A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions

Microbial autotrophic biorefineries: Perspectives for biopolymer production

The use of autotrophic microorganisms to fabricate biochemical products has attracted much attention in both academia and industry. Unlike heterotrophic microorganisms that require carbohydrates and amino acids for growth, autotrophic microorganisms have evolved to utilize either light (photoautotrophs) or chemical compounds (chemolithotrophs) to fix carbon dioxide (CO₂) and drive metabolic processes. Several biotechnological approaches, including synthetic biology and metabolic engineering, have been proposed to harness autotrophic microorganisms as a sustainable/green production platform for commercially essential products such as biofuels, commodity chemicals, and biopolymers. Here, we review the recent advances in natural autotrophic microorganisms (photoautotrophic and chemoautotrophic), focusing on the biopolymer production. We present current state-of-the-art technologies to engineer autotrophic microbial cell factories for efficient biopolymer production

Acetate-Inducing Metabolic States Enhance Polyhydroxyalkanoate Production in Marine Purple Non-sulfur Bacteria Under Aerobic Conditions

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that a variety of microorganisms accumulate as carbon and energy storage molecules under starvation conditions in the presence of excess carbon. Anoxygenic photosynthetic bacteria exhibit a variety of growth styles and high PHA production activity. Here, we characterized PHA production by four marine purple non-sulfur bacteria strains (Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakhapatnamense) under different growth conditions. Unlike the well-studied PHA-producing bacteria, nutrient limitation is not appropriate for PHA production in marine purple non-sulfur bacteria. We found that marine purple non-sulfur bacteria did not accumulate PHA under aerobic conditions in the presence of malate and pyruvate. Interestingly, PHA accumulation was observed upon the addition of acetate under aerobic conditions but was not observed upon the addition of reductants, suggesting that an acetate-dependent pathway is involved in PHA accumulation. Gene expression analysis revealed that the expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions and increased with the addition of acetate, indicating that TCA cycle activity is involved in PHA production under aerobic conditions. We also found that expression of PdhRrs, which belongs to the GntR family of transcription regulators, in Rhodovulum sulfidophilum was upregulated upon the addition of acetate. Taken together, the results show that the changes in the metabolic state upon the addition of acetate, possibly regulated by PdhR, are important for PHA production under aerobic conditions in marine purple non-sulfur bacteria

Testing Orem’s self-care deficit theory : dependent care agency for duration of breast-feeding

Aims : The purpose of this study was to test of Orem’s theory of self-care deficit. Specifically, limiting factors influencing the duration of breast-feeding were analyzed with Orem model to clarify the ability needed by a mother for continuing breast-feeding. Background : The mean incidence of mothers with breast-feeding, mixtrophic breast-feeding or bottled milk is 52.6%, 43.2%, and 4.8%, respectively until one month after birth. However, the incidence of mothers with breast-feeding decreases to 46.2% just one month after birth. Although midwives instruct the importance of breast-feeding and promote breast-feeding by using various kinds of empirical care, there is not the standardized care with a high effect for continuation of breastfeeding. Method : descriptive study. Design Methodology : One hundred thirty-three mothers who delivered their babies in the University Hospital were included in this research. The hospital experienced around 400 birth in a year. Sixty-nine mothers of them fed a baby with breast milk (the breast-feeding group). The other mothers fed a baby with bottled milk or with bottled milk and breast milk (the mixed group). Results : According to Orem model, comments obtained by interviews were categorized, and we found 3 factors impairing continuation of breast-feeding, “crying of a child”, “inappropriate advice” and “previous experience”. The limiting factors were related to each other, and inexperience of a life with a baby, the time-consuming life without composure, and unskilled responses to the shortage of milk enhanced restriction of adaptability caused by a difference between an imagined baby and a real baby. Conclusions:This study support’s Orem’s model. Factors influencing the duration of breast-feeding were clarifyed by Orem’s limitaitions factors

Engineered Nanogel Particles Enhance the Photoautotrophic Biosynthesis of Polyhydroxyalkanoate in Marine Photosynthetic Bacteria

