An apparatus based on an atomic force microscope for implementing tip-controlled local breakdown.

Solid-state nanopores are powerful tools for sensing of single biomolecules in solution. Fabrication of solid-state nanopores is still challenging, however; in particular, new methods are needed to facilitate the integration of pores with larger nanofluidic and electronic device architectures. We have developed the tip-controlled local breakdown (TCLB) approach, in which an atomic force microscope (AFM) tip is brought into contact with a silicon nitride membrane that is placed onto an electrolyte reservoir. The application of a voltage bias at the AFM tip induces a dielectric breakdown that leads to the formation of a nanopore at the tip position. In this work, we report on the details of the apparatus used to fabricate nanopores using the TCLB method, and we demonstrate the formation of nanopores with smaller, more controlled diameters using a current limiting circuit that zeroes the voltage upon pore formation. Additionally, we demonstrate the capability of TCLB to fabricate pores aligned to embedded topographical features on the membranes.

A Novel Diagnostic System for Infectious Diseases Using Solid-State Nanopore Devices.

Nanopore-based diagnostic systems are a promising tool for counting viruses in a specimen one by one. However, despite intensive R&D efforts, it remains difficult to recognize virus subtypes by nanopore devices. We thus propose a novel diagnostic system that combines a specialized virus recognition procedure with a nanopore detection procedure. This recognition procedure consists of three steps: 1) capture target viruses using specific probes for recognition; 2) release captured targets; and 3) detect released targets by nanopore. Proof-of-concept tests are conducted using avidin-modified fluorescent particles (as a model for viruses) and biotin-modified alkane thiol (as a model for probes). The avidin-modified particles are confirmed to be captured on electrode by biotin-modified probes and then, the particles are electrochemically released from the electrode. Consequently, the released particles are successfully detected by nanopore devices. Furthermore, the concept is also proved by using human influenza viruses (H1N1, A/PR/8/34) and sugar chain (6'-sialyllactose)-modified probes. This suggests that our concept is applicable to various infectious diseases by changing probes (ligands).

Heterocyclic boronic acids display sialic acid selective binding in a hypoxic tumor relevant acidic environment.

Boronic acids are well known for their ability to reversibly interact with the diol groups found in sugars and glycoproteins. However, they are generally indiscriminate in their binding. Herein we describe the discovery of a group of heterocyclic boronic acids demonstrating unusually high affinity and selectivity for sialic acids (SAs or N-acetylneuraminic acid), which are sugar residues that are intimately linked with tumor growth and cancer progression. Remarkably, these interactions strengthen under the weakly acidic pH conditions associated with a hypoxic tumoral microenvironment. In vitro competitive binding assays uncovered a significantly higher ability of 5-boronopicolinic acid, one of the derivatives identified in this work as a strong SA-binder, to interact with cell surface SA in comparison to a gold-standard structure, 3-propionamidophenylboronic acid, which has proven to be an efficient SA-binder in numerous reports. This structure also proved to be suitable for further chemical conjugation with a well-preserved SA-binding capability. These findings suggest an attractive alternative to other ongoing boronic acid based chemistry techniques aiming to achieve tumor-specific chemotherapies and diagnoses.

Viper bite during pregnancy: case report.

Viper bites in pregnant women have rarely been reported thus far. Moreover, there is no consensus regarding the treatment of such cases. In this paper, the authors report the successful treatment of viper bite during pregnancy without using antivenom.

One-to-one spatially matched experiment and atomistic simulations of nanometre-scale indentation.

We have carried out nanoindentation studies of gold in which the indenter is atomically characterized by field-ion microscopy and the scale of deformation is sufficiently small to be directly compared with atomistic simulations. We find that many features of the experiment are correctly reproduced by molecular dynamics simulations, in some cases only when an atomically rough indenter rather than a smooth repulsive-potential indenter is used. Heterogeneous nucleation of dislocations is found to take place at surface defect sites. Using input from atomistic simulations, a model of indentation based on stochastic transitions between continuum elastic-plastic states is developed, which accurately predicts the size distributions of plastic 'pop-in' events and their dependence on tip geometry.

Effect of using stencil masks made by focused ion beam milling on permalloy (Ni81Fe19) nanostructures.

