Diagnostic performance of a multiplexed gastrointestinal PCR panel for identifying diarrheal pathogens in children undergoing hematopoietic stem cell transplant.

BACKGROUND: Diarrhea is a common complication of hematopoietic stem cell transplantation (HSCT) and is associated with substantial morbidity, but its etiology is often unknown. Etiologies of diarrhea in this population include infectious causes, chemotherapy- or medication-induced mucosal injury and graft-versus-host disease (GVHD). Distinguishing these potential causes of diarrhea is challenging since diarrheal symptoms are often multifactorial, and the etiologies often overlap in transplant patients. The objectives of this study were to evaluate whether the FilmArray gastrointestinal (GI) panel would increase diagnostic yield and the degree to which pre-transplantation colonization predicts post-transplantation infection. METHODS: From November 2019 to February 2021, a total of 158 patients undergoing HSCT were prospectively included in the study. Stool specimens were obtained from all HSCT recipients prior to conditioning therapy, 28 ± 7 days after transplantation and at any new episode of diarrhea. All stool samples were tested by the FilmArray GI panel and other clinical microbiological assays. RESULTS: The primary cause of post-transplantation diarrhea was infection (57/84, 67.86%), followed by medication (38/84, 45.24%) and GVHD (21/84, 25.00%). Ninety-five of 158 patients were colonized with at least one gastrointestinal pathogen before conditioning therapy, and the incidence of infectious diarrhea was significantly higher in colonized patients (47/95, 49.47%) than in non-colonized patients (10/63, 15.87%) (P < 0.001). Fourteen of 19 (73.68%) patients who were initially colonized with norovirus pre-transplantation developed a post-transplantation norovirus infection. Twenty-four of 62 (38.71%) patients colonized with Clostridium difficile developed a diarrheal infection. In addition, FilmArray GI panel testing improved the diagnostic yield by almost twofold in our study (55/92, 59.78% vs. 30/92, 32.61%). CONCLUSIONS: Our data show that more than half of pediatric patients who were admitted for HSCT were colonized with various gastrointestinal pathogens, and more than one-third of these pathogens were associated with post-transplantation diarrhea. In addition, the FilmArray GI panel can increase the detection rate of diarrheal pathogens in pediatric HSCT patients, but the panel needs to be optimized for pathogen species, and further studies assessing its clinical impact and cost-effectiveness in this specific patient population are also needed.

Metagenomic next-generation sequencing in detecting pathogens in pediatric oncology patients with suspected bloodstream infections.

BACKGROUND: Studies on mNGS application in pediatric oncology patients, who are at high risk of infection, are quite limited. METHODS: From March 2020 to June 2022, a total of 224 blood samples from 195 pediatric oncology patients who were suspected as bloodstream infections were enrolled in this study. Their clinical and laboratory data were retrospectively reviewed, and the diagnostic performance of mNGS was assessed. RESULTS: Compared to the reference tests, mNGS showed significantly higher sensitivity (89.8% vs 32.5%, P < 0.001) and clinical agreement (76.3% vs 51.3%, P < 0.001) in detecting potential pathogens and distinguishing BSI from non-BSI. Especially, mNGS had an outstanding performance for virus detection, contributing to 100% clinical diagnosed virus. Samples from patients with neutropenia showed higher incidence of bacterial infections (P = 0.035). The most identified bacteria were Escherichia coli, and the overall infections by gram-negative bacteria were significantly more prevalent than those by gram-positive ones (90% vs 10%, P < 0.001). Overall, mNGS had an impact on the antimicrobial regimens' usage in 54.3% of the samples in this study. CONCLUSIONS: mNGS has the advantage of rapid and effective pathogen diagnosis in pediatric oncology patients with suspected BSI, especially for virus. IMPACT: Compared with reference tests, mNGS showed significantly higher sensitivity and clinical agreement in detecting potential pathogens and distinguishing bloodstream infections (BSI) from non-BSI. mNGS is particularly prominent in clinical diagnosed virus detection. The incidence of bacterial infection was higher in patients with neutropenia, and the overall infection rate of Gram-negative bacteria was significantly higher than that of Gram-positive bacteria. mNGS affects the antimicrobial regimens' usage in more than half of patients.

