevisiae [et al. constructed whole genome recombination maps for wildtype, endonuclease defective, and null mlh3 yeast mutants. Both the endonuclease defective and null yeast mutants for mlh3 showed increases in the number of NCO events, consistent with recombination intermediates being resolved through alternative recombination pathways [We demonstrate that an intact endonuclease domain within MLH3 is not required for DSB or synaptonemal complex formation in early prophase I, similar to that seen in /- males . Howeverrevisiae ,53. In ypathways . Thus, iMlh3-/-Mus81-/- double mutant animals revealed a very small (<1 on average), but consistent, number of residual chiasmata, indicating the existence of other resolvase complexes [The absence of either component of MutLγ results in the loss of 90–95% of chiasmata, consistent with the established dogma that class I COs account for the majority, but not all, chiasmata in mammalian meiosis ,29,39,68omplexes ,11. TakeMlh3DN/DN males show normal accumulation of both MLH1 and MLH3 at pachynema, but this results in an increase in the residual chiasmata count at diakinesis relative to that seen in Mlh3-/- mice: approximately 78% of COs are lost in Mlh3DN/DN spermatocytes, leading to 22% residual chiasmata. This suggests several possibilities: either that the endonucleolytic function of MLH3 does not account for the resolution of all class I COs under wildtype situations, and/or that other resolvases can be recruited under certain circumstances once MutLγ loads, irrespective of whether this complex is endonucleolytically competent. Alternatively, the point mutation in the endonuclease domain does not completely eliminate endonucleolytic activity in the mouse, resulting in partial class I resolvase activity. We find this latter possibility unlikely due to the severity of the defect in endonuclease activity in the S. cerevisiae Mlh1-mlh3-D523N complex [In the current study, we show that spermatocytes from  complex ,33, but Mlh3DN/DN and Mlh3-/- mice is that, in the former, the existence of a defective MutLγ prevents most class I-type COs, but facilitates the resolution of recombination intermediates through alternative pathways. Thus, despite the presence of the MLH3DN protein, some class I CO events can be processed by other CO machinery under conditions of normal accumulation of pro-crossover factors, MutSγ, HEI10 and CDK2. While we cannot assess the recruitment of the class II machinery to sites of DSB repair in prophase I in the mouse (due to the lack of available reagents), there is evidence to support the idea that MUS81-EME1 might participate in this CO resolution crosstalk. Specifically, double mutants lacking Mus81 and bearing a homozygous Mlh3DN allele show reduced chiasmata relative to Mlh3DN/DN males, indicating that MUS81 may account for at least some of the increase in chiasmata above that of Mlh3-/- males. Arguing against this idea is the observation that the loss of Mus81 on the Mlh3-/- background results in a similar drop in residual chiasmata from Mlh3-/- single mutant males alone to that observed in Mlh3DN/DNMus81-/- double mutant animals compared to Mlh3DN/DN single mutant animals.Another explanation for the difference in chiasmata counts between Mus81-/- single mutant males revealed a compensatory increase in MutLγ in pachynema that resulted in normal crossover numbers as a result of upregulation of class I CO events [Mus81-/- males suggested that the class II-destined DSBs were not repaired, but that additional COs were derived from increased CO designation from the larger pool of MutSγ-loaded DSB repair intermediates, suggesting some crosstalk between CO pathways to achieve CO homeostasis. Further evidence for crosstalk between the two major crossover pathways is provided in the current study. However, this elevated MLH1 focus frequency is not observed in Mlh3DN/DNMus81-/- and Mlh3+/DNMus81-/- males, suggesting that a functional MutLγ is required for this crosstalk. Intriguingly, loss of Mus81 on the Mlh3DN/DN and Mlh3+/DN backgrounds results in altered distribution of MLH1 across the genome, resulting in elevated numbers of chromosomes lacking an MLH1 focus entirely. This increase in “E0” chromosomes does not occur in either Mus81-/- or Mlh3DN/DN single null males, indicating that CO distribution is dependent on the functionality  of both pathways. While we cannot fully explain the reason for altered MLH1 distribution in both Mlh3DN/DNMus81-/- and Mlh3+/DNMus81-/- males, these