Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

Identification of novel neuroblastoma biomarkers in urine samples.

Urine is a complex liquid containing numerous small molecular metabolites. The ability to non-invasively test for cancer biomarkers in urine is especially beneficial for screening child patients. This study attempted to identify neuroblastoma biomarkers by comprehensively analysing urinary metabolite samples from children. A total of 87 urine samples were collected from 54 participants (15 children with neuroblastoma and 39 without cancer) and used to perform a comprehensive analysis. Urine metabolites were extracted using liquid chromatography/mass spectrometry and analysed by Metabolon, Inc. Biomarker candidates were extracted using the Wilcoxon rank sum test, random forest method (RF), and orthogonal partial least squares discriminant analysis (OPLS-DA). RF identified three important metabolic pathways in 15 samples from children with neuroblastoma. One metabolite was selected from each of the three identified pathways and combined to create a biomarker candidate (3-MTS, CTN, and COR) that represented each of the three pathways; using this candidate, all 15 cases were accurately distinguishable from the control group. Two cases in which known biomarkers were negative tested positive using this new biomarker. Furthermore, the predictive value did not decrease in cases with a low therapeutic effect. This approach could be effectively applied to identify biomarkers for other cancer types.

Quantifying late-stage host-seeking behaviour of Anopheles gambiae at the insecticidal net interface using a baited-box bioassay.

BACKGROUND: Insecticide-treated nets (ITNs) are losing efficacy against pyrethroid-resistant malaria vector populations throughout Africa. Safeguarding bed net efficacy, vital for effective malaria control, requires greater knowledge of mosquito-ITN interactions and how this impacts on the mosquito. METHODS: A purpose-built benchtop apparatus with a closed 10 cm cubic chamber (the 'Baited-box') was used to video record behaviour of individual free-flying female Anopheles gambiae during approach and blood-feeding on a human hand through untreated nets and ITNs at close range. Time and duration of defined behavioural events, and knockdown and mortality at 1- and 24-h post-exposure respectively, were recorded for pyrethroid susceptible and resistant mosquitoes. RESULTS: Using three human volunteers differing in relative attractiveness to mosquitoes, 328 mosquitoes were individually tested. There were no significant differences between response rates to ITNs and untreated nets (P > 0.1) or between resistant (Tiassalé) and susceptible (Kisumu) mosquito strains, at untreated nets (P = 0.39) or PermaNet 2.0 (P = 1). The sequence of behavioural events from host-seeking to completion of blood-feeding was consistent in all tests but duration and start time of events involving net contact were reduced or delayed respectively with ITNs. Blood-feeding durations at untreated nets (means from 4.25 to 8.47 min (95% confidence interval (CI) = 3.39-9.89) at 3 human volunteers) were reduced by 37-50% at PermaNet 2.0, in susceptible (mean 2.59-4.72 min, 95% CI = 1.54-5.5, P = < 0.05) and resistant (mean 4.20 min, 95% CI = 3.42-4.97, P = 0.01) strains. Total accumulated net contact was approximately 50% lower at PermaNet and Olyset ITNs (P < 0.0001) in susceptible (two of the three volunteers) and resistant mosquitoes. Times prior to first net contact were similar at untreated nets and ITNs (P > 0.2), and neither ITN type showed detectable spatial repellency. After initial contact, blood-feeding commenced later at Olyset (mean 2.76 min, 95% CI = 1.74-3.76, P = 0.0009) and PermaNet (mean 2.4 min, 95% CI = 1.52-3.33, P = 0.0058) than untreated netting (mean 0.68 min, 95% CI = 0.42-0.94). CONCLUSIONS: The baited box offers a simple method for detailed characterization of mosquito behavioural responses to insecticidal nets, for comparing entomological modes of action between nets and for defining the behavioural responses of particular mosquito strains or populations. The device has potential as a screening assay in the search for novel net treatments and for investigations into behavioural resistance mechanisms.

Selective regulation of type II interferon-inducible genes by NPM1/nucleophosmin.