Focused ion beam (FIB) milling is a common fabrication technique to make nanostencil masks which has the unintended consequence of gallium ion implantation surrounding milled features in silicon nitride membranes. We observe major changes in film structure, chemical composition, and magnetic behaviour of permalloy nanostructures deposited by electron beam evaporation using silicon nitride stencil masks made by a FIB as compared to stencil masks made by regular lithography techniques. We characterize the stenciled structures and both types of masks using transmission electron microscopy, electron energy loss spectroscopy, energy dispersive x-ray spectroscopy, magnetic force microscopy and kelvin probe force microscopy. All these techniques demonstrate distinct differences at a length scale of a 1-100 nm for the structures made using stencil mask fabricated using a FIB. The origin of these differences seems to be related to the presence of implanted ions, a detailed understanding of the mechanism however remains to be developed.

Anti-TCRß mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.

TCR specific antibodies may modulate the TCR engagement with antigen-MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRß (H57-597), TCRα or CD3 promptly reduced the number of CD4(+) and CD8(+) T cells in normal mice, but H57-597 mAb most potently increased the frequency of CD4(+) Foxp3(+) Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57-597 mAb, the expansion of SEB-reactive Vß8(+) T cells was completely abrogated while SEB-nonreactive Vß2(+) T cells remained unaffected. More importantly, transient H57-597 mAb treatment exerted long-lasting effect in preventing T cell responses to alloantigens, and produced long-term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor-specific long-term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57-597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin-sensitized recipients. Thus, transient modulation of the TCRß chain by H57-597 mAb exhibits potent, long-lasting therapeutic effects to control alloimmune responses.

siRNA-mediated silencing of PD-1 ligands enhances tumor-specific human T-cell effector functions.

Adoptive cell therapy using tumor-specific T cells is a promising strategy for treating patients with malignancy. However, accumulating evidences have demonstrated that optimal function of tumor-reactive T cells is often attenuated by negative regulatory signal(s) delivered through receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and their cognate ligands. Although systemic blocking of these molecules needs careful attention on the risk of uncontrolled immune activation, selective inhibition of negative signals in tumor-specific T cells by their genetic modification is an attractive approach to overcome immunological suppression in cancer patients. Here, we demonstrate the improved effector functions of tumor-specific CD4(+) and CD8(+) human T cells by small interfering RNA (siRNA) -mediated silencing of PD-1 ligands, PD-L1 or PD-L2. Tumor antigen MAGE-A4-specific human T-cell clones upregulated the expression of PD-1 ligands upon activation. siRNA-mediated knockdown of PD-L1 or -L2 enhanced the interferon-γ production and antigen-specific cytotoxicity of these cells. Peripheral blood mononuclear cells transduced with a retroviral vector encoding MAGE-A4-specific T-cell receptor α/ß chains also increased their effector functions by this modification. These results suggest that siRNA-mediated knockdown of PD-1 ligands is an attractive strategy to inhibit a negative regulatory mechanism of tumor-specific T cells resulting in enhanced efficacy of adoptive T-cell therapy of cancer using genetically modified autologous lymphocytes.

Field deposition from metallic tips onto insulating substrates.

The deposition of gold ions from atomic force microscope cantilever tips onto bulk insulating substrates with nearby surface electrodes is discussed. Numerical models of the potential distribution are used to estimate potential barriers for the desorption process. These models indicate deposition height thresholds of 7-10 nm with the tip 20-25 nm from the metallic electrode edge over a KBr surface but greater than 20 nm high for InP/GaAs/InP substrates with a two-dimensional electron gas (2DEG) as the back electrode. Experimental results for the deposition of gold clusters over KBr surfaces near metal electrodes in ultra-high vacuum (UHV) are presented and show promising agreement with calculations of the deposition threshold heights. Deposition of clusters over InP is discussed for comparison and indicates similar trends.

Lung diseases directly associated with rheumatoid arthritis and their relationship to outcome.

The outcome and cause of death of each lung disease directly associated with rheumatoid arthritis (RA-LD) have been poorly investigated. A retrospective study was conducted of 144 patients with RA-LD, in whom the median follow-up period after the initial visit for a respiratory examination was 4.5 yrs. A total of 57 patients were identified with usual interstitial pneumonia (UIP), 31 with bronchiectasis, 16 with nonspecific interstitial pneumonia (NSIP), 11 with bronchiolitis, five with organising pneumonia (OP), five with diffuse alveolar damage (DAD) and 19 with combined disease. The 5-yr survival rates were 36.6% in the UIP group, 87.1% in the bronchiectasis group, 93.8% in the NSIP group, 88.9% in the bronchiolitis group, 60.0% in the OP group and 20.0% in the DAD group. Survival of patients with DAD was worse than that of patients with UIP. Overall, survival of patients with UIP was worse than that of patients with bronchiectasis, NSIP or bronchiolitis. Of the 144 patients, 71 (49.3%) died, of whom 58 (81.7%) died due to respiratory lesions. Of patients with RA-LD, patients with DAD experienced the highest mortality, and the survival of patients with UIP was worse than that of patients with NSIP.

