Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific protease 25, a key member of the deubiquitinating enzyme family that is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown aetiology. Five heterozygous USP25 variants, including two de novo and three co-segregated variants, were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared with the East Asian population and all populations in the gnomAD database. The mean age at onset of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom, except that one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was expressed ubiquitously in mouse brain with two peaks, on embryonic Days 14-16 and postnatal Day 21, respectively. In human brain, likewise, USP25 is expressed in the fetus/early childhood stage and with a second peak at ∼12-20 years old, consistent with the seizure onset age in patients during infancy and in juveniles. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knockout mice, which showed increased seizure susceptibility compared with wild-type mice in a pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we used multiple functional detections. In HEK293 T cells, the variant associated with a severe phenotype (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed stable truncated dimers with an increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del variants increased neuronal excitability in mouse brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating that USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play an epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have a profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

[Clinicopathological features of children with lupus nephritis with positive antineutrophil cytoplasmic antibody].

OBJECTIVE: To study the clinicopathological features of children with lupus nephritis (LN) with positive anti-neutrophil cytoplasmic antibody (ANCA). METHODS: A retrospective analysis was performed for the children who were diagnosed with LN in the First Affiliated Hospital of Sun Yat-sen University from January 2003 to December 2019. According to the results of serum ANCA, they were divided into two groups: ANCA-positive group (n=59) and ANCAnegative group (n=454). The two groups were compared in terms of clinical manifestations, histopathological features, remission rate, and prognosis. RESULTS: Compared with the ANCA-negative group, the ANCA-positive group had a significant reduction in leukocytes and a significant increase in erythrocyte sedimentation rate (P < 0.05). There were no significant differences between the two groups in serum creatinine, urine protein, and urine red blood cell count (P > 0.05). A total of 308 children underwent kidney biopsy. The results on light microscopy showed that compared with the ANCAnegative group, the ANCA-positive group had a significantly higher proportion of children with cellular fibrous crescents (P < 0.05) and a significantly lower proportion of children with immune complex deposition (P < 0.05). There were no significant differences between the two groups in the remission rate and survival rate (P > 0.05). CONCLUSIONS: Children with ANCA-positive LN tend to have more severe renal pathological injury, which is not exactly parallel with clinical manifestations, suggesting that timely renal biopsy is of great importance.

Dihydropyrazothiazole derivatives as potential MMP-2/MMP-8 inhibitors for cancer therapy.

MMP-2/MMP-8 is established as one of the most important metalloenzymes for targeting cancer. A series of dihydropyrazothiazole derivatives (E1-E18) bearing a salicylaldehyde group linked to Pyrazole ring were designed, synthesized, and evaluated for their pharmacological activity as MMP-2/MMP-8 inhibitors. Among them, compound E17 exhibited most potent inhibitory activity (IC50 = 2.80 µM for MMP-2 and IC50 = 5.6 µM for MMP-8), compared to the positive drug CMT-1 (IC50 = 1.29 µM). Compounds (E1-E18) were scrutinized by CoMFA and CoMSIA techniques of Three-dimensional quant. structure-activity relationship (3D-QSAR), as well as a docking simulation. Moreover, treatment with compound E4 could induce MCF-7 cell apoptosis. Overall, the biological profile of E1-E18 may provide a research basis for the development of new agents against cancer.

High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris.

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.

Preliminary Extraction and Identification of the 44.5 kDa Outer Membrane Proteins Isolated from Bovine Fusobacterium necrophorum (AB).

Fusobacterium necrophorum (AB) in the pharynx, respiratory tract, female reproductive tract or urinary system is the causative agent of footrot and hepatic abscesses in animals and acute Lemierre's syndrome in humans. Current methods do not effectively protect animals and humans against F. necrophorum (AB). The outer membrane proteins (OMP) of F. necrophorum (AB) can be used as new material to protect against the diseases induced by F. necrophorum (AB). The aim of this study was to extract OMP and examine the immunogenic response of OMP. The preliminary extraction of OMP of F. necrophorum (AB) was identified by SDS-PAGE and stained by Coomassie Brilliant Blue R-250 (CB B R-250) and silver staining methods. The results showed that only a major band of 44.5 kDa was observed when staining the gel using CB B R-250. This band represented the target protein. In contrast, many small bands were observed by the silver staining method. The OMP also exhibited immune biological activities according to western blot analysis. The brightest band among the multi-banding observed was the OMP. Thus, the OMP was obtained and had immunogenic activity. The results provide a new direction to protect animals and humans against F. necrophorum (AB) in the clinical setting.

