Calcium Involved Directional Organization of Polymer Chains in Polyester Nanogranules in Bacterial Cells.

Soil bacteria accumulate polyesters (typically poly([R]-3-hydroxybutyrate (PHB), in which one end of the chain terminates with a carboxyl group) in the form of hydrated, amorphous nanogranules in cells. However, it is not clear what drives the structure of these biomaterials inside bacterial cells. Here, we determined that calcium guides intracellular formation of PHB nanogranules. Our systematic study using the surface zeta potential measurement and the carboxyl-specific SYTO-62 dye binding assay showed that the terminal carboxyl is not exposed to the granule surface but is buried inside native "unit-granules" comprising the mature granule. Extracellular Ca2+ was found to mediate the formation of these PHB unit-granules, with uptaken Ca2+ stored inside the granules. Comparative [Ca2+]-dependent fluorescence spectroscopy revealed that the native granules in Cupriavidus necator H16 act as a Ca2+ storage system, presumably for the regulation of its cytosolic Ca2+ level, but those from recombinant Escherichia coli do not. This study reveals intimate links between Ca2+ and native granule formation, and establishes a novel mechanism that intracellular PHB granules function as Ca2+ storage in order to relieve soil bacteria from Ca2+ stress.

Monochorionic dizygotic twins with discordant sex and confined blood chimerism.

UNLABELLED: Monochorionic (MC) pregnancy in humans is usually considered to be associated only with monozygotic twinning. However, several reports have revealed that dizygotic (DZ) twins can also share a chorion during pregnancy. A chimera is defined as an organism that contains different cells derived from two or more distinct zygotes. As artificial reproductive techniques develop, it can be predicted that the occurrence of MC DZ twins will increase, and DNA-fingerprinting methods, such as short tandem repeat (STR) analysis, will be essential for their accurate diagnosis. We report the first Korean case of MC DZ twins with blood chimerism, 46,XX/46,XY, as a consequence of in vitro fertilization/embryo transfer. The clinical phenotypes of the twins' genitalia were complete female and male, respectively. Monochorionicity was confirmed by pathological analysis of the placenta after delivery. The dizygosity and confined blood chimerism of the twins were confirmed by STR analysis using their peripheral lymphocytes and skin fibroblasts. The confined blood chimerism of the twins can be considered similar to the status of the hematopoietic system in patients after allogenic bone marrow transplantation. CONCLUSION: When MC twins with discordant sex are expected during pregnancy, it is important to consider the possibility of DZ twins showing normal sexual development, especially in twins who were fertilized using artificial reproductive techniques.

Elevated Pentraxin 3 in bone metastatic breast cancer is correlated with osteolytic function.

Pentraxin 3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies, but its exact role remains unclear. This study found that PTX3 expression was up-regulated in distant bone metastases of breast cancer compared to lung, liver, and brain metastases in 64 human breast cancer patients. Elevated expression of PTX3 was correlated with poor survival in patients with breast cancer. PTX3 expression was also up-regulated in a bone metastatic breast cancer cell line and further enhanced by pro-inflammatory cytokine TNFα. Administration of PTX3 promoted the migratory potential of breast cancer cells and the mobilization of macrophages, a precursor of osteoclasts (OCs), toward breast cancer cells. In addition, elevated expression of PTX3 by TNFα led to enhanced OC formation, implying the distinct role of PTX3 in osteolytic bone metastasis of breast cancer cells. Furthermore, PTX3 silencing using PTX3-specific siRNA prevented breast cancer cell migration, macrophage chemotaxis, and subsequent OC formation. These findings provide an important insight into the key role of PTX3 in inflammation-associated osteolytic complications of breast cancer.

Nanoscale poly(4-hydroxybutyrate)-mPEG carriers for anticancer drugs delivery.

