An interaction between mTOR and Myc in cell size control

Regulation of cell size is controlled by multiple biochemical pathways, such as the mammalian target of rapamycin (mTOR) signalling pathway and the transcription factor Myc. The loss of tightly regulated control of cell size is known to be associated with multiple diseases such as cancer. Understanding how key molecular components in such pathways is necessary to elucidate the mechanisms behind cell size control. Since pathways consist of large networks of interacting molecules, changes in the expression of such molecules may have a large impact on the signalling cascade output, therefore investigating the impact of how the growth-regulating signalling cascade is affected by hampering expression of effectors involved in the pathway is of great interest. This thesis shows that chemical and siRNA downregulation of central pathway effectors of the mTOR pathway reduces cell size and volume, which also change the cell cycle distribution. Furthermore, RNAseq analysis from a dataset similar to our experiments showed that differentially expressed genes from cells exposed to rapamycin seem to be involved in ribosomal biogenesis and metabolism. We observed that over-expression of Myc lead to notable increases in cell size and volume which were linked to higher levels of protein and rRNA content. RNAseq analysis of this cells showed that differentially expressed genes seemed to be involved in different cellular mechanisms related to cell size. Remarkably, Myc over expressing cells that were exposed to the mTOR inhibitor Rapamycin had a size like non-treated cells but with higher content of protein and rRNA. Sequencing analysis from these cells revealed many down-regulated genes involved in cell metabolism, translation, and other cellular process. A combination of Rapamycin and the rRNA synthesis inhibitor, Actinomycin D reduced the size and rRNA content of the Myc overexpressing cells, suggesting that ribosome biogenesis could be the mechanism behind the size control. To identify an alternative effector involved in cell size and ribosome biogenesis, we show that incubation of cells with a p38 MAPK inhibitor further reduces cell size and rRNA content, suggesting a possible interaction between the PI3K/AKT/mTOR pathway, with the p38 MAPK pathway and Myc

Corrigendum: Variability of Bacterial Essential Genes Among Closely Related Bacteria: The Case of Escherichia coli

The definition of bacterial essential genes has been widely pursued using different approaches. Their study has impacted several fields of research such as synthetic biology, the construction of bacteria with minimal chromosomes, the search for new antibiotic targets, or the design of strains with biotechnological applications. Bacterial genomes are mosaics that only share a small subset of gene-sequences (core genome) even among members of the same species. It has been reported that the presence of essential genes is highly variable between closely related bacteria and even among members of the same species, due to the phenomenon known as “non-orthologous gene displacement” that refers to the coding for an essential function by genes with no sequence homology due to horizontal gene transfer (HGT). The existence of dormant forms among bacteria and the high incidence of HGT have been proposed to be driving forces of bacterial evolution, and they might have a role in the low level of conservation of essential genes among related bacteria by non-orthologous gene displacement, but this correlation has not been recognized. The aim of this mini-review is to give a brief overview of the approaches that have been taken to define and study essential genes, and the implications of non-orthologous gene displacement in bacterial evolution, focusing mainly in the case of Escherichia coli. To this end, we reviewed the available literature, and we searched for the presence of the essential genes defined by mutagenesis in the genomes of the 63 best-sequenced E. coli genomes that are available in NCBI database. We could not document specific cases of non-orthologous gene displacement among the E. coli strains analyzed, but we found that the quality of the genome-sequences in the database is not enough to make accurate predictions about the conservation of essential-genes among members of this bacterial species

The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks

<p>Abstract</p> <p>Background</p> <p>The Ala-Pro-rich <it>O</it>-glycoprotein known as the 45/47 kDa or APA antigen from <it>Mycobacterium tuberculosis </it>is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant <it>O</it>-glycoprotein APA was produced by the non-pathogenic filamentous bacteria <it>Streptomyces lividans</it>, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to <it>M. tuberculosis</it>.</p> <p>Results</p> <p>Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of <it>S. lividans </it>and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of <it>O</it>-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks.</p> <p>Conclusions</p> <p>The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the <it>O</it>-mannosylation of rAPA in <it>S. lividans</it>. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant <it>O</it>-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.</p

The microRNA landscape of cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived skin tumor. It is the second-most-common cancer affecting the Caucasian population and is responsible for >20% of all skin-cancer-related deaths. The estimated incidence of non-melanoma skin cancer in the USA is >1000000 cases per year, of which roughly 20-30% are squamous cell carcinoma. To better understand and treat this challenging cancer, current research focuses on development of novel strategies to improve the understanding of tumor biogenesis on an individual basis. microRNAs are becoming important biomarkers in the diagnosis, prognosis and treatment of cSCC. This review describes the current knowledge on miRNA expression in cSCC and its role as a biomarker for personalized medicine

