[Hospitalization costs of pediatric community-acquired pneumonia in Shanghai].

Objective: To investigate the epidemiology and hospitalization costs of pediatric community-acquired pneumonia (CAP) in Shanghai. Methods: A retrospective case summary was conducted on 63 614 hospitalized children with CAP in 59 public hospitals in Shanghai from January 2018 to December 2020. These children's medical records, including their basic information, diagnosis, procedures, and costs, were extracted. According to the medical institutions they were admitted, the patients were divided into the children's hospital group, the tertiary general hospital group and the secondary hospital group; according to the age, they were divided into <1 year old group, 1-<3 years old group, 3-<6 years old group, 6-<12 years old group and 12-18 years old group; according to the CAP severity, they were divided into severe pneumonia group and non-severe pneumonia group; according to whether an operation was conducted, the patients were divided into the operation group and the non-operation group. The epidemiological characteristics and hospitalization costs were compared among the groups. The χ2 test or Wilcoxon rank sum test was used for the comparisons between two groups as appropriate, and the Kruskal-Wallis H test was conducted for comparisons among multiple groups. Results: A total of 63 614 hospitalized children with CAP were enrolled, including 34 243 males and 29 371 females. Their visiting age was 4 (2, 6) years. The length of stay was 6 (5, 8) days. There were 17 974 cases(28.3%) in the secondary hospital group, 35 331 cases (55.5%) in the tertiary general hospital group and 10 309 cases (16.2%) in the children's hospital group. Compared with the hospitalizations cases in 2018 (27 943), the cases in 2019 (29 009) increased by 3.8% (1 066/27 943), while sharply declined by 76.2% (21 281/27 943) in 2020 (6 662). There were significant differences in the proportion of patients from other provinces and severe pneumonia cases, and the hospitalization costs among the children's hospital, secondary hospital and tertiary general hospital (7 146 cases(69.3%) vs. 2 202 cases (12.3%) vs. 9 598 cases (27.2%), 6 929 cases (67.2%) vs. 2 270 cases (12.6%) vs. 9 397 cases (26.6%), 8 304 (6 261, 11 219) vs. 1 882 (1 304, 2 796) vs. 3 195 (2 364, 4 352) CNY, χ2=10 462.50, 9 702.26, 28 037.23, all P<0.001). The annual total hospitalization costs of pediatric CAP from 2018 to 2020 were 110 million CNY, 130 million CNY and 40 million CNY, respectively. And the cost for each hospitalization increased year by year, which was 2 940 (1 939, 4 438), 3 215 (2 126, 5 011) and 3 673 (2 274, 6 975) CNY, respectively. There were also significant differences in the hospitalization expenses in the different age groups of <1 year old, 1-<3 years old, 3-<6 years old, 6-<12 years old and 12-18 years old (5 941 (2 787, 9 247) vs. 2 793 (1 803, 4 336) vs. 3 013 (2 070, 4 329) vs. 3 473 (2 400, 5 097) vs. 4 290 (2 837, 7 314) CNY, χ2=3 462.39, P<0.001). The hospitalization cost of severe pneumonia was significantly higher than that of non-severe cases (5 076 (3 250, 8 364) vs. 2 685 (1 780, 3 843) CNY, Z=109.77, P<0.001). The cost of patients who received operation was significantly higher than that of whom did not (10 040 (4 583, 14 308) vs. 3 083 (2 025, 4 747) CNY, Z=44.46, P<0.001). Conclusions: The number of children hospitalized with CAP in Shanghai decreased significantly in 2020 was significantly lower than that in 2018 and 2019.The proportion of patients from other provinces and with severe pneumonia are mainly admitted in children's hospitals. Hospitalization costs are higher in children's hospitals, and also for children younger than 1 year old, severe cases and patients undergoing operations.

[Effects of vaccines on the viral negative conversion of children with COVID-19].

