Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro , P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB) staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.     

脐带间充质干细胞与系统性红斑狼疮

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of immune cells and production of autoantibodies by plasma cells and release of cytokines. Mesenchymal stem cells (MSC) are widely studied as an alternative cell source for their ability to differentiate into multiple mesenchymal lineages. An important function for MSC for autoimmune diseases is their immunomodulatory effect on various activated lymphoid cells, such as T cells B cells, NK cells, and dendritic cells; and on cytokines , such as IL-10, TGF-β, TNF-α, et al. Umbilical cord MSCs (UC-MSCs) had a higher proliferation capacity and lower immunogenicity, indicating that it may be a novel alternative source of human MSC for clinical application. In this review, we introduce the immunomodulatory effect and clinical application of UC-MSC in SLE based on recent findings in human and animal models.     

Effect of Alternative Splicing of VLDL Receptor on its Ligand Binding and Internalization Capability

1 Introduction Very low density lipoprotein receptor (VLDL-R) is a main receptor mediating the uptake of triglyceride-rich lipoprotein(TRL), so it is in all probability to play an important role in the development of atherosclerosis(AS). On account of alternative splicing of O-linked carbohydrate chains in extracellular fragment, VLDL-R can be classified into two isoforms: VLDL-R Ⅰ with O-linker sugar region, while VLDL-R Ⅱ without this domain[1].But so far, the difference of their function and biological significance between two isoforms, especially those of VLDL-R Ⅱ has not been clarified. In our research, ldlA7 cell strains stably expressing two isoforms of VLDL-R were obtained through gene clone technology. Binding and internalization of the natural ligands (VLDL and β-VLDL)of two types VLDL-R and their roles in the formation of foam cells were compared to clarify the difference between two isoforms of VLDL-R, and elucidate their roles in the metabolism of lipoprotein and development of AS.     

An Alternative Derivation for Bronnikov’s Reconstruction Algorithm in X-ray Phase Contrast Tomography

In this article, we present an alternative derivation for Bronnikov’s reconstruction algorithm in X-ray phase contrast tomography with holographic measurements. A two-step method was used in the alternative derivation. The phase shift induced by the object was obtained by Fourier transform and the real part of the complex refractive index of the object was retrieved by applying the conventional filtered backprojection method. The alternative derivation provides an easier way to understand the reconstruction formula.     

IRE 1α is essential for Xenopus pancreas development

Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon the endoplasmic reticu- lum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventionally splicing XBP1 mRNA, which activates the unfolded protein response （UPR） to restore ER homeostasis. In mice, IREla inactivity leads to embryonic death and IREla plays an essential role in extraembryonic tissues and the placenta. However, its precise action in the embryo proper is still unknown. In this study, the loss of function ana/ysis was performed to investigate the function of Xenopus IREla （xlREla） during pancreas development. Firstly, the complete open reading frame of xIRE1α was amplified and the expression pattern was detected. The effects of Xenopus IRE1α and XBP1 during embryo development were detected with whole-mount in situ hybridization. The results demonstrated that xIRE1α was much closer to human IREla when compared with their sequence alignment, xlREla was expressed strongly in developing pancreas and the knockdown of xIREla inhibited the differentiation and specification of the pancreas, xlREltt, which was required for cytoplasmic splicing of XBP1 pre-mRNA and XB- P1MO, also showed inhibitory effects on pancreas development. These results suggest that xlREla is essential for pancreas development during embryogenesis and functions via the XBP1 dependent pathway.     

肝移植及腹部多器官移植手术的成分输血

BACKGROUND: The liver transplantation and abdominal multiple organ transplantation are complicated surgeries, characterized by massive blood loss and high blood transfusion requirements. OBJECTIVE: To explore the characteristics of blood loss and blood transfusion in liver transplantation and abdominal multiple organ transplantation and post-operative survival rate. METHODS: Clinical data from 192 patients were retrospectively analyzed, including blood transfusion data with the first 24 hours after surgery and post-operative survival rate. RESULTS AND CONCLUSION: These 192 patients included 177 patients receiving liver transplantation,    2 patients receiving liver and kidney transplantation and 13 patients receiving abdominal multiple organ transplantation. The average intra-operative blood loss of each patient was (2 401.5±3 239.5) mL. The average infusion of red blood cells, platelet, cryoprecipitate and frozen plasma of each patient at the first 24 hours after surgery was (11.3±11.9), (0.8±0.9), (10.7±11.7) U and (2 805.5±1 393.1) mL, respectively. All kinds of blood infusion in the liver cancer group were obviously less than those in the hepatic failure group. The infusion of cryoprecipitate and frozen plasma in the cirrhosis group was obviously less than that in the hepatic failure group, but the infusion of platelet in the cirrhosis group was significantly more than that in the liver cancer group. The infusion of red blood cells from July 2013 to June 2015 was significantly less than that from July 2012 to June 2013. The blood loss, infusion of red blood cells and frozen plasma in the liver transplantation group of cirrhosis were significantly more than those in the abdominal multiple organ transplantation group of cirrhosis (all P < 0.05). In conclusion, diagnosis of liver diseases, and the maturity of surgery exert an effect on the blood loss and blood infusion. As the development of liver transplantation and abdominal multiple organ transplantation, both the blood loss and blood infusion are decreased. Besides, compared with liver transplantation, the blood loss and blood infusion show no increase in the abdominal multiple organ transplantation. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Effect of Alternative Splicing of VLDL Receptor on its Ligand Binding and Internalization Capability