A Novel Prognostic Model Predicts Outcomes in Non-Metastatic Nasopharyngeal Carcinoma Based on Inflammation, Nutrition, and Coagulation Signature.

Purpose: This study aimed to assess the prognostic and predictive value of a circulating hematological signature (CHS) and to develop a CHS-based nomogram for predicting prognosis and guiding individualized chemotherapy in non-metastatic nasopharyngeal carcinoma (NPC) patients. Patients and Methods: NPC patients were recruited between January 2014 and December 2017 at the Jiangxi Cancer Hospital. The CHS was constructed based on a series of hematological indicators. The nomogram was developed by CHS and clinical factors. Results: A total of 779 patients were included. Three biomarkers were selected by least absolute shrinkage and selection operator regression, including prognostic nutritional index, albumin-to-fibrinogen ratio, and prealbumin-to-fibrinogen ratio, were used to construct the CHS. The patients in the low-CHS group had better 5-year DMFS and OS than those in the high-CHS group in the training (DMFS: 85.0% vs 56.6%, p<0.001; OS: 90.3% vs 65.4%, p<0.001) and validation cohorts (DMFS: 92.3% vs 43.6%, p<0.001; OS: 92.1% vs 65.5%, p<0.001). The nomogram_CHS showed better performance than clinical stage in predicting distant metastasis (concordance index: 0.728 vs 0.646). In the low-TRS (total risk scores) group, the patients received RT alone, CCRT and IC plus CCRT had similar 5-year DMFS and OS (p>0.05). In the middle-TRS group, the patients received RT alone had worse 5-year DMFS (58.7% vs 80.8% vs 90.8%, p=0.002) and OS (75.0% vs 94.1% vs 95.0%, p=0.001) than those received CCRT or IC plus CCRT. In the high-TRS group, the patients received RT alone and CCRT had worse 5-year DMFS (18.6% vs 31.3% vs 81.5%, p<0.001) and OS (26.9% vs 53.2% vs 88.8%, p<0.001) than those received IC plus CCRT. Conclusion: The developed nomogram_CHS had satisfactory prognostic accuracy in NPC patients and may individualize risk estimation to facilitate the identification of suitable IC candidates.

Risk factors for renal outcomes in children with antineutrophil cytoplasmic antibody-associated vasculitis: a nationwide retrospective study in China.

BACKGROUND: Pediatric antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a life-threatening systemic vasculitis featured by liability to renal involvement. However, there are few studies on the risk factors and predictive models for renal outcomes of AAV in children. METHODS: Data from 179 AAV children in multiple centers between January 2012 and March 2020 were collected retrospectively. The risk factors and predictive model of end-stage renal disease (ESRD) in AAV were explored. RESULTS: Renal involvement was the most typical manifestation (95.5%), and the crescent was the predominant pathological lesion (84.9%). The estimated glomerular filtration rate (eGFR) was evaluated in 114 patients, of whom 59.6% developed ESRD, and the median time to ESRD was 3.20 months. The eGFR [P = 0.006, odds ratio (OR) = 0.955, 95% confidence interval (CI) = 0.924-0.987] and the percentages of global glomerulosclerosis (pGGS; P = 0.018, OR = 1.060, 95% CI = 1.010-1.112) were independent risk factors for ESRD of renal biopsy. Based on the pGGS and eGFR at renal biopsy, we developed three risk grades of ESRD and one predictive model. The Kaplan‒Meier curve indicated that renal outcomes were significantly different in different risk grades (P < 0.001). Compared with serum creatinine at baseline, the predictive model had higher accuracy (0.86 versus 0.58, P < 0.001) and a lower coefficient of variation (0.07 versus 0.92) in external validation. CONCLUSIONS: Renal involvement is the most common manifestation of pediatric AAV in China, of which more than half deteriorates into ESRD. The predictive model based on eGFR at renal biopsy and the pGGS may be stable and accurate in speculating the risk of ESRD in AAV children. Supplementary file 2 (MP4 18937 KB).

