Uncovered Defects Research Articles

Immunity-linked genes are stimulated by a membrane stress pathway linked to Golgi function and the ARF-1 GTPase.

Infection response and other immunity-linked genes (ILGs) were first named in Caenorhabditis elegans-based expression after pathogen challenge, but many are also up-regulated when lipid metabolism is perturbed. Why pathogen attack and metabolic changes both increase ILGs is unclear. We find that ILGs are activated when phosphatidylcholine (PC) levels change in membranes of secretory organelles in C. elegans. RNAi targeting of the ADP-ribosylation factor arf-1, which disrupts the Golgi and secretory function, also activates ILGs. Low PC limits ARF-1 function, suggesting a mechanism for ILG activation via lipid metabolism, as part of a membrane stress response acting outside the ER. RNAi of selected ILGs uncovered defects in the secretion of two GFP reporters and the accumulation of a pathogen-responsive complement C1r/C1s, Uegf, Bmp1 (CUB) domain fusion protein. Our data argue that up-regulation of some ILGs is a coordinated response to changes in trafficking and may act to counteract stress on secretory function.

The large GTPase AtGBPL3 links nuclear envelope formation and morphogenesis to transcriptional repression.

Guanylate binding proteins (GBPs) are prominent regulators of immunity not known to be required for nuclear envelope formation and morphogenesis. Here we identify the Arabidopsis GBP orthologue AtGBPL3 as a lamina component with essential functions in mitotic nuclear envelope reformation, nuclear morphogenesis and transcriptional repression during interphase. AtGBPL3 is preferentially expressed in mitotically active root tips, accumulates at the nuclear envelope and interacts with centromeric chromatin as well as with lamina components transcriptionally repressing pericentromeric chromatin. Reduced expression of AtGBPL3 or associated lamina components similarly altered nuclear morphology and caused overlapping transcriptional deregulation. Investigating the dynamics of AtGBPL3-GFP and other nuclear markers during mitosis (1) revealed that AtGBPL3 accumulation on the surface of daughter nuclei precedes nuclear envelope reformation and (2) uncovered defects in this process in roots of AtGBPL3 mutants, which cause programmed cell death and impair growth. AtGBPL3 functions established by these observations are unique among dynamin-family large GTPases.

Targeting the Oncogenic Functions of an RNA-Binding Protein Involved in Hematologic Malignancies

hnRNP K (heterogeneous ribonucleoprotein K) is an RNA-binding protein that binds to conserved poly-C rich tracks in RNA and influences a diverse set of molecular pathways involved in tumorigenesis. Our previous studies identified hnRNP K overexpression in patients with diffuse large B-cell lymphoma (46/75, 61%) and acute myeloid leukemia (45/160, 28%). This overexpression correlates with dismal clinical outcomes and a lack of therapeutic responses to standard treatment. To explore hnRNP K's in vivo functions, we generated Hnrnpk-transgenic mouse models. These mice develop lymphoma phenotypes through activation of the c-Myc pathway. In pre-clinical settings, bromodomain inhibitors disrupted hnRNP K-mediated c-Myc activation, demonstrating that hnRNP K overexpression mediated-pathways are amenable to therapeutic intervention.To further our studies, we used IP-mass spectrometry, RNA-sequencing, RNA immunoprecipitation, reverse phase protein analyses, and polysome profiling to identify novel pathways associated with changes in hnRNP K expression. Here, we observed that alterations in hnRNP K expression result in an impairment of ribosomal biogenesis and activation of pathways directly responsible for global translation. Using both knockdown and overexpression systems, we observed a direct correlation between hnRNP K expression and expression of S6, S6K, phosphorylated S6, eIF and mTOR pathways and uncovered defects in rRNA splicing. Collectively, these data indicate that impairment of cap-dependent loading and alterations in ribogenesis may be a driving force in the clinical manifestations of hnRNP K-driven malignancies. Furthermore, these results suggest that translational-inhibitors may be useful in exploiting hnRNP K-dependent vulnerabilities. To examine this aspect, we are currently using FDA-approved translation inhibitors and disruptors of ribogenesis (e.g. homoharringtonineand mTOR-inhibitors) and KTP- compounds, respectively.While these indirect targeting strategies are interesting, our results indicate that hnRNP K also regulates cellular programs outside of translation. Thus, potential therapies that effectively target hnRNP K overexpression will require direct inhibition of its RNA binding functions. To this end, we used several screening assays including fluorescence anisotropy (FA), surface plasmon resonance, SYPRO-orange thermal shift assays, and cell proliferation assays to screen 80,000 small molecule compounds which led to the identification of 9 candidates that disrupt hnRNP K-mRNA interactions and cause cell death in an hnRNP K-dependent manner. Further, cellular thermal shift assays revealed these lead compounds engage hnRNP K within cells and most critically, result in reduced expression of hnRNP K targets in vivo. These candidate compounds as well as potentially more potent structural analogs are currently being evaluated.Collectively, our results demonstrate that the oncogenic functions of hnRNP K are amenable to both indirect therapeutic intervention using FDA-approved agents as well as direct inhibition through newly identified small molecule compounds, signifying that there may be a roadmap to effective therapies for hnRNP K-dependent malignancies. DisclosuresNo relevant conflicts of interest to declare.

Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia

Identifying biomarkers is important for assessment of disease progression, prediction of symptom development, and determination of treatment effectiveness. While unbiased analyses of differential gene expression using next-generation sequencing methods are now routinely conducted, proteomics studies are more challenging because of traditional methods predominantly being low throughput and offering a limited dynamic range for simultaneous detection of hundreds of proteins that drastically differ in their intracellular abundance. We utilized a sensitive and high-throughput proteomic technique, reverse phase protein array (RPPA), to attain protein expression profiles of primary fibroblasts obtained from patients with Friedreich's ataxia (FRDA) and unaffected controls (CTRLs). The RPPA was designed to detect 217 proteins or phosphorylated proteins by individual antibody, and the specificity of each antibody was validated prior to the experiment. Among 62 fibroblast samples (44 FRDA and 18 CTRLs) analyzed, 30 proteins/phosphoproteins were significantly changed in FRDA fibroblasts compared with CTRL cells (p < 0.05), mostly representing signaling molecules and metabolic enzymes. As expected, frataxin was significantly downregulated in FRDA samples, thus serving as an internal CTRL for assay integrity. Extensive bioinformatics analyses were conducted to correlate differentially expressed proteins with critical disease parameters (e.g., selected symptoms, age of onset, guanine–adenine–adenine sizes, frataxin levels, and Functional Assessment Rating Scale scores). Members of the integrin family of proteins specifically associated with hearing loss in FRDA. Also, RPPA data, combined with results of transcriptome profiling, uncovered defects in the retinoic acid metabolism pathway in FRDA samples. Moreover, expression of aldehyde dehydrogenase family 1 member A3 differed significantly between cardiomyopathy-positive and cardiomyopathy-negative FRDA cohorts, demonstrating that metabolites such as retinol, retinal, or retinoic acid could become potential predictive biomarkers of cardiac presentation in FRDA.

TUSC2(FUS1)-erlotinib Induced Vulnerabilities in Epidermal Growth Factor Receptor(EGFR) Wildtype Non-small Cell Lung Cancer(NSCLC) Targeted by the Repurposed Drug Auranofin.

Expression of the TUSC2/FUS1 tumor suppressor gene in TUSC2 deficient EGFR wildtype lung cancer cells increased sensitivity to erlotinib. Microarray mRNA expression analysis of TUSC2 inducible lung cancer cells treated with erlotinib uncovered defects in the response to oxidative stress suggesting that increasing reactive oxygen species (ROS) would enhance therapeutic efficacy. Addition of the thioredoxin reductase 1 inhibitor (TXNRD1) auranofin (AF) to NSCLC cells treated with combination of TUSC2 forced expression with erlotinib increased tumor cell apoptosis and inhibited colony formation. TXNRD1 overexpression rescued tumors from AF-TUSC2-erlotinib induced apoptosis. Neutralizing ROS with nordihydroguaiaretic acid (NDGA) abrogated cell death induced by AF-TUSC2-erlotinib, indicating a regulatory role for ROS in the efficacy of the three drug combination. Isobologram-based statistical analysis of this combination demonstrated superior synergism, compared with each individual treatment at lower concentrations. In NSCLC tumor xenografts, tumor growth was markedly inhibited and animal survival was prolonged over controls by AF-TUSC2-erlotinib. Microarray mRNA expression analysis uncovered oxidative stress and DNA damage gene signatures significantly upregulated by AF-TUSC2-erlotinib compared to TUSC2-erlotinib. Pathway analysis showed the highest positive z-score for the NRF2-mediated oxidative stress response. Taken together these findings show that the combination of TUSC2-erlotinib induces additional novel vulnerabilities that can be targeted with AF.