observations point to complex interplay between crossover pathways in achieving normal distribution of crossover events in mammalian meiosis. Indeed, our recent studies involving a mouse model harboring a point mutation within Msh5 indicates that altered MutSγ function affects both crossover pathways [Previous analysis of O events . The perpathways .Mlh3-/- [Mlh3DN/DN (current work) males, prophase I spermatocytes show increased and persistent accumulation of BLM helicase through until late pachynema. A similar increase in BLM localization was also noted in Mus81-/- spermatocytes [S. cerevisiae, loss of class I CO pathway components  is suppressed by mutation of the BLM ortholog, Sgs1, highlighting the role of Sgs1 as an anti-crossover factor. However, the additional CO events that arise in these double mutant yeast strains are presumed to be processed via Mus81-dependent resolution, and thus via class II CO events [msh4/5 sgs1 double mutant strains, the restoration of COs occurs without any concomitant decrease in NCO events, suggesting either that other CO pathways account for the non-class I COs, or that these DSBs are repaired via inter-sister repair processes. In this sense, Sgs1 has been proposed to be master orchestrator of recombination pathway choice [Mlh3-/- and Mlh3DN/DN males with respect to BLM persistence may be similar to that seen in yeast for Sgs1, in that up-regulation of BLM foci is observed in both Mlh3 mutant lines from zygonema onwards, but is significantly higher in Mlh3DN/DN males at pachynema. Thus, residual chiasmata counts in Mlh3-/- and Mlh3DN/DN males are proportional to pachytene BLM focus counts . Thus, we can postulate that the loss of MLH3 protein entirely in Mlh3-/- males results in a compensatory, but ineffective, increase in BLM that cannot overcome the failure to process class I COs sufficiently (a situation that is different to yeast). In the presence of intact, but catalytically inert MutLγ, on the other hand, the availability of additional BLM foci can then direct DSB repair in favor of other CO pathways in a similar fashion to the situation in yeast, where the engagement of Sgs1 promotes alternative repair mechanisms, primarily through the recruitment of structure specific nucleases, and the resolution of some dHJs through a class II (or other) CO pathway [Our previous studies, along with the current one, indicate a role for BLM helicase in modulating the pathway choice in DSB repair during mouse meiosis. In Mlh3-/-  and Mlh3atocytes . In S. cO events ,36,70. Iy choice ,24. The  pathway ,36,70–72Mlh3DN/DN spermatocytes in a manner that may be dependent on the MUS81-EME1 endonuclease, or on other resolvase complexes that have yet to be determined in mammalian meiosis. Indeed, our previous analysis of Mlh3-/-Mus81-/- males indicated the existence of additional CO events that were independent of the class I and class II pathways [We provide evidence that COs are achieved in pathways . Additiopathways ,73,74. Tpathways . Thus, wWork performed in this manuscript was approved by the Cornell Institutional Animal Care and Use Committee, under protocol 2004–0063.Mlh3-D1185N point mutation in the potential endonuclease domain and a loxP-neo-loxP cassette in intron 5–6 of Mlh3 was incorporated into an embryonic stem cell line. Mlh3DN transgenic mice were crossed with a Spo11-Cre mouse line to remove the neo cassette [Mlh3+/DN, and Mlh3DN/DN mice was performed using the following PCR primer pairs: forward (5’-AAGCCAAGTCTGCATGAGTA-3’) and reverse (5’-TAAATGTGCCACTGACTAAAT-3’) followed by a restriction enzyme digestion with Sau96I (New England Biolabs) at 37°C for 2–3 hours, which results in 439-bp and 263-bp fragments from the WT allele and a 702-bp fragment from the mutant allele. Fertility tests were performed by breeding Mlh3DN/DN adult males with WT females. At least 3 males of each genotyped were evaluated. Presence of a copulation plug the following morning counted as a successful mating event. Pregnancy was confirmed by gentle palpation of the abdomen after gestation day 11 or on delivery date of litters. Mus81-/- animals were generated from our breeding stock of such mice, as previously described [A PL253 targeting vector containing the cassette , and theTestes from adult mice were fixed in Bouin’s solution overnight at room temperature and then washed 3 x 10 min with 70% ethanol at room temperature with agitation. Fixed and paraffin-embedded testes were section at 5 μm. H&E staining was