Nucleophosmin (NPM1) is a multifunctional nucleolar protein. Here, we analyze the role of NPM1 in gene expression using our previous microarray data and find a relationship between NPM1 and interferon (IFN)-γ-inducible genes. We show that NPM1 selectively regulates the expression of a subset of IFN-γ-inducible genes and directly binds to two important transcription factors in the type II IFN pathway: signal transducer and activator of transcription 1 and interferon regulatory factor 1 (IRF1). Furthermore, NPM1 is found to regulate the IFN-γ-inducible promoter activity of major histocompatibility complex class II transactivator (CIITA), and mutation of the IRF1-binding site on the CIITA promoter abolishes the effect of NPM1. Our results suggest a novel mechanism for IFN-γ-mediated gene expression by NPM1.

Host-seeking activity of a Tanzanian population of Anopheles arabiensis at an insecticide treated bed net.

BACKGROUND: Understanding how mosquitoes respond to long lasting insecticide treated nets (LLINs) is fundamental to sustaining the effectiveness of this essential control tool. We report on studies with a tracking system to investigate behaviour of wild anophelines at an LLIN, in an experimental hut at a rural site in Mwanza, Tanzania. METHODS: Groups of adult female mosquitoes (n = 10 per replicate) reared from larvae of a local population, identified as predominantly (95%) Anopheles arabiensis, were released in the hut. An infrared video tracking system recorded flight and net contact activity over 1 h as the mosquitoes attempted to reach a supine human volunteer within a bed net (either a deltamethrin-treated LLIN or an untreated control net). A range of activities, including flight path, position in relation to the bed net and duration of net contact, were quantified and compared between treatments. RESULTS: The total time that female An. arabiensis spent in flight around LLINs was significantly lower than at untreated nets [F(1,10) = 9.26, p = 0.012], primarily due to a substantial reduction in the time mosquitoes spent in persistent 'bouncing' flight [F(1,10) = 18.48, p = 0.002]. Most activity occurred at the net roof but significantly less so with LLINs (56.8% of total) than untreated nets [85.0%; Χ2 (15) = 234.69, p < 0.001]. Activity levels at the bed net directly above the host torso were significantly higher with untreated nets (74.2%) than LLINs [38.4%; Χ2 (15) = 33.54, p = 0.004]. 'Visiting' and 'bouncing' rates were highest above the volunteer's chest in untreated nets (39.9 and 50.4%, respectively) and LLINs [29.9 and 42.4%; Χ2 (13) = 89.91, p < 0.001; Χ2 (9) = 45.73, p < 0.001]. Highest resting rates were above the torso in untreated nets [77%; Χ2 (9) = 63.12, p < 0.001], but in LLINs only 33.2% of resting occurred here [Χ2 (9) = 27.59, p = 0.001], with resting times spread between the short vertical side of the net adjacent to the volunteer's head (21.8%) and feet (16.2%). Duration of net contact by a single mosquito was estimated at 204-290 s on untreated nets and 46-82 s on LLINs. While latency to net contact was similar in both treatments, the reduction in activity over 60 min was significantly more rapid for LLINs [F(1,10) = 6.81, p = 0.026], reiterating an 'attract and kill' rather than a repellent mode of action. CONCLUSIONS: The study has demonstrated the potential for detailed investigations of behaviour of wild mosquito populations under field conditions. The results validate the findings of earlier laboratory studies on mosquito activity at LLINs, and reinforce the key role of multiple brief contacts at the net roof as the critical LLIN mode of action.

Pds5 Regulates Sister-Chromatid Cohesion and Chromosome Bi-orientation through a Conserved Protein Interaction Module.

