Degree of Freedom of Gene Expression in Saccharomyces cerevisiae.

The complexity of genome-wide gene expression has not yet been adequately addressed due to a lack of comprehensive statistical analyses. In the present study, we introduce degree of freedom (DOF) as a summary statistic for evaluating gene expression complexity. Because DOF can be interpreted by a state-space representation, application of the DOF is highly useful for understanding gene activities. We used over 11,000 gene expression data sets to reveal that the DOF of gene expression in Saccharomyces cerevisiae is not greater than 450. We further demonstrated that various degrees of freedom of gene expression can be interpreted by different sequence motifs within promoter regions and Gene Ontology (GO) terms. The well-known TATA box is the most significant one among the identified motifs, while the GO term "ribosome genesis" is an associated biological process. On the basis of transcriptional freedom, our findings suggest that the regulation of gene expression can be modeled using only a few state variables. IMPORTANCE Yeast works like a well-organized factory. Each of its components works in its own way, while affecting the activities of others. The order of all activities is largely governed by the regulation of gene expression. In recent decades, biologists have recognized many regulations for yeast genes. However, it is not known how closely the regulation links each gene together to make all components of the cell work as a whole. In other words, biologists are very interested in how many independent control factors are needed to operate an artificial "cell" that works the same as a real one. In this work, we suggested that only 450 control factors were sufficient to represent the regulation of all 5800 yeast genes.

Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins.

BACKGROUND: Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. RESULTS: In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2-Ire1-Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. CONCLUSIONS: Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

Can domperidone decrease transit time of pediatric video capsule endoscopy? A randomized controlled trial.

BACKGROUND: The complete examination rate of video capsule endoscopy can be increased by reduced gastric transit time (GTT) and or small bowel transit time (SBTT). This study aims to examine whether the prokinetic domperidone reduces GTT and/or SBTT in pediatric patients undergoing video capsule endoscopy (VCE). METHODS: We performed a single-center randomized controlled trial (n=200) to evaluate the effect of domperidone on GTT and SBTT among pediatric patients in a tertiary university-affiliated hospital for children. We explored whether patients randomized to domperidone had increased GTT, SBTT (primary outcomes) or higher complete examination rate (secondary outcome). The safety outcomes were the adverse effects in the domperidone group. This study was registered on ClinicalTrials.gov (NCT03662113). RESULTS: Demographic features including gender and age were similar between the 100 patients of the domperidone group and the 100 patients of the control group. The median GTT was 67.5 minutes (44.8-117.5) in the domperidone group and 80.0 minutes (42.0-128.0) in the control group, while the median SBTT was 317 minutes (231-436) and 323 minutes (225-426), respectively. There were no significant differences in GTT (P=0.49) and SBTT (P=0.52) between the two groups. The complete examination rate was 97% and 98% in the domperidone and control groups, respectively (P=1.00). CONCLUSIONS: Domperidone shows no effect on GTT, SBTT and complete examination rate in pediatric patients receiving VCE.

Thalidomide Inhibits Angiogenesis via Downregulation of VEGF and Angiopoietin-2 in Crohn's Disease.

Immune-mediated angiogenesis is important in the pathogenesis of inflammatory bowel disease and targeted treatment could alleviate the disease. Thalidomide is an effective drug in inflammatory bowel disease, which might be related to its multiple role in anti-inflammatory, immunoregulatory, and anti-angiogenesis. This study is to investigate the effect of thalidomide on angiogenesis in tissues from patients and in vitro cells. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), VEGF, and CD31 expressions in intestinal mucosa from pediatric CD patients before and after thalidomide treatment were measured by immunohistochemistry. Western blotting and polymerase chain reaction were performed to characterize the change of angiogenic factors before and after treatment in remission. Human umbilical vein endothelial cells (HUVECs) treated by thalidomide were used to examine its effect on endothelial cell proliferation and migration and capillary-like structures. Results showed that VEGF and Ang-2 levels were significantly greater in CD patients over controls. Thalidomide produced a significant reduction in protein expression of Ang-2 and VEGF, along with a decrease in mRNA expression of Ang-2. While, Ang-1 level did not show a statistically significant change. Thalidomide significantly inhibited cell proliferation in a dose-dependent manner. It also suppressed VEGF- and Ang-2-induced cell migration and capillary-like tube formation in HUVECs. Therefore, our study suggests that VEGF and Ang-2 levels are up-regulated in pediatric CD patients. It also indicated that thalidomide can be able to deactivate endothelium by the downregulation effect on angiogenic factors by targeting VEGF and Ang-2.

Fecal Microbial Signatures Are Associated With Engraftment Failure Following Umbilical Cord Blood Transplantation in Pediatric Crohn's Disease Patients With IL10RA Deficiency.