performed on Bouin’s fixed testes using standard methods. At least 6 males of each genotyped were evaluated.2. After incubation, 20 μL of sperm suspension was re-suspended in 480 μL of 10% formalin. Sperm counts were performed with a hemocytometer. At least 10 males of each genotype were evaluated for sperm counts and testis weights.Caudal epididymides were removed from adult males and placed in pre-warmed 1X PBS containing 4% bovine serum albumin. Sperm were released into solution by squeezing epididymis with tweezers and incubated for 20 min at 32°C/5% CO2O for 2 x 5 min, then air-dried for approximately 10 min and stored in -80°C or used immediately for staining. Primary antibodies used were: anti-γH2AX , anti-SYCP3 , anti-SYCP1 , anti-RAD51 , anti-BLM , anti-CDK2 , anti-MLH3 . Images were captured with a high-resolution microscopy camera AxioCam MRM  and processed with ZEN Software .Focus counts were performed manually by at least two people, and the results averaged before analysis. For RPA, we employed and ImageJ algorithm for automated counting, as described , and comMlh1 and Mlh3 open reading frames were PCR amplified from cDNA using Expand High Fidelity DNA polymerase using primer pairs AO3365 (5’GCTAGCAGCTGATGCATATGGCGTTTGTAGCAGGAG) and AO3366 (5’TACCGCATGCTATGCATTAACACCGCTCAAAGACTTTG) for Mlh1, and AO3367 (5’ACGTCGACGAGCTCATATGCATCACCATCACCATCACCATCACCATCACATCAGGTGTCTATCAGATGAC) and AO3368 (5’CGAAAGCGGCCGCGATCATGGAGGCTCACAAGG) for His10-Mlh3. Each fragment was cloned into the Spe1 site of pFastBac1 (ThermoFisher) using Gibson assembly PCR (NEB) to create pEAE393 (Mlh1) and pEAE397 (His10-Mlh3). Constructs were verified by DNA sequencing with NCBI reference sequences NM_026810.2 and NM_175337.2 for Mlh1 and Mlh3, respectively. These constructs were then modified as follows:cDNA was synthesized from total testis RNA from wildtype C57B/6J adult males using the SuperScript III Reverse Transcriptase Kit from ThermoFisher. Mlh1 in pEAE393 to create pEAE395.The MBP–TEV sequence was inserted at the N-terminus of mlh3-D1185N mutation was introduced into pEAE397 by Quick Change (Agilent) to create pEAE413.The Sf9 cells were transfected with pEAE397 (His10-Mlh3), pEAE413 (His10-Mlh3-D1185N) and pEAE395 (MBP-Mlh1) using the Bac-to-Bac baculovirus infection system (Invitrogen). Fresh Sf9 cells were co-infected with both viruses (containing Mlh1 and Mlh3 or Mlh3-D1185N). Cells were harvested 60 hours post infection, washed with phosphate buffered saline, and kept at -80°C until use.2, 1 mM PMSF and EDTA free protease inhibitor mixture from Roche and Thermo Scientific) and incubated for 15 min on ice. The suspension was adjusted to 250 mM NaCl, 15 mM imidazole, 10% glycerol, 2 mM ß-mercaptoethanol (BME), and clarified by centrifugation at 17,000 g for 20 min at 4°C. The supernatant was mixed with 6 ml of 50% nickel-nitrolotriaceticacid-agarose (Ni-NTA) resin and allowed to bind for 2 hours or overnight followed by centrifugation to remove the unbound fraction. The resin was packed onto a column and washed with 7–10 column volumes of wash buffer . Protein was eluted with 15 ml of 300 mM imidazole in 50 mM HEPES-KOH pH 7.5, 250 mM NaCl, 40 mM imidazole, 10% glycerol, 2 mM BME and 1 mM PMSF. Elution fractions containing MLH1-MLH3, determined by SDS-PAGE, were pooled and loaded onto 1 ml 100% amylose resin (NEB). The resin was washed with 10 column volumes of wash buffer  and eluted with 6 ml wash buffer containing 10 mM maltose. Fractions containing MLH1-MLH3 were pooled and aliquots were flash frozen and stored in -80°C. The protein yield, following amylose chromatography, was similar for wild-type and mutant complexes (approximately 120–150 μg per 250 ml cells).Cell pellets from 250 ml of cells as thawed, resuspended in 60 ml hypotonic lysis buffer 2E(X)4E, shows the conservation of this domain across these species. Asterisk refers to the conserved aspartic acid (D) that was targeted for a point mutation and converted to asparagine (N) to generate the Mlh3-DN mouse. (B) Mus musculus MLH3 is composed of a 1411 amino acid long sequence that results in an ~158 kDa sized protein . MLH3 contains a globular N-terminal domain  and C-terminal domain  connected by a flexible linker arm (white). The NTD (light gray) contains ATP binding motifs (dark gray) that are conserved across species. The CTD consists of the MLH1 interacting domain (light blue) and the conserved endonuclease motif (dark blue). The aspartic acid (D) in the conserved endonuclease motif in mouse, DQHAAHERIRLE, was converted to an asparagine  at amino acid site 1185.