To analyze the clinical audiological characteristics of X-Linked Alport syndrome (XLAS) in males and their relationships with genotypes.COL4A5 and the pathogenic variants were analyzed. The relationships between auditory phenotypes and genotypes were analyzed.The clinical data of 87 male patients with AS were reviewed. Hearing levels were evaluated using pure tone audiometry (PTA) testing, acoustic immittance, and otoacoustic emissions (OAE) testing. The genotypes of COL4A5 pathogenic variants. Of the 49 patients who were followed-up for more than 2 years, 28 cases presented a decreasing trend in the hearing level of about 5 dB per year. The degree of hearing loss was positively correlated with gene mutation type and renal function.Among the 87 patients, the number of patients with normal hearing and hearing loss were 32 and 55, respectively. In all cases, the hearing loss was characterized as bilateral symmetrical sensorineural deafness. Majority of the patients had mild-to-moderate hearing loss. Hearing loss usually started in the middle frequency range and gradually affected high frequencies, at school age and gradually increased with increasing age. However, it maintained a relatively steady level of 50–60 dB HL during the teenage years. The audiometric curves included groove-type in 51 cases (92.73%). Patients were identified to have 60 different Hearing loss in males with XLAS is symmetrical sensorineural, and progressive with increasing age. There is a significant correlation between the degree of hearing loss and genotype, renal function, and age. COL4A5 mutations, autosomal recessive of COL4A3 or COL4A4 mutations, and autosomal dominant of COL4A3 or COL4A4 mutations, with a percentage of 80, 15, and 5%, respectively . D. D8]. DeEach alpha chain of type IV collagen contains both collagen and non-collagen regions . The colCOL4A5 gene resulted in the abnormality or absence of the corresponding coding product. Therefore, the normal IV collagen network structure cannot be constructed, resulting in basement membrane injury and hearing loss [COL4A5 gene have been identified [Earlier studies –13 have ing loss , 14. DifGenotype–phenotype correlations in the kidney have been studied for X-linked AS disease , 16; howTill date, there is no trend in hearing changes or possible influencing factors on the hearing level have been reported. Therefore, this study aimed to explore the clinical audiological characteristics in male patients with AS and the auditory genotype–phenotype correlations. The results of this study might be useful in predicting the trends in hearing changes and in providing timely interventions for such patients.patients = 16, nears tested = 32), school-age children , school-age children , teenage children , teenage children , and adults . None of the patients had end-stage renal disease. Diagnosis of X-linked AS is highly possible if glomerular hematuria coincides with one of the following criteria: (i) a family history of AS, and no other cause of hematuria, (ii) characteristic clinical features, including lenticonus or retinopathy, and (iii) lack of expression of the collagen α5(IV) chains in the glomerular basement membrane (GBM) or epidermal basement membrane (EBM). The diagnosis is confirmed if there is a lamellated GBM or a pathogenic mutation in the COL4A5 gene [Eighty-seven patients diagnosed with X-linked Alport syndrome (XLAS), with an age range of 2–33 years and a median age of 13 years were studied between March 2013 and March 2017. They were divided into 6 different age groups: pre-school age , 1000 Genomes Project (http://www.1000genomes.org/), Exome sequencing project (ESP6500. http://evs.gs.washington.edu/EVS/), Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/) and BGI in-house database. SIFT, PolyPhen 2 and MutationTaster and Human Splicing Finder were used. The pathogenicity of variants identified was assessed according to the ACMG  standards [The identified variants were checked in the following databases: NCBI dbSNP build 141 , and renal function . The time interval between the hearing test, the renal function test, and the urine protein test did not exceed 3 months.COL4A3–6 genes. Targeted next generation sequencing (NGS) was performed by BGI-Tianjin, China as published before. All pathogenic mutations identified with NGS were confirmed by Sanger sequencing [Total RNA were extracted from skin fibroblasts and analyzed according to the previous report . Genomicquencing .COL4A5 were divided into two types: missense mutations (including Gly and non-Gly substitutions) and other mutations . The relationships between audiological phenotype and mutation of COL4A5 gene were analyzed.Mutations of All patients underwent a normal otolaryngological physical examination. Audiological testing including pure tone audiometry (PTA) testing, acoustic immittance and otoacoustic emissions (OAE) testing were performed. OAE testing was conducted in 4 children who did not cooperate with PTA due to young age.To observe the characteristics of audiological curves of hearing loss in these patients, the average hearing thresholds  at 500, 1000, and 2000 Hz were taken in pure tone audiometry for statistical analysis. The degree of deafness was referred to the World Health Organization (WHO) 1997 criterion, wherein the PTA was the average hearing threshold at 500, 1000, 2000 and 4000 Hz. The hearing loss was divided into four levels, mild 26–40 dB HL), moderate (41–60 dB HL), severe (61–80 dB HL), and profound (≥81 dB HL). Symmetrical hearing loss was present if the difference in hearing loss at the same frequency in both ears was less than 15 dB. The audiometric curves included ascending-type, groove-type, descending-type, flat-type, and high frequency steep drop-type [–40 dB HLP < 0.05 was considered to be statistically significant.Statistical analysis was performed using SPSS 20.0 . All continuous data were displayed as means ± standard deviation. The unpaired Student’s t-test was used to compare the quantitative variables among different groups. Pearson’s chi-squared test was used to compare the categorical variables among different groups. Multiple logistic regression analysis was used to evaluate the significance of independent variables. Among the 87 patients, 83 received pure tone audiometry tests and 4 patients (2–3 years old) received OAE tests. All patients received acoustic impedance tests. The results of OAE testing were normal. The acoustic immittance of all patients was type A. The number of patients with normal and abnormal hearing was 32 and 55, respectively, with an incidence of hearing loss of 63.2% (55/87). The auditory results were characterized by bilateral symmetrical sensorineural deafness. Majority of the patients presented with mild to moderate hearing loss. The hearing loss was usually started in the middle frequency range and gradually affected the higher frequencies Fig. . In 55 pA total of 87 children were followed up for 5 years, and 175 audiometric data were obtained. We analyzed the relationship between hearing and corresponding age.Hearing was normal before school age. Hearing loss usually started at school age and was gradually increased with increasing age. However, it maintained a relatively steady platform level during the teenage years (13–18 years) with a value of 50–60 dB HL Fig. . These rNext, we analyzed the relationship between hearing threshold and age at a single frequency (1 kHz and 2 kHz). The relationship between hearing and age of 1 k Hz was shown in Fig. Hearing loss usually started at school age and was gradually increased with increasing age. Initially, hearing level dropped gradually and then more rapidly, reaching a platform period during the teenage years (13–18 years old) with a level of 50–60 dB HL. After the teenage years, the hearing level was continued to decrease with increasing age. These results suggested that the hearing level was declined with increasing age.The relationship between hearing threshold at 2 kHz and age was analyzed. We found that the hearing loss at 2 kHz was earlier than at 1 kHz, which then gradually reached 50 dB HL level in the platform period, and then continued to increase with increasing age.We concluded that the hearing changes with age at 1 kHz and 2 kHz are similar to the changes in PTA’.COL4A5/6 genotype, where 60 different COL4A5 pathogenic variants were identified in 63 patients.All 87 patients were tested for In this study, 17 gene variants Table . MutatioWe studied the pathogenicity of the 43 unreported variants, and it can be seen that the unreported gene variants in this study are basically pathogenic, and these nucleic acid sequences are highly conserved.Among the 87 children, 63 children were identified to have clear gene mutations. Of the 32 patients with missense pathogenic variants, 9 presented hearing loss (28.1%). Of the 31 patients with indels, splicing, or nonsense mutations, 26 presented hearing loss (83.9%). Of the 55 patients, 35 patients with hearing loss presented gene pathogenic variants, and 26 with severe pathogenic variants .For mild mutations, hearing was normal before school age, hearing loss began at school age and gradually increased with increasing age Fig. . For sevThese results suggested a significant correlation between the degree of hearing and genotype.The more serious the mutation type was, the greater the hearing loss was. However, normal hearing was still present in those patients with indels, splicing and nonsense mutations. For missense mutations, hearing was normal, but severe hearing loss was still present in these patients. Among patients with hearing loss, missense mutations were identified and glycine substitutions were the most common type Fig. .Fig. 5ReThese findings suggested that the hearing level was associated with the severity of gene mutations and to the types of amino acid changes.The relationship between hearing loss and distribution of missense mutation sites  was presented in Table p value of less than 0.05 was considered to be statistically significant).Chi-squared test: P>0.05; not statistically significant .The possible factors that could influence hearing were age, variant type, Up/24 h, and renal function (Ccr). All the variables listed were analyzed using multiple regression analysis. The linear regression equation was as follows:PTA’ = − 12.238 + 5.628*Renal function+ 18.458*Variant type.The results showed a positive correlation between hearing and type of mutation and renal function. The more severe the type of mutation was, the worse the renal function was, and the poorer the hearing was.There are three types of inheritance in AS: X-linked dominant inheritance, autosomal recessive and autosomal dominant inheritance. Among them, X-linked inheritance is the most important type of inheritance. An earlier study  showed tThis study showed that the hearing impairments of X-linked male patients were characterized as bilateral symmetrical sensorineural deafness, with an incidence of 63.2% (55/87), and was consistent