Biological response of protists Haematococcus lacustris and Euglena gracilis to conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate.

Improving the growth and pigment accumulation of microalgae by electrochemical approaches was considered a novel and promising method. In this research, we investigated the effect of conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) dispersible in water on growth and pigment accumulation of Haematococcus lacustris and Euglena gracilis. The results revealed that effect of PEDOT:PSS was strongly cell-dependent and each cell type has its own peculiar response. For H. lacustris, the cell density in the 50 mg·l-1 treatment group increased by 50·27%, and the astaxanthin yield in the 10 mg·l-1 treatment group increased by 37·08%. However, under the high concentrations of PEDOT:PSS treatment, cell growth was significantly inhibited, and meanwhile, the smaller and more active zoospores were observed, which reflected the changes in cell life cycle and growth mode. Cell growth of E. gracilis in all the PEDOT:PSS treatment groups were notably inhibited. Chlorophyll a content in E. gracilis decreased while chlorophyll b content increased in response to the PEDOT:PSS treatment. The results laid a foundation for further development of electrochemical methods to promote microalgae growth and explore the interactions between conductive polymers and microalgae cells.

Crossbow injury to the neck.

Crossbow injuries are uncommon among penetrating trauma. The tendency for a crossbow bolt to remain in situ appears to limit catastrophic haemorrhage despite the involvement of major vessels.1 Here we report our experience with an injury to the left internal jugular vein by a crossbow bolt. The injury was successfully treated by emergency neck exploration.

Reductions of docosahexaenoic acid-containing phosphatidylcholine levels in the anterior horn of an ALS mouse model.

In this study, we analyzed the spatiotemporal alterations of phospholipid composition in the spinal cord of an amyotrophic lateral sclerosis (ALS) mouse model (G93A-mutated human superoxide dismutase 1 transgenic mice [SOD1(G93A) mice]) using imaging mass spectrometry (IMS), a powerful method to visualize spatial distributions of various types of molecules in situ. Using this technique, we deciphered the phospholipid distribution in the pre-symptomatic stage, early stage after disease onset, and terminal stages of disease in female SOD1(G93A) mouse spinal cords. These experiments revealed a significant decrease in levels of docosahexaenoic acid (DHA)-containing phosphatidylcholines (PCs), such as PC (diacyl-16:0/22:6), PC (diacyl-18:0/22:6), and PC (diacyl-18:1/22:6) in the L5 anterior horns of terminal stage (22-week-old) SOD1(G93A) mice. The reduction in PC (diacyl-16:0/22:6) level could be reflecting the loss of motor neurons themselves in the anterior horn of the spinal cord in ALS model mice. In contrast, other PCs, such as PC (diacyl-16:0/16:0), were observed specifically in the L5 dorsal horn gray matter, and their levels did not vary between ALS model mice and controls. Thus, our study showed a significant decrease in DHA-containing PCs, but not other PCs, in the terminal stage of ALS in model mice, which is likely to be a reflection of neuronal loss in the anterior horns of the spinal cords. Given its enrichment in dorsal sensory regions, the preservation of PC (diacyl-16:0/16:0) may be the result of spinal sensory neurons being unaffected in ALS. Taken together, these findings suggest that ALS spinal cords show significant alterations in PC metabolism only at the terminal stage of the disease, and that these changes are confined to specific anatomical regions and cell types.

Assessment of four methodologies (microparticle enzyme immunoassay, chemiluminescent enzyme immunoassay, affinity column-mediated immunoassay, and flow injection assay-tandem mass spectrometry) for measuring tacrolimus blood concentration in Japanese liver transplant recipients.

Therapeutic drug monitoring (TDM) and subsequent dosage adjustment for individual patients in the treatment with tacrolimus are required after liver transplantation to prevent rejection and over-immunosuppression, which leads to severe infection and adverse reactions including nephrotoxicity. The purpose of this study was to evaluate the analytical performance among commercially available immunoassay methods, which were microparticle enzyme immunoassay (MEIA), chemiluminescent enzyme immunoassay (CLIA), and affinity column-mediated immunoassay (ACMIA), compared with an assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the flow injection assay (FIA-MS/MS) was also evaluated to determine whether it could be available as a new method of analysis in tacrolimus therapy. The blood tacrolimus concentrations in samples from liver transplant recipients (n = 102) were measured using MEIA, CLIA, ACMIA, and LC-MS/MS. Additional blood samples from liver transplant recipients (n = 54) were analyzed using both FIA-MS/MS and LC-MS/MS. Because the assay performance and characteristics of MEIA, CLIA, ACMIA, and FIA-MS/MS are relatively different, the measured data should be carefully considered depending on the methodology.

