Analysis of the expression pattern of the BCL11B gene and its relatives in patients with T-cell acute lymphoblastic leukemia

<p>Abstract</p> <p>Background</p> <p>In a human T-cell acute lymphoblastic leukemia (T-ALL) cell line (Molt-4), siRNA-mediated suppression of <it>BCL11B </it>expression was shown to inhibit proliferation and induce apoptosis, functions which may be related to genes involved in apoptosis (such as <it>TNFSF10 </it>and <it>BCL2L1</it>) and TGF-β pathways (such as <it>SPP1</it>and <it>CREBBP</it>).</p> <p>Methods</p> <p>The expression levels of the above mentioned genes and their correlation with the <it>BCL11B </it>gene were analyzed in patients with T-ALL using the TaqMan and SYBR Green I real-time polymerase chain reaction technique.</p> <p>Results</p> <p>Expression levels of <it>BCL11B, BCL2L1</it>, and <it>CREBBP </it>mRNA in T-ALL patients were significantly higher than those from healthy controls (<it>P <</it>0.05). In T-ALL patients, the <it>BCL11B </it>expression level was negatively correlated with the <it>BCL2L1 </it>expression level (<it>r</it>_s= -0.700; <it>P </it><it><</it>0.05), and positively correlated with the <it>SPP1 </it>expression level (<it>r</it>_s= 0.683; <it>P </it><it><</it>0.05). In healthy controls, the <it>BCL11B </it>expression level did not correlate with the <it>TNFSF10</it>, <it>BCL2L1</it>, <it>SPP1</it>, or <it>CREBBP </it>expression levels.</p> <p>Conclusions</p> <p>Over-expression of <it>BCL11B </it>might play a role in anti-apoptosis in T-ALL cells through up-regulation of its downstream genes <it>BCL2L1 </it>and <it>CREBBP</it>.</p

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

BACKGROUND:Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes. METHODOLOGY/PRINCIPAL FINDINGS:In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis. CONCLUSIONS/SIGNIFICANCE:The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs

Extract of Sheng-Mai-San Ameliorates Myocardial Ischemia-Induced Heart Failure by Modulating Ca2+-Calcineurin-Mediated Drp1 Signaling Pathways

Sheng-Mai-San (SMS) is a well-known traditional Chinese medicine (TCM) complex prescription used to treat heart failure (HF) and angina in clinic. However, its potential therapeutic mechanisms remain unclear. The present study evaluated the cardioprotection of extract of SMS (ESMS) on myocardial ischemia (MI)-induced HF, and explored the underlying molecular mechanisms. The results demonstrated that ESMS (728.0 mg/kg) significantly attenuated MI injury-induced HF by improving cardiac function and pathological changes, decreasing lactate dehydrogenase (LDH), creatine kinase (CK) activities, and brain natriuretic peptide (BNP) levels; increasing ATPase activity; and reducing intracellular Ca2+ levels in MI-induced HF mice model. It also significantly decreased the apoptotic index. In vitro, ESMS (400 μg/mL) inhibited mitochondrial-dependent myocardial apoptosis by modulating the expression of caspase-3 and the Bcl-2/Bax ratio, and improved mitochondrial function through increasing mitochondrial membrane potential and cellular ATP content. ESMS restored intracellular Ca2+ and downregulated the expression of Calcineurin A (CnA), thus inhibiting phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 and increasing phosphorylation of Drp1 at Ser637 to prevent cardiomyocyte mitochondrial fission. Above-mentioned results demonstrated ESMS suppressed mitochondrial-mediated apoptosis in oxygen glucose deprivation (OGD) injured H9c2 cardiomyocytes. These findings suggested that ESMS attenuated MI-induced HF by regulating Ca2+ homeostasis and suppressing mitochondrial mediated apoptosis through the modulation of Ca2+-calcineurin-mediated Drp1 signaling pathways. Our results provide insight into the mechanism and clinical applications of SMS and suggest a potential therapeutic strategy for HF

Identification of Reliable Reference Genes for the Expression of <i>Hydrangea macrophylla</i> ‘Bailmer’ and ‘Duro’ Sepal Color

Hydrangea spp. is renowned for its variety of color changes in its developmental stage and before and after aluminum treatment. We analyzed gene expression in hydrangeas sepals to study the causes of color change. The accuracy of quantitative RT-qPCR analysis depends on the reliability of reference genes. We selected reference genes for hydrangea of varying cultivars, at different developmental stages, and in aluminum treatment groups. We chose ‘Bailmer’ and ‘Duro’ as subject species. We selected eight candidate genes, all of which were ranked by geNorm, NormFinder, BestKeeper, and RefFinder. CCR, NHX1, and LODX were used to verify the exactitude of reference genes. According to the ranking result of RefFinder, the top-ranked reference genes in each group were different; the top four candidate reference genes in each group mostly included EF1-β, RPL34, GADPH, and RPL10. EF1-β and RPL34 ranked top in the ‘all materials’ group, and their expression trends, obtained from the analysis of CCR, NHX1, and LODX, were consistent. From the results, we gather that EF1-β and RPL34 can be used as reference genes to quantify target gene expression. In this study, we screened for reference genes in hydrangeas to provide a technical basis for hydrangea sepal formation and transformation for further experiments