1 IntroductionVery low density lipoprotein receptor (VLDL-R) is a main receptor mediating the uptake of triglyceride-rich lipoprotein(TRL), so it is in all probability to play an important role in the development of atherosclerosis (AS). On account of alternative splicing of O-linked carbohydrate chains in extracellular fragment, VLDL-R can be classified into two isoforms: VLDL-RⅠ with O-linker sugar region, while VLDL-RⅡ without this domain~([1]). But so far, the difference of th…     

Interleukin-7 Receptor Signaling Network： An Integrated Systems Perspective

Interleukin-7 （IL-7） is an essential cytokine for the development and homeostatic maintenance of T and B lymphocytes. Binding of IL-7 to its cognate receptor, the IL-7 receptor （IL-7R）, activates multiple pathways that regulate lymphocyte survival, glucose uptake, proliferation and differentiation. There has been much interest in understanding how IL-7 receptor signaling is modulated at multiple interconnected network levels. This review examines how the strength of the signal through the IL-7 receptor is modulated in T and B cells, including the use of shared receptor components, signaling crosstalk, shared interaction domains, feedback loops, integrated gene regulation, multimerization and ligand competition. We discuss how these network control mechanisms could integrate to govern the properties of IL-7R signaling in lymphocytes in health and disease. Analysis of IL-7 receptor signaling at a network level in a systematic manner will allow for a comprehensive approach to understanding the impact of multiple signaling pathways on lymphocyte biology. Cellular ＆ Molecular Immunology.     

Treatment Registration and Nuclide Decay Calculation System

Objective ：To design a software to do the complicated and multiple calculations automatically in routine internal radionuelide irradiation therapy to avoid mistakes and shorten patients waiting times. Methods：The software is designed on the Microsoft Windows XP operating system. Visual Basic 5.0 and Microsoft Access 2000 are used respectively as the programming language and database system here. The data and DBGrid controls and VB data window guide of Visual Basic were used to control access to and Access database. Results ： Not only can the radioactivity of any radionuelide be calculated, but also the administered total iodine dose of therapy for hyperthyroidism or thyroid cancer and the total administered 153Sm-EDTMP solutions for remedy of bone metastasis of malignant tumor can be ciphered out. Conclusion ：The work becomes easier, faster, more correct and interesting when the software can make the complicated and multiple calculations automatically. Patients＇ information, diagnosis and treatment can be recorded for further study.     

Familial cardiac myxoma with multifocal recurrences: a case report and review of the literature

We report a case of myxoma with multiple recurrences in both the atrium and ventricle in a 26-year-old woman five years after the surgical removal of left atrial myxoma.Her 52-year-old mother had a similar medical history.To our knowledge,this was the first familial case who suffered multifocal cardiac myxoma recurrences without any sign of the myxoma complex.Based on our understanding of the mechanism of recurrence,the approaches to prevent the recurrence,and markers to predict recurrence,we propose that multifocal recurrences,as reported herein,may result from a combination of familial predisposition and multifocal onset.The bi-atrial surgical approach and transesophageal echocardiography are preferred for patients with recurrent cardiac myxomas,especially for those with multiple recurrences and familial myxoma.Immunological and genetic screenings may help to identify family members at risk for developing this disease.     

The Development of Animal Models of Inflammatory Bowel Disease

Alternative splicing is a fine-tuned process known for generating multiple functional variants from individual genes leading to protein diversity. The immune system utilizes pre-mRNA splicing to expand its gene function. Numerous immunologically relevant genes have been found to undergo alternative splicing, thus revealing a new source of complexity in the immune gene network. This review attempts to summarize the general features of alternative splicing and its role in the immune system along with a special focus on the available reports of alternative splicing in cytokines, mainly interleukins and their receptors and their regulatory significance.     