with that of the literature . The majHearing loss in male XLAS patients initially involved the middle frequency range and was gradually increased with increasing age. Middle frequency hearing was relatively stable when it had dropped to a certain level, and then high frequency hearing began to decline. It is speculated that this change in audiology is related to cochlear middle basement and basal membrane disease. During the early stages of the disease, damage to middle basement membrane was more obvious than to the basal membrane, and the lesion on the basal membrane was not sufficient to cause high-frequency hearing loss. With increasing time and age, the middle basement and basal membrane lesions gradually became more severe, with the development of hearing loss at higher frequencies. We hypothesized that there was a different expression and metabolism of type IV collagen regulatory genes in the middle and basal membrane of the cochlea, resulting in different manifestations of early middle frequency hearing loss.COL4A5 gene mutation is the cause of XLAS, and so far, more than 300 COL4A5 mutation sites have been found [COL4A5/6 genotype, and 60 different COL4A5 pathogenic variants were identified in 63 patients. There are various forms of COL4A5 gene mutations, including rearrangement, deletion, insertion and point mutations, leading to splicing mutations, frameshift mutations, missense mutations or truncation mutations. Mutations such as rearrangement, nonsense and frameshift mutations led to the abnormal structure or function of type IV collagen, and are also known to be severe mutations in AS [en found . All 87 ns in AS , 16. OurIt has been reported that in patients with mild gene mutations, hearing loss remains mild . HoweverCOL4A5 collagen region, such as the replacement of glycine or arginine, usually result in a more severe disease in the kidney [The missense mutations in e kidney . In thise kidney . TherefoAt the same time, a total of 49 patients were followed up for more than 2 years, where 28 cases showed a decreasing trend in the hearing level of about 5 dB per year. Through observation, it was found that there was no significant difference in the degree of progression of hearing loss between patients with mild mutations and those with severe mutations after hearing loss had occurred. The European Union’s AS Collaborative Group conducted a study on this phenomenon to evaluate the genetic heterogeneity of diseases among different families of the disease and to study the relationship between genotype differences and end-stage renal disease progression. Therefore, it is necessary for us to further expand the sample size and follow-up time to study the relationship between the auditory genotypes and the phenotypes.Through family studies, we identified the phenomenon of co-segregation of clinical phenotypes and genotypes. The pathogenicity of 17 kinds of gene mutations found in this study has already been reported in the previous studies. We conducted nucleic acid conservation and pathogenicity analysis and amino acid conservation analysis for unreported gene mutations in this study. The PhastCons and PhyloP software showed that the amino acid sequences were almost highly conserved. Polyphen-2 and SIFT gene prediction softwares were used to predict the possible pathogenicities of 20 unreported missense mutations. Polyphen-2 prediction software could not be able to predict the outcome in 2 patients. In one of these 2 cases, SIFT predicted the pathogenicity, while the other patient was non-pathogenic. The remaining 18 unreported missense mutations were likely to be pathogenic. The severe mutations were predicted to be pathogenic. Further cytological and animal experimental studies should be conducted for those novel mutations that discovered gene loci in this study.AS is a rare disease. We have conducted a preliminary study on the clinical audiological characteristics and the relationships between auditory genotypes and phenotypes in AS, and finally reached to certain conclusions. In conclusion, this study contributed to the timely diagnosis of the disease, and has important clinical implications in estimating the progression and prognosis of hearing loss. The limitations of the study include small sample size and short follow-up time. Our professional group will continue to collect the data to further study the correlation between auditory phenotypes and genotypes. This is a broad research field for future work, and genetically modified animals or animal models should be employed to understand the disease mechanisms involved in hearing loss.


Of the 10 patients with adenoviral type 54 keratoconjunctivitis examined at Nojima Hospital, 2 developed stellate keratitis and mutton-fat keratic precipitates (KPs) following acute symptoms.We encountered 10 cases of epidemic keratoconjunctivitis from August to October 2017. All patients were adults with a mean age of 60.9 ± 10.0 years. The species D human adenovirus (HAdV)-54 was detected in the conjunctival scrapings of these patients. Fluorometholone instillation was administered during the first week for acute symptomatic relief.Case 1: A 64-year-old female was prescribed with fluorometholone instillation, which was discontinued after 1 week when her symptoms alleviated. One week after discontinuation of the instillation, she presented with blurred vision in her left eye with KPs and multiple stellate keratitis. The anterior chamber had no apparent cells. Her symptoms disappeared after 1 week of betamethasone instillation.Case 2: A 66-year-old female was prescribed with 0.1% fluorometholone instillation, which was discontinued within10 days. Three months after the appearance of initial symptoms, multiple subepithelial corneal infiltrates (MSI) appeared in her eyes. Stellate keratitis and dark-brown pigmentation were observed in the centres of MSI, with several cells in the anterior chamber. Betamethasone was prescribed, and MSI and stellate keratitis improved within 1 week. However, KPs were observed in the left eye. The instillation was continued for 3 more weeks until symptoms improved.MSI is an immune reaction that occurs after the disappearance of acute symptoms. Here, corneal findings and KPs were observed after improvement in eye redness and discontinuation of steroids. These symptoms were presumed to be secondary inflammation due to immune response to the adenoviral antigen.The clinical features of HAdV-54 keratoconjunctivitis on the ocular surface are initially moderate, but become active in the subacute to chronic phases. This may develop atypical findings, including stellate keratitis with KPs. Although early steroid administration can relieve acute symptoms, it may facilitate chronic corneal immunological reaction. Epidemic keratoconjunctivitis (EKC) is most commonly caused by species D human adenoviruses (HAdV), including types HAdV-8, HAdV-37, HAdV-56, HAdV-64 and HAdV-85 –4. In JaThe occurrence of superficial punctate keratitis (SPK) during the acute phase of adenoviral keratoconjunctivitis and that of multiple subepithelial corneal infiltrates (MSI) during the subacute and chronic phases have been well reported. Clinical features are typically moderate in the early phase of HAdV-54 keratoconjunctivitis; however, the occurrence rate of MSI was higher than that in previous epidemics of several HAdV types , 7. UemuAkiyoshi et al. reported that the clinical presentation in the early stage of HAdV-54 infection resembles that of acute allergic keratoconjunctivitis and that the typical features of severe EKC subsequently appear . Of 10 pWe encountered 10 cases of patients with EKCs at Nojima Hospital from August to October 2017. All patients were adults, and the mean age was 60.9 ± 10.0 years. Most patients visited the hospital within a few days of symptom onset  because her symptoms alleviated. However, 1 week after discontinuation she presented with blurred vision in her left eye. Examination revealed a visual acuity of 10/20 in the left eye with mutton-fat KPs and multiple stellate keratitis  after the initial symptoms, she presented with MSI with a foreign body sensation and blurred vision in both eyes . Examination revealed stellate keratitis-like fluorescein staining and dark-brown pigmentation in the centres of MSI with a few cells in the anterior chamber Figs. . The patMSI and stellate keratitis improved within 1 week; however, mutton-fat KPs were observed in the left eye Fig. . The betAfter healing, the second steroid instillation was gradually reduced over a period of 6 and 8 weeks in cases 1 and 2, respectively. Unpleasant symptoms, such as photophobia or blurred vision, were not observed over an 8-month observation period.Uveitis is a rare finding and has been described only in patients with severe keratitis in adenoviral keratoconjunctivitis , 10. TwoA study analysing the symptoms of different adenoviral serotypes showed that HAdV-8 was characteristic. SPK in HAdV-8 infection leads to coarse SPK with a slightly delayed onset  and can progress into subepithelial lesions. Of the 21 patients in the study, 3 presented with mild uveitis, 3 with coarse SPK and 2 with diffuse stromal keratitis .In the cases presented here, corneal stromal oedema was not observed. However, mild uveitis appeared after the symptoms of conjunctivitis alleviated and showed similar progression. Although it is unclear whether SPK coarseness described in the aforementioned study is similar to the stellate keratitis observed in the present cases, the consistency in their onset times suggests a possible similarity.Diffuse SPK is often accompanied with acute adenoviral keratoconjunctivitis. However, in the cases presented here, epithelial damage appeared after improvement of acute symptoms. In case 1, multiple stellate keratitis was observed without MSI. In case 2, stellate keratitis-like staining was observed in the centre of several MSI lesions and pigmentation was also observed in the centres of several MSI lesions. This suggests that stellate keratitis in case 1 was either a precursory symptom or a mild finding of MSI. In case 2, stellate keratitis was presumed to have progressed into MSI with the healing process causing pigmentation. The site around the stellate keratitis-like staining appeared slightly elevated, and a lack of fluorescein staining resulted in a focal dry up  was reported by Uemura et al  This sugEarly administration of corticosteroids for adenoviral keratoconjunctivitis remains controversial because chronic adenoviral conjunctivitis is associated with corticosteroid use . AforemeThe clinical features of HAdV-54 keratoconjunctivitis on the ocular surface may seem moderate in the early phase. In contrast, the immunological reaction in the subacute to chronic phases is more active leading to the development of atypical findings, including stellate keratitis and mutton-fat KPs. Although early administration of steroids can relieve acute symptoms, it may also facilitate chronic corneal immunological reactions.


