[Clinical phenotype and genotype analysis of progressive familial intrahepatic cholestasis type 3 caused by novel ABCB4 gene mutation].

Objective: To investigate the pathogenic mechanism and clinical characteristics of the novel splicing variant of ATP-binding cassette subfamily B member 4 (ABCB4) and provide a basis for subsequent genetic diagnosis. Methods: The clinical data of a 5-year-old child with cholestatic liver disease admitted to the Beijing Children's Hospital of Capital Medical University was retrospectively analyzed. The pathogenic variations were detected by whole exome sequencing and verified by Sanger sequencing, and bioinformatics was used to predict the pathogenicity of the mutation sites. Possible pathogenic variations were verified in vitro by Minigene assay. The clinical outcome was followed after discharge from hospital. Results: The 5-year-old boy had developed cholestasis at the age of 11 months. His physical examination showed obvious enlargement of the liver and spleen. Cholestatic cirrhosis was diagnosed by liver function tests, abdominal ultrasonography, liver biopsy and pathology. The results of genetic analysis showed that the patient was a complex heterozygote of the ABCB4 gene, with a pathogenic mutation c.2860G>A and a novel mutation c.2065-8T>G, derived from the mother and father respectively. The conservative prediction of the c.2065-8T>G site showed that this region was highly conserved and may affect splicing. Minigene assay results confirmed that the c.2065-8T>G mutation resulted in a 7 bp retention of intron 16 in the mature mRNA. In the absence of nonsense-mediated mRNA decay, the amino acid frameshift forms a truncated protein, which is represented by p.Glu689ValfsTer19. The patient was diagnosed as progressive familial intrahepatic cholestasis type 3 (PFIC3) and treated with ursodeoxycholic acid (UDCA). His clinical symptoms improved during 18 months of follow-up. Conclusions: The c.2065-8T>G variant is confirmed to affect the splicing process and exhibits complex heterozygosity with c.2860G>A, which is identified as the cause of the disease. PFIC3 children with this variant showed cholestatic liver disease as the main manifestation with a slow progression and was sensitive to treatment with UDCA.

[Clinical features and risk factors for early relapse of pediatric ulcerative colitis].

Objective: To investigate the clinical features of pediatric ulcerative colitis (UC) and analyze the risk factors of disease relapse. Methods: The clinical data of 79 children with UC diagnosed in Beijing Children's Hospital, Capital Medical University from January 2016 to February 2021 were retrospectively analyzed. They were divided into early relapse group and non-early relapse group according to the clinical relapse within 12 months after diagnosis. T-test, rank sum test, χ2 test or Fisher's exact test were used to compare the variables between the 2 groups, including the clinical features, laboratory examination results and treatments. The Logistic regression was used to analyze the risk factors of early relapse. The cumulative relapse rate during follow-up was calculated by Kaplan-Meier method. Results: Among the 79 UC children, 46 were males and 33 were females, and the age of onset was 10.6 (6.4, 12.7) years. The children were mainly characterized by extensive disease (E3) and pancolitis (E4) (51/79, 65%), moderate to severe activity (48/79, 61%) and moderate to severe inflammation of colonic mucosa (71/79, 90%). Thirty-eight (48%) patients had atypical phenotype and 17 (22%) had extraintestinal manifestations. The follow-up period was 43.9 (22.8, 61.3) months, and of the 41 patients rechecked with colonoscopy, 7 (17%) had disease progression. According to Kaplan-Meier analysis, the cumulative relapse rate of the 79 cases at 3 months, 6 months, 1 year and 2 years after diagnosis were 27% (21/79), 47% (37/79), 57% (45/79) and 73% (53/73), respectively. There were 45 children (57%) in early relapse group and 34 (43%) in non-early relapse group. In early relapse group, hemoglobin and mucosal healing rate were both significantly lower (105 (87, 122) vs. 120 (104, 131) g/L, 28% (7/25) vs. 7/9, Z=-2.38, χ²=4.87, both P<0.05). The rate of steroid-dependent, E3 and step-up therapy during the induction period were all significantly higher than those in non-early relapse group (11/19 vs. 1/12, 24% (11/45) vs. 6% (2/34), 29% (13/45) vs. 6% (2/34), χ²=5.67, 4.85, 6.66, all P<0.05). Multivariate Logistic regression analysis showed that extraintestinal manifestations (OR=4.33, 95%CI 1.05-17.83), E3 (OR=8.27, 95%CI 1.47-46.46) and step-up therapy during the induction period (OR=5.58, 95%CI 1.01-30.77) were independent risk factors for early relapse. Conclusions: Pediatric UC is usually extensive and severe, with atypical phenotype, a high rate of relapse and a risk of disease progression. Extraintestinal manifestations, E3 and step-up therapy during the induction period are independent risk factors for early relapse.

[Epidemiological investigation of tinnitus in Sichuan and Chongqing].

Objectives: To investigate the prevalence and associated risk factors of tinnitus in Sichuan and Chongqing. Methods: We designed a tinnitus epidemiological questionnaire. The multi-stage stratified cluster random sampling methods was applied to obtain study subjects in six areas (Nanchong, Jiangjin, Fengdu, Yunyang, Suining and Ya'an), which were selected for epidemiological investigation. Home visit completion of epidemiological questionnaires was conducted. The trained investigators guided the respondents to fill in the tinnitus epidemiological questionnaires, and the epidemiological status of six areas on prevalence and risk factor was investigated. SPSS 22.0 software was used for statistical analysis. Results: Sampling population were 10 289, in which 9 273 were valid questionnaires. There were 4 281 males and 4 992 females, with an average age of 47.3 years, among which 34.83% (3 230/9 273) had tinnitus. 3.99% (370/9 273) were diagnosed with bothersome tinnitus. In a multivariable logistic regression mod, the following factors were associated with onsetting of tinnitus: sleep disorder [Odds Ratio(OR)=3.74] and noise exposure(OR=1.99). The risk of disease was lowest in the age of 30-40 years old, while the risk of disease was higher for people under 30 and over 40. In another multivariable logistic regression mode, the following factors were associated with having bothersome tinnitus: older people were more likely to suffer from tinnitus, sleep disorders (OR=4.68) and noise exposure (OR=1.56). Conclusions: The prevalence of tinnitus in Sichuan and Chongqing is about 34.83%, but most of the tinnitus is short-lived and has low loudness, which will not affect the patients. Only a small number of patients with tinnitus (3.99%) persist and affect their health and need treatment. The occurrence and exacerbation of tinnitus may be related to sleep, age, and noise exposure.

[Mutation analysis and prenatal diagnosis of MYO7A gene in a case of Usher syndrome type 1].

Objective: To analyze the clinical characteristics and identify the causative gene of a case with congenital deafness. Methods: Detailed medical history and clinical examination of a 4-year-old male child with congenital deafness were conducted in the First Affiliated Hospital of Army Military Medical University in June 2016. He was diagnosed with sensorineural deafness. The venous blood of the child and his parents was drawn, and genomic DNA was extracted. Proband's DNA was performed with targeted capture of high-throughput sequencing, then Sanger sequencing was used to verify the suspected mutation and segregation in this pedigree. According to the genetic diagnosis of the proband's deafness, ophthalmic examinations were performed. Genetic prenatal diagnosis was performed when the proband's mother was pregnant again. Results: The patient was detected with p.Trp1466Ter/p.Tyr2042Ter compound heterozygous mutations of MYO7A gene with targeted high-throughput sequencing. The mutation of p.Trp1466Ter was a reported mutation, while p.Tyr2042Ter has not been reported. In addition to congenital deafness, retinitis pigmentosa was also found by ophthalmologic examination, and the patient was clinically diagnosed with Usher syndrome type 1. Amniocentesis and fetal DNA sequencing were performed on the repregnancy fetus of this family at 18 weeks of gestation. The heterozygous mutation of MYO7A gene p.Tyr2042Ter was found, and the other allele was the wild type, indicating that the child will not exhibit clinical manifestations of Usher syndrome type 1. Indeed, the second child passed neonatal hearing screening. Conclusions: The clinical features and genetic variants were delineated in this family with Usher syndrome type 1. The results of the current study have enriched the phenotype and genotype data of the disease and provided a basis for genetic counseling.

Clonal integration facilitates spread of Paspalum paspaloides from terrestrial to cadmium-contaminated aquatic habitats.

Cadmium (Cd) is a hazardous environmental pollutant with high toxicity to plants, which has been detected in many wetlands. Clonal integration (resource translocation) between connected ramets of clonal plants can increase their tolerance to stress. We hypothesised that clonal integration facilitates spread of amphibious clonal plants from terrestrial to Cd-contaminated aquatic habitats. The spread of an amphibious grass Paspalum paspaloides was simulated by growing basal older ramets in uncontaminated soil connected (allowing integration) or not connected (preventing integration) to apical younger ramets of the same fragments in Cd-contaminated water. Cd contamination of apical ramets of P. paspaloides markedly decreased growth and photosynthetic capacity of the apical ramets without connection to the basal ramets, but did not decrease these properties with connection. Cd contamination did not affect growth of the basal ramets without connection to the apical ramets, but Cd contamination of 4 and 12 mg·l-1 significantly increased growth with connection. Consequently, clonal integration increased growth of the apical ramets, basal ramets and whole clones when the apical ramets were grown in Cd-contaminated water of 4 and 12 mg·l-1 . Cd was detected in the basal ramets with connection to the apical ramets, suggesting Cd could be translocated due to clonal integration. Clonal integration, most likely through translocation of photosynthates, can support P. paspaloides to spread from terrestrial to Cd-contaminated aquatic habitats. Amphibious clonal plants with a high ability for clonal integration are particularly useful for re-vegetation of degraded aquatic habitats caused by Cd contamination.

