A simple enzymatic assay for the quantification of C1-specific cellulose oxidation by lytic polysaccharide monooxygenases.

OBJECTIVE: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). RESULTS: In combination with a ß-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. CONCLUSIONS: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

AOA Critical Issues Symposium: So, You Want to Be a Department Leader: Essentials for Success.

ABSTRACT: Most health systems are vertically integrated, and the leaders of orthopaedic surgery departments or service lines must have a comprehensive understanding of their role in the strategic plan of the health system. Orthopaedic surgery departments must be profitable while supporting the tripartite mission of excellence in clinical care, research, and education. This symposium had 4 specific objectives: to discuss how to (1) create synergy between the department or service line and the health system, (2) develop a strategy to enhance financial stability and revenue growth, (3) develop a comprehensive plan to enhance recruitment and retention of a diverse faculty, and (4) consider alternative strategies to foster education and research, even when the health system may be more focused on revenue generation.

Graphene field-effect transistors as room-temperature terahertz detectors.

The unique optoelectronic properties of graphene make it an ideal platform for a variety of photonic applications, including fast photodetectors, transparent electrodes in displays and photovoltaic modules, optical modulators, plasmonic devices, microcavities, and ultra-fast lasers. Owing to its high carrier mobility, gapless spectrum and frequency-independent absorption, graphene is a very promising material for the development of detectors and modulators operating in the terahertz region of the electromagnetic spectrum (wavelengths in the hundreds of micrometres), still severely lacking in terms of solid-state devices. Here we demonstrate terahertz detectors based on antenna-coupled graphene field-effect transistors. These exploit the nonlinear response to the oscillating radiation field at the gate electrode, with contributions of thermoelectric and photoconductive origin. We demonstrate room temperature operation at 0.3 THz, showing that our devices can already be used in realistic settings, enabling large-area, fast imaging of macroscopic samples.

A ballistic quantum ring Josephson interferometer.

We report the realization of a ballistic Josephson interferometer. The interferometer is made from a quantum ring etched in a nanofabricated two-dimensional electron gas confined in an InAs-based heterostructure laterally contacted to superconducting niobium leads. The Josephson current flowing through the structure shows oscillations with h/e flux periodicity when threading the loop with a perpendicular magnetic field. This periodicity, in sharp contrast with the h/2e one observed in conventional dc superconducting quantum interference devices, confirms the ballistic nature of the device in agreement with theoretical predictions. This system paves the way for the implementation of interferometric Josephson π-junctions, and for the investigation of Majorana fermions.

A few-layer graphene for advanced composite PVDF membranes dedicated to water desalination: a comparative study.

Membrane distillation is envisaged to be a promising best practice to recover freshwater from seawater with the prospect of building low energy-consuming devices powered by natural and renewable energy sources in remote and less accessible areas. Moreover, there is an additional benefit of integrating this green technology with other well-established operations dedicated to desalination. Today, the development of membrane distillation depends on the productivity-efficiency ratio on a large scale. Despite hydrophobic commercial membranes being widely used, no membrane with suitable morphological and chemical feature is readily available in the market. Thus, there is a real need to identify best practices for developing new efficient membranes for more productive and eco-sustainable membrane distillation devices. Here, we propose engineered few-layer graphene membranes, showing enhanced trans-membrane fluxes and total barrier action against NaCl ions. The obtained performances are linked with filling polymeric membranes with few-layer graphene of 490 nm in lateral size, produced by the wet-jet milling technology. The experimental evidence, together with comparative analyses, confirmed that the use of more largely sized few-layer graphene leads to superior productivity related efficiency trade-off for the membrane distillation process. Herein, it was demonstrated that the quality of exfoliation is a crucial factor for addressing the few-layer graphene supporting the separation capability of the host membranes designed for water desalination.

Tumor therapy with targeted atomic nanogenerators.

A single, high linear energy transfer alpha particle can kill a target cell. We have developed methods to target molecular-sized generators of alpha-emitting isotope cascades to the inside of cancer cells using actinium-225 coupled to internalizing monoclonal antibodies. In vitro, these constructs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels. Injection of single doses of the constructs at kilobecquerel (nanocurie) levels into mice bearing solid prostate carcinoma or disseminated human lymphoma induced tumor regression and prolonged survival, without toxicity, in a substantial fraction of animals. Nanogenerators targeting a wide variety of cancers may be possible.

