Vibrio sp. ArtGut-C1, a polyhydroxybutyrate producer isolated from the gut of the aquaculture live diet Artemia (Crustacea)

BACKGROUND: Vibrio species display variable and plastic fitness strategies to survive and interact with multiple hosts, including marine aquaculture species that are severely affected by pathogenic Vibrios. The culturable Vibrio sp. strain ArtGut-C1, the focus of this study, provides new evidence of such phenotypic plasticity as it accumulates polyhydroxybutyrate (PHB), a biodegradable polymer with anti-pathogen activity, particularly in the marine larviculture phase. The strain was isolated from the gut of laboratory-reared Artemia individuals, the live diet and PHB carrier used in larviculture. Its main phenotypic properties, taxonomic status and genomic properties are reported based on the whole-genome sequencing. RESULTS: Vibrio sp. ArtGut-C1 yielded 72.6% PHB of cells' dry weight at 25 C. The genomic average nucleotide identity (ANI) shows it is closely related to V. diabolicus (ANI: 88.6%). Its genome contains 5,236,997- bp with 44.8% GC content, 3,710 protein-coding sequences, 96 RNA, 9 PHB genes functionally related to PHB metabolic pathways, and several genes linked to competing and colonizing abilities. CONCLUSIONS: This culturable PHB-accumulating Vibrio strain shows high genomic and phenotypic variability. It may be used as a natural pathogen biocontrol in the marine hatchery and as a potential cell factory for PHB production.

Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers

Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.

Characterization and assessment of the genotoxicity and biocompatibility of poly (hydroxybutyrate) and norbixin membranes

Abstract Purpose To synthesize and characterize poly(hydroxybutyrate) (PHB) and norbixin membranes to evaluate them for genotoxicity in rats and wound healing in mice by histological staining. Methods For the evaluation of genotoxicity, male rats ( Rattus novegicus ) were divided into three groups (n= 5): 5% PHB/Norbixin membrane introduced into the peritoneum by laparotomy; B - negative control; C - positive control (intraperitoneal dose of cyclophosphamide 50 mg/kg). For the evaluation of biocompatibilty, a cutaneous wound was induced on the back of males mice ( Mus musculus ) divided into two experimental treatment groups: control and membrane that underwent euthanasia after 7 and 14 days treatment. Statistical analysis ware made by One Way Anova post hoc Tukey Test (p<0.05). Results Regarding the incidence of polychromatic erythrocytes, there was no difference between negative control and 5% PHB/Norbixin membrane; however, when compared to the positive control represented by cyclophosphamide, there was a significant difference (p <0.001). As for DNA damage, the changes induced in the first 4h were repaired in 24h. In addition, the membrane was effective in abbreviating the inflammatory process and served as a scaffold due to the stimulus to reepithelialization mainly on the 7 days of treatment. Conclusion The non-genotoxic PHB/Norbixin 5% membrane presented promising results that suggest its effectiveness as a guide for tissue regeneration given its biocompatibility.

Effect of low intensity photobiomodulation associated with norbixin-based poly (hydroxybutyrate) membrane on post-tenotomy tendon repair. In vivo study

Abstract Purpose: To evaluate the in vivo response of photobiomodulation therapy associated with norbixin-based poly(hydroxybutyrate) membrane (PHB) in tenotomized calcaneal tendon. Methods: Thirty rats were randomly allocated to six groups (n=5 each): LED groups (L1, L2 and L3) and membrane + LED groups (ML1, ML2 and ML3). The right calcaneal tendons of all animals were sectioned transversely and were irradiated with LED daily, one hour after surgery every 24 hours, until the day of euthanasia. At the end of the experiments the tendons were removed for histological analysis. Results: The histological analysis showed a significant reduction in inflammatory cells in the ML1, ML2 and ML3 groups (p=0.0056, p=0.0018 and p<0.0001, respectively) compared to those in the LED group. There was greater proliferation of fibroblasts in the ML1 (p<0.0001) and L3 (p<0.0001) groups. A higher concentration of type I collagen was also observed in the ML1 group (p=0.0043) replacing type III collagen. Conclusion: Photobiomodulation in association with norbixin-based PHB membrane led to control of the inflammatory process. However, it did not favor fibroblast proliferation and did not optimize type I collagen formation in the expected stage of the repair process.