Despite several advancements in chemotherapy, cancer is still the second most frequent cause of mortality worldwide. Drug delivery to solid tumors is one of the most challenging aspects in cancer therapy. In pharmaceutical industries biodegradable polymeric nanoparticles as drug carriers have attracted great research interest because of their biocompatibility, biodegradability and sustained release of drugs. In our study we prepared poly(4-hydroxybutyrate)-mPEG (P(4HB)-mPEG) nanocarriers for the delivery of cisplatin as anticancer drug to mouse hippocampal HT22 cells. P(4HB) is more suitable candidate to be utilized in pharmaceutical industries due to its wide medical applications. P(4HB) is a homopolymer of 4-hydroxybutyrate (4HB), and belongs to a diverse class of materials called polyhydroxyalkanoates (PHA) produced by microorganisms inside the cells as energy storage materials. P(4HB) has certain unique properties such as biocompatibility and rapid in vivo degradation, which differentiate it from others PHA based polymers. Novel amorphous amphiphilic block copolymer P(4HB)-mPEG nanocarriers were prepared and characterized. Flow cytometry, and confocal microscopy revealed a suppression effect by the cisplatin loaded nanocarriers on HT22 cell growth, and enhancement of apoptotic process of the cells compared to free drug treated cells. The amorphous polymeric nanocarriers could be effective vehicles for the sustained delivery of toxic anticancer drugs for the therapy of cancer.

Clinical and radiological features of pituitary stalk lesions in children and adolescents.

PURPOSE: The diagnosis of pituitary stalk lesion has been based on clinical feature, radiologic assessment for its critical location and role. This study aimed to investigate clinical symptoms, endocrine disturbance, magnetic resonance imaging (MRI) findings of pituitary stalk lesions in children and adolescents and to evaluate differences between neoplastic lesions with the others. METHODS: We performed a retrospective review of patients under 18 years old with pituitary stalk lesions diagnosed at the Seoul National University Children's Hospital between 2000 and 2013, by a text search for head MRI reports by using 'pituitary stalk', 'infundibulum', and 'infundibular stalk', as keywords. RESULTS: For the 76 patients, sixteen patients (21.1%) had congenital lesions, and 52 (68.4%) had neoplasms. No inflammatory lesions were found. Diabetes insipidus (DI) was the most common endocrine defect, diagnosed in 38 patients (50%). There was male predominance especially in neoplastic group. Thickened pituitary stalk was, but enhancement of lesion was not, associated with neoplasm. DI was more prevalent in neoplastic stalk lesions. Anterior pituitary dysfunction such as growth hormone and adrenocorticotropic hormone deficiencies were less prevalent in neoplastic lesions of pituitary stalk. CONCLUSION: In conclusion, the etiology of pituitary stalk lesions in children and adolescents is diverse and different from that in adults. Neoplastic pituitary stalk lesions can be differentiated from nonneoplastic lesions by systemic evaluation of clinical, hormonal, radiological findings.

Phosphorus in the young supernova remnant Cassiopeia A.

Phosphorus ((31)P), which is essential for life, is thought to be synthesized in massive stars and dispersed into interstellar space when these stars explode as supernovae (SNe). Here, we report on near-infrared spectroscopic observations of the young SN remnant Cassiopeia A, which show that the abundance ratio of phosphorus to the major nucleosynthetic product iron ((56)Fe) in SN material is up to 100 times the average ratio of the Milky Way, confirming that phosphorus is produced in SNe. The observed range is compatible with predictions from SN nucleosynthetic models but not with the scenario in which the chemical elements in the inner SN layers are completely mixed by hydrodynamic instabilities during the explosion.

Simultaneous inhibition of rhamnolipid and polyhydroxyalkanoic acid synthesis and biofilm formation in Pseudomonas aeruginosa by 2-bromoalkanoic acids: effect of inhibitor alkyl-chain-length.

Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

Haptic guided virtual reality simulation for targeted drug delivery using nano-containers manipulation.

