The decision to perform surgery for patients with T1 colorectal cancer hinges on the estimated risk of lymph node metastasis, residual tumour and risks of surgery. The aim of this observational study was to compare surgical outcomes for T1 colorectal cancer with those for more advanced colorectal cancer.This was a population‐based cohort study of patients treated surgically for pT1–3 colorectal cancer between 2009 and 2016, using data from the Dutch ColoRectal Audit. Postoperative complications  were compared using multivariable logistic regression. A risk stratification table was developed based on factors independently associated with severe complications  after elective surgery.versus 9·5 per cent respectively; odds ratio (OR) 0·89, 95 per cent c.i. 0·80 to 1·01, P = 0·061), surgical complications  or mortality . Male sex, higher ASA grade, previous abdominal surgery, open approach and type of procedure were associated with a higher severe complication rate in patients with pT1 colorectal cancer.Of 39 813 patients, 5170 had pT1 colorectal cancer. No statistically significant differences were observed between patients with pT1 and pT2–3 disease in the rate of severe complications (8·3 Elective bowel resection was associated with similar morbidity and mortality rates in patients with pT1 and those with pT2–3 colorectal carcinoma. The introduction of population‐based colorectal carcinoma screening programmes aims to reduce mortality from colorectal cancer. Screening‐detected colorectal cancers have a more favourable stage distribution than those that are symptom‐detected, but it remains unclear whether early diagnosis following screening results in better surgical outcomesA proportion of colorectal cancers limited to the submucosa (pT1) can be treated with minimally invasive endoscopic resection techniques, in contrast to more advanced colorectal cancersThe aim of this study was to compare short‐term postoperative outcomes after elective bowel resection in patients with pT1 and those with pT2–3 colorectal cancer, and to identify the key clinical features associated with severe complications after surgery for pT1 colorectal cancer from which a risk stratification table could be developed to help clinicians guide treatment decisions in patients with pT1 colorectal cancer.This was a population‐based cohort study of patients who underwent colorectal surgery for pT1–3 stage colorectal cancer between January 2009 and December 2016 in the Netherlands. The total population in the Netherlands was estimated as 16·6 million people in 2010, according to Statistics Netherlands. Patients were identified from the Dutch ColoRectal Audit (DCRA), formerly known as the Dutch Surgical Colorectal Audit. The DCRA is a web‐based national audit, in which information on all patients undergoing surgery for a primary tumour is recorded prospectivelyPatients who had an elective oncological resection were included in the study. Those who underwent neoadjuvant treatment, urgent or emergency surgery, or only a local procedure were excluded, as were patients with metastatic disease or synchronous colorectal cancer. Patients treated with a local surgical procedure before bowel resection were not excluded. As all data in the DCRA are coded, no ethical approval or informed consent was required for this study under Dutch lawTable Main outcome measures were overall, surgical and severe complications, and mortality. Definitions are shown in versus III–V), history of abdominal surgery, BMI, preoperative complications , tumour location (colon or rectum), detection method (non‐screening‐detected versus screening‐detected), year of surgery, type of procedure , type of surgery , abdominoperineal resection (APR), (sub)total colectomy or other), lymph node yield (less than 12 or 12 or more nodes) and pN category . Ileocaecal and transverse resections were also categorized as right colectomy. Panproctocolectomy and subtotal colectomy were categorized together as (sub)total colectomy. When information on co‐morbidity was missing, it was interpreted as absent. For all patients, tumour stage was defined according to the fifth edition of the TNM classification of malignant tumours for colorectal cancerPatient‐ and tumour‐related risk factors associated with morbidity and mortality following elective colorectal surgery in previous literature were used in analyses2 test for categorical variables and the Kruskal–Wallis test for continuous variables. Missing data were assumed to be missing at random. For all logistic regression analyses, multiple imputation using a Markov chain Monte Carlo method was performed to adjust for missing values Baseline characteristics were compared between patients with pT1, pT2 and pT3 colorectal cancer using the χversus pT2–3 colorectal cancer) and short‐term postoperative outcomes was evaluated with univariable logistic regression analysis, expressed as odds ratios (ORs) with 95 per cent confidence intervals. Multivariable logistic regression analysis was performed to adjust for possible confounding factors. Age, BMI and year of surgery were analysed continuously in regression analyses; the remaining variables were analysed as categorical.The association between pT category , type of operation (right colectomy versus left colectomy versus sigmoid resection versus LAR versus APR) and ASA grade (I–II versus III–V). Bootstrapping was performed to calculate 95 per cent confidence intervals.To identify risk factors associated with severe complications after elective surgery for pT1 colorectal cancer, logistic regression analyses were performed. Independent variables with P < 0·050.GraphPad Prism® version 7.02  and Microsoft Visio® version 2010  were used to draw figures. All analyses were performed in IBM SPSS® version 23.0 software . Statistical significance was defined as Fig. Table P < 0·001). pT1 cancers were more often screening‐detected, more frequently diagnosed in 2015–2016 and more often located in the rectum . Patients with pT2–3 tumours more often had preoperative complications and underwent open surgery more frequently (both P < 0·001). Patients treated with a local surgical procedure before bowel resection accounted for 1·3 per cent of the complete cohort. Ileocaecal and transverse resections accounted for 0·6 and 2·1 per cent of operations respectively; these were recategorized as right colectomies. Panproctocolectomy and subtotal colectomy accounted for 0·3 and 1·3 per cent respectively, and were recategorized as (sub)total colectomies.Of 51 470 surgically treated patients with colorectal cancer identified, 39 813 fulfilled the inclusion criteria (P < 0·001)  . This finding remained statistically significant after adjusting for confounders . Rates of surgical complications , severe complications  and mortality  did not significantly differ between the two groups . Surgical complications occurred in 13·4 per cent (5334 patients) and severe complications in 9·3 per cent (3711). The 30‐day mortality rate was 2·4 per cent. The overall complication rate was significantly lower following surgery for pT1 cancer compared with surgery for pT2–3 disease , previous abdominal surgery , open approach , conversion from a laparoscopic to an open procedure  and subtotal colectomy  were independently associated with an increased risk of severe complications. Sigmoid resection was associated with a lower risk of severe complications . Using these risk factors, severe complication risk was stratified  of surgically treated patients with colorectal carcinoma (2009–2016)Table S2 Univariable and multivariable analyses of variables associated with severe complication rate following colorectal surgery for pT1 colorectal cancerClick here for additional data file.


Abnormal neural activity in the cerebellum has been implicated in hearing impairments, but the effects of long-term hearing loss on cerebellar function are poorly understood. To further explore the role of long-term bilateral sensorineural hearing loss on cerebellar function, we investigated hearing loss-induced changes among neural networks within cerebellar subregions and the changes in cerebellar-cerebral connectivity patterns using resting-state functional MRI. Twenty-one subjects with long-term bilateral moderate-to-severe sensorineural hearing loss and 21 matched controls with clinically normal hearing underwent MRI scanning and a series of neuropsychological tests targeting cognition and emotion. Voxel-wise functional connectivity (FC) analysis demonstrated decreased couplings between the cerebellum and other cerebral areas, including the temporal pole (TP), insula, supramarginal gyrus, inferior frontal gyrus (IFG), medial frontal gyrus, and thalamus, in long-term bilateral sensorineural hearing loss patients. An ROI-wise FC analysis found weakened interregional connections within cerebellar subdivisions. Moreover, there was a negative correlation between anxiety and FC between the left cerebellar lobe VI and left insula. Hearing ability and anxiety scores were also correlated with FC between the left cerebellar lobe VI and left TP, as well as the right cerebellar lobule VI and left IFG. Our results suggest that sensorineural hearing loss disrupts cerebellar-cerebral circuits, some potentially linked to anxiety, and interregional cerebellar connectivity. The findings contribute to a growing body showing that auditory deprivation caused by cochlear hearing loss disrupts not only activity with the classical auditory pathway but also portions of the cerebellum that communicates with other cortical networks. According to the World Health Organization (WHO), hearing loss (HL) of sensorineural origin affects approximately 466 million people (5% of the world's population) and ranks as the fifth leading age-related disability . It is eThe cerebellum has traditionally been thought of as being involved in motor control; however, emerging evidence indicates that the cerebellum also plays important roles in sensory, cognitive, and emotional processes –14. All From a global perspective, the cerebellum lies within independent sets of closed cortico-cerebellar loops, including “the motor loop” and “prefrontal loop,” which provide the structural and functional foundations linking the cerebellum to higher-order behaviors . AbnormaFunctional imaging and electrophysiological studies using an animal model of drug-induced HL and tinnitus identified an auditory-cerebellar network, in which some neurons in the parafloccular lobe of the cerebellum (PFL) and vermis respond to sound through direct and indirect connections with neurons in the cochlear nucleus (CN), inferior colliculus (IC), and auditory cortex (ACx) , 40–42. SNHL has been ranked into five severity levels in the Global Burden of Disease (GBD) 2013 . In the present study, twenty-one long-term bilateral, moderately severe SNHL subjects and 21 normal hearing subjects matched for age, sex, body mass index (BMI), and education level were recruited from the