Improving polyhydroxyalkanoate (PHA, a biodegradable plastic) production under photoautotrophic cultivation is challenging for sustainable bioproduction. In this study, we demonstrated the use of engineered nanogel particles to enhance PHA accumulation in the marine photosynthetic bacterium Rhodovulum sulfidophilum under photoautotrophic culture. We screened the effect of 13 engineered nanogel particles on the cell growth and PHA accumulation of R. sulfidophilum. The addition of anionic nanogel particles significantly enhanced PHA accumulation in R. sulfidophilum up to 157-fold compared to that without nanogel particles. By performing ¹³C tracer experiments and gas chromatography–mass spectrometry analysis, we confirmed that HCO₃⁻ was assimilated throughout the central carbon metabolism and that the accumulated PHA was indeed incorporated from HCO₃⁻. Our results indicate successful PHA production with the supplementation of engineered nanogel particles under photoautotrophic cultivation in R. sulfidophilum. Furthermore, the strategy of using engineered nanoparticles demonstrated in this study may be applicable to other microbial cell factories to produce other commodity metabolites

Engineered Mutants of a Marine Photosynthetic Purple Nonsulfur Bacterium with Increased Volumetric Productivity of Polyhydroxyalkanoate Bioplastics

Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer

Relationship between mammary gland structures during pregnancy and breast-feeding

Study Purpose Diagnostic indices for prediction of breast milk production and assessment of care necessary for mammary gland structure during the pregnancy period were investigated. Methods Development of mammary gland during the pregnancy period was examined via ultrasound methodology. Each subject underwent three measurements once per trimester. Quantity of post-partum breast milk production was also measured ; furthermore, the relationship between mammary gland image and production quantity was evaluated. Results and conclusion As previously reported, mammary gland reaches a certain level of development during the course of pregnancy. Four distinct types of developmental pattern were observed. Mammary gland exhibited remarkable development up to the 15th week of pregnancy in comparison with non-pregnant adult females in type 1. In type 2, mammary gland displayed significant development from the 16th to the 35th week of pregnancy. In types 3 and 4, mammary gland tissue development, which was apparent from 35 weeks, was characterized by gland thickness of > 20 mm and < 20 mm, respectively. Among these four types, type 1 mammary gland demonstrated the largest level of breast milk production. Therefore, we suggest that understanding with respect to mammary gland structure during pregnancy contributes toward individual support in breast-feeding following childbirth

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants

Basic study of new diagnostic modality according to noninvasive measurement of the electrical conductivity of tissues

The purposes of this study were to estimate the electrical conductivity of tissues by non-invasively measuring the electrical bio-impedance, to develop a new method for tissue diagnosis, i.e., electrical impedance tomography (EIT). Tissue models were first designed taking into consideration the distribution of the fat tissue, muscle and bone in the human forearm, and then the intra-tissue distributions of electrical potential and field, and the electrical impedance in the models was theoretically analyzed by the three-dimensional finite element method. The electrical impedance of both forearms was measured in healthy human subjects, and estimated the electrical conductivity of individual local tissues. The results of the analysis showed that the distributions of electrical potential and field were affected by the presence of fat tissue but not by the presence or absence of bone. In addition, as a result of calculation of the electrical resistance of the extracellular fluid (Re) in each model, it was found that the value of bio-impedance was influenced by the presence of fat tissue, and the value of bio-impedance was increased by the intervention of a fat layer. The electrical conductivity estimated by fitting the observed values to the values obtained by finite element analysis was 0.40 S/m and 0.15 S/m for male muscle and fat tissue, and 0.35 S/m and 0.11 S/m for female muscle and fat tissue, respectively. The sex difference in the slope of linear approximation in the estimation of electrical conductivity of the males and females was thought to be due to sex differences in the properties and structure of fat tissue. These results suggest that local tissues can be diagnosed differentially and electrically by percutaneous measurement of local bio-impedance and subsequent estimation of the electrical conductivity of each tissue