When dealing with nano targeted drug delivery process the significant area of virtual reality application can be visualizing real time process and simulating it at nano-scale, since the effectiveness of a drug primarily depends on the affected cell and targeted doze. This paper proposes virtual reality (VR) as a tool to analyze and simulate nanoparticles (NPs) manipulation, in this paper amorphous NPs are analyzed and simulated in virtual environment. Haptic guides virtualizing the atomic force microscope (AFM) is applied in the virtual environment which allows the operators to sense and touch the NPs when evaluating its structure, drug release time, and behavioral study. Cisplatin was loaded as a modal drug to the self-assembled amorphous copolymer P(3HV-co-4HB)-b-mPEG NPs, where the efficiency and bioavailability of Cisplatin was further investigated. The prepared NPs when simulated in virtual environment proved to show good biocompatibility. Results showed that amorphous polymeric NPs could be efficient vehicles for the constant and targeted delivery of toxic anticancer drugs.

A seroepidemiological survey of toxocariasis among eosinophilia patients in Chungcheongnam-do.

There have been several reports on the relationship between toxocariasis and eosinophilia, but all have been limited to the areas of Seoul or Gangwon-do. In the present study, we investigated the seroprevalence of toxocariasis among eosinophilia patients in Chungcheongnam-do, the central district of Korea. Among the 101 patients tested, 51 (50.5%) were identified as positive by Toxocara ELISA, and 46 (45.5%) were confidently diagnosed with toxocariasis because of absence of any other cause of eosinophilia. Whereas 22 of 42 seropositive patients (52.3%) had a recent history of consuming raw livers, especially the cow liver, only 1 of 25 seronegative patients (4%) had done so (P<0.01). From these results, we could confirm that toxocariasis is related to eosinophilia, and infer that ingestion of raw cow liver plays a vital role in the transmission of toxocariasis in Chungcheongnam-do.

Amorphous amphiphilic P(3HV-co-4HB)-b-mPEG block copolymer synthesized from bacterial copolyester via melt transesterification: nanoparticle preparation, cisplatin-loading for cancer therapy and in vitro evaluation.

Cisplatin is a chemotherapeutic agent used against a variety of tumors. We determined the efficacy and bioavailability of cisplatin in the form of cisplatin-loaded self-assembled amphiphilic copolymer nanoparticles (NPs). Non-crystallizing bacterial copolyester was employed as hydrophobic segment to increase drug loading efficiency. Novel amorphous amphiphilic block copolymer P(3HV-co-4HB)-b-mPEG was synthesized from bacterial copolyester poly(3-hydroxyvalerate-co-4-hydroxybutyrate) coupled via transesterification reaction using bis(2-ethylhexanoate) tin catalyst to monomethoxypoly(ethylene glycol). The product was characterized, and core-shell particles with nanometer size range were prepared by emulsification-solvent evaporation method. Transmission electron microscopy (TEM) examination revealed that the NPs took the shape of spheres with inner concealed core of hydrophobic P(3HV-co-4HB) polymer and the outer shell formed by hydrophilic mPEG segment. The in vitro release profile of cisplatin from the core hydrophobic domain showed a sustained release of the drug. TEM and confocal microscopy examination revealed clearly the internalization of cisplatin-loaded NPs into the tumor cells. MTT assay, flow cytometry, western blot and confocal microscopy revealed a suppression effect by the NPs on tumor cell growth, and enhancement of apoptotic process of the tumor cells compared to free drug treated cells. The amorphous polymeric NPs could be effective vehicles for the sustained delivery of toxic anticancer drugs.

Synthesis and characterization of PHV-block-mPEG diblock copolymer and its formation of amphiphilic nanoparticles for drug delivery.