OBJECTIVES: Umbilical cord blood transplantation (UCBT) is associated with a relatively high rate of engraftment failure. This study aimed at exploring whether any fecal microbiota could be associated with engraftment failure following UCBT in Crohn's disease patients with IL10RA deficiency. METHODS: Thirteen patients were recruited and their 230 fecal samples were collected longitudinally from immediately before conditioning chemotherapy to 8 weeks post the UCBT. The V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR and sequenced, followed by bioinformatics analyses. RESULTS: Following the UCBT, 7 out of 13 patients achieved neutrophil and platelet engraftment with a median of 21 and 28 days, respectively (S group), while 6 patients failed to achieve engraftment (F group). In comparison with that in the S group, significantly lower Shannon diversity values on the UCBT day (P = 0.0176) and less abundance of Bifidobacterium longum, Bifidobacterium pseudolongum, Enterobacteriaceae_538000, and one taxon of Lachnospiraceae family was detected in the F group, accompanied by significantly higher abundances of four taxa including Lautropia, Pseudomonas, and species Microvirgula aerodenitrificans during the chemotherapy period as well as UCBT. The abundances of thirty OTUs were correlated significantly with clinical indices. CONCLUSIONS: Microbial indicators of reduced diversity of microbiota and signatures of specific bacterial abundances, such as a lower abundance of Bifidobacterium longum, for engraftment failure would require validation. These indicators may help for the risk stratification in patients with IL10RA deficiency undergoing UCBT.

Clinical features of capsule endoscopy in 825 children: A single-center, retrospective cohort study.

Video capsule endoscopy (VCE) can detect mucosal lesions in the intestine, especially in the small bowel.Our study aims to evaluate the applications of VCE for pediatric gastrointestinal diseases.In this retrospective study, we included all patients who underwent VCE between December 2012 and December 2018. Clinical information and VCE data were analyzed.Among 828 patients, the completion rate was 99.6% (n = 825), with an average age of 10.2 ±â€Š3.3 years old. A total of 459 VCE procedures showed abnormalities, and the overall diagnostic yield was 55.6%. The most common indications for VCE were abdominal pain among 505 (61.2%) patients and hematochezia (10.1%) among 83. Among the positive results of VCE, small bowel ulcers accounted for the highest percentage (57.7%), of which 164 cases were diagnosed as inflammatory bowel disease. For obscure gastrointestinal bleeding, 12 cases were diagnosed as Meckel's diverticulum. In terms of the small bowel transit time of VCE, compared with the negative group [288 (216.5, 390.3) min] and the enteritis group [277 (192.5, 374.8) min], a longer transit time was needed in the small bowel ulcer group [332.5 (240, 451.5) min, P < .01]. There were no correlations of positive VCE findings with anemia, the white blood cell count, the C-reactive protein level or the small bowel transit time according to Spearman rank analysis.VCE is relatively well tolerated and safe in children and has great value for the diagnosis and treatment of abdominal pain, especially inflammatory bowel disease and obscure gastrointestinal bleeding.

[Value of capsule endoscopy in children with small intestinal diseases with hematochezia as the chief complaint].

OBJECTIVE: To evaluate the value of capsule endoscopy in children with small intestinal diseases with hematochezia as the chief complaint. METHODS: A retrospective analysis was performed on the clinical data and capsule endoscopy findings of 93 children with hematochezia who were admitted to Children's Hospital of Fudan University from May 2015 to January 2019 and underwent capsule endoscopy. According to the capsule endoscopy findings of the jejunum and the ileum, they were divided into a positive lesion group with 39 patients and a negative lesion group with 54 patients. Related clinical data and the features of lesion on capsule endoscopy were analyzed for the two groups. RESULTS: There were no significant differences in age, sex, duration of capsule endoscopy, gastric transit time, and small intestinal transit time between the positive lesion and negative lesion groups (P>0.05). The positive lesion group had a significantly lower level of hemoglobin than the negative lesion group (P<0.05). Hemoglobin level was negatively correlated with the rate of positive lesions on capsule endoscopy (r=-0.342, P=0.001). Among the 39 patients with positive lesions on capsule endoscopy, the detection of Meckel's diverticulum was the highest (41%), followed by inflammatory bowel disease (21%). CONCLUSIONS: Capsule endoscopy has a certain value in detecting small intestinal diseases, especially diseases in the jejunum and the ileum, in children with lower gastrointestinal hemorrhage.

Intestinal dysbiosis in pediatric Crohn's disease patients with IL10RA mutations.