Influence of femoral stem geometry, material and extent of porous coating on bone ingrowth and atrophy in cementless total hip arthroplasty: an iterative finite element model.

This work presents a computational model for the concurrent study of bone remodelling and ingrowth around cementless femoral stems in total hip arthroplasty. It is assumed that biological fixation depends upon the magnitude of relative displacement at the bone-stem interface as well as an ongoing updating of interface conditions during the remodelling process. The remodelling model determines the distribution of bone density by producing the stiffest structure for a given set of biological conditions at the point of equilibrium in bone turnover. Changes in bone density and patterns of ingrowth are compared for different stem geometries, materials and lengths of surface coating. Patterns of bone ingrowth on the tapered stem were independent of extent of porous coating, while ingrowth varied with the length of coating on the cylindrical stem. This model integrates knowledge of under what mechanical conditions bone ingrowth occurs on prosthetic stem surfaces with remodelling behaviour over time.

Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate.

Plant biomass is a low-cost and abundant source of carbohydrates for production of fuels, "green" chemicals and materials. Currently, biochemical conversion of the biomass into sugars via enzymatic hydrolysis is the most viable technology. Here, the role of carbohydrate binding modules (CBMs) in the disruption of insoluble polysaccharide structures and their capacity to enhance cellulase-promoted lignocellulosic biomass hydrolysis was investigated. We show that CBM addition promotes generation of additional reducing ends in the insoluble substrate by cellulases. On the contrary, bovine serum albumin (BSA), widely used in prevention of a non-specific protein binding, causes an increase in soluble reducing-end production, when applied jointly with cellulases. We demonstrate that binding of CBMs to cellulose is non-homogeneous, irreversible and leads to its amorphisation. Our results also reveal effects of CBM-promoted amorphogenesis on cellulose hydrolysis by cellulases.

Ultralow friction of ink-jet printed graphene flakes.

We report the frictional response of few-layer graphene (FLG) flakes obtained by the liquid phase exfoliation (LPE) of pristine graphite. To this end, we inkjet print FLG on bare and hexamethyldisilazane-terminated SiO2 substrates, producing micrometric patterns with nanoscopic roughness that are investigated by atomic force microscopy. Normal force spectroscopy and atomically-resolved morphologies indicate reduced surface contamination by solvents after a vacuum annealing process. Notably, the printed FLG flakes show ultralow friction comparable to that of micromechanically exfoliated graphene flakes. Lubricity is retained on flakes with a lateral size of a few tens of nanometres, and with a thickness as small as ∼2 nm, confirming the high crystalline quality and low defects density in the FLG basal plane. Surface exposed step edges exhibit the highest friction values, representing the preferential sites for the origin of the secondary dissipative processes related to edge straining, wear or lateral displacement of the flakes. Our work demonstrates that LPE enables fundamental studies on graphene friction to the single-flake level. The capability to deliver ultralow-friction-graphene over technologically relevant substrates, using a scalable production route and a high-throughput, large-area printing technique, may also open up new opportunities in the lubrication of micro- and nano-electromechanical systems.

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells.

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.

An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer.

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.

Immunotoxins against CD19 and CD22 are effective in killing precursor-B acute lymphoblastic leukemia cells in vitro.

Monoclonal antibodies (Mabs) conjugated to toxins or their subunits (immunotoxins or ITs) are undergoing clinical testing in adults with a variety of malignancies. The potential impact of this form of therapy in pediatric precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) has yet to be determined. Mabs directed against the cell surface antigens, CD19 and CD22 conjugated to deglycosylated ricin A chain (dgRTA) have been tested in patients with non-Hodgkin's lymphoma (NHL), but not in patients with pre-B ALL. Because of the encouraging performance of these ITs in phase I trials, we evaluated the specific cytotoxicity of anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) ITs or their combination (Combotox) on patient-derived pre-B ALL cells maintained in vitro on a stromal feeder layer. After 48 h in culture, cytotoxicity to tumor cells was determined by flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated anti-CD10, 19, and 22. Both RFB4-dgRTA and HD37-dgRTA induced a statistically significant reduction in the number of viable leukemic cells, and Combotox was even more effective. Our results demonstrate that these ITs are specifically cytotoxic to primary pre-B ALL cells and that they should be further evaluated for the therapy of B-lineage ALL.

Radioimmunotherapy for model B cell malignancies using 90Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies.