Sister-chromatid cohesion is established by the cohesin complex in S phase and persists until metaphase, when sister chromatids are captured by microtubules emanating from opposite poles [1]. The Aurora-B-containing chromosome passenger complex (CPC) plays a crucial role in achieving chromosome bi-orientation by correcting erroneous microtubule attachment [2]. The centromeric localization of the CPC relies largely on histone H3-T3 phosphorylation (H3-pT3), which is mediated by the mitotic histone kinase Haspin/Hrk1 [3-5]. Hrk1 localization to centromeres depends largely on the cohesin subunit Pds5 in fission yeast [5]; however, it is unknown how Pds5 regulates Hrk1 localization. Here we identify a conserved Hrk1-interacting motif (HIM) in Pds5 and a Pds5-interacting motif (PIM) in Hrk1 in fission yeast. Mutations in either motif result in the displacement of Hrk1 from centromeres. We also show that the mechanism of Pds5-dependent Hrk1 recruitment is conserved in human cells. Notably, the PIM in Haspin/Hrk1 is reminiscent of the YSR motif found in the mammalian cohesin destabilizer Wapl and stabilizer Sororin, both of which bind PDS5 [6-12]. Similarly, and through the same motifs, fission yeast Pds5 binds to Wpl1/Wapl and acetyltransferase Eso1/Eco1, in addition to Hrk1. Thus, we have identified a protein-protein interaction module in Pds5 that serves as a chromatin platform for regulating sister-chromatid cohesion and chromosome bi-orientation.

Regulation of Cellular Dynamics and Chromosomal Binding Site Preference of Linker Histones H1.0 and H1.X.

Linker histones play important roles in the genomic organization of mammalian cells. Of the linker histone variants, H1.X shows the most dynamic behavior in the nucleus. Recent research has suggested that the linker histone variants H1.X and H1.0 have different chromosomal binding site preferences. However, it remains unclear how the dynamics and binding site preferences of linker histones are determined. Here, we biochemically demonstrated that the DNA/nucleosome and histone chaperone binding activities of H1.X are significantly lower than those of other linker histones. This explains why H1.X moves more rapidly than other linker histones in vivo Domain swapping between H1.0 and H1.X suggests that the globular domain (GD) and C-terminal domain (CTD) of H1.X independently contribute to the dynamic behavior of H1.X. Our results also suggest that the N-terminal domain (NTD), GD, and CTD cooperatively determine the preferential binding sites, and the contribution of each domain for this determination is different depending on the target genes. We also found that linker histones accumulate in the nucleoli when the nucleosome binding activities of the GDs are weak. Our results contribute to understanding the molecular mechanisms of dynamic behaviors, binding site selection, and localization of linker histones.

Infrared video tracking of Anopheles gambiae at insecticide-treated bed nets reveals rapid decisive impact after brief localised net contact.

Long-lasting insecticidal bed nets (LLINs) protect humans from malaria transmission and are fundamental to malaria control worldwide, but little is known of how mosquitoes interact with nets. Elucidating LLIN mode of action is essential to maintain or improve efficacy, an urgent need as emerging insecticide resistance threatens their future. Tracking multiple free-flying Anopheles gambiae responding to human-occupied bed nets in a novel large-scale system, we characterised key behaviours and events. Four behavioural modes with different levels of net contact were defined: swooping, visiting, bouncing and resting. Approximately 75% of all activity occurred at the bed net roof where multiple brief contacts were focussed above the occupant's torso. Total flight and net contact times were lower at LLINs than untreated nets but the essential character of the response was unaltered. LLINs did not repel mosquitoes but impacted rapidly: LLIN contact of less than 1 minute per mosquito during the first ten minutes reduced subsequent activity; after thirty minutes, activity at LLINs was negligible. Velocity measurements showed that mosquitoes detected nets, including unbaited untreated nets, prior to contact. This is the most complete characterisation of mosquito-LLIN interactions to date, and reveals many aspects of LLIN mode of action, important for developing the next generation of LLINs.

[Current status and issues of anti-smoking measures in the workplace in Japan].

Compared with developed foreign countries, anti-smoking measures in Japan is lagging behind. As a country that has signed the Framework Convention on Tobacco Control (FCTC), it should be run the appropriate tobacco control. For example, in many stores of the service industry that smoking is allowed, employees are working while being exposed to second-hand smoke. Even in workplace air polluted environment, employees will not be able to leave there. Such a harsh environment to ignore health and safety, it must be eliminated as soon as possible. In order to protect the health of workers, the workplace should be smoke free.

[Problems of tobacco smoke and positive effects of quit-smoking on human health].