Blockade of IL-6 signaling by MR16-1 inhibits reduction of docosahexaenoic acid-containing phosphatidylcholine levels in a mouse model of spinal cord injury.

The interleukin (IL)-6 pathway plays an important role in recovery after spinal cord injury (SCI). The anti-IL-6 receptor antibody MR16-1 has been shown to suppress inflammation after SCI and promote recovery of motor function. The purpose of this study was to analyze the effects of MR16-1 on the expression patterns of phospholipids in the spinal cord in a mouse model of SCI. Eight-week-old C57BL/6JJmsSlc mice were used in this study. Laminectomy was performed at the ninth and tenth thoracic levels (T9-T10), and contusion injury of the spinal cord was induced at level T10. Immediately after SCI, mice were intraperitoneally injected with a single dose of MR16-1 (MR16-1 group) or a single dose of phosphate-buffered saline of the same volume (control group). Imaging mass spectrometry was performed to visualize phosphatidylcholine (PC) expression in the spinal cord 7 days after SCI. We found that MR16-1 treatment suppressed the infiltration of immune cells after SCI, and was able to increase the locomotor function post-injury. Phospholipid imaging revealed that the MR16-1 was able to prevent the reduction of docosahexaenoic acid (DHA)-containing PC in comparison with the control group. We also observed high levels of glial fibrillary acidic protein (GFAP) at the site of DHA-containing PC expression in the MR16-1 group. These results suggest that MR16-1 treatment influences the DHA-containing PC composition of GFAP-positive cells at the injury site as early as 7 days post-SCI.

Role of aquaporin-5 in gallbladder carcinoma.

BACKGROUND/PURPOSE: Aquaporins (AQPs) are important in controlling bile formation. However, the exact role in human gallbladder carcinogenesis has not yet been defined. METHODS: AQP-5-expressing gallbladder carcinoma (GBC) cell lines (NOZ) were transfected with anti-AQP-5 small interfering RNA (siRNA). Growth, migration, invasion assay, and drug susceptibility tests were performed. Next, microRNA (miRNA) expression was analyzed by miRNA oligo chip (3D-Gene®). AQP-5 and AQP-5-related miRNA target gene expressions were also analyzed using tissue microarray (TMA) in 44 GBC samples. RESULTS: Treatment with AQP-5 siRNA decreased cell proliferation, migration, and invasion. On the other hand, those cells increased IC50 of gemcitabine. By performing miRNA assays, miR-29b, -200a, and -21 were shown to be highly overexpressed in cells treated with AQP-5 siRNA NOZ. When focusing on miR-21, phosphatase and tensin homolog (PTEN) was found to be a target of miR-21. In the TMA, AQP-5/PTEN coexpression was significantly associated with the depth of invasion and MIB-1 index (p = 0.003, 0.010). Survival of patients with a high AQP-5/PTEN coexpression was longer than that of patients with a low coexpression (p = 0.003). CONCLUSIONS: Our result suggested that miR-21 and PTEN may contribute to the role of AQP-5 in GBC. AQP-5 and PTEN cascades are favorable biomarkers of GBC.

Monosaccharide composition of the outer membrane lipopolysaccharide and O-chain from the freshwater cyanobacterium Microcystis aeruginosa NIES-87.

AIMS: Bacterial lipopolysaccharide (LPS) protruding from the outermost layer of the outer membrane is expected to play an important role in cell physiology by interacting with molecules in the extracellular milieu; however, the structural and functional characteristics of these components in cyanobacteria remain largely unknown. We isolated water-soluble fractions of LPS and O-chain from the bloom-forming freshwater cyanobacterium Microcystis aeruginosa NIES-87 and identified their monosaccharide compositions. METHODS AND RESULTS: SDS-PAGE followed by silver staining demonstrated that the isolated total LPS was the smooth type with different numbers of repeating sugar units in the O-chain region. GC/MS analysis after acid hydrolysis, reduction and acetylation treatments indicated that the neutral monosaccharide components of the total LPS include glucose, rhamnose, mannose, galactose and xylose (in decreasing order of weight percentage), while only glucose was detected in the purified O-chain fraction. MALDI-TOF MS analysis suggested that the O-chain fraction is composed of repeating glucose and methylated glucose disaccharide units. CONCLUSIONS: Our results indicate that the monosaccharide composition of M. aeruginosa O-chain is relatively simple. SIGNIFICANCE AND IMPACT OF THE STUDY: Although further studies are necessary, these findings provide fundamental information for understanding the structural and functional properties of cyanobacterial LPS and O-chain.