In recent years, radioimmunotherapy (RIT) with beta(-) particle emitting radionuclides targeting the CD20 antigen on B cells in the treatment of non-Hodgkin's lymphoma has provided the most compelling human clinical data for the success of RIT. CD19, like CD20, is an antigen expressed on the surface of cells of the B lineage, and CD19 may provide an alternative target for radioimmunotherapy of B cell neoplasms. CD19 has been largely overlooked as a target for conventional 131I RIT, because the antigen rapidly internalizes upon binding of antibody, resulting in catabolism and significant release of 131I. Such modulation may be an advantage to RIT with radiometals such as 90Y, 177Lu, 213Bi and 225Ac. Herein, we have compared beta(-) particle RIT with antibodies targeting either CD19 or CD20. The anti-CD19 and anti-CD20 antibodies, B4 or C2B8, respectively, were appended with the SCN-CHX-A''-DTPA bifunctional chelating agent and labeled with 90Y. In the tumor model used, there were three times as many CD20 target sites on lymphoma cells as compared to CD19 sites (62000 vs 20000 binding sites, respectively). We compared the efficacy of the 90Y-labeled antibodies to reduce lymphoma in a nude mouse xenograft solid tumor model, after measurable lymphoma appeared. Reduction in tumor size began at day 3 in all three 90Y-treated groups, but tumor began to recur in many animals 9 days after the treatments. There was one cure in each specific treatment group. In contrast, the tumor in the two control groups showed no regression. There was a significant prolongation of median survival time from xenograft (P < 0.0001) in all the 90Y-labeled antibody construct-treated groups (32 days for 0.15 mCi 90Y-B4; 26 days for 0.20 mCi 90Y-C2B8, and 23 days for 0.15 mCi 90Y-C2B8) in comparison to the two control groups (11 days for 0.02 mg of C2B8 and 9 days for untreated growth controls). Specificity of the radioimmunotherapy was also shown. In conclusion, 90Y-labeled anti-CD19 antibody has efficacy comparable to 90Y-labeled anti-CD20 antibody in the treatment of mice bearing human lymphoma xenografts. These data suggest that CD19-targeted RIT merits further study.

A molecular model of artificial glycoprotein with predetermined multiple immunodeterminants for gram-positive and gram-negative encapsulated bacteria.

An artificial molecule was synthesized by covalently linking the oligosaccharide haptens derived frm Streptococcus pneumoniae type 6A and Neisseria meningitidis group C capsular polysaccharides to the non-toxic mutant protein CRM197, serologically related to diphtheria toxin. Immunochemical analysis using polyclonal and monoclonal antibodies showed in the glycoprotein the presence of specific immunodeterminants of the native polysaccharides and of the carrier protein. The immunological activity of this hybrid molecule tested in two animal models gave evidence for anamnestic induction of serum antibodies specifically directed to the three distinct native molecules. They neutralized the toxicity of diphtheria toxin, recognized the polysaccharide capsule of S. pneumoniae type 6A and 6B (group 6) strain and killed the N. meningitidis group C bacteria by complement-mediated bacterial lysis. These findings support the possibility of using in humans a multivalent antigen with immunogenic activity for several epidemiologically significant Gram-positive and Gram-negative encapsulated bacterial strains.

Antithrombin III prophylaxis of venous thromboembolic disease after total hip or total knee replacement.

The use of antithrombin III (ATIII) replacement in combination with low-dose heparin therapy in prevention of postoperative venous thrombosis in patients following total hip replacement or total knee replacement was evaluated. A randomized prospective venographically controlled trial in hip replacement compared treatment with dextran 40 with a regimen of ATIII (1,500 units preoperatively and 1,000 units daily for five days) and low-dose heparin. Patients receiving ATIII/heparin had significantly higher postoperative ATIII concentrations than dextran-treated patients and also had a low incidence of venous thromboembolic disease (7 percent). The ATIII/heparin regimen was well tolerated with no increase in bleeding or significant prolongation of the activated partial thromboplastin time. Two cohorts of patients undergoing total knee replacement were studied using different doses of ATIII in combination with heparin. An initial 10 patients were treated with the same ATIII dose used for patients undergoing total hip replacement, with a 50 percent incidence of venous thrombosis. A second group of 11 patients was treated with twice the dose of ATIII, and an incidence of venous thrombosis of 27 percent was found. The higher ATIII dose resulted in significantly higher ATIII concentrations and maintained the postoperative ATIII concentration above normal. Among patients receiving prophylaxis with either warfarin. dextran, or ATIII/heparin, no clear association was found between reduced ATIII concentrations and occurrence of venous thrombosis. It is concluded that ATIII replacement following total hip or knee replacement corrects the postoperative ATIII deficiency and that the combination of ATIII and low-dose heparin is an effective prophylactic regimen following total hip replacement.