High Q optical fiber tips for NC-AFM in liquid.

Non-contact atomic force microscopy is rapidly expanding from ultra-high vacuum to include the study of surfaces and biomolecules in liquids by high resolution imaging and force spectroscopy. This is despite the additional frequency shift noise due to the inherently low Q factor of the cantilever oscillating in a liquid. In this paper we present a tip based on an optical fiber which can operate in liquid with Q factors in excess of 100 using a 'diving bell' arrangement which allows only a small portion of the tip to be submerged. We demonstrate stable imaging and force spectroscopy using this set-up. The tips are based on scanning near-field optical microscopy tips and, when used with NC-AFM, provide a method of combining both high resolution mechanical and fluorescence studies of biomolecules and cells.

Determination of the local contact potential difference of PTCDA on NaCl: a comparison of techniques.

There has been increasing focus on the use of Kelvin probe force microscopy (KPFM) for the determination of local electronic structure in recent years, especially in systems where other methods, such as scanning tunnelling microscopy/spectroscopy, may be intractable. We have examined three methods for determining the local apparent contact potential difference (CPD): frequency modulation KPFM (FM-KPFM), amplitude modulation KPFM (AM-KPFM), and frequency shift-bias spectroscopy, on a test system of 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) on NaCl, an example of an organic semiconductor on a bulk insulating substrate. We will discuss the influence of the bias modulation on the apparent CPD measurement by FM-KPFM compared to the DC-bias spectroscopy method, and provide a comparison of AM-KPFM, AM-slope detection KPFM and FM-KPFM imaging resolution and accuracy. We will also discuss the distance dependence of the CPD as measured by FM-KPFM for both the PTCDA organic deposit and the NaCl substrate.

Toll-like receptors and immune regulation: implications for cancer therapy.

Toll-like receptors (TLRs) function as pathogen pattern recognition molecules that sensor and initiate innate and adaptive immune responses against microbes and cancer cells. Recognition of pathogen-derived ligands by TLRs expressed on many types of cells, including dendritic cells and T cells, triggers the nuclear factor (NF)-kappaB and type-1 interferon pathways, leading to the production of proinflammatory cytokines that are essential in stimulating CD4(+) T cells to differentiate to T helper (Th) 1, Th2 Th17 and regulatory T (Treg) cells. Recent studies indicate that Treg cells play a critical role in suppressing immune responses and inducing immune tolerance to cancer and infectious diseases. Of particular interest, the human TLR8 signaling pathway is essential for reversing the suppressive function of Treg cells. Thus, TLRs regulate cancer immunity and tolerance through innate immune responses mediated by Treg, dendritic and other immune cells. In this review, we focus on the current understanding of TLRs and Treg cells with emphasis on their roles in cancer immunity. Related information on non-TLR immune receptors will be briefly discussed.

Improved COBAS TaqMan hepatitis C virus test (Version 2.0) for use with the High Pure system: enhanced genotype inclusivity and performance characteristics in a multisite study.

We have evaluated the COBAS TaqMan hepatitis C virus (HCV) test (version 2.0) for use with the High Pure system (HCVHPS V2), a new, revised real-time reverse transcription-PCR assay developed to improve the genotype quantitation of version 1.0 (HCVHPS V1). Revisions were made in the wash buffer and in the reverse transcription temperature. The genotype inclusivity of HCVHPS V2 was evaluated at three different sites, using HCVHPS V2, HCVHPS V1, and the COBAS AMPLICOR HCV MONITOR test (version 2.0) (CAHCM). The fully automated COBAS Ampliprep/COBAS TaqMan HCV test was also used in one of the participating laboratories. The mean differences in HCV RNA values between HCVHPS V2 and CAHCM and between HCVHPS V2 and HCVHPS V1 ranged from -0.21 to 0.13 log and from 0.24 to 1.27 log, respectively, with >0.5-log differences for genotypes 2, 3, 4, and 5. With a NIBSC panel of HCV genotypes 1 through 6, the measured HCVHPS V2 values were within 0.25 log of the nominal values for all 6 genotypes. When serial dilutions of genotype-specific clinical HCV specimens were tested, the assay showed a limit of detection between 10 and 20 IU/ml and a linear range of 25 IU/ml to 3.91 x 10(8) IU/ml. Clinical and analytical specificities of 100% were demonstrated with 100 HCV-seronegative specimens as well as with 12 non-HCV members of Flaviviridae and 22 additional microorganisms. These data indicate that HCVHPS V2 is a robust and accurate test for the quantitation of all six HCV genotypes and useful in monitoring viral load in all clinical HCV specimens.