Objective: To explore the effect of vaccination on viral negative conversion of children with COVID-19. Methods: A retrospective cohort study was conducted. A cohort of 189 children aged 3-14 years with COVID-19 admitted to Renji Hospital (South branch) of Shanghai Jiao Tong University School of Medicine from April 7th to May 19th 2022 was enrolled in the study. According to the vaccination status, the infected children were divided into an unvaccinated group and a vaccinated group. Age, gender, severity, clinical manifestations, and laboratory tests, etc. were compared between groups, by rank sum test or chi-square test. The effects of vaccination on viral negative conversion were analyzed by a Cox mixed-effects regression model. Additionally, a questionnaire survey was conducted among the parents of unvaccinated children to analyze the reasons for not being vaccinated. Results: A total of 189 children aged 3-14 years were enrolled, including 95 males (50.3%) and 94 females (49.7%), aged 5.7 (4.1,8.6) years. There were 117 cases (61.9%) in the unvaccinated group and 72 cases (38.1%) in the vaccinated group. The age of the vaccinated group was higher than that of the unvaccinated group (8.8 (6.8, 10.6) vs. 4.5 (3.6, 5.9) years, Z=9.45, P<0.001). No significant differences were found in clinical manifestations, disease severity, and laboratory results between groups (all P>0.05), except for the occurrence rate of cough symptoms, which was significantly higher in the vaccinated group than in the non-vaccinated group (68.1% (49/72) vs. 50.4% (59/117),χ2=5.67, P=0.017). The Kaplan-Meier survival curve and Cox mixed-effects regression model showed that the time to the viral negative conversion was significantly shorter in the vaccinated group compared with the unvaccinated group (8 (7, 10) vs. 11 (9, 12) d, Z=5.20, P<0.001; adjusted HR=2.19 (95%CI 1.62-2.97)). For questionnaire survey on the reasons for not receiving a vaccination, 115 questionnaires were distributed and 112 valid questionnaires (97.4%) were collected. The main reasons for not being vaccinated were that parents thought that their children were not in the range of appropriate age for vaccination (51 cases, 45.5%) and children were in special physical conditions (47 cases, 42.0%). Conclusion: Vaccination can effectively shorten the negative conversion time of children with COVID-19 and targeted programs should be developed to increase eligible children's vaccination rate for SARS-CoV-2 vaccination.

[Comparison on the prognosis of severe aortic stenosis patients treated with transcatheter aortic valve replacement versus surgical aortic valve replacement: a systematic review and meta-analysis].

Objective: To systematically review the prognosis of transcatheter aortic valve replacement (TAVR) versus surgical aortic valve replacement (SAVR) in patients with severe aortic stenosis. Methods: A systematic search of PubMed, EMBASE, Scopus, Cochrane Library, China biomedical literature database, China journal full text database (CNKI), Wanfang database and VIP database from January 2012 to February 2022 was conducted for randomized controlled trial (RCT) that comparing TAVR and SAVR in the treatment of severe aortic stenosis. The primary outcomes were the incidence of all-cause mortality, stroke incidence, reoperation rate and complications (pacemaker implantation, atrial fibrillation) at 1 month and 1, 2, 5 years after operation. Jadad scale was used to evaluate the literature quality of RCTs. All statistical analyses were performed using the standard statistical procedures provided in RevMan 5.4.1. Results: A total of 17 studies including 11 712 patients were identified, including 6 007 patients treated with TAVR and 5 705 patients treated with SAVR. There were 4 high-quality studies and 13 medium-quality studies. The results of meta-analysis showed that the rate of new onset atrial fibrillation was lower in TAVR group than that in SAVR group (RR=0.28, 95%CI 0.21-0.38, P<0.001), and there was no significant difference in all-cause death, stroke, pacemaker implantation and reoperation rate (all P>0.05) at 30 days follow-up. At one year after TAVR and SAVR treatment, all-cause mortality (RR=0.85, 95%CI 0.74-0.97, P=0.01) and new onset atrial fibrillation (RR=0.28, 95%CI 0.20-0.39, P<0.001) were lower in TAVR group than SAVR group. However, the pacemaker implantation rate was higher in TAVR group than that of SAVR group (RR=1.79, 95%CI 1.11-2.89, P=0.02), while there was no significant difference in the incidence of stroke and reoperation between the two groups (P>0.05). At two years after TAVR and SAVR treatment, the pacemaker implantation rate was higher in TAVR group than that in SAVR group (RR=2.23, 95%CI 1.28-3.86, P=0.004), and the rate of new atrial fibrillation was lower in TAVR group than that in SAVR group (RR=0.46, 95%CI 0.38-0.56, P<0.001). There was no significant difference in all-cause death, stroke and reoperation rates between the two groups (P>0.05). At five years after TAVR and SAVR treatment, the pacemaker implantation rate (RR=1.89, 95%CI 1.13-3.17, P=0.02) and reoperation rate (RR=3.64, 95%CI 1.75-7.58, P=0.000 5) were higher in TAVR group than those in SAVR group, while the rate of new onset atrial fibrillation was lower in TAVR group than that in SAVR group (RR=0.45, 95%CI 0.37-0.55, P<0.001). There was no significant difference in all-cause death and stroke incidence between the two groups (all P>0.05). Conclusions: The all-cause mortality and the incidence of new onset atrial fibrillation after TAVR are lower than SAVR, and TAVR is a preferred therapy for patients with aortic stenosis.