Despite the recent research interest in the field of nanoparticles delivery system, their structure modification and transport behavior of various hydrophobic drugs is poorly developed. In this article the synthesis of novel amphiphilic diblock copolymer poly([R]-3-hydroxyvalerate)-block-monomethoxy poly(ethylene glycol) (PHV-block-mPEG) was undertaken by modifying the structure of biodegradable and hydrophobic poly([R]-3-hydroxyvalerate) (PHV) with hydrophilic monomethoxy poly(ethylene glycol) (mPEG). The chemical combination of the two blocks was carried out in the melt using bis(2-ethylhexanoate) tin as transesterification catalyst. The synthesized product was characterized by gel permeation chromatography (GPC), 1H nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) analysis. The block copolymer self-assembled into amphiphilic nanoparticles with a core of hydrophobic PHV and a shell of hydrophilic mPEG in aqueous solution. Characterization of the nanoparticles showed the formation of discrete, spherically shaped nanoparticles with mean particle size of 200 +/- 1 nm and zeta potential of -14 +/- 1 mV. A hydrophobic drug thymoquinone was efficiently incorporated into the core hydrophobic domain of the nanoparticles and its release kinetics was studied in vitro. The amphiphilic PEGylated nanoparticles showed biocompatibility when checked in neuronal hippocampal cells of prenatal rat. Our results suggest that the amphiphilic nanoparticles with core-shell structures are potentially useful to develop novel drug carriers.

Metabolic relationship between polyhydroxyalkanoic acid and rhamnolipid synthesis in Pseudomonas aeruginosa: comparative ¹³C NMR analysis of the products in wild-type and mutants.

Polyhydroxyalkanoic acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty acids were also investigated using octanoic-1-¹³C acid. ¹³C NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-¹³C being converted from the octanoic acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-¹³C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-¹³C via fatty acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depended on the type of carbon sources. The rhlB mutant could be exploited as an efficient producer of the important biosurfactant HAAs (e.g., ~700 mg/L HAAs was obtained when grown on 60 mM octanoic acid).

Amphiphilic PHA-mPEG copolymeric nanocontainers for drug delivery: preparation, characterization and in vitro evaluation.

Amphiphilic biodegradable core-shell nanoparticles were prepared by emulsification-solvent evaporation technique from diblock copolymers which were synthesized by chemical coupling of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) to monomethoxy poly(ethylene glycol) (mPEG) through transesterification reaction. The nanoparticles were found to be assembled in aqueous solution into an outer hydrophilic shell of mPEG connected to the interior hydrophobic polyhydroxyalkanoate (PHA) copolymer core, which was identified by a comparative analysis of enzymatic degradation of the mPEG-coupled and non-coupled PHA nanoparticles. Morphological examination under atomic force microscope showed the formation of smooth spherically shaped nanoparticles. The average particle sizes and zeta potentials of amphiphilic nanoparticles were in the range of 112-162 nm and -18 to -27 mV, respectively. A hydrophobic drug thymoquinone was encapsulated in the nanoparticles and its release kinetics was studied. The in vitro cytotoxicity evaluation of the nanoparticles on prenatal rat neuronal hippocampal and fibroblast cells revealed that biocompatibility of the amphiphilic nanoparticles was generally independent of the ratio of comonomer units in the PHA block. In conclusion, the amphiphilic nanoparticles contained the hydrophobic PHA segments buried in the core and could thus be used as safe carriers for the controlled release of variety of hydrophobic drugs.

Production of poly-N-acetylglucosamine by Staphylococcus saprophyticus BMSZ711: characterization and production optimization.

This is the first report on the characterization and production optimization of poly-N-acetylglucosamine (PNAG) in Staphylococcus saprophyticus. A strain producing glucosamine exopolysaccharide was isolated and characterized by biochemical test and 16S rRNA gene sequence homology analysis and named as S. saprophyticus BMSZ711. The molecular mass of the purified exopolymer was about 12 kDa. Digestion of the PNAG with DispersinB proved that it has beta-1,6 linkage. BMSZ711 can only produce PNAG when grown in M1 minimal medium but not in nutrient rich medium with optimum temperature of 30 degrees C and pH of 7. Glycerol and ammonium sulfate were found to be the best carbon and nitrogen source, respectively. Maximum PNAG production was obtained when glycerol 100mM, ammonium sulfate 0.3%, yeast extract 1.5 g/L, sodium chloride 10 g/L and valine 2mM were used.

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens BM07 enhancing the production of polyhydroxyalkanoic acid but deficient in cold-induced exobiopolymer production.