Colorectal endoscopic submucosal dissection (ESD) is a technically difficult procedure with a higher risk of complications, especially for large colorectal epithelial neoplasms. This study aimed to report our experience and clinical outcomes, and to estimate the factors associated with incomplete resection and complications.One hundred forty one colorectal epithelial neoplasms in 130 consecutive patients treated by ESD at the endoscopy center of Tianjin Medical University General Hospital from January 2013 to January 2016 were included. Factors associated with the incomplete resection and perforation were evaluated.P = .002) and perforation  of colorectal ESD.The mean colorectal epithelial neoplasm size was 26.5 ± 9.5 (15.0–60.0) mm, the mean procedure time of colorectal ESD was 76.1 ± 48.7 (36.5–195.0) minutes. The en bloc resection rate, the en bloc R0 resection rate, and the curative resection rate, were 93.6% (132/141), 91.5% (129/141), and 88.7% (125/141), respectively. Perforation during colorectal ESD occurred in 7 patients (4.9%), postoperative bleeding occurred in 4 patients (2.8%). There was no recurrence occurred in all patients during follow periods of 13.2 ± 8.6 (6.0–36.0) month. Submucosal fibrosis was the only independent factor related to both incomplete resection (odds ratio [OR] 12.425; 95% confidence interval [CI] 2.501–61.734; Colorectal ESD is a safe and effective technique for en bloc resection of large colorectal epithelial neoplasms. Submucosal fibrosis was independently related to incomplete resection and perforation. Recently, the Japan Gastroenterological Endoscopy Society has established a guideline for colorectal ESD and endoscopic mucosal resection (EMR) in 2015, which standardized the diagnostic and therapeutic strategies and stipulations of endoscopic procedures for colorectal tumors.Endoscopic submucosal dissection (ESD), which was originally invented for early gastric cancer, has facilitated the en bloc resection with tumor-free margins of large superficial tumors in recent years.,6 However, colorectal ESD resulted in an increased risk of perforation because of the complex and challenging nature of the technique. Numerous previous studies investigated the risk factors for perforation of colorectal ESD.–9 Submucosal fibrosis in particular is not only 1 major contributor to perforation during colorectal ESD, but also 1 major cause for increasing the probability of incomplete resections.,11Colorectal ESD is a technically difficult procedure because the colonic wall is thin and endoscopic maneuverability is limited caused by colonic flexure and extensibility. Colorectal ESD has been reported to achieve a higher en bloc resection rate and lower recurrence rate than EMR, meanwhile, enable detailed pathological evaluation with less invasive treatment.As colorectal ESD has been widely available to treat the increasing number of epithelial neoplasms in Eastern nations, therefore, we aimed to report our experience, clinical outcomes, and prognosis of large colorectal epithelial neoplasms treated by ESD in a setting of north China. Furthermore, we aimed to estimate the potential factors of incomplete resection and complications for colorectal ESD procedures.22.1We retrospectively reviewed the electronic database and medical charts of 141 colorectal epithelial neoplasms treated by ESD in 130 consecutive patients from January 2013 to January 2016. This study was carried out at the endoscopy center of Tianjin Medical University General Hospital, Tianjin, China, and was approved by the institutional ethical committee.2.2An initial endoscopy, chromoendoscopy, endoscopic ultrasonography (EUS) , and abdominal contrast-enhancement computed tomography (CT) were used concurrently for all patients to predict the invasion depth and metastasis before colorectal ESD procedures.Indications for colorectal ESD are as follows: depth of invasion limited to the mucosa or submucosa with a noninvasive pattern on chromoendoscopy and EUS. Large lesions in which en bloc resection using EMR are difficult. Lesions with submucosal fibrosis caused by previous endoscopic treatment or biopsy. Local residual of early carcinoma after endoscopic resection.Exclusion criteria included lesions with evidence of submucosal deep invasion diagnosed by chromoendoscopy and EUS; lesions with evidence of local or distant metastasis diagnosed by contrast-enhancement CT; lesions of submucosal tumor; lesions with inflammatory bowel disease.2.3All patients were admitted to hospital and underwent fasting and bowel preparation of drinking 2 L of polyethylene glycol solution the day before the procedure. The patients were generally anesthetized by intravenous injection of midazolam and/or propofol. The cardiopulmonary function was monitored by an anesthetist following endotracheal intubation in an endoscopy unit. Intravenous scopolamine was administered to reduce colonic movements. All ESD procedures were performed by 3 experienced endoscopists .Colorectal ESD was performed with carbon dioxide insufflation, using a single-channel endoscope . A transparent hood  was attached to the tip of the endoscope for a good visualization. The electrosurgical current was applied using an electrosurgical generator . The tumor was marked with several dots around the lesion using argon plasma coagulation (APC) . A saline solution combined with epinephrine, glycerin fructose, and methylene blue was injected into the submucosa with an injection needle . A Dual-knife  was used for circumferential incision along the margin of the targeted lesion. A Dual-knife, or a hybrid knife , or an insulated-tip knife , was used to dissect the surrounding tissue at the level of the deepest submucosal layer and shell the connective muscular fibers and stalks along the capsule of the tumor. An electrosurgical knife, or a snare  sometimes was used to remove the tumor at the root completely. When perforation occurred during the procedure, metallic clips  were carried out as soon as possible, regardless of the location. Coagulation (APC) or clipping was carried out for bleeding associated with the procedure.2.4 Lesions were classified as protruding large tumors and laterally spreading tumors (LSTs). LSTs are classified into granular type (LST-G) and nongranular type (LST-NG). Tumor locations were grouped into cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Histopathological assessment was evaluated in accordance with the Vienna classification, grouped into adenoma, intramucosal carcinoma, SM1 (<1000 mm) carcinoma, and SM2 (>1000 mm) carcinoma.–4The specimen was pinned on a cork sheet flatly then fixed with 10% formaldehyde solution. The macroscopic tumor type of colon superficial neoplastic lesions was classified according to the Paris endoscopic classification.2.5: no fibrosis (F0), manifested as a blue transparent layer; mild fibrosis (F1), appearing as a white web-like structure in the blue submucosal layer; and severe fibrosis (F2), manifested as a white muscle-like structure without a blue transparent layer in the submucosal layer.Submucosal fibrosis was classified into 3 groups on the basis of findings obtained at the time of submucosal dissection after the injection. Criteria were as followsEn bloc resection was defined as a 1-piece resection of an entire lesion as observed endoscopically. En-bloc R0 resection was defined as tumor removal in a 1-piece resection with tumor-free lateral and vertical margins. Curative resection was obtained when tumor removal in a 1-piece resection with tumor-free lateral and vertical margins, and there was no submucosal invasion of 1000 mm or more, lymphatic invasion, vascular involvement, or poorly differentiated component.,10,14,15 Additional endoscopic treatment was recommended for the patient who could not suffer the risk of surgery, considering the body age, life expectancy, and comorbidities.Surgery was recommended when the tumor was diagnosed as invasive carcinoma with deep submucosal invasion (>1000 mm) or exhibited risk factors for lymph node metastasis, vascular involvement, budding, or poor differentiation.,7 Postoperative bleeding was defined as clinical evidence of bleeding manifested by hematochezia within 14 days after the procedure that required endoscopic hemostasis.,16Perforation during an ESD was defined as immediate perforation which was deemed as a full-thickness defect during the ESD. Delayed perforation was defined as any perforation occurring after completion of the procedure.2.6All patients received follow-up endoscopy at 3, 6, and 12 months after the initial ESD therapy during the first year and thereafter annually. Any suspicious lesions were confirmed by biopsy.2.7t test and categorical variables were analyzed by using the chi-squared test or Fisher exact test as appropriate. Risk factors were analyzed by univariate logistic regression analysis. Factors with a P value <.1 were included into the multiple logistic regression analysis. SPSS software version 22.0  was used for statistical