Fluorescent Dye Paper-Based Method for Assessment of Pesticide Coverage on Leaves and Trees: A Citrus Grove Case Study.

Crop disease management depends on efficient and adequate pesticide distribution to reduce pest population. Instrument-based methods to evaluate the spatial distribution of pesticides are available, but they are not field-compatible because of instrument size, cost, and extensive sample preparation. The current gold standard of water-sensitive papers is field-compatible; however, these papers often produce false positives due to reaction with water from sources other than the pesticide mixture. Thus, we developed a novel method in which a fluorescent dye is sprayed over a crop with circles of filter paper (samplers) attached to the leaves. After collection, a lightbox is used to take pictures of the samplers, and an algorithm analyzes each image for percent coverage to visualize the pesticide distribution within the crop. Our method produces results quickly and inexpensively compared to current methods and can be applied to any crop to inform best pesticide application strategies.

Rotary manifold for automating a paper-based Salmonella immunoassay.

Foodborne pathogens are responsible for hundreds of thousands of deaths around the world each year. Rapid screening of agricultural products for these pathogens is essential to reduce and/or prevent outbreaks and pinpoint contamination sources. Unfortunately, current detection methods are laborious, expensive, time-consuming and require a central laboratory. Therefore, a rapid, sensitive, and field-deployable pathogen-detection assay is needed. We previously developed a colorimetric sandwich immunoassay utilizing immuno-magnetic separation (IMS) and chlorophenol red-ß-d-galactopyranoside for Salmonella detection on a paper-based analytical device (µPAD); however, the assay required many sample preparation steps prior to the µPAD as well as laboratory equipment, which decreased user-friendliness for future end-users. As a step towards overcoming these limitations in resource-limited settings, we demonstrate a reusable 3D-printed rotational manifold that couples with disposable µPAD layers for semi-automated reagent delivery, washing, and detection in 65 minutes. After IMS to clean the sample, the manifold performs pipette-free reagent delivery and washing steps in a sequential order with controlled volumes, followed by enzymatic amplification and colorimetric detection using automated image processing to quantify color change. Salmonella was used as the target pathogen in this project and was detected with the manifold in growth media and milk with detection limits of 4.4 × 102 and 6.4 × 102 CFU mL-1 respectively. The manifold increases user friendliness and simplifies immunoassays resulