Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Dynamic ultrasound-guided short-axis needle tip navigation technique vs. landmark technique for difficult saphenous vein access in children: a randomised study.

Dynamic ultrasound-guided short-axis needle tip navigation is a novel technique for vascular access. After venipuncture, the needle and catheter are further advanced within the vessel lumen under real-time ultrasound guidance with constant visualisation of the needle tip in the short-axis view. This can minimise the risk of transfixing the cannulated vessel. We compared two techniques for non-visible saphenous vein cannulation under general anaesthesia in children weighing ≥ 3 kg and less than four years of age: dynamic ultrasound-guided short-axis needle tip navigation technique (ultrasound group) vs. landmark technique. Venous cannulation was performed by three experienced anaesthetists. The primary outcome measure was first-attempt success rate. Success rate within 10 min was a secondary outcome. A total of 102 patients were randomly allocated to either the ultrasound group or the landmark group. First-attempt success rate was 90% in the ultrasound group compared with 51% in the landmark group, p<0.001, difference 39%, 95% confidence interval (CI) of the difference 23-55%. Success rate within 10 min was 92% in the ultrasound group compared with 63% in the landmark group, p = 0.001, difference 29%, 95%CI of the difference 14-45%. We conclude that, when performed by experienced anaesthetists, the dynamic ultrasound-guided short-axis needle tip navigation technique improved non-visible saphenous vein cannulation in children compared with the landmark technique.

Interpretation of chest fluoroscopy: the risk of misdiagnosing atelectasis as pneumothorax due to greyscale inversion.

Anaesthetists may be required to work in hybrid theatres for procedures using fluoroscopic imaging. Adequate knowledge of fluoroscopic images allows prompt and effective emergency management of complications which arise during procedures. Here, we present a case of severe hypotension and hypoxia occurring shortly after induction of anaesthesia. Atelectasis was mistaken for a pneumothorax due to misinterpretation of fluoroscopic imaging, which demonstrated a dark pleural cavity peripheral to a partially collapsed left lung, leading to an incorrect diagnosis. This case highlights the importance of understanding greyscale inversion in fluoroscopy.

Photoinduced properties of anodized Ti alloys for biomaterial applications.

The photocatalytic properties of anodic oxides on a newly developed TiNbSn and commonly used Ti6Al4V alloys as biomaterials were investigated. The alloys were anodized in an electrolyte of sodium tartrate acid with H2O2 at a high voltage and the mechanism of the photocatalytic and antiviral activities was studied. The anodized TiNbSn and Ti6Al4V exhibited highly crystallized rutile TiO2 and poorly crystallized anatase TiO2, respectively. X-ray photoelectron spectroscopy analysis revealed the presence of oxides of the alloying elements in addition to TiO2. The anodized TiNbSn exhibited higher activities than Ti6Al4V, and electron spin resonance spectra indicated that the number of hydroxyl radicals (⋅OH) generated from the anodized TiNbSn was higher than that from the anodized Ti6Al4V. The results can be explained by two possible mechanisms: the higher crystallinity of TiO2 on TiNbSn than that on the Ti6Al4V reduces the number of charge recombination sites and generates abundant ⋅OH; charge separation in the anodic oxide on TiNbSn due to the electronic band structure between TiO2 and the oxides of alloying elements enhances photo activities. The excellent photoinduced characteristics of the anodized TiNbSn are expected to contribute to the safe and reliable implant treatment.

Anoxygenic photosynthesis and iron-sulfur metabolic potential of Chlorobia populations from seasonally anoxic Boreal Shield lakes.