Phylogenetic analysis of the bovine parainfluenza virus type 3 from cattle herds revealing the existence of a genotype A strain in China.

In 2009, a bovine parainfluenza virus (BPIV3), named as NM09, was isolated using MDBK cell culture from the nasal swabs of normal cattle in China. The NM09 isolate was characterized by RT-PCR and nucleotide sequence analysis. Its complete genome was 15,456 nucleotides in length. Similar to other sequenced PIV strains, the NM09 virus consisted of six non-overlapping genes, which were predicted to encode nine proteins with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and trinucleotide intergenic sequences. Nucleotide phylogenetic analysis of matrix and hemagglutinin-neuraminidase gene demonstrated that this NM09 isolate belonged to BPIV3 genotype A instead of the previously reported BPIV3 genotype C in China. It is implicated that the different genotypes A and C might coexist infection for a long time in China.

Novel PANK2 Mutations in Patients With Pantothenate Kinase-Associated Neurodegeneration and the Genotype-Phenotype Correlation.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare genetic disorder caused by mutations in the mitochondrial pantothenate kinase 2 (PANK2) gene and displays an inherited autosomal recessive pattern. In this study, we identified eight PANK2 mutations, including three novel mutations (c.1103A > G/p.D368G, c.1696C > G/p.L566V, and c.1470delC/p.R490fs494X), in seven unrelated families with PKAN. All the patients showed an eye-of-the-tiger sign on the MRI, six of seven patients had dystonia, and two of seven patients had Parkinsonism. Biallelic mutations of PANK2 decreased PANK2 protein expression and reduced mitochondrial membrane potential in human embryonic kidney (HEK) 293T cells. The biallelic mutations from patients with early-onset PKAN, a severity phenotype, showed decreased mitochondrial membrane potential more than that from late-onset patients. We systematically reviewed all the reported patients with PKAN with PANK2 mutations. The results indicated that the early-onset patients carried a significantly higher frequency of biallelic loss-of-function (LoF) mutations compared to late-onset patients. In general, patients with LoF mutations showed more severe phenotypes, including earlier onset age and loss of gait. Although there was no significant difference in the frequency of biallelic missense mutations between the early-onset and late-onset patients, we found that patients with missense mutations in the mitochondrial trafficking domain (transit peptide/mitochondrial domain) of PANK2 exhibited the earliest onset age when compared to patients with mutations in the other two domains. Taken together, this study reports three novel mutations and indicates a correlation between the phenotype and mitochondrial dysfunction. This provides new insight for evaluating the clinical severity of patients based on the degree of mitochondrial dysfunction and suggests genetic counseling not just generalized identification of mutated PANK2 in clinics.

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China.

A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.

Codon Optimization Significantly Improves the Expression Level of α -Amylase Gene from Bacillus licheniformis in Pichia pastoris.

α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use.

Isolation and Sequence Analysis of Highly Pathogenic Porcine reproductive and respiratory syndrome virus from Swine Herds in the Jilin Province of China.

The aim of the present study was to determine the causative agent of infected swines in the Jilin province of China and assess its genetic characteristics. Virus was isolated from tissues suspected of being infected by porcine reproductive and respiratory syndrome virus (PRRSV) and inoculated onto MARC-145 cells. Virus detection was carried out by RT-PCR, immunofluorescence, electron microscopy and sequencing. The results showed that the isolate was the North American genotype PRRSV, termed the JL-04/12 strain, with a 15,320 bp genome. The homology of the amino acid sequences in two nonstructural proteins and GP2 to GP5, between strains JL-04/12 and HUN4, ranged from 97.2 to 99.3 %. However, JL-04/12 GP6 and N protein were identical in HP-PRRSV JXA1 and HUN4. JL-04/12 was characterized by two discontinuous deletions in Nsp2. We speculate that the isolate is a variant of highly pathogenic porcine reproductive and respiratory syndrome derived from strains in 2006-2008. Altogether, these results indicate that highly pathogenic porcine reproductive and respiratory syndrome virus still exists in the Jilin province of China.