Effect of norbixin-based poly(hydroxybutyrate) membranes on the tendon repair process after tenotomy in rats

Abstract Purpose: To determine the efficacy of norbixin-based poly(hydroxybutyrate) (PHB) membranes for Achilles tendon repair. Methods: Thirty rats were submitted to total tenotomy surgery of the right Achilles tendon and divided into two groups (control and membrane; n = 15 each), which were further subdivided into three subgroups (days 7, 14, and 21; n = 5 each). Samples were analyzed histologically. Results: Histological analysis showed a significant reduction in inflammatory infiltrates on days 7, 14 (p < 0.0001 for both), and 21 (p = 0.0004) in the membrane group compared to that in the control group. There was also a significant decrease in the number of fibroblasts in the control group on days 7, 14 (p < 0.0001), and 21 (p = 0.0032). Further, an increase in type I collagen deposition was observed in the membrane group compared to that in the control group on days 7 (p = 0.0133) and 14 (p = 0.0107). Conclusion: Treatment with norbixin-based PHB membranes reduces the inflammatory response, increases fibroblast proliferation, and improves collagen production in the tendon repair region, especially between days 7 and 14.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Usos terapéuticos de sustancias prohibidas: LSD, MDMA, THC, GHB, DMT, psilocybina y mescalina / Therapeutic uses of prohibited substances: LSD, MDMA, THC, GHB, DMT, psilocybin, mescaline

El presente texto hace un breve recorrido sobre los usos terapéuticos que han tenido la LSD, MDMA, THC, GHB, DMT, Psilocybina y Mescalina en la historia, así como también refiere algunos de los beneficios para la salud física y mental que se considera tienen en la actualidad. Esta información científica se contrapone a la normativa internacional en materia de drogas, que las clasifica como sustancias prohibidas en la Lista I, debido a su falta de uso médico aceptado por Estados Unidos y a su alto potencial de abuso. En este trayecto también se intenta comprender a qué hace referencia dicho potencial, así como las motivaciones que podrían existir detrás de la prohibición del uso terapéutico de estas drogas. En este marco, se consideran consecuencias para la salud de la población, las que atentan contra los Derechos Humanos de las personas que podrían requerir alguna de estas sustancias.

This paper makes a brief of the therapeutic uses have had the LSD, MDMA, THC, GHB, DMT, Psilocybin and Mescaline in history, as well as some of the benefits referred to physical and mental health that are considered today. This scientific information seems contrary to international legislation on drugs, which classifies as prohibited substances in Schedule I, due to its lack of acceptance medical use by the United States and its high potential for abuse. In this way also try to understand what makes this potential reference, and the reasons that could be behind the ban on therapeutic use of these drugs. In this framework, we consider health consequences of the population, which violate the human rights of people who may require some of these substances.

Emprego experimental da placa de compósito poli-hidroxibutirado/hidroxiapatita na fixação femoral em gatos / Experimental employment of polyhydroxybutyrate/hydroxyapatite composite plates in femoral fixation in cats

Avaliou-se o compósito de poli-hidroxibutirado (PHB) 70 por cento e hidroxiapatita (HA) 30 por cento na forma de placas para fixação óssea em gatos. Foram usadas placas do compósito com 60mm de comprimento por 10mm de largura e espessura variando de 3mm no centro a 5mm nas extremidades, com seis orifícios. A placa do compósito foi empregada na fixação de osteotomia de fêmur em quatro gatos, totalizando seis intervenções. Verificou-se a ruptura de cinco placas (83,3 por cento) até o quarto dia e de uma placa (16,7 por cento) aos 21 dias, quando se observou um calo ósseo exuberante. O resultado da implantação da placa no gato mostrou que o compósito não possui resistência suficiente para ser empregado como placas de fixação de fêmur em gatos.