Aquatic environments with high levels of dissolved ferrous iron and low levels of sulfate serve as an important systems for exploring biogeochemical processes relevant to the early Earth. Boreal Shield lakes, which number in the tens of millions globally, commonly develop seasonally anoxic waters that become iron rich and sulfate poor, yet the iron-sulfur microbiology of these systems has been poorly examined. Here we use genome-resolved metagenomics and enrichment cultivation to explore the metabolic diversity and ecology of anoxygenic photosynthesis and iron/sulfur cycling in the anoxic water columns of three Boreal Shield lakes. We recovered four high-completeness and low-contamination draft genome bins assigned to the class Chlorobia (formerly phylum Chlorobi) from environmental metagenome data and enriched two novel sulfide-oxidizing species, also from the Chlorobia. The sequenced genomes of both enriched species, including the novel "Candidatus Chlorobium canadense", encoded the cyc2 gene that is associated with photoferrotrophy among cultured Chlorobia members, along with genes for phototrophic sulfide oxidation. One environmental genome bin also encoded cyc2. Despite the presence of cyc2 in the corresponding draft genome, we were unable to induce photoferrotrophy in "Ca. Chlorobium canadense". Genomic potential for phototrophic sulfide oxidation was more commonly detected than cyc2 among environmental genome bins of Chlorobia, and metagenome and cultivation data suggested the potential for cryptic sulfur cycling to fuel sulfide-based growth. Overall, our results provide an important basis for further probing the functional role of cyc2 and indicate that anoxygenic photoautotrophs in Boreal Shield lakes could have underexplored photophysiology pertinent to understanding Earth's early microbial communities.

Bioactive TiNbSn alloy prepared by anodization in sulfuric acid electrolytes.

The bioactivity of anodized near-ß TiNbSn alloy with low Young's modulus prepared in sulfuric acid electrolytes was examined to explore the osseointegration mechanism with a focus on the role of anodic oxide. Hydroxyapatite (HA) precipitated on the surface of anodic oxide following immersion in Hank's solution, and precipitation accelerated with increase in the sulfuric acid concentration of the electrolyte. HA is formed on the surface of as-anodized oxide without subsequent annealing or hot water (HW) treatment. This outcome differs from that of a previous study using anodized TiNbSn alloy prepared in acetic acid electrolytes requiring for subsequent HW treatment. It was found that the oxide anodized in sulfuric acid electrolyte contains a large amount of internal pores and is highly crystallized thick TiO2, whereas the same prepared in the acetic acid electrolyte is low crystalline thin TiO2 containing a small amount of pores. The present anodized TiNbSn alloy is preferred for maintaining the low Young's modulus of the alloy and eliminating the subsequent treatment to increase the Young's modulus. A model to rationalize the bioactivity of the present anodic oxide is proposed based on the series of studies. It is concluded that the sulfuric acid electrolyte is favorable for both HA formation and low Young's modulus, and the bioactivity is attributed to the anodic TiO2 that facilitates incorporation of bone ingredients.

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors.

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC.

Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor.

Single-chain immunotoxins anti-Tac(Fv)-PE40 and anti-Tac(Fv)-PE40KDEL, composed of variable domains of the anti-Tac monoclonal antibody and truncated forms of Pseudomonas exotoxin, have shown potent cytotoxic activity against malignant peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia (ATL) patients originating from the Caribbean. However, several clinically important issues have not previously been addressed. These include the potential of soluble interleukin 2 receptor in ATL patients to block immunotoxin effectiveness, the relative sensitivity of malignant lymph node cells (LNCs) versus PBMCs, the effect of an immunotoxin with a prolonged half-life, and finally whether ATL cells from patients in Japan have toxin sensitivity equal to those of the Caribbean patients. To resolve these questions, we studied 32 malignant PBMC and LNC samples from 30 ATL patients from Japan. PBMCs from 27 of 27 patients were very sensitive with 50% inhibition of protein synthesis achieved with 0.02-0.85 ng/ml (0.3-13 pM) of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fv)-PE40. LNCs had sensitivity very similar to that of PBMCs in the five patients tested. The fully recombinant immunotoxin, anti-Tac(Fab)-PE40, which has 8-10 times the t1/2 alpha and beta compared to the Fv-immunotoxins, was also very cytotoxic toward cells from 27 of 27 patients tested with 50% inhibition of protein synthesis of 0.08-25 ng/ml. It was found that purified soluble interleukin 2 receptor added to the cytotoxicity assay decreased the cytotoxic activity of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fab)-PE40, but that 1 x 10(4) units/ml or less had minimal competitive effects. It was found that ATL patients who have responded even incompletely to conventional chemotherapy have soluble interleukin 2 receptor levels lower than this at posttreatment. We conclude that recombinant immunotoxins containing anti-Tac(Fv) are effective against Japanese ATL PBMCs or LNCs and might be most effective if used in vivo after conventional chemotherapy. If it is found in humans that the effectiveness of single-chain recombinant toxins is limited by short half-life, anti-Tac(Fab)-PE40 should be considered as an alternative agent.