[Value of metagenomic next-generation sequencing in the etiology diagnosis of bacterial meningitis in children].

Objective: To explore the value of metagenomic next-generation sequencing (mNGS) in the etiology diagnosis of bacterial meningitis in children. Methods: The etiological results of 189 children diagnosed with "bacterial meningitis" or "purulent meningitis" or "central nervous system infection" in the Children's Hospital of Fudan University from 1st January 2019 to 31st December 2020 were analyzed retrospectively. The cerebrospinal fluid (CFS) of the children with bacterial meningitis was detected by culture and mNGS respectively, and the difference of pathogen detection rate between the 2 methods was analyzed. According to the age at the time of visit, the children were divided into neonatal group (≤28 days of age) and non-neonatal group (>28 days of age), and χ2 test was used to compare the positive rate between the 2 groups. Taking CFS culture as the gold standard, the sensitivity and specificity of mNGS in the diagnosing of bacterial meningitis in children were analyzed. Results: Among these 189 children with bacterial meningitis, 116 were males and 73 were females. A total of 76 strains of pathogens were detected in blood and (or) CSF cultures, of which 50 strains (65.8%) were Gram-positive bacteria; among those, 18 strains (23.7%) of Streptococcus agalactiae, 17 strains (19.7%) of Escherichia coli and 15 strains (19.7%) of Streptococcus pneumoniae were detected with higher detection rate. The infection rate of Gram-positive bacteria in the non-neonatal group was higher than that in the neonatal group (76.0% (38/50) vs. 50.0% (13/26), χ2=5.24, P=0.020).The same CSF samples of 48 cases were tested by mNGS and culture at the same time, and the detection rate of mNGS was higher than that of CSF culture (20 cases (41.7%) vs. 12 cases (25.0%), χ2=16.45, P<0.001). The consistency of mNGS and culture results was 79.2% (38/48), and the same pathogen was detected in 11 children with both positive mNGS and CSF culture. Taking the results of CSF culture as the gold standard, the sensitivity of mNGS in the diagnosing of bacterial meningitis was 91.7%, and the specificity was 75.0%. Conclusions: The mNGS technology can improve the pathogen detection rate of bacterial meningitis in children, and has a high consistency with CSF culture. In suspected cases where the pathogen cannot be identified by traditional methods, CSF mNGS should be considered timely.

[Developmental and epileptic encephalopathy 85 caused by SMC1A gene truncating variation: 4 cases report and literature review].

Objective: To summarize the clinical phenotype of patients with developmental and epileptic encephalopathy 85 caused by SMC1A gene truncating variation. Methods: The clinical data of 4 patients with epileptic encephalopathy caused by SMC1A gene truncating variation from August 2016 to June 2020 were analyzed retrospectively. Related literatures up to October 2021 with the key words "SMC1A" "Developmental and epileptic encephalopathy 85" "SMC1A, epilepsy" and "SMC1A, truncating" in PubMed, CNKI, and Wanfang databases were searched. Relevant literature was summarized and reviewed. Results: These 4 patients were all female. The onset age of seizure were all in the infantile period. They were admitted to the hospital at 3, 2, 11 and 18 months respectively. Focal seizures occurred in all 4 patients, while 1 of them experienced infantile spasm. The characteristic of cluster was observed in all of them with an interval of 14 days to 5.0 months. The seizures were all refractory to different kinds of anti-seizure medications. All 4 patients had severe developmental retardation with microcephaly (head circumference<-2 s). The interictal electroencephalogram (EEG) was characterized by diffuse slow wave. The 4 SMC1A gene variants were p.Gly655fs, p.Glu811fs, p.Arg412fs and p.Ile143fs, all of which were de novo frameshift variation after parental validation. There were another 17 cases with SMC1A gene truncating variation reported in 6 English articles and 1 Chinese article. Among these 21 patients, who were all female, the onset of seizures occurred between 0.5 and 18.0 months of age. Seventeen cases (81%) had the characteristics of cluster attacks, and the intervals of attack cycles were different. Seizure types included generalized tonic-clonic seizure (12 cases (57%)), focal seizure (11 cases(52%)), myoclonic(4 cases(19%)), spasm (4 cases(19%)), atypical absence (3 cases(14%)), tonic seizure (2 cases (10%)), and atonia (1 case(5%)). In addition, 4 cases (19%) had status epilepsy. All patients had moderate to severe mental retardation. Microcephaly was found in all patients. Among 18 cases,EEG in 8 cases had diffuse slow wave background. Brain magnetic resonance imaging (MRI) was normal in 13 cases (62%). Other MRI changes included cerebellar atrophy (3 cases), thin corpus callosum (3 cases), and lateral ventricular enlargement (2 cases). Twenty patients did not respond well to antiepileptic drugs. Conclusions: The clinical phenotypes of patients with epilepsy encephalopathy 85 caused by SMC1A gene truncating variation are characterized by female, early-onset, clustering of seizures, development delay and microcephaly. Diffuse slow waves are shown in interictal EEG in partial. Response to treatment and prognosis are poor.