Staphylococcal superantigen-like protein 10 induces necroptosis through TNFR1 activation of RIPK3-dependent signal pathways.

Staphylococcal aureus (S. aureus) infection can lead to a wide range of diseases such as sepsis and pneumonia. Staphylococcal superantigen-like (SSL) proteins, expressed by all known S. aureus strains, are shown to be involved in immune evasion during S. aureus infection. Here, we show that SSL10, an SSL family protein, exhibits potent cytotoxicity against human cells (HEK293T and HUVEC) by inducing necroptosis upon binding to its receptor TNFR1 on the cell membrane. After binding, two distinct signaling pathways are activated downstream of TNFR1 in a RIPK3-dependent manner, i.e., the RIPK1-RIPK3-MLKL and RIPK3-CaMKII-mitochondrial permeability transition pore (mPTP) pathways. Knockout of ssl10 in S. aureus significantly reduces cytotoxicity of the culture supernatants of S. aureus, indicating that SSL10 is involved in extracellular cytotoxicity during infection. We determined the crystal structure of SSL10 at 1.9 Å resolution and identified a positively charged surface of SSL10 responsible for TNFR1 binding and cytotoxic activity. This study thus provides the description of cytotoxicity through induction of necroptosis by the SSL10 protein, and a potential target for clinical treatment of S. aureus-associated diseases.

Evaluation of acute kidney injury by urinary tissue inhibitor metalloproteinases-2 and insulin-like growth factor-binding protein 7 after pediatric cardiac surgery.

BACKGROUND: With adult patients, the measurement of [TIMP-2]*[IGFBP7] can predict the risk of moderate to severe AKI within 12 h of testing. In pediatrics, however, the performance of [TIMP-2]*[IGFBP7] as a predictor of AKI was less studied and yet to be widely utilized in clinical practice. This study was conducted to validate the utility of [TIMP-2]*[IGFBP7] as an earlier biomarker for AKI prediction in Chinese infants and small children. METHODS: We measured urinary [TIMP-2]*[IGFBP7] using NEPHROCHECK® at eight perioperative time points in 230 patients undergoing complex cardiac surgery and evaluated the performance of [TIMP-2]*[IGFBP7] for predicting severe AKI within 72 h of surgery. RESULTS: A total of 50 (22%) of 230 developed AKI stages 2-3 within 72 h after CPB initiation. In the AKI stage 2-3 patients, two patterns of serum creatinine (SCr) elevations were observed. The patients with only a transient increase in SCr within 24 h (< 24 h, early AKI 2-3) did not experience a worse outcome than patients in AKI stage 0-1. AKI stage 2-3 patients with SCr elevation after 24 h (24-72 h, late AKI 2-3), as well as AKI dialysis patients (together designated severe AKI), did experience worse outcomes. Compared to AKI stages 0-1, significant elevations of [TIMP-2]*[IGFBP7] values were observed in severe AKI patients at hours T2, T4, T12, and T24 following CPB initiation. The AUC for predicting severe AKI with [TIMP-2]*[IGFBP7] at T2 (AUC = 0.76) and maximum T2/T24 (AUC = 0.80) are higher than other time points. The addition of the NEPHROCHECK® test to the postoperative parameters improved the risk assessment of severe AKI. CONCLUSIONS: Multiple AKI phenotypes (early versus late AKI) were identified after pediatric complex cardiac surgery according to SCr-based AKI definition. Urinary [TIMP-2]*[IGFBP7] predicts late severe AKI (but not early AKI) as early as 2 h following CPB initiation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Structural insights into the recognition of histone H3Q5 serotonylation by WDR5.

Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins that specifically recognize H3Q5ser remain unknown. Here, we found that WDR5 interacts with the N-terminal tail of histone H3 and functions as a "reader" for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that H3K4me3 modification is hampered upon disruption of WDR5-H3Q5ser interaction. WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes in neuroblastoma cells, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser-mediated epigenetic regulation apparently promotes tumorigenesis.

The Effectiveness of Postoperative Chemotherapy on pT1bN0 and pT2N0 Gastric Cancer Patients with Risk Factors: An International Dual-Center Analysis.

PURPOSE: This study aimed to investigate the effectiveness of postoperative chemotherapy in pT1bN0 and pT2N0 gastric cancer patients with high risk factors. MATERIALS AND METHODS: Clinicopathological data of gastric cancer patients, who had undergone gastrectomy in high volume centers in Korea and China and were finally diagnosed with pT1bN0 and pT2N0 between 2006 and 2010, were analyzed retrospectively. Survival analyses stratified by risk factors and multivariable analyses were performed. RESULTS: A total of 1509 patients were enrolled, with 41 (2.7%) patients receiving adjuvant chemotherapy after gastrectomy and 1468 (97.3%) patients undergoing surgery alone. The adjuvant chemotherapy group showed higher percentages of tumor with maximal diameter >3 cm (51.2% vs. 25.8%), poor differentiation (68.3% vs. 49.8%), and less harvested lymph nodes (17.1% vs. 5.2%) compared to the surgery alone group. The overall survival rates were 95.1% in the adjuvant chemotherapy group and 93.3% in the surgery alone group, without significant difference. In multivariable analysis, age was found to be an independent prognostic factor. However, there were no difference in the overall survival between patients with risk factors and those without risk factors, even in terms of age. Meanwhile, patients with more than two risk factors who received chemotherapy showed better survival trend, especially for pT2N0 patients, compared to the surgery alone group, although no significant differences were observed. CONCLUSION: In pT1bN0 and pT2N0 patients, age was found to be an independent prognostic factor. However, adjuvant chemotherapy seemed to be unnecessary, while postoperative chemotherapy might offer survival benefits to pT2N0 patients with more than two risk factors.

Fecal microbiota transplantation before hematopoietic stem cell transplantation in a pediatric case of chronic diarrhea with a FOXP3 mutation.

BACKGROUND: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by mutation of the forkhead box protein 3 (FOXP3) gene, often leading to intractable and life-threatening diarrhea. Fecal microbiota transplantation (FMT), has been regarded in recent years as an available approach to reconstruct disrupted gut microbiome and successfully used to attenuates diarrhea induced by different underlying diseases. Therefore, FMT may have curative potential on the symptoms of enteropathy in patients with IPEX syndrome. METHODS: Physical and laboratory examinations were performed, and clinical data were collected. FMT was administered via frozen fecal microbial solution, and the fecal microbiota composition was analyzed using 16S rDNA sequencing before and after FMT. RESULTS: The patient was diagnosed with IPEX syndrome with a mutation detected in the FOXP3 gene, which was identified as c.767T > C (p.M256T). He presented with recurrent watery diarrhea and respiratory infections after birth and developed a significant failure to thrive. Disturbances in the gut microbiota composition and marked decreased bacterial diversity were observed to be involved in the persistent and refractory diarrhea. After receiving FMT treatment, the patient responded with remission of the diarrhea without apparent side effects. His stool output significantly decreased, corresponding to increased microbial diversity and modification of his microbiota composition. The patient finally achieved full recovery after hematopoietic stem cell transplantation (HSCT). CONCLUSION: Our data suggest an association between the gut microbiota and clinical symptoms of patient with IPEX syndrome and demonstrate FMT as an alternative therapy for severe diarrhea unresponsive to routine therapy in these patients.

Polymicrobial anaerobic bacterial meningitis secondary to dermal sinus: a case report.

