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Thomas Jefferson University Science Outreach Program brings the scientific method into the classroo

D-glucuronyl C5-epimerase acts in dorso-ventral axis formation in zebrafish

BACKGROUND: Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated. RESULTS: Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity. CONCLUSION: Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development

p53 Activation by Knockdown Technologies

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies

Descending thoracic aorta to iliofemoral artery bypass grafting: A role for primary revascularization for aortoiliac occlusive disease?

AbstractPurpose: Bypass grafts that originate from the descending thoracic aorta to the iliac or femoral arteries are well described but are not commonly used as primary procedures, and the long-term results remain unknown. A 15-year experience with 50 descending thoracic aorta to iliofemoral artery bypass grafts for aortoiliac occlusive disease is the basis of this report. Methods: From January 1983 to December 1997, patients who underwent bypass grafting procedures from the descending thoracic aorta to the iliac or femoral arteries were identified. Surgical indications, morbidity and mortality rates, primary and secondary patency rates, limb salvage rates, and survival rates were determined. Results: Fifty descending thoracic aorta to iliofemoral artery bypass grafting procedures were performed—24 (48%) for severe claudication, 22 (44%) for rest pain, and 4 (8%) for ischemic ulceration. A primary procedure was performed in 31 patients (62%) for complete occlusion (21 patients) and severe atherosclerotic disease (10 patients) of the infrarenal aorta. The indications for 19 secondary revascularizations (38%) were prior aortic or extra-anatomic graft failure in 17 cases and aortic graft infection in 2 cases. The follow-up periods ranged from 1 to 150 months (mean, 39 months). The cumulative life-table 5-year primary patency, secondary patency, limb salvage, and survival rates were 79%, 84%, 93%, and 67%, respectively. An improved patency trend was observed for patients who underwent operation for severe claudication as compared with limb-threatening ischemia (92% and 69%; P = .07). However, there was no difference between primary and secondary operations in primary patency rates (81% and 79%; P = NS) or survival rates (72% and 62%; P = NS). Conclusion: Descending thoracic aorta to iliofemoral artery bypass grafting has excellent overall long-term results. These results support its more liberal use for primary revascularization, especially for patients with severe atherosclerotic disease or complete occlusion of the infrarenal aorta. (J Vasc Surg 1999;29:249-58.

Descending thoracic aorta to iliofemoral artery bypass grafting: A role for primary revascularization for aortoiliac occlusive disease?

AbstractPurpose: Bypass grafts that originate from the descending thoracic aorta to the iliac or femoral arteries are well described but are not commonly used as primary procedures, and the long-term results remain unknown. A 15-year experience with 50 descending thoracic aorta to iliofemoral artery bypass grafts for aortoiliac occlusive disease is the basis of this report. Methods: From January 1983 to December 1997, patients who underwent bypass grafting procedures from the descending thoracic aorta to the iliac or femoral arteries were identified. Surgical indications, morbidity and mortality rates, primary and secondary patency rates, limb salvage rates, and survival rates were determined. Results: Fifty descending thoracic aorta to iliofemoral artery bypass grafting procedures were performed—24 (48%) for severe claudication, 22 (44%) for rest pain, and 4 (8%) for ischemic ulceration. A primary procedure was performed in 31 patients (62%) for complete occlusion (21 patients) and severe atherosclerotic disease (10 patients) of the infrarenal aorta. The indications for 19 secondary revascularizations (38%) were prior aortic or extra-anatomic graft failure in 17 cases and aortic graft infection in 2 cases. The follow-up periods ranged from 1 to 150 months (mean, 39 months). The cumulative life-table 5-year primary patency, secondary patency, limb salvage, and survival rates were 79%, 84%, 93%, and 67%, respectively. An improved patency trend was observed for patients who underwent operation for severe claudication as compared with limb-threatening ischemia (92% and 69%; P = .07). However, there was no difference between primary and secondary operations in primary patency rates (81% and 79%; P = NS) or survival rates (72% and 62%; P = NS). Conclusion: Descending thoracic aorta to iliofemoral artery bypass grafting has excellent overall long-term results. These results support its more liberal use for primary revascularization, especially for patients with severe atherosclerotic disease or complete occlusion of the infrarenal aorta. (J Vasc Surg 1999;29:249-58.

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation

Prospective screening for postoperative deep venous thrombosis in patients undergoing infrainguinal revascularization

AbstractPurpose: The incidence of deep venous thrombosis (DVT) in patients undergoing infrainguinal bypass graft procedures has not been well documented, and the need for routine prophylaxis remains controversial. The purpose of this study was to prospectively evaluate the risk of postoperative DVT complicating infrainguinal revascularization. Methods: Seventy-four patients undergoing infrainguinal bypass graft procedures during a 12-month period were prospectively screened for DVT. Bilateral lower extremity venous duplex scan imaging was performed preoperatively and within 1 week and 6 weeks, postoperatively. Routine DVT prophylaxis was not used, with anticoagulation reserved for specific indications. Results: Of the 74 patients screened, three patients (4.1%) had DVT identified on preoperative venous duplex scan imaging and were excluded from the study. Of the remaining 71 patients enrolled, only two patients (2.8%) had postoperative DVT. Postoperative DVT was ipsilateral to the bypass graft extremity in both patients, with involvement of the peroneal vein in one patient and the femoral vein in the other. Although routine prophylaxis was not used, 18 of these patients (25%) were anticoagulated for other indications, with DVT occurring in one patient (5.6%). Of the remaining 53 patients who did not receive postoperative anticoagulation, only one patient (1.8%) had DVT. Conclusions: According to this prospective study, the risk of postoperative DVT in patients undergoing infrainguinal revascularization is low. Routine prophylaxis is not recommended, with postoperative anticoagulation reserved for specific indications. (J Vasc Surg 2000;32:669-75.

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Funder: G. Harold and Leila Y. Mathers Charitable Foundation; funder-id: http://dx.doi.org/10.13039/100001229Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology

Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made