clinic of the E.N.T. Department of Zhongda Hospital and local community. All subjects in the two groups were right-handed and had completed at least 8 years of education. Diagnosis of long-term SNHL was based on pure tone audiometry (PTA) at frequencies of 0.25, 0.5, 1, 2, 4, and 8 kHz. Detailed information regarding the assessment of the subjects is shown in None of the participants were excluded due to excessive head motion which needed to be less than 2.0 mm in translation and 2.0 degrees in rotation. Inclusion criteria were average PTA values of >25 dB , duratioWritten informed consent was provided by all subjects before their participation in the study. All experimental procedures were approved by the ethics committee of Zhongda Hospital, Southeast University (2016ZDSYLL031.0), and in accordance with the declaration of Helsinki .All participants underwent a detailed battery of neuropsychological tests. Mini-Mental State Examination (MMSE), Auditory Verbal Learning Test (AVLT), and Symbol Digit Modalities Test (SDMT) targeting cognitive function. The Self-Rating Anxiety Scale (SAS) and Hamilton Depression Scale (HAMD) were used to evaluate mental status.3) T1-weighted images .The functional and structural imaging data were acquired at the Radiology Department of Zhongda Hospital using 3.0 Tesla MRI  with a standard head coil. Foam padding and earplugs  were used to attenuate scanner noise by approximately 32 dB. Subjects were instructed to stay still, keep their eyes closed, remain awake, and avoid thinking of anything in particular during the scan. The whole-brain fMRI dataset which took about 8 min and 6 sec to collect was based on BOLD signals. Images were obtained axially using a gradient echo-planar imaging (EPI) sequence. Parameters were as follows: 36 slices, volume = 240, repetition time (TR) = 2000 ms, echo time (TE) = 25 ms, section thickness = 4 mm, gap = 0 mm, field of view FOV = 240 mm × 240 mm, acquisition matrix = 64 × 64, and flip angle (FA) = 90°. We then used 3D MPRAGE sequence to acquire high-resolution (1 mmhttp://rfmri.org/DPARSF), which is based on statistical parametric mapping . The preprocessing steps were as follows: (a) removing the first 10 time points for signal equilibrium of initial magnetic resonance signals and adaption of the subjects to the scanner; (b) slice timing adjustment; (c) realignment for head motion correction; (d) segmentation and normalization to the Montreal Neurological Institute (MNI) template by linear transformation (resampling to 3 × 3 × 3 mm3 isotropic voxels); (e) regressing 6 motion parameters, 6 temporal derivatives, and 12 corresponding squared items using the Friston-24 parameter model to minimize the effect of head motion  correction). A two-sample t-test was performed to test for differences between SNHL patients and HCs. The mask was combined with the significant clusters of both groups. The significance threshold was set at p < 0.01 by FWE correction with age, sex, education level, and motion corrected. Furthermore, interregional seed-based matrices were also constructed among the nine chosen ROIs of the cerebellum, quantifying the unique functional relationships between each pair of ROIs. The uncorrected p value threshold was set at <0.01.On the basis of previous findings , we sepahttp://www.restfmri.net). A Pearson correlation analysis was then conducted between mean z values of FC and each clinical feature  using SPSS software. A p value < 0.05 was considered statistically significant.To study the relationships between the FC results and the clinical variables, we extracted the signals from each significant cluster using REST software . Compared to normal hearing controls, the SNHL group showed [No significant differences were found in GM and WM volume between the SNHL and normal hearing groups. However, there were significant differences between the SNHL group and HC group in terms of FC among the nine ROIs within the cerebellum  and other brain regions as shown in p showed  reduced p showed  reduced p showed  reduced p < 0.01, uncorrected); however, after FWE correction, these differences disappeared. No other significance between group differences in rs-FC was observed among the remaining cerebellar regions   .z values between left crus VI and left TP were lower than that in the control group     and nega= 0.019) .z-scores between left crus VI and left IFG were lower than that in the normal hearing control group  and also negatively correlated with SAS anxiety scores . In contrast, none of the correlations between FC values and the clinical scores were statistically significant in the normal hearing control group.Also, in the SNHL group, mean FC ol group . The redmisphere . FC z-scty of HL  . Clinical evidence suggests that the cerebellum functions in cognition and emotion patterns, lesions of the cerebellar posterior lobe could result in cognitive and affective impairments . Indepenr = 0.240, p = 0.295), in contrast to other that found a positive correlation between severity of HL and anxiety [Weakened left cerebellar crus VI-SMG FC and left cerebellar crus VI-TP FC were observed in deafness. SMG  can not only be activated by pure tones  but is a anxiety . HeterogIn our study, the cerebellar subdivisions had weakened connectivity with the thalamus and insula, compared with the controls. The thalamus is considered as the main relay station of cerebral-cerebellar circuits, regulating information between deep cerebellar nuclei and cerebral cortex . As a seSeveral limitations of this study need to be acknowledged in addition to the relatively small population size of adult participants. First, the duration and medication of SNHL were quite variable in our study. In order to minimize the effects of confounding factors, further longitudinal studies with a larger sample size are needed to identify dynamic alternations in related brain areas and cognitive deficits in SNHL patients. Second, we cannot completely rule out the contribution of MR scanner noise which ranges from 103-113 dB SPL in 3 T MRI scanners , althougTo conclude, this preliminary study is the first to discover widespread decreased cerebellar-cerebral connections and abnormal internal cerebellum function resulting from long-term bilateral moderately severe SNHL. Some of the weakened connectivity patterns were negatively correlated with severity of HL and neuropsychological scores. Our study provides novel insights into the role of the cerebellum and a better understanding of the neuropathology of cerebellar-cerebral interactions following sensory deprivation, proving a therapeutic target for SNHL-related emotional impairments.


TCOF1, POLR1D, and POLR1C, have been identified as the pathogenic genes for about 90% TCS patients, the pathogenic variants of about 8–11% cases remain unknown. The object of this study is to describe the molecular basis of 14 clinically diagnosed TCS patients from four families using Whole-exome sequencing (WES) followed by Sanger sequencing confirmation, and to analyze the effect of bone conduction hearing rehabilitation in TCS patients with bilateral conductive hearing loss.Treacher Collins syndrome  is an autosomal disorder of craniofacial development with an incidence rate of 1/50,000 live births. Although TCOF1 gene, one in each of the four families. Sanger sequencing in family members confirmed co-segregation of the identified TCOF1 variants with the phenotype. The mean pure-tone threshold improvements measured 3 months after hearing intervention were 28.8 dB for soft-band BAHA, 36.6 ± 2.0 dB for Ponto implantation, and 27.5 dB SPL for Bonebridge implantation. The mean speech discrimination improvements measured 3 months after hearing intervention in a sound field with a presentation level of 65 dB SPL were 44%, 51.25 ± 5.06, and 58%, respectively. All six patients undergoing hearing rehabilitation in this study got a satisfied hearing improvement.Four previously unreported heterozygous pathogenic variants  were identified in the WES combined with Sanger sequencing enables the molecular diagnosis of TCS and may detect other unknown causative genes. Bone conduction hearing rehabilitation may be an optimal option for TCS patients with bilateral conductive hearing loss. Treacher Collins syndrome  is an autosomal disorder of craniofacial development that has an incidence rate of 1/50,000 live births . TCS is TCOF1 (OMIM 606847), POLR1D (OMIM 613715), and POLR1C (OMIM 610060). More than 200 distinct mutations have been reported in TCOF1, accounting for about 70–93% of TCS individuals, which are inherited via an autosomal dominant pattern, while POLR1D and POLR1C mutations have been found in about 11–23% of the remaining patients, which are inherited via autosomal dominant and autosomal recessive patterns, respectively [Historically, a diagnosis of TCS has been based on the clinical identification of a minimal clinical phenotype: downward slanting palpebral fissures and hypoplasia of the zygomatic arch. However, this can overlook some mildly affected patients. The use of molecular diagnosis could enable the range of TCS phenotypes to be determined with less bias . TCS is D OMIM 61715, and TCOF1, POLR1D, and POLR1C. In this study, we used WES combined with Sanger confirmation to screen for causative genes in TCS families in China.To date, molecular diagnosis of TCS has focused on Sanger sequencing of these three known pathogenic genes, a method that is now the recommended first-tier test for TCS. The causative pathogenic variants of ~ 8–11% of TCS cases remain unknown, suggesting that there may be other TCS-related genes . AlthougTCOF1 variant in each family, followed by Sanger sequencing to perform familiar segregation analysis. Our findings provide relevant information to diagnose TCS patients and counsel their families.The occurrence rate of TCS in China is low, which has hindered genetic counseling for Chinese TCS patients. Although there have been several genetic studies of TCS in Chinses populations , 10, 11,TCS is not a progressive disease. The primary concern in a newborn TCS patient is respiratory failure arising from craniofacial malformation-associated airway narrowing. Early interventions may be required to clear and maintain the airway, enable feeding, protect the eyes, improve the auditory ability, and allow for the development of speech. Later operations may include aesthetic and functional reconstructions of the mouth, face, and external ear . With reThe present study included nine female and four male patients from four families, each of which included at least two patients and were of Han nationality. The major clinical features of all patients were evaluated Table . DownwarTCOF1 were identified in the four families  reported the clinical findings and molecular diagnostics of a Chinese family with TCS. They concluded that the progeny of the proband and her mother have a 50% risk of suffering from TCS and therefore genetic counseling is necessary . Howevers