Visuomotor impairments have been demonstrated in preclinical AD in individuals with a positive family history of dementia and APOE e4 carriers. Previous behavioral findings have also reported sex-differences in performance of visuomotor tasks involving a visual feedback reversal. The current study investigated the relationship between grey and white matter changes and non-standard visuomotor performance, as well as the effects of APOE status, family history of dementia, and sex on these brain-behavior relationships.n = 49) with no cognitive impairments completed non-standard visuomotor tasks involving a visual feedback reversal, plane-change, or combination of the two. Participants with a family history of dementia or who were APOE e4 carriers were considered at an increased risk for AD. T1-weighted anatomical scans were used to quantify grey matter volume and thickness, and diffusion tensor imaging measures were used to quantify white matter integrity.Older adults . This finding was replicated in the diffusion data, where regression analyses revealed that lower white matter integrity  was a significant predictor of worse visuomotor performance in the forceps minor, forceps major, cingulum, inferior fronto-occipital fasciculus (IFOF), inferior longitudinal fasciculus (ILF), superior longitudinal fasciculus (SLF), and uncinate fasciculus (UF). Some of these tracts overlap with those important for visuomotor integration, namely the forceps minor, forceps major, SLF, IFOF, and ILF.These findings suggest that measuring the dysfunction of brain networks underlying visuomotor control in early-stage AD may provide a novel behavioral target for dementia risk detection that is easily accessible, non-invasive, and cost-effective. The results also provide insight into the structural differences in inferior parietal lobule that may underlie previously reported sex-differences in performance of the visual feedback reversal task. AD pathology begins several decades before the onset of clinical dementia, and symptoms appear only after there has already been significant damage to the brain , which mAs an alternative to current diagnostic tools and procedures, measuring the dysfunction of brain networks underlying visuomotor transformations in early-stage AD may provide a novel behavioral target for dementia risk detection that is easily accessible and cost-effective. Default or “standard” visually-guided arm movements typically involve looking towards the intended target of a reach, followed by an arm movement to the same location . For exaVisuomotor integration relies on interconnected brain regions within the frontoparietal network . There aGiven the known functional substrates of non-standard visuomotor transformations, several white matter tracts that connect these areas of the frontoparietal network may be implicated in visually-guided reaching. The superior longitudinal fasciculus (SLF) has been shown to be important for visuomotor integration  and to aLittle evidence exists regarding the role of the medial temporal lobe in visual guidance of movements and in visuomotor or motor tasks. Some areas of the medial temporal lobe have been implicated in visuomotor coordination. The parahippocampal cortex may be an important relay for the transformation between visual inputs and hand kinematics (outputs), and the hippocampus, parahippocampus, and entorhinal cortex appear to encode the position of the hand in space . The retDiffusion tensor imaging measures  provide information about white matter microstructure and pathology , 2003, aIn the present study, we first examined whether individuals at an increased risk for AD (due to a family history or a genetic risk) showed grey matter atrophy or declines in white matter integrity in brain regions and tracts typically affected early in disease progression. However, the main objective was to explore the neural underpinnings to previous behavioral findings, where individuals with a positive family history of dementia and APOE e4 carriers demonstrated performance deficits on non-standard visuomotor tasks . There w2.2.1.n = 12 females), and 24 individuals with no family history of dementia , e3/e4 (n = 11). The breakdown of the APOE genotyping in male participants was as follows: e2/e3 (n = 1), e3/e3 (n = 18), e2/e4 (n = 1), e3/e4 (n = 4). One female and one male participant were excluded from statistical analyses looking at the APOE genotype as their genotyping results were inconclusive.A total of 2 ml of saliva were collected from each participant in microtubes from Diamed Lab Supplies Inc. using collection aids from Cedarlane Labs. Samples were sent to DNA Genotek Inc.  for DNA extraction and APOE genotyping. DNA was isolated from samples according to the manufacturer’s protocols. Genotyping for APOE involved single nucleotide polymorphism (SNP) genotyping, and tested for SNPs rs429358 and rs7412. The proteins that are produced by the APOE gene are either E2, E3, or E4 combinations . The breakdown of the APOE genotyping in female participants was as follows: e3/e3  a yellow central (home) target was presented on the vertical tablet, (2) participants moved a white cursor to the central target, changing its color to green once they reached it, (3) after holding the central target for 4,000 ms, one of four red peripheral targets appeared and the central target disappeared, serving as the ‘Go’ signal for initiation of a movement, (4) participants were told to look towards the visual target and slide their finger along the touchscreen to direct the cursor towards the target, (5) once the peripheral target was reached and the participant held it for 500 ms, it disappeared, signaling the end of the trial, (6) the next trial began with the presentation of the central target after an inter-trial interval of 2000 ms . In the PC condition (non-standard), participants needed to move on the horizontal screen while looking at the vertical screen in order to direct the cursor towards the visual target displayed on the vertical screen. In the FR condition (non-standard), the cursor moved in the opposite direction of the participant’s finger movements, requiring them to slide their finger on the vertical screen away from the visual target in order to move the cursor towards it. Finally, in the PC + FR condition (non-standard), movements needed to be made in the opposite direction and on a different plane from the visual target in order to direct the cursor towards it.2.3.2.o in either direction from a straight line to the target) were coded as direction reversals. Direction reversals were not included in metrics from correct trials, but instead were analyzed as a separate variable. All scored data were then processed to compute 7 different timing, accuracy, and precision measures described below. Any trials exceeding 2 standard deviations from the participant’s mean for any of the outcome measures were eliminated from final outcome calculations.Kinematic measures, including timing, finger position , and error data were recorded for each trial and converted into a MATLAB readable format using a custom written (C++) application. Custom analysis software  was used to process individuals’ finger trajectories with a fourth-order  low-pass Butterworth filter at 10 Hz. Finger trajectories were generated from these filtered paths for each successful trial and displayed on a Cartesian plot illustrating finger location data superimposed on central and peripheral target locations. Movement onsets and ballistic movement offsets  were scored at 10% peak velocity. Total movement offsets were scored as the final 10% peak velocity point once the finger position was within the correct peripheral target. If the initial movement successfully resulted in the finger reaching the peripheral target, then ballistic and total movement offsets were the same. These movement profiles were then verified by visual inspection, and manually corrected when necessary. Unsuccessful trials (error data) were detected by the data collection software by meeting the following criteria: finger left the home target too early , reaction time (RT) was <150 or >8,000 ms, or total movement time was >10,000 ms. Trials in which the first ballistic movement exited the boundaries of the central target in the wrong direction (>90The kinematic outcome measures were as follows: (1) Reaction time (RT), the time interval (in ms) between the central target disappearance and movement onset; (2) Full movement time (MTf), the time (in ms) between movement onset and offset; (3) Peak velocity (PV), the maximum velocity obtained during the ballistic movement, and used to calculate the 10% threshold used for determining movement onsets and offsets; (4) Path length (PL), the total distance  travelled between movement onset and offset; (5) Absolute error (AE), a measure of end-point accuracy, and the average distance (in mm) from the individual ballistic movement endpoints  to the actual target location; (6) Variable error (VE), a measure of end-point precision, and the distance (in mm) between the individual ballistic movement endpoints (σ) from their mean movement; and (7) Direction reversals (DR), recorded as a percentage of total completed trials. Corrective path length (CPL) represents corrective movements and was quantified by subtracting the PLb  from the PLf (full movement offset).2.4.2.4.1.o. The MP-RAGE scan was used to quantify grey matter volume and thickness. For assessing white matter integrity, a whole-brain diffusion-weighted scan was acquired with 64 encoding directions using diffusion-weighted spin-echo single-shot echo planar imaging. The diffusion tensor imaging (DTI) sequence used the following acquisition parameters: 56 axial slices , FOV of 192 × 192 mm, matrix size of 128 × 128 resulting in a voxel resolution of 1.5 × 1.5 × 2.0 mm, TE = 86 ms, TR = 6,900 ms, b-value = 1,000 s/mm2 .MRI data were acquired using a 3 Tesla (3 T) Siemens Trio scanner at York University. Participants received a T1-weighted anatomical scan using a sagittal volumetric magnetization-prepared rapid gradient echo (MP-RAGE) sequence. The MP-RAGE consisted of the following acquisition parameters: 192 sagittal slices , field of view (FOV) of 256 × 256 mm, matrix size of 256 × 256 resulting in a voxel resolution of 1 × 1 × 1 mm, echo time (TE) = 2.96 ms, repetition time (TR) = 2,300 ms, flip angle = 92.4.2.Preprocessing: The cortical surface was reconstructed using FreeSurfer 7.1  with individual T1-weighted MR images serving as input. The main Freesurfer reconstruction pipeline  was used to parcellate and segment the brain into anatomically distinct regions of the cortex and subcortical nuclei, respectively. Visual inspection of each participant’s parcellation and segmentation was performed to ensure that there were no obvious errors.Grey matter volume and thickness , molecular layer, fimbria, hippocampal fissure, hippocampus-amygdala-transition-area (HATA), and hippocampal tail. Visual inspection of each participants’ hippocampal subfield segmentation was performed to ensure that there were no obvious errors, with the caveat that precise visualization of the distinct subfield boundaries at the spatial resolution used in this study is difficult. The following subfields were excluded: the parasubiculum (volume of <100 μl and may be more prone to noise), the molecular later , HATA (volume of <100 μl and may be more prone to noise), fimbria , the hippocampal fissure , and the hippocampal tail  adjustment is an important step as it takes individuals’ variations in head size into account . ICV was calculated from the T1-weighted images using Statistical Parametric Mapping (SPM) 12 software. The hippocampal subfield, parahippocampal, entorhinal, perirhinal, precuneus, inferior parietal lobule, and retrosplenial cortical volumes of interest (VOI) were ICV-corrected using the residuals method. This method was originally described by , the PC (posterior commissure), and the mid-sagittal plane. Diffusion-weighted images were preprocessed in FMRIB’s Software Library (FSL) using FMRIB’s Diffusion Toolbox (FDT) for eddy current correction and head motion correction. The preprocessed diffusion-weighted images and the ACPC-aligned T1-weighted images were then input into the automated fiber quantification (AFQ) software  Fiber tract segmentation is performed based