The Importance of Foodservice in Higher Education: A Business Anthropological Case Study in China

This research employed business anthropological methods (participant observation and in-depth interviews) in addition to a quantitative survey in order to investigate the quality of food service operations at a Chinese university. Food service quality’s impacts on student satisfaction, student learning, and student health were explored. In addition, the research uncovered defects in food service operations, and suggestions were made for improvement. Finally, this paper recommended that food service quality should be included in the Chinese government’s performance evaluation of universities, which would greatly encourage improvements of university food service.

Integrated Test Frame Work for defect free Embedded Product delivery

Now a days, the industries are looking for zero defect embedded products.Of late the companies are recalling the products due to the non functioning and performance of the products up to their expectations as per the design conditions and estimations. So many uncovered defects are highlighted even their operation which eventually triggers the products recall later times. Recently M/s Toyota recalled 60 lacks vehicles globally for non functioning wheel cable.With this we can get a question that “Are all delivered products are defect free?” Probably, answer would be Not. Even Though organizations spending lot off investment during product life cycle . Due to this embedded product margin being minimized to the manufacturing Industries and is going to be a non profit product.Hence there is immense need to deliver defect free product by the manufacturers .There is a need to maintain or adopt sophisticated/test Proven test process, test coverage techniques.This paper proposes an integrated test frame work which fills the gap among Test levels,process,Testcoverage,Prototype testing, Virtual testing model based testing etc,... to deliver defect free embedded product

Condensin and the Spindle Midzone Prevent Cytokinesis Failure Induced by Chromatin Bridges in C. elegans Embryos

During cell division, chromosomes must clear the path of the cleavage furrow before the onset of cytokinesis. The abscission checkpoint in mammalian cells stabilizes the cleavage furrow in the presence of a chromatin obstruction. This provides time to resolve the obstruction before the cleavage furrow regresses or breaks the chromosomes, preventing aneuploidy or DNA damage. Two unanswered questions in the proposed mechanistic pathway of the abscission checkpoint concern factors involved in (1) resolving the obstructions and (2) coordinating obstruction resolution with the delay in cytokinesis. We found that the one-cell and two-cell C. elegans embryos suppress furrow regression following depletion of essential chromosome-segregation factors: topoisomerase II(TOP-2), CENP-A(HCP-3), cohesin, and to a lesser degree, condensin. Chromatin obstructions activated Aurora B(AIR-2) at the spindle midzone, which is needed for the abscission checkpoint in other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase and to the midbody during normal cytokinesis. Interestingly, condensin I is enriched on chromatin bridges and near the midzone/midbody in an AIR-2-dependent manner. Disruption of AIR-2, the spindle midzone, or condensin leads to cytokinesis failure in a chromatin-obstruction-dependent manner. Examination of the condensin-deficient embryos uncovered defects in both the resolution of the chromatin obstructions and the maintenance of the stable cleavage furrow. We postulate that condensin I is recruited by Aurora B(AIR-2) to aid in the resolution of chromatin obstructions and also helps generate a signal to maintain the delay in cytokinesis.