of TCS , 16. ConOver 50% of TCS patients have bilateral conductive hearing loss due to abnormal development of the external/middle ear , 17. TheTCOF1 as the major causative gene, and a series of mutations within its coding sequence have been identified [TCOF1, and thus might cause dominant-negative effects. Alternatively, it has been suggested that mutation of one TCOF1 allele may cause TCS by haploinsufficiency [TCOF1, mutations in genes encoding two subunits of POLR1C and POLR1D have also been associated with TCS [Most cases of TCS represent an autosomal dominant disorder of craniofacial development. Positional cloning enabled researchers to identify entified . The mosentified . Splicinentified , 20, 21.ficiency , 23. In with TCS .TCOF1. The high detection rate of pathogenic variants achieved in this study suggests that WES combined with Sanger sequencing may be a useful method for detecting pathogenic variants in TCS families. Four different pathogenic TCOF1 variants were identified and all haven’t been previously reported, which broadens the mutational spectrum in the Asian population. Among the studied patients, 71.4% (10/14) had one nucleotide deletion that were predicted to yield haploinsufficiency of the treacle protein. This finding is consistent with most of the previous studies. In the present work, each family harbored a different mutation. The most frequent mutation was a deletion (C. 648delC) in exon 6A, which was found in eight patients of the same family. Although the literature suggests that exons 10, 15, 16, 23, and 24 are mutational hotspots in TCOF1, the mutations identified in the present work were located in exon 6A, exon 5, intron 14, and intron 17 [The 14 patients studied herein were all found to have pathogenic variants in ntron 17 .TCOF1, as did her affected mother and other affected individuals of this family. The clinical diagnosis was missed in the proband’s mother because she showed mild downslanting palpebral fissures that were rendered nearly inconspicuous by the droop of her aging eyelids. This serves to emphasize that the severity of the TCS phenotype is highly variable, and some subjects are so mildly affected that it is almost impossible to make a clinical diagnosis without molecular analysis.The proband of one family (F4) was initially suspected to have autosomal recessive (AR) inheritance at the time of clinical diagnosis, as both of his parents showed normal physical features. Our work ruled out this potential AR inheritance, however, as we found that the proband’s seemingly normal mother carried a 1-bp deletion C in exon 6A of TCOF1, which is located at 5q32–q33.1.11–18 and has an open reading frame that encodes 4465 base pairs and 28 exons. The gene product, treacle, contains at least 1411 amino acids and acts as a nucleolar phosphoprotein that travels between the nucleolus and the cytoplasm. Treacle is a low-complexity protein with a 14-residue N-terminus followed by 11 repeated units with potential phosphorylation sites and a C-terminus with multiple putative nuclear and nucleolar localization signals. It has been suggested that correct expression of treacle is essential for the survival of cephalic neural crest cells. The pathogenic TCOF1 mutations can reduce the number of neural crest cells (NCCs), which are needed for craniofacial embryological development, by impacting the involvement of treacle in ribosomal DNA gene transcription. Nonsense mutations of TCOF1 can lead to an immediate termination of translation, producing a shortened protein. The location of the mutation affects the length of the produced protein, and all of the shortened proteins are likely to be degraded via nonsense-mediated decay. In addition, the C-terminus of treacle contains multiple putative nuclear localization signals, which can be disrupted by two constructs that split the C-terminal region [The majority of TCS patients are heterozygous for mutations in l region , 26, 27.POLR1C or POLR1D, which encode proteins that are important in the ribosomal transcription of RNA and affect ribosomal biogenesis [POLR1C or POLR1D.No clear phenotype-genotype correlation is seen in TCS, but the severity is related to the type of mutation. Although penetrance is high, there is intra- and interfamilial variation. 11–23% of patients have mutations in ogenesis . HoweverTCOF1, POLR1D, and POLR1C. The major causative gene, TCOF1, has a total of 27 coding exons and adjacent splice junctions, rendering such analysis time-consuming and costly. We thus set out to use WES to quickly screen the causative exons, followed by Sanger sequencing of specific exons that appeared to confirm mutations.Most suspected cases of TCS can be molecularly confirmed by Sanger sequencing of three causative genes: The pathogenic variants in about 8–11% of TCS cases remain undetected. There are four main possible explanations for this. First, some of these cases may have been clinically misdiagnosed. However, we note that in most such cases, CT scans and clinical analysis have been used to confirm the diagnosis. Second, the causative mutations might be located in untranslated (and thus unexamined) regions of the three known TCS genes. Although such pathogenic variants are rare in the literature, these regions should be checked in future studies. Third, the causative mutations may be large deletions or insertions within the known TCS genes, which may not be detected by Sanger sequencing. This is especially true in dominant diseases, in which patients also possess the normal allele. Finally, there may be additional, yet-undiscovered genes responsible for TCS. These could potentially be identified by WES.TCS, Goldenhar syndrome, Miller syndrome, and Nagar syndrome exhibit overlaps in their variable phenotypic expression, asymmetric involvement of facial structures, and familial occurrence of microtia or related anomalies . This complicates the diagnosis of such diseases according to the patient’s clinical manifestation. WES could help overcome this limitation. The common phenotypes and causative genes of these syndromes are shown in Table TCS is characterized by a complex presentation of mandibulofacial dysplasia that requires a multidisciplinary intervention from birth to adulthood. Although the presenting features are predictable, there is considerable individual variability, and the functional, aesthetic, and psychosocial needs of each patient will differ. Patients can be given a general scheme of operations and a broad description of the potential help that is available, but a more individually tailored approach is needed –14, 28. It has been suggested that genetic or pharmacological blockade of the p53 gene could reduce neuroepithelial apoptosis during embryogenesis and restore the migrating population of NCCs, potentially preventing the TCS phenotype. However, this would also block the ability of p53 to act as a tumor suppressor, so it would be necessary to interrogate its downstream targets to find a safe point for intervention. This would have to occur in the first trimester, which would make it challenging to detect the need for and properly time the treatment , 29.We herein show that mutational analysis based on WES was useful for the definitive diagnosis of TCS Chinese families and may also provide more information for molecular diagnosis. We also report that bone conduction hearing rehabilitation was consistently helpful for TCS patients with bilateral conductive hearing loss.This single-center prospective study, which involved four Chinese families with 14 clinically diagnosed TCS patients, was performed at Peking Union Medical College Hospital (PUMCH) in Beijing, China. Approval was obtained from the Institutional Review board of PUMCH and written informed consent was obtained from each studied family member. A comprehensive clinical history was taken and a complete physical examination was performed on all subjects to exclude Goldenhar, Nager, and Miller syndromes. The scoring system developed by Ozge Altug and Teber was used to clarify the phenotypic expression of TCS in these patients . All patGenomic DNA was extracted from peripheral blood samples using a TIANamp Blood DNA Kit  according to the manufacturer’s protocol. WES was performed on the four TCS probands at Beijing Allwegene . Exome enrichment was performed using a Sure Select Human All Exon v6 kit (65 Mb) , which yielded an average sequencing depth of 100-fold and a coverage of 97.7%. Enriched shotgun libraries were sequenced on a HiseqX platform .http://genome.ucsc.edu/) using the Burrows-Wheeler Aligner. Genotypes were called using SAMtools, Picard, and GATK. Sequence variants, including single-nucleotide variants (SNVs) and small insertions or deletions (InDels), were annotated using the ANNOVAR software (http://annovar.openbioinformatics.org) (TCOF1 reference: NM_001135243). For coding or splice-site mutations, the conservation at the variant site and the predicted effect on protein function were evaluated using the in silico tools, SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org/), and CADD (http://cadd.gs.washington.edu/).Sequenced reads were collected, filtered for quality, and aligned to the human reference sequence ; the 1000 Genomes Project; the HapMap CHB  population; the National Heart, Lung, and Blood Institute Exome Sequencing Project (ESP); and the Exome Aggregation Consortium (ExAC) Browser. Finally, missense variants were excluded if they were not predicted to be deleterious by the SIFT, PolyPhen-2, MutationTaster, or CADD analyses.http://www.hgvs.org/mutnomen) when naming the identified variants.The mutations identified in the four families were prioritized for Sanger confirmation. The relevant sequences were PCR amplified from the probands and their family members, and the amplified fragments were purified using an Agencourt AMPure XP kit . Sanger sequencing was performed with an ABI3730xl DNA Sequencer  and the results were analyzed using the Sequencing Analysis 5.2 software . We referred to the HGVS nomenclature guidelines , four received Ponto implantation, and one received bonebridge implantation. Pure-tone audiograms and speech discrimination tests were performed before and after the hearing interventions. Loudspeakers were placed 1 m in front of each subject and free sound field hearing thresholds were evaluated at 0.5, 1, 2 and 4 kHz frequencies. Speech discrimination scores (in quiet) were measured using the Mandarin Speech Test Materials (MSTM) , which c