analysis. A P value <.05 was considered to indicate statistical significance.Continuous variables were tested by using independent 2-side 33.1The clinicopathological characteristics of colorectal ESD were shown in Table 3.2The treatment results of colorectal ESD procedures were shown in Table P = .035), and submucosal fibrosis  were significantly associated with the incomplete resection of colorectal ESD (Table P = .002) was the only independent factor related to incomplete resection of colorectal ESD.Submucosal fibrosis was identified in 42 (29.8%) lesions during colorectal ESD, including 25 (17.7%) mild fibrosis (F1) lesions and 17 (12.1%) severe fibrosis (F2) lesions. Univariate logistic regression analysis revealed that tumor size exceeding 30 mm , and submucosal fibrosis  were significantly associated with the perforation of colorectal ESD (Table P = .035) was the only independent factor related to perforation of colorectal ESD.Perforation occurred in 7 patients 4.9%). All of them were detected during the ESD procedures and were successfully closed by endoscopic clipping. After closure of the perforation, all patients were hospitalized to receive fasting and intravenous antibiotic conservative treatment, avoiding of emergency surgery. There was no case of delayed perforation in all colorectal ESD procedures. Postoperative bleeding occurred in 4 patients (2.8%), and all were cured conservatively by coagulation or clipping, without blood transfusion or surgical intervention. Univariate logistic regression analysis revealed that tumor size exceeding 30 mm , 91.5% (129/141), and 88.7% (125/141), respectively, in the presented study. Previous multicenter study from Asian countries revealed that ESDs were amenable to colorectal tumors with an overall en bloc resection rate and curative resection rate of 95.4% and 89.1%, respectively.–11,18–20 In the present study, the univariate logistic regression analysis revealed that both tumor size and submucosal fibrosis were significantly associated with incomplete resection; while the multivariate logistic regression analysis identified that only submucosal fibrosis was the independent factor related to incomplete resection of colorectal ESD. As a matter of fact, ESD can be performed in en bloc manner irrespective of tumor size, however, in regard to tumor size as an independent factor affecting the en bloc resection of colorectal ESD, the results are still in controversy. The increasing improvement in devices and technique for ESD and different proficiency of endoscopists may explain the different results regarding to the risk associated with tumor size. In our present study, the mean colorectal neoplasm size was 26.5 mm, range from 15 to 60 mm, with a high en bloc resection rate of 93.6%. The explanation for the relatively higher en bloc resection rate for large neoplasms might be, firstly, that we included data from recent period, when the devices and technique for ESD were all advanced and mature in our department. Secondly, the three endoscopists in our study, were all have extensive training and professional experience in gastric ESD procedure, and were all skilled in various electric knife, hemostatic, and stitching instruments. Thirdly, the explanation might be the comprehensive preoperative evaluation of lesions and the selection of appropriate procedures. Therefore, we are confident that, as the constantly improvement of colorectal ESD equipment and technique, ESD has already enabled the en bloc resection for large colorectal superficial tumors.As for the factors related to the en bloc resection, several previous studies illustrated that, tumor size, submucosal fibrosis, invasive depth, and procedure time were associated with incomplete resection.,6,7,9–11 In the present study, the perforation rate of colorectal ESD was 4.9%, and all cases were closed successfully with endoscopic clipping and following intravenous antibiotic conservative treatment. There was no additionally surgery for perforation, because perforations were tiny enough to be closed by clips and treated conservatively. Previous reports revealed that, larger size, location, invasive depth, fibrosis, and less experienced ESD colonoscopists were considered as the risk factors for perforation during colorectal ESD.,7,9,11 Hong et al recently developed a score consisting of simple clinical factors, which based on tumor size, tumor location, endoscopist experience, and submucosal fibrosis, to estimate the risk of colorectal ESD-induced perforation. Similar variables have been analyzed in this present study, the univariate logistic regression analysis revealed that tumor size and submucosal fibrosis were significantly associated with perforation of colorectal ESD. While multivariate logistic regression analysis identified that submucosal fibrosis was the only independent factor related to perforation of colorectal ESD, which was consistent with the results reported previously.–9,11 As for tumor size, which differs from the results of others, the different in colorectal ESD treatment periods may explain the difference, as well as the extensive trained and experienced colonoscopists.Perforation is the main complication of colorectal ESD. Because of the much more thinner of the colonic wall, the risk of perforation during the procedure is higher than that of the stomach. The perforation rate of colorectal ESD was reported to range from 1.4% to 10.4%.,11,13,21 As mentioned before, submucosal fibrosis is caused by the peristaltic motion or forceps biopsy, and severe fibrosis can complicate the separation of the submucosa from the muscular layer. In such cases, after injection the solution rapidly into submucosal layer, a blue transparent layer cannot be easily exfoliated in the ESD procedure. According to previous studies, we judged submucosal fibrosis on the basis of findings obtained at the time of submucosal dissection after the injection. In the present study, submucosal fibrosis was identified in 29.8% lesions during colorectal ESD. The en-bloc resection rate and the perforation rate for lesions with submucosal fibrosis in the present study were consistent with those in previous reports. From the histological point of view, Lee et al evaluated the histologic submucosal fibrosis of colorectal ESD samples through a pathologic review by using Masson's trichrome staining, which verified that fibrosis was the most powerful risk factor for en bloc resection and complications. Makino et al tried to predict the presence of fibrotic lesions preoperative in the colon by using endoscopic ultrasound sonography, which sensitivity and specificity were 77.8% and 57.1%. Hence, evaluation of submucosal fibrosis might be useful to predict the technical difficulties of colorectal ESD, and further studies should be focus on the preoperative factors predicting fibrosis in lesions treated by colorectal ESD. Therefore, lesions with submucosal fibrosis should be addressed cautiously by experienced endoscopists owing to the increased risk for incomplete resection and perforation.In this present study, we have drew a conclusion that, in cases of lesions with submucosal fibrosis for colorectal ESD, the en bloc resection rate was low and the perforation rate was high, which were consistent with those in previous reports. Our outcomes indicated that ESD significantly reduced the local recurrence rate of large colorectal neoplasms, but follow up should be pay attention to piecemeal resected lesions intensively.In the present study, the en bloc resection rate was 93.6%, while the curative resection rate was 88.7%. The additional surgery rate was 2.8% and there was no recurrence occurred in all patients during the intensively follow periods. A multicenter prospective study stated that the local recurrence rates were 4.3% (65/1524), 6.8% (55/808), and 1.4% (10/716) for the entire cohort, for piecemeal EMR, and for ESD, which demonstrated that piecemeal resection was the most important risk factor for local recurrence.Indeed, our study still has some limitations. Firstly, it was a retrospective, single-center study. Secondly, our study included a relatively small sample size; thirdly, the extent of the tissue fibrosis in our study was assessed by endoscopists based on endoscopic findings, not by pathological findings. Prospective studies including larger number of patients are needed to confirm our results.In conclusion, we confirm that ESD is a very safe and effective technique for en bloc resection of large superficial colorectal neoplasms. Colorectal ESD is becoming increasingly standardized technique worldwide. Submucosal fibrosis is an independent risk factor for both incomplete resection and perforation of colorectal ESD, and the evaluation of submucosal fibrosis might be useful to predict the technical difficulties of colorectal ESD. Further improvement will be required for safe and complete resection of colorectal neoplasms with submucosal fibrosis.The authors wish to thank the endoscopy staff for their assistance.