(A) Amino acid protein sequence of the (TIF)Click here for additional data file.S2 FigMlh3DN/DN prophase I cells exhibit grossly normal DSB formation and signaling as observed by γH2AX staining (green) on synaptonemal complex protein SYCP3 (red) as compared to WT and Mlh3-/- cells. Images show abundant γH2AX signal in leptonema, following by diminished signal in zygonema, with the absence of signal in pachynema and diplonema, except at the sex body, due to MSCI.  Over exposure of the γH2AX signal results in γH2AX foci or flares on the autosomes in WT, Mlh3DN/DN, as well as Mlh3-/- cells (white arrows), suggesting that this signal may not be representative of true un-repaired DSBs. (P-W) Mlh3DN/DN prophase I cells have normal synapsis as observed by the localization of synaptonemal complex protein SYCP1 (green) and SYCP3 (red) on the chromosomes when compared to WT. SYCP3 forms as short patches along the chromosomes in leptonema, extending into filaments in zygonema along with the appearance of SYCP1, full synapsis with the co-localization of SYCP1 and SYCP3 are observed in pachynema, followed by desynapsis in diplonema with the degradation of SYCP1.(A-O) (TIF)Click here for additional data file.S3 FigMlh3DN/DN spermatocytes. However, at leptonema and diplonema, the RPA focus counts are significantly elevated in Mlh3DN/DN spermatocytes compared to WT littermate controls . Values given are number of foci per nucleus ± s.d. (B-I) Example spread images of RPA staining at different prophase I stages, including leptonema , zygonema , pachynema , and diplonema  from Mlh3+/+ (B-E) and Mlh3DN/DN (F-I) male mice. Chromosome spreads were stained with antibodies against SYCP3 (red) and RPA (green).(A) Quantitation of RPA foci in spermatocytes from leptonema through diplonema shows initially elevated RPA numbers, with a progressive decline through prophase I for both WT and (TIF)Click here for additional data file.S4 FigMlh3DN/DN (E-H), and Mlh3-/- (I-L) adult males. Panel M shows the quantitation of RNF212 foci in all three genotypes at early (EP) and late pachynema (LP). RNF212 accumulates on chromosome cores at zygonema in high numbers and these foci diminish gradually through pachynema with only one or two foci remaining in late pachynema. (N-Z) MSH4 (green) localization on chromosome cores stained with antibodies against SYCP3 (red) through early prophase I . Representative images of spermatocytes from WT (M-P), Mlh3DN/DN (Q-T), and Mlh3-/- (U-X) adult males. Panel Z shows the quantitation of MSH4 foci in all three genotypes at early (EP) and late pachynema (LP). A similar pattern of MSH4 foci accumulation and loss is observed for MSH4 as for RNF212: accumulation of high numbers of foci in zygonema, diminishing to one or two foci per chromosome in late pachynema. In all cases, statistical analysis was performed using unpaired t-test with Welch’s correction , with Bonferroni’s adjustment for multiple comparisons where necessary. For all chromosome imaging and foci counts, at least three mice of each genotype were observed for each staining set.(A-M) RNF212 (green) localization on chromosome cores stained with antibodies against SYCP3 (red) through early prophase I . Representative images of spermatocytes from WT (A-D), (TIF)Click here for additional data file.S5 FigE0 chromosomes are highlighted with a white box. The XY bivalent is indicated .(TIF)Click here for additional data file.


To know the prevalence, associated factors and temporal trends of disabilities for basic and instrumental activities of daily living in older people in Spain from 2009 to 2017.Disability in older people is associated with health problems, increased health costs and low quality of life. There are no updated data in Spain with a representative sample about disability.Cross-sectional study with 25,465 non-institutionalized older people who participated in the European Health Survey in 2009 and 2014 and the National Health Survey in 2011/12 and 2017 in Spain. The prevalence rates of disability were evaluated using the Katz Scale and Lawton and Brody Scale. Logistic regression was used to determine if there was an association between basic and instrumental activities of daily living and sociodemographic characteristics.More individuals had disability for instrumental activities of daily living (31.9%) than disability for basic activities of daily living (11.1%). The most predominant