[Etiology and clinical characteristics of fever of unknown origin in 357 pediatric patients].

Objective: To explore the etiologies and clinical characteristics of fever of unknown origin (FUO) and to provide clues for early diagnosis of FUO. Methods: The data about etiology, age, sex, clinical course, length of hospital stays and the expression levels of inflammatory factors in fever phase of 357 pediatric inpatients who were diagnosed with FUO in Children's Hospital of Fudan University from 1 January 2016 to 31 December 2020 were collected and retrospectively analyzed. Participants were grouped into infectious disease, inflammatory disease, malignancy and others and according to the classification of diseases and also grouped into those aged<1 year, 1-<3 years,3-<6 years, 6-<12 years and 12-<18 years. Comparisons between groups were performed using the Mann-Whitney U test, Kruskal-Wallis H test and χ² test. Results: Among the 357 patients (217 males and 140 females). The age of onset was 3.9 (1.3, 9.2) years and visiting age was 5.1 (2.0, 9.3) years. The time-consuming of diagnosis was 94 (66, 213) days. The hospital stay was 8 (6, 14) days. The most frequently identified cause of FUO was infectious diseases (163 cases, 45.7%), followed by non-infectious inflammatory diseases (133 cases, 37.2%), malignancy (21 cases, 5.9%) and others (40 cases, 11.2%). The patients at younger age were more likely to be attacked by malignancy, oncologic diagnoses, and others, nevertheless patients at older age were more likely to be attacked by non-infectious inflammatory diseases oppositely (9.8 (3.6, 11.5) vs. 3.0 (1.2, 7.0), 2.3 (1.0, 5.2), 0.9 (0.5, 1.8) years, U=41.30, 15.94, 37.08, all P<0.01);106 (65%) patients were male, and 57 (35%) patients were female. This result indicated that boys were more susceptible to infectious diseases (χ²=14.73, P<0.01). Analysis of inflammatory factors in serum among 103 patients, interleukin (IL)-6 level in 40 infectious diseases patients (9 (2, 38) ng/L) was significantly lower than those of 6 tumor patients (89 (64, 599) ng/L) and 57 non-infectious inflammatory diseases patients (25 (8, 78) ng/L, U=51.05, 15.70, both P<0.05), no significant difference was observed in IL-2, IL-4, IL-10, tumor necrosis factor α and interferon among the groups (all P>0.05). The patients grouped into those aged 1-<3 years and 3-<6 years were more likely to be attacked by infectious diseases (51.3% (59/115) and 57.1% (40/70)), while patients grouped into those aged 6-<12 years and 12-<18 years were more likely to be attacked by non-infectious inflammatory diseases (55.6% (65/117) and 72.4% (21/29)). Conclusions: Infectious disease is still the main cause of FUO in children and the boys are more susceptible to infectious diseases. However, the morbidity of non-infectious inflammatory diseases increases to number 1 in FUO of children over 6 years of age.

[Interleukin-1 receptor associated kinase 4 deficiency: a case report and literature review].