Pseudomonas fluorescens BM07 is known to produce cold-induced exobiopolymer, which is mainly composed of water-insoluble hydrophobic polypeptides (up to 85%) and saccharides (8%), by decreasing the culture temperature down to as low as 10 degrees C. We screened for transposon insertion mutants of P. fluorescens BM07 that were unable to produce the exobiopolymer. Among the eight mutants that showed the deficiency of exobiopolymer and O-lipopolysaccharide, one mutant BM07-59 that had the highest polyhydroxyalkanoates (PHA) production was selected. The transposon inserted gene in BM07-59 was identified as galU. The disruption of the gene galU coded for the putative product, UDP-glucose pyrophosphorylase (GalU), resulted in 1.5-fold more accumulation of PHA compared with the wild-type strain from 70 mM fructose or galactose at 30 degrees C. Electrophoretic analysis of lipopolysaccharide showed that the mutant lacked the O-antigen lipopolysaccharide bands. The glycosyl composition of the lipopolysaccharide produced by the mutant strain was significantly different from that of the wild-type strain. We suggest that the deletion of galU could be a way to shift carbon flux efficiently from exobiopolymer toward PHA in P. fluorescens BM07.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07.

Correlating metal ionic characteristics with biosorption capacity of Staphylococcus saprophyticus BMSZ711 using QICAR model.

Quantitative Ion Character-Activity Relationship (QICAR) was used for correlating metal ionic properties with maximum biosorption capacity (q(max)). Heat inactivated biomass of Staphylococcus saprophyticus BMSZ711 was studied for biosorption of nine metal ions. Influence of contact time and initial pH was checked. q(max) was determined by Langmuir isotherm and followed a descending sequence (in mmol/g): Pb(2+)>Cd(2+)>Cr(3+)>Zn(2+)>Hg(2+)>Cu(2+)>Co(2+)>Ni(2+)>K(+). q(max) values was modeled with 20 metal ionic characteristics, among these covalent index (X(m)(2)r) was best fitted with q(max) for all metal ions tested, in the following model: q(max)=0.09+0.11(X(m)(2)r) (R(2)=0.73, AIC=-4.14). Classification of metal ions according to valence or soft/hard improved QICARs modeling and more characteristics significantly correlated with q(max) which revealed that covalent bonding played major role in biosorption of soft metal ions and ionic bonding for borderline and hard ions. Biosorption capacity was most effectively predicted (R(2)=0.99, AIC=-8.04) with a two variable model containing electro-negativity (X(m)) and softness index (o(rho)(')).

Discriminative cytotoxicity assessment based on various cellular damages.

There are several assays currently available for the assessment of cell cytotoxicity, including trypan blue exclusion, lactate dehydrogenase (LDH) release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays. Trypan blue exclusion and LDH release assays are appropriate for evaluating cell membrane damage and a colorimetric MTT assay is available for measuring mitochondrial-related reduction capacity. As these assays were randomly utilized to assess the extent of cell damage, we suggest herein that the assay should be selected in accordance with the prevailing cellular situation. This can be determined by using a variety of cell types with differing reduction status, exogenous and endogenous oxidative stressors, and several different oxidized/reduced molecules. Although the trypan blue exclusion and released LDH assay have proven useful for assessments of necrotic and apoptotic cell death with membrane damage, the LDH assay is not appropriate for the measurement of the number of varied cells without membrane damage. In addition, when the cells were treated with exogenous and endogenous oxidative stressors, MTT reduction was shown to be sensitive to a shift to a more oxidizing cellular environment within a narrow range without loss of membrane integrity, and this effect increased in a linear fashion, dependent on the dosage of cytosolic extracts containing various physiological reductants, small reductive molecules (NADPH and GSH), and artificial DTT reducing agent. Finally, we noted that the MTT assay is available for the determination of small-scale oscillations in cellular reduction status and changes in mitochondrial functional activity, but not for evaluating the cytotoxicity of cells with a higher cellular reduction capacity. Altogether, the findings of this study indicate that tools for the testing of cytotoxicity should be selected differently by considering the correlation between the cellular conditions for various stimuli and the principle underlying the assay system.