Samples were then collected after incubation for 120 days and analyzed for concentrations and half-life (t1/2) of TCS and CBZ, and soil physicochemical properties, together with enzyme activities. The results showed that TCS was degraded completely during the incubation period. In contrast, only about 5.82–21.43% CBZ was degraded. CSS amendment inhibited TCS and CBZ degradation and prolonged t1/2 compared to the control, and the t1/2 of TCS and CBZ increased with CSS addition amount in all treatments except for CBZ in the garden soil amended with 10% CSS. Correlation studies showed a significantly positive relationship between t1/2 of TCS and CBZ and total organic carbon (TOC), while a significantly negative relationship between t1/2 of the two PPCPs and pH was observed. Alkaline phosphatase showed a significantly negative relationship with the Ct/C0 of TCS in garden soil amended with 25% CSS and CBZ in the control. The urease activity was negatively correlated with the Ct/C0 of TCS in 10% and 25% CSS treatments and CBZ in 10% CSS treatment for garden soil.Composted sewage sludge (CSS) has been extensively used in agriculture and landscaping, offering a practical solution for waste disposal. However, some pharmaceutical and personal care products (PPCPs) like triclosan (TCS) and carbamazepine (CBZ) have restricted its land application. In this study, CSS was added to agricultural soil and garden soil at 0%, 5%, 10%, and 25% ( PPCPsmg kg−1) ,5. Hencemg kg−1) . Once inmg kg−1) , transpomg kg−1) , degradamg kg−1) , accumulmg kg−1) . Also, img kg−1) ,12.−1 [−1 and in biosolids up to 258 μg kg−1 [Among many PPCPs, triclosan (TCS), an anti-bacterial agent, has been found in numerous household and healthcare products like soaps, toothpaste, cosmetics, shampoo, and textiles. TCS has been designated as one of the high-production-volume chemicals by the U.S. Environmental Protection Agency (U.S. EPA) ,13,14 an−1 . Researc−1 ,17,18. C−1 . CBZ hasAs one of the main carriers of PPCPs into soil, biosolids have been widely used in foreign countries. For example, the production of biosolids in the USA and Europe is about 7.2 and 4.7 million tons of dry materials per year, respectively . ApproxiHowever, biosolids amendment tends to result in the accumulation of PPCPs in soil to some extent. PPCPs in soil may be degraded or transformed as a result of abiotic reactions. Previous studies have confirmed that the degradation PPCPs was closely correlated with soil properties ,23. Whent1/2 of PPCPs in agricultural soil was frequently analyzed [t1/2 of PPCPs was less involved. Additionally, more studies focused on the effects of PPCPs on enzyme activities [t1/2 of PPCPs.To date, a number of studies have considered the degradation or persistence of TCS and CBZ in soil with or without biosolids amendment. Most of them focused on the degradation of PPCPs in biosolids-amended agricultural soils ,27 whileanalyzed . Howevertivities ,29, but t1/2 of TCS and CBZ and soil TOC, pH, and soil enzyme activities.The aims of the present study were to: (1) evaluate the degradation of TCS and CBZ in agricultural and garden soils amended with CSS, determining the impact of CSS on the degradation of TCS and CBZ; (2) analyze the relationships between Two types of soil were selected as the test soil. The agricultural soil was collected from a farmland with a wheat–corn rotation system, located in Jiyang County of Jinan, Shandong Province of China . Jiyang County has a population of 552,400 and is located in the alluvial plain of the Yellow River, in which agriculture is the main industry and there is no pharmaceutical factory. About 10-kg composite samples were obtained from the 0–0.2 m surface layer by mixing ten random pure samples from each regional sampling point. The garden soil was collected from the 0–0.1 m surface layer of an existing typical recreational garden  in the Shandong Academy of Sciences in Jinan, using a plastic trowel. The CSS was supplied by Guangdong Huayang Environmental Science and Technology Co., Ltd., in Zhaoqing, China. The CSS has been used in commercial tree planting to supply nutrients and organic matter for years. The soil and CSS were air-dried, gently crushed, and passed through a 2-mm stainless steel sieve to remove gravels, plant materials, and other debris.w/w), which was labelled as A0, A5, A10, A25, G0, G5, G10, and G25, respectively. An aliquot of 400 g (dry weight) soil and CSS mixture was placed in a 600 mL plastic bottle. First, the soil moisture was adjusted to 30% of the soil water holding capacity using deionized water. After a 7 day pre-incubation, 40 g of soil sample of each bottle was taken and spiked with 4 mL of CBZ and TCS mixed solution (1000 mg/L in methanol). Then, the plastic bottles were left open in a fume hood until the solvent was evaporated. Afterward, the 40 g of contaminated soil was mixed thoroughly with the remaining soil using a stainless steel spatula, and distilled water was added to adjust the soil moisture to 60% of the water holding capacity. The soil bottles  with air holes on their top cap were placed in a 25 °C incubator and kept in the dark. The samples were collected at the 1st, 10th, 30th, 60th, and 120th day, and one part of the collected soil was freeze-dried for PPCPs determination. Another part was kept at 4 °C for enzyme activity determination, and the remaining soil was air-dried for physical and chemical properties determination.The CSS was added to the agricultural soil and the garden soil at 0%, 5%, 10%, 25% on a dry mass basis (w/v) suspension method using a calibrated pH meter  and an EC meter  [Sample pH and electrical conductivity (EC) were determined by the 1:5 solid/water  ,32.Freeze-dried soil (2 g) was added to a centrifuge tube and supplemented with 5 mL of methanol. The tube was securely closed, agitated for 2 min on a vortex mixer, and extracted for 20 min with ultrasonication, then centrifuged for 10 min at 4000 rpm. Soil was sequentially extracted a further two times, the supernatants pooled and passed through a 0.22-μm PTFE filter membrane. All final samples were stored at −20 °C prior to instrumental analysis. Instrumental analysis was performed on a Ultimate 3000 high-performance liquid chromatography system . The chromatographic column was a C18 reversed phase column  with 80% methanol in water and water as the mobile phases B and A, respectively, and the system was programmed (with respect to mobile phase B) for 0–15 min, at a flow rate of 0.8 mL/min. The injection volume was 10 μL and the column temperature was 30 °C. Quality control for PPCPs extraction tests was assessed by the use of a soil spiked with 4 mL of PPCPs (1000 mg/L in methanol), and duplicate samples. The recovery rates for TCS and CBZ in two soils varied from 87% to 97%.Dehydrogenase was determined by measuring the rate of reduction of 2,3,5-triphenyltetrazolium chloride (TTC) as a substrate, which was described by Serra-Wittling et al. . Moist s2 (0.5 mol/L) and 4 mL of NaOH (0.5 mol/L) were added, then the mixture was filtered. The five times dilution of the filtrate was conducted before it was quantified with the spectrophotometer at 420 nm. Blanks without the incubation were carried out in the same manner.Soil alkaline phosphatase enzyme activity was determined using the method described by Lu . Briefly4+ ions produced from urea hydrolysis was determined at 578 nm as a blue-colored complex to represent urease activity.Soil urease activity was determined by the following method described by Guan et al. . Air-dri2O2 and shaking for 20 mins. After that, 5 mL 1.5 mol/L H2SO4 was added to stop the enzymatic activity. The solution was filtered and a 25 mL aliquot was titrated with 0.01 M KMnO4. A blank was conducted with a mix of 40 mL of distilled water, 5 mL of 0.3% H2O2, and 5 mL of 1.5 M H2SO4, and 25 mL of this mixture was titrated with KMnO4.Catalase activity was assayed according to Guan et al. . It was C0 and tC are the concentrations of the target pollutants at time 0 and t (ug/kg); k is the first order degradation rate constant (1/day for the biodegradation tests), and t is the experimental time (in days for biodegradation experiments) [The results obtained in the biodegradation assays were fitted to the first order kinetic model described by Equation (1):riments) . Since et1/2 is the half-life for each compound under the experimental conditions (days) and k is the first-order degradation rate constant (1/day) [In the case of biodegradation tests, the half-life was determined for each pollutant in the tested conditions by using Equation (2): (1/day) .r) by Statistical Product and Service Solutions (SPSS) software for the relationships between degradation half-lives of TCS and CBZ and soil properties and enzymatic activities.Statistical analysis was performed using IBM SPSS Statistics 18  and Excel 2010 . Correlations between various variables were determined using Pearson’s product moment correlation coefficient (p < 0.05), but positively correlated with TOC in the two soils . In the garden soil, CEC was highest in the 5% CSS treatment, followed by the 25% CSS treatment, and the value in the control was the lowest. The clay content in the agricultural soil increased firstly and then decreased with the increase of CSS added amount. The clay content was highest in 10% CSS treatment.k) in t1/2 of TCS increased with CSS application rate. The t1/2 of TCS in the agricultural control and garden control was 4.85 and 3.70 days, respectively. The value increased to 55.45 days in the agricultural soil and 70.73 days in the garden soil at a CSS application rate of 25%. This was generally in agreement with the findings from the earlier studies, where t1/2 varied from 11.5 to 73 days for TCS [t1/2 of TCS were prolonged by 2.06 days in the agricultural soil and 2.90 days in the garden soil. The t1/2 of TCS prolonged by the CSS was different from the findings in previous studies. For example, Fu et al. [t1/2 of TCS was prolonged by 7.5 days. However, Wu et al. [t1/2 of TCS. The difference may be attributed to the types of biosolids used in the studies. The solid biosolids used by Fu et al. contained higher organic carbon than that used by Wu et al. and the CSS used in our experiment.As shown in  for TCS ,37. Whenu et al.  found whu et al.  discovert1/2 [t1/2 of TCS increased with the CSS addition rate. Moreover, the t1/2 of TCS was relatively longer in the garden soil than in the agricultural soil. A significantly positive correlation between the t1/2 of TCS and CSS application rate was observed . Hence, the CSS prolonged the degradation of TCS in the agricultural and garden soils. Moreover, more TCS was degraded in the agricultural soil compared to the garden soil.The addition of CSS increased the soil TOC content, as shown in t1/2 . This prBy contrast, CBZ degradation was much slower compared to TCS, as shown in t1/2 of CBZ had a significantly positive correlation with CSS application rate , see t1/2 of CBZ in the agricultural control and garden control was 533.19 and 407.73 days, respectively. The value reached the highest in the 25% CSS treatment, which was 1386.29 days in agricultural soil and 1732.87 days in garden soil. Our results were similar to the dissipation half-lives (355–1624 days) published by Dalkmann et al. and Kodešová et al. [t1/2 of CBZ, increasing 1% CSS addition would prolong t1/2 by 46.60 days in the agricultural soil and 76.81 days in the garden soil. This finding was