Cigarette smoking dominates among Japanese smokers. A smoker takes a small amount of nicotine into the body by one aspiration of cigarette; this amount is not large enough to cause acute symptoms. The way of consuming one cigarette by about 10 aspirations may supply a sufficient amount of nicotine to the body without producing unpleasant symptoms of acute toxicity. Cigarette is very effective goods of nicotine delivery to increase the nicotine-dependent patients. In addition, owing to the tricky image strategy by a tobacco company, the citizens in Japan have not become sufficiently aware of hazards of tobacco. For health hazards of smoking, people do not fully understand cigarette smoking may affect the whole body because of the strong impression of "cancer" particularly "lung cancer" as the disease of smoking. Therefore, we, healthcare professionals, should treat diseases with accurate knowledge of tobacco hazards and also should play a positive role to enlighten people about the truth of tobacco hazards, which do not mean cancer alone.

A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells.

tRNase Z(L)-utilizing efficacious gene silencing is a gene control technology, which is based on the property that tRNase Z(L) can cleave any target RNA under the direction of an appropriate small guide RNA (sgRNA). To find therapeutic sgRNAs to cure hematological malignancies, we investigated behavior of heptamer-type sgRNA. We demonstrated that a heptamer, mh1(Bcl-2), which targets the human Bcl-2 mRNA, can be taken up by cells without any transfection reagents and that it can induce apoptosis of the leukemia cells. Mouse xenograft experiments showed that a median survival of the mh1(Bcl-2)-treated mice was longer than that of the control mice.

Elimination of specific miRNAs by naked 14-nt sgRNAs.

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

Expression of angiogenesis-related factors and inflammatory cytokines in placenta and umbilical vessels in pregnancies with preeclampsia and chorioamnionitis/funisitis.

We hypothesized that gene expression in placenta and umbilical vessels are affected by intrauterine environment and some of the expression in umbilical vessels originating from the fetus could reflect fetal condition of these complicated pregnancies. Expression of angiogenesis-related factors and inflammatory cytokines were examined in placenta and umbilical vessels to clarify the effects of intrauterine environment of pregnancies complicated by preeclampsia and chorioamnionitis/funisitis. Forty-six preterm cesarean section deliveries were classified into three groups based on maternal condition during prenatal monitoring: preeclampsia (PE) (n = 11), chorioamnionitis/funisitis (CAM) (n = 8), and preterm control (PC) (n = 27). Angiogenesis-related factors and inflammatory cytokines in placentas, umbilical arteries and umbilical veins were analyzed by RT-PCR and immunohistochemistry. We demonstrated that Ang-2, Tie-2, and Dll4 increase in the placentas of PE compared to PC for the first time, and we confirmed the findings of previous reports showing the high expression of HIF-1α, sFlt-1, endoglin, leptin, and AT1R. Expression of angiogenesis-related factors, including HIF-1α, VEGF, angiopoietin, and TGF-ß systems, and inflammatory cytokines, such as TNF-α and IL-6, increased in umbilical vessels of PE. Umbilical veins of CAM showed a higher Dll4 level than did PC. In preeclampsia, abnormal expressions of angiogenesis-related factors related to lifestyle diseases in adulthood were seen in the placenta and umbilical vessels as compared to PC. Chorioamnionitis/funisitis showed only upregulation of DII4 in umbilical veins.

Prognostic items for the last 10 and 3 days of life of cancer patients at home.

BACKGROUND: Prognostic indices are needed to optimize end-of-life care for cancer patients at home, but few prognostic indices predict the last 10 days. OBJECTIVE: The purpose of this study was to identify predictors for the last 10 and 3 days of life in patients with lung, gastric, or colorectal cancer at home. METHODS: Symptoms and signs were initially identified by literature review, and questionnaire was developed. Evaluation of these items and identification of additional items were then performed by 72 visiting nurses using the 3-round Delphi approach. RESULTS: The evaluation of 31 third-round responses is reported. The items for gastric and colorectal cancers were almost same; these cancers were treated as gastrointestinal cancer. To predict the last 10 and 3 days, there were 6 and 0 specific items for lung cancer, respectively, and 5 and 13 specific items for gastrointestinal cancer, respectively. There were 9 common items to predict the last 10 days and 29 common items to predict the last 3 days. CONCLUSION: The specific and common items that could be used to predict the last 10 and 3 days in patients with lung or gastrointestinal cancer were identified. The prognostic items for the last 3 days of life were more numerous among the gastrointestinal cancers than those for the last 10 days. IMPLICATIONS FOR PRACTICE: Specific prognostic items for each cancer are useful for visiting nurses to offer individualized care to patients and families. Using the specific and common prognostic items, end-of-life care may be improved.