on the waypoint ROI procedure described in  Fiber tract clipping to the central portion that spans between the two defining ROIs for that tract; and finally (6) Fiber tract quantification to calculate diffusion measures at 100 equidistant nodes along the trajectory of the fiber group. The diffusion measures are calculated at each node by taking the weighted average of each fiber making up the tract based on its distance from the tract core. Thus, the AFQ pipeline produces a set of “tract profiles” that measure the tissue properties  at equidistant sample positions  from the start to the end of the tract core. There is one profile for each tract and tissue property combination . The 20 fiber tracts identified with AFQ are: bilateral anterior thalamic radiation, bilateral corticospinal tract, bilateral cingulum cingulate, bilateral cingulum hippocampus, callosum forceps minor, callosum forceps major, bilateral inferior fronto-occipital fasciculus, bilateral inferior longitudinal fasciculus, bilateral superior longitudinal fasciculus, bilateral uncinate fasciculus, and bilateral arcuate fasciculus (n = 47) and right (n = 46) cingulum cingulate (CGC), callosum forceps minor (n = 49), callosum forceps major (n = 49), left (n = 48) and right (n = 47) inferior fronto-occipital fasciculus (IFOF), left (n = 48) and right (n = 49) inferior longitudinal fasciculus (ILF), left (n = 49) and right (n = 48) superior longitudinal fasciculus (SLF), and left (n = 48) and right (n = 49) uncinate fasciculus , APOE e4 positive vs. e4 negative participants (p = 0.64), and female vs. male participants (p = 0.82). There were also no statistically significant differences observed between groups on MoCA scores , years of education , computer experience , and touchscreen experience .All statistical analyses were carried out using open-source R software v4.1.0 . All par2.5.1.p < 0.05.Three-way ANOVAs were used to compare differences in structural MRI and DTI measures between three groups: (1) individuals with a family history of dementia and individuals without a family history of dementia, (2) females and males, and (3) APOE e4 carriers and non-carriers. Specifically, the neuroimaging measures being compared were (1) grey matter volumes of the hippocampal subfields, parahippocampal cortex, entorhinal cortex, perirhinal cortex, retrosplenial cortex, inferior parietal lobule, and precuneus, (2) grey matter thickness of the parahippocampal cortex, entorhinal cortex, perirhinal cortex, retrosplenial cortex, inferior parietal lobule, and precuneus, and (3) mean diffusivity measures  across the white matter tracts of interest. Post-hoc analyses were adjusted for multiple comparisons using the Holm correction method. Corrections were considered statistically significant at an alpha of 2.5.2.Some of the kinematic measures were combined into composite scores to decrease the number of comparisons made in the data analysis, and the procedure was previously described in detail . Brieflyp-values were adjusted for multiple comparisons using the Holm correction method and were considered statistically significant at p < 0.05. For example, the regression models in the plane-change feedback reversal condition were set up as follows:Multiple regression analyses previously revealed both sex and family history as significant predictors of greater endpoint error scores (indicative of worse visuomotor performance) in the visual feedback reversal condition. The regression analyses also provided preliminary evidence that having an APOE e4 allele was a significant predictor of greater endpoint error scores in the plane-change feedback reversal condition, and greater corrective path lengths (indicative of worse visuomotor performance) in both plane-change conditions. Based on these previous behavioral findings, the current study used multiple linear regression analysis to explore whether visuomotor performance declines are associated with grey and white matter structural neuroimaging measures. The white matter tracts of interest were the cingulum cingulate, callosum forceps minor, callosum forceps major, IFOF, ILF, SLF, and UF. The grey matter regions of interest were the retrosplenial cortex, inferior parietal lobule, and precuneus, all shown to be involved in visually-guided reaching. Regression models were set up with grey or white matter measures as the predictor variable, visuomotor performance measures as the dependent variable, and were controlled for (1) sex and family history in the feedback reversal condition, and (2) APOE e4 status in the two plane-change conditions. The where: Y is the continuous dependent variable,X is the continuous independent variable,Z is the dichotomous independent variable,X*Z is the interaction term calculated as X multiplied by Z,0 is the intercept,β1 is the effect of X on Y,β2 is the effect of Z on Y, and.β3 is the effect of XZ on Y.βA sample model looking at whether FA in the right ILF is predictive of endpoint error scores in the plane-change feedback reversal condition, moderated by APOE e4 status:If a significant moderated relationship has been identified , we used simple slopes analysis to examine the effects of X on Y within the individual levels of Z. A simple slope is the regression of Y on X at a specific value of Z. In this study, all moderator variables  were binary - therefore, we tested the regression of Y on X at 0  and 1 . Simple slopes analysis was used to determine whether either of the slopes differed significantly from zero .3.3.1.1,43 = 6.903, p < 0.05). There was a significant main effect of family history on parahippocampal cortical volume . Conversely, there was a significant main effect of APOE status on parahippocampal cortical volume . Lastly, there was a significant main effect of sex on inferior parietal lobule thickness . There were no statistically significant differences between (1) positive and negative family history, (2) females and males, and (3) APOE e4 carriers and non-carriers found for grey matter volume or thickness in the hippocampal subfields, entorhinal cortex, and perirhinal cortex.There was a significant main effect of family history on retrosplenial cortical volume , where il volume , where il volume , where Ahickness , where m3.2.There were no significant interaction effects between the inferior parietal lobule, precuneus, or restrosplenial cortex and family history, sex, or APOE status in the multiple regression models that were based on previous behavioral findings. Multiple linear regression analyses revealed that presubiculum, subiculum, and parahippocampal volumes, as well as entorhinal cortical thickness were predictors of visuomotor performance in several of the non-standard conditions.Hippocampal subfields (p < 0.05) on endpoint error scores in the feedback reversal condition. The results of the regression indicated that the predictors explained 35.8% of variance . Simple slopes analysis showed that smaller left presubiculum volume was a significant predictor of greater endpoint error scores only in males . There was a significant interaction effect between right presubiculum volume and APOE status  on corrective path length in the plane-change condition, where the two predictors explained 43.1% of variance . Simple slopes analysis showed that larger left presubiculum volume was a significant predictor of greater corrective path length only in APOE e4 carriers . There was a significant interaction effect between right subiculum volume and APOE status  on corrective path length in the plane-change condition, where the two predictors explained 32.5% of variance . Simple slopes analysis showed that larger right subiculum volume was a significant predictor of greater endpoint error scores only in APOE e4 carriers .ubfields –C: ThereEntorhinal cortex (p < 0.05) on endpoint error scores in the plane-change feedback reversal condition. The results of the regression indicated that the predictors explained 34.8% of variance . Simple slopes analysis showed that smaller right entorhinal thickness was a significant predictor of greater endpoint error scores only in APOE e4 carriers .l cortex : There wParahippocampal cortex (p < 0.05) on corrective path length in the plane-change condition. The results of the regression indicated that the predictors explained 35.2% of variance . Simple slopes analysis showed that smaller left parahippocampal volume was a significant predictor of greater corrective path length only in APOE e4 carriers . There was also a significant interaction effect of left parahippocampal volume and APOE status  on corrective path length in the plane-change feedback reversal condition. The results of the regression indicated that the predictors explained 36.2% of variance , however simple slopes analysis was not significant, indicating that APOE e4 carriers and non-carriers were significantly different from each other but not from zero.l cortex : There w3.3.There were no statistically significant differences between (1) positive and negative family history, (2) females and males, and (3) APOE e4 carriers and non-carriers found for any of the diffusion measures in the major white matter tracts.3.4.Multiple linear regression analyses revealed that lower FA, higher MD, higher RD, and higher AxD were significant predictors of worse visuomotor performance only in the plane-change feedback reversal condition. Simple slopes analyses revealed that the relationship between the diffusion measures and visuomotor performance was only statistically significant in APOE e4 carriers  as well as associated with non-standard visuomotor deficits. Converging evidence suggests that by the time clinical impairment is detectable in AD, extensive neurodegeneration has already taken place . However4.2.Studies examining white matter diffusion characteristics have demonstrated that white matter may be independently affected from amyloid pathology  and greyOur findings suggest that lower white matter integrity in several major tracts in the brain may underlie the non-standard visuomotor deficits we previously demonstrated in APOE e4 carriers compared to non-carriers. In the brain, the APOE protein plays a central role in cholesterol transport , which i4.3.We previously showed that sex was a significant predictor of visuomotor performance in the feedback reversal condition, where males had greater endpoint error scores compared to females after controlling for family history and APOE status . In the 4.4.One of the limitations of the current study was that hippocampal subfield segmentation was obtained from T1-weighted MRI scans, which have limited tissue contrast of hippocampal structures . The num4.5.in advance of any cognitive deficits. We also found structural grey matter differences in the inferior parietal lobule that may underlie the sex-related differences we have previously demonstrated in non-standard visuomotor performance.Our results suggest that a visuomotor task may have potential for detecting the early effects of the APOE e4 allele on white matter microstructure and medial temporal lobe grey matter early in dementia progression, as the white matter and grey matter declines observed here were associated with visuomotor difficulties only in e4 carriers. Importantly, the visuomotor deficits were seen prior to group differences in volumetric and diffusion measures due to genetic risk, and The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Participants Review Sub-Committee of York University’s Ethics Review Board. The patients/participants provided their written informed consent to participate in this study.AR: conceptualization, methodology, software, formal analysis, investigation, data curation, writing – original draft, writing – review and editing, and visualization. DG: conceptualization, methodology, software, formal analysis, resources, data curation, and writing – review and editing. KH: conceptualization, methodology, and investigation. LS: conceptualization, methodology, formal analysis, resources, writing – review and editing, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This work was supported by a Canadian Institutes of Health Research operating grant (grant number MOP-125915 to LS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this population-based, cross-sectional study, we investigated vertebral fracture (VF) prevalence among