Objective: To summarize the clinical characteristics of children with interleukin-1 receptor associated kinase 4 (IRAK4) deficiency. Methods: The clinical data of a child with IRAK4 deficiency who was admitted to the Department of Neurology of Shenzhen Children's Hospital for several times from June 2019 to August 2020 were retrospectively analyzed. Related literature up to January 2021 with the key words "IRAK4 gene variation", and "interleukin-1 receptor-associated kinase 4 deficiency" in PubMed, CNKI, Wanfang, and CQVIP databases were searched. The clinical characteristics of this disease were summarized and analyzed. Results: The boy was 6 years of age and had recurrent respiratory tract infections. He was improved after antibiotic treatment. His clinical manifestation included Streptococcus pneumoniae meningoencephalitis, multiple sclerosis, invasive discitis and inflammatory bone destruction. Family-based whole exome sequencing showed that the boy had a homozygous frameshift variation in the IRAK4 gene, NM_016123.3:C.540del (p.Phe180leufs*26), and both parents were heterozygous. A total of 23 cases were reported in ten English articles. Together with this case, there were 24 cases, including 13 males and 11 females. The age of onset was 8 days to 7 years. The main manifestations were recurrent invasive bacterial infection, including 11 cases with Streptococcus pneumoniae meningitis, 9 cases with Streptococcus pneumoniae and (or) Staphylococcus aureus septicemia, 1 case with Pseudomonas aeruginosa meningitis, 1 case of salmonella infection, and 1 case with Staphylococcus aureus skin abscess. Only 1 case had recurrent virus infection. There were 2 patients with autoimmune diseases, 1 with autoimmune encephalitis and the other one with juvenile idiopathic arthritis. Among the 24 cases, 10 died (9 in infancy). Most of the surviving children were diagnosed early and received antibiotics preventively and intravenous immunoglobulin (IVIG). Their susceptibility to infection decreased year by year, and could be close to normal children at the age of 14 years. Among the 24 cases, 21 cases had homozygous variation of IRAK4 gene and 3 cases had complex heterozygous variation. There were 15 kinds of variation, including 9 kinds of frameshift variation, 4 kinds of nonsense variation and 2 kinds of missense variation. One candidate variation hotspot was c.877 c>T (3 cases). Conclusions: IRAK4 deficiency mainly manifest as recurrent and invasive bacterial infection, with Streptococcus pneumoniae meningitis or septicemia being the most common. A few patients are complicated with autoimmune diseases. The mortality rate is high in infancy, early diagnosis and treatment can avoid severe illness or death.

Polymorphism of the androgen receptor gene CAG repeat sequence and male climacteric syndrome.

The aim of this study was to analyze the association between polymorphism of the androgen receptor (AR) gene CAG repeat sequence and the climacteric syndrome in men. The study was performed in 103 males with climacteric syndrome and 111 males without the clinical syndrome of climacteric, aged between 40 and 70 years. DNA sequencing of the CAG repeat sequence in the N-terminal domain of the first exon of the AR gene was analyzed. The AR allele length ranged from 18-34 CAG repeats in males with climacteric syndrome. The average value of CAG repeat was 24.7±2.58. However, the corresponding values ranged from 15-24 CAG repeat in control group and the average value of CAG repeat was 21.25±2.63. There was a significant difference of the number of CAG repeat between the two groups. The occurrence of male climacteric syndrome was related to the CAG repeat number of androgen receptor gene, and the male patients with more CAG repeats had higher risk of clinical syndrome of climacteric. The detection of CAG repeat number of AR gene might be helpful for the prediction of clinical syndrome of climacteric.

[Value of albumin in diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency].

Objective: To investigate the clinical value of albumin (Alb) in the diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Methods: A retrospective analysis was performed for the clinical data of 90 children with NICCD who visited Children's Hospital of Fudan University from January 2007 to December 2014, and according to the content of Alb, these children were divided into Alb < 30 g/L (LA) group with 20 children and Alb ≥30 g/L (NA) group with 70 children. The clinical manifestations, results of laboratory examination, results of blood tandem mass spectrometry and urine gas chromatography-mass spectrometry, and gene detection results were compared between the two groups. The t-test and the chi-square test were used for statistical analysis.. Results: There were significant differences between the LA group and the NA group in splenomegaly degree (3.28±1.95 cm vs 1.92±1.06 cm, P = 0.030), aspartate aminotransferase/alanine aminotransferase ratio [3.15 (0.38-5.93) vs 2.14 (0.26-6.67), P = 0.010], activated partial thromboplastin time (53.27±11.68 s vs 45.06±9.79 s, P = 0.003), and international normalized ratio (1.92±1.35 vs 1.29±0.33, P = 0.001). The SLC25A13 mutation I 851_854del4 was associated with Alb (χ2 = 4.76, P = 0.025). Conclusion: As for the children with Alb < 30g/L who are highly suspected of having NICCD, SLC25A13 gene detection and blood/urine mass spectrometry should be performed as early as possible, in order to initiate intervention treatment as soon as possible, prevent and treat complications, and improve prognosis.