consistent with other studies. For example, Li et al. [t1/2 at 108 days, as compared to 46 days in the same soil without the amendment.The á et al. ,38 and wá et al.  and Grost1/2 of CBZ was relatively longer in agricultural soil than in garden soil when the CSS application rates were 0% and 10%. As for the 5% and 25% CSS treatments, the opposite occurred. It is worth mentioning that the t1/2 of CBZ was longer in the 5% CSS treatment than in the 10% CSS treatment for garden soil. Although a 10% CSS treatment contained more organic carbon than a 5% CSS treatment, it seemed that the high organic carbon did not lead to inhibition of CBZ degradation in two treatments. As we mentioned above, increasing organic carbon probably enhanced sorption of PPCPs by soil, and thereby inhibited PPCPs degradation. However, soil OM likely played a dual role in the degradation of PPCPs; it served as an alternative nutrition source for the microorganisms involved in the degradation, and there was strong evidence that the total soil microbial activity increased with increasing soil OM content [t1/2 [t1/2 of CBZ differed with soils and CSS application rate.Moreover, the  content , and thent [t1/2 ,10,41,42t1/2 for TCS , as shown in t1/2 of CBZ was significantly correlated with organic carbon content in a positive way , as shown in t1/2 of PPCPs, thereby inhibiting the degradation and removal of pollutants. This was probably ascribed to the increase of organic carbon content which promoted the sorption of PPCPs by soil and hence decreased the bioavailability of PPCPs [Soil physicochemical properties, such as organic carbon content, play an important role in the degradation of PPCPs ,7. Thereof PPCPs ,6,10,43.of PPCPs .t1/2 of TCS and CBZ , as shown in t1/2 of TCS and CBZ, and promote the degradation of TCS and CBZ. The most likely reason was that increasing the pH probably lead to the enhancement of soil microbial and enzymatic activities, and promoted PPCPs degradation. It has been confirmed that soil pH was an important factor in determining soil microbial and enzymatic activities [In contrast with OM, soil pH was significantly negatively correlated with tivities ,46. Anottivities ,48,49. Ftivities . Varioustivities ,50,51,52tivities . This fitivities ,44.t1/2 of TCS and CBZ varied in different soils amended with different CSS rates, and various other factors might affect their degradation behaviors. Firstly, the chemical structures and physicochemical properties  of PPCPs might significantly affect their degradation pathways [t1/2 of TCS and CBZ were positively correlated with organic carbon in our study, suggesting that organic carbon played an important role in PPCPs degradation.The pathways . Grossbepathways  observedpathways  and Xu epathways  reportedt1/2 of TCS and CBZ was positively related to the CSS addition amount . The inhibitory effect of CSS was probably ascribed to organic carbon, which was found in various studies [When CSS was applied to soil, the TCS and CBZ degradation behavior was also affected by it. It was reported that biosolids amendment generally inhibited the degradation of PPCPs ,24; ther studies ,7,10. Th studies ,55, and  studies . Hence, The use of compost sewage sludge should be strictly controlled. Once it is applied to soil, PPCPs introduced by it may eventually lead to the deterioration of the soil and groundwater quality. Hence, the use amount and purpose of land use should be carefully considered. Moreover, when assessing ecological risks or human exposure of TCS and CBZ, the increased persistence due to biosolids amendment should be considered.Soil enzyme activities were greatly affected by CSS, as shown in t/C0 of TCS and CBZ and soil catalase, dehydrogenase, and alkaline phosphatase activities in almost all different CSS application treatments, see t/C0 of TCS in G25 treatment  and CBZ in A0 treatment . For urease, in the agricultural soil, it showed a positive relationship with the Ct/C0 of TCS and CBZ, while in the garden soil a negative correlation between urease activity and Ct/C0 of TCS and CBZ was observed. A significantly negative correlation between the urease activity and the Ct/C0 was found in G10 and G25 for TCS  and in G10 for CBZ .Correlation studies showed negative relationships between the Ct1/2. Correlation studies showed a significantly positive relationship between t1/2 of TCS and CBZ and TOC, while a significantly negative relationship between t1/2 and pH was observed. As for the role of enzyme activities in the degradation of TCS and CBZ, the alkaline phosphatase showed a significantly negative relationship with the Ct/C0 of TCS in G25 and CBZ in A0 treatments. The urease activity was significantly negatively correlated with the Ct/C0 of TCS in G10 and G25 treatments and CBZ in G10 treatment.TCS was degraded totally during the incubation period, while only approximately 5.82–21.43% of CBZ was degraded. The degradation of TCS and CBZ decreased with CSS application rate in almost all treatments except for the garden soil amended with 10% CSS. This finding suggested that the CSS addition inhibited the degradation of TCS and CBZ and prolonged their


People with hand-related rheumatoid arthritis (RA) experience problems performing activities of daily living (ADL). Compensatory strategies to improve ADL ability have shown effective. Similarly, hand exercise has shown effect on pain, grip strength, and self-reported ability. A combination has shown positive effects based on self-report, but self-report and observation provide distinct information about ADL. The purpose of this study was to examine whether hand exercise as add on to compensatory intervention (CIP) will improve observed ADL ability in RA.n = 55) with hand-related RA were randomized to CIPEXERCISE (intervention) or CIP only (control). CIP is focused on joint protection, assistive devices, and alternative ways of performing AD. The hand-exercise program addressed range of motion and muscle strength.Women (Primary outcome was change in observed ADL motor ability measured by the Assessment of Motor and Process Skills (AMPS). Baseline measures were repeated after 8 weeks.EXERCISE  and CIPCONTROL  were statistically significant, with no differences between groups . Thirteen (46.4%) participants in the CIPEXERCISE and 12 (44.4%) in the CIPCONTROL obtained clinically relevant improvements (≥ 0.30 logits) in ADL motor ability; this group difference was not significant .Improvements in ADL motor ability in CIPAdding hand exercise to a compensatory intervention did not yield additional benefits in women with hand-related RA.ClinicalTrials.gov 16th of May 2014 (NCT02140866).The study was approved by the ethics committee 14th of April 2014 (H-3-2014-025) and registered at The online version of this article (10.1186/s13075-019-1924-9) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of synovium . In 90% Hand-exercise programs may have positive effects on hand function in RA –9. A ranCompensatory programs based on individualized joint protection and education in coping strategies to improve performance of ADL tasks have shown to be effective –13. HammClinical guidelines recommend that patients with RA receive hand-exercise programs focusing on increasing muscle strength and movements and programs on compensatory strategies to overcome difficulties in ADL task performance related to hand impairments .However, existing evidence of the effectiveness of hand exercise on functional ability is based on self-reported data, typically questionnaires. While self-report represents the patient’s perspective, it is documented that measures of self-reported ADL ability have low to moderate relationship to observation-based ADL ability measures , 15. ThuIt remains to be tested whether hand-exercise therapy as an add on to a compensatory intervention program (CIP) will improve the observable performance of ADL tasks requiring hand function in patients with RA. Moreover, as suppression of disease activity is essential to avoid progression of joints destruction , 19, it It was hypothesized that hand-exercise therapy as an add on to CIP in patients with RA-related hand impairments would result in larger improvements in observed ADL ability as compared to CIP alone, without increasing disease activity.Participants were females with RA reporting ADL task performance problems involving the hands, recruited from May 2014 to January 2016 through rheumatologists at outpatient clinics in Copenhagen and announcements in daily press. Potential participants were given further information and pre-screened for eligibility via telephone. If inclined to participate, they were examined by a rheumatologist to determine if they fulfilled inclusion criteria.> 18 years; diagnosed with RA (ACR/EULAR 2010 Criteria) [Inclusion criteria are as follows: females aged ClinicalTrials.gov (NCT02140866). The study was conducted in accordance with standards of the responsible committee on human experimentation and the Declaration of Helsinki. If participants experienced increased disease activity during the study period, US and rheumatologist examinations were made to determine if modifications or withdrawal were needed.Participants gave written informed consent. The study was approved by the ethics committee of the Capital Region of Denmark (H-3-2014-025) and registered at EXERCISE) or control group  with a 1:1 equal allocation ratio utilizing a concealment process. Randomization was made using sealed envelopes and carried out by the project secretary. The outcome assessors and data analysts were kept blinded to the allocation, and participants were instructed not to convey their group allocation. The persons performing US examination and evaluation were blinded to results of clinical examination and group allocation.The investigator-initiated study was designed as an RCT with parallel groups. Participants entered an 8-week program and were randomly assigned to either intervention group , assistive devices, and alternative methods of performing ADL , 22. TheThe hand-exercise program lasted for 8 weeks as a strength increase is possible within this time frame and was designed based on recent research –25. FirsPrimary outcome was observed, ADL motor ability measured by Assessment of Motor and Process Skills (AMPS).Secondary outcomes were observed, ADL process ability (AMPS), self-reported ADL ability , self-reported disability , overall disease activity (DAS28), grip strength, and pain. Exploratory outcome was disease activity assessed by US.