Chinese postmenopausal women. We found 14.7% of population had VFs, which increased with age. Age ≥ 65 years, hip fracture, and densitometric osteoporosis were significantly associated with VFs. The prevalence of osteoporosis was remarkably high.To investigate VF prevalence among Chinese postmenopausal women in this population-based, randomized-sampling, cross-sectional study.The investigator obtained lists of women from communities. Randomization was performed using SAS programming based on age group in each region. Postmenopausal women aged ≥ 50 years in the urban community were included. The investigator interviewed subjects to collect self-reported data and measured BMD. Spine radiographs were adjudicated by Genant’s semi-quantitative method. VFs were defined as fractures of at least one vertebra classified by Genant’s score 1–3 and were analyzed using descriptive statistics.p < 0.0001) and was significantly higher in women aged ≥ 65. VF prevalence did not differ between North  and South China . In women with no prior VFs, prevalent VFs were 12.4% . Age ≥ 65 years , hip fracture , and densitometric osteoporosis  were significantly associated with prevalent VFs. Prevalence of osteoporosis was 32.9% measured by BMD and 40.8% using NOF/IOF clinical diagnosis criteria.A total of 31,205 women listed for randomized sampling from 10 Tier-3 hospitals at 5 regions. Of 2634 women in the full analysis set, 14.7%  had prevalent VFs. VF prevalence increased with age (Cochran–Armitage test VFs are prevalent among Chinese postmenopausal women who were ≥ 50 years and community-dwelled. Osteoporosis prevalence is remarkable when fragile fractures were part of clinical diagnosis.The online version contains supplementary material available at 10.1007/s11657-022-01158-x. Osteoporosis is prevalent worldwide  and causVertebral fractures increase with age among postmenopausal women. Published literature has suggested an overall prevalence between 10–20% –20 and 3This was a population-based, randomized sampling, cross-sectional study investigating the prevalence of VFs among urban-dwelling postmenopausal women in China . The study selected 5 geographic regions  in China to recruit postmenopausal women. Subject enrollment commenced in January 2017 and ended in July 2018 at outpatient endocrinology or orthopedics clinics from 10 Tier-3 hospitals in 5 geographic regions , masked all identifiable bio-information, and presented them to the study statistician who generated a new list to resequence residents by stratum using SAS randomization programming. The investigator used the original resident number retained in the randomization list to find the residents’ contact details to invite them by post, phone calls, or other electronic methods. The resident who accepted the invitation was asked to undergo on-site formal study screening procedures.The investigator assessed the subject eligibility at the site. Women were eligible if they were Chinese aged 50 years or above, postmenopausal, willing to consent, and lived in an urban community > 6 months. Menopause was defined as no menses naturally for at least a year by self-reporting, or 6 months of spontaneous amenorrhea with serum FSH levels > 40 mIU/mL, or at least 6 months after a surgical bilateral oophorectomy with or without hysterectomy. Women were excluded if they were not Asian and had cognitive impairment , physical impediment, or other potential reasons  affecting the completion of the study procedure. The recruitment was independent of a seasonal or social impact and planned to occur at any time point. All subjects or their legal representatives provided written informed consent before any study screening procedure.The subject paid a single visit to the site for all study-related procedures. Upon inclusion, the investigator performed an interviewer-administered questionnaire. The subject was required to have BMD measurement and vertebral radiologic assessment after the completion of the interview. The investigator may perform a physical examination for fracture-related medical history whenever necessary.The questionnaire was interviewer-administered and designed based on previous studies –33 with A dual-energy x-ray absorptiometry (DXA) device  measured BMD at each subject’s lumbar spine, the femoral neck, and the total hip. The study did not allow a DXA measurement on a mobile health van. Quality control and calibration of DXA machines  were based on the site routine practice as recommended by the manufacturer (GE Lunar and Hologic) at each site. BMD measurement was done by the same machine or machine type from the manufacturer for all subjects at each site.All sites used the same scanning protocol on patient positioning. Patient was positioned straight on the DXA table and centered in the field with no rotation. The spine can be shown straight  on the image from the testing report (spine is straight on the image). The scan included at least the lowest vertebra with ribs (T12) and L4 and L5. The investigators reviewed the DXA BMD report to check the patient positioning and scanning area.The site’s radiologists followed the study protocol to perform lateral radiographs centered on the thoracic (T)7 and the lumbar (L)2 vertebrae. Radiographs were taken with the subject in the left lateral position by breathing to blur the overlying ribs and lung by motion. The investigator sent radiographs to the central adjudication team , who standardized fracture assessment using Genant’s semi-quantitative method with excellent inter- and intra-observer agreements . Two (2)A vertebral fracture is defined as the presence of at least one vertebra morphometrically classified as grade 1 (mild), 2 (moderate), or 3 (severe) on Genant’s score  for vertDensitometric osteoporosis is defined as a BMD T-score ≤  − 2.5 in at least one of the anatomic sites, including the lumbar spine, the femoral neck, and the total hip  (WHO). TA fragile fracture is defined as a fracture that occurred with minimal trauma at a typical site of osteoporotic fracture that would be unlikely to cause a fracture in a non-osteoporotic adult , 33.The sample size was calculated based on previous prevalence data per age group –17, 20. Descriptive statistics addressed the analysis of prevalence in VFs as the primary objective. Percentages of prevalence with a two-sided 95% CI were calculated in overall study population, age groups, and geographic regions. The 95% CI calculation used a more conservative Wilson Score method , 36. TheThe statistical analysis plan (SAP) specified all variables (risk factors) associated with prevalent VFs among postmenopausal women. A risk factor with a p-value ≤ 0.1 in univariate logistic regression entered the multivariate logistic regression model . The multivariate analysis gave an adjusted OR and corresponding 95% CI for each risk factor in the model. A ROC (Receiver Operating Characteristics) AUC (Area Under the Curve) showed the model prediction strength for prevalent VFs.The full analysis set (FAS) included all subjects who had vertebral radiologic procedures in the study. The per protocol set (PP) included all subjects who had per-protocol vertebral radiologic procedures in the study. Subjects who did not complete both thoracic and lumbar radiologic assessments were excluded from the PP. The FAS serves the analysis population for the primary objective.The statistical analysis did not impute missing data. All analyses were performed by using SAS 9.4, SAS JMP, and 15.2 . A p-value of 0.05 or less at 2-sided significance was considered statistically significant whenever applicable. Statistical comparisons on prevalence were made using data visualization. No overlap between the two 95% CIs for prevalence concluded a significant difference.Figure Table p < 0.0001). In women who were ≥ 80 years of age, VF prevalence was 35.7% .Table p < 0.01). In women who had densitometric osteoporosis, prevalent VFs accounted for 24.2% ; in women who had normal BMD, prevalent VFs accounted for 7.0% .Figure Table The prevalence of densitometric osteoporosis was 32.9%  among Chinese postmenopausal women. Women who were ≥ 65 years (42.5%), who lived in South China (38.9%), and who had medium (39.1%) and high (66.0%) risks of osteoporosis defined by OSTA had significantly higher prevalence of densitometric osteoporosis (no overlaps between 95% CIs to compare references). Prevalent osteoporosis was high using NOF/IOF , CSOBMR , or AACE  clinical diagnosis criteria. This trend in prevalent osteoporosis was similar in the subgroups. Supplementary Table p < 0.0001), dominantly labor work , height decreased , fall on the flat ground after 50 years old , hip fracture , and densitometric osteoporosis  were associated with an increased risk of prevalent VFs. Women who received osteoporosis treatment had a 1.93-fold likelihood of having a prevalent VF . The multivariate logistic regression exihibited a good model fit of all independent variables (p = 0.92). A complete analysis of risk factors associated with prevalent VFs using a two-step logistic regression model is displayed in Supplementary Table In the multivariate logistic model Fig. , adjusteFindings from this population-based, randomized-sampling, cross-sectional study suggested VFs were prevalent and increased with age among Chinese postmenopausal women who were 50 years or above and community-dwelling. The study did not suggest a geographic difference in prevalent VFs. The prevalence of osteoporosis was remarkably high among Chinese postmenopausal women.The study obtained lists of 31,205 residents from local communities to randomize and sample by geographic region and age group. The study adopted an interviewer-administered questionnaire, had BMD measurements at three anatomical sites, used a standard X-ray protocol, and adjudicated all vertebral fractures. This study is among the few Chinese population-based studies on osteoporotic VFs and has been the only nationwide study. The study has limitations. Incident vertebral fractures were not studied because of a cross-sectional design. The analysis of the temporal relationship between osteoporosis and VFs was not feasible. Randomized sampling was performed to recruit women free of volunteer effect. Nevertheless, this procedure inherently brought non-response to study invitation, which was higher than in a previous study in Beijing [The study investigated VF prevalence as the primary objective. Most women (98.9%) followed protocol-specified procedures for radiologic imaging. Study analysis indicated that 14.7% of postmenopausal women had prevalent vertebral fractures. In women with no history of fragile or vertebral fractures, prevalent VFs did not differ significantly compared to the overall population. The prevalence increased with age, in which the highest (35.7%) was observed in women who were 80 years or above. The overall and age-specific VF prevalence was generally comparable to results from studies in the USA  and diffPublished studies supported the difference in prevalent VFs among countries –20. One Vertebral fractures are clinically asymptomatic and usually require a radiologic diagnosis. Specific radiologic assessments, including quantitative or semi-quantitative methods, are time-consuming, and trained radiologists are needed. In routine practice, clinical diagnosis missed approximately three-fourths of vertebral fractures in East Asia , 41. Thi(30) and AACE [Many studies have demonstrated the high prevalence of osteoporosis in the USA and Europe, and Asian countries, including China . Treatme and AACE and AACE2 showed a moderate association with the increased risk of prevalent VFs, which differed from previous findings [Women enrolled in the study have medical history representing the general Chinese women population. They were generally healthy, had a low proportion of smoking or drinking, had outdoor exercise, and had low calcium and vitamin D intake , 26, 42.findings . Last, wIn conclusion, vertebral fractures are prevalent among postmenopausal women who were 50 years or above and community-dwelled in China. The prevalence of osteoporosis is remarkably high among postmenopausal women when major fragile fractures were part of the clinical diagnosis as recommended by NOF/IOF, CMA/CSOBMR, and AACE.Supplementary file1 (DOCX 1441 KB)Below is the link to the electronic supplementary material.