Radiotherapy negates the effect of retroperitoneal nonclosure for prevention of lymphedema of the legs following pelvic lymphadenectomy for gynecological malignancies: an analysis from a questionnaire survey.

Using patient questionnaires, we studied the long-term effect of leaving the peritoneum open on the incidence of lymphedema of the legs in patients following pelvic lymphadenectomy for gynecological malignancies. The patients were retrospectively assigned to one of two groups, depending on whether the retroperitoneum was closed or left open at surgery. Three years after surgery, we obtained valid questionnaire responses from 101 patients (43 cervical, 46 endometrial, and 12 ovarian cancers) in the closure group and 83 patients (34 cervical, 40 endometrial, and 9 ovarian cancers) in the nonclosure group. In patients' self-analysis, the overall incidence of lymphedema of the legs was significantly lower in the nonclosure group than in the closure group (25.3% and 50.5%, respectively; P<0.01). The incidence of lymphedema of the legs was significantly increased by postoperative radiotherapy. Especially in the nonclosure group, the incidence of lymphedema was only 15.8% in patients who did not have radiotherapy, but it increased significantly (44.4%) (P<0.05) when patients underwent radiotherapy. In conclusion, this questionnaire survey suggested that leaving the retroperitoneum open after lymphadenectomy is significantly effective in reducing the incidence of leg lymphedema, which impairs patients' quality of life more than expected by physicians.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model.

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells.

The human ovarian surface epithelium (hOSE) is a single layer of mesothelial-type primitive epithelial cells that are potential estrogen targets. It has been reported that hOSE cells can produce estrogen. However, the mechanisms that regulate estrogen level(s) in hOSE cells are not yet known. To elucidate the enzymes involved in these reactions, we examined gene expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in primary hOSE (POSE) and OSE2a cells using RT-PCR. We found that POSE cells and cells of the immortalized hOSE line, OSE2a, bidirectionally converted estrone (E1) and 17beta-estradiol (E2). Both cell types expressed mRNA for 17beta-HSD type 1 (17beta-HSD1), suggesting that the enzyme is involved in the E1 to E2 conversion. Interestingly, both cells expressed 17beta-HSD4 mRNA but not 17beta-HSD2 mRNA. We prepared an antibody against the carboxyl terminal of 17beta-HSD4 (anti-17beta-HSD4 antibody), which recognized the 80 and 48 kDa proteins in POSE and OSE2a cells based on immunoblot analysis. Furthermore, immunohistochemical study revealed the presence of 17beta-HSD4 in hOSE cells in the human ovary. These results suggest that 17beta-HSD4 is involved in estrogen inactivation and may protect against an excessive accumulation of E2 in hOSE cells.

Effect of cis-diammine dichloroplatinum on vascular endothelial growth factor expression in uterine cervical carcinoma.

PURPOSE OF INVESTIGATION: In this study, we investigated the effects of cis-diammine dichloroplatinum (CDDP) on VEGF mRNA expression and VEGF production in uterine cervical carcinoma tissues obtained from patients with locally advanced disease and in CaSki cells cultured in vitro. METHODS: VEGF in cultured CaSki cells and in the culture media was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) before and 24 h, 48 h and 72 h after 3 h exposure to CDDP. VEGF mRNA expression in CaSki cells was assessed by the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and 24 h and 48 h after 3 h exposure to CDDP. We also examined the effect of CDDP on microvessel counts in uterine cervical carcinoma tissues obtained before and after high-dose CDDP intraarterial chemotherapy. Immunohistochemical staining using a monoclonal antibody against CD34 was carried out with cervical carcinoma tissue specimens, and microvessel counts were quantified by counting vessels. RESULTS: CDDP treatment resulted in significant increases in not only VEGF concentrations in cultured CaSki cells and culture media but also in VEGF mRNA expression levels in cultured CaSki cells in a time-dependent and dose-dependent manner compared to untreated controls (p < 0.05, n = 5). On the other hand, VEGF concentrations and microvessel counts in cervical carcinoma tissues were significantly lower in cases with complete response (CR) and partial response (PR), compared to those before treatment (p < 0.05, n = 5 ). By contrast, in cases with no change (NC) to CDDP, both VEGF concentrations and microvessel counts did not decrease and rather showed a somewhat increase compared with levels prior to the treatment. CONCLUSIONS: These results suggest that CDDP-induced increases in VEGF production by cervical carcinoma cells may stimulate angiogenesis in the tumor lesion after CDDP treatment.