Establishment and characterization of a new human megakaryoblastic cell line (SET-2) that spontaneously matures to megakaryocytes and produces platelet-like particles.

A new factor-independent megakaryoblastic cell line, designated SET-2, was established from the peripheral blood of a patient with leukemic transformation of essential thrombocythemia (ET). SET-2 expressed CD 4, 7, 13, 33, 34, 36, 38, 41, 61, 71, 117, 126, 130 and c-mpl. In addition, it spontaneously produced numerous platelet-like particles in liquid culture. These particles were shown to be the same size as normal platelets, and to express CD 36, 38, 41, 61 and 71. Proliferation of SET-2 was not influenced by thrombopoietin (TPO) and other hemopoietic cytokines. SET-2 was found to express the platelet-specific proteins such as platelet factor 4 and beta-thromboglobulin. The levels of expression were not altered by TPO. SET-2 also secreted interleukin-6 into the supernatants, as well as normal megakaryocytes. These results suggest that SET-2 spontaneously matures to megakaryocytes and produces platelet-like particles. These findings indicate that SET-2 may be useful for investigating the proliferation and differentiation mechanisms of leukemia cells and the role of c-mpl on megakaryoblasts, megakaryocytes, and platelets in ET. Leukemia (2000) 14, 142-152.

Prognostic significance of multidrug resistance protein in adult T-cell leukemia.

The response of adult T-cell leukemia (ATL) to chemotherapy is poor, and a major obstacle to successful treatment is intrinsic or acquired drug resistance. To determine the clinical significance of multidrug resistance protein (MRP) 1 in ATL, we studied MRP1 expression and its association with clinical outcome. The expression of MRP1 mRNA in leukemia cells from 48 ATL patients was studied by slot blot analysis. The expression level of MRP1 mRNA in chronic-type ATL was significantly higher than that in lymphoma-type ATL (P = 0.033). There was no correlation between MRP1 expression and age, gender, WBC count, LDH, hypercalcemia, blood urea nitrogen, or performance status. However, the expression of MRP1 mRNA correlated only with peripheral blood abnormal lymphocyte counts (P = 0.008). The transporting activity of MRP1 was assessed using membrane vesicles. Membrane vesicles prepared from ATL cells with high expression of MRP1 mRNA showed a higher ATP-dependent leukotriene C(4) uptake than did those with low expression of MRP1 mRNA. This uptake was almost completely inhibited by LTD(4) antagonists ONO-1078 and MK571. In acute- and lymphoma-type ATL, high expression of MRP1 mRNA at diagnosis correlated with shorter survival, and Cox regression analysis revealed that MRP1 expression was an independent prognostic factor. These findings suggest that functionally active MRP1 is expressed in some ATL cells and that it is involved in drug resistance and has a possible causal relationship with poor prognosis in ATL. Multidrug resistance-reversing agents, such as ONO-1078 and MK571, that directly interact and inhibit the transporting activity of MRP1 may be useful for treating ATL patients.

[Hemothorax associated with congenital pulmonary arteriovenous fistula; report of a case].

A 20-year-old man was admitted to our hospital with a sudden onset of dyspnea, caused by right pleural effusion. The patient was in a state of shock. Thoracentesis was revealed hemothorax. Right thoracotomy revealed pulmonary arteriovenous fistula projecting into the intrapleural space from the right upper lobe. Partial resection was performed. His postoperative course was uneventful. Intrapleural rupture of pulmonary arteriovenous fistula was reported rarely; therefore we described here our case.

[Surgery for a pericardial diverticulum; report of a case].

We report a case with pericardial diverticulum in the mediastinum. A 64-year-old woman visited our hospital because of cough and dyspnea. Chest X-ray showed a smooth-bordered mass in the right superior mediastinum and computed tomography (CT) showed a homogeneous cystic mass in the paratracheal area. During right mini-thoracotomy, a thin-walled cystic lesion was found involving the trachea, the superior vena cava, and the azygos vein. The cyst extended a duct toward the pericardium. We performed total excision and pathological examination confirmed the diagnosis of a pericardial diverticulum. Mini-thoracotomy provides an effective and minimally invasive means of excising the mediastinal mass, relieving symptoms, and allowing pathological examination.