> 0.30 logits on the AMPS ADL motor and/or ADL process scale defines a clinically relevant change [The AMPS is a standardized observation-based tool used to measure a person’s observed ADL task performance , 28. TheSelf-reported ADL ability was assessed using the ADL-Q, a standardized instrument with 47 ADL tasks developed to measure a person’s perceived quality of ADL performance , 30. TheThe HAQ-DI is developed to assess disability in RA . The queOverall disease activity was assessed using the DAS28 which is based on count of 28 joints for swelling and tenderness, C-reactive protein level in the blood and the patient’s self-reported impact of disease on a visual analog scale . The DAS28 score range from 0 to 9.4 .The maximal grip strength was measured in kilogram using a digital hand Dynamometer . The grip strength was measured three times in both hands. The maximal force performed in each hand was used in the analysis.Hand pain during activity and in rest was measured in both hands on a visual analog scale (VAS), where zero was no pain and 100 was maximal pain.Synovial hypertrophy and increased synovial perfusion are indications of disease activity assessed by US . Gray sct-test of a normal mean difference with pooled variances  and a two-sided significance level of 5% (p ≤ 0.05), assuming a common standard deviation (SD) of 0.36 logits, a sample size of n = 32 participants per group was required to obtain a power of at least 90% to detect a group mean difference of 0.3 logits. It was decided to include n = 45 participants in each group.Sample size calculation was based on previous data on AMPS ADL motor ability in women with RA . For a tData analyses were carried out according to a pre-established statistical analysis plan (SAP); all analyses were done applying SAS . Descriptive statistics and tests are reported in accordance to the “Enhancing the QUAlity and Transparency Of health Research” (EQUATOR) network , 39. To Intention-to-treat (ITT) analyses were made, i.e., analyzing participant outcomes according to the group to which they were allocated, even if participants did not receive allocated intervention. The ITT principle was done by replacing missing data with the value obtained at baseline.EXERCISE group was compared with the CIPCONTROL group using general linear model  for mean changes from baseline and t tests for comparison of least squares means between groups. The model included change as the dependent variable (Δ), with treatment group as a main effect and the baseline score as an additional covariate. Results were expressed as the difference between the group means and 95% CI with the associated p values, based on the general linear model.At week 8, the CIPFor sensitivity purposes, the analyses were repeated with further adjustment for disease duration and hand pain at baseline as there were group imbalances in these variables at baseline  was analyzed using EXERCISE (n = 28) or CIPCONTROL (n = 27); 22 and 25 participants, respectively, completed the trial  years, and mean disease duration was 12.6 (SD = 11.1) years. Mean baseline tender and swollen joint count (28 joints) was 5.0 (SD = 4.8) and 1.4 (SD = 1.7), respectively; the mean hand pain in activity was 42.7 (SD = 26.1) (right) and 36.2 (SD = 27.6) (left) mm VAS. Mean baseline AMPS ADL motor ability measure was 1.4 (SD = 0.5) logits. Mean baseline HAQ score was 1.1 (SD = 0.6). For other baseline characteristics, see Table Fifty-five were randomized to CIPial Fig. . Reason 3.7 SD = 3.0 yearsEXERCISE group attended a mean of 2.4 (SD = 1.2) CIP sessions, whereas the participants in the CIPCONTROL attended 2.7 (SD = 1.0) CIP sessions. The average number of hand-exercise sessions was 20.2 (SD = 10.3). Exercise diary was received from 24 of the 27 participants. The hand-exercise program was delivered as intended  and CIPCONTROL , however with no statistically significant difference between groups . A significant increase in grip strength in the right hand was seen in the CIPEXERCISE group and ESR increased significantly in the CIPCONTROL group  in the CIPCONTROL obtained clinically relevant improvements (≥ 0.30) in ADL motor ability, and this difference was not significant .The responder analysis revealed that 13 (46.4%) participants in the CIPBased on the results of this study, it was not possible to confirm the hypothesis that hand-exercise therapy as add on to a compensatory intervention program in patients with decreased ADL ability, following RA-related hand impairments, would result in larger improvements in observed ADL ability as compared to CIP alone.> 0.3 logits) increase in observed ADL ability. These results, and the fact that the number of responders in each group was almost the same, suggest that a large percentage of the participants benefitted from the CIP intervention.While no statistically significant differences in changes in primary and secondary outcomes were seen between groups, both groups obtained statistically significant improvements in the primary outcome, observed ADL motor ability. While the mean increases in ADL motor ability in both groups were not clinically relevant, responder analysis revealed that almost half of the participants (46.3%) achieved a clinically relevant  was seen between the two groups strongly indicating the groups were comparable.One limitation of the study was not reaching the intended sample size. Still, the risk of overlooking a real group difference in primary outcome is minor, as both groups obtained a statistically significant increase in ADL motor ability with no indication of a group difference. Another limitation is that the disease duration was longer in the CIPCONTROL and CIPEXERCISE improved their ADL motor ability, but no statistically significant difference between groups was seen. Moreover, no differences between groups were seen in secondary outcomes. Thus, based on our results, it cannot be concluded that hand exercise as an add on to compensatory intervention further improves observed ADL ability in persons with RA-related impaired hand function.In conclusion, participants in both CIPAdditional file 1:Table S1. Changes from baseline in primary and secondary outcomes analyses based on the intention-to-treat-population adjusted for baseline value, disease duration, and hand pain at baseline. (DOCX 20 kb)Additional file 2:Patient handout exercise program. (PDF 1.48 kb)Additional file 3:Detailed description of the hand-exercise programme. (PDF 386 kb)


Escherichia coli diversity does not provide sufficient resolution to identify strains present at low levels. Here, we target the hypervariable gnd gene and describe a database containing 534 distinct partial gnd sequences and associated O groups for use with culture-independent E. coli community analysis.The use of culture methods to detect  Escherichia coli diversity does not provide sufficient resolution to identify strains present at low levels. Here, we target the hypervariable gnd gene and describe a database containing 534 distinct partial gnd sequences and associated O groups for use with culture-independent E. coli community analysis.The use of culture methods to detect  Escherichia coli diversity in fecal and environmental samples often fail to identify strains that are present in low numbers. Our previous work using sequencing of metabarcoded amplicons has targeted the hypervariable gnd gene to provide a comprehensive analysis of E. coli community structure from complex samples such as feces   , 6, a rebination , which ignd polymorphisms and adopting culture-independent methods, our previous work provided an indication of intestinal E. coli diversity and in parallel developed a gnd database for cross-referencing purposes using O-AGC DNA sequence data from distinct O groups (E. coli (and Shigella) isolates have provided new insights as to the range of different E. coli O groups according to incongruent wzx and wzy or wzm and wzt  gene sequences and the identification of six novel O groups  with novel O groups has provided evidence for the identification of 534 distinct gnd sequence types (gSTs), each 284 bp in length, forming the core of this database.By targeting O groups . HoweverO groups . By isol studies , 10 and  studies , 6, 8, wgnd gene of 1,407 bp precludes its use in its entirety as a tool for culture-independent amplicon sequencing studies using the Illumina platform; therefore, the 284-bp region spanning nucleotide positions 443 to 727 is targeted for use in E. coli community analysis studies. The hypervariable nature of gnd also restricts suitable PCR primer sites for the generation of amplicons of a suitable size for routine high-throughput sequencing using the Illumina platform. This gnd database may also provide some assistance in the identification of novel O groups and offer a resource for E. coli subtyping using conventional dideoxy Sanger sequencing methods as a primary screen for subsequent WGS analysis  and are accompanied by a spreadsheet which provides matching O groups for each gST and a representative accession number of matching draft genome assemblies or submitted nucleotide sequences. The database and list of matching O groups will be curated and updated as further WGS data are made available.The DNA sequences of the 534 distinct gSTs are available in FASTA format from GitHub (


Therapeutic strategies to restore hearing and balance in mouse models of inner ear disease aim to rescue sensory function by gene replacement, augmentation, knock down or knock out. Modalities to achieve therapeutic effects have utilized virus-mediated transfer of wild type genes and small interfering ribonucleic acids; systemic and focal administration of antisense oligonucleotides (ASO) and designer small molecules; and lipid-mediated transfer of Cas 9 ribonucleoprotein (RNP) complexes. This work has established that gene or drug administration to the structurally and functionally immature, early neonatal mouse inner ear prior to hearing onset is a prerequisite for the most robust therapeutic responses. These observations may have significant implications for translating mouse inner ear gene therapies to patients. The human fetus hears by gestational week 19, suggesting that a corollary window of therapeutic efficacy closes early in the second trimester of pregnancy. We hypothesize that fetal therapeutics deployed prior to hearing onset may be the most effective approach to preemptively manage genetic mutations that cause deafness and vestibular dysfunction. We assert that gene therapy studies in higher vertebrate model systems with fetal hearing onset and a comparable acoustic range and sensitivity to that of humans are an essential step to safely and effectively translate murine gene therapies to the clinic. Gene replacement, augmentation, knock down and knock out strategies have been elucidated to ameliorate the effects of deleterious mutations that cause hearing loss and vestibular dysfunction in mouse models of human inner ear disease. Table Vglut3 knockout mouse after round window membrane inoculation at postnatal day 1–3 , compound action potentials (CAP) and acoustic startle -mediated gene transfer to the sensory epithelium by delivery through the round window membrane  introduces a cryptic splice site in exon 3 of the PDZ  domain scaffolding protein, harmonin and generates a frameshift with premature stop codon. The splicing machinery strongly prefers the cryptic splice