Fabrication methods for infrared resonant devices.

Photonic metastructures operating in the 8-13 µm mid-infrared are fast becoming a topic of active research. However, literature describing techniques for their fabrication, which lie on a scale between nanofabrication and microelectromechanical systems technology, is scant. Here, we present detailed fabrication blueprints for achieving robust and repeatable results for devices in this region. Applying a Ge device layer on a ZnS substrate, we fabricate a materially robust transverse-magnetic-polarized wideband reflector with high reflectance in a 2.4 µm band. We then regrow a conformal layer on the device, boosting its performance to yield a ∼3 µm 90% reflectance band from 8.1 µm to 11.1 µm and a band of 98% reflectance spanning 8.7-10.4 µm. The generalized methods presented are applicable in most labs with ordinary fabrication resources.

Osteosarcoma in a Patient Receiving Long-Term TNF Inhibitor Therapy.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing malignancy. The use of immunosuppressive therapies and tumour necrosis factor (TNF) inhibitors in these patients may provide a further risk for the development of malignancy. We report the clinical and pathological findings of a high grade osteosarcoma in a patient with Crohns disease receiving TNF inhibitor therapy. In this case, a 32-year old female presented with a painful right knee after receiving maintenance adalimumab for Crohns disease for a period of six years. There is a substantial hypothetical link between TNF inhibitor regimens such as adalimumab and an increased risk of malignancy. TNF inhibitor therapy should be ceased and chemotherapy and surgery is an effective combined modality approach in these patients. The role of TNF inhibitors in patients after cancer diagnosis is uncertain and further research is required to assess efficacy and safety.

A multicentre retrospective comparison of central nervous system prophylaxis strategies among patients with high-risk diffuse large B-cell lymphoma.

BACKGROUND: Central nervous system (CNS) relapse in diffuse large B-cell lymphoma (DLBCL) is a devastating complication; the optimal prophylactic strategy remains unclear. METHODS: We performed a multicentre, retrospective analysis of patients with DLBCL with high risk for CNS relapse as defined by two or more of: multiple extranodal sites, elevated serum LDH and B symptoms or involvement of specific high-risk anatomical sites. We compared three different strategies of CNS-directed therapy: intrathecal (IT) methotrexate (MTX) with (R)-CHOP 'group 1'; R-CHOP with IT MTX and two cycles of high-dose intravenous (IV) MTX 'group 2'; dose-intensive systemic antimetabolite-containing chemotherapy (Hyper-CVAD or CODOXM/IVAC) with IT/IV MTX 'group 3'. RESULTS: Overall, 217 patients were identified (49, 125 and 43 in groups 1-3, respectively). With median follow-up of 3.4 (range 0.2-18.6) years, 23 CNS relapses occurred (12, 10 and 1 in groups 1-3 respectively). The 3-year actuarial rates (95% CI) of CNS relapse were 18.4% (9.5-33.1%), 6.9% (3.5-13.4%) and 2.3% (0.4-15.4%) in groups 1-3, respectively (P=0.009). CONCLUSIONS: The addition of high-dose IV MTX and/or cytarabine was associated with lower incidence of CNS relapse compared with IT chemotherapy alone. However, these data are limited by their retrospective nature and warrant confirmation in prospective randomised studies.

Archaeological evidence of an ethnographically documented Australian Aboriginal ritual dated to the last ice age.

In societies without writing, ethnographically known rituals have rarely been tracked back archaeologically more than a few hundred years. At the invitation of GunaiKurnai Aboriginal Elders, we undertook archaeological excavations at Cloggs Cave in the foothills of the Australian Alps. In GunaiKurnai Country, caves were not used as residential places during the early colonial period (mid-nineteenth century CE), but as secluded retreats for the performance of rituals by Aboriginal medicine men and women known as 'mulla-mullung', as documented by ethnographers. Here we report the discovery of buried 11,000- and 12,000-year-old miniature fireplaces with protruding trimmed wooden artefacts made of Casuarina wood smeared with animal or human fat, matching the configuration and contents of GunaiKurnai ritual installations described in nineteenth-century ethnography. These findings represent 500 generations of cultural transmission of an ethnographically documented ritual practice that dates back to the end of the last ice age and that contains Australia's oldest known wooden artefacts.

Limited role for surveillance PET-CT scanning in patients with diffuse large B-cell lymphoma in complete metabolic remission following primary therapy.