Anaerobic bacterial meningitis is a rare infectious disease, and there are some special predisposing factors for it. We report a case of polymicrobial anaerobic bacterial meningitis in a nine-month-old boy who visited our hospital due to "fever with drowsiness and vomiting for 2 days". It was confirmed by the method of sanger sequencing after polymerase chain reaction (PCR) that the purulent meningitis was caused by a mixture of four anaerobic bacteria (Finegoldia magna, Campylobacter ureolyticus, Bacteroides fragilis and Porphyromonas bennonis). Even though there was no obvious structural abnormality on the skin surface, magnetic resonance imaging (MRI) examination suggested the presence of a sacrococcygeal dermal sinus. It was proven that anaerobic bacterial meningitis was secondary to retrograde infection of the dermal sinus. Finally, he was cured by a combination of anti-infection measures and surgical treatment. In conclusion, using appropriate molecular diagnostic techniques may quickly and accurately determine the pathogenic bacteria of anaerobic bacterial meningitis. When anaerobic bacterial meningitis occurs, the presence of structural abnormalities such as dermal sinus needs to be ruled out to avoid recurrence of the disease. In addition to anti-infective treatment, patients with dermal sinuses should undergo surgery as soon as possible to address abnormal structures and their root causes.

CD200 is overexpressed in neuroblastoma and regulates tumor immune microenvironment.

Patients with pediatric cancers such as neuroblastoma (NB) are often unresponsive to checkpoint blockade immunotherapy. One major factor in pediatric tumor resistance to immunotherapy is considered to be the low mutation rate of pediatric tumors. Another factor may be the overexpression of additional inhibitory pathways. While analyzing the RNA-sequencing database TARGET, we found that human NB tumors overexpress immune checkpoint molecule CD200. To determine its significance and impact on tumor immune microenvironment, we analyzed 49 cases of previously untreated, surgically removed NB tumors using immunohistochemistry and multi-color flow cytometry (FACS). We found that CD200 is overexpressed in more than 90% of NB tumors. In the tumor microenvironment of NB, CD200 is mainly overexpressed in CD45- NB tumor cells, while its cognate receptor (CD200R) is mainly expressed in HLA-DR+CD14+ myeloid cells and CD11c+ dendritic cells. Low-level expression of CD200R is also observed in tumor-infiltrating CD4+ and CD8+ T cells. In NB tumors with higher CD200 expression (CD200high), we observed lower numbers of HLA-DR+CD14+ myeloid cells and less tumor-infiltrating CD4+ and CD8+ T cells. Moreover, we found that CD4+ and CD8+ T cells produced less IFN-γ and/or TNF-α in CD200high NB tumors. Thus, CD200-CD200R pathway appears to downregulate anti-tumor immunity in the tumor microenvironment of NB tumors, and blockade of this pathway may be beneficial for NB patients.

A Diagnostic Panel of DNA Methylation Biomarkers for Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the most common cancers and lethal diseases in the world. Recognition of the undetermined lung nodules at an early stage is useful for a favorable prognosis. However, there is no good method to identify the undetermined lung nodules and predict their clinical outcome. DNA methylation alteration is frequently observed in LUAD and may play important roles in carcinogenesis, diagnosis, and prediction. This study took advantage of publicly available methylation profiling resources and a machine learning method to investigate methylation differences between LUAD and adjacent non-malignant tissue. The prediction panel was first constructed using 338 tissue samples from LUAD patients including 149 non-malignant ones. This model was then validated with data from The Cancer Genome Atlas database and clinic samples. As a result, the methylation status of four CpG loci in homeobox A9 (HOXA9), keratin-associated protein 8-1 (KRTAP8-1), cyclin D1 (CCND1), and tubby-like protein 2 (TULP2) were highlighted as informative markers. A random forest classification model with an accuracy of 94.57% and kappa of 88.96% was obtained. To evaluate this panel for LUAD, the methylation levels of four CpG loci in HOXA9, KRTAP8-1, CCND1, and TULP2 of tumor samples and matched adjacent lung samples from 25 patients with LUAD were tested. In these LUAD patients, the methylation of HOXA9 was significantly upregulated, whereas the methylation of KRTAP8-1, CCND1, and TULP2 were downregulated obviously in tumor samples compared with adjacent tissues. Our study demonstrates that the methylation of HOXA9, KRTAP8-1, CCND1, and TULP2 has great potential for the early recognition of LUAD in the undetermined lung nodules. The findings also exhibit that the application of improved mathematic algorithms can yield accurate and particularly robust and widely applicable marker panels. This approach could greatly facilitate the discovery process of biomarkers in various fields.