Randomized trial comparing single dose versus fractionated irradiation for prevention of heterotopic bone: a preliminary report.

Radiation therapy has been shown to prevent heterotopic bone formation in high risk patients undergoing total hip replacement. A number of doses have been used without a randomized trial comparing one dose regimen against another. A prospective randomized trial was undertaken comparing 10 Gy in 5 fractions versus 8 Gy in 1 fraction. Forty-seven patients have been randomized at the time of this evaluation with 37 patients eligible for analysis. The pre-operative, immediate post-operative and 2 month post-operative radiographs were graded. At the time of this analysis, 17 patients were randomized to the 8 Gy arm with 20 patients in the 10 Gy arm. Patients were treated with limited fields so as to only cover the area at risk for development of heterotopic bone to prevent adverse effects on biologic fixation of uncemented implants. When comparing the pre-operative, operative, and 2 month post-operative radiographs, only four patients (1 patient in the 8 Gy arm and 3 patients in the 10 Gy arm), had an increase in the score. No patient had an increase in score to a clinically significant level, usually grade 3 or 4. These preliminary results appear to show that 8 Gy in a single fraction can be as effective as 10 Gy in 5 fractions in preventing heterotopic bone in susceptible individuals. Further follow-up of the remaining patients may confirm this.

Comparison of two warfarin regimens in the prevention of venous thrombosis following total knee replacement.

A prospective, randomized trial was conducted to compare the effectiveness and safety of warfarin given in two regimens in prevention of venous thrombosis after total knee replacement. Adult patients scheduled for primary or revision total knee replacement were randomly assigned to receive either a "two-step" warfarin regimen beginning 10-14 days pre-operatively or, alternatively, to begin warfarin the night before surgery. Post-operatively, the dose was adjusted in both groups to achieve a target International Normalized Ratio (INR) of 2.2 and prophylaxis was continued until venography on post-operative days five through nine. Bleeding was assessed by surgical blood loss, transfusion requirements, changes in hematocrit, and clinically identified bleeding complications. The occurrence of deep vein thrombosis was nearly the same in the two treatment groups, 39% in patients randomized to the two-step regimen as compared to 38% in those beginning the night before surgery. The occurrence of proximal vein thrombosis was also similar, 5% versus 7% (p = NS). Patients in the two-step group received 1.33 +/- 1.26 transfusions compared to 0.95 +/- 1.22 in the night before group (p < 0.05) and also had a lower nadir post-operative hematocrit of 26.7 +/- 3.1 as compared to 28.5 +/- 3.2 (p < 0.0001). Major bleeding complications were associated with excessively prolonged INRs and occurred in five patients in the two-step group and two in the night before group. Patients in both groups who developed thrombosis had a significantly lower INR on post-operative days two and three compared to those without thrombosis. We conclude that a prophylactic warfarin regimen for prevention of deep vein thrombosis after total knee replacement beginning the night before surgery is more convenient and may be associated with less bleeding than a regimen beginning warfarin 10-14 days pre-operatively. Careful control of anticoagulant intensity is needed to achieve maximum effectiveness and avoidance of bleeding complications.

Assessment of inactivation of hepatitis A vaccine by compound PCR.

Assuring the complete inactivation of hepatitis A virus (HAV) vaccine commonly requires prolonged tissue culture amplification, followed by detection of virus antigen in cell lysates. A reliable, but faster, alternative procedure is highly desirable since it will permit the prescreening of experimental batches of killed HAV, prior to tissue-culture amplification. We established experimental conditions for simultaneous, polymerase chain reaction (PCR)-based amplification of viral and cellular mRNA sequences from infected cell RNA (compound PCR). Under these conditions, the presence of virus-specific amplified sequences, as detected by Southern blot, allows the identification of incompletely inactivated vaccine batches with a threshold practically identical to that of the more time-consuming subculture and ELISA. Compound PCR is, by its nature flexible enough for adaption to different requirements and it should prove useful for rapid prescreening of vaccine batches and pilot studies for improvement of inactivation protocols.

The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: local fluorescence study at the single-molecule level.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.