ARSG) gene have been reported to underlie USH type IV. This distinct type of USH is characterized by late-onset RP with predominantly pericentral and macular changes, and late onset SNHL without vestibular dysfunction. In this study, we describe the USH type IV phenotype in three unrelated subjects. We identified three novel pathogenic variants, two novel likely pathogenic variants, and one previously described pathogenic variant in ARSG. Functional experiments indicated a loss of sulfatase activity of the mutant proteins. Our findings confirm that ARSG variants cause the newly defined USH type IV and support the proposed extension of the phenotypic USH classification.Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is highly heterogeneous both clinically and genetically. Recently, variants in the arylsulfatase G (The online version contains supplementary material available at 10.1007/s00439-022-02441-0. Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is the leading cause of inherited deaf-blindness with an estimated prevalence of 3–17 per 100,000 individuals  was described to be associated with USH type I but this was later refuted  has been suggested to be a modifier gene of retinal disease and a contributor to digenic USH  gene (OMIM 610008) was associated with an atypical USH phenotype characterized by late-onset RP with a distinctive retinal phenotype of ring-shaped atrophy along the arcades, late-onset progressive SNHL, and no vestibular involvement   in the arylsulfatase G   and 4-methylumbelliferyl sulfate , the University Medical Center Freiburg (Germany) and the Mater Misercordiae University Hospital . All included subjects were previously seen at the out-patient clinic of these hospitals.To obtain a complete description of the subjects' phenotypes, we retrospectively collected demographic data and general medical history, ophthalmic data, audiological data, and data on vestibular function. Demographics and medical history included age, descent, comorbidities, medication use, and medical family history. For ophthalmic phenotyping, we collected ocular history, age of onset and progression of ophthalmic symptoms, best corrected visual acuity, refractive error, slit lamp examination findings, and multimodal imaging including color fundus photography, optical coherence tomography (OCT), visual field tests, electroretinography (ERG), and fundus autofluorescence. Audiological data included age of onset, type and progression of audiological symptoms, medical history of the ear, otoscopic assessment, pure tone and speech audiometry, and speech-in-noise testing. The medical history of the ear included recurrent ear infections, previous noise exposure, the use of ototoxic medication, meningitis, severe head injuries, and ear surgeries. Finally, we collected data on vestibular function by identifying whether vestibular symptoms were present and whether vestibular function had been examined. Incomplete data were supplemented with additional diagnostics if possible.0.5–4 kHz) for each subject was determined. A two-parameter logistic function was used to fit the data, i.e. agemid describes the age at which the hearing thresholds are halfway between 0 dB and the asymptotic value (fixed at 130 dB). The parameter scale represents the slope of the function at this midpoint, which was used as an estimate for the annual threshold deterioration. The calculated age of onset was defined as when the calculated hearing threshold exceeds 25 dB, defined by the WHO as abnormal hearing, and subjects could benefit from amplification  was used to analyze and plot the thresholds for pure-tone audiometry. Air conduction thresholds were used to analyze the observed severity and degree of symmetry of hearing loss and audiogram configuration. In case of an air–bone gap, the bone conduction thresholds of all audiograms of the subject in question were used instead. In addition, age-related typical audiograms (ARTA) were derived from linear regression analysis of all available audiometric data of included and previously reported subjects as described to provide insight into the rate of progression of hearing loss  and hearing impairment  was performed in the ISO15189 accredited Genome Diagnostic Laboratory of the Radboud university medical center  according to routine diagnostic procedures , CNGA1 (OMIM 123825), EYS (OMIM 612424), PDE6A (OMIM 180071), PDE6B (OMIM 180072), and RPE65 (OMIM 180069).Subject D and F were pre-screened to exclude biallelic variants in other USH-associated genes in Trinity College Dublin (Ireland) as part of the Target 5000 study . Both patient-derived missense variants were introduced by site-directed mutagenesis as previously described in the WT ARSG . The PCR product was then inserted in a pCI-NEO vector, adapted to the Gateway® cloning system as previously described . His visual acuity was relatively spared (OD (right eye) 20/25 and OS (left eye) 20/32 Snellen). Ophthalmoscopy revealed waxy pallor of the optic disc, mild attenuation of the vessels, and a preserved fovea with perifoveal retinal pigment epithelial mottling. Atrophy was present around the vascular arcades; adjacent to the atrophy, predominantly located nasally and superior to the optic disc, intraretinal bone spicules were noted . The far periphery appeared relatively preserved. OCTs showed foveal sparing with only paracentral loss of the outer retina, corresponding to the relatively mild loss of visual acuity. On fundus autofluorescence, a ring of hyperautofluorescence was visible in the posterior pole at the transition zone. The mid-periphery showed confluent areas of hypoautofluorescence with preserved autofluorescence in the far periphery. ERG revealed lower scotopic than photopic responses; both were diminished. Goldmann perimetry showed an absolute ring scotoma with intact central vision and a relatively preserved peripheral field in accordance with a normal V-4e Isopter. At the age of 42, before his RP diagnosis, subject N was diagnosed with bilateral SNHL for which he is rehabilitated with bilateral conventional hearing aids . In addition, he experienced tinnitus. Physical examination and otoscopy showed no abnormalities. Pure tone audiometry revealed bilateral moderate to severe SNHL, with air and bone conduction PTAs (0.5–4.0 kHz) of 49 and 46 dB hearing level (dB HL) (right) and 55 and 53 dB HL (left) at age 46. At age 49, there was asymmetry in the air conduction thresholds, mainly in the high frequencies to the detriment of the right. An air–bone gap was observed primarily in the right ear, which could not be explained by otoscopy or CT. The latter revealed no congenital temporal bone abnormalities. At that time, air and bone conduction PTAs were 70 and 51 dB HL (right) and 64 and 55 dB HL (left). Based on the bone conduction PTAs, the progression in three years was 5 (right) and 3 dB HL (left). The subject did not report any vestibular problems.Subject F is a 72-year-old woman of Caucasian origin, recruited at the Eye Center of the University of Freiburg, Germany. She was diagnosed with hypothyroidism earlier in life. There was no relevant medical or family history related to hearing, vision, or vestibular function. Her parents were not known to be consanguineous. This individual experienced central vision loss from the age of 40 . Visual acuity deteriorated to hand movement vision only. Ophthalmoscopy revealed a pale optic disc, mild attenuation of the vessels, and atrophy around the arcades with intraretinal bone spicules predominantly located nasally and superior to the atrophy. The far periphery appeared intact. OCT showed complete loss of the outer retinal layers. Fundus autofluorescence showed extensive hypoautofluorescence in the periphery that extended to the inner part of the vascular arcades, leaving a small ring of relative normal autofluorescence in the posterior pole. ERG revealed absence of both cone and rod responses. As the disease progressed, concentric restriction of the peripheral visual field and a central scotoma developed. Subject F also experienced progressive, bilateral SNHL since the age of 20 for which she has been using bilateral conventional hearing aids since age 31–40 . At the age of 60 and 72, pure-tone audiometry showed moderate to severe SNHL with a flat audiogram configuration and PTAs of 73 dB HL (both ears) and 90 (right) and 85 dB HL (left). There were no additional problems, notably no reported vestibular symptoms. Bilateral bithermal vestibular caloric stimulation revealed a symmetrical response of the lateral semicircular canals.Subject D is an 87-year-old man of Caucasian origin seen at the Mater Misercordiae University Hospital in Dublin, Ireland. He had pulmonary tuberculosis when he was 19 years old. Later in life, he developed gastro-esophageal reflux disease, atrial fibrillation, and benign prostatic hyperplasia. This subject was diagnosed with exotropia of the left eye in his early youth. When he was over 60 years old, a posterior subcapsular cataract was observed bilaterally. Medical and family history was uneventful, his parents were not known to be consanguineous. At the age of 51–60, he experienced night blindness and photophobia . Over time, visual acuity deteriorated to light perception in the right eye and no-light perception in the left eye at age 86. Retinal imaging revealed excavation of the nerve heads, mild attenuation of the vessels, and central atrophy connecting to atrophy around the vascular arcades. This was surrounded by a ring of intraretinal bone-spicule hyperpigmentation, nasally and superior to the disc. Fundus autofluorescence showed central hypoautofluorescence and large confluent areas of hypoautofluorescence adjacent and posterior to the vascular arcades. OCT revealed complete loss of outer retinal layers. At the age of 31–40, subject D developed bilateral SNHL for which he was rehabilitated by bilateral conventional hearing aids . No other audiological data were available or could be obtained. He reported no vestibular symptoms.Audiograms of the right and left ears of included and previously reported subjects showed a predominantly bilateral moderate to severe high-frequency SNHL . The ARTA displayed an average progression of hearing loss of 1.0–1.5 dB HL per year ; NM_014960.5:c.275T > C NP_055775.2:p.(Leu92Pro). Segregation analysis showed that these variants were present in a compound heterozygous state  as there were no unaffected relatives with both ARSG variants. The c.275T > C p.(Leu92Pro) variant is a missense variant that was predicted to be pathogenic by all used in-silico prediction tools ; NM_014960.5:c.1024C > T NP_055775.2:p.(Arg342Trp). The c.1326del p.(Ser443Alafs*12) variant is a previously described pathogenic stop-gain variant ) was identified in ARSG (NM_014960.5:c.588C > A NP_055775.2:p.(Tyr196*)), a non-canonical splice site variant in ADGRV1 ), a missense variant in MYO7A , PhyloP: 2.5 (threshold: 2.7), Grantham: 29 (threshold: 80), MutationTaster: 'Benign' and SIFT: 'Tolerated') and the SpliceAI predictions for the ADGRV1 and PCDH15 variants were low (ADGRV1: acceptor splice site loss at + 11nt (0.01/1); PCDH15: acceptor splice site gain at + 124nt (0.24/1), acceptor splice site loss at −54nt (0.02/1), donor splice site gain at + 3nt (0.19/1)). Also, SpliceAI predicted no pseudo-exon inclusion for the PCDH15 variant. Therefore, the ARSG variant was considered the strongest candidate in a gene associated with USH. Manual inspection of the sequencing reads (CRAM files) revealed indications for a deletion spanning exons 7 and 8 of ARSG. PCR and Sanger sequencing confirmed the deletion and breakpoints: NM_014960.5:c.705-3940_982 + 2952del NP_055775.2:p.