Effective intravenous thrombolytic therapy in a patient with cerebral infarction associated with left atrial myxoma.

A 70-year-old man presented with sudden onset of global aphasia and right hemiplegia. Brain MRI revealed occlusion of the left middle cerebral artery. He was diagnosed as having a hyperacute cerebral infarction. Intravenous thrombolytic therapy was started, and the neurological symptoms were resolved after 11 h. Echocardiography showed a mobile mass in the left atrium, suspicious of a myxoma. The tumor was resected and pathologically diagnosed as a myxoma. In this patient, intravenous thrombolytic therapy was effective, and no adverse effects were observed. This suggests that, even with complications of myxoma, thrombolytic therapy can be considered if there are no contraindications.

Cut-offs for screening prolonged QT intervals from Fridericia's formula in children and adolescents.

BACKGROUND: The corrected QT interval (QTc) according to Bazett's formula (QTc = QT/RR(1/2)) has been used in clinical practice. Bazett's formula, however, overcorrects the QT interval at fast heart rates and undercorrects it at low heart rates. Guidelines and some investigators have recommended using Fridericia's formula (QTc = QT/RR(1/3)) in these cases, especially in tachycardic subjects. The aim of the present study was to determine cut-offs for QTc suitable for screening pediatric subjects with prolonged QT intervals, based on manually measured values corrected by Fridericia's formula in a large number of subjects. METHODS AND RESULTS: Three consecutive QT and RR intervals were measured in 4,655, 4,655, and 5,273 1st, 7th, and 10th graders, aged 6, 12, and 15 years, respectively. Each QT interval was corrected by Fridericia's formula, and mean values were calculated. Determination of the cut-offs for screening was based on the prevalence of abnormal electrocardiographic phenotypes of 1:1,164 and on the upper 0.025 percentile in the QTc distribution derived from previous studies. The tentative cut-offs suitable for screening subjects with prolonged QT intervals were 430 ms for 1st graders, 445 ms for 7th graders, and 440 and 455 ms for 10th grade boys and girls, respectively. CONCLUSIONS: These tentative cut-offs can be used to screen subjects with prolonged QT intervals in the clinical setting. Further studies are needed to confirm their validity.

Therapeutic angiogenesis by implantation of a capillary structure constituted of human adipose tissue microvascular endothelial cells.

OBJECTIVE: We previously reported a novel technology for the engineering of a capillary network using an optical lithographic technique. To apply this technology to the therapy of ischemic diseases, we tested human omental microvascular endothelial cells (HOMECs) as an autologous cell source and decellularized human amniotic membranes (DC-AMs) as a pathogen-free and low immunogenic transplantation scaffold. METHODS AND RESULTS: Human umbilical vein endothelial cells were aligned on a patterned glass substrate and formed a capillary structure when transferred onto an amniotic membrane (AM). In contrast, HOMECs were scattered and did not form a capillary structure on AMs. Treatment of HOMECs with sphingosine 1-phosphate (S1P) inhibited HOMEC migration and enabled HOMEC formation of a capillary structure on AMs. Using quantitative RT-PCR and Western blot analyses, we demonstrated that the main S1P receptor in HOMECs is S1P(2), which is lacking in human umbilical vein endothelial cells, and that inhibition of cell migration by S1P is mediated through an S1P(2)-Rho-Rho-associated kinase signaling pathway. Implantation of capillaries engineered on DC-AMs into a hindlimb ischemic nude mouse model significantly increased blood perfusion compared with controls. CONCLUSIONS: A capillary network consisting of HOMECs on DC-AMs can be engineered ex vivo using printing technology and S1P treatment. This method for regeneration of a capillary network may have therapeutic potential for ischemic diseases.