Influence of hemodialysis on echocardiographic Doppler indices of the left ventricle: changes in parameters of systolic and diastolic function and Tei index.

AIMS: Numbers of previous studies have evaluated the influence of dialysis-induced altered loading condition on Doppler-echocardiographic indices of left ventricle in patients with chronic renal failure. It has been suggested that most of Doppler-derived indices are preload-dependent. On the other hand, there are no studies that have evaluated the influence of hemodialysis on Tei index; a new Doppler-derived index obtained by isovolumetric contraction time plus isovolumetric relaxation time divided by ejection time. The aim of this study is to evaluate whether Tei index is also influenced by dialysis-induced altered loading condition as well as other Doppler-derived indices, and to assess the possibility that Tei index is also preload-dependent. PATIENTS AND METHODS: Thirty-two patients with chronic renal failure (21 men and 11 women, aged 48-93 years) on maintenance hemodialysis were evaluated for Doppler-derived indices before and after hemodialysis. We studied parameters of diastolic function (peak velocities of mitral inflow in early diastole (E) and late diastole from atrial filling (A), ratio of A to E (A/E), deceleration time (DT), and isovolumetric relaxation time (IRT)), parameters of systolic function (ejection time (ET), pre-ejection period (PEP), ratio of PEP to ET (PEP/ET), and isovolumetric contraction time (ICT)) and Tei index. RESULTS: Hemodialysis resulted in significant decreases in E, increase in A/E, prolongation of IRT, no change in A and DT; significant prolongation of ICT and PEP, shortening of ET, and increase in PEP/ET and a significant increase in Tei index (0.42 +/- 0.16 vs 0.51 +/- 0.16, p < 0.0001). When patients were subdivided into 2 groups based on weight loss after hemodialysis (> or = 1.5 kg and < 1.5 kg), only the group that lost > or = 1.5 kg had significant change in Tei index before and after hemodialysis (0.40 +/- 0.15 vs 0.52 +/- 0.17, p = 0.0002). CONCLUSION: This study demonstrates that not only most of Doppler-derived indices but also Tei index is affected by dialysis-induced altered loading condition and suggests that Tei index is possibly preload-dependent.

Transient increase in squamous cell carcinoma antigen expression in cultured cervical carcinoma CaSki cells in response to exposure to cis-diamminedichloroplatinum.

The present study was conducted to elucidate the molecular mechanism underlying the transient increase in circulating squamous cell carcinoma antigen (SCC Ag) levels in response to CIS-diamminedichloroplatinum (CDDP) infusion using an in vitro model. The uterine cervical squamous carcinoma CaSki cells were cultured for 72 h after 3 h exposure to 5.0 microg/ml CDDP. The effects of CDDP exposure on the proliferative activity and apoptosis in cultured CaSki cells were determined by bromodeoxyuridine (BrdU) uptake and cell counting and by the TUNEL assay, respectively. SCC Ag levels in cultured CaSki cells and culture media were determined with the use of SCC-RIA kit. The expression of SCC Ag-1 mRNA and SCC Ag-2 mRNA in cultured CaSki cells was assessed using semiquantitative RT-PCR with Southern blot analysis. The number of BrdU-positive CaSki cells significantly decreased 6 h after exposure to CDDP, whereas the apoptosis-positive rate of cultured CaSki cells significantly increased 12 h after the CDDP exposure. The number of cultured CaSki cells significantly decreased 72 h after the CDDP exposure. The total SCC Ag protein levels in both cultured CaSki cells and the culture media after the 3-hour CDDP exposure increased in a time-dependent manner during the subsequent incubation for 48 h. Semiquantitative RT-PCR revealed that the expression levels of both SCC Ag-1 and SCC Ag-2 mRNA increased (1.7- and 2.7-fold, respectively) 12 h after the exposure to CDDP relative to those before the subsequent cultures. Exposure of uterine cervical squamous carcinoma CaSki cells to CDDP resulted in a transient increase in SCC Ag protein and mRNA expression in those cells during the initial 12 h after the exposure, being associated with decreased proliferative activity and increased apoptosis of those cells.