Democracia en el Estado de México: fortalezas y debilidades

En toda interacción humana puede estar presente el poder. Por tanto, todas las formas de interacción humana son susceptibles de catalogarse como democráticas o como autoritarias (e incluso híbridas, cuando conjugan elementos democráticos y autoritarios). En la interacción democrática o bien está ausente el poder o bien éste es ejercido con tolerancia y con apego a normas preestablecidas. En la interacción autoritaria prevalece la intolerancia, la arbitrariedad y la violencia

Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design

A Conserved Inverted Repeat, the LipR Box, Mediates Transcriptional Activation of the Streptomyces exfoliatus Lipase Gene by LipR, a Member of the STAND Class of P-Loop Nucleoside Triphosphatases

Expression of the Streptomyces exfoliatus lipA gene, which encodes an extracellular lipase, depends on LipR, a transcriptional activator that belongs to the STAND class of P-loop nucleoside triphosphatases. LipR is closely related to activators present in some antibiotic biosynthesis clusters of actinomycetes, forming the LipR/TchG family of regulators. In this work we showed that purified LipR protein is essential for activation of lipA transcription in vitro and that this transcription depends on the presence of a conserved inverted repeat, the LipR box, located upstream of the lipA promoter. Mutagenesis of the lipA promoter region indicated that most transcription depends on LipR binding to the proximal half-site of the LipR box in close proximity to the −35 region of the promoter. Our experiments also indicated that LipR establishes contact with the RNA polymerase on both sides of the LipR box, since some activation was observed when only the distal half-site was present or when the entire LipR box was moved further upstream. We also showed that the LipR proteins of S. exfoliatus and Streptomyces coelicolor are functionally interchangeable both in vitro and in vivo, revealing the functional conservation of the regulatory elements in these two species

Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design

Consensus on Criteria for Potential Areas for Wolf Reintroduction in Mexico

Given the conflict with human interests that in many cases results in the extirpation of large carnivores, acceptance of their reintroduction is a considerable challenge. By the 1980s Mexican wolves (Canis lupus) were extinct in the wild. In 1998 a population was reintroduced in the Blue Range Mountains of New Mexico (U.S.A.). Efforts to reintroduce the species in Mexico have been ongoing since the late 1980s. Four teams working independently identified 6 areas in northern Mexico in the historic range of Mexican wolves, where reintroductions could potentially be successful. Each team used different methods and criteria to identify the areas, which makes it difficult to prioritize among these areas. Therefore, members of the different teams worked together to devise criteria for use in identifying priority areas. They identified areas with high, intermediate, and low potential levels of conflict between wolves and humans. Areas with low potential conflict had larger buffers (i.e., distance from human settlement to areas suitable for wolves) around human settlements than high- and intermediate-conflict areas and thus were thought most appropriate for the first reintroduction. High-conflict areas contained habitat associated with wolf presence, but were closer to human activity. The first reintroduction of Mexican wolves to Mexico occurred in October 2011 in one of the identified low-conflict areas. The identification of suitable areas for reintroduction represents a crucial step in the process toward the restoration of large carnivores. Choice of the first reintroduction area can determine whether the reintroduction is successful or fails. A failure may preclude future reintroduction efforts in a region or country. © 2012 Society for Conservation Biology.Peer Reviewe

Expression of the Azotobacter vinelandii Poly-β-Hydroxybutyrate Biosynthetic phbBAC Operon Is Driven by Two Overlapping Promoters and Is Dependent on the Transcriptional Activator PhbR

The Azotobacter vinelandii phbBAC genes encode the enzymes for poly-β-hydroxybutyrate (PHB) synthesis. The phbR gene, which is located upstream of and in the opposite direction of phbBAC, encodes PhbR, a transcriptional activator which is a member of the AraC family of activators. Here we report that a mutation in phbR reduced PHB accumulation and transcription of a phbB-lacZ fusion. We also report that phbB is transcribed from two overlapping promoters, p(B)1 and p(B)2. The region corresponding to the −35 region of p(B)1 overlaps the p(B)2 −10 region. In the phbR mutant, expression of phbB from the p(B)1 promoter is significantly reduced, whereas expression from the p(B)2 promoter is slightly increased. Two phbR promoters, p(R)1 and p(R)2, were also identified. Transcription from p(R)2 was shown to be dependent on σ(S). Six conserved 18-bp sites, designated R1 to R6, are present within the phbR-phbB intergenic region and are proposed to be putative binding targets for PhbR. R1 overlaps the −35 region of the p(B)1 promoter. A model for the regulation of phbB transcription by PhbR is proposed