[Single center retrospective study of 184 children with inflammatory bowel disease seen from 2000-2014].

Objective: To investigate the clinical data of children with inflammatory bowel disease (IBD) retrospectively, including Crohn's disease (CD) and ulcerative colitis (UC) and identify the clinical characteristics and trends of change. Method: Clinical data of hospitalized patients diagnosed as IBD in Beijing Children's Hospital from January 2000 to December 2014 were collected and retrospectively analyzed. Patients were divided into six groups based on type of disease and year of admission: Group A1(CD, 2000-2004) included 12 patients, Group B1(CD, 2005-2009) included 11, Group C1(CD, 2010-2014) included 51; Group A2(UC, 2000-2004) included 17, Group B2(UC, 2005-2009) included 25, Group C2(UC, 2010-2014) included 68. Result: A total of 184 IBD patients were included in the study, 74 had CD and 110 had UC. The hospitalization constituent ratio of CD increased from 0.6/10 000 in Year 2000 to 2.9/10 000 in Year 2014. The hospitalization constituent ratio of UC increased from 0.5/10 000 in Year 2001 to 3.9/10 000 in Year 2014. The hospitalization constituent ratios of CD and UC both increased gradually(P<0.05). Up to 61.4%(113/184) of IBD patients belong to early onset IBD, furthermore the very early onset IBD and infantile IBD accounted for 41.8%(77/184) and 26.6%(49/184) respectively. For CD, ileocolonic type(47.3%, 35/74) and non-structuring, non-penetrating type (67.6%, 50/74) were more common. Perianal disease occured in 31.1%(23/74) of CD patients; 81.1%(60/74) of CD patients had moderate/severe activity. For UC, pancolitis type(59.1%, 65/110) was more common. There were no significant changes for location of pathological change, disease behavior, activity degree of CD, extent of UC lesion and incidence of surgery, intestinal perforation and hemorrhage of gastrointestinal tract for IBD in the past 15 years(P>0.05). Severe UC(S3) was more common in Group A2(64.7%, 11/17), but moderate UC(S2) was more common in Group C2(48.5%, 33/68), the difference was statistically significant (P=0.001 7). Conclusion: During the past 15 years, the hospitalization constituent ratio for IBD in our hospital showed a growing trend. The ratio of infantile IBD and very early onset IBD was high. For CD, perianal disease was commonly seen and most patients had moderate/severe activity. The surgery rate and incidence of intestinal obstruction and perforation were higher in the CD patients than UC patients. For UC, the lesions were more extensively combined with higher disease activity.

[Clinical analysis of 11 children with pancreatic cystic fibrosis].

Objective: To increase the recognition of pancreatic cystic fibrosis (PCF) in children and facilitate diagnosing and treatment of this rare entity. Method: This is a retrospective analysis of children who presented to Beijing Children's Hospital affiliated to Capital Medical University from January 2010 to December 2015. We describe their clinical features, laboratory testing and management. Result: Eleven children were diagnosed with PCF by genetic testing or sweat chloride test during these 5 years, including 4 boys and 7 girls. Their age ranged from 0.5-14.3 (mean 9.0±3.9) years. Family history was positive in 3 children. Significant clinical findings on presentation were: malnutrition 6, including 2 cases of mild, moderate and severe malnutrition each; diarrhea 4 (yellow mushy or watery stool with frequency ranging from 2-5 times a day), including 1 case of acute diarrhea and 3 of chronic diarrhea, 3 of them had steatorrhea; abdominal pain 3. All of them had pancreatic lesions shown by abdominal ultrasound. Blood tests showed 6 cases had elevated serum amylase and lipase. The main treatment was pancreatic replacement therapy and nutritional support. Conclusion: PCF is rare in children. Malnutrition, diarrhea and abdominal pain are the main clinical manifestations. Treatment is mostly pancreatic enzymes replacement and supportive care.

Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells.

Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

Medical radiation usage and exposures from medical X ray diagnosis in Shandong province of China.

