EFFECTS OF ICE ON ASCOPHYLLUM NODOSUM WITHIN THE GREAT BAY ESTUARY SYSTEM OF NEW HAMPSHIRE-MAINE12

The, effects of ice on Ascophyllum nodosum (L.) Le Jolis within the Great Bay Estuary System of New Hampshire-Maine, U.S.A. were assessed during the winter of 1980–81. Approximately 50% of the plants late fall standing crop, or an estimated 136 tonnes dry weight, was removed by ice-rafting. Pieces of Ascophyllum removed averaged 22 cm in length, 15 g in fresh weight and generally represented 2–3 years of growth. Although the winter of 1980–81 wax somewhat extreme, a similar pruning process probably occurs each year contributing to the characteristic bushy habit of attached inner estuarine plants. Ascophyllum fragments rafted in ice and deposited into salt marshes are suggested as a major source of the, ecad scorpioides (Hornemann) Hauck.     

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.     

Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production

The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.     

A Familial Factor Independent of CAG Repeat Length Influences Age at Onset of Machado-Joseph Disease

Machado-Joseph disease (MJD) is a late-onset, progressive, neurodegenerative disorder caused by the expansion of an unstable trinucleotide (CAG) repeat sequence in a novel gene (MJD1) on chromosome 14. Previous studies showed that age at onset is negatively correlated with the number of CAG repeat units, but only part of the variation in onset age is explained by CAG repeat length. Ages at onset and CAG repeat lengths of 136 MJD patients from 23 kindreds of Portuguese descent were analyzed, to determine whether familial factors independent of CAG repeat length modulate age at onset of MJD. Correlation among sibs for onset age adjusted for CAG repeat length was .43, which indicates that an environmental or genetic factor common to sibs influences onset age. Positive correlations were also observed for avuncular (r = .22) and first-cousin pairs (r = .28), which supports the hypothesis that a genetic factor is influencing age at onset. Commingling analysis of onset ages adjusted for CAG repeat length identified three distributions in this population of affected individuals. Further studies of a much larger sample are needed to determine whether these distributions represent the influence of a genetic or environmental factor.     

Strain design optimization using reinforcement learning

Engineered microbial cells present a sustainable alternative to fossil-based synthesis of chemicals and fuels. Cellular synthesis routes are readily assembled and introduced into microbial strains using state-of-the-art synthetic biology tools. However, the optimization of the strains required to reach industrially feasible production levels is far less efficient. It typically relies on trial-and-error leading into high uncertainty in total duration and cost. New techniques that can cope with the complexity and limited mechanistic knowledge of the cellular regulation are called for guiding the strain optimization.In this paper, we put forward a multi-agent reinforcement learning (MARL) approach that learns from experiments to tune the metabolic enzyme levels so that the production is improved. Our method is model-free and does not assume prior knowledge of the microbe’s metabolic network or its regulation. The multi-agent approach is well-suited to make use of parallel experiments such as multi-well plates commonly used for screening microbial strains.We demonstrate the method’s capabilities using the genome-scale kinetic model of Escherichia coli, k-ecoli457, as a surrogate for an in vivo cell behaviour in cultivation experiments. We investigate the method’s performance relevant for practical applicability in strain engineering i.e. the speed of convergence towards the optimum response, noise tolerance, and the statistical stability of the solutions found. We further evaluate the proposed MARL approach in improving L-tryptophan production by yeast Saccharomyces cerevisiae, using publicly available experimental data on the performance of a combinatorial strain library.Overall, our results show that multi-agent reinforcement learning is a promising approach for guiding the strain optimization beyond mechanistic knowledge, with the goal of faster and more reliably obtaining industrially attractive production levels.     

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth,glutathione level and causes proteome alterations outside mitochondria

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.     

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world’s most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All F_ST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.     

Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure, while this loop is disordered in the D. guianensis lectin structure. A single amino acid difference at position 131 (histidine in D. grandiflora and asparagine in D. guianensis) drastically reduces interdimer contacts, accounting for the disordered loop. Further, this amino acid change yields a conformation that may explain why a pH-dependent dimer-tetramer equilibrium exists for the D. guianensis lectin but not for the D. grandiflora lectin.