Genotyping Cancer-Associated Genes in Chordoma Identifies Mutations in Oncogenes and Areas of Chromosomal Loss Involving CDKN2A,PTEN, and SMARCB1

The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis.     

Clock Polymorphisms and Circadian Rhythms Phenotypes in a Sample of the Brazilian Population

A Clock polymorphism T to C situated in the 3′ untranslated region (3′‐UTR) has been associated with human diurnal preference. At first, Clock 3111C had been reported as a marker for evening preference. However these data are controversial, and data both corroborating and denying them have been reported. This study hypothesizes that differences in Clock genotypes could be observed if extreme morning‐type subjects were compared with extreme evening‐type subjects, and the T3111C and T257G polymorphisms were studied. The possible relationship between both polymorphisms and delayed sleep phase syndrome (DSPS) was also investigated. An interesting and almost complete linkage disequilibrium between the polymorphisms T257G in the 5′ UTR region and the T3111C in the 3′ UTR region of the Clock gene is described. Almost always, a G in position 257 corresponds to a C in position 3111, and a T in position 257 corresponds to a T in position 3111. The possibility of an interaction of these two regions in the Clock messenger RNA structure that could affect gene expression was analyzed using computer software. The analyses did not reveal an interaction between those two regions, and it is unlikely that this full allele correspondence affects Clock gene expression. These results show that there is no association between either polymorphism T3111C or T257G in the Clock gene with diurnal preference or delayed sleep phase syndrome (DSPS). These controversial data could result from the possible effects of latitude and clock genes interaction on circadian phenotypes.     

Amaranthus caudatus subsp. mantegazzianus: A new host of ‘Candidatus Phytoplasma hispanicum’ (subgroup 16Sr XIII‐A)

In Argentina, amaranth is a promising crop due to high nutritional quality and ability to grow in a diversity of environments. In areas cultivated with amaranth, were observed plants exhibiting slow growth, deformed leaves, proliferation of shoots and malformed lateral panicles. Field survey revealed up to 96% disease incidence and 92% of the seeds collected from mother plants produced diseased seedlings. A phytoplasma was detected in association with seedlings and adult plants using nested PCR assays. Molecular identification by computer‐simulated RFLP and phylogenetic analysis evidenced the occurrence of a ‘Candidatus Phytoplasma hispanicum’‐related strain, affiliated with 16SrXIII‐A subgroup. The findings implicate amaranth as a new host for this subgroup and as a potential reservoir of the pathogen for other cultivated species. In addition, to the best of our knowledge, this study reports for the first time the presence of 16SrXIII‐A phytoplasma in Argentina and in South America. Furthermore, transmission assays pointed that naturally infected seed is an important vehicle of dissemination of the pathogen, threatening the expansion of the crop for new geographical areas.     

HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels

The level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra-optimal temperatures: the total amount of this RNA per cell is increased at 37°C, and slightly decreased at 40°C relative to its level at 29°C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat-shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37°C and 40°C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37°C, in addition, an increase in the total amount of this template is observed in the cells.     

Signal transduction inTrypanosoma cruzi: opposite effects of adenylcyclase and phospholipase C systems in growth control

Fetal calf serum (FCS), which is mitogenic for the pathogenic protozoaT. cruzi, inhibits cAMP production in basal and forskolin-stimulated epimastigotes. It also activates phosphoinositides hydrolysis yielding diacylglycerol and inositol phosphates (Ins-P). Ins-P production is enhanced by AlF₄-, GTP or --methylene-GTP, thus implying G proteins mediation in the phenomenon. An enzyme with phospholipase C activity which may be involved in the phospholipid metabolism was partially characterized.     

Control of tubulin gene expression during metacyclogenesis of Trypanosoma cruzi

During differentiation of the dividing epimastigote to the non-dividing metacyclic trypomastigote form of the parasitic protozoan Trypanosoma cruzi there is a marked reduction in the rate of synthesis of the major proteins alpha- and beta-tubulin. Our results indicate that the control of synthesis of these proteins during the differentiation event is exerted at the level of alpha- and beta-tubulin mRNA accumulation.     

Developmental regulation,induction, and embryonic tissue specificity of sea urchin metallothionein gene expression

Metallothionein (MT) is shown to be present in sea urchin embryos on the basis of its characteristic properties as a small protein (6–7 Da) of extraordinarily high cysteine content, whose biosynthesis is readily induced by heavy metals. Induction by Zn²⁺ results in the accumulation of the cysteine-rich MT protein, a 0.8 kb MT mRNA and a 2.9 kb nuclear RNA. The amount of MT mRNA is regulated intrinsically through the course of embryogenesis to the pluteus stage: A maternal MT mRNA is poly(A)-deficient and is polyadenylated after fertilization. New MT mRNA begins to accumulate between the seventh and eighth cell cleavage, reaches a maximum at the mesenchyme blastula stage, decreases during gastrulation, and rises again in the early pluteus stage. “Animalizing” embryos with Zn²⁺ during early embryogenesis causes a sustained accumulation of MT mRNA to levels greater than 25 times the normal amount. MT mRNA is present in high amount in the ectoderm of the pluteus, but is barely detectable in the mesoderm-endoderm tissue fraction. Treatment of either the pluteus or its isolated tissue fractions with Zn²⁺ results in the induction of MT mRNA accumulation in the mesoderm-endoderm but not in the already MT mRNA-enriched ectoderm. Furthermore, differences in Zn²⁺ induction of the MT gene in the blastula and gastrula are consistent with a developmental pattern in which MT gene expression is maintained constitutively at a high level in the ectoderm and at a low level in the mesoderm-endoderm tissues, which are, however, preferentially inducible by Zn²⁺.     

- and β-tubulin mRNAs of Trypanosoma cruzi originate from a single multicistronic transcript

The cluster of alternated - and β-tubulin genes in the genome of Trypanosoma cruzi was shown to be transcribed into a single RNA molecule which upon processing gives rise to the mature - and β-tubulin mRNAs. This conclusion was based on: (i) nuclear RNA species with the same molecular mass hybridize to both - and β-tubulin cDNA probes; (ii) S₁ nuclease assay of the clustered tubulin genes has shown protected DNA fragments of the same size and of greater molecular mass than that corresponding to the mRNAs, hybridizable to both - and β-tubulin cDNA probes; (iii) β-tubulin hybrid selected RNA is still able to hybridize to -tubulin probe.     

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20–50?°C), gelatinization time (30–60 min) and broken rice concentration in culture medium (40–80 g L^?1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L^?1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31?°C temperature, 45 min gelatinization time and 79 g L^?1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.