Non-structural protein 2 of the porcine reproductive and respiratory syndrome (PRRS) virus: a crucial protein in viral pathogenesis, immunity and diagnosis.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of significant economic importance that causes reproductive and respiratory problems in pigs. The replicase non-structural protein 2 (Nsp2) of the porcine reproductive and respiratory syndrome virus (PRRSV) is recognized as the most variable region within the PRRSV genome. This review discusses the molecular characteristics and biological and immunological functions of the PRRSV Nsp2 and its involvement in the virus's pathogenesis. The role of Nsp2 in cell and tissue tropism, replication and growth, and variation and pathogenicity of PRRSV and the differences in virulence among different strains are described in the present review. Nsp2 is an ideal marker for monitoring genetic variation and for developing differential diagnostic tests.

[Anti-depression effect of acupunctrue and moxibustion based on SSRIs medication].

OBJECTIVE: To verify the anti-depression effect of acupuncture and moxibustion based on the medication with selective serotonin reuptake inhibitors (SSRIs). METHODS: Eighty cases of depression were randomly divided into an acupuncture-moxibustion-medication group (25 cases), an acupuncture-medication group (25 cases) and a medication group (30 cases). SSRIs medication was administered in all of the three groups. Complementarily, in acupuncture-moxibustion-medication group, the needling technique of qi conduction in the Governor Vessel was applied to Baihui (GV 20), Fengfu (GV 16), Dazhui (GV 14), etc. Additionally, mild moxibustion was added at Dazhui (GV 14) and Baihui (GV 20). In acupuncture-medication group, acupuncture for qi conduction in the Governor Vessel was only adopted. Hamilton Depression Scale (HAMD) was used for the evaluation of the total score, the score of each factor before and after treatment separately, and the therapeutic effects were observed among 3 groups. RESULTS: Compared with medication group, the scores of the factors as retardation, sleep, and anxiety/somatization, as well as the total score were all apparently improved in the other two groups (P < 0.05, P < 0.01). Compared with acupuncture-medication group, the scores of sleep and cognition factors as well as the total score in HAMD were much improved in acupuncture-moxibustion-medication group (P < 0.05, P < 0.01). The remarkable effective rates were 100.0% (25/25), 84.0% (21/25) and 56.7% (17/30) in the three groups separately, in which, the result in acupuncture-moxibustion-medication group was superior to acupuncture-medication group (P < 0.05), and the results of these two groups were superior to medication group (both P < 0.01). CONCLUSION: Either acupunctrure or moxibustion has a definite anti-depression effect based on SSRIs medication, but the coordination of acupuncture and moxibustion achieves a superior efficacy as compared with simple acupuncture therapy.

Role of non-structural protein 2 in the regulation of the replication of the porcine reproductive and respiratory syndrome virus in MARC-145 cells: effect of gene silencing and over expression.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas. Many vaccine strategies have been developed to control the disease, but none have yet been completely successful. The development of a cell line that can produce large yields of PRRSV vaccine is very necessary. In order to determine the role of Nsp2 in the replication of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in MARC-145 cells, we used an RNA interference-based short hairpin RNA of Nsp2 and constructed cell lines expressing the HP-PRRSV Nsp2 gene. Conserved HP-PRRSV Nsp2 sequences were used to design short interfering RNAs and test their ability to silence PRRSV transcript expression and replication in cells in vitro transfection. Nsp2, ORF7, and ß-actin mRNA expression were determined using semi-quantitative real-time PCR. Infection with siRNA targeting Nsp2 was found to reduce the Nsp2 expression in MARC-145 cells infected with PRRSV. Both MARC-145-TJ Nsp2 and MARC-145-TJM Nsp2 cell lines were screened by G418, which were infected with HP-PRRSV, normal MARC-145 cells for mock, and then virus titers were calculated by TCID(50) after the CPE showing up. The downregulation of Nsp2 induced a remarkable decrease in PRRSV replication, causing the reduction of structural protein. The Nsp2-targeted siRNA was found to downregulate the expression of Nsp2 in MARC-145 cells and inducing replication reduce of PRRSV in MARC-145 cells. The shRNA vectors S-1 and S-2 could effectively induce the inhibition of viral replication in MARC-145. Results showed that cells expressing the Nsp2 gene of the highly pathogenic PRRSV TJ and attenuated TJM remained stable. PRRSV replication was faster in these cells than in MARC-145 cells, especially during the early stage. This shows that Nsp2 plays a positive role in PRRSV proliferation.