Analyses of mixed melanogenesis in tyrosinase cDNA-transfected human amelanotic melanoma cells.

In the pigment cell the synthesis of tyrosinase and the formation of premelanosomes are independent, yet coordinated, processes. However, the interrelationship between the two processes has not been elucidated previously. In this study, an expression plasmid for human tyrosinase cDNA was constructed and transfected into a human amelanotic melanoma cell line, G-361. Stable transfected cells (G-CMHT-3) were obtained with high tyrosinase activity and distinct melanization occurred. As for the type of melanin, both pheo- and eumelanin contents increased in G-CMHT-3 cells. Interestingly, catalase activity as one of the other melanogenic enzymes was decreased in G-CMHT-3 cells. The decrease of catalase activity was considered to play a role in melanin-polymer formation, resulting in the increase of both pheo- and eumelanin contents. Under electron microscopic observation, dopa-oxidase-positive Golgi-associated endoplasmic reticulum of lysosome, coated vesicles, and premelanosomes were observed in pigmented G-CMHT-3 cells, and the expressed tyrosinase was considered to be well translocated to these organelles. In addition, the number of premelanosomes (stages I-III) as well as melanosomes (stage IV) increased in G-CMHT-3 cells compared to those in G-361 cells. It is also noted that G-CMHT-3 cells showed more normal phenotype premelanosomes with occasional transitional premelanosomes exhibiting partial melanin polymer formation within their concentric whorl-like internal membranes. Furthermore, the number of eumelanosomes in G-CMHT-3 cells was much larger than that in G-361 cells. These results suggest that the tyrosinase introduced by its cDNA transfection induced active and structurally non-aberrant premelanosome formation resulting in the upregulated pheo- and eumelanogenesis.

Decreased serotonin S2 and increased dopamine D2 receptors in chronic schizophrenics.

Serotonin S2 and dopamine D2 receptors in the prefrontal cortex and caudate nucleus of postmortem brains of chronic schizophrenics were studied using 3H-ketanserin and 3H-spiperone, respectively. In the prefrontal cortex of schizophrenics, we found a significant decrease in the maximum number of 3H-ketanserin binding sites (Bmax), with no change in the dissociation constant (Kd). Conversely, both Bmax and Kd of 3H-spiperone binding to the caudate nucleus were significantly increased in the schizophrenic patients. There were no differences in receptor indices between patients who were taking neuroleptics until their death and those who had taken none for 2 months or more prior to death. These findings suggest that alterations in S2 receptors in the prefrontal cortex may reflect the disease process, per se, and that the increase in the number of D2 receptors in the caudate nucleus of schizophrenics is not due solely to neuroleptic medication.

An enzyme-linked immunosorbent assay for immune complex of HTLV-I.

A sandwich enzyme-linked immunosorbent assay (ELISA) for immune complexes of human T cell leukemia virus type I (HTLV-I) was developed using monoclonal antibody (MoAb) 3G1 which recognizes a different epitope on HTLV-I to that with which natural human anti-HTLV-I antibody binds. The assay was capable of titrating artificial immune complexes not only at antigen-antibody equivalence but also at antibody excess. Although the antigen-antibody ratios could not be determined in the individual sera from patients with overt ATL, the level of immune complexes in three out of four sera was estimated to be 250 +/- 36 ng/ml. Immune complexes of HTLV-I could not be identified in sera obtained from one patient with overt ATL, three healthy HTLV-I carriers and three normal human controls.

Identification of interleukin-6 producing fibroblastoid cells in cerebrospinal fluid from patients with leukemic meningitis.

Cytokine producing native cells in cerebrospinal fluid (CSF) have not been identified. So, we investigated the cytokine producing ability of floating cells in CSF from patients with leukemic meningitis. Morphologic study revealed that established cell lines were polygonal or elongated in shape and had an abundant and irregular branched cytoplasm. Immunocytochemical analysis demonstrated positive reactivity with monoclonal anti-fibroblast antibody only. Interleukin-6 (IL-6) was constitutively produced in vitro by these cell lines; both interleukin-1 and lipopolysaccharides significantly increased its synthesis. These findings imply that these fibroblastoid cells are floating in CSF of patients with leukemic meningitis and produce IL-6 in response to various inflammatory stimulations in vivo.