site though wild type splicing is not precluded. The Ush1c c.G216A knock-in mouse accurately models the hearing and vestibular abnormalities seen in USH1C patients  to interfere with pre-mRNA splicing from the cryptic splice site and enhance wild type splicing. ASO therapy restored functionally relevant levels of protein expression, improved hair cell survival, rescued ABR thresholds at low and intermediate frequencies, and corrected vestibular dysfunction . The French-Acadian mutation  of the transmembrane channel-like 1 (TMC1) gene is dominantly inherited and causes progressive hearing loss in mice  from 8 kHz to 22.6 kHz  thresholds, a sensitive measure of outer hair cell function  protein joins SANS cause Usher syndrome type 1G (Ush1g) characterized by congenital profound sensorineural hearing loss and vestibular dysfunction at birth with onset of retinitis pigmentosa by adolescence -GFP at P2.5 improved stereociliary bundle morphology in auditory and vestibular hair cells. In addition, Sans rescued mechanoelectrical transduction currents in inner and outer hair cells, modestly improved ABR thresholds from 5 kHz to 15 kHz, and completely restored vestibular function measured by circling behavior and vestibulo-ocular reflex (VOR) testing  designed against the Beethoven allele was delivered by lipid-mediated transfer to the mouse inner ear at P0–P2 -GFP mouse cochlea at 6 weeks of age to test the efficiency of inner ear genome editing in adult hair cells. Encouragingly, GFP expression was perturbed in 25% of the adult, Atoh1-GFP hair cells /Cas9 nuclease genome editing system further validates the early neonatal window of efficacy while offering potential for adult inner ear genetic modulation. The The literature discussed thus far demonstrates that AAV-, ASO- and CRISPR/Cas9 RNP-based therapies achieve maximal benefit for recessively and dominantly inherited forms of deafness and vestibular dysfunction when initiated from P0–P5. Furthermore, initiating therapeutic strategies after P5 shows reduced efficacy or the elimination of therapeutic benefit entirely Table . The thoClarin 1 is expressed in inner and outer hair cells as well as spiral ganglion neurons in the mouse cochlea  mouse under the control of the Atoh1 3’ enhancer/β-globin basal promoter sequence  family are potent transcriptional repressors of Atoh1 expression preventing hair cell fate commitment  is one of four connexins expressed in the inner ear that link all supporting cells with adjacent epithelial cells but not hair cells  and the auditory sensory epithelium degrades at P18  and the EP was significantly improved (n = 5 mutant mice). An alternative model system was developed due to the difficulty in obtaining sufficient numbers of mutant mice for extensive analysis. A plasmid encoding four short hairpin RNAs (shRNAs) against Cx-30 was electroporated into wild type mice at E11.5 that knocked down Cx-30, elevated ABR thresholds to over 90 dB SPL from 4 KHz to 20 KHz, and depleted the EP. Co-electroporation of a shRNA-resistant sequence encoding Cx-30 along with the shRNAs against wild type Cx-30 in wild type mice resulted in improved ABR thresholds and EP  is a nuclear-localized, long noncoding RNA robustly expressed in a diverse array of tissues that is nonessential for mouse pre- and postnatal development (Zhang et al., MALAT1 RNA effectively reduced expression in most mouse tissues analyzed (Wheeler et al., MALAT1 ASO into the E13-E13.5 mouse amniotic cavity reduced MALAT1 RNA abundance in the inner ear through P15 establishing that the ASO gains entry into embryonic tissues and exerts its bioactive effects through the second postnatal week (Depreux et al., Ush1c mutant mice generated correctly spliced harmonin RNA in the inner ear at P22 (Depreux et al., While direct injection of bioactive reagents into the developing inner ear is formally a plausible gene therapy strategy, it is unlikely that targeted injections will be conducted into the human otic vesicle which first forms by GA 6 not long after most pregnancies are first verified (Streeter, The therapeutic modulation of inner ear gene expression by systemic and focal injections of bioactive reagents has defined authentic gene therapy strategies in mouse models of hearing loss and vestibular dysfunction. The literature establishes that therapeutic intervention in the functionally immature mouse inner ear by 5 days postnatal age leads to optimal recovery of hearing and balance. This early neonatal window of therapeutic efficacy holds irrespective of the molecular strategy, delivery modality, or viral vector serotype deployed. Moreover, a corollary prenatal window of therapeutic efficacy in humans is predicted to close by 19 weeks of GA before the onset of fetal hearing. The efficacy of fetal gene therapy to preemptively correct deleterious genetic mutations in the organogenesis stage mouse embryo before the onset of inner ear disease is an emerging research focus that has promising potential. The process of defining and validating fetal and postnatal gene therapies will be critically enhanced by rigorous evaluation in higher vertebrate model systems in which the fetal onset of hearing is a defining feature.LW, JK and JB identified the need for a comprehensive review of the literature on gene therapy to treat inner ear disease. LW, JK and JB compiled the necessary literature and wrote the manuscript.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.


Hearing loss is among the leading causes of disability in persons 65 years and older worldwide and is known to have an impact on quality of life as well as social, cognitive, and physical functioning. Our objective was to assess statewide prevalence of self-reported hearing ability in Arizona adults and its association with general health, cognitive decline, diabetes and poor psychosocial health.n = 6462). Logistic and linear regression were used to estimate the associations between self-reported hearing loss and health outcomes.A self-report question on hearing was added to the 2015 Behavioral Risk Factor Surveillance System (BRFSS), a telephone-based survey among community-dwelling adults aged > 18 years , with responses ranging from “a little trouble hearing” to being “deaf.” Adults reporting any trouble hearing were at nearly four times higher odds of reporting increased confusion and memory loss  and decreased odds of reporting good general health  as compared to participants reporting no hearing difficulty. Those reporting any trouble hearing also reported an average 2.5 more days of poor psychosocial health per month . After adjusting for sex, age, questionnaire language, race/ethnicity, and income category the association between diabetes and hearing loss was no longer significant.Self-reported hearing difficulty was associated with report of increased confusion and memory loss and poorer general and psychosocial health among Arizona adults. These findings support the feasibility and utility of assessing self-reported hearing ability on the BRFSS. Results highlight the need for greater inclusion of the full range of hearing disability in the planning process for public health surveillance, programs, and services at state and local levels. Hearing loss is among the leading causes of disability in persons 65 years and older worldwide . Projectany severity has been associated with dementia, depression, diabetes, the risk of falls, and a number of other health concerns related to healthy aging [Beginning in 2016, the Centers for Disease Control and Prevention (CDC) added a question on severe hearing loss or deafness to the core module of the Behavioral Risk Factor Surveillance System (BRFSS) in 2016 . In Arizhy aging .all degrees of self-reported hearing loss in Arizona in 2015 and to investigate the associations between any trouble hearing with four self-reported health outcomes: general health, diabetes, cognitive functioning, and mental/psychosocial health. We hypothesized that hearing loss would be associated with each of these outcomes based on the current evidence [The purpose of this study was to use the BRFSS to determine the prevalence of evidence , 11–14.The first aim of the study was to estimate the prevalence of self-reported hearing loss in Arizona. The second aim was to examine the hypothesized associations between self-reported hearing loss and self-reported health outcomes: general health, cognitive functioning, and mental/psychosocial health. The BRFSS is a cell phone/landline conducted survey that collects state-based data in collaboration with the CDC on self-reported individual health status, risk-related behaviors, and prevention activities . Core moIn addition to adding a question about hearing loss, the Arizona Department of Health Services, responsible for administering the survey, also reviewed the standard BRFSS survey procedures for inclusive participation and equal access by people with hearing loss. This review led to accommodations developed in partnership with the University of Arizona and the Arizona Commission for the Deaf and the Hard of Hearing. Accommodations included: a) announcement of the availability of accommodations for full participation by persons with hearing loss in the pre-notification letter routinely sent to all randomly selected survey participants; b) a dedicated phone number to contact the state BRFSS coordinator to schedule an interview time and facilitate accommodations as requested; and c) approval from the CDC to allow those with a hearing impairment to use another household member or an American Sign Language interpreter to provide their answers to the survey. In addition, the Arizona Commission for the Deaf and Hard of Hearing prepared a captioned video in American Sign Language to introduce the BRFSS survey and communicate instructions for participation, which was made available on the BRFSS website.n = 6462).Participants included respondents aged 18 to 99 years who answered the state-added hearing question and self-reported their age  from the 2015 Arizona BRFSS ? Excellent, good, a little trouble, moderate trouble, a lot of trouble, or deaf?” We categorized self-reported hearing status as a binary variable with responses of ‘excellent’ and ‘good’ categorized as “no hearing loss” and ‘a little trouble,’ ‘moderate trouble,’ ‘a lot of trouble,’ and ‘deaf’ as “hearing loss,” following the definition used in other previous studies of self-reported hearing loss , 16.3 Conversely, we included diabetes as a variable given the evidence that it may be a risk factor for hearing loss [The outcome variables were selected based on the rationale that hearing health is known to have impact on quality of life as well as associations with social, cognitive, and physical functioning.ing loss , 18. Out1 Income was categorized in increments of $5000, from < $10,000 to >$75,000.Our covariates included age, sex, race/ethnicity, household income and the respondent’s primary language (English or Spanish) measured