BACKGROUND: Several studies have employed animal models to explore the association between microbiota and interleukin (IL) 10 signaling; however, limited information is available about the human microbiome. AIM: To characterize the microbiome in patients with IL10RA mutations and to explore the association between gut dysbiosis and disease severity. METHODS: Fecal samples were collected from patients who were diagnosed with loss-of-function mutations in the IL10RA gene between January 2017 and July 2018 at the Children's Hospital of Fudan University. Age-matched volunteer children were recruited as healthy controls. Patients with Crohn's disease (CD) were used as disease controls to standardize the antibiotic exposure. Microbial DNA was extracted from the fecal samples. All analyses were based on the 16S rRNA gene sequencing data. RESULTS: Seventeen patients with IL10RA mutations (IL10RA group), 17 patients with pediatric CD, and 26 healthy children were included. Both patients with IL10RA mutations and those with CD exhibited a reduced diversity of gut microbiome with increased variability. The relative abundance of Firmicutes was substantially increased in the IL10RA group (P = 0.02). On further comparison of the relative abundance of taxa between patients with IL10RA mutations and healthy children, 13 taxa showed significant differences. The IL10RA-specific dysbiosis indices exhibited a significant positive correlation with weighted pediatric CD activity index and simple endoscopic score for CD. CONCLUSION: In patients with IL10RA mutations and early onset inflammatory bowel disease, gut dysbiosis shows a moderate association with disease severity.

Long-term outcomes of thalidomide in pediatric Crohn's disease.

BACKGROUND AND AIM: In this largest pediatric cohort to date in Asian population, we aimed to report our long-term real-life experience with thalidomide treatment in pediatric Crohn's disease (CD). METHODS: A retrospective single-center analysis of pediatric CD patients treated by thalidomide was conducted. The clinical characteristics and outcomes were extracted. Primary outcomes were clinical response and remission rate at different time points, especially comparing the difference between monogenic and non-monogenic mutation patients. We also evaluated the long-term safety of thalidomide. RESULTS: A total of 62 patients met the inclusion criteria. The median follow-up period was 30.5 months. Among all, 19 patients (30.6%) were diagnosed with monogenic mutation during treatment. Clinical remission rate was 53.2% (33/62) at 6 months, 54.8% (34/62) at 12 months, and 33.9% (21/62) at the end of follow-up. Clinical remission rates between non-monogenic and monogenic groups at the end were statistically different (44.2% [19/43] vs 10.5% [2/19], P < 0.05). At 12 months, 66.7% (30/45) were with normalized C-reactive protein level. Most patients (95.4%, 21/22) discontinued steroids with a median time of 4.4 months. Twelve patients relapsed, but no risk factor was identified to be significantly associated with relapse. A total of 45.2% (28/62) patients experienced an adverse event, in which 22 patients stopped thalidomide due to safety concern. Cumulative dose was not associated with abnormal electromyography but with the occurrence of adverse events. CONCLUSIONS: Thalidomide was clinically efficacious and safe among pediatric CD. Our results suggest that it is an alternative therapy in monogenic mutation patients.

Phenotypic Characterization of Very Early-Onset Inflammatory Bowel Disease with Interleukin-10 Signaling Deficiency: Based on a Large Cohort Study.

BACKGROUND: Interleukin-10 (IL10)/interleukin-10 receptor (IL10R) deficiency is a rare disease with life-threatening infantile-onset colitis. We sought to accurately phenotype this disorder based on a large cohort of patients with a proven defect of IL10 signaling and to clarify the effects of allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: We analyzed the phenotypes of our 61 patients and reviewed 78 other previously reported cases with identified mutations in the genes encoding IL10 or IL10R. We also compared the clinical features of patients with interleukin-10 receptor B (IL10RB), interleukin-10 receptor A (IL10RA), and IL10 mutations. The therapeutic effects of allogeneic HSCT were evaluated. RESULTS: We found that the disease onset time was extremely early: 70.3% within 30 days postnatal and 94.9% within the first 6 months of life. In addition, 94.2% of patients typically presented with perianal lesions. Oral ulcers and skin rash were common extra-intestinal manifestations (33.8% and 51.8%, respectively). There was no statistically significant difference in disease onset time, perianal lesion involvement, or mortality rate among patients with IL10RB, IL10RA, or IL10 deficiency. However, the surgery rate was higher in patients with IL10RB mutations than in those with IL10 or IL10RA mutations (P < 0.05). Compared with those with IL10RA deficiency, a higher percentage (32%, 9 of 28) of patients with IL10RB mutations developed B-cell lymphoma (P < 0.01). Compared with other regions, a higher percentage (98.7%) of IL10RA mutations was detected among patients in East Asia countries (P < 0.01), with hot-spot mutation sites of c.C301T and c.G537A. Allogeneic HSCT is efficacious but has a high mortality rate (17.5%, 7 of 40). CONCLUSIONS: Our study expands the current knowledge on the genotype-correlated phenotypes with a defect of IL10 signaling. B-cell lymphoma was more frequent than would be expected in patients with IL10RB mutations. There may be a unique genetic architecture among Eastern Asia compared with other populations. Although allogeneic HSCT represents a causal therapeutic approach for IL10-and IL10R-deficient patients, a word of caution is warranted.