Alternative splicing of SV40 early pre-mRNA is determined by branch site selection

Splicing of SV40 early pre-mRNA to alternative large-T and small-t mRNAs involves the utilization of multiple lariat branch sites. To determine the functional significance of these sites, we constructed and analyzed a set of base substitution mutants in which the major branch acceptors were altered, either singly or in combination. The ratio of large-T to small-t mRNAs produced in vivo was found to vary by over 100-fold between different mutants. When splicing was assayed in vitro with a standard pre-RNA, which results in splicing almost exclusively to large-T mRNA, the patterns of branch site utilization were altered dramatically, although the mutations were essentially without effect on splicing efficiency. However, use of a 5' truncated pre-RNA, which results in a splicing pattern that reflects the in vivo alternative splicing potential of this pre-RNA, revealed a strong correlation between the effects of the base substitutions on branch site selection and alternative splice-site utilization. An RNase protection analysis of factor interactions with the 5' splice sites and branch sites in wild-type and mutant pre-RNAs suggests that a competition for different branch sites plays a crucial role in the assembly of 'alternative' spliceosomes, thereby controlling alternative splice-site selection.     

Splice variants of the receptor for advanced glycosylation end products (RAGE) in human brain

Previous studies indicate that the receptor for advanced glycosylation end products (RAGE) plays an important role in multiple pathological processes, including Alzheimer's disease. Currently there are three established isoforms of the RAGE receptor, with each isoform generated as the result of alternative splicing. It is presently unclear which of the RAGE isoforms are normally expressed in the human brain, nor has it been determined if additional RAGE isoforms exist in the human brain. In the present study we demonstrate for the first time that each of the three established RAGE isoforms, as well as three previously unidentified RAGE splicing variants, are normally expressed in the human brain. These data suggest that RAGE may have multiple functions in the human brain, mediated by the individual or coordinated efforts of the different RAGE isoforms, with alternative splicing generating individual RAGE isoforms that specifically interact with the various ligands present in the brain.     

A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.     

Processing of fish Ig heavy chain transcripts: diverse splicing patterns and unusual nonsense mediated decay

While the diversification of the antigen-binding sites is realized by genomic VDJ rearrangements during B cell differentiation, different forms of immunoglobulin (Ig) heavy (H) chains can be produced through multiple splicing pathways. In most vertebrates, the secreted (S) and membrane (Mb) forms of IgM chain are created by alternative splicing through usage of a cryptic splice site in Cμ4 allowing the junction to the TM exon. The processing pattern for Igμ is different in teleosts, which generally use the Cμ3 donor site instead. In ancient fish lineages, multiple unusual splicing patterns were found for Ig H chain, involving donor sites that do not always follow the classical consensus. The production of IgD versus IgM H chains seems to be generally realized by alternative splicing in all vertebrates, but typical teleost IgD H chains are chimeric and contains a Cμ1 domain. Together, these observations raise questions on how different fish regulate RNA splicing and if their splicing machinery is especially complex. A preliminary scan of the zebrafish and stickleback genomes provides evidence that gene orthologs to the mammalian main splice factors are highly conserved as single copy genes, while the snRNPs U repertoire may be different and may explain other particular features of RNA processing in fish.     

Alternative RNA splicing in the nervous system

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.     

Expression of tumor-promoting Cyr61 is regulated by hTRA2-β1 and acidosis

The matricellular protein Cysteine rich 61 (Cyr61) displays a remarkable diversity of multiple cellular functions involved in significant physiologic and pathologic processes. Cyr61 is known as an important player in tumor progression, promoting neovascularization and metastasis. Our prior investigations elucidated an oxygen-dependent Cyr61 alternative splicing process characterized by retention of its intron 3, regulating its biological function in a hypoxia-driven on/off switch mechanism. In this work, we identified extracellular acidosis as a potent inducer for altered Cyr61 alternative splicing pattern regulating Cyr61 expression. Intriguingly, splicing factor hTRA2-beta1 displayed an opposite effect on Cyr61 expression. Nuclear hTRA2-beta1 protein expression was found markedly reduced under acidic conditions. In keeping with these conclusions, we show that hTRA2-beta1 can specifically bind a 'GAAG' motif in Cyr61 exon 3 RNA, that the splicing factor displays acidosis-dependent protein localization in cellular compartments, and shRNA-mediated hTRA2-beta1 knock-down triggers the same effects on Cyr61 alternative splicing like acidosis or hypoxia. Our findings strongly support the hypothesis of a specific regulation of Cyr61 expression by hTRA2-beta1.