CDKL5 kinase is associated with a human neurodevelopmental condition commonly referred to as CDKL5 deficiency disorder.§ Two recent phosphoproteomics studies identify the first physiological substrates of mammalian CDKL5 and evaluate functional consequences of their phosphorylation and its loss in cells lacking functional CDKL5, highlighting potential roles for this kinase in regulating neuronal microtubule dynamics.Mutation or inactivation of  Muñoz and colleagues initially employed a clean CRISPR knockout/in approach in human U2OS cells ± CDKL5, which was coupled to a quantitative (6‐plex TMT) phosphoproteomic workflow  was impaired in microtubule binding et al, Another exciting outcome from these studies is the finding that catalytically active CDKL5 phosphorylates Ser/Thr in a sharply defined, specific amino acid consensus sequence in its target substrates Fig A, as haset al, The functional consequences of pathological CDKL5 mutations were previously uncertain, with evidence for inhibition and preservation of activity, alongside Tyr phosphorylation in the TEY activation segment  in the activation segment Thr‐Glu‐Tyr motif. This autophosphorylation event appears to be intimately associated with an active kinase conformation when evaluated et al, et al, C. elegans, where CeCDKL1 (most similar to human CDKL1/4) regulates cilium length, and Chlamydomonas, where CrCDKL5 is also required for controlling aspects of cilial dynamics  CDKL5 activity in systems relevant to CDD, such as the brain. Further detective work will be required to uncover the complete set of cellular CDKL5 substrates (many more being predicted in these phosphoproteomics studies) and to decipher regulatory networks associated with CDKL5 catalytic output that are central to physiological function and underlie CDKL5 loss‐of‐function in CDD. Unravelling the molecular mechanisms of CDKL5 activation and inactivation could also help link subset(s) of the CDKL5 network that contribute to disease phenotypes to potential therapeutics. In terms of targetable outputs, factors that control CDKL5 activation include pharmacological agents linked to activity of the NMDA receptor (Tramarin et al, et al, et al, Finally, very recent studies have begun to analyse small molecule kinase inhibitors, including the pre‐clinical GSK3α inhibitor tideglusib, which exhibits some promise for restoring memory function in immature CDKL5 knockout mice, although the specific intracellular target that mediates these effects is unknown (Fuchs


Sphingopyxis) and resistant (Pseudoxanthomonas) bacterial populations. The analysis also revealed a significant reduction of core activated sludge members, Chitinophagaceae  and Comamonadaceae , potentially affecting ecosystem functions  directly associated with system performance . Overall, our study provides new findings that inform the mechanisms underlying the community structure and diversity of activated sludge, which not only advances the current understanding of microbial ecology in activated sludge, but also has practical implications for the design and operation of environmental bioprocesses for treatment of antimicrobial-bearing waste streams.Ecological processes shaping the structure and diversity of microbial communities are of practical importance for managing the function and resilience of engineered biological ecosystems such as activated sludge processes. This study systematically evaluated the ecological processes acting during continuous exposure to a subinhibitory level of antimicrobial triclosan (TCS) as an environmental stressor. 16S rRNA gene-based community profiling revealed significant perturbations on the community structure and dramatic reduction (by 20–30%) in species diversity/richness compared to those under the control conditions. In addition, community profiling determined the prevalence of the deterministic processes overwhelming the ecological stochasticity. Analysis of both community composition and phenotypes in the TCS-exposed communities suggested the detailed deterministic mechanism: selection of TCS degrading ( TCS is readily detectable in a variety of environmental settings due to its extensive use, although it has been recently banned in consumer soaps by the US Food and Drug Administration4. TCS is ubiquitous in natural aquatic ecosystems and occurs at substantial levels (about hundreds of micrograms per liter) in wastewater-associated environments where it represents a health hazard with presumable ecological risks5. TCS can also promote broad resistance phenotypes in microorganisms and thus it is possible that it contributes to the spread of antibiotic resistance in the environment6. Because a substantial amount of TCS can be carried in urban waste streams , wastewater treatment plants (WWTPs) may represent one of the major sources where TCS can select for antibiotic-resistant bacteria and genes in the environment. Accordingly, many previous studies have recommended appropriate management and monitoring campaigns for TCS in the urban water cycle.Triclosan (TCS) is a popular ingredient commonly added to personal care products  and biocides8. Recent studies have identified core (highly frequently occurring) bacterial populations across many WWTPs that are thought to carry out key ecosystem functions10. Microbial interactions can drive deterministic processes governing the dynamics of microbial communities, helping infer the biological performance and functional stability of the ecosystem9. Accordingly, advancing our understanding of ecological processes shaping such engineered microbial communities and developing mathematical models that describe these communities are highly desired to better manage system resilience and effluent quality of WWTPs.Over the past 100 years, activated sludge processes have been one of the most successful biotechnologies to be widely applied in municipal and industrial WWTPs across the world. Many engineering studies have improved wastewater treatment efficiency and waste management; however, these works primarily focused on manipulating operational parameters of the process, with less attention to the ecology of microbial communities involved. Understanding the assembly patterns of activated sludge microbial communities is essential for predicting the function and resilience of activated sludge ecosystems18. Exposure to TCS is known to enrich antibiotic resistance genes  in activated sludge19; however, the impacts of TCS on the phenotypes and community structure/diversity of activated sludge have not been systematically undertaken. Since the majority of TCS is likely collected into urban WWTPS where high levels of TCS potentially cause ecological disturbances on activated sludge, this study examined activated sludge as a model community in laboratory microcosm studies. The TCS levels tested in this study were related to those from environmentally relevant (hundreds of micrograms per liter) to subinbitory (a few miligrams per liter). A total of 12 16S rRNA gene sequence datasets were analyzed to document time-course changes in phylogenetic diversity and composition in the TCS-exposed communities. In addition to describing the TCS-mediated shifts in community structure, this work conducted time-course physiological characterization of triplicate reactors exposed to TCS, validating the 16S rRNA gene-based findings. Overall, our results provide quantitative assessments on the type and relative importance of ecological processes shaping community assembly largely acting under an environmental stressor exerted by a commonly used antimicrobial. The results of this work will help answer a fundamental question in microbial ecology of activated sludge, which has important implications in the treatment of diverse micropollutants in urban WWTPs.In the present study, we addressed the ecological mechanisms involved in controlling community diversity, succession, and function of microbial communities under the presence of TCS as the environmental stressor. Previous studies documented the effects of TCS on microbial communities associated with sink drain biofilms, anaerobic digestion, wetlands, seawater, soil, and gut microbiome (human and fish)2PO4 (0.34 g), K2HPO4 (0.6 g), CaCl2 (0.05 g), MgSO4 7H2O (0.27 g), NH4Cl (0.03 g), and FeSO4 7H2O (0.09 g) and 10 mL of 100 × trace mineral stock solution . One-third of the mixed culture suspension was discarded from the reactor twice a week  and was replaced with the same volume of 2 g COD/L of fresh substrate. The semi-continuous reactors operated in fed-batch mode were aerated (3–4 mg/L dissolved oxygen) and maintained at room temperature (25 °C) and pH 6.9 ± 0.1. After one month of reactor operation, the reactors displayed stable biomass (about 0.4 g/L mixed liquor volatile suspended solids [VSS]) and organic matter removal  every feeding cycle. The operational parameters at steady state were 10.5 days of solid retention time, 0.4 kg COD/kg VSS·d of organic loading rate, and 0.2 kg COD/m·d of food-to-microorganism ratio. After observing the steady state, additional two sets of triplicate batch reactors were further established as subcultures from the control (glucose-fed) cultures by feeding 0.5 mg/L and 5 mg/L of TCS, respectively, in addition to the substrate that was maintained for more than two months. Since TCS occurs at up to 0.08–0.33 mg/L in water environments , 0.5–5 mg/L of TCS tested in this study represented environmental maxima . Fig. Activated sludge was sampled from an aeration tank of a local municipal wastewater treatment plant where biological nitrogen removals (nitrification followed by denitrification) occur at aerobic and anoxic tanks. The sludge was inoculated into 1-liter semi-continuous laboratory reactors (600 mL working volume) in triplicate. The substrate  was composed of 1.8 g/L of glucose and the mineral medium solution containing KH2PO4 buffer adjusted to pH 2.5 with phosphoric acid. The mobile phase was delivered at 1.8 mL/min through the column. The sample of 50 µL volume was injected and detected at the wavelength of 281 nm. The detection limit of TCS using the HPLC system was at 50 µg/L. VSS and COD were measured following standard methods20.Triclosan (99% purity) was purchased from Alfa Aesar Inc. . A 50 g/L TCS stock solution was prepared in acetone and stored at 4°C until use. TCS concentration was measured using a high-performance liquid chromatography (HPLC) system equipped with a Shim-Pack GIST Phenyl, 5 μm, 4.6 × 250 mm column (Shimadzu Asia Pacific Pte Ltd) and a UV-Vis detector. The mobile phase was prepared as follows: 60% acetonitrile and 40% 25 mM NaH0 and A correspond to the initial and time-dependent TCS concentrations, respectively, and ▵t is the given time (h).To determine presumable routes  of TCS removal in the semi-continuous reactors, four sets of triplicate batch experiments were established, separately from the semi-continuous reactors. The four sets were as follows: Abiotic (supplemented with no inoculum + TCS + glucose), Inactive (inactivated inoculum + TCS + glucose), T (inoculum + TCS), and TG (inoculum + TCS + glucose). The inoculum was taken from the mixed culture suspension of the TCS-fed reactors at day 70 and washed twice with 1 × phosphate saline buffer prior to inoculation. The Abiotic condition lacked an inoculum, which was intended to assess the physicochemical losses of TCS such as those via photolysis, hydrolysis, and evaporation. The inactivated inoculum was prepared