Identification of Seven Key Structural Genes in the Anthocyanin Biosynthesis Pathway in Sepals of <em>Hydrangea macrophylla</em>

Under specific cultivation conditions, the sepal color of Hydrangea macrophylla (H. macrophylla) changes from red to blue due to the complexation of aluminum ions (Al3+), delphinidin 3-glucoside, and copigments. However, this phenomenon cannot occur in all cultivars despite the presence of sufficient Al3+ and copigments. To explore the mechanism of sepal bluing in H. macrophylla, there is an urgent need to study the molecular regulation of the anthocyanin biosynthesis pathway. However, the key structural genes, other than CHS, regulating anthocyanin biosynthesis in the sepals of H. macrophylla have not been identified. In this study, based on full-length transcriptome data from H.macrophylla ‘Bailmer’, the key structural genes regulating anthocyanin biosynthesis in the sepals of H. macrophylla were isolated and investigated. Ultimately, seven key structural genes, HmCHS1, HmCHI, HmF3H1, HmF3′H1, HmF3′5′H, HmDFR2, and HmANS3, were demonstrated to show high expression levels in colored sepals. The expression levels of these seven genes increased gradually with the development of sepals and were highest in the full-bloom stage. The trend of gene expression was consistent with the trend of anthocyanin contents. It was concluded that the seven selected genes were involved in anthocyanin biosynthesis in the sepals of H. macrophylla. The full-length sequence data have been deposited into the NCBI Sequence Read Archive (SRA) with accession number PRJNA849710. This study lays a good foundation for the further elucidation of the molecular mechanism of sepal coloration in H. macrophylla

Characteristics of New Onset Herpes Simplex Keratitis after Keratoplasty

Purpose. To observe clinical characteristics and treatment outcomes of new onset herpes simplex keratitis (HSK) after keratoplasty. Methods. Among 1,443 patients (1,443 eyes) who underwent keratoplasty (excluding cases of primary HSK) in Shandong Eye Hospital, 17 patients suffered postoperative HSK. The clinical manifestations, treatment regimens, and prognoses of the patients were evaluated. Results. The incidence of new onset HSK after keratoplasty was 1.18%. Epithelial HSK occurred in 10 eyes, with dendritic epithelial infiltration in 6 eyes and map-like epithelial defects in 4 eyes. Nine eyes had lesions at the junction of the graft and recipient. Stromal necrotic and endothelial HSK occurred in 7 eyes, presenting map-shaped ulcers in the entire corneal graft and recipient (two eyes) or at the graft-recipient junction (five eyes). Confocal microscopy revealed infiltration of a large number of dendritic cells at the junction of the lesion and transparent cornea. All 10 eyes with epithelial lesions and two eyes suffering stromal lesions of ≤1/3 corneal thickness healed after systematic and local antiviral treatment. Best-corrected visual acuity and corneal graft transparency were restored. For stromal HSK with an ulcer of >1/3 corneal thickness, amniotic membrane transplantation was performed, and visual acuity and graft transparency decreased significantly. Conclusion. New onset HSK after keratoplasty primarily resulted in epithelial and stromal lesion, involving both the graft and recipient. Effective treatments included antiviral medications and amniotic membrane transplantation. Delayed treatment may lead to aggravated graft opacification

Outcomes of Wound Dehiscence after Penetrating Keratoplasty and Lamellar Keratoplasty

Objective. To investigate the incidence, causes, occurrence time, and range of wound and outcomes of wound dehiscence in patients treated by penetrating keratoplasty (PK) or lamellar keratoplasty (LK). Methods. We retrospectively reviewed medical records of keratoplasty in Shandong Eye Hospital from January 2006 to June 2017. Thirty-one eyes of 30 patients had sustained wound dehiscence (WD) after surgical treatment. The surgical type, causes, occurrence time, extent of the wound, treatment, and outcomes were recorded. Results. The study population consisted of 26 men and 4 women. The mean age at the occurrence of WD was 44.6 years old (range: 12–78 years), and the mean time from keratoplasty to WD was 45.9 months (range: 1–204 months). WD occurred in 23 eyes (23/1385, 1.66%) after PK and 8 eyes (8/1632, 0.49%) after LK (p<0.05). Twenty-seven eyes (27/31, 87.0%) had trauma-induced dehiscence. The mean range of dehiscence was 5.5 o’clock. The vision ranged from 20/50 to light perception after wound suture. The eyes receiving LK had fewer serious complications than PK. Conclusions. Compared with LK, PK seems to be more prone to result in wound dehiscence. The WD after LK may be less severe. The visual acuity after treatment of WD can be worse in the eyes with PK than LK