(Ser235Argfs*29). PCR with a reverse primer designed to bind nucleotides deleted in the c.705-3940_982 + 2952del allele demonstrated that the c.588C > A p.(Tyr196*) variant and the deletion are in trans.Genome sequencing was initiated in subject D because no variants could be identified in genes associated with USH. Initially, four heterozygous variants in USH-associated genes were identified: a stop-gain variant in ARSG variants were either not detected or detected at low frequencies (< 0.01%) in gnomAD (Table The identified ies < 0.0% in gnomAD Table  and accoies < 0.0% in gnomAD Table . Three nAD Table  (Mistry AD Table .ARSG variant of subject N (c.275T > C p.(Leu92Pro)), followed by immunoblot analysis, revealed a modestly increased ARSG expression level compared to the WT construct , showed that the original splice donor site was lost, and that an alternative splice donor site at + 13nt in intron 10 was used. This resulted in an out of frame elongation of exon 10 and a premature stop codon   ARSG (subject F) previously revealed similar ARSG expression levels as found in constructs encoding WT ARSG, but with a slightly lower molecular weight, in line with a truncated protein. Also, a significantly lower sulfatase activity was observed than for WT ARSG  and c.705-3940_982 + 2952del p.(Ser235Argfs*29) identified in subject D confirmed the truncated proteins to be of lower molecular weight than the WT ARSG, within their respective expected ranges  mouse was shown to accumulate heparan sulfate in enlarged lysosomes and develop typical mucopolysaccharidosis-like features  involvement, as would be expected from the phenotype of the KO mice and ARSG-deficient dogs. Remarkably, a similar situation has been described for other genes causative for lysosomal storage diseases. While variants in genes associated with neuronal ceroid lipofuscinosis (CLN3 (OMIM 607042), CLN5 (OMIM 608102), and MFSD8/CLN7 (OMIM611124)) typically lead to severe neuronal dysfunction and lysosomal storage in neurons and the peripheral tissues, some variants present with isolated retina disease  are associated with mucopolysaccharidosis in the vast majority of patients, but isolated retina degeneration has also been described   had a medical history of hypothyroidism, which was not reported in previous CDH23  nor in the literature to the best of our knowledge. Second, there are cases with variants in known USH-associated genes and phenotypes that differ from the corresponding type of USH, such as an USH II phenotype caused by CDH23 variants or a phenotype with vestibular dysfunction caused by USH2A variants Supplementary file2 (DOCX 16 KB)Supplementary file3 (DOCX 24 KB)Supplementary file4 (DOCX 27 KB)S Fig. 1A) Fundus photo  showing intraretinal bone-spicules predominantly located nasally and superior to the optic disc. (B) Fundus autofluorescence image  showing a ring of hyperautofluorescence in the posterior pole at the transition zone and confluent areas of hypoautofluorescence in the mid-periphery with preserved autofluorescence in the far periphery. (C) Optical coherence tomography  showing foveal sparing with only paracentral loss of the outer retina, corresponding to the relatively mild loss of visual acuity (OD 0.8 and OS 0.6 Snellen). (D) Goldmann perimetry (at age 49 years) showing an absolute ring scotoma with intact central vision and a relatively preserved peripheral field. OD, right eye; OS left eye. Supplementary file5 (EPS 8991 KB)Retinal imaging of patient N. ; subject MOL0120 III:2, MOL0737 II, and TB55 II (Khateb 2018). dB HL, decibel hearing level. Supplementary file6 (EPS 3432 KB)S Fig. 3 SPP2 and MCM2 variants were also found in unaffected relatives. The ARSG variants were shown to be present in a compound heterozygous state. *Subject II:1 was not tested. Supplementary file7 (EPS 770 KB)Segregation analysis of subject N. The initially identifiedBelow is the link to the electronic supplementary material.


Longjing43 and Zhongcha108 are two widely planted tea varieties in China. Although Zhongcha108 shows higher disease resistance than Longjing43, the potential role of beneficial tea rhizosphere microbes in disease resistance is largely unknown. In this study, the structure and function of rhizosphere microbial communities of these two tea varieties were compared by using the Illumina MiSeq sequencing (16S rRNA gene and ITS) technologies. Rhizosphere soil was collected from four independent tea gardens distributed at two locations in Hangzhou and Shengzhou cities in eastern China, Longjing43 and Zhongcha108 are planted at both locations in separate gardens. Significant differences in soil physicochemical properties as demonstrated by ANOVA and PCA, and distinct rhizosphere microbial communities by multiple-biotech analyses  between both locations and tea varieties (p < 0.01) were found. Functions of bacteria were annotated by the FAPROTAX database, and a higher abundance of Nitrososphaeraceae relating to soil ecological function was found in rhizosphere soil in Hangzhou. LDA effect size showed that the abundance of arbuscular mycorrhizal fungi (AMF) was higher in Zhongcha108 than that in Longjing43. Field experiments further confirmed that the colonization rate of AMF was higher in Zhongcha108. This finding testified that AMF could be the major beneficial tea rhizosphere microbes that potentially function in enhanced disease resistance. Overall, our results confirmed that locations affected the microbial community greater than that of tea varieties, and fungi might be more sensitive to the change in microenvironments. Furthermore, we found several beneficial microorganisms, which are of great significance in improving the ecological environment of tea gardens and the disease resistance of tea plants. These beneficial microbial communities may also help to further reveal the mechanism of disease resistance in tea and potentially be useful for mitigating climate change-associated challenges to tea gardens in the future.Soil microenvironments and plant varieties could largely affect rhizosphere microbial community structure and functions. However, their specific effects on the tea rhizosphere microbial community are yet not clear. Beneficial microorganisms are important groups of microbial communities that hold ecological functionalities by playing critical roles in plant disease resistance, and environmental stress tolerance.  Rhizosphere microbial communities play crucial roles in facilitating plant nutrient uptake, defense and immunity . As soilBeneficial microorganisms are important groups of microbial communities that hold ecological functionalities by playing critical roles in plant disease resistance and environmental stress tolerance . MutualiCamellia sinensis (L.) O. Ktze.) is a perennial evergreen broad-leaved cash crop, and distributed in areas with high temperature and humidity  is more sensitive to the change in microenvironments?Zhongcha108?What are the beneficial microbial groups in the tea rhizosphere? Are there any potential beneficial microorganisms that could contribute to the higher disease resistance of Sampling was conducted from two locations with different soil nutrient conditions in Zhejiang, China: tea gardens of the Tea Research Institute of the Chinese Academy of Agricultural Sciences, Hangzhou  and the tea gardens of the Shengzhou integrated experimental base, Shengzhou . Two tea varieties  were being cultivated in both sampling sites since 2013 and were managed under similar management practices and fertilization routines. Briefly, there were four tea gardens in this study, two of them were located in Hangzhou City and the rest two were located in Shengzhou City. In the same location, both two tea varieties were planted in different tea gardens. Urea (450 kg·hm-2) was used as germination-accelerating fertilizer in the middle of February, and compound fertilizers  were applied before the heavy pruning of spring tea in May, and rapeseed cake  was used as base fertilizer in the first 10 days of October every year. Tea plants were light pruned after the spring tea harvesting, the second pruning was done in mid-July, and the third topping pruning was performed at the end of November each year.In each tea garden, we randomly selected eight sampling plots, and in each plot (6*6 m2)，five tea plants were randomly selected for rhizosphere soil sampling. The rhizosphere soil of each tea garden was mixed into three samples for the analysis of soil physicochemical properties. All samples were collected and stored in the ice box, then brought them back to the laboratory and stored at −80°C for subsequent DNA extraction in 4 h. The rhizosphere soils of Longjing43 and Zhongcha108 in Hangzhou were recorded as HZ-LJ43 and HZ-ZC108, respectively. The rhizosphere soils of Longjing43 and Zhongcha108 in Shengzhou were recorded as SZ-LJ43 and SZ-ZC108, respectively.+, Ca2+, and Mg2+ were extracted using 1 M KCl (1:10), and determined by using atomic absorption spectrophotometry .Soil pH was determined by a combination of glass electrodes using a 1: 2.5 (w: v) ratio of soil to distilled water . Soil toRhizophagus irregularis (syn. Globus intraradices). Corn plants were used to propagate fungal spores. After 3 months of low phosphorus cultivation, corn roots were cut up, mixed with sand, and stored at 20°C for later use. Sixhundred spores/pot (25 g) were added to potted tea varieties Longjing43 and Zhongcha108. After 60 days, the root length colonization rate (RLC%) was detected.The AMF used in the experiment is The RLC% of tea roots was detected by trypan blue staining . The roo2O. The PCR system was amplified according to the following reaction conditions: pre-denaturation 98°C for 30s, followed by 25 cycles: denaturation 98°C for 15 s annealing 58°C for 15 s extension 72°C for 15 s. The final extension is 72°C for 1 min. PCR products were purified with AMPure XP Beads . Quantification was performed by using PicoGreen dsDNA Assay Kit . After quantification, Illumina Novaseq 6,000 pin-end 2 × 150 bp platform was used for further sequencing.SDS cracking fluid freezing and thawing method was used for DNA extraction, genomic DNA extraction was accomplished by PowerMax extraction kit , and thereafter the DNA gel was stored at −20°C. DNA quantity and quality were determined by using NanoDrop ND-1000 luminance meter . The primer sequences of bacteria and fungi are two sets: 1) 16S: forward primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and reverse primers 806R (5′-GGACTACHVGGGTWTCTAAT-3′); 2) ITS: forward primers ITS1 (5’-CTTGGTCATTTAGagGaAGTAA-3′) and reverse primers ITS2 (5 ‘-GCTGCGTTCTTCATCGATGC-3′). Barcode was synthesized into the sequence by using a specific 7-BP specific sequence. Phusion high-fidelity PCR Master Mix with HF Buffer was used for the PCR reaction system 50 μl: 25 μl. Three microliter (10 μM) F/R primers, 10 μl DNA sample, 6 μl ddHThe Quantitative Insights Into Microbial Ecology  pipeline was employed to process the sequencing data, as previously described . BrieflyThe OTU table was further generated to record the abundance of each OTU in each sample and the taxonomy of these OTUs. OTUs containing less than 0.001% of total sequences across all samples were discarded. To minimize the difference in sequencing depth across samples, an average, rounded rarefied OTU table was generated by averaging 100 evenly resampled OTU subsets under 90% of the minimum sequencing depth for further analysis.Sequence data analyses were mainly performed by using QIIME and R (v3.2.0) packages. OTU-level alpha diversity indices  were calculated. OTU-level ranked abundance curves were generated to compare the richness and evenness of OTUs among samples.via principal coordinate analysis (PCoA)  unless otherwise stated.LEfSe  was performed to detect differentially abundant taxa across groups using the default parameters  . Differes (PCoA) . Taxa abrameters . FAPROTArameters , the sigThe experimental data  were statistically analyzed by SPSS19.0 and figures were drawn by R and GraphPad prism8.