by autoclaving at 121 °C and 15 psi for 15 min. The Inactive setting included an inoculum that was pre-autoclaved . The losses of TCS in the Inactive condition could be attributable to sorption to biomass  in addition to those  of the Abiotic setting. The other experimental conditions  established in the batch experiments were identical to those of the semi-continuous reactors. The time-course removal from each experimental condition was monitored at 0, 0.5, 1, 2, 4, 8, 12, 16, 24, and 48 h. The removal rate of TCS was calculated by the first-order kinetic equation as follows:The mixed culture suspensions were sampled in triplicate from the control (no TCS added) reactors at days 0 and 42 and from the TCS-exposed communities (fed by glucose plus 5 mg/L of TCS) at day 56 when steady-state performance regarding removal of TCS was demonstrated. Total genomic DNA was extracted from the mixed culture suspension sampled using the MO BIO PowerSoil DNA isolation kit  according to the manufacturer’s protocol. The concentration and purity of DNA were measured using a NanoDrop One Spectrophotometer , showing >1 μg/μL and >1.8 absorbance ratios (A260/A280). Total genomic DNA was stored at −20°C prior to sequencing. The sequencing libraries was prepared Macrogen , according to the standard Illumina library protocol. In brief, the DNA quantity was measured by PicoGreen and input gDNA (about 10 ng) was amplified by PCR. The primer sequences used for the first PCR amplifications were as follows: V3-F: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, V4-R: 5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′. The final purified product was then quantified, according to the Illunima qPCR Quantification Protocol Guide  and qualified using the TapeStation DNA screentape . The paired-end (2 × 300 bp) sequencing was performed by Macrogen using the MiSeq™ platform .21. The commands and parameters used for sequence analysis were described previously23. In brief, raw sequences were filtered using the following parameters: maxambig = 0, minimum length = 200, maximum length of homopolymer = 8, and all other parameters at their default settings. High quality sequences after preprocessing were chimera-checked using chimera.vsearch with the default settings, after which the remaining sequences were taxonomically classified using classify.seqs with a cut of 80. Sequences taxonomically assigned to chloroplasts, mitochondria, unknown, archaea, and eukaryotes were excluded. The remaining sequences were clustered into representative operational taxonomic units (OTUs) based on a 97% nucleotide identity cutoff, using dist.seqs with a cutoff of 0.03 and cluster with the default settings. The Mann-Whitney U test was employed to assess differential community characteristics  across communities.The 16S rRNA gene sequence was analyzed according to the MiSeq SOP pipeline0_1 (SRS2340183), Control0_2 (SRS2340176), Control0_3 (SRS2340220), Control42_1 (SRS2340175), Control42_2 (SRS2340198), Control42_3 (SRS2340197), TCS3.5_1 (SRS2340205), TCS3.5_2 (SRS2340170), TCS3.5_3 (SRS2340172), TCS56_1 (SRS2340171), TCS56_2 (SRS2340173), and TCS56_3 (SRS2340174).The 16S rRNA gene sequence datasets used in this study were deposited in GenBank under the following accession numbers: ControlFigure 3.5, sampled at the end of the first feeding cycle) and day 56 . To assess the degree of random variability in the community structure and diversity, triplicate 16S rRNA gene datasets were also obtained from the control reactors (fed by glucose) at days 0 and 42. MiSeq sequencing generated a total of 40,000–64,000 sequences per sample. The total number of OTUs of the 12 16S rRNA gene datasets ranged from 150 to 300 per sample . The OTU composition data across datasets were used to perform non-metric multidimensional scaling (NMDS) analysis  and 0.17 ± 0.03 (control vs. TCS56). A permutational multivariate analysis of variance (PERMANOVA) test24 indicated significant difference in phylogenetic structure among the three communities (Bonferroni-corrected p < 0.05). These results suggest that exposure to TCS could result in significant alteration of the community structure under TCS exposure even in a short period (3.5 days).To document the time-course community dynamics, triplicate 16S rRNA gene sequence datasets were obtained from the 5 mg/L TCS-exposed reactors (fed both TCS and glucose) at day 3.5  can create significant variability in phylogenetic structure  across communities even under homogeneous environmental conditions. Our quantitative assessments on the strength of ecological processes showed that the deterministic factor  overwhelmed (Bonferroni-corrected p < 0.05) the stochastic (reflected from random variability within six control reactors). Further, the intra-group community structure under TCS exposure was similar in TCS3.5 (0.65 ± 0.41 within group) and TCS56 (0.79 ± 0.25 within group). In contrast, there was substantial temporal variation (0.57 ± 0.36 within group) generated within triplicate control reactors, indicating considerable stochastic variation, although they were strictly controlled under identical laboratory settings. Accordingly, for future studies investigating the degree of consequences of a deterministic factor on community structure, we strongly recommend considering the substantial stochastic variability in the structure of replicate communities generated even under a homogeneous laboratory condition, the relative importance of which many previous studies have ignored. While many studies have investigated the deterministic consequences in microbial community structure by varying operational conditions and environmental factors, considering the random temporal changes created in the control condition for the same period of operation with treatment  will help more accurately estimate (and not overestimate) the significance of a deterministic factor relative to ecological stochasticity.Ecological processes that can affect the structure and diversity of microbial communities include, but not limited to, diversification, selection, dispersal, and ecological drift. Diversification, dispersal, and ecological drift are considered stochastic processes , while selection is a deterministic process3.5) and 188 ± 3 (TCS56), suggesting dramatic reduction by more than 30%. The alpha diversity indices21, representing species diversity (Shannon and Simpson) and richness (Chao1 and Ace), are shown in Fig. p < 0.05) in both TCS-exposed communities by more than 20%. The Shannon index was 3.20 ± 0.29 in the control communities and decreased to 2.55 ± 0.83 (p = 0.11) in the TCS3.5 and 2.51 ± 0.20 (p < 0.05) in the TCS56, although the p-value between the control and TCS3.5 did not reach significance. Similar results (significant reduction in species diversity) were obtained based on the Simpson indices. Overall, both short-term (3.5 days) and long-term (56 days) exposure to TCS significantly decreased species richness, while long-term exposure could pose more pronounced perturbations, reflected in the species diversity indices.The TCS-mediated shifts in the community phylogenetic structure (beta diversity) motivated us to examine the effects of TCS on alpha diversity indices among the communities. The number of OTUs found in the control communities was 247 ± 31. The number decreased to 177 ± 17 , diversity, and equitability (evenness) of taxa. Our results clearly show that both species richness and diversity were dramatically reduced  in response to TCS. While there are many alpha diversity metrics, no index is without bias, and each metric has its own strengths and weaknessesXanthomonadaceae, Pseudomonadaceae, Sphingomonadaceae, and Verrucomicrobiaceae were significantly enriched through exposure to TCS, among which Xanthomonadaceae was selected even within the first feeding cycle. The community composition at the genus level showed significant enrichment of five genera in the TCS-exposed communities , Pseudoxanthomonas (5-fold), Luteolibacter (2-fold), and Azotobacter (6-fold).Taxonomic analysis using the MOTUHR pipeline identified 14 major families among the three communities. Figure ies Fig. . While SSphingopyxis, which are aerobic alphaproteobacteria capable of accumulating poly-β-hydroxyalkanoates. Although the exact role in activated sludge remains unclear, Sphingopyxis are known to detoxify many xenobiotics such as TCS and chlorophenol. In particular, a Sphingopyxis population (strain KCY1) originating from wastewater could degrade TCS at a wide range of concentrations (0.005–5 mg TCS/L)31. The 16S rRNA gene data analysis identified one OTU sequence (OTU078) phylogenetically affiliated with Sphingopyxis  between OTU078 and KCY1 with the demonstrated TCS metabolic capacity.Since our experiments were conducted under identical conditions with no invasion/movement of new organisms on a relatively short time scale, we speculated that selection of organisms due to fitness differences, rather than other mechanisms such as dispersal and diversification, might prevail under the selective pressure (TCS exposure). TCS selected for Sphingopyxis under exposure to TCS) were highly consistent with the physiological characterization results   Fig. . The lev00) Fig. . In the 00) Fig. . These rPseudomonas, Alcaligenes, Nitrosomonas, and Sphingomonas as well as Sphingopyxis34. Pseudomonas and Alcaligenes carry out dioxygenase-mediated catabolic pathways on halogenated phenolic compounds and are able to grow by utilizing TCS as a sole carbon source33. Sphingopyxis and Sphingomonas break down TCS via a meta-cleavage pathway using 2,3-dioxygenase34. In addition to Sphingopyxis, 16S rRNA gene data analysis revealed the abundance of two taxa, Pseudomonas (0.01 ± 0.02%) and Sphingomonas (0.07 ± 0.07%), in the control communities, whose TCS degradation activities were previously reported. However, why Sphingopyxis could be selectively enriched under the TCS exposure and the others were not enriched remains a question. It should be noted that the TCS utilization rates of bacterial populations reported in the literature are very different34, indicating that the differential TCS-utilizing activity might confer a different degree of competitive fitness for succession under TCS exposure. Of note was higher first order degradation rate constants (>8 × 10−2/h) of two Sphingomonadaceae populations (Sphingopyxis and Sphingomonas) on TCS utilization34 compared to those (<3 × 10−2/h) of Pseudomonas33. These previous estimates support more successful succession (competition) of the Sphingopyxis population than Pseudomonas that was observed during our 16S rRNA gene sequencing and analysis  observed in our study was higher than other previous estimates ( < 10 × 10−2/h) of TCS degradation by activated sludge at similar levels (1–5 mg/L of TCS)36. Accordingly, the higher TCS utilization rate observed in our study was likely due to the microbial community adaptation  that occurred within the first month of reactor operation  to genus Pseudomonas that is capable of degrading TCS40. Since Azotobacter carries an array of diverse oxygenase genes  potentially associated with enzymatic degradation of TCS31, these organisms might be involved in metabolism of TCS and thus were potentially selected upon TCS exposure, although this argument awaits further experimental examination.Pseudomoxanthomonas and Luteolibacter, in addition to the demonstrated (Sphingopyxis) and potential (Azotobacter) TCS degraders  with 