disability for instrumental activities of daily living was performing severe housework (34%). The prevalence of disabilities decreased from 2009 to 2017. In general, disability was associated with female gender, advanced age, lower education, restricted daily activity, being bedridden and higher pain levels.There is a considerable prevalence of disabilities for basic and instrumental activities of daily living in older people in Spain. Although the disability prevalence has decreased slowly from 2009 to 2017, it continues to remain a health problem. Gender may influence the disabilities for basic and instrumental activities of daily living. Health policymakers should establish prevention strategies and effective interventions  for prevention and reduction of the disabilities for basic and instrumental activities of daily living, particularly in older females. The world's population is an increasingly aging population . Spain, Older people also may have decreased functional capacity due to alterations in physiology during their lifespan; as a result, they demonstrate disabilities in the Basic Activities of Daily Living (ADL) and Instrumental Activities of Daily Living (IADL) . The ADLFunctional capacity can be defined as the potential of elderly people to decide and act independently in their daily living ; disabilDisability in the elderly is particularly measured by the capacity for individuals to carry out ADL and IADL ,9. When Healthy aging correlates with good performance of the ADL and IADL . TherefoAge likewise is associated with the major probability of having a disability; this can have repercussions in health and social systems , becauseStudies from the United States  and SwedThe aim of this study was to estimate the prevalence of disabilities for ADL and IADL in people over 65 years old in Spain, the associated factors for disability, and the temporal trends for functional dependence from 2009 to 2017.We conducted a cross-sectional analysis using publicly available sample survey data. Information sources were microdata from the European Health Survey in Spain (EHSS) 2009  and the EHSS and NHSS utilized a probabilistic multi-stage sampling method with stratification of the first- and second-stage units. The units of the first-stage were the census section, the units of the second-stage were the main family dwellings, and the units of the third-stage were individuals who were present in the household; those individuals were selected by random sampling and quotas based on sex and age. Each participant was assigned a weighted coefficient to ensure the representativeness of the sample.Microdata were anonymized and are available on the NHI of Spain website –23. MicrWe utilized the following sociodemographic variables (contained in each record): age, gender, nationality, cohabitation with a partner, marital status, educational level, pension contribution, self-perceived health status in the prior 12 months, chronic diseases, restriction of habitual activity during the past 2 weeks, bedridden status during the past 2 weeks, use of glasses or hearing aids, difficulty seeing or hearing, difficulty walking 500 meters or up 12 stairs, pain during the past 4 weeks, social class and body mass index (BMI).I am unable to do it by myself” or “I have a lot of difficulty” for one or more items. IADL independence was assessed using seven items based on the Lawton and Brody Scale [“I cannot do it at all” or “I have a lot of difficulty” for one or more items. These classifications for disability have been used in previous studies [The main variable in this study was disability, which included ADL and IADL. ADL independence was assessed using five items based on the Katz Scale   was used to compare categorical variables between groups. Logistic regression analyses were used to estimate the association of the independent variables with ADL disability and IADL disability. The Wald test was used, and the variables for which p≥0.15 were eliminated one by one from the model. The results are presented as odds ratios (ORs) and their respective 95% confidence intervals (95% CI). Statistical significance was established at p<0.05 .Descriptive measures for all variables of interest were used, utilizing count (n), percentage (%), mean (m) and standard deviation (SD). The chi-square test  licensed by the University of Castilla La-Mancha.The total sample was 25,465 individuals over 65 years old, with a mean age of 75.85 years (SD±7.44). Of them, 60.6% were