BACKGROUND: The usefulness of positron emission tomography with computed tomography (PET-CT) in the surveillance of patients with diffuse large B-cell lymphoma (DLBCL) in complete metabolic remission after primary therapy is not well studied. METHODS: We performed a retrospective review of our database between 2002 and 2009 for patients with de novo DLBCL who underwent surveillance PET-CT after achieving complete metabolic response (CMR) following primary therapy. RESULTS: Four-hundred and fifty scans were performed in 116 patients, with a median follow-up of 53 (range 8-133) months from completion of therapy. Thirteen patients (11%) relapsed: seven were suspected clinically and six were subclinical (all within first 18 months). The positive predictive value in patients with international prognostic index (IPI) <3 was 56% compared with 80% in patients with IPI ≥3. Including indeterminate scans, PET-CT retained high sensitivity 95% and specificity 97% for relapse. CONCLUSION: Positron emission tomography with computed tomography is not useful in patients for the majority of patients with diffuse large B-cell lymphoma in CMR after primary therapy, with the possible exception of patients with baseline IPI ≥3 in the 18 months following completion of primary therapy. This issue could be addressed by a prospective clinical trial.

An assessment of DNA extraction methods from blood-stained soil in forensic science.

In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline.

A method validation for the hemoglobin chemical reagent test strip with the addition of ethylenediaminetetraacetic acid to increase the selectivity of this presumptive test for residual blood.

Tetramethylbenzidine based chemical reagent test strips are often used in forensic science as a presumptive test for blood. These tests are designed as urinalysis test strips and include brands such as Combur®, HENSOTest®, Hemastix®, MultiStix® and Chemstrip®. They are used because they are simple to apply, stable, temperature tolerant and cost effective. The addition of a chelating agent, ethylenediaminetetraacetic acid increases the selectivity of this presumptive test for blood. This is a method validation for the hemoglobin chemical reagent test strip with EDTA. A range of substances, metal compounds, chemical solutions, blood and mixtures were tested in this method validation. The chelation with EDTA successfully prevented non-blood (false) positive results from all the substances tested and consistently produced a positive result for blood on a variety of surfaces. This study has shown that this method is capable of discriminating a blood stain on copper metal surfaces and eliminate the positive results generated by clean-up solutions such as hydrogen peroxide, which usually produce a positive result for most other presumptive tests for blood. This modified method is a simple, effective and reliable test for blood stains. A variety of variations were evaluated in this study. The simplest method of application was spraying the surface of the stain with a 0.5 M EDTA solution and testing the surface of the stain, and only requires a spray bottle of 0.5 M EDTA and the chemical reagent test strip. This spray approach is rugged and can be applied to horizontal, vertical and underside surfaces and requires little additional training. Overall, this study provides the forensic science community with an improved method more easily used, stored, transported and selective for blood, than luminol and safer than TMB.

Composition of trace residues from the contents of 11th-12th century sphero-conical vessels from Jerusalem.

The residues from the internal surface of four archaeological ceramic sherds, excavated from the Armenian Gardens, Jerusalem were analysed to characterise the contents of the original vessel. The sherds derive from four small, thick-walled, sphero-conical vessels recovered from a destruction layer, dating between the 11th and 12th century, Jerusalem. The residue has been analysed using light microscopy, biochemical characterisation, gas chromatography mass spectroscopy, inductively coupled plasma atomic emission spectroscopy and cold vapour atomic fluorescence spectrometry. This analysis established the presence of various compounds including fatty acids and notable levels of mercury, sulphur, aluminium, potassium, magnesium, nitrates and phosphorous. The contents and probable functions of the four vessels were characterised from the residues on these sherds as different from each other, reflecting their different decoration, manufacture and ceramic typologies. One of these vessels contains residue that indicate the vessel held oils. The residue of the second vessel is consistent with either scented materials or medicinal contents, while a third probably contained medicinal material. The unique fourth sherd is from a stoneware sphero-conical vessel with very thick walls, no decoration and the residue supports the possibility it was used for the storage of chemicals or may have held the chemical ingredients for an explosive device, consistent with a medieval grenade. This residue analysis of Mamluk sphero-conical vessels provides insight into luxury items, medicines, technology and trade in medieval Jerusalem.

Inorganic phosphate induces mammalian growth plate chondrocyte apoptosis in a mitochondrial pathway involving nitric oxide and JNK MAP kinase.

Chondrocytes in the hypertrophic zone of the growth plate undergo apoptosis during endochondral bone development via mechanisms that involve inorganic phosphate (Pi) and nitric oxide (NO). Recent evidence suggests that Pi-dependent NO production plays a role in apoptosis of cells in the resting zone as well. This study examined the mechanism by which Pi induces NO production and the signaling pathways by which NO mediates its effects on apoptosis in these cells. Pi decreased the number of viable cells based on MTT activity; the number of TUNEL-positive cells and the level of DNA fragmentation were increased, indicating an increase in apoptosis. Blocking NO production using the NO synthase (NOS) inhibitor L: -NAME or cells from eNOS(-/-) mice blocked Pi-induced chondrocyte apoptosis, indicating that NO production is necessary. NO donors NOC-18 and SNOG both induced chondrocyte apoptosis. SNOG also upregulated p53 expression, the Bax/Bcl-2 expression ratio, and cytochrome c release from mitochondria, as well as caspase-3 activity, indicating that NO induces apoptosis via a mitochondrial pathway. Inhibition of JNK, but not of p38 or ERK1/2, MAP kinase was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Pi elevates NO production via eNOS in resting zone chondrocytes, which leads to a mitochondrial apoptosis pathway dependent on JNK.

Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer.

We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.

The frequency, manifestations, and duration of prolonged cytopenias after first-line fludarabine combination chemotherapy.

BACKGROUND: Fludarabine-based chemoimmunotherapy has well-recognised efficacy and short-term toxicity in the treatment of lymphoid malignancies. However, the presence and significance of prolonged cytopenias after completion of treatment have not been thoroughly quantified. METHODS: Sixty-one patients receiving initial therapy with fludarabine-based regimens were categorised according to the presence of post-treatment cytopenias (haemoglobin <110-130 g/l depending on sex and age, neutrophils <2.0 x 10(9)/l, or platelets <140 x 10(9)/l) lasting >3 months. RESULTS: Persistent cytopenias unrelated to persistent disease were found in 43% of patients. Cytopenias were associated with clinically important rates of infection and transfusion requirement (P = 0.03) and predicted for worse overall survival (61% versus 96% at 60 months, P = 0.05). Increasing age predicted for persistent cytopenias (P = 0.02), but the presence of pretreatment cytopenias and delivered dose intensity were not predictive. The median times to resolution of anaemia, neutropenia, and thrombocytopenia were 7, 9, and 10 months, respectively. CONCLUSIONS: Cytopenias often persist >3 months after first-line fludarabine combination therapy and can lead to important clinical sequelae. Although cytopenias generally resolve over time, treating physicians should be aware of these factors when considering fludarabine combination chemotherapy and when documenting treatment response status in chronic lymphocytic leukaemia.

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway.

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50microM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7microg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7microg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.

125I-thrombin binds to clustered receptors on noncoated regions of mouse embryo cell surfaces.

We used electron microscope autoradiography (EMAR) to visualize the interaction of 125I-thrombin with its surface receptors on mouse embryo (ME) cells. Autoradiographic grains were spaced over the surface of cells in a periodic nonrandom pattern, indicating 125I-thrombin association with clusters of thrombin receptors. The grain spacing varied slightly from cell to cell, indicating subpopulations of cells with different numbers of thrombin receptors. The average distance between grains on ME cells after binding 125I-thrombin (125 ng/ml) at 37 degrees C was 1.65 +/- 0.49 microns. The average distance between grains on prefixed cells and cells incubated with 125I-thrombin at 4 degrees C was not significantly different from that observed at 37 degrees C. This indicates that thrombin receptors are clustered before thrombin binding and that the thrombin receptor aggregates do not redistribute into large aggregates on the surface of cells subsequent to thrombin binding. The number of grains per cluster also does not change under these three binding conditions. Thus, the number of occupied receptors in each cluster appears to be constant. On the basis of the average grain number and spacing, we estimate that each cluster is approximately 400 nm in diameter containing approximately 550 thrombin-binding sites. These receptor-clusters are not associated with specialized structures or coated regions of the membrane. Additionally, grains observed within cells were not found associated with coated vesicles. Therefore, neither the clustering patterns nor internalization of 125I-thrombin are characteristic of molecules which bind to receptors and are internalized by receptor-mediated endocytosis.

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells.

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.

High levels of intracellular bombesin characterize human small-cell lung carcinoma.

"Small cells" or "oat cells" characterize a virulent form of lung cancer and share many biochemical properties with peptide-secreting neurones. The neuropeptide bombesin is present in all small-cell lines examined, but not in other lung cancer cell lines, suggesting that bombesinergic precursor cells in lung may give rise to this disease.

Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23).

A specific, acquired chromosomal abnormality (deletion 3p) has been found in at least one chromosome 3 in 100 percent of the metaphases in 12 of 12 cell lines cultured from human small-cell lung cancer tissue and in 2-day tumor culture specimens from three patients. Analysis of the shortest region of overlap shows the deletion to be 3p(14-23). This specific change was not seen in five of five lung cancer cell lines other than small-cell lung cancer or in two lymphoblastoid lines cultured from cells of small-cell lung cancer patients whose tumors had the 3p deletion.

Technical note: removal of metal ion inhibition encountered during DNA extraction and amplification of copper-preserved archaeological bone using size exclusion chromatography.

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results.