The composite of polyhydroxybutyrate (PHB) 70 percent and hydroxyapatite (HA) 30 percent was evaluated as plate for bone fixation in cats. The employed composite plates presented six orifices and measured 60 x 10mm, length and width, respectively, with thickness ranging from 3 to 5mm according to the region. The composite plate was used in the fixation of femoral osteotomy in four cats, in a total of six interventions. There were ruptures in five plates (83.3 percent) until day 4 and in one plate (16.7 percent) on the day 21, when it was possible to observe an exuberant osseous callus. The result of the plate deployment in the cat showed that the composite does not have sufficient strength to be used as plate of femoral fixation in cats.

Macromolecular composition and anaerobic degradation of the sludge produced in a sequencing batch reactor

The effect of sequencing batch reactor (SBR) operating conditions on sludge macromolecular composition and the effect of sludge macromolecular composition on the anaerobic degradation of the sludge produced in SBR was investigated in this work. A SBR, fed with synthetic wastewater, was operated at different air flow rates. The resulting sludge was analyzed in terms of protein, carbohydrate, phospholipid and polyhydroxybutyrate concentrations. Methane production during anaerobic digestion of the sludge was also measured. Ammonium, nitrite, nitrate, dissolved oxygen and chemical oxygen demand (COD) track studies in the SBR were carried out in order to relate SBR performance and sludge macromolecular composition. The lowest air flow rate at which the SBR was operated was 2 l min-1, in which case the dissolved oxygen concentration was lower than 0.5 mg l-1 in the SBR and partial denitrification occurred during the feeding phase. An increased air flow rate caused a decrease in protein concentration, as well as an increase in carbohydrate concentration. Polyhydroxybutyrate (PHB) concentration in the sludge was independent of air flow rate. At different air flows, the methane production rates were similar, but the total volume of methane was greater during anaerobic digestion of the sludge produced at low air flow rates. These results indicate a strategy by which changes in sludge composition can optimize the operation of anaerobic sludge digesters.

Nutrient optimization for production of polyhydroxybutyrate from halotolerant photosynthetic bacteria cultivated under aerobic-dark condition

Three halotolerant bacterial strains; Rhodobacter sphaeroides ES16 (the wild type) and the two mutant strains of R. sphaeroides ES16, namely N20 and U7, were cultivated in glutamate-malate (GM) medium and screened for production of polyhydroxybutyrate (PHB). The mutant strains N20 and U7 were found to accumulate PHB (53.9 and 42.0 percent of DCW, respectively) 3.6 and 2.8 times higher than the wild type strain (19.5 percent of DCW), respectively. R. sphaeroides N20 were selected for studies on the effects of nutrient and environmental conditions on PHB accumulation. The optimal condition was 4 g/l acetate, 0.02 g/l (NH4)2SO4, C/N ratio of 6:1, 1.0 g/l K2HPO4, 1.0 g/l KH2PO4 and 3 percent NaCl with initial pH at 7.0. Under this optimal condition, the maximum PHB accumulation increased from 53.9 percent to 88 percent of DCW and 9.11 ± 0.08 g/l biomass, 8.02 +/- 0.10 g/l PHB concentration were achieved after 60 hrs cultivation at 37ºC. These results are the highest values ever obtained from photosynthetic bacteria reported so far.

Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10 percent fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70 percent) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45 percent) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated

Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-ß-hidroxibutirato (PHB) / Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-ß-hydroxybutyrate (PHB)

La centrifugación en gradientes de sacarosa permite la separación de células según su densidad boyante de acuerdo al contenido en poli-ß-hidroxibutirato (PHB). En este trabajo este método se evaluó y se adaptó para detectar mutantes deficientes en la síntesis de PHB de B. megaterium analizando un bajo porcentaje de la población mutagenizada