The Effectiveness of Barrier Membranes on Bone Regeneration in Localized Bony Defects: A Systematic Review

The aim of this study was to assess the effectiveness of barrier membranes in bone augmentation through a systematic review of the literature. Electronic data banks and hand searching were used to find relevant articles on the reconstruction of localized bone defects published up to May 2011. Controlled animal and human studies with more than 4 weeks of follow-up were included; studies of periodontal lesions, extraction sockets, and maxillary sinus grafts were excluded. Applications of recombinant growth factor or assessments of membranes' effects on implant osseointegration were also considered exclusion criteria. Defects filled with bone graft/bone substitute material and covered with a membrane were considered the test group, while uncovered defects were considered the control group. Thereafter, human and animal studies were evaluated separately by meta-analysis. Of the 3,986 articles found in the initial search, 34 studies met the inclusion criteria. Four animal studies concluded that the use of barrier membranes would increase the amount of vertical augmented bone (mean difference 0.32 mm; P = .006). Qualitative results regarding horizontal bone augmentation were controversial. Membranes do not increase the risk of improper healing, according to both human studies (odds ratio 5.67; P = .32) and animal studies (odds ratio 3.35; P = .12). There is limited evidence to support the effectiveness of barrier membranes in the treatment of bone deficiencies. Membranes do not increase postoperative infection, wound dehiscence, or membrane/bone graft exposure in either human or animal models. Because a majority of the results are based on animal studies, more randomized clinical trials are needed to objectively measure the efficacy of membranes in human bone augmentation.

Shank3 mutant mice display autistic-like behaviours and striatal dysfunction

Autism spectrum disorders (ASDs) comprise a range of disorders that share a core of neurobehavioural deficits characterized by widespread abnormalities in social interactions, deficits in communication as well as restricted interests and repetitive behaviours. The neurological basis and circuitry mechanisms underlying these abnormal behaviours are poorly understood. Shank3 is a postsynaptic protein, whose disruption at the genetic level is thought to be responsible for development of 22q13 deletion syndrome (Phelan-McDermid Syndrome) and other non-syndromic ASDs. Here we show that mice with Shank3 gene deletions exhibit self-injurious repetitive grooming and deficits in social interaction. Cellular, electrophysiological and biochemical analyses uncovered defects at striatal synapses and cortico-striatal circuits in Shank3 mutant mice. Our findings demonstrate a critical role for Shank3 in the normal development of neuronal connectivity and establish causality between a disruption in the Shank3 gene and the genesis of autistic like-behaviours in mice.

Coverage of Fetal Skin Defects With a Collagen Biomatrix Loaded With Growth Factors in a Sheep Model

Purpose Patients with spina bifida suffer from severe morbidity. In former animal studies we have shown that coverage of a surgically created spina bifida during the fetal period can result in decreased morbidity after birth: less incontinence and less paralysis of the hindlimbs.1 However, to enhance skin regeneration, i.e. more angiogenesis and cell attraction, the biomatrix needs to be improved. Aim of this study is to evaluate the coverage of fetal skin defects with a collagen biomatrix, a biomatrix loaded with heparin and growth factors (VEGF and FGF) and wound healing without a biomatrix. Material and Methods At 79 days' gestation, 3 skin defects (O 10 mm) were created on the back of 11 fetal lambs. One defect was left uncovered, one was covered with a collagen biomatrix, and one with a biomatrix loaded with glycosaminoglycans (heparin) and growth factors (VEGF and FGF-2). Lambs were sacrificed at 93 (n = 4), 107 (n = 3) and 138 (n = 4) days' gestation. The skin defects were macroscopically and histologically examined. Results Macroscopic evaluation revealed wound healing mainly by wound contraction for the uncovered defects. The covered defect with plain biomatrix showed partially contraction and the covered defect with growth factor loaded biomatrix showed minor contraction. Histological evaluation revealed increased angiogenesis and keratinocyt growth in the growth-factor-loaded biomatrix. Additionally, decreased degradation of the biomatrix was seen in these defects, i.e. at 138 days' gestation the biomatrix was still largely present. Overall, improved maturation of skin tissue was seen in time. Conclusions Coverage of fetal skin defects with a VEGF and FGF-2 loaded collagen biomatrix resulted in less wound contraction, increased angiogenesis and keratinocyt growth. This biomatrix can be useful in coverage of large spina bifida defects to improve neonatal outcome. 1:Eggink et al. Fetal Diagnosis and Therapy 2005 Sep-Oct;20(5):335-40.