A single amino acid substitution in the 126-kDa protein of pepper mild mottle virus controls replication and systemic movement into upper non-inoculated leaves of bell pepper plants.

Previously, we generated attenuated variants of pepper mild mottle virus by replacing residue 649 in the 126-kDa replicase protein with various amino acids. Here, we examined the biological properties of the 16 variants that caused either mild mosaic or no mosaic. All but one (A649N) of the mild-mosaic-inducing strains replicated at higher levels in pepper plants and systemically moved at higher rates into the upper non-inoculated leaves than the no-mosaic strains. C1421, previously selected for practical use, not only caused mild symptoms but also had an especially high replication rate in pepper plants and spread more efficiently into the upper non-inoculated leaves.

Effect of light on iron uptake by the freshwater cyanobacterium Microcystis aeruginosa.

Visible light was observed to induce reductive dissociation of organically complexed Fe and dramatically increase the short-term uptake rate of radiolabeled Fe by Microcystis aeruginosa PCC7806 in Fraquil* medium buffered by a single metal chelator, ethylenediaminetetraacetic acid (EDTA). Only wavelengths <500 nm activated Fe uptake indicating that Fe photochemistry rather than biological factors is responsible for the facilitated uptake. The measured rate of photochemical Fe(II) production combined with a significant decrease in (55)Fe uptake rate in the presence of ferrozine (a strong ferrous iron chelator) confirmed that photogenerated unchelated Fe(II) was the major form of Fe taken up by M. aeruginosa under the conditions examined. Mathematical modeling based on unchelated Fe(II) uptake by concentration gradient dependent passive diffusion of Fe(II) through nonspecific transmembrane channels (porins) could account for the magnitude of Fe uptake and a variety of other observations such as the effect of competing ligands on Fe uptake. Steady-state uptake rates indicated that M. aeruginosa acquires Fe predominantly during the light cycle. This study confirms that Fe photochemistry has a dominant impact on Fe acquisition and growth by M. aeruginosa in EDTA-buffered culture medium.

Norovirus-binding proteins recovered from activated sludge micro-organisms with an affinity to a noroviral capsid peptide.

AIMS: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro-organisms could be totally different from that of those particles moving freely, micro-organisms with natural affinity ligands such as virus-binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus-binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. METHODS AND RESULTS: NoVBPs were recovered from activated sludge micro-organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus-like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two-dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N-terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. CONCLUSIONS: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.

A single residue in the 126-kDa protein of pepper mild mottle virus controls the severity of symptoms on infected green bell pepper plants.

Infectious cDNA clones originally derived from a mild strain of Pepper mild mottle virus were constructed by replacing residue 649, a critical point for attenuation of this virus, with all possible amino acids. All clones were infectious to pepper plants and induced a variety of symptoms, including no visible symptoms. The results of this study showed that a single amino acid mutation at residue 649 could control the function of the 126- and 183-kDa proteins, replicases with multiple roles in the life cycle of this virus.

Molecular analysis of the genome segments S1, S4, S6, S7 and S12 of a Rice gall dwarf virus isolate from Thailand; completion of the genomic sequence.

The complete nucleotide sequences of the double-stranded RNA segments S1, S4, S6, S7 and S12 of the genome of a Rice gall dwarf virus (RGDV) isolate from Thailand were determined. The segments consisted of 4505, 2622, 1648, 1652 and 853 nucleotides, encoding putative proteins of 1458, 725, 489, 511 and 206 amino acids with molecular masses of approximately 166, 80, 53, 59 and 24 kDa, respectively. Homology searches indicated that each of the putative proteins has a counterpart in isolates of Rice dwarf virus (RDV) and Wound tumor virus, two other species in the genus Phytoreovirus. However, no similarities were found to other registered sequences, including those of other viruses that belong to the family Reoviridae. The identities between homologous structural proteins of RGDV and RDV ranged from 34 to 51% and were thus higher than those between homologous non-structural proteins of RGDV and RDV (16-37%). Among the nonstructural proteins, the highest amino acid sequence identity (37%) was observed for RGDV Pns11 and RDV Pns10, a constituent of tubular inclusions. This observation suggests that a specific amino acid backbone might be required for maintaining not only the three-dimensional structure of virions but also that of inclusions. The entire sequence of the RGDV genome is now available.

