Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses / 中华儿科杂志

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

Analysis of 4 children with DYNC1H1 gene related spinal muscular atrophy with lower extremity predominant 1 / 中华儿科杂志

Objective: To investigate the clinical features and gene variation characteristics of children with dynein cytoplasmic 1 heavy chain 1 (DYNC1H1) gene associated spinal muscular atrophy with lower extremity predominant (SMALED) 1. Methods: The clinical data of 4 SMALED1 children admitted to Peking University First Hospital from December 2018 to May 2021, who were found to have pathogenic variation of DYNC1H1 gene through genetic testing, except for other genes known to be related to motor retardation, were retrospectively summarized to analyze the phenotype and genotype characteristics. Results: There were 3 males and 1 female. The age of onset was 1 year, 1 day, 1 day and 4 months, respectively. The age of diagnosis was 4 years and 10 months, 9 months, 5 years and 9 months, and 3 years and 1 month, respectively. The clinical manifestations were muscle weakness and muscular atrophy of lower limbs, 2 cases with foot deformity, 1 case with early non progressive joint contracture, 1 case with hip dislocation and 1 case with mental retardation. De novo heterozygous missense variations in DYNC1H1 gene were found in all 4 children. According to the rating of American College of medical genetics and genomics, they were all possible pathogenic and pathogenic variations, with p.R598C, p.P776L, p.Y1109D variations had been reported, and p.I1086R variation had not been reported. Conclusions: For those with unexplained lower limb muscle weakness, muscle atrophy, joint contracture and foot deformity, upper limb motor ability related retention, with or without mental retardation, as well as the motor ability progresses slowly, it is necessary to consider the possibility of SMALED1 and the detection of DYNC1H1 gene when necessary.

Value of urine IL-8, NGAL and KIM-1 for the early diagnosis of acute kidney injury in patients with ureteroscopic lithotripsy related urosepsis / 中华创伤杂志(英文版)

PURPOSE@#To investigate the clinical value of urine interleukin-18 (IL-8), neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) for the early diagnosis of acute kidney injury (AKI) in patients with ureteroscopic lithotripsy (URL) related urosepsis.@*METHODS@#A retrospective study was carried out in 157 patients with urosepsis after URL. The patients were divided into AKI group and non-AKI group according to the Kidigo guideline and urine IL-8, NGAL and KIM-1 levels were detected by enzyme-linked immunosorbent assay at 0, 4, 12, 24 and 48 h after the surgery. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of these three biomarkers for postoperative AKI.@*RESULTS@#The level of urine IL-8, NGAL and KIM-1 in AKI group was significantly higher than that in non-AKI group at 4, 12, 24 and 48 h (p < 0.01). The ROC analysis showed the combined detection of urine IL-8, NGAL and KIM-1 at 12 h had a larger area under curve (AUC) than a single marker (0.997, 95% CI: 0.991-0.998), and the sensitivity and specificity were 98.2% and 96.7%, respectively. Pearson correlation analysis showed that the levels of urine NGAL at 4, 12, 24 and 48 h in AKI patients were positively correlated with the levels of urine KIM-1 and IL-18 (p < 0.01).@*CONCLUSION@#AKI could be quickly recognized by the elevated level of urine IL-8, NGAL and KIM-1 in patients with URL-related urosepsis. Combined detection of the three urine biomarkers at 12 h after surgery had a better diagnostic performance, which may be an important reference for the early diagnosis of AKI.

Study on Anti-inflammatory Effect and Mechanism of Jingulian Capsule on Inflammatory Model Rats / 中国药房

