Abstract Background It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications. Methods In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region. Results MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3 , MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003). Conclusion MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.
The rising success of cancer immunotherapy has produced immense interest in defining the clinical contexts that may benefit from this therapeutic approach. To this end, there is a need to ascertain how the therapeutic modulation of intrinsic cancer cell programs influences the anticancer immune response. For example, the role of autophagy as a tumor cell survival and metabolic fitness pathway is being therapeutically targeted in ongoing clinical trials that combine cancer therapies with antimalarial drugs for the treatment of a broad spectrum of cancers, many of which will likely benefit from immunotherapy. However, our current understanding of the interplay between autophagy and the immune response remains incomplete. Here, we have evaluated how autophagy inhibition impacts the antitumor immune response in immune-competent mouse models of melanoma and mammary cancer. We observed equivalent levels of T cell infiltration and function within autophagy-competent and -deficient tumors, even upon treatment with the anthracycline chemotherapeutic doxorubicin. Similarly, we found equivalent T cell responses upon systemic treatment of tumor-bearing mice with antimalarial drugs. Our findings demonstrate that antitumor adaptive immunity is not adversely impaired by autophagy inhibition in these models, allowing for the future possibility of combining autophagy inhibitors with immunotherapy in certain clinical contexts.
Abstract Genetic mutations leading to premature termination of protein translation, nonsense mutations, result in aberrant precursor mRNA processing and subsequent UPF1-dependent exosomal degradation of mRNA. The significance of this surveillance mechanism in controlling the expression of key mutated tumor suppressor genes in cancer in vivo has remained unclear. We have recently shown that TP53 mutations are almost invariably present in high-grade serous ovarian cancers (Ahmed at al., Journal of Pathology, 2010;221:49) and that about one third of such mutations are nonsense. We have now tested the hypothesis that nonsense mutations induce nonsense mediated decay resulting in TP53 mRNA depletion. We analyzed microarray expression profiling data from 136 epithelial ovarian cancers with known TP53 mutation status and found a strikingly statistically significant underexpression of TP53 mRNA transcript in cancers with truncating mutations (p<0.0001) compared to other types of mutations. This effect allowed the identification of a class of TP53-non-expressing serous epithelial cancers that comprised all TP53 truncating mutations. We next conducted a systematic analysis of 18 publically available expression profiling ovarian cancer studies published at the Gene Expression Omnibus database. We validated the results by showing that cancers that had nonsense mutations had a significantly lower TP53 expression irrespective of stage. Importantly, this systematic analysis of multiple independent data set established that lack of TP53 expression was, almost exclusively, a feature of high grade serous cancers and invasion as opposed to low grade disease or tumors of low malignant potential (p<0.0001 and p=0.002, respectively). Expression profiling analysis revealed that cancers with truncating mutations had a distinct gene expression signature compared to those with missense mutations. This signature was enriched for genes that regulated angiogenesis such as CSPG4 and metabolism. Surprisingly, however, the magnitude of expression differences was not high. Based on these results, we propose a model for the biogenesis of a subset of high-grade serous ovarian cancers that is defined by nonsense mediated decay of nonsense TP53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4822. doi:10.1158/1538-7445.AM2011-4822
This study describes a system for quantifying paclitaxel activity using the C-terminus of α-tubulin as a biomarker. Following stabilization of microtubules with paclitaxel, a specific detyrosination reaction occurs at the C-terminus of α-tubulin which could be used to assess efficacy. A fluorescence resonance energy transfer (FRET) based biosensor was synthesized comprising a short peptide that corresponded to the C-terminus of α-tubulin, a fluorophore (Abz), and a quencher (Dnp). The fluorophore added to the end of the peptide can be released upon enzymatic detyrosination. In addition, a single fluorophore-tagged peptide was also conjugated to mesoporous silica nanoparticles to examine the feasibility of combining the drug with the peptide biomarker. As a proof of concept, we found that the degree of peptide cleavage, and therefore enzymatic activity, was directly correlated with exogenous bovine carboxypeptidase (CPA) an enzyme that mimics endogenous detyrosination. In addition, we show that cell lysates obtained from paclitaxel-treated cancer cells competed with exogenous CPA for biosensor cleavage in a paclitaxel dose-dependent manner. Our work provides strong evidence for the feasibility of combining paclitaxel with a novel biosensor in a multi-load nanoparticle.
Quarantine plant pests are socially, economically and environmentally important due to their impact on food security, human health, global trade and crop production costs. The increase in global trade and tourism, frequent occurrence of natural disasters and climate changes have exacerbated the rate of entry, establishment and spread of plant pests regionally and globally. It has, therefore, become exigent to develop a list of pests of quarantine importance at the regional and national levels to prioritise and allocate the limited available resources to manage the associated risks. In the present study, the Technical Committee on the Formulation and Prioritisation of a Regional Priority Pest List for the Caribbean, in collaboration with the National Plant Protection Organisation of the Caribbean countries and the United States Department of Agriculture - Animal and Plant Health Inspection Service (USDA-APHIS), developed and prioritised a quarantine pest list using a multi-criteria decision-making approach. The technical committee successfully evolved the process in 2014 and 2018 and developed a list of the top 10 pests of quarantine importance for the Caribbean Region, employing the Delphi Technique (DT) and Analytical Hierarchy Process (AHP) through the assignment of criteria that are relevant to the region. The Mediterranean fruit fly ( Ceratitis capitata ), frosty pod rot ( Moniliophthora roreri ) and the tomato leaf miner ( Tuta absoluta ), listed as top quarantine pest threats, were subsequently detected in the region. This exercise guided the authorities in advance to allocate resources and to develop response plans including capacity building for surveillance and detection of priority pests. This has demonstrated the significance and appropriateness of the multi-criteria decision approach to determine priority pest lists and prepare the region for development of better management practices.
ABSTRACT Autophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay the deleterious effects induced by compromised autophagy. Neurons expressing common familial Parkinson’s disease (PD)-associated mutations in LRRK2 kinase exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human iPSC-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2 G2019S neurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesized that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised, likely to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and immunocytochemistry demonstrated that secretory autophagy was upregulated in LRRK2 G2019S neurons. We also found that LRRK2 G2019S neurons upregulate the release of exosomes containing miRNAs. Live-cell imaging confirmed that this upregulation of exosomal release was dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate the compensatory release of secreted autophagosomes and exosomes, to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to increased neuroinflammation over the longer term. SIGNIFICANCE A hallmark feature of many neurodegenerative diseases is autophagy dysfunction, resulting in the accumulation of damaged proteins and organelles that is detrimental to neuronal health. The late onset of neurodegenerative diseases, however, suggests alternative quality control mechanisms may delay neuronal degeneration. Here, we demonstrate that neurons expressing a Parkinson’s Disease-causing mutation upregulate the release of two extracellular vesicle populations. First, we observe the increased expulsion of secreted autophagosomes to mediate cellular waste disposal. Second, we observe the increased release of exosomes, likely to facilitate transcellular communication. Thus, we propose that increases in secretory autophagy and exosome release are a homeostatic response in neurons undergoing chronic stress.
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