Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs

Inter and intra-tumoral heterogeneity as a platform for personalized therapies in medulloblastoma

Medulloblastoma is the most common malignant CNS tumor of childhood, affecting ~350 patients/year in the USA. In 2020, most children are cured of their disease, however, survivors are left with life-long late-effects as a consequence of intensive surgery, and application of chemo- and radio-therapy to the developing brain. The major contributor to improvements in patient survival has been the development of risk-stratified treatments derived from a better understanding of the prognostic value of disease biomarkers. The characterization and validation of these biomarkers has engendered a comprehensive understanding of the extensive heterogeneity that exists within the disease, which can occur both between and within tumors (inter- and intra-tumoral heterogeneity, respectively). In this review, we discuss inter-tumoral heterogeneity, describing the early characterization of clinical and histopathological disease heterogeneity, the more recent elucidation of molecular disease subgroups, and the potential for novel therapies based on specific molecular defects. We reflect on the limitations of current approaches when applied to a rare disease. We then review early investigations of intra-tumoral heterogeneity using FISH and immunohistochemical approaches, and focus on the application of next generation sequencing on bulk tumors to elucidate intra-tumoral heterogeneity. Finally, we critically appraise the applications of single-cell sequencing approaches and discuss their potential to drive next biological insights, and for routine clinical application

Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death

Epigenetic alterations, including methylation, have been shown to be an important mechanism of gene silencing in cancer. Ependymoma has been well characterized at the DNA copy number and mRNA expression levels. However little is known about DNA methylation changes. To gain a more global view of the methylation profile of ependymoma we conducted an array-based analysis. Our data demonstrated tumors to segregate according to their location in the CNS, which was associated with a difference in the global level of methylation. Supratentorial and spinal tumors displayed significantly more hypermethylated genes than posterior fossa tumors, similar to the ‘CpG island methylator phenotype’ (CIMP) identified in glioma and colon carcinoma. This hypermethylated profile was associated with an increase in expression of genes encoding for proteins involved in methylating DNA, suggesting an underlying mechanism. An integrated analysis of methylation and mRNA expression array data allowed us to identify methylation-induced expression changes. Most notably genes involved in the control of cell growth and death and the immune system were identified, including members of the JNK pathway and PPARG. In conclusion, we have generated a global view of the methylation profile of ependymoma. The data suggests epigenetic silencing of tumor suppressor genes is an important mechanism in the pathogenesis of supratentorial and spinal, but not posterior fossa ependymomas. Hypermethylation correlated with a decrease in expression of a number of tumor suppressor genes and pathways that could be playing an important role in tumor pathogenesis

Discovery of induced point mutations in maize genes by TILLING

BACKGROUND: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project

Amplification and Overexpression of Hsa-miR-30b, Hsa-miR-30d and KHDRBS3 at 8q24.22-q24.23 in Medulloblastoma

Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.We performed a SNP-array based genome-wide copy number analysis in medulloblastoma cell lines, to identify regions of genomic amplification and homozygous deletion, which may harbour critical disease genes. A series of novel and established medulloblastoma defects were detected (MYC amplification (n = 4), 17q21.31 high-level gain (n = 1); 9p21.1-p21.3 (n = 1) and 6q23.1 (n = 1) homozygous deletion). Most notably, a novel recurrent region of genomic amplification at 8q24.22-q24.23 was identified (n = 2), and selected for further investigation. Additional analysis by interphase fluorescence in situ hybridisation (iFISH), PCR-based mapping and SNP-array revealed this novel amplification at 8q24.22-q24.23 is independent of MYC amplification at 8q24.21, and is unique to medulloblastoma in over 800 cancer cell lines assessed from different tumour types, suggesting it contains key genes specifically involved in medulloblastoma development. Detailed mapping identified a 3Mb common minimal region of amplification harbouring 3 coding genes (ZFAT1, LOC286094, KHDRBS3) and two genes encoding micro-RNAs (hsa-miR-30b, hsa-miR-30d). Of these, only expression of hsa-miR-30b, hsa-miR-30d and KHDRBS3 correlated with copy number status, and all three of these transcripts also displayed evidence of elevated expression in sub-sets of primary medulloblastomas, measured relative to the normal cerebellum.These data implicate hsa-miR-30b, hsa-miR-30d and KHDRBS3 as putative oncogenic target(s) of a novel recurrent medulloblastoma amplicon at 8q24.22-q24.23. Our findings suggest critical roles for these genes in medulloblastoma development, and further support the contribution of micro-RNA species to medulloblastoma pathogenesis

Combinatorial Markov chains on linear extensions

We consider generalizations of Schuetzenberger's promotion operator on the set L of linear extensions of a finite poset of size n. This gives rise to a strongly connected graph on L. By assigning weights to the edges of the graph in two different ways, we study two Markov chains, both of which are irreducible. The stationary state of one gives rise to the uniform distribution, whereas the weights of the stationary state of the other has a nice product formula. This generalizes results by Hendricks on the Tsetlin library, which corresponds to the case when the poset is the anti-chain and hence L=S_n is the full symmetric group. We also provide explicit eigenvalues of the transition matrix in general when the poset is a rooted forest. This is shown by proving that the associated monoid is R-trivial and then using Steinberg's extension of Brown's theory for Markov chains on left regular bands to R-trivial monoids.Comment: 35 pages, more examples of promotion, rephrased the main theorems in terms of discrete time Markov chain