+ (AK), Ca2+ (ACa), and Mg2+ (AMg), of different tea varieties in different locations. Results revealed that soil pH, and the contents of AK, ACa and AMg in Shengzhou gardens were significantly higher than that in Hangzhou (p < 0.01). There is no significant difference in the contents of TOC and TON between the tea gardens in Shengzhou and Hangzhou. Even the TOC and TON contents were much higher in Longjing 43, than that in Zhongcha108 in Hangzhou, however, there is no difference between those two varieties in the Shengzhou location. The contents of AP in Hangzhou were significantly higher than that in Shengzhou gardens  and N (TON), available phosphorus (AP), soil available KTo assess the rhizosphere microbial community structure, we compared the composition and abundance of rhizosphere microorganisms in tea plants and the co-occurrence of microbial communities in different locations and varieties. Sequences were clustered into Operational taxonomic units (OTU) with a 97% threshold, and diluted to the same sequencing depth. These sequences were divided into nine dominant bacteria phyla, including Proteobacteria(38%), Crenarchaeota (4%), Acidobacteriota (22%), Actinobacteriota (11%) , and fivp < 0.01; We compared the differences in bacterial phyla between the four gardens, and found that Crenarchaeota was the phylum with higher relative abundance in Hangzhou, the relative abundance of Crenarchaeota in HZ-LJ43 (4.1%) and HZ-ZC108 (4.1%) were higher than that in SZ-LJ43 (1.9%) and SZ-ZC108 (2.5%). The relative abundance of Acidobacteriota in HZ-LJ43 (21.9%) and HZ-ZC108 (30.5%) were higher than SZ-LJ43 (16.8%) and SZ-ZC108 (17.7%); The relative abundance of Chloroflexi was higher in Shengzhou  than that in Hangzhou   and SZ-LJ43 (30%) was higher than that in HZ-ZC108 (23.4%) and SZ-ZC108 (27.7%). However, there was no bacterial phylum with significantly higher relative abundance in p < 0.01; p < 0.01; Next, we compared the differences in fungal phyla between the four gardens, and found that Ascomycota was the phylum with higher relative abundance in Hangzhou gardens, the relative abundance of Ascomycota in HZ-LJ43 (17.4%) and HZ-ZC108 (22.5%) were higher than SZ-LJ43 (13.2%) and SZ-ZC108 (9.1%); the relative abundance of Zygomycota in HZ-LJ43 (0.05%) and HZ-ZC108 (0.1%) were higher than that in SZ-LJ43 (0.03%) and SZ-ZC108 (0.01%)   . For theZhongcha108 than that in Longjing43 , HZ-ZC108 (610), SZ-LJ43 (212), SZ-ZC108 (125). There were 458 identical OTUs in fungal communities. The unique OTU of each fungal community was HZ-LJ43 (248), HZ-ZC108 (324), SZ-LJ43 (189), SZ-ZC108 (125). The number of specific OTU was higher in Hangzhou than that in Shengzhou, and the number of specific Fungal OTU was higher in ngjing43 .Longjing43 and Zhongcha108, and the influence of different locations on the fungal community was greater than the influence of variety  to screen biomarkers (species with significant differences between groups) among samples. The results showed that compared with Zhongcha108 was significantly higher than that in Longjing43 (p < 0.01). The relative abundance of Glomerella and Colletotrichum was also significantly different among locations and varieties (p < 0.05). Especially, the relative abundance of Colletotrichum in Longjing43 was significantly higher than that of Zhongcha108 . Subgroup_13 was negatively correlated with nitrate respiration, nitrate reduction, nitrogen respiration, photoautotrophy-related functions and photonutrition (p < 0.01). Gaiellales was positively correlated with nitrate reduction (p < 0.05). Chitinophagaceae was positively correlated with the degradation of aromatic compounds (p < 0.05). Paenibacillus was negatively correlated with urea degradation. Edaphobacter bacteria was positively correlated with urea degradation . The relative abundance of Paenibacillus was significantly higher in longjing43 than that in zhongcha108 (p < 0.05). The relative abundance of Subgroup_13 was significantly higher in Hangzhou than that in Shengzhou, and was significantly higher in longjing43 than that in zhongcha108  was higher than that of Longjing43 (27.54%) , and the microbial abundance of the two locations is also significantly different (p < 0.05). We found that the rhizosphere microorganisms of tea plants were affected at different pH values, for example, the abundance of microorganisms such as Acidobacteriota and Ascomycota was higher in the tea gardens with lower pH at Hangzhou. It is consistent with the results from previous studies, which have also confirmed that these microorganisms are more abundant in acidic soils  ,D. Moreop < 0.01) (p = 0.05)  . Huang e < 0.01)  and Begu < 0.01) . We also < 0.01) . It was  < 0.01) . Previou < 0.01) , and the < 0.01) . Althoug < 0.01) . However = 0.05) ,B. BasedThere were significant differences in soil physical and chemical properties and rhizosphere microbial communities between the two tea locations, and there were also significant differences in rhizosphere microorganisms between the two tea varieties in the same location. The analysis of PCoA and Co-occurrence confirmed that the influence of different locations on tea rhizosphere microorganisms is greater than that of different tea varieties, and fungi are more sensitive to the change in microenvironments. LDA Effect Size analysis of fungi showed that the abundance of AMF was higher in Zhongcha108 than that in Longjing43, and the difference in colonization rate of AMF between the two varieties was verified by field experiments. The rhizosphere microbial community could be influenced by both locations and varieties, however, the location is a more impactful driving factor, suggesting that rhizosphere fungi are more sensitive to changes in microenvironments. Among various microbial communities, Nitrososphaeraceae are related to the ecological function of tea gardens and AMF are related to the disease resistance of tea plants. Thus, the use of beneficial microbial communities is of great significance for the improvement of the ecological environment of tea gardens and the disease resistance of tea plants.http://www.ncbi.nlm.nih.gov/bioproject/872891.The data presented in the study are deposited in the BioProject database, accession number PRJNA872891. XL, L-CF, and W-YH conceived and designed the research. ZZ, S-BG, QH, GJA, PY, L-PZ, Z-ZL, J-YF, and J-YZ performed the experiments and analyzed the data. XL, J-YF, W-YH, ZZ, and L-CF discussed the data. ZZ, L-CF, GJA, and XL wrote the manuscript with the contributions from the other authors. All authors contributed to the article and approved the submitted version.This work was funded by the National Key R&D Program of China (2020YFD10007); the Opening Fund of Provincial Key Lab of Tea Refining and Innovation Key Laboratory of Sichuan Province (SCTOF202202); Hangzhou Science and Technology Development Project (2020ZDSJ0632); and the Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2019-TRICAAS). L-CF thanks to the Scientific research startup fee for introduced talents of NWAFU (Z1090222024).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Posterior minimally invasive surgery has been increasingly used in in recent years for the clinical treatment of cervical spondylosis. However, this treatment remains challenging and has not been comprehensively reported. The aim of this study was to provide a systematic review of posterior minimally invasive treatment for cervical spondylosis to demonstrate the clinical efficacy and safety of this procedure.We collected information from patients with myelopathy or radiculopathy cervical spondylosis who underwent posterior minimally invasive surgery and verified the clinical efficacy and safety of these surgeries with different measurement indicators from five electronic databases: the Nurick, visual analog scale score, Japanese Orthopaedic Association (JOA) score, Neck Disability Index (NDI), EuroQol Five Dimensions Questionnaire (EQ-5D) score, Short-Form Health Survey Physical Component Summary (SF12-PCS) questionnaire score, Short-Form Health Survey Mental Component Summary (SF12-MCS) questionnaire score, and the MOS item short form health survey (HF-36) score. The decompression effect, cervical spine stability, average surgery time, surgical blood loss volume, length of hospital stay, and related complications were included in the descriptive analysis. Reporting of this protocol followed the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines checklist.P < 0.05), and satisfactory clinical significance was acquired in the descriptive indicators . Additionally, we showed that there was a 4.9% postoperative complication rate and the types of complications that may occur.We identified 14 observational studies of cervical spondylosis with 479 patients, mainly including 197 cases of myelopathy and 207 cases of radiculopathy. Channel and endoscopic techniques were used. This study was certified by PROSPERO: CRD42021290074. Significant improvements in the quantitative indicators  were observed between pre- and postoperation (Posterior minimally invasive surgery is an effective and safe method for the treatment of cervical spondylosis and is a recommended optional surgical procedure for single-segment myelopathy and radiculopathy. Degenerative cervical spondylosis (DCS) can be divided into a number of subdiseases, including cervical spondylotic radiculopathy (CSR) and cervical spondylotic myelopathy (CSM) –3. CSR iRecently, minimally invasive surgeries have been developed in spine surgery and have shown the same clinical effects as traditional open surgery for certain degenerative diseases in the lumbar spine , 11. MinBy analyzing previous research, we argue in favor of the clinical efficacy of posterior minimally invasive surgery in DCS, which includes endoscopy technology, channel technology keyhole surgery, minimally invasive laminectomy, and so on. Many clinical indicators and whether these procedures can help patients avoid certain complications related to the traditional open surgical approach are further explored in this paper.This protocol is reported following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines checklist and is certified by PROSPERO: CRD42021290074.Articles were excluded if they (1.) had a sample size of less than 10 consecutive patients or (2.) were an editorial, conference abstract, review, comment, letter, or animal study. The language was restricted to English.The main patient inclusion criterion was CSR or CSM at a single level or multiple levels (documented using magnetic resonance imaging (MRI) and microcomputed tomography). Any patients with instability, significant anterior pathology, cervical spondylosis secondary to a tumor, trauma, severe ossification of the posterior longitudinal ligament, rheumatoid arthritis, septic spondylitis, and destructive spondylosis were excluded from the study.The