Restoration of shoulder function and elbow flexion by nerve transfer for poliomyelitis-like paralysis caused by enterovirus 71 infection.

We report the case of an eight-month-old girl who presented with a poliomyelitis-like paralysis in her left upper limb caused by enterovirus 71 infection. She recovered useful function after nerve transfers performed six months after the onset of paralysis. Early neurotisation can be used successfully in the treatment of poliomyelitis-like paralysis in children.

Membrane separation of indigenous noroviruses from sewage sludge and treated wastewater.

In this study, feasibility of membrane separation for the removal of indigenous noroviruses (NVs) is evaluated. The indigenous NV gene was never detected from ultrafiltration (UF) permeates of sewage sludge and treated wastewater. Indigenous NV gene was also not detected from permeates of sewage sludge and treated wastewater by microfiltration (MF) with a pore size of 0.1 microm (MF0.1). Even though the pore size of MF (0.1 microm) was much larger than the diameter of virus particle (approximately 30-40nm), more than 4-log10 reduction value (LRV) at maximum was achieved by membrane separation with MF0.1. NV genes were often detected from permeates of sewage sludge and treated wastewater by MF with a pore size of 0.45 microm (MF0.45), although the maximum log10 reduction values were more than 3.59 for sewage sludge and more than 2.90 for treated wastewater. It is important to verify factors determining the removal efficiency of viruses with MF membranes.

The relationship between pre-operative symptoms, operative findings and postoperative complications in schwannomas.

This study presents a retrospective review of the management of schwannomas in the limbs and examines the relationship between pre-operative clinical examination, operative findings and postoperative neurological complications. Eighteen tumours with a histological diagnosis of schwannoma in 17 patients who underwent surgery between 1998 and 2004 were the basis of this study. Enucleation of the tumour was possible in 14 cases. None of these patients had neurological complications pre-operatively but eight had mild neurological complications postoperatively. The complications consisted of sensory deficit in five cases, motor weakness in one and both in two. Enucleation of the tumours was impossible in four cases. These schwannomas originated in the brachial plexus in three cases and the ulnar nerve in the proximal arm in one case. Tumours with pre-operative symptoms and masses located at a proximal site in the limb were more likely to be impossible to enucleate completely.

Heavy metal-binding proteins from metal-stimulated bacteria as a novel adsorbent for metal removal technology.

Water pollution with toxic heavy metals is of growing concern because heavy metals could bring about serious problems for not only ecosystems in the water environment but also human health. Some metal removal technologies have been in practical use, but much energy and troublesome treatments for chemical wastes are required to operate these conventional technologies. In this study, heavy metal-binding proteins (HMBPs) were obtained from metal-stimulated activated sludge culture with affinity chromatography using copper ion as a ligand. Two-dimensional electrophoresis revealed that a number of proteins in activated sludge culture were recovered as HMBPs for copper ion. N-termini of five HMBPs were determined, and two of them were found to be newly discovered proteins for which no amino acid sequences in protein databases were retrieved at more than 80% identities. Metal-coordinating amino acids occupied 38% of residues in one of the N-terminal sequences of the newly discovered HMBPs. Since these HMBPs were expected to be stable under conditions of water and wastewater treatments, it would be possible to utilize HMBPs as novel adsorbents for heavy metal removal if mass volume of HMBPs can be obtained with protein cloning techniques.

Pns4 of rice dwarf virus is a phosphoprotein, is localized around the viroplasm matrix, and forms minitubules.

Rice dwarf virus (RDV), a member of the family Reoviridae, has a 12-segmented dsRNA genome. Seven segments, designated S1, S2, S3, S5, S7, S8, and S9, encode structural proteins, while the remainder encode nonstructural proteins. One of the nonstructural proteins, Pns4, which is encoded by S4, was characterized. Pns4 was a phosphorylatable substrate in a phosphorylation assay in vivo; it associated with large cytoplasmic fibrils and formed novel minitubules in infected cultured cells of its leafhopper insect vector, as revealed by immunofluorescence and immunoelectron microscopy. Early in infection, Pns4 was detected at the periphery of the viroplasm, and it was then observed on amorphous or fibrillar inclusions, which were identified as bundles of minitubules, at later stages of infection. Since viroplasms are believed to be the site of RDV replication, the intracellular location of Pns4 suggests that this protein might be involved in the process of assembly of the RDV virion.

Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia.

OBJECTIVE: To examine the effects of microbubble destruction with ultrasound (MB) combined with bone marrow derived mononuclear cell transplantation (BMT) into ischaemic tissues in rat hind limb ischaemia. METHODS AND RESULTS: Unilateral hind limb ischaemia was surgically induced in Lewis rats. At postoperative day 7, rats were randomly divided into three groups: a vehicle treated group, an ultrasound treated group, and an MB treated group. MB treatment increased vascular endothelial growth factor mRNA as assessed by real time polymerase chain reaction (3.0-fold, p < 0.05). At four weeks, the MB group had increases in laser Doppler blood flow index (LDBFI; 1.2-fold, p < 0.05), angiographically detectable collateral vessels (angiographic score: 1.4-fold, p < 0.01), and capillary to muscle fibre ratio (1.4-fold, p < 0.01) in ischaemic limbs compared with the vehicle treated group. No differences were seen between the vehicle and ultrasound treated groups. Secondly, rats were allocated to vehicle treatment, BMT (5 x 10(6) cells/rat), or a combination of MB and BMT (MB+BMT) at seven days after hind limb ischaemia. BMT treatment significantly increased LDBFI, angiographic score, and capillary to muscle fibre ratio compared with vehicle treatment. Interestingly, MB+BMT treatment produced significantly greater LDBFI (1.2-fold, p < 0.01), angiographic score (1.5-fold, p < 0.01), and capillary to muscle fibre ratio (1.5-fold, p < 0.05) than BMT treatment alone. CONCLUSIONS: MB may be a useful technique to enhance BMT induced neovascularisation.

Angiotensin converting enzyme inhibitor prevents left ventricular remodelling after myocardial infarction in angiotensin II type 1 receptor knockout mice.

BACKGROUND: It is well known that angiotensin converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT1) receptor blockers (ARBs) prevent left ventricular (LV) remodelling after myocardial infarction (MI). However, it is still not clear whether inhibition of the AT1 receptor is enough to prevent LV remodelling after MI. OBJECTIVE: To elucidate the effects of ACE inhibitors that are not mediated by the AT1 receptor on LV remodelling, MI was experimentally induced in wild-type (WT-MI) mice and AT1 receptor knockout (KO-MI) mice. METHODS: Mice were divided into six groups: WT-control, KO-control, WT-MI, KO-MI, WT-MI treated with an ACE inhibitor, and KO-MI treated with an ACE inhibitor. Four weeks after MI, cardiac function was assessed by Doppler echocardiography and non-infarcted myocardial mRNA expression by northern blot analysis. RESULTS: Cardiac function decreased significantly in the MI groups compared with the sham operated groups. Additionally, in the MI groups end diastolic dimension, E wave velocity, the ratio of peak velocity of E wave to A wave, deceleration rate of E wave, and mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, and collagens I and III increased significantly compared with the sham groups. LV remodelling after MI was prevented in KO-MI mice compared with WT-MI mice. ACE inhibitor administration significantly attenuated progressive LV remodelling in both WT and KO-MI groups. CONCLUSION: ACE inhibitors can prevent the LV remodelling process that accompanies cardiac dysfunction after MI, even in AT1 KO mice. These findings suggest that ACE inhibitors prevent LV remodelling after MI by mechanisms other than inhibition of angiotensin AT1 receptor mediated effects.

Integration of mutations responsible for the attenuated phenotype of Pepper mild mottle virus strains results in a symptomless cross-protecting strain.

An enhanced attenuated strain of Pepper mild mottle virus (PMMoV) was constructed by incorporating mutations that affect viral attenuation from three reported attenuated strains of PMMoV, which causes serious economic losses in the production of green pepper in Japan. The new strain caused no symptoms on pepper plants and protected them from infection by a wild-type strain. The mutations responsible for viral attenuation were located in the intervening region (IR) of the 126-kDa/183-kDa proteins. The mutations had synergistic effects in terms of the attenuation of symptoms and decreased the accumulation of the viral coat protein in infected pepper plants. In this paper, we propose an efficient method for the improvement of attenuated viruses by reverse genetics in plant viruses.