Transcription Impairment and Cell Migration Defects in Elongator-Depleted Cells: Implication for Familial Dysautonomia

Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.

The effects of guided tissue regeneration (GTR) on modified Le Fort I osteotomy healing in rabbits

Osteogenesis following surgery depends on the osteoblasts at the wound site. Fibrous nonunions may be the result of differential and rapid migration of fibroblasts compared to osteoblasts into the wound. The present study was designed to test this hypothesis through the use of guided tissue regeneration (GTR) in a rabbit model. Bilateral, Le Fort I osteotomies ( n = 20) were produced in the maxillae of 10 New Zealand White rabbits. The segments were advanced 6 mm and rigidly fixed using microplates and screws. One side was covered with a resorbable collagen membrane or left uncovered. Rabbits were followed for four weeks with radiographs and the maxillae were harvested for histology. Cephalometry revealed that membrane-covered defects had significantly ( P < 0.01) reduced defect area (by approximately 70%) compared to uncovered defects. Histologically, membrane-covered defects showed more organized osteogenesis and less fibrous tissue than uncovered defects. Histomorphometry revealed that membrane covered defects had significantly ( P < 0.05) reduced defect areas (by approximately 20%) compared to uncovered defects. While findings suggest that GTR can facilitate osseous wound healing in Le Fort I osteotomies, results also caution against relying exclusively on two-dimensional radiography to assess bony wound healing in lieu of three-dimensional imaging and evaluations.

Mutations of Tubulin Glycylation Sites Reveal Cross-talk between the C Termini of α- and β-Tubulin and Affect the Ciliary Matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.

Enhanced bone regeneration and formation around implants using guided bone regeneration.

This study investigated the use of a prototype expanded polytetrafluoroethylene membrane attached to bone with butyl-cyanoacrylate, in facilitating guided bone regeneration into bone defects and around titanium screws in rabbit femora. Two experimental models were used to assess bone growth. The first model investigated two unicortical defects in each femora. The second was bone growth in a 500-micron engineered space around one transcortical titanium screw. In the first model there was a significant increase in bone formation at 1 and 2 months in the membrane groups (p < 0.01) as compared to the controls. In the second model the percentage of bone in contact with the implant was significant at 1 and 2 months in the defects covered with membrane compared to the uncovered defects. The uncovered defects had fibrous tissue adherent to the implant continuous with the overlying soft tissue. Our study demonstrated three points: this membrane can be used to increase bone regeneration into defects, this technique allows bone to grow directly around an implant, and butyl-cyanoacrylate can be used in deep soft tissue and bone applications without any apparent deleterious effects.

Synthetic skin replacement for temporary wound coverage in eyelid surgery

Large uncovered defects of lid-skin which extend beyond the margin of the orbital rim frequently cause wound contracture and malposition of the lid. Between April 1989 and October 1992 we have successfully used synthetic skin in severe cases of traumatic skin ablation (n = 3) and in complicated tumor surgery of the lids and of the medial canthal area (n = 14). Polyurethan foam material was used for temporary skin replacement (Epigard). The synthetic skin stimulated wound granulation and vascularization of the host area before final closure of the defect was achieved. No wound contracture occurred. Temporary bandage with synthetic skin is recommended if large skin defects can not be closed within 48 hours.

Surgically modelled reduced ridge in the beagle dog

A reproducible surgical method for the production of reduced alveolar ridge in dogs using guided tissue regeneration principles is described. 3 defects resembling a reduced ridge were formed on both sides of the mandible in 4 dogs. In each of the dogs, a polytetrafluoroethylene membrane was placed on 4 defects, while 2 defects were covered by gingival flaps only. Half of the defects were allowed to heal for 6 weeks and half for 12 weeks. Bucco-lingual width measurements revealed healing up to the membrane. In the uncovered defects, the bony wall width increased during the 12-week healing period. Histological examination showed close adaptation between bone and membrane in well-sealed defects. In cases of incomplete sealing, connective tissue had penetrated into the defect, resulting in unpredictable bone healing. The use of guided tissue regeneration techniques is suggested as a valuable tool in creating experimentally reduced ridges.