Ischemic preconditioning reduces deep hypothermic circulatory arrest cardiopulmonary bypass induced lung injury.

PURPOSE: Ischemic preconditioning (IP) has been used to reduce ischemia-reperfusion injury in several models. It remains unknown whether IP is sufficient to prevent deep hypothermic circulatory arrest (DHCA) cardiopulmonary bypass (CPB) induced lung injury. MATERIALS AND METHODS: Twenty-four piglets were randomly divided into four groups: routine CPB (CPB), CPB + DHCA (DHCA), CPB + IP + DHCA (IP-1) and CPB + hypoxia-ischemia preconditioning + DHCA (IP-2). Lung static compliance (Cstat) and pulmonary vascular resistance (PVR) were measured as indicators of lung function at three points during CPB. TNF-α, IL-8 and IL-10 expressions were detected by radioimmunoassay. CD18 expression was determined by flow cytometer. Some lung tissues were excised to measure the wet/dry weight ratio (W/D) and some were fixed to observe pathological changes. RESULTS: Cstat significantly decreased whereas PVR increased in DHCA group. IP prevented DHCA-induced lung functional impairment, especially IP-2 treatment. More cytokines were produced after CPB in all groups, but with varying level. Left atrium/pulmonary artery ratio of CD18 expression on monocytes decreased only in DHCA group, whereas which on polymorphonuclear neutrophils decreased in DHCA group, IP-1 group at 1h post-CPB and IP-2 group. Although lung W/D was increased in IP-2 group compared with pre-CPB, but significantly lower than that in DHCA group. Histological findings showed less lung injuries in IP groups than DHCA group. CONCLUSIONS: DHCA aggravates lung inflammatory injury and IP may reverse this injury. Maintaining ventilation with pulmonary artery perfusion in the lung IP process during CPB seems to be more superior to single pulmonary artery perfusion.

Thermal nociception in adult Drosophila: behavioral characterization and the role of the painless gene.

Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult Drosophila avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in Drosophila larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult Drosophila as a useful model to study the genetic mechanisms of thermal nociception.

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling.

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L). Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P(3) site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.

[The expression patterns of genes in different biotypes by genome addition].

The patterns of SOD, EST and haemoglobin genes expression among the different combinations conducted with genome addition were investigated. The erythrocyte expression of these isozymes and protein were visualized by histochemical staining after polyacrylamide gradient gel electrophoresis. The results of our electrophoresis analysis have revealed that biochemical polymorphism exists in the combinations, and these genetic variance have resulted from the contribution of different genomes. This study provides additional evidence that gene dosages can be interpreted from the enzyme patterns and electrophoretic staining intensities existed in different ploidies, which suggests that subunit interactions for SOD are co-operative and additive. This would indicate that each allele could be differentially regulated so that even one-third genome can be expressed in triploid individuals. These genetic data could be used to construct genome specific varieties, and would not only help to maximize efficiency in the use of different biotypes identification, but also help to maximize studying the relationship between gene dosage, expression and regulation.

Effects of ICAM-1 antisense oligonucleotide on the tubulointerstitium in mice with unilateral ureteral obstruction.