intervention analyzed was posterior minimally invasive surgery for patients with different types of DCS. These include endoscopy technology, channel technology, and keyhole technology. The indication for posterior minimally invasive surgery was based on the current guidelines.We used the Nurick score, visual analog scale (VAS), JOA (Japanese Orthopaedic Association (JOA) score, Neck Disability Index (NDI), EuroQol Five Dimensions Questionnaire (EQ-5D) score, Short-Form Health Survey Physical Component Summary questionnaire (SF-12 PCS) score, Short-Form Health Survey Mental Component Summary questionnaire (SF-12 MCS) score, and the MOS item short from health survey (HF-36) score as quantitative indicators. The decompression effect, cervical spine stability, average surgery time, surgical blood loss volume, length of hospital stay, and complications were also evaluated for a descriptive analysis.Four electronic databases were searched for potential eligible studies: The Cochrane Library, PubMed, EMBASE, and Web of Science. Additionally, we manually searched the references from ClinicalTrials.gov and the gray literature. The search for the relevant literature was conducted from database inception until November 2021. We performed a literature search with the keywords “posterior approach,” “cervical spondylosis” and “minimally invasive surgery.” The full list used in the search strategy for PubMed was as follows:  OR ) OR (Thoracic Spondylosis)) OR ) OR ) OR ) OR (Spondylosis Deformans)) AND  OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR ) OR (channel)) OR (tunnel))) AND (posterior approach). This strategy was also applied for the other databases searched.Two researchers independently screened the literature and extracted and cross-checked the data; in cases of disagreement, they discussed and resolved the disagreement, or a third researcher was consulted to assist in the determination. For studies lacking information, the original authors were contacted. The extracted information included the following: (1) basic information on the included studies, including author names, year of publication, and country; (2) basic characteristics of the study population, including sample size and age; (3) specific details of the interventions; (4) key elements of the risk of bias evaluation; and (5) data on the outcome indicators of interest.The risk of bias in the included studies was evaluated using the risk of bias evaluation tool recommended by the METHODOLOGICAL INDEX FOR NONRANDOMIZED STUDIES (MINORS): (1.) A clearly stated aim: The question addressed should be precise and relevant in light of the available literature. (2.) Inclusion of consecutive patients: All patients potentially fit for inclusion (satisfying the criteria for inclusion) should be included in the study during the study period (no exclusion or details about the reasons for exclusion). (3.) Prospective data collection: Data should be obtained according to a protocol established before the beginning of the study. (4.) Endpoints appropriate to the aim of the study: Unambiguous explanation of the criteria used to evaluate the main outcome should be in accordance with the question addressed by the study. Additionally, the endpoints should be assessed on an intention-to-treat basis. (5.) Unbiased assessment of the study endpoint: Blind evaluation of objective endpoints and double-blind evaluation of subjective endpoints should be performed. Otherwise, the reasons for not blinding should be stated. (6.) Follow-up period appropriate to the aim of the study: The follow-up should be sufficiently long to allow the assessment of the main endpoint and possible adverse events. (7.) Loss to follow-up less than 5%: All patients should be included in the follow-up. Otherwise, the proportion lost to follow-up should not exceed the proportion experiencing the major endpoint. (8.) Prospective calculation of the study size: Information on the size of the detectable difference in interest should be obtained, the 95% confidence interval should be calculated according to the expected incidence of the outcome event, and information about the level of statistical significance and estimates of power should be obtained when comparing the outcomes. The risk of bias evaluation was performed independently by 2 evaluators, and the results were cross-checked and discussed.If evidence of clinical homogeneity could be established, then the study was included in the meta-analysis. We performed a qualitative synthesis of findings from scientifically admissible studies to develop evidence statements according to the principles of best-evidence synthesis. GraphPad Prism 8.0 was used for statistical analysis and graphing.A PRISMA diagram was used to report the search results and screening process. We identified a total of 176 studies: 98 studies in PubMed, 10 studies in Web of Science, 61 studies in Embase, 2 studies in the Cochrane Library, 2 studies in ClinicalTrials.gov, and 3 studies from the manual search. After the initial screening, a total of 147 studies remained . After reviewing the titles and abstracts of the 147 studies, 31 studies remained . Finally, we assessed the full text, and a consensus was reached regarding the inclusion of 14 articles (13 case series studies and 1 cohort study) , and no patients experienced postoperative cervical instability. Eleven studies reported average surgery times ranging from 44 to 163.6 min (94.56 ± 37.26 min). Nine studies reported blood loss volumes ranging from 18 to 360 ml (68.78 ± 103.31 ml), and 5 of the studies did not clearly indicate the amount of blood loss. In addition, only 6 studies reported the average length of hospital stay; the longest average length of stay was 4.66 days, and the shortest average length of stay was 1.1 days (2.39 ± 1.20 days). A total of 12 studies recorded postoperative complications, with 92 patients in five studies experiencing no complications. In the remaining 7 studies, 18 patients experienced complications; the most common was dural tears (4 patients), the least common was disk herniation recurrence (1 patient) and a loose screw (1 patient); other reported complications included bleeding (3 patients), decreased muscle strength (3 patients), dysesthesia (2 patients), pain and numbness (2 patients), and root injury (2 patients). Finally, only 6 studies reported MacNab criteria, and the proportion of patients with a poor outcome was less than 10% (Table P < 0.05) and Dahdaleh [p < 0.05) (Table P > 0.05), Qiu [P < 0.001), Yu [P < 0.001), Zhang [p < 0.001), Yabuki [p < 0.05), Haijun [P < 0.001), Oshima, S [P < 0.001), Liu [p < 0.001), Zhang [p < 0.001), and Siemionow [p < 0.001)) (Table p < 0.001), Zhang [p < 0.001), Yabuki [p < 0.05), Qiu [P < 0.001), Oshima [P < 0.001), Zhang [P < 0.001), and Ross [P < 0.01)) (Table P < 0.001), Luo [P < 0.001), Haijun [P < 0.001), Oshima, S [P < 0.001), and Siemionow [p < 0.001)) (Table P < 0.01) and Siemionow [p < 0.0001)) (Table P < 0.01) and Oshima [P = 0.05) (Table p < 0.0001)). Patient SF12-MCS scores were reported in 1 study [p < 0.0001)). Patient HF-36 scores were reported in 1 study [P < 0.05).The patients’ Nurick scores were reported in 2 studies , 25. TheDahdaleh  (p < 0.005), Qiu  (P < 0.0001), Yu  (P < 0.0), Zhang  , Liu  (p < 0.0), Zhang  (p < 0.0iemionow  (p < 0.0)) Table . The JOA), Zhang  , Qiu  , Zhang  (P < 0.0and Ross  (P < 0.0)) Table . The NDI01), Luo  ) Table . Patientiemionow    . However, we found that by comparing the quantitative indicators of posterior minimally invasive surgery and two types of cervical spondylosis, low-quality evidence showed the differences in the channel and endoscopy techniques between the pre- and postoperation study data. At the same time, among these indicators, all of the indicators for radiculopathy and most of the indicators for myelopathy showed differences, except for VAS  Table , Fig. 5.More importantly, we compared the quantitative indicators of patients with CSM treated with different surgical approaches. There was lower quality evidence showing that the most significant surgical approach for improving the change rate of JOA was removal of the herniated nucleus, and the least significant was unilateral laminectomy. Similarly, the most significant surgical approach for reducing the change rate of Neck-VAS was bilateral partial laminectomy, and the least significant was bilateral laminoplasty with spinous process ligament complex and deep extensor muscle retroposition Table , Fig. 6.This systematic review mainly focuses on the clinical efficacy and safety of posterior minimally invasive surgery for DCS. Fourteen or articles were included in the study, including 13 case series studies and 1 cohort study. In our review, patients with CSM and CSR accounted for 85% of the total number of patients, and the number of patients with the two types (CSM and CSR) was similar. Posterior minimally invasive surgery is widely used in single-segment DCS, especially in CSR, and in recent years, researchers have paid more attention to the role of different posterior minimally invasive procedures in CSM, which is also reflected in our study. From the results of the analysis, it can be concluded that there were significant differences in the data on pain, function and quality of life after posterior minimally invasive approaches when compared with those before surgery. At the same time, some results showed that posterior minimally invasive surgery in DCS had some long-term effects on reducing pain and improving local disability. Interestingly, average surgery time, surgical blood loss volume, and length of hospital stay were also satisfactory. Postoperative complications are a paramount concern, with an approximately 4.9% complication rate reported after posterior minimally invasive approaches among the 365 patients.There are limitations to the conclusions drawn from the current body of evidence, as none of the studies were RCTs. Additionally, the sample sizes are too small and may be difficult to follow-up in surgical cases. Although we classified the studies in terms of the different posterior minimally invasive procedures, each surgical team had subtle differences in surgical style, which may have increased the heterogeneity of the study. Our findings are at risk of publication bias: R. J. Luo et al. reported outliers on the Neck-VAS, for example, while K. Siemionow reported the NDI and Neck-VAS at 24 months. This may be because surgeons are reluctant to publish studies that adversely affect surgical outcomes.Surgical intervention is required for patients with DCS to relieve symptoms after failing to respond to systematic conservative treatment. For CSR patients, pain is the most significant factor affecting quality of life, and the most significant benefit of posterior minimally invasive approaches is the reduction in pain radiating to the neck and upper limbs . For sevThe posterior minimally invasive posterior surgery in DCS reduces the impact on the homeostasis of the internal environment and reduces the patient's own recovery burden, making the procedure more acceptable to patients. At the same time, this surgical approach causes less damage to the soft tissues at the back of the neck, requiring less soft tissue to be stripped away and greatly reducing postoperative axial symptoms . InteresPosterior minimally invasive surgery is an effective and safe method for the treatment of cervical spondylosis myelopathy and radiculopathy patients, especially simple, single-segment cervical spondylosis.