HLA-A*26, HLA-B*4002, HLA-B*4006, and HLA-B*4801 alleles predispose to adult T cell leukemia: the limited recognition of HTLV type 1 tax peptide anchor motifs and epitopes to generate anti-HTLV type 1 tax CD8(+) cytotoxic T lymphocytes.

Genetic risk for adult T cell leukemia (ATL) has been implicated by ethnic and familial segregation of ATL patients from HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the genetic risk for ATL, we characterized HLA class I alleles of ATL patients and analyzed the anchor motifs of HTLV-1 peptides binding to HLA class I molecules, using 291 lines of anti-HTLV-1 CD8(+) cytotoxic T lymphocytes (CTLs) generated in vitro with a total of 165 synthetic peptides for HTLV-1 Tax and Env proteins. Allele frequencies of HLA-A*26, B*4002, B*4006, and B*4801 were significantly higher in ATL patients than in HAM/TSP patients and asymptomatic HTLV-1 carriers in southern Japan. CD8(+) CTL analysis revealed the HTLV-1 Tax peptide sequence to completely lack anchor motifs of peptides binding to HLA-A*26,B*4002, and B*4006 molecules but to possess one anchor for HLA-B*4801, while the HTLV-1 Env peptide sequence had many anchor motifs for HLA-A*26, B*4002, B*4006, and B*4801 molecules. Most ATL patients featured heterozygous HLA class I alleles composed of HLA-A*26, B*4002, B*4006, and B*4801, with a lower number of HTLV-1 Tax peptide anchor motifs and epitopes generating anti-HTLV-1 Tax CD8(+) CTLs than individuals possessing other HLA alleles. The relationship between Tax epitope and ATL incidence was verified by the significantly decreased number of HTLV-1 Tax epitopes in ATL patients compared with asymptomatic HTLV-1 carriers (p < 0.01) as well as late onset ATL patients (p < 0.001). These results indicate that HLA-A*26, B*4002, B*4006, and B*4801 alleles predispose to ATL because of the limited recognition of HTLV-1 Tax peptide anchor motifs and epitopes capable of generating anti-HTLV-1 Tax CD8(+) CTLs.

Improved outcome of adult T cell leukemia/lymphoma with allogeneic hematopoietic stem cell transplantation.

Adult T cell leukemia/lymphoma (ATL) is a poor prognosis T cell malignancy. In order to improve the outcome, we employed allogeneic stem cell transplantation (allo-SCT) for ATL in 10 patients, nine of whom were from HLA-identical siblings and one from an unrelated donor. Conditioning regimens varied among the patients except that all received total body irradiation. The patients tolerated the regimens well with mild, if any toxicity, and engraftment occurred in all cases. Median leukemia-free survival after allo-SCT was 17.5+ months (range 3.7-34.4+). Six of the 10 patients developed acute GVHD (one case each with grade I, III or IV, and three cases with grade II) and three patients developed extensive chronic GVHD. Four patients died after allo-SCT during the study period from either acute GVHD (grade IV), pneumonitis, gastrointestinal bleeding or renal insufficiency. Two of the 10 cases with no symptoms of GVHD relapsed with clinical ATL. These results strongly suggest that allo-SCT may improve the survival in ATL if a controlled degree of GVHD develops.

Isolation and characterization of malic enzyme from the pupa of Bombyx mori.

Malic enzyme, which requires NADP+ as a coenzyme, was isolated and purified from pupae of the silkworm, Bombyx mori. The purified enzyme appeared homogeneous and had a molecular weight of 195,000 on polyacrylamide gel electrophoresis. The optimum pH for the oxidative decarboxylation of malate, measured in terms of the increase of NADPH (MH activity) and CO2 (MC activity), was pH 7.5, while that for the decarboxylation of oxaloacetate measured in terms of the increase of CO2 (OC activity) was pH 4.6. Several differences between MH and OC activity were investigated.