OBJECTIVE：To study the anti- inflammatory effect and mechanism of Jingulian capsule on inflammatory model rats. METHODS ：Totally 48 rats were randomly divided into blank control group ，model group ，Jingulian capsule low-dose ， medium-dose and high-dose groups （0.66，1.32，2.64 g/kg），dexamethasone group （positive control ，0.945 mg/kg），with 8 rats in each group. Blank control group and model group were given constant volume of water intragastrically ，and other groups were given relevant medicine intragastrocally ，twice a day ，for consecutive 3 days. Thirty minutes after last administration ，model group and administration groups were given lipopolysaccharide （10 mg/kg）intraperitoneally to induce inflammatory model. Six hours after intraperitoneal injection ，the contents of TNF-α，IL-1β，IL-6，PGE2 in serum were detected by ELISA. The wet to dry weight （W/D）ratio of lung tissue were determined. HE staining was used to observe the pathological changes of lung tissue . RT-PCR was used to detect the mRNA expression of TNF-α，IL-6，PGE2 and IL- 1β in lung tissue. Western blot assay was used to detect the phosphorylation of NF-κB p65 protein and the expression of IκBα protein in lung tissue. RESULTS：Compared with blank ； control group ，the contents of TNF-α，IL-1β，IL-6 and PGE 2 in serum ，the W/D ratio of lung tissue ，the expression of TNF-α，IL-1β，IL-6 and PGE 2 mRNA and the phosphorylation level of NF-κB p65 protein in lung tissue of model group weresignificantly increased ，and the expression of IκBα proteinwas significantly decreased （P＜0.05 or P＜0.01）；a large number of alveolar atrophy and collapse ，alveolar wall thickening ，lung consolidation ，and a large number of inflammatory cell infiltration could be seen in lung tissue. Compared with model group ，the contents of TNF-α，IL-1β（except for low-dose group ）， IL-6 and PGE 2 in serum ，as well as the expression of TNF-α（except for high-dose group ），IL-1β，IL-6（except for low-dose ， high-dose groups ）and PGE 2 mRNA in lung tissue were decreased significantly in Jingulian capsule groups （P＜0.05 or P＜0.01）； the W/D ratio of lung tissue was decreased significantly in Jingulian high-dose group （P＜0.05 or P＜0.01）；the expression of phosphorylation level of NF-κB p65 protein in lung tissue of Jingulian medium-dose group were decreased significantly （P＜0.05 or P＜0.01），while the expression of IκBα protein was increased significantly（P＜0.05）；the alveolar structure was clear ，the alveolar wall was slightly thickened ， and a small amount of inflammatory cell infiltration was seen in lung tissue. CONCLUSIONS：Jingulian capsule has good anti-inflammatory effect on inflammatory model rats ，the mechanism of which may be related to the inhibition of NF-κB signal pathway.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Report of 5 cases of Griesinger syndrome in the elderly / 中国血吸虫病防治杂志

There were 5 elderly patients with Griesinger syndrome.There was no specificity in the clinical manifestations of Griesinger syndrome in the elderly.The positive rate of stool hookworm eggs was low,but the gastroscopy could help the diagno-sis.

Relationship of impaired brain function after sevoflurane-nitrous oxide anesthesia and CREB signal pathway / 局解手术学杂志

Objective To explore the relationship of impaired brain function after emergency inhalation anesthesia of sevoflurane com -bined with nitrous oxide and cyclic AMP response element binding protein(CREB)signal pathway.Methods A total of 442 patients who were admitted into our hospital from January 2014 to April 2017 were selected as the object of this study.And these patients were divided into 3 groups according to different anesthesia ways,namely the inhalation anesthesia group(118 cases),the local anesthesia group(206 cases), and the control group(118 cases).The blood cyclic adenosine monophosphate(cAMP)concentration were examined by the mini mental state examination(MMSE)score scale.At the same time,the 60 rats were randomly divided into the anesthesia group(n=30)and the con-trol group(n=30).The learning and memory function of the two groups were evaluated by Morris water maze test,and the expressions of cAMP and CREB were measured.Results The blood cAMP concentration and MMSE score in the inhalation anesthesia group were signifi -cantly decreased after inhalation anesthesia(P<0.05).In the place navigation test,rats of the anesthesia group cost much more time to find the platform compared with rats in the control group, and rats of the anesthesia group encountered less times of crossing the platform com-pared with rats in the control group,and the difference was significant(P<0.05).Western blot showed that patients of the anesthesia group had lower cAMP and expression of p-CREB protein,with significant difference(P<0.05).Conclusion Brain function decline after sevoflu-rane inhalation anesthesia combined with nitrous oxide may induced by increasing the nucleus of the second messenger CAMP /Ca2+pathway and decreasing the expression of CREB.

Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect / 中国实验动物学报

Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.

Relevant research on expression of C5b-9 and disease in gastric adenocarcinoma / 局解手术学杂志

Objective To evaluate the correlation between the expression of C5b-9 and the gastric carcinoma.Methods In this study, the human gastric adenocarcinoma (HGAC)tissues (n =32 cases)from 32 patients were evaluated by immunohistochemistry and tissue chip.The result was analyzed and evaluated by Multinomial logistic regression and likelihood ratio.Results Multinomial logistic regression and likelihood ratio testing showed that over-expression of C5b-9 in HGAC tissue was significantly correlated with clinical stage (P =0.007) and tumor stage (P =0.005),but not with tumor metastasis,lymphoid nodal status,sex or age.Conclusion C5b-9 can be the criteria of di-agnosis and clinical staging for gastric adenocarcinoma,which may help in diagnosis and assessment disease severity of human gastric carcinoma.

4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.

Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.

Researching on fingerprint of Inulacappa by HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>This study is to establish the fingerprint and find out the common chromatographic peaks of Inula cappa by HPLC.</p><p><b>METHOD</b>The HPLC analysis was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 150 mm, 1.8 μm) with 0.1% fomic acid aqueous solution-0.1% fomic acid acetonitrile solution as mobile phase at a flow rate of 0.3 · mL(-1) · min(-1); The detective wavelength is 325 nm; The column temperature is 45 °C.</p><p><b>RESULT</b>The results indicated that 5 of 17 common peaks were identified . The similarity about 10 groups of Inulacappais is over 0.95.</p><p><b>CONCLUSION</b>This method is able to be a scientific basis of quality assessment according to its convenient and reliable.</p>

Surveillance of low pathogenicity avian influenza viruses in major live poultry wholesale markets around Dongting Lake Region,China,2009-2011 / 中国人兽共患病学报

The distribution and prevalence of low pathogenic avian influenza virus in major live poultry wholesale markets around the Dongting Lake region ,China were investigated in our study to propose prevention and control measures on low pathogenic avian flu in the area of live poultry wholesale market .The samples were injected to SPF chicken embryos by allanto-ic cavity ,and then the allantoic fluid were harvested and used for hemagglutination (HA) .If it was positive by HA ,subtypes of the virus would be determined by hemagglutination inhibition (HI) and RT-PCT .We isolated 627 low pathogenic avian in-fluenza viruses in major live poultry wholesale market around Dongting Lake region systematically in winter and spring during 2009-2011 ,and the total separation rate was 22 .2% .The duck swab separation rate of low pathogenic avian influenza was the highest ,which was 24 .6% ,and the following was chicken swab that reached 21 .5% ,and the goose swab separation rate was 11% .We isolated 6 HA subtypes including H3 ,H4 ,H6 ,H9 ,H10 ,and H11 in every live poultry wholesale market ,and the separation rate of H9 ,H6 and H4 subtypes was relatively high ,which could reach 11% ,6 .3% and 3 .4% ,respectively . Those results indicated that recessive infection of low pathogenic avian influenza virus was serious in live poultry wholesale mar-ket around the Dongting Lake area ,and it was a great threat to the occurrence of avian flu .

Phenotype/genotype analysis of 4 cases of LMNA related congenital muscular dystrophy with inflammatory changes / 中华实用儿科临床杂志