Shandong province is situated in the eastern part of China with a population of 88 million. A collaborative survey was conducted in 1999 to investigate the medical radiation usage and the patients' exposures from medical X ray diagnosis in this province. There were three stages in this survey: general survey, sampling survey and dose survey. Patients' entrance surface doses (ESD) were measured using thermoluminescence dosemeters (TLDs) attached to the patient's skin. All the hospitals in this province and 2153 patients were included in this survey. The frequencies of tele- and brachytherapy were 1.450 and 1.685 per 1000 population in 1996 and 1998 respectively. The frequency of diagnostic nuclear medicine was 0.325 and 0.412 per 1000 population in 1996 and 1998 respectively; for therapeutic nuclear medicine, it was 0.015 and 0.021. The total annual frequencies of every type of X ray examination and interventional radiology (IVR) in 1996 and 1998 were 179.8 and 200.0 per 1000 population respectively. The main type of X ray examination was chest fluoroscopy, which made up about 39% of the total. The average entrance surface doses were comparatively high for the examinations of CT, gastrointestinal tract, lumbarspine, urography, angiography, hip and cerebral angiography.

Proline-rich motifs of the Na+/H+ exchanger 2 isoform. Binding of Src homology domain 3 and role in apical targeting in epithelia.

The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and 786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2.

Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms.

We recently identified a region within the cytoplasmic C-terminal tail of the Na+/H+ exchanger NHE3 isoform (residues 579 to 684) which is essential for inhibition of transport activity by cAMP-dependent protein kinase (PKA) (Cabado, A. G., Yu, F. H., Kapus, A., Gergely, L., Grinstein, S., and Orlowski, J. (1996) J. Biol. Chem. 271, 3590-3599). To further define determinants of PKA regulation, six serine residues located in potential recognition sequences for PKA within, or adjacent to, this region (positions 552, 605, 634, 661, 690, and 691) were altered either independently or in various combinations using site-directed mutagenesis. Wild type and mutant NHE3s tagged with the influenza virus hemagglutinin epitope were stably expressed in exchanger-deficient Chinese hamster ovary cells (AP-1) for functional studies. Of the individual mutations examined, only substitutions at Ser605 or Ser634 affected sensitivity to forskolin, an activator of adenylate cyclase, although partial inhibition of NHE3 activity by forskolin remained. By contrast, simultaneous mutation of both these serines completely abolished cAMP-mediated inhibition of NHE3 without greatly affecting basal transport activity. Two-dimensional analysis of tryptic digests of immunoprecipitated NHE3 labeled in vivo with [32P]orthophosphate revealed several phosphopeptides under basal conditions. Phosphorylation was increased approximately 3-fold in one of these peptides following forskolin treatment, and this change was eliminated by mutation of residue Ser605. Thus, phosphorylation of Ser605 is essential for cAMP-mediated inhibition of NHE3. In addition, Ser634 is also required for the effect of cAMP, even though this residue does not become phosphorylated upon activation of PKA.

Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.

Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.

Effects of hydroxyflutamide on rats treated with a superovulatory dose of pregnant mare serum gonadotropin.

Immature female rats treated with superovulatory doses of pregnant mare serum gonadotropin (PMSG) were used to study the effects of the antiandrogen hydroxyflutamide on steroid production, particularly the biologically active androgens, in two experiments. In the first experiment, animals were given either 5 mg hydroxyflutamide or vehicle alone at 30 and 36 h following 40 IU PMSG. Compared with the vehicle group, hydroxyflutamide treatment significantly reduced the percentage of degenerate oocytes recovered from oviducts (p less than 0.05). Serum levels of testosterone and androstenedione, and their aromatized product 17 beta-estradiol, significantly decreased (p less than 0.05) in the hydroxyflutamide-treated group; however, nonaromatizable androgen, 5 alpha-dihydrotestosterone, was not affected. In the second experiment, ovaries obtained 48 h after stimulation with 4 or 40 IU PMSG were incubated with and without hydroxyflutamide (10(-5) M) and (or) testosterone (10(-7) M) to study [4-14C]pregnenolone metabolism to major steroids. In 40 IU stimulated ovaries, hydroxyflutamide significantly decreased the metabolism of pregnenolone to progesterone (p less than 0.01) and androstenedione (p less than 0.01), while the production of 17 beta-estradiol increased significantly (p less than 0.05); however, pregnenolone conversions to testosterone and 5 alpha-dihydrotestosterone were not affected. Testosterone completely reversed the hydroxyflutamide-induced alteration of pregnenolone metabolism. In contrast, there was no difference in the pregnenolone conversion patterns between untreated and hydroxyflutamide or hydroxyflutamide plus testosterone groups in 4 IU stimulated ovaries. Present results confirm our previous finding that hydroxyflutamide decreases the percentage of abnormal oocytes recovered from superovulating rats and indicates that this hydroxyflutamide effect may be partly mediated by altered ovarian steroidogenesis following inhibition of androgen binding in the ovary.