UNLABELLED: Effects of ICAM-1 antisense oligonucleotide on the renal tubulointerstitium in mice with unilateral ureteral obstruction. BACKGROUND: To extend our previous study of the therapy of the renal lesions of unilateral ureteral obstruction (UUO) in mice by an inhibitor of intercellular adhesion molecule-1 (ICAM-1), we investigated the blocking effects of ICAM-1 antisense oligonucleotides (ASONs) on the ICAM-1 expression in mouse kidney. METHODS: First, ICAM-1 ASON was transducted into mouse renal tubular epithelial cells to investigate the effects of ICAM-1 ASON in vitro. Second, fluorescein isothiocyanate (FITC)-labeled ICAM-1 ASON was injected intravenously to determine the distribution of the ASON in vivo. Third, the expression of ICAM-1 in kidney and the changes of renal morphology were observed to investigate the therapeutic effects of ICAM-1 ASON on the UUO mice in vivo. RESULTS: The expressions of ICAM-1 in the epithelial cells induced by interleukin-1beta were inhibited by ICAM-1 ASON at the dosages of 100 and 200 nmol/L. Twenty-four hours after an introvenous injection with FITC-labeled ICAM-1 ASON, the highest level of fluorescein was detected within the proximal tubules in mouse kidney. Results of immunohistology and Northern blot showed that the ICAM-1 expression was markedly reduced in the obstructed kidney after treatment with ICAM-1 ASON. The ASON also alleviated the infiltration of inflammatory cells and accumulation of the extracellular matrix in the tubulointerstitium of UUO mice without apparent side effects. CONCLUSION: Our data demonstrate that ICAM-1 ASON is taken up primarily by the proximal tubular cells of mouse kidney. ICAM-1 ASON can selectively inhibit the ICAM-1 expression of the renal tubular cells both in vitro and in vivo.

[Expression of citrate transporter mRNA in the kidneys of rats with metabolic acidosis].

The purpose of the present study was to examine whether metabolic acidosis affects the expression of rat renal Na(+)/citrate cotransporter. Female Wistar rats were pair-fed with normal rat chow and drinking water (control) or water with 0.28 mol/L NH4Cl (metabolic acidosis). The mRNA of two renal Na(+)/citrate cotransporters, which were respectively expressed on apical and basolateral membrane, were measured by Northern blot with two probes, SDCT1 and SDCT2. Animals were sacrificed on day 1,3 and 7. On the 1st day, the blood plasma HCO(-)3 of acidosis group decreased significantly (P<0.01), but the mRNA abundance did not change. On the 3rd day in the acidosis group, the blood plasma HCO(-)3 increased slightly more than that on the 1st day, but was still significantly lower than that of the control group (P<0.01). Both the probes detected some increase in mRNA of brush border and basolateral Na(+)/citrate cotransporter. On the 7th day, the blood plasma HCO(-)3 of the acidosis group continuously increased and there was no significant difference between the two groups. The abundance of brush border and basolateral Na(+)/citrate cotransporter mRNA increased, but there was no difference between those of the 3rd day and the 7th day. It is concluded that metabolic acidosis can induce increase of Na(+)/citrate cotransporter mRNA, which may be responsible for hypocitraturia.

Anti-nitrotyrosine antibodies for immunohistochemistry.

Nitrotyrosine is an important marker for the formation of peroxynitrite and possibly other reactive nitrogen species derived from nitric oxide in vivo (1). Pathological conditions can substantially increase the production of nitric oxide, yet this molecule itself does not generally yield nitration of tyrosine residues in proteins when added to biological samples (1,2). However nitric oxide reacts at near diffusion-limited rates with superoxide (O(2) (-)) to form the strong oxidant peroxynitrite (ONOO(-)) (3). Nitration on the 3-position of tyrosine is a major product of peroxynitrite attack on proteins (4,5). Certainly, small amounts of nitrotyrosine can be produced in vivo by other mechanisms (6), but peroxynitrite is by far the most efficient mechanism for nitrating tyrosine under biologically relevant conditions with natural antioxidants and alternative targets present.

Microtubule dysfunction by posttranslational nitrotyrosination of alpha-tubulin: a nitric oxide-dependent mechanism of cellular injury.

NO2Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO2Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO2Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of alpha-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of alpha-tubulin, incorporation of NO2Tyr shows apparent irreversibility. Nitrotyrosination of alpha-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of alpha-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of alpha-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO2Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the alpha-tubulin tyrosination/detyrosination cycle plays in microtubule function.

Immunohistochemical methods to detect nitrotyrosine.

The immunohistochemical detection of nitrotyrosine is a robust method for detecting peroxynitrite and other reactive nitrogen species. Success depends on optimizing conditions for the particular tissue and experimental design under investigation and the use of positive and negative controls to verify specificity. The two controls of dithionite reduction and blocking with nitrotyrosine are a powerful combination to demonstrate specificity. The pathological significance of tyrosine nitration in proteins can also be approached. Generally, nitrated proteins can be isolated from diseased tissues by immunoprecipitation and Western blotting. The sites of nitration on specific proteins can be determined by mass spectrometry, which has revealed surprising specificity in which tyrosines and/or proteins are nitrated in vivo. This provides important evidence concerning the functional consequences of peroxynitrite formation in vivo.