MicroRNA-15a - cell division cycle 42 signaling pathway in pathogenesis of pediatric inflammatory bowel disease.

AIM: To determine whether cell division cycle (Cdc)42 is regulated by microRNA (miR)-15a in the development of pediatric inflammatory bowel disease (IBD). METHODS: We cultured 293T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor (TNF)-α. We then employed lentiviruses to alter the expression of miR-15a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15a and Cdc42 in pediatric IBD patients. RESULTS: Target Scan and dual-luciferase assay revealed that Cdc42 was a miR-15a target gene. MiR-15a expression increased (P = 0.0038) and Cdc42 expression decreased (P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens (ZO)-1 (P < 0.05) and E-cadherin (P < 0.001) decreased. Cdc42 levels decreased in miR-15a-mimic cells (P < 0.001) and increased in miR-15a inhibitor cells (P < 0.05). ZO-1 and E-cadherin decreased in miR-15a-mimic cells (P < 0.001) but not in the miR-15a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α (P < 0.05) or miR-15a-mimic cells (P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease (CD) active (AC) group, seven in the CD remission (RE) group, and seven in the ulcerative colitis (UC) group. MiR-15a increased and Cdc42 decreased in the CD AC group compared to the control group (P < 0.05). miR-15a decreased and Cdc42 increased in the CD RE group compared to the CD AC group (P < 0.05). miR-15a was positively correlated with the Pediatric Crohn's disease Activity Index (PCDAI) (P = 0.006), while Cdc42 was negatively correlated with PCDAI (P = 0.0008). Finally, miR-15a expression negatively correlated with Cdc42 in pediatric IBD patients (P = 0.0045). CONCLUSION: MiR-15a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.

Children with cyclic vomiting syndrome: phenotypes, disease burden and mitochondrial DNA analysis.

BACKGROUND: Cyclic vomiting syndrome (CVS) is characterized by repeated, stereotypical vomiting episodes. It is possibly associated with mitochondrial DNA (mtDNA) variants. We examined the phenotype, disease burden, treatment and performed mtDNA analysis in pediatric CVS. METHODS: This retrospective study included 42 children with CVS in a tertiary care center. Information regarding medical history, clinical features, laboratory tests, and treatment were collected. mtDNA sequencing was performed among 13 patients. RESULTS: Mean age of onset among patients was 4.0±3.4 years, and mean age at diagnosis was 6.7±4.2 years. CVS episodes in onset and features were stereotypic. Recognizable prodromes were reported in 54.8% patients. Neuroimaging showed previously unknown intracranial abnormalities. Gastrointestinal infection was found in four patients. Mean duration of hospitalization was 7.0±2.4 days, and mean hospitalization cost was 10,891 RMB. Sequencing showed that 4/13 patients had C16519T mtDNA polymorphism, and 2/13 patients had G3010A mtDNA polymorphism. CONCLUSIONS: Cyclic vomiting syndrome is a disabling disorder, which causes huge disease burdens to the patients and their families. Early clinical suspicion and prompt diagnosis are crucial. mtDNA polymorphisms were found in some patients, but they were not significantly associated with pediatric CVS.

Infantile Onset Intractable Inflammatory Bowel Disease Due to Novel Heterozygous Mutations in TNFAIP3 (A20).

Background: Mutations in tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key player in the negative feedback regulation of nuclear factor-κB signaling, have recently been recognized as leading to early onset autoinflammatory and autoimmune syndrome. Here, we have reported the phenotypes of 3 infantile onset intractable inflammatory bowel disease (IBD) patients with TNFAIP3 mutations and reviewed previously reported cases to establish phenotypic features associated with TNFAIP3 monogenicity. Methods: From January 1, 2015, to December 31, 2017, we recruited 58 infantile-onset IBD patients. Targeted sequencing and whole-exome sequencing were performed. Sanger sequencing confirmed the variants and determined the parental origin. We followed all the patients with TNFAIP3 mutations in our cohort and analyzed their clinical data. Results: Genetic screening in all 58 patients with infantile-onset IBD revealed 44 (75.9%) cases of monogenic disorders, and 3 de novo TNFAIP3 mutations were identified, including 1 nonsense and 2 frame shift mutations. All the mutations resulted in premature stop codon. All 3 patients had multiple systemic involvements, with predominant gastrointestinal diseases. Conclusions: Most infantile-onset IBD was associated with monogenetic mutation, and in addition to the 50 reported genes, other rare genetic variants need to be determined. TNFAIP3 may be an important candidate gene. The treatment of TNFAIP3-associated infantile-onset-IBD was challenging. 10.1093/ibd/izy165_video1izy165.video15789607089001.