Diagnostic support in pediatric craniopharyngioma using deep learning.

PURPOSE: We studied a pediatric group of patients with sellar-suprasellar tumors, aiming to develop a convolutional deep learning algorithm for radiological assistance to classify them into their respective cohort. METHODS: T1w and T2w preoperative magnetic resonance images of 226 Chilean patients were collected at the Institute of Neurosurgery Dr. Alfonso Asenjo (INCA), which were divided into three classes: healthy control (68 subjects), craniopharyngioma (58 subjects) and differential sellar/suprasellar tumors (100 subjects). RESULTS: The PPV among classes was 0.828±0.039, and the NPV was 0.919±0.063. Also explainable artificial intelligence (XAI) was used, finding that structures that are relevant during diagnosis and radiological evaluation highly influence the decision-making process of the machine. CONCLUSION: This is the first experience of this kind of study in our institution, and it led to promising results on the task of radiological diagnostic support based on explainable artificial intelligence (AI) and deep learning models.

The Maize Hairy Sheath Frayed1 (Hsf1) Mutation Alters Leaf Patterning through Increased Cytokinin Signaling.

Leaf morphogenesis requires growth polarized along three axes-proximal-distal (P-D) axis, medial-lateral axis, and abaxial-adaxial axis. Grass leaves display a prominent P-D polarity consisting of a proximal sheath separated from the distal blade by the auricle and ligule. Although proper specification of the four segments is essential for normal morphology, our knowledge is incomplete regarding the mechanisms that influence P-D specification in monocots such as maize (Zea mays). Here, we report the identification of the gene underlying the semidominant, leaf patterning maize mutant Hairy Sheath Frayed1 (Hsf1). Hsf1 plants produce leaves with outgrowths consisting of proximal segments-sheath, auricle, and ligule-emanating from the distal blade margin. Analysis of three independent Hsf1 alleles revealed gain-of-function missense mutations in the ligand binding domain of the maize cytokinin (CK) receptor Z. mays Histidine Kinase1 (ZmHK1) gene. Biochemical analysis and structural modeling suggest the mutated residues near the CK binding pocket affect CK binding affinity. Treatment of the wild-type seedlings with exogenous CK phenocopied the Hsf1 leaf phenotypes. Results from expression and epistatic analyses indicated the Hsf1 mutant receptor appears to be hypersignaling. Our results demonstrate that hypersignaling of CK in incipient leaf primordia can reprogram developmental patterns in maize.

First description of a lesion in the upper digestive mucosa associated with a novel gammaherpesvirus in a striped dolphin (Stenella coeruleoalba) stranded in the Western Mediterranean Sea.

BACKGROUND: A wide variety of lesions have been associated with herpesvirus in cetaceans. However, descriptions of herpesvirus infections in the digestive system of cetaceans are scarce. CASE REPORT: A young female striped dolphin stranded in the Valencian Community (Spain) on the 6th August 2021. The animal showed external macroscopic lesions suggestive of an aggressive interaction with bottlenose dolphins (rake marks in the epidermis). Internally, the main findings included congestion of the central nervous system and multiple, well-defined, whitish, irregularly shaped, proliferative lesions on the oropharyngeal and laryngopharyngeal mucosa. Histopathology revealed lymphoplasmacytic and histiocytic meningoencephalitis, consistent with neuro brucellosis. The oropharyngeal and laryngopharyngeal plaques were comprised histologically of focally extensive epithelial hyperplasia. As part of the health surveillance program tissue samples were tested for cetacean morbillivirus using a real-time reverse transcription-PCR, for Brucella spp. using a real-time PCR, and for herpesvirus using a conventional nested PCR. All samples were negative for cetacean morbillivirus; molecular positivity for Brucella spp. was obtained in pharyngeal tonsils and cerebrospinal fluid; herpesvirus was detected in a proliferative lesion in the upper digestive mucosa. Phylogenetic analysis showed that the herpesvirus sequence was included in the Gammaherpesvirinae subfamily. This novel sequence showed the greatest identity with other Herpesvirus sequences detected in skin, pharyngeal and genital lesions in five different species. CONCLUSIONS: To the best of the authors' knowledge, this is the first report of a proliferative lesion in the upper digestive mucosa associated with gammaherpesvirus posititvity in a striped dolphin (Stenella coeruleoalba).

Conformational stability, dynamics and function of human frataxin: Tryptophan side chain interplay.

In humans, the loss of frataxin results in Friedreich's Ataxia, a neurodegenerative disease, in which a deficit in the iron-sulfur cluster assembly is observed. In this work, we analyzed three frataxin variants in which one tryptophan was replaced by a glycine: W155G, W168G and W173G. As expected, given its localization in the assembly site, W155G was not able to activate the desulfurase activity of the supercomplex for iron-sulfur cluster assembly. In turn, W168G, which was significantly more unstable than W155G, was fully active. W173G, which was highly unstable as W168G, showed a significantly decreased activity, only slightly higher than W155G. As W168G and W173G were highly sensitive to proteolysis, we investigated the protein motions by molecular dynamic simulations. We observed that W173G may display altered motions at the Trp155 site. Furthermore, we revealed a H-bond network in which Trp155 takes part, involving residues Gln148, Asn151, Gln153 and Arg165. We suggest that this motion modulation that specifically alters the population of different Trp155 rotamers can be directly transferred to the assembly site, altering the dynamics of the ISCU His137 key residue. This hypothesis was also contrasted by means of molecular dynamic simulations of frataxin in the context of the complete supercomplex. We propose that the supercomplex requires very definite motions of Trp155 to consolidate the assembly site.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering.