Exosomal hsa-miR199a-3p Promotes Proliferation and Migration in Neuroblastoma.

Neuroblastoma (NB) is the most common pediatric extra-cranial solid tumor with heterogeneous characteristics, and the prognosis of patients with high-risk NB is usually poor. Discovery of novel biomarkers for early detection and investigation of the underlying mechanisms governing invasion and metastasis of NB are urgently needed. Recently, exosomal microRNAs (miRNAs) have been shown to play vital regulatory or communication roles in the process of various types of cancers. However, the roles and mechanisms of exosomal miRNAs in NB remain unknown. Thus, the present study aims to investigate the detailed functions of tumor-derived exosomal miRNAs in progression and migration of NB in vivo and in vitro. By examining different exosomal miRNA expression profiles in the plasma of NB patients, we identified that the expression of hsa-miR199a-3p from exosomes was significantly upregulated, which was correlated with the severity of NB patients. Furthermore, we confirmed that exosomal hsa-miR199a-3p could facilitate proliferation and migration of NB via regulating NEDD4 expression. In summary, our data, for the first time, revealed that exosomal hsa-miR199a-3p could promote tumor proliferation and migration via decreasing NEDD4 expression in NB, suggesting that exosomal hsa-miR199a-3p may be applicated as a fast, easy, and non-invasive detection biomarker and contribute to the development of novel therapeutic strategies for NB in the future.

Rapid detection of respiratory organisms with the FilmArray respiratory panel in a large children's hospital in China.

BACKGROUND: Respiratory tract infections (RTIs) are the most common illness in children, and rapid diagnosis is required for the optimal management of RTIs, especially severe infections. METHODS: Nasopharyngeal swab or sputum specimens were collected from children aged 19 days to 15 years who were admitted to a hospital in Shanghai and diagnosed with RTIs. The specimens were tested with the FilmArray Respiratory Panel, a multiplex PCR assay that detects 16 viruses, Mycoplasma pneumoniae (M. pneumoniae), Bordetella pertussis (B. pertussis) and Chlamydophila pneumoniae (C. pneumoniae). RESULTS: Among the 775 children studied, 626 (80.8%, 626/775) tested positive for at least one organism, and multiple organisms were detected in 198 (25.5%). Rhinoviruses/enteroviruses (25.5%, 198/775) were detected most often, followed by respiratory syncytial virus (19.5%, 151/775), parainfluenza virus 3 (14.8%, 115/775), influenza A or B (10.9%), adenovirus (10.8%), M. pneumoniae (10.6%) and B. pertussis (6.3%). The prevalence of organisms differed by age, and most of the viruses were more common in winter. Of the 140 children suspected of having pertussis, 35.0% (49/140) tested positive for B. pertussis. CONCLUSIONS: FilmArray RP allows the rapid simultaneous detection of a wide number of respiratory organisms, with limited hands-on time, in Chinese pediatric patients with RTIs.

Identification of sparganosis based on next-generation sequencing.