by the language used to conduct the questionnaire. We used the imputed race variable provided by the 2015 data and collapsed the six categories into: 1) White, non-Hispanic, 2) Black, non-Hispanic, 3) Hispanic, and 4) Other race, non-Hispanic.All analyses were performed in SAS version 9.4 . The survey sampling design was accounted for in all analyses by using the survey, cluster and strata weights provided in the dataset, in SAS’s survey procedures . Survey weighted descriptive statistics were calculated. The prevalence of self-reported hearing loss in Arizona was estimated using a proportion and 95% confidence interval. We used logistic regression to test the association of hearing with general health status, diabetes, and cognitive functioning. Linear regression was used to model the number of days of poor psychosocial health. We fit unadjusted and adjusted models for the four outcomes. Adjusted models were pre-specified and included sex, income level, age, race/ethnicity, and questionnaire language. We also performed sensitivity analyses to address missing income data, which was missing 20% of its values. In the sensitivity analysis, we used education level as a proxy for socio-economic status in each of the adjusted models. Education was missing only 0.4% of its values, and was categorized as: “less than high school,” “high school graduate,” “some college” and “college graduate.”A total of 7946 participants responded to the 2015 Arizona BRFSS and of these, 6462 participants answered the state-added question about hearing status and were included in our sample for measuring the prevalence of self-reported hearing loss in the state of Arizona.Of the 6462 participants with complete data for the state-added hearing question on the 2015 Arizona BRFSS, 1919 reported some hearing loss. The estimated prevalence, accounting for the sampling weights, was 23.2% . The lower confidence limit is well above the US prevalence of 16% based on responses to this question on the NHIS [Results from the regression models are shown in Table This study represents the first effort to gain state-specific population-based information in Arizona about hearing loss and associations with health outcomes. There is limited data from other states with which to compare. A published study from Michigan estimated the prevalence of self-reported hearing loss as 19% using a state-added BRFSS question in 2003: Do you now have deafness of trouble hearing in one or both ears?  StartingOur findings confirmed that increased self-reported hearing trouble is associated with the hypothesized subjective health outcomes: poorer general health, change in cognitive functioning, and psychosocial health, and possibly diabetes. Adults reporting trouble hearing, even of a mild degree, were at lower odds of reporting good general health, higher odds of reporting confusion or memory loss, worse psychosocial health and possibly greater prevalence of self-reported diabetes. Our findings are supported by other cohort and longitudinal studies documenting independent associations between hearing loss and cognition, , 12 as wOur findings demonstrate the importance of state level efforts to document self-reported levels of hearing loss by severity for two reasons. First, the inclusion of a question addressing mild to moderate hearing loss captured a higher prevalence of hearing loss in Arizona than in national estimates of serious trouble hearing or deafness. Second, in our findings, any trouble hearing was associated with increased risk for negative health outcomes. Taken together, these findings encourage ongoing monitoring of disability to assist public health programs in prioritizing hearing loss as a risk factor. The data support increased attention to all degrees of hearing loss within initiatives for healthy aging for older adults and effective communication access in healthcare settings.The inclusion of a core question in the BRFSS with response categories for mild to moderate hearing impairment would enable states to identify functional disability and consider the allocation of appropriate resources. State governments and agencies of the Deaf and the hard-of-hearing advocate for and implement policies that impact distribution of resources related to health care, education, and telecommunications. Analyses of current public health agency resources and spending generally have demonstrated that priorities vary by region . ApproxiThese issues are particularly important with regard to access to hearing health care, also variable across the states. Access to hearing health care and hearing aids for individuals eligible for Medicaid varies significantly from state to state . ArizonaEquitable communication access in education, public, and government settings is protected under the Americans with Disabilities Act of 1990 and Section 504 of the Rehabilitation Act of 1973. The BRFSS is essential in complying with the act given that a lack of data sources including persons with hearing loss makes it otherwise impossible to consider how being Deaf or hard of hearing may impact access to care and health outcomes at a state level. We found that it was feasible to engage state public health authorities to increase accommodations for people with hearing loss to access to a state-level public health survey. These survey data were collected over a single year, but the outcomes demonstrate feasibility and success of providing accommodations to access the survey for the implementation of the question and process in future years. Subsequent efforts to further increase accessibility in future years could include translation of survey items into American Sign Language such as has been conducted in the Deaf Health Survey or the Health Information National Trends Survey in American Sign Language or through follow-up study based on an individual’s responses related to the hearing question [Some limitations of our study are inherent to challenges of the BRFSS as a statewide population study. The BRFSS is a cross sectional study, and therefore cannot establish causality across variables. The BRFSS protocol randomly selects individuals with personal cell phones and/or landlines, and therefore does not capture individuals living in nursing homes or other institutions who may be significantly impacted by hearing loss. Another potential limitation is the use of a telephone study with people with hearing loss. We attempted to mitigate this concern by providing accommodations in the announcement, instructions, and administration of the survey. Plans for on-line administration of the BRFSS may eliminate this barrier in the future.It is important to note that our survey relied on self-reported hearing loss not verified by audiometric testing. A known limitation of self-report data collected through surveys such as the BRFSS is that the results are not perfectly correlated with audiometric assessments of hearing loss . In termYet, there are at least two arguments supporting the use of self-report methods and our result for the purposes of our study. First, based on prior research, interpretation of self-report of hearing ability tends to underestimate overall prevalence among older adults, so use of this data represents a conservative estimate of the population health impacts. Secondly, self-report of hearing disability has been shown to be associated with all major domains of hearing aid outcomes, including: help-seeking, uptake of amplification, use of hearing aids in daily life (treatment adherence), and satisfaction with care . Thus, iThis statewide prevalence study of self-reported hearing ability in Arizona and association with health outcomes found that hearing loss was associated with subjective reports of change in cognitive functioning, poorer psychosocial health, and lower self-rated general health among Arizona adults. These findings support the feasibility of assessing self-reported hearing ability on the BRFSS and the usefulness of the results in informing planning processes for programs and services at state and local level.


While sudden hearing loss after stereotactic radiosurgery has been demonstrated in some cases a recent article by Linge et al. and have demonstrated the need for further discussion on this topic. We attempt to delineate the fact that the type of dosing regimen or technology used will not affect the hearing or radio-graphical control outcomes and thus should not be a consideration while administering treatment. Also we discuss the role of location of the lesion and vascularity and potential new therapies for this unexpected outcome after radiosurgery. Dear Editor,We read with great interest the article by Linge et al.  regardinVS comprise 8% of all primary brain neoplasms and 16% of all benign brain lesions and are inherently slow growing in nature thus allowing the great potential of treatment by radiosurgery. Perhaps no other intracranial pathology garnered such enthusiasm as VS after its remarkable track record of functional preservation and tumor growth control with gamma knife radiosurgery (GKRS). Regarding the radiosurgical technology used, there is no difference in the radiographic tumor control rate among the options available. Radiographic control ranged from 88.5–100% in LINAC-based series, and 71–100% in GK series . With loAnalogous to radiographic control, hearing preservation decreased with longer follow-up irrespective of the technology. Combs et al.  reportedIn addition to the often-discussed mechanism of cochlear spearing, other factors influence hearing preservation. A higher auditory function at baseline and young age can favorably contribute to higher rates of hearing preservation after SRS while an injury to the vasa nervosa of the cochlear nerve can secondarily cause radiation-induced tumor edema and lead to acute hearing loss. Hasegawa et al.  reportedIn this article for single-dose treatments, a dose of 12 Gy was prescribed at the 80% isodose surrounding the PTV. For fractionated treatments, a dose of 54 Gy was prescribed at the 100% isodose with the 95% isodose surrounding the PTV. Here, FSRT was delivered in 30 daily fractions of 1.8 Gy over a period of 6 weeks. Within a range of doses used in various series, a lower dose had little to no appreciable difference in progression-free survival, and generally high rates of progression-free survival were reported across a wide range of delivered doses [In addition to the effect of steroids on the treatment of acute hearing loss, the role of Bevacizumab needs to be highlighted. Treatment with Bevacizumab results in a clinically relevant tumor-volume reduction and hearing improvement in some but not all patients treated with some possible treatment-related side effects. It is also useful to note that NF2 pathologic specimens stain for VEGF. Although the mechanism of action is unknown, it is presumed that bevacizumab inhibits VEGF-mediated angiogenesis within the tumor, resulting in its effect . With ou