women and 39.4% were men. Although the total sample lived in Spain, only 98.7% had Spanish nationality. Individuals were most frequently married (49.6%), had a primary or secondary educational level (46.6%), had a pension contribution (95.8%), had a good self-perceived health status (37%), had a chronic disease (84.7%), did not have difficulty walking 500 meters (69.2%), did not have difficulty going up or down 12 stairs (59.9%), had pain in the past 4 weeks (61.1%), belonged to the V social class (skilled workers in the primary sector and other semi-skilled workers) (35.6%) and were overweight (39.1%). The majority (85.9%) of older people used glasses or contact lenses, although the majority had no difficulty seeing (78.9%). Only 8.5% were using hearing aids, but 26.7% had difficulty hearing. Demographic characteristics are shown in A minority (11.1%) had an ADL disability, with a mean number of 2.69 (SD±1.54). ADL with the most disabilities were bathing (9.9%) and dressing/undressing (6.6%). More subjects had an IADL disability (31.9%), with a mean number of 2.88 (SD±2.13), as compared to subjects who had an ADL disability. The largest categories of IADL disabilities were performing severe housework (34%), performing light housework (16.6%) and going shopping (14.6%). Figs The ≥ 85 years old age group demonstrated more ADL and IADL disabilities than that of the other age groups (65–74 years old and 75–84 years old) Figs  and 2. AThe logistic regression analysis  showed tThe logistic regression analysis  showed tDisability can be either a result of physiopathology and/or biological alterations in older individuals. It is an important health issue as society ages. From a general point of view, incapacity for performing the activities of daily living in older people decreases their quality of life, increases sanitary costs and contributes to premature death –29. ReceOur study showed that there was a large decrease in ADL and IADL disabilities in 2011/12 that later rebounded. This might be due to the impact of the economic crisis. This could also be possibly due to the fact that more older persons lived in institutions for the 2011/12 year, but our study population only included non-institutionalized individuals. Nevertheless, older people who live in institutions have more disability as compared to those who are non-institutionalized ,32. HoweIn our study, in 2017, the ADL disability was 11.3%, while the IADL disability was 35.1%. This prevalence is important because disability status is an indicator in the prediction of adverse results and mortality risk . These dIn our study, disability is associated, as in other studies, with female gender ,14,16,33In Spain and Mediterranean countries, in general, older people are not institutionalized. The help that older people with disabilities need is provided in many cases by informal caregivers. It is possible for older people to improve their ability to perform ADL and/or to reduce their limitations . Some inEarly detection of disability can be beneficial for individuals who need more attention ; this reThe present study has some limitations. First, the data are self-reported. Although this could be a possible bias, studies have shown that self-reported disability is congruent with disabilities diagnosed by medical services . Second,The main strength of our study is that the data came from a representative, valid and reliable Spanish survey. Therefore, it is beneficial in offering representative data about disabilities in older individuals in Spain, which may be useful for health professionals in developing preventive programs and interventions.In conclusion, this study provides valid data about disability in Spanish non-institutionalized older people. The prevalence of ADL and IADL disabilities is considerable, and almost one-third of people over 65 years old have some form of IADL disability, with IADL disabilities having a higher prevalence than ADL disabilities. ADL and IADL disabilities are associated with female gender, being older, having lower educational status, having activity restrictions, being bedridden and experiencing pain. Although the disability prevalence decreased slowly from 2009 to 2017, disability remains a health problem that is associated with negative outcomes and poorer quality of life among older people.Therefore, it is necessary to improve prevention strategies and to establish effective interventions for reducing or reversing disability status in older people.