The incidence of sparganosis, a parasitic disease caused by plerocercoid larvae of the genus Spirometra, has gradually risen worldwide (especially in remote areas) in recent years. Pulmonary and pleural sparganosis, as well as other sites of infestation, including the subcutaneous tissues, the abdominal viscera, brain and eyes, has been reported. In clinical practice, due to the atypical signs and symptoms as well as limited laboratory approaches for the specific detection of sparganum, sparganosis is often misdiagnosed. In the present study, an 11-year-old girl visited the Department of Infectious Diseases in Shanghai Children's Medical Center for recurrent shoulder and chest pain and shortness of breath. Imaging tests demonstrated bilateral pleural and pericardial effusion, enlarged lymph nodes in front of the tracheal carina, and infection of the left lower lobe. Sparganum were not observed in the dissected soft tissue at the root of the right thigh with naked-eye and light microscopy examination. Histologic examination revealed granulomatous inflammation and tunnel-like necrosis with eosinophilic, neutrophilic and lymphocytic infiltration. Although the patient's serum was positive for sparganum antibodies, the diagnosis of sparganosis was not confirmed for more than three months. Ultimately, genomic DNA of Spirometra erinaceieuropaei was detected in the mass at the root of the right thigh using next-generation sequencing (NGS), confirming the diagnosis of sparganosis. The patient was treated with praziquantel (150 mg/kg/day) without recurrence after an eight-month follow-up. We present, for the first time, a study of human sparganosis diagnosed using NGS, which provided a clinically actionable diagnosis of a specific infectious disease from an uncommon pathogen.

Deficiency of SUMO-specific protease 1 induces arsenic trioxide-mediated apoptosis by regulating XBP1 activity in human acute promyelocytic leukemia.

Small ubiquitin-like modifier (SUMO)/sentrin-specific protease 1 (SENP1), a member of the SENP family, is highly expressed in several neoplastic tissues. However, the effect of SENP1 in acute promyelocytic leukemia (APL) has not been elucidated. In the present study, it was observed that SENP1 deficiency had no effect on the spontaneous apoptosis or differentiation of NB4 cells. Arsenic trioxide (As2O3) could induce the upregulation of endoplasmic reticulum (ER) stress, resulting in the apoptosis of NB4 cells. Additionally, knockdown of SENP1 significantly increased As2O3-induced apoptosis in NB4 cells transfected with small interfering RNA targeting SENP1. SENP1 deficiency also increased the accumulation of SUMOylated X-box binding protein 1 (XBP1), which was accompanied by the downregulation of the messenger RNA expression and transcriptional activity of the XBP1 target genes endoplasmic reticulum-localized DnaJ 4 and Sec61a, which were involved in ER stress and closely linked to the apoptosis of NB4 cells. Taken together, these results revealed that the specific de-SUMOylation activity of SENP1 for XBP1 was involved in the ER stress-mediated apoptosis caused by As2O3 treatment in NB4 cells, thus providing insight into potential therapeutic targets for APL treatment via manipulating XBP1 signaling during ER stress by targeting SENP1.

Circulating histones for predicting prognosis after cardiac surgery: a prospective study.

OBJECTIVES: The objective of this study was to assess the perioperative changes in circulating histones and their relationships with other biomarkers and clinical outcomes after cardiac surgery with cardiopulmonary bypass (CPB) in patients. METHODS: Forty-eight patients with congenital cardiac diseases undergoing corrective procedure with CPB were prospectively enrolled in this study. Circulating histones, N-terminal pro-brain natriuretic peptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) were measured preoperatively (T0) and at 0 (T1), 24 (T2), 48 (T3) and 72 (T4) h postoperatively. The relationships between biomarkers and clinical outcomes were analysed. RESULTS: Circulating histones, NT-proBNP, PCT and CRP increased significantly postoperatively, with histones reaching the peak value earliest at T1. Circulating histone levels were higher in patients with adverse events. Receiver operating characteristic curve analysis showed that peak histone levels had a better predictive value for adverse events postoperatively. Peak histone levels correlated with the peak level of NT-proBNP (r = 0.563, P < 0.01), PCT (r = 0.551, P < 0.01), CRP (r = 0.606, P < 0.01) and clinical parameters such as ventilation time (r = 0.601, P < 0.01) and intensive care unit time (r = 0.623, P < 0.01). CONCLUSIONS: Circulating histones reached peak levels faster than NT-proBNP, PCT and CRP. Furthermore, peak histone levels correlated with biomarkers and postoperative clinical outcomes. Circulating histones may be used as a prognostic indicator for patients after cardiac surgery with CPB. CLINICAL TRIALS: ClinicalTrials.gov (ID: NCT02325765).