0.125–8 mg/L of TCS was normalized to that of the control to provide the relative cell growth, respectively. The relative community growth at day 0 was inhibited by 88–45% at 0.25–8 mg/L TCS. In contrast, the relative growth at the same level of TCS was 95–80% compared to the control at days 42–56, showing significant reduction (p < 0.05) in susceptibility to TCS in the tested range (0.25–8 mg/L). These results corroborated the significant increase of antimicrobial resistance phenotypes upon exposure to TCS, which is consistent with the selection of potential TCS-resistant bacterial populations observed in Figs Pseudoxanthomonas and Luteolibacter might partly account for the increased resistance against TCS, other bacterial populations might also acquire broad-spectrum resistance phenotypes through adaptive evolution via point mutations and/or horizontal gene transfer of multi-drug resistant genes under TCS exposure. Our recent works showed good examples of how omics methods can be used to identify genetic mechanisms of community members involved in degradation of and resistance to toxic xenobiotics43. We are currently employing omics technologies  with a particular focus on elucidating the genomic elements responsible for the induction of antimicrobial resistance to TCS, as observed in this study.The extent of antimicrobial resistance susceptibility of the activated sludge communities in response to TCS exposure was examined over time at days 0, 42, and 56 Fig. . TCS susChitinophagaceae and Comamonadaceae were significantly decreased (p < 0.05) among the 14 major families  and 2.0 ± 1.4% (TCS56), which was significantly reduced from that of the control (5.0 ± 2.2%). Comamonadaceae also showed a significant reduction in response to TCS at days 3.5 (1.0 ± 0.2%) and 56 (0.7 ± 0.5%) compared to the control (2.9 ± 0.7%). Chitinophagaceae and Comamonadaceae represent core bacterial members of activated sludge that frequently occur across many full-scale, geographically differentially located WWTPs and are found to be relatively sensitive to antimicrobials44. Members of the Chitinophagaceae family dominating activated sludge are aerobic heterotrophs capable of degrading organic matter and are involved in biosynthesizing and exporting extracellular polymeric substances (EPSs)45. Bacteria can aggregate to form biofilms, flocs, and granules in activated sludge via physical interactions such as adhesion and cohesion. Bacterial EPSs can initiate the attachment of cells to solid particles and are essential for structural integrity by helping cells establish and colonize biofilms of the particulate supports. EPSs account for 50–90% of the total organic matter in biofilms and are essential for the formation of activated sludge flocs46. Floc formation is of great practical importance because it can significantly affect the performance of many unit processes  in WWTPs. In addition, EPSs can act as protective barriers for biofilm cells against harmful environmental perturbations . The surface properties (hydrophilicity/hydrophobicity) of EPSs offer additional benefits for contaminant removal by adsorption of diverse metals and organic/inorganic compounds47. On the other hand, Comamonadaceae are often associated with nitrogen removal in activated sludge. Some members of the Comamonadaceae family  are acetate-utilizing denitrifiers and are predominant in activated sludge49. Fig. Chitinophagaceae and Comamonadaceae, respectively. In addition to the selective decrease of Acidovorax phenotypically related to denitrification aforementioned, Ferruginibacter, a genus of Chitinophagaceae, were found to be significantly reduced under exposure to TCS. Previous works reported that Ferruginibacter are highly enriched in activated sludge and potentially associated with bioflocculation and biofilms52, consistently supporting the suggested role in floc formation. In sum, the significant reduction in core activated sludge members observed in this study highlighted the potential disruptions on the ecosystem functions of activated sludge directly associated with process performance , which therefore suggests future experiments assessing the effects of TCS on biofilm formation and nutrient removal.± 0.3% TC.5 and 2.This work quantitatively assessed microbial community dynamics during exposure to a high level of TCS (5 mg/L). Although the toxicity threshold of TCS can be variable across organisms, a few milligrams per liter (or higher) of TCS pose perturbations to the functions of activated sludge ecosystems. For example, our data suggested that 50% of cell growth activities of activated sludge communities were reduced between 4 and 8 mg/L of TCS. The results of this study were in a good agreement with a previous study reporting that about 2 mg/L of TCS is 50%-inhibitory to activated sludge . The previous work highlighted that activated sludge could resist against TCS much more than other aquatic organisms  by a factor of 5–1800. Accordingly, the TCS-mediated shifts in microbial community diversity and structure systematically described in this study were relevant to those at subinhibitory levels of TCS capable of disrupting important ecosystem functions of activated sludge such as oxygen uptake and biomass growth activities.Time course community profiling using 16S rRNA gene sequencing revealed the importance of the deterministic ecological process relative to the stochastic for controlling the structure and diversity of activated sludge communities under exposure to subinhibitory levels of TCS. The results of this work provided not only quantitative estimates on the consequence of TCS exposure to community structure and diversity, but also the mechanism of the ecological process mediating the deterministic shifts in the community assembly . Physiological characterization  of the activated sludge could provide experimental validation on the 16S rRNA gene-based findings. Overall, this work advanced the mechanistic understanding of how the antimicrobial TCS shapes the structure and diversity of activated sludge communities, which will be of great importance for managing and predicting microbial communities in engineered biological systems to provide better services to human society.Figs. S1-3


Wolfram syndrome (WFS) is a rare autosomal recessive disease with clinical manifestations of diabetes mellitus (DM), diabetes insipidus (DI), optic nerve atrophy (OA) and sensorineural hearing loss (SNHL). Although SNHL is a key symptom of WFS, there is limited information on its natural history using standardized measures. Such information is important for clinical care and determining its use as an outcome measure in clinical trials.Standardized audiologic measures, including pure-tone testing, tympanometry, speech perception, and the unaided Speech Intelligibility Index (SII) were assessed in patients with confirmed WFS annually. Mixed model analyses were used to examine main effects of age, time or interactions for pure tone average (PTA), high frequency average (HFA) and SII.n = 10), mild-to-severe (n = 24) or profound SNHL (n = 6). Mean age of diagnosis for SNHL was 8.3 years (SD = 5.1) with 75% prevalence. HFA worsened over time for both ears, and SII worsened over time in the worse ear, with greater decline in both measures in younger patients. Average estimated change over 1 year for all measures was in the subclinical range and power analyses suggest that 100 patients would be needed per group (treatment vs. placebo) to detect a 60% reduction in annual change of HFA over 3 years. If trials focused on just those patients with SNHL, power estimates suggest 55 patients per group would be sufficient.Forty WFS patients were evaluated between 1 and 6 times. Mean age at initial enrollment was 13.5 years (SD = 5.6). Patients were classified as having normal hearing  was small and would not be detectable for at least 2 years in an individual. Relatively large sample sizes would be necessary to detect significant impact on hearing progression in a clinical trial. Hearing function should be monitored clinically in WFS to provide appropriate intervention. Because SNHL can occur very early in WFS, audiologists and otolaryngologists should be aware of and refer for later emerging symptoms.The online version of this article (10.1186/s13023-018-0852-0) contains supplementary material, which is available to authorized users. Wolfram syndrome (WFS) is an autosomal recessive neurodegenerative disease with an estimated prevalence worldwide from one in 55,000 to one in 770,000 –4. The mWFS1 gene on chromosome 4p16.1 [WFS2 (CISD2) [The genetic cause for WFS has been described with pathogenesis attributed to mutations in the e 4p16.1 –16 and l (CISD2) . Althoug (CISD2) –17.The typical sequence of WFS has been described as DM followed by OA in the first decade with DI and SNHL potentially developing in later years , 18–20. There is growing appreciation for variability in the relative onset and severity of symptoms in WFS, which has implications for referring healthcare providers , 24–29. The majority of reports that include information regarding hearing in WFS do not include adequate descriptions of their methods or detail the type or severity of hearing loss. However, there are several notable exceptions , 21, 39.Accurate assessment of hearing in individuals with WFS is necessary to define this key symptom over the lifespan. The primary purpose of this study was to assess auditory status longitudinally in a relatively large cohort of 40 participants with WFS using a standardized test battery with commercially available instrumentation. The multidisciplinary nature of the Washington University Wolfram Syndrome Research Clinic afforded the opportunity to compare SNHL onset relative to the other cardinal WFS symptoms of DM, DI and OA.WFS participants were recruited through direct or physician referral and the International Wolfram Syndrome Registry. Recruitment occurred in accordance with the approved procedures of the Human Research Protection Office of Washington University in Saint Louis. All participants provided informed consent prior to participation. Children under age 18 provided assent, and their parent or guardian provided written consent. In most cases the guardians or participants initiated joining the registry and thus were self-selected to participate in the research clinic. Participants were assessed during a multidisciplinary clinic held annually from 2010 to 2017. All participants were not tested each year.Audiologic assessment was comprised of pure-tone testing, tympanometry, speech reception thresholds (SRTs) and speech discrimination testing in quiet and noise. Audiologic measures and calculations were conducted as previously described . The minPure tone, SRT, and speech in noise testing (BKB-SIN) were conducted in a sound-treated examination room . Stimuli were presented with a Madsen Orbiter-922, Grason Stadler Audiostar or 61 audiometer calibrated to American National Standards Institute (ANSI S3.6–1996) via sound field, insert earphones (ER-3A), or a bone conduction oscillator (Radioear B71). If excess cerumen precluded use of insert earphones, Telephonics TDH50 supraural headphones were used. Tympanometric measures of physical volume, static admittance and peak pressure were obtained with an Interacoustics AT235 or Grason Stadler tympanometer. The unaided SII was calculated on the Audioscan Verifit based on pure tone AC thresholds.Our goal was to determine