Pigpen, a nuclear coiled body component protein, is involved in angiogenesis.

We previously reported that puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) is required for vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced angiogenesis and for endothelial differentiation from embryonic stem (ES) cells via the aminopeptidase activity of PILSAP. In this study, we searched for molecules that function during angiogenesis with PILSAP. We performed proteome analysis of nuclear extracts from embryoid bodies (EBs) made from ES cells transfected with mutant PILSAP lacking aminopeptidase activity and mock EBs. We identified pigpen, a 67-kDa nuclear coiled body component protein. Immunoprecipitation and western blotting demonstrated the binding of PILSAP and pigpen in endothelial cells (ECs), and this interaction was enhanced by VEGF and bFGF. Pigpen was reported to be expressed in actively growing ECs such as those in embryos and tumors. However, whether Pigpen is involved in angiogenesis is not known. Therefore, we examined the effect of pigpen on angiogenesis by silencing pigpen with siRNA (siPigpen). Compared with scrambled RNA (scrPigpen), transfection of siPigpen into mouse ECs inhibited proliferation, migration, and network formation. These results were confirmed with other two siRNAs. Moreover, siPigpen suppressed bFGF-induced angiogenesis in a Matrigel plug assay, and injection of siPigpen into Lewis lung carcinoma cell tumors implanted subcutaneously into 5-week-old C57/BL male mice prevented tumor growth and tumor angiogenesis. These data indicate that Pigpen is involved in angiogenesis and that pigpen may be a target for blocking tumor angiogenesis.

Spatial distribution and pyrethroid susceptibility of mosquito larvae collected from catch basins in parks in Nagasaki city, Nagasaki, Japan.

We investigated the spatial distribution and pyrethroid susceptibility of the mosquito larvae belonging to Aedes albopictus and Culex pipiens group in catch basins located in parks in Nagasaki city, Nagasaki, Japan. Among the 308 parks located in the central regions of the city, 194 were investigated. Cx. pipiens group larvae were collected from 31 sites; larvae of Ae. albopictus, from 34 sites. The Cx. pipiens group larvae were identified by PCR: 93.4% were found to belong to Cx. pipiens pallens, and 0.9%, to Cx. pipiens form molestus. A bioassay was performed by observing the knockdown of larvae during 30-min exposures to 0.4- and 0.1-ppm solutions of d-allethrin. High tolerance to d-allethrin (susceptibility index = 36) was observed in only 1 colony of Cx. pipiens pallens across 24 sites. On the other hand, Ae. albopictus showed high tolerance (susceptibility index > 30) in 8 of 22 sites; this indicated that Ae. albopictus populations tolerant to pyrethroids were spreading widely in Nagasaki city. The organized and massive larvicidal treatment of graveyard containers with DDT in the 1950s was thought to be one of the main causes for the development of pyrethroid resistance in Ae. albopictus.

Implantation of capillary structure engineered by optical lithography improves hind limb ischemia in mice.

We previously reported a novel optical lithographic technique for the construction of a capillary network consisting of endothelial cells. To investigate the feasibility of clinical application in the treatment of ischemic diseases, capillary structures were formed on scaffolds made from amniotic membrane (AM) and implanted into mice. The capillary network remained in place for at least 5 days and blood perfusion through the implanted capillaries was histologically detected in an ear flap model. Moreover, blood was observed flowing through the capillary network implanted in abdominal subcutaneous tissue of mice at 5 days after insertion. Implantation of the AM capillary structure into the ischemic hind limbs of mice significantly increased reperfusion compared with controls (AM only). Blood flow was restored in the ischemic limbs to the level of corresponding nonischemic limbs as early as 9 days after surgical implantation. The treatment reversed ischemic symptoms, and ambulatory impairment was significantly improved. Thus, the implantation of a capillary network engineered ex vivo could have therapeutic potential for ischemic diseases.