A novel CHD7 mutation in a Chinese patient with CHARGE syndrome.

In Genetics Out-patient Department of Shanghai Children's Medical Center, we consulted a 3-year-old boy with multiple anomaly syndrome (congenital heart disease, cryptorchidism, congenital deafness, mental retardation, exophthalmos, laryngeal cartilage dysplasia and high arched palate). We ruled out the possibility of multiple deformities caused by genomic imbalances. The patient was then clinically considered to have CHARGE syndrome, an autosomal dominant multi-system disorder involving defects in multiple organs, and CHD7 is the only known gene associated with the syndrome. Sequencing analysis of CHD7 of the proband identified a de novo heterogeneous mutation (c.2916_2917del, p.Gln972HisfsX22), a two-nucleotide deletion causing reading frame shift and resulting in a truncated CHD7 protein. Computational structure analysis suggests that the truncated protein only contains the chromodomains of CHD7, but lacks the SWI2/SNF2-like ATPase/helicase domain and the DNA binding domain, which are indispensable for the proper function of the protein, especially on chromatin remodeling. The patient then received follow up treatment in different clinical departments in a long period. To our best knowledge, this is the first CHARGE syndrome in Chinese patients diagnosed by gene analysis. In summary, the clinical symptoms and the description of treatment in the present case, combined with genetic test and functional prediction of CHD7, are helpful for further understanding and genetic counseling of the CHARGE syndrome.

Effects of rosiglitazone on the growth and lymphangiogenesis of human gastric cancer transplanted in nude mice.

Gastric cancer mainly metastasizes via lymphatic vessels. Thus, it is critical to identify efficacious chemopreventive agents for lymphangiogenesis. The present study was undertaken to explore the effects of rosiglitazone (ROSI) on the growth and lymphangiogenesis of human gastric cancer. We established a model of gastric cancer by subcutaneously inoculating the human gastric cancer cell line SGC-7901 into nude mice. Mice were randomly divided into 4 groups and each group received a different agent by oral gavage. The control group received normal saline and treatment groups received different doses of ROSI once every 2 days. The growth of the tumor in vivo was assessed by measuring tumor volume. After 42 days, the mice were sacrificed and the tumors were removed. H&E staining was used to observe the histomorphological features; immunohistochemistry staining for lymphatic vessel density (LVD) was used to evaluate tumor lymphangiogenesis, RT-PCR was performed to determine the mRNA expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3), and western blotting was used to detect the protein expression of VEGF-C and VEGFR-3. Compared with the control group, all treatment groups had smaller tumor volume and higher tumor growth inhibitory rate every day. The number of typical tumor cells in the control group was higher compared to that in the treatment groups, and the highest level of LVD was found in the control group. Furthermore, both the expression of VEGF-C and VEGFR-3 mRNA and proteins in the control group were significantly higher compared to those in the treatment groups. Markedly, these changes were correlated in a dose-dependent manner with ROSI. These results demonstrated that, through simultaneously blocking the expression of VEGF-C and VEGFR-3, ROSI suppresses lymphangiogenesis. This may represent a powerful therapeutic approach for controlling gastric cancer cell growth and metastasis.

Survivin up-regulates the expression of breast cancer resistance protein (BCRP) through attenuating the suppression of p53 on NF-κB expression in MCF-7/5-FU cells.