Bacteroides in human faecal samples. Bacteria from faecal samples were cultured anaerobically on selective media. Sonication-based DNA extraction was performed, followed by almost complete 16S rRNA gene polymerase chain reaction amplification and MinION sequencing with the Flongle adapter. Sequence analysis was performed using NanoCLUST, while RStudio was used for graphics. For 110 of the 125 colonies investigated, 100% of reads were attributed to a single species, while the remaining 15 colonies consisted of mixtures of up to three different species. The proposed bacterial identification method is advantageous for isolating particular bacteria for which there are no exclusively selective media, as it avoids lengthy colony purification and DNA purification methods, and yields a quick colony identification with high accuracy. Therefore, this method can be used for directly screening for pure cultures of target microorganisms and is suitable for the identification of bacteria in culturomics studies.Most bacterial identification methods require extensive culturing, strain purification and DNA extraction protocols. This leads to additional expenses and time lags when isolating specific bacteria from complex microbiological ecosystems. This study aimed to develop a fast and robust method for identification of lactobacilli, bifidobacteria and The online version contains supplementary material available at 10.1007/s00284-023-03201-7. Non-selective microbiological growth media can support the growth of a wide variety of microorganisms by supplying a rich nutrient source. On the other hand, selective media often include growth inhibiting substances that select for the types of bacteria that can grow in the presence of these and inhibit or eliminate the growth of unwanted microbiota . The useBacillus spp. can grow on this medium, as can some yeasts and moulds  gel in 1 × TAE buffer for 1 h at 100 V and bands were checked for products of the expected size (ca. 1500 bp) under UV light using the GelRed Nucleic Acid Stain (Merck). The PCR products were purified using the Mag-Bind® TotalPure NGS Kit (Omega) with a ratio of 1:1 (v/v). Purified PCR products were then quantified using the Qubit™ dsDNA BR Assay Kit (Invitrogen™) and a Tecan Spark multimode microplate reader (Tecan) followed by equimolar pooling of the samples.200 ng of the equimolar pool was used for sequencing on an ONT MinION Flongle  following the “Genomic DNA by Ligation (SQK-LSK109)” protocol for Flongle flow cells (Version: GDE_9063_v109_revAI_14Aug2019). These flow cells allow for the sequencing of up to 96 samples per run. The final library was quantified using the Qubit™ dsDNA HS Assay Kit (Invitrogen™) and a Qubit 3.0 Fluorometer (Invitrogen™). A final library concentration of 20 fmol was loaded on the Flongle flow cell and sequenced for 24 h.Once the raw data files were obtained, basecalling was done using the GPU Guppy Basecalling Software (version 3.4.4) with an nvidia RTX 2080. Q-score was assessed using pycoQC (v2.5.2) and was between 8.57 and 10.49. The resulting fastq files were demultiplexed using Porechop (v0.2.4) with customized barcodes . The demIn order to check the species identification obtained with our 16S rRNA gene-based method at the whole genome level, we randomly selected four isolates  and sequenced their genomic DNA using Illumina short-read sequencing. Isolates 10670603MRS5 and 10693850MRS7 were sequenced using the Illumina TruSeq Nano DNA LT library Preparation Kit , while isolates 10685605MRS4 and 10693850MRS2 were sequenced using the Illumina DNA Prep Tagmentation Kit (Illumina) according to the manufacturer’s protocol. All isolates were sequenced on an Illumina MiSeq sequencer using the MiSeq Reagent Nano Kit v2 (Illumina) (500-cycles) for paired end sequencing with 2 × 251 cycles according to the manufacturer’s instructions, but on separate runs for the different protocols.The raw WGS data were trimmed using the Trimmomatic pipeline   and de nBifidobacterium spp. (n = 24), lactobacilli (different genera based on the reclassification of the genus Lactobacillus [n = 19) and Enterococcus spp. (n = 12), while only few were identified as Streptococcus (n = 2), Weissella (n = 1), Alteribacillus (n = 1) and Dialister (n = 1). Regarding the LP-MRS agar used to isolate bifidobacteria, the majority of the colonies could be identified as Bifidobacterium (n = 22), lactobacilli (n = 18) and Enterococcus (n = 16), while one of each could be identified as Blautia, Nitrosomonas, Pantoea and Ruminococcus. In case of the BBE agar for isolation of Bacteroides spp., the bacteria were identified as Bacteroides (n = 42), Parabacteroides (n = 13), Phocaeicola (n = 3) and Coprococcus (n = 2)  are shown in Fig. bacillus ) (n = 19Bacteroides (Ba.) spp. isolated from BBE agar were Ba. fragilis and Ba. xylanisolvens, while Ba. uniformis, Ba. ovatus and Ba. thetaiotamicron were also isolated frequently. Only a few isolates of Ba. caccae and Ba. cellulosilyticus could be obtained , the genera Lactobacillus, Lacticaseibacillus, Latilactobacillus and Ligilactobacillus were identified. Interestingly, L. acidophilus could only be isolated from MRS medium  spp. isolated from both LP-MRS and MRS included mainly E. faecalis and E. faecium, while bacteria from two colonies were identified as E. mundtii. Bifidobacteria could be isolated from both MRS and LP-MRS in most cases, except for Bifidobacterium (Bf.) dentium, for which only few isolates were obtained. The most frequently isolated bifidobacteria were Bf. longum, Bf. adolescentis and Bf. animalis, while Bf. bifidum and Bf. dentium were rarely isolated  may be considered to be of food origin and associated with westernized populations, as previously described [Bf. longum, Bf. adolescentis and Bf. animalis. This is in agreement with the literature, which shows that Bf. adolescentis and Bf. longum are predominant bifidobacteria in adults [Bacteroides spp. identified have also been described to be part of the human faecal community [Although the aim of the study was not an in-depth characterization of the LAB, bifidobacteria and escribed . For bifn adults . The Bacommunity .Bacteroides bacteria isolated from BBE agar were Parabacteroides distasonis, Phocaeicola vulgatus, Phocaeicola dorei and Coprococcus eutactus. The non-lactobacilli isolated from MRS agar included Alteribacillus bidgolensis, Dialister succinatiphilus, Streptococcus anginosus, Streptococcus mutans and Weissella viridescens. The non-bifididobacteria isolated from LP-MRS included Blautia obeum, Nitrosomonas oligotropha, Pantoea agglomerans and Ruminococcus faecis (basonym of Mediterraneibacter faecis). As expected, our results show that the selective media used for the isolation of lactobacilli, bifidobacteria and Bacteroides are not exclusively selective and supported the growth of other faecal bacteria such as Dialister, Alteribacillus, Blautia, Nitrosomonas, Pantoea, Coprococcus, Phocaeicola and Parabacteroides spp.The non-Bacteroides. Out of these, 110 showed 100% purity  and Akkermansia municiphila , Rubrobacter bracarensis and Alteribacillus bidgolensis , Bf. bifidum LMG 11041 T (acc. no. JGYO01000000), Bf. dentium Bd1T (acc. no. JDUY00000000) and L. rhamnosus DSM 20021 T (acc. no. AZCQ01000000). When compared with the type strains, the ANI and dDDH values exceeded the cut-off values for species delineation and indicated the very same identification as NanoCLUST with ONT MinION sequencing .In addition, we randomly selected four isolates  for precise strain identification through WGS by Illumina short-read sequencing. Strain identification was performed by calculation of the ANI and the dDDH with closely related type strains, i.e. Our aim was to identify bacteria present in complex samples and to select specific species for which there are no exclusively selective media. Both of our goals were achieved successfully and efficiently by combining and adapting several existing technologies. First, this method requires no more than two culturing steps, namely the culturing of the samples on agar plates and the single-step culturing of the selected resulting colonies in broth medium. Second, our DNA extraction method worked for all bacterial taxa, and it took less than 25 min regardless of the number of samples (limited only by the space in the ultrasonicator) and without increasing the handling workload. This is highly advantageous compared to all other methods that we tested, especially for Gram-positive bacteria. Bead beating and heat-based techniques were unsuccessful, and kit-based extractions are more expensive and require a higher workload.Enterococcus strains that we found. Nonetheless, we performed WGS on four randomly selected isolates to check for the accuracy of the identification, and we obtained consistent results between the two techniques. Finally, NanoCLUST has outperformed state-of-the-art software both in terms of identification accuracy and abundance profile estimation at the species level when classifying mock communities [Furthermore, 16S rRNA gene sequencing has been shown to provide an accurate bacterial taxonomic classification . The 16Smunities . This anmunities . AdditioAlternative methods such as MALDI-TOF require pure colonies, costly equipment such as the MALDI-TOF mass spectrometer, and extensive databases of protein profiles, often developed in-house , 29. In The method here described allows for an accurate bacterial identification as well as an insight into the purity of the isolates. Therefore, it can be useful for having a fast, unexpensive and robust overview of the bacteria extracted from complex microbiological samples before selecting them for further studies.Supplementary file1 (DOCX 70 KB)Below is the link to the electronic supplementary material.