Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Red and White Wine Lees Show Inhibitory Effects on Liver Carcinogenesis.

SCOPE: Wine has shown anticarcinogenic benefits in hepatocarcinoma and polyphenols seem to be responsible for these effects. Wine lees are the sediments produced during fermentation and they endow wine with organoleptic and physicochemical properties. However, the anticarcinogenic role of these compounds is still unknown. Thus, the purpose of this work is to determine the phytochemical profiles of wine lees and then to analyze their anticarcinogenic effect and DNA methylation on a model of hepatocarcinogenesis. METHODS AND RESULTS: The phytochemical composition of lees is determined by the Folin-Ciocalteu method and high-performance liquid chromatography. An in vivo study using a diethyl nitrosamine-hepatocarcinogenesis-induced model is performed to investigate the hepatoprotective properties of different doses of wine lees. For the DNA methylation analysis, a bisulfite-based method is used. Both types of lees mostly contain pyrogallol, gallic, and syringic acid with a high content of catechins in red lees. The carcinogen hypermethylates the Alu-M2 repetitive sequence and white lees decreases the hypermethylation at all tested concentrations. Low concentration of red and white lees and high concentration of white lees significantly improve the hepatocellular architecture and decrease the mitotic index in the murine model. CONCLUSION: These findings suggest that wine lees are promising agents for chemoprevention of hepatocarcinoma.

Ecocardiografía clínica básica en urgencias: ¿podemos mejorar el pronóstico en algunos pacientes? / Focused cardiac ultrasound in the emergency department: Can we improve prognosis in selected patients?

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Initial accuracy of bedside ultrasound performed by emergency physicians for multiple indications after a short training period.

PURPOSES: Emergency physician-performed ultrasonography holds promise as a rapid and accurate method to diagnose multiple diseases in the emergency department (ED). Our objective was to assess the initial diagnostic accuracy (first 55 explorations) of emergency physician-performed ultrasonography for multiple categories of ultrasound use after a short training period. BASIC PROCEDURES: This was a prospective observational study conducted at an urban ED from June 2010 to March 2011 in patients with suspected cholecystitis, hydronephrosis, deep vein thrombosis, and different cardiovascular problems. Five physicians had a 10-hour training session before enrolling patients. The test characteristics of bedside ultrasonography were determined with the final radiologist/cardiologist interpretation. MAIN FINDINGS: A total of 275 ultrasonographic examinations were performed (78 abdominal explorations, 80 renal explorations, 76 2-point compression ultrasonographic examinations in patients with suspected deep vein thrombosis, and 41 echocardiograms in patients with different acute cardiovascular problems). Radiologists/cardiologists detected 28 cases of cholecystitis, 26 cases of deep vein thrombosis, 49 cases of hydronephrosis, and 15 cases of significant cardiovascular alterations. The overall diagnostic accuracy of ED ultrasonograms yielded a sensitivity, specificity, positive predictive value, and negative predictive value of 92.6% (95% confidence interval [CI], 90%-99%), 89% (95% CI, 84%-94%), 86.2 % (95% CI, 82%-93%), and 94.2% (95% CI, 92%-99%), respectively. Nineteen (6.9%) false-positive results and 6 false-negative results (2.1%) were obtained. PRINCIPAL CONCLUSIONS: Emergency physicians in our institution attained reasonably high initial accuracy in the performance of ultrasonography for a variety of clinical problems after a 10-hour training period.

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis.

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (~37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: assessment of geographic variation and its implication on snakebite management.

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA(2) crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for approximately 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted ( approximately 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.

Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America.

We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A(2) (PLA(2) crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn(2+)-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD(50)), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North-South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America. The paedomorphic trend is supported by a decreasing LNC (lethal neurotoxicity coefficient, defined as the ratio between the average LD(50) of the venom and the crotoxin + crotamine concentration) along the North-South axis, coincident with the evolutionary dispersal pattern of the Neotropical rattlesnakes. The indistinguisable immunoreactivity patterns of Costa Rican and Venezuelan polyvalent antivenoms toward C. simus and C. durissus venoms strongly suggest the possibility of using these antivenoms indistinctly for the management of snakebites by adult C. simus and by certain C. d. cumanensis populations exhibiting a hemorrhagic venom phenotype. The antivenomic results also explain why the antivenoms effectively neutralize the hemorrhagic activity of adult C. simus venoms but does not protect against adult C. durissus sp. and newborn C. simus envenomations. The identification of evolutionary trends among tropical Crotalus, as reported here, may have an impact in defining the mixture of venoms for immunization to produce an effective pan-American anti-Crotalus antivenom.

Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism.

The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA(2) molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA(2) molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.