Both breast cancer resistance protein (BCRP, ABCG2) and apoptosis-related molecules are involved in the development of multidrug resistance (MDR) in cancer cells. However, the association of BCRP with apoptosis-related molecules in the development of MDR is unknown. In this study, we investigated the changes in expression levels of BCRP, Survivin, p53, Bcl-2, Bcl-xL or Bax in cultured MCF-7 and MCF-7/5-FU cells, and explored whether these changes affected the expressions of BCRP. Our data showed that the protein and mRNA expression levels of BCRP, Survivin and Bcl-2 were significantly higher in MCF-7/5-FU cells than in MCF-7 cells, while p53 expression lower in MCF-7/5-FU cells than in MCF-7 cells. Knockdown of Survivin or Bcl-2 in MCF-7/5-FU cells and overexpression of Survivin in MCF-7 cells revealed that Survivin had significant association with BCRP expression. Luciferase reporter gene assays showed that Survivin up-regulated BCRP expression at transcriptional level and this response was mediated through NF-κB(p50) pathway. However, may be due to the physical interaction between p53 and Survivin, p53 directly affected Survivin-regulated BCRP expressions. Interestingly, we found that Survivin would suppress p53 expression. Furthermore, our data revealed that Survivin itself had no apparent effect on NF-κB(p50) and BCRP expression when knockdown of p53 in MCF-7 cells; whereas p53 exerted significant inhibitory action on these when knockdown of Survivin. In conclusion, through down regulation of p53 expression, Survivin attenuates the suppressing effect of p53 on NF-κB(p50) expression and then enhances BCRP expression. This may represent a novel strategy for reversal of BCRP drug transporter activity to modulate MDR in cancer cells.

Effect of silencing of high mobility group A2 gene on gastric cancer MKN-45 cells.

AIM: To investigate the effect of high mobility group A2 (HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro. METHODS: HMGA2 short hairpin RNA (shRNA) expression plasmids were constructed, including a pair of random scrambled sequences. Human gastric cancer cell line MKN-45 cells were divided into three groups: blank control group (non-transfected cells), transfected group (cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group (transfected with random scrambled plasmid). Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro, and the cells transfection efficiency was assayed by fluorescence microscopy. The HMGA2 messenger RNA (mRNA) expression was detected by reverse transcription polymerase chain reaction, gastric cancer cells apoptosis was detected by flow cytometry, cell proliferation was detected by methyl thiazol tetrazolium, and the protein expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), P27, caspase-9 and B-cell leukemia/lymphoma-2 (Bcl-2) were analyzed by Western blotting. RESULTS: Compared with the blank control group and the scrambled sequence group, the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced (P < 0.05). The relative HMGA2 mRNA expression levels of the blank control group, transfected group and scrambled sequence group were 0.674 ± 0.129, 0.374 ± 0.048 and 0.689 ± 0.124, respectively. The relative HMGA2 protein expression levels of the blank control group, transfected group and scrambled sequence group were 0.554 ± 0.082, 0.113 ± 0.032 and 0.484 ± 0.123, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of HMGA2. After being transfected with shRNA for 24, 48 and 72 h, the cell apoptotic rates of the transfected group were 21.65% ± 0.28%, 39.98% ± 1.82% and 24.51% ± 0.93%, respectively, which significantly higher than those of blank control group (4.72% ± 1.34%, 5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group (4.28% ± 1.33%, 7.87% ± 1.43% and 6.71% ± 0.92%). After 24, 48 and 72 h, the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%, 39.45% ± 2.07% and 37.56% ± 2.32%, respectively; the most obvious cell proliferation inhibition appeared at 48 h after transfection. Compared with the blank control group and scrambled sequence group, after transfection of shRNA for 72 h, the protein expression levels of PI3K (0.042 ± 0.005 vs 0.069 ± 0.003, 0.067 ± 0.05), Akt (0.248 ± 0.004 vs 0.489 ± 0.006, 0.496 ± 0.104) and Bcl-2 (0.295 ± 0.084 vs 0.592 ± 0.072, 0.594 ± 0.109) were significantly reduced. The protein expression levels of P27 (0.151 ± 0.010 vs 0.068 ± 0.014, 0.060 ± 0.013) and caspase-9 (0.136 ± 0.042 vs 0.075 ± 0.010, 0.073 ± 0.072) were significantly upregulated. CONCLUSION: HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.