Objective To analyze the clinical characteristics,muscle pathological features and pathogenic gene mutation in 4 cases with LMNA-related congenital muscular dystrophy (L-CMD).Methods Clinical data of the probands and the parents were collected.Skeletal muscle specimens were biopsied from the probands for pathological analysis.Genomic DNA and RNA were extracted from peripheral blood leukocytes,and PCR,reverse transcription(RT)-PCR and DNA direct sequencing were employed to analyze the LMNA gene to determine the gene mutation and confirm the pathogenicity.Results Four patients had symptoms from fetal period to several months after birth.They presented with motor retardation,muscle weakness with prominent the proximal upper limbs,distal lower limbs and neck extensor,hypotonia,contractures,with mild to moderate elevation of CK level.The muscle biopsies showed muscular dystrophic and with inflammatory changes,and the abnormal nuclear morphology was observed with transmission electron microscopy.Genetic analysis of them detected 4 dominant de novo mutations.Three of them had unreported pathogenic mutations.The same sites of the LMNA gene were wild type in their parents.Conclusions Four cases of L-CMD are genetically identified.Genetic counseling of the family can be possible.The patients should be considered LMNA gene mutation of they present themselves with muscle weakness with the proximal upper limbs,distal lower limbs and neck extensor,hypotonia,contractures,mild to moderate elevation of CK level,and if the biopsies show muscular dystrophic changes but also with inflammatory changes should be considered LMNA gene mutation.Genetic analysis is the most reliable method for diagnosing L-CMD.

A family with autosomal dominant Emery-Dreifuss muscular dystrophy and literature review / 中华实用儿科临床杂志

Objective To analyze the clinical characteristics,muscle pathological features and pathogenic gene mutation of a family with autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD).Methods Clinical data of the proband and her family members,a Chinese family of AD-EDMD,were collected.Skeletal muscle specimens were collected from the proband for pathological analysis.Genomic DNA from the proband and her parents was extracted using standard procedures from the peripheral blood leukocytes.PCR and DNA direct sequencing were employed to analyze all of the 12 exons of the LMNA gene to determine the gene mutation,and the case was summarized along with related literature review.Results The proband,female,4 years and 5 months old now,presented with muscle weakness during her early childhood,the proximally was more prominent,mild pectus excavatum.Her CK level was elevated,her electromyogram showed myogenic injuries,the muscle biopsy showed myopathy changes.Her father had the same symptom,with disease progressed,showed elbow contractures in early stage,stiff neck,tight achilles tendon,slowly progressive muscle weakness of the limbs,sinus bradycardia.A heterozygous missense mutation c.1580G ＞ C (p.Arg527Pro) was identified in exon 9 of the LMNA gene in the proband and her father,but not in her mother.This heterozygous missense mutation had been reported as a pathogenic gene mutation.Conclusions The patient who has elbow contractures in early stage,limited neck flexion,spine stiffness,muscle weakness with the proximal upper limbs and distal lower limbs,and arrhythmia,should have an analysis of the LMNA gene.It's important for the early diagnosis of EDMD,assessment of the prognosis,timely and effectively monitoring the changes of arrhythmia,then taking interventions to improve the quality of life and prolong life.So genetic analysis is most reliable method to diagnose EDMD.

Absorption and pharmacokinetics of radix rehmanniae in rats / 药学学报

In this paper, absorption and pharmacokinetic study of Radix Rehmanniae was studied by liquid chromatography coupled with mass spectrometry method after oral administration to rats. By comparing the chromatograms of ultraviolet, full scan, extracted ion and selective reaction monitoring (SRM) of standard solution, Radix Rehmanniae, blank plasma and rat plasma post drug administration, catalpol and ajugol were found to be the main compounds absorbed from Radix Rehmanniae. Plasma concentrations of aucubin, dihydrocatalpol, rehmannioside A (or rehmannioside B/ melittoside) and rehmannioside D were very low. Quantitative method for catalpol and aucubin and semi-quantitative method for other compounds in rat plasma were established. The pharmacokinetic study of those absorbed components was conducted after oral administration of 6 g x kg(-1) Radix Rehmanniae water extract to rats. Cmax, t(1/2) and AUC(0-infinity) of catalpol and ajugol were (2349.05 +/- 1438.34) and (104.25 +/- 82.05) ng x mL(-1), (0.86 +/- 0.32) and (0.96 +/- 0.37) h, (4407.58 +/- 2734.89) and (226.66 +/- 188.38) ng x h x mL(-1), respectively. tmax was at 1.00 h for catalpol and ajugol. Both catalpol and ajugol were absorbed and excreted rapidly.