if any of the primary audiology metrics  changed over time in patients with WFS, and whether the degree of change differed due to age or sex. We asked these questions for each ear separately. For each patient we determined which ear had worse hearing based on the unaided SII. If unaided SII was the same for both ears, the worse ear was determined by the HFA and, if necessary, PTA. Identical analyses were performed for worse and better ears separately, pooling data for all patients. In addition, as an exploratory analysis, we examined the same effects in the subset of patients with defined hearing loss at most recent hearing test.Analyses assumed a linear relationship between the outcome variables  and time and used a random slope model (mixed model) to predict average annual rate of change of the outcomes. This model allows slopes  to vary randomly between patients and fits a separate regression line for each patient. Interaction between time and age at the first testing session was examined to determine if the average annual rate of change significantly differs with patient age at the first testing session. The estimated annual rate of change of the outcomes are plotted against patient age at the first testing session. All mixed model analyses were conducted using a two-sided test at a significance level of 0.05 using SAS 9.4 .2) and homoscedastic error distributed N with both δ2 and Ɛ2 estimated from the WFS group data. Means of audiologic measures were assumed to be equal at baseline for the two groups. We assumed equal allocation between the two groups (treatment vs. placebo) and 1000 simulated trials were analyzed with empirical power calculated.Using the estimated annual rate of change in audiologic metrics, we performed power analyses to determine the sample size necessary to detect a significant effect of an intervention. Extensive simulations were conducted to determine the sample size to achieve at least 80% power to detect a 50% or 60% reduction in mean annual rate of progression in the audiologic measure with the fastest progression at a significance level of 0.05. We assumed that a trial would measure the outcome every six months during a 3-year follow-up. Mean trajectories were simulated using the random slope model assuming mean annual rate of progression in placebo group estimated from the WFS group data and a random slope distributed N. Thirty-five participants were tested at least once . Data from year 1 of the 11 participants previously reported  are inclp = .27).Age at onset or diagnosis for SNHL relative to the onset of DM, DI and OA varied, with hearing loss as the first symptom in 7 and as the second symptom in 7 of the 40 participants. SNHL was found in 30 patients, for a prevalence of 75% and an average onset at 8.3 years. With respect to sex, 12 of 18 males (67%) and 18 of 22 females (82%) had SNHL. Sex distribution did not differ between those with SNHL and those without  and Fig. Transient conductive hearing loss was present for 3 audiograms and 1 audiogram was judged to be invalid because it was inconsistent with prior and subsequent test results. These audiograms were excluded from statistical analysis. After these exclusions, we analyzed data from 35 patients with 1 or more sessions, 25 with 2 or more sessions, 19 with 3 or more, 13 with 4 or more sessions and 9 with 5 sessions. See Table n = 10), mild-to-severe hearing loss (n = 24), and profound hearing loss of early onset (n = 6). Audiometric threshold data for octave frequencies from 250 to 8000 Hz may be found in Additional file Based on case history and the most recent clinic hearing test, patients were placed in 3 hearing status categories: normal hearing n = 0, mild-tn = 5) and/or being non-native English speakers (n = 8). Administration of the BKB-SIN for the remaining 27 participants yielded SNR 50 results within normal limits for all but 2 participants who exhibited mildly reduced BKB-SIN performance that was within expectations for the magnitude of their hearing loss (unaided SII = 41 and 8%).Of the 10 participants with normal hearing on their most recent hearing test, 4 were beginning to show signs of impending hearing loss, with thresholds greater than 20 dB HL at 8 kHz. Of the remaining 6 participants, 2 were among the younger participants in the cohort: 6.0 and 8.9 years of age. Of note, one patient had completely normal AC responses from 250 Hz to 8 kHz and was one of the oldest participants in the cohort (age = 23.7 years). Twenty-seven participants were tested with the BKB-SIN. The other participants could not be tested due to cochlear implants . Six participants (15%) had SNHL by 3 years of age, with SNHL as the first symptom of WFS. This early onset included all 5 cochlear implant users.Of the 15 participants with an unaided SII less than 75% for the better ear, 14 use amplification devices  and SII (p = .01) with hearing becoming worse over time. No main effect of time was found for PTA  and unaided SII (p = .03), such that younger individuals tended to show more change in hearing than older individuals  and borderline interaction between time and age at first session (p = .05) for HFA, but not for the other measures  and SII (p = .01)   Fig. . Estimatp = .003) and an interaction between time and age at first session (p = .03) for HFA. The estimated change over 1 year was a 2.17 dB increase for HFA.There was a main effect of time (p > .19).There were no main effects of sex or interactions between sex and session for any of the three measures for either ear or for either group  to detect a 50% change and 100 WFS patients per group to detect a 60% slowing of progression of HFA. For the subgroup with SNHL, we estimate that it would take 75 WFS patients per group to detect a 50% change and 55 patients per group to detect a 60% slowing of progression of HFA.We examined prevalence, age of onset, severity and quantitative progression for SNHL in children, adolescents and young adults with WFS. In this relatively young cohort, we found that the mean SNHL diagnosis was 8.3 years SD = 5.10) with 75% prevalence. Although there was substantial individual variability across our cohort , mild-to-severe SNHL (n = 24) and profound SNHL (n = 6). Because no universally accepted classification scheme for hearing loss could be identified, we provided the threshold data for individual participants in Additional file Within our cohort three categories of hearing sensitivity were observed: normal hearing n = 0, mild-tThe average age of SNHL onset for our cohort was 8.3 years, which is younger than most previous reports. Plausible explanations for this difference include differences in diagnostic criteria, age of the cohort and variable expression or penetrance of the mutated gene. Adding to reports of early-onset SNHL , 29–33, It is likely that periodic audiologic evaluation, rather than relying on patient or parent report, increases sensitivity for early identification of SNHL. In support of this explanation, 5 participants thought to have normal hearing based on case history were identified with SNHL at an annual Washington University Wolfram Syndrome Research Clinic. Another factor is our diagnostic criteria for SNHL of greater than 20 dB HL for the PTA or 2 or more test frequencies worse than 20 dB HL, which may be stricter than those used by others.Examining relative onset of SNHL to DM and OA in our cohort of 40 participants, SNHL was the first symptom identified for 7 participants 18%) and the second symptom identified for 7 participants (25%) This percentage of SNHL as the first symptom is higher than the 6.4% (first symptom) reported for a much larger cohort of 392 patients with WFS [% and theWith the exception of early onset SNHL, progression of hearing loss in WFS appears to occur slowly. Although HFA and unaided SII worsened over time numerically on average, progression would be clinically undetectable for several years. For example, within the entire cohort, there was an estimated average 1.77 dB change over 1 year in HFA in the worse ear Table . Within With regard to amplification, we explored the role of the unaided SII as a possible guide for counseling. Fourteen of the 15 patients with SNHL and unaided SIIs less than 75% routinely used amplification. In clinical practice, the recommendation for a hearing aid evaluation would be based on the unaided SII in conjunction with the audiogram  and speech perception results. Further investigation of the unaided SII as a guideline for amplification candidacy in this population is needed.Strengths of our study include use of standardized audiologic procedures with calibrated commercially available equipment and test administration by experienced, licensed audiologists. Inclusion of a speech in noise test (BKB-SIN) and calculation of the unaided SII allowed us to examine hearing care and treatment options for patients with WFS. The use of experienced audiologists facilitated diagnosis of conductive hearing loss and resolution of the one case involving inconsistent test results. In addition, few studies of hearing with WFS have repeated hearing measures over several years for many participants. Limitations of the current study include use of a small sample size and lack of representation at the older ages. In addition, selection criteria may have biased the sample because participants had to have the interest and ability to attend the WFS clinic.To more fully elucidate hearing loss trajectories for WFS, it would be necessary to pool longitudinal data across clinics and countries , 12, 48.Early diagnosis of WFS allows for appropriate counseling and treatment to improve quality of life for patients with WFS. In concordance with other recent reports, both the presence and onset of the more frequent symptoms of WFS  are quite variable. Therefore, it is important for health care professionals who examine and diagnose diabetes, hearing loss and visual impairment to consider referral for genetic assessment of WFS when more than one symptom appears and/or family history includes individuals with more than one of the symptoms associated with WFS. With respect to hearing loss, it is best practice to make sure that vision is examined and corrected to the extent possible, as these patients may rely more heavily on visual cues to compensate for hearing loss.Accurate identification of the type and severity of SNHL in patients with WFS requires periodic audiologic assessment with a standardized test battery administered by qualified personnel from the onset of WFS diagnosis. SNHL is prevalent in WFS and preliminary results suggest that in addition to routine audiologic measures, the unaided SII may be a useful measure for determining amplification candidacy. Patients with WFS who acquired SNHL beyond early childhood experienced a slow progression on an annual basis. Our results suggest that HFA may be the most useful metric of change in hearing, but only over long periods of time  and with relatively large sample sizes, making its use in clinical trials for a rare disorder such as WFS challenging. It is likely that other measures  may be more sensitive and thus require fewer patients to adequately power clinical trials. However, since SNHL may be the first symptom in some patients with WFS, hearing tests should be administered when patients present with a concomitant symptom such as DM or OA.Additional file 1:Table S1. Raw data for the better ear for each participant and time point. (DOCX 43 kb)