[Application of "regulating spirit and conducting qi" therapy in treatment of psychological diseases].

Taking the clinical curative effects of psychological diseases treated with "regulating spirit and conducting qi" therapy as the starting point, the manipulation and technology method of "regulating spirit and conducting qi" therapy are introduced in the paper. Based on analysis of "conducting qi" therapy of Neijing (Internal Classic), it is summarized that, in "regulating spirit and conducting qi" therapy, tardiness and tenderness are taken as technical core of manipulation, individualization of patients is taken as basis, and regulation on spirit as key issue of technology, so as to rapidly, effectively and safely regulate the functional status of emotion and organism. It is aq effective technology method for treating psychological diseases, especially for insomnia, anxiety and somatic symptoms, with better curative effects.

[Efficacy and safety of cyclosporine A in treatment of refractory nephrotic syndrome in children: a systematic review of randomized controlled trials].

OBJECTIVE: To evaluate the efficacy and safety of cyclosporine A(CsA) in the treatment of refractory nephrotic syndrome (RNS) in children. METHODS: The Cochrane library, PubMed, EMBASE, CBMdisk, CNKI and VIP were searched from the time when the databases were established to December 31, 2008. Reports on RCTs on treating RNS in children with CsA were collected. Data were extracted and assessed independently by three reviewers. The methodological quality of included RCTs was assessed by the revised Jadad-scale (including randomization, allocation concealment, blinding method and withdrawal). Meta-analysis of homogenous RCTs was managed by using RevMan4.2.3. RESULT: Nine RCTs involving 293 participants were included. Six RCTs were assessed as high-quality studies with scores from 4 to 7 and 3 RCTs were assessed as low-quality studies with scores from 1 to 3. Sub-category meta-analysis was based on different clinical types and interventions of RNS in children. Meta-analysis based on included RCTs showed the following results. (1) In children with steroid-dependent or frequent relapse nephrotic syndrome: the short-term efficacy of CsA plus prednisone was better than that of prednisone alone [OR 0.14, 95% CI (0.03, 0.71)]; the short-term efficacy of CsA, cyclophosphamide (CTX) and mycophenolate mofetil had no significant differences, but compared with chlorambucil, CsA had a worse short-term efficacy [OR 6.93, 95% CI (1.53, 31.38)] and a higher relapse rate [OR 0.06, 95% CI (0.01, 0.58)]; maintaining a blood level of CsA between 60 and 80 microg/L during remission period could reduce the long term relapse rate [OR 6.43, 95% CI (1.21, 34.19)]; the incidence of end-stage renal disease (ESRD) or mortality was zero in both groups. (2) In children with steroid-resistant nephrotic syndrome, the short-term efficacy of CsA was better than that of placebo or supportive treatment and CTX, OR and 95% CI were 0.15 (0.02, 0.96) and 0.41 (0.03, 5.00), respectively, but no significant differences were found in the relapse rate and the incidence of ESRD or mortality. (3) Side effects of CsA: the incidence of nephrotoxicity, hypertrichosis and gum hypertrophy was higher in the CsA group than in that of control group, OR and 95% CI were 0.19 (0.05, 0.79), 0.06 (0.02, 0.19), 0.05 (0.02, 0.18), respectively, but no significant differences were found in the incidence of hypertension and liver toxicity. CONCLUSIONS: Available evidence showed that CsA could improve short term efficacy in RNS in children, but could not improve long term and endpoint efficacy, therefore CsA could be one of the ideal second-line drugs for RNS in children. There was a trend that the effect of CsA on steroid-dependent or frequent relapse nephrotic syndrome was superior to that on steroid-resistant nephrotic syndrome.