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.

Relationship between activity and stability: Design and characterization of stable variants of human frataxin.

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.

Alpha- and gammaherpesviruses in stranded striped dolphins (Stenella coeruleoalba) from Spain: first molecular detection of gammaherpesvirus infection in central nervous system of odontocetes.

BACKGROUND: Herpesvirus infections in cetaceans have always been attributed to the Alphaherpesvirinae and Gammaherpesvirinae subfamilies. To date, gammaherpesviruses have not been reported in the central nervous system of odontocetes. CASE PRESENTATION: A mass stranding of 14 striped dolphins (Stenella coeruleoalba) occurred in Cantabria (Spain) on 18th May 2019. Tissue samples were collected and tested for herpesvirus using nested polymerase chain reaction (PCR), and for cetacean morbillivirus using reverse transcription-PCR. Cetacean morbillivirus was not detected in any of the animals, while gammaherpesvirus was detected in nine male and one female dolphins. Three of these males were coinfected by alphaherpesviruses. Alphaherpesvirus sequences were detected in the cerebrum, spinal cord and tracheobronchial lymph node, while gammaherpesvirus sequences were detected in the cerebrum, cerebellum, spinal cord, pharyngeal tonsils, mesenteric lymph node, tracheobronchial lymph node, lung, skin and penile mucosa. Macroscopic and histopathological post-mortem examinations did not unveil the potential cause of the mass stranding event or any evidence of severe infectious disease in the dolphins. The only observed lesions that may be associated with herpesvirus were three cases of balanitis and one penile papilloma. CONCLUSIONS: To the authors' knowledge, this is the first report of gammaherpesvirus infection in the central nervous system of odontocete cetaceans. This raises new questions for future studies about how gammaherpesviruses reach the central nervous system and how infection manifests clinically.

Frataxin Structure and Function.

Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich's Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.

Exploiting the natural poly(3-hydroxyalkanoates) production capacity of Antarctic Pseudomonas strains: from unique phenotypes to novel biopolymers.

Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we isolated two Antarctic bacterial strains able to produce poly(3-hydroxyalkanoates) (PHAs), which were classified after 16S rRNA analysis as Pseudomonas sp. MPC5 and MPC6. The MPC6 strain presented nearly the same specific growth rate whether subjected to a temperature of 4 °C 0.18 (1/h) or 30 °C 0.2 (1/h) on glycerol. Both Pseudomonas strains produced high levels of PHAs and exopolysaccharides from glycerol at 4 °C and 30 °C in batch cultures, an attribute that has not been previously described for bacteria of this genus. The MPC5 strain produced the distinctive medium-chain-length-PHA whereas Pseudomonas sp. MPC6 synthesized a novel polyoxoester composed of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate). Batch bioreactor production of PHAs in MPC6 resulted in a titer of 2.6 (g/L) and 1.3 (g/L), accumulating 47.3% and 34.5% of the cell dry mass as PHA, at 30 and 4 °C, respectively. This study paves the way for using Antarctic Pseudomonas strains for biosynthesizing novel PHAs from low-cost substrates such as glycerol and the possibility to carry out the bioconversion process for biopolymer synthesis without the need for temperature control.

[Place of Death in Chile: A descriptive study of a time series from 1990 to 2014]. / Lugar de muerte en Chile: un estudio descriptivo de serie temporal entre 1990-2014.

BACKGROUND: With the aim to inform end of life public policies, the place of death in Chile, its trends and associated factors were analysed. MATERIALS AND METHODS: A cross sectional using publically available death database from 1990 to 2014 was conducted. The proportion of hospital deaths was selected as the main outcome. A logistic regression was used to assess the association between place of death, age, and main diagnosis at death. Also, a Prais-Winsten regression and a Chi2 test were used to assess a time series and regional analysis, respectively. RESULTS: 2,063,615 deaths were analysed. Overall, deaths 898,871 (43.6%) occurred at hospital. Those who died over 85 years (OR 2,52 IC95% 2.49-2.55) and those who died from cancer (OR 2.43 IC95% 2.42-2.45) had higher risk for dying outside de hospital. For the general population and those who die form cancer, there is no evidence for an increase or decrease trend in the proportion of hospital deaths over time (p = 0,75 and p = 0.68, respectively). However, there is an increase of the proportion of hospital deaths in those who died over 85 years (p < 0.001,27% in 1990 to 32% in 2014). Also, there are geographic differences between country regions (p < 0.001) (Eg. Magallanes 52.9%). CONCLUSIONS: The proportion of hospital deaths has been stable over time in the general population and has increased in those over 85 years.

Multi-level evaluation of Escherichia coli polyphosphate related mutants using global transcriptomic, proteomic and phenomic analyses.

BACKGROUND: Polyphosphate (polyP) is a linear biopolymer found in all living cells. In bacteria, mutants lacking polyphosphate kinase 1 (PPK1), the enzyme responsible for synthesis of most polyP, have many structural and functional defects. However, little is known about the causes of these pleiotropic alterations. The link between ppk1 deletion and those numerous phenotypes observed can be the result of complex molecular interactions that can be elucidated via a systems biology approach. METHODS: By integrating different omics levels (transcriptome, proteome and phenome), we described the functioning of various metabolic pathways among Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and ΔpolyP). Bioinformatic analyses reveal the complex metabolic and regulatory bases of the phenotypes unique to polyP mutants. RESULTS: Our results suggest that during polyP deficiency (Δppk1 mutant), metabolic pathways needed for energy supply are up-regulated, including fermentation, aerobic and anaerobic respiration. Transcriptomic and q-proteomic contrasting changes between Δppk1 and Δppx mutant strains were observed in those central metabolic pathways and confirmed by using Phenotypic microarrays. In addition, our results suggest a regulatory connection between polyP, second messenger metabolism, alternative Sigma/Anti-Sigma factors and type-II toxin-antitoxin (TA) systems. CONCLUSIONS: We suggest a broader role for polyP via regulation of ATP-dependent proteolysis of type II toxin-antitoxin system and alternative Sigma/Anti-Sigma factors, that could explain the multiple structural and functional deficiencies described due to alteration of polyP metabolism. GENERAL SIGNIFICANCE: Understanding the interplay of polyP in bacterial metabolism using a systems biology approach can help to improve design of novel antimicrobials toward pathogens.

Phosphorylation of targeting protein for Xenopus kinesin-like protein 2 (TPX2) at threonine 72 in spindle assembly.

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.

Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.

BACKGROUND: Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network. RESULTS: Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol. CONCLUSION: This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This knowledge will form the basis for the development of future metabolically engineered hyper-PHA-producing strains derived from the versatile bacterium P. putida KT2440.

A generic methodology for the optimisation of sewer systems using stochastic programming and self-optimizing control.

The design of sewer system control is a complex task given the large size of the sewer networks, the transient dynamics of the water flow and the stochastic nature of rainfall. This contribution presents a generic methodology for the design of a self-optimising controller in sewer systems. Such controller is aimed at keeping the system close to the optimal performance, thanks to an optimal selection of controlled variables. The definition of an optimal performance was carried out by a two-stage optimisation (stochastic and deterministic) to take into account both the overflow during the current rain event as well as the expected overflow given the probability of a future rain event. The methodology is successfully applied to design an optimising control strategy for a subcatchment area in Copenhagen. The results are promising and expected to contribute to the advance of the operation and control problem of sewer systems.

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions.

BACKGROUND: Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source. RESULTS: Among all tested strains, P. putida KT2440 most efficiently synthesized mcl-PHA under nitrogen-limiting conditions, amassing more than 34% of its cell dry weight as PHA. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% PHA (%wt/wt). The low biomass and PHA titer found in the mutant strain and the wild-type strain KT2440 seems to be triggered by the high production of the side-product citrate during the fermentation process which shows a high yield of 0.6 g/g. CONCLUSIONS: Overall, this work demonstrates the importance of choosing an appropriate microbe for the synthesis of mcl-PHA from waste materials, and a close inspection of the cell metabolism in order to identify undesired compounds that diminish the availability of precursors in the synthesis of biopolymers such as polyhydroxyalkanoates. Future metabolic engineering works should focus on reducing the production of citrate in order to modulate resource allocation in the cell's metabolism of P. putida, and finally increase the biopolymer production.

Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint.

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida.

BACKGROUND: Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation. RESULTS: A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms. CONCLUSION: Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.

Thermoplastic polymers as water substitutes.

Background. New applications of 3D printing have recently appeared in the fields of radiotherapy and radiology, but the knowledge of many radiological characteristics of the compounds involved is still limited. Therefore, studies are needed to improve our understanding about the transport and interaction of ionizing radiation in these materials.Purpose. The purpose of this study is to perform an analysis of the most important radiation interaction parameters in thermoplastic materials used in Fused Deposition Modeling 3D printing. Additionally, we propose improvements to bring their characteristics closer to those of water and use them as water substitutes in applications such as radiodiagnosis, external radiotherapy, and brachytherapy.Methods. We have calculated different magnitudes as mass linear attenuation, mass energy absorption coefficients, as well as stopping power and electronic density of several thermoplastic materials along with various compounds that have been used as water substitutes and in a new proposed blend. To perform these computations, we have used the XCOM and ESTAR databases from NIST and the EGSnrc code for Montecarlo simulations.Results. From the representation of the calculated interaction parameters, we have been able to establish relationships between their properties and the proportion of certain chemical elements. In addition, studying these same characteristics in different commercial solutions used as substitutes for water phantoms allows us to extrapolate improvements for these polymers.Conclusion. The radiological characteristics of the analyzed thermoplastic materials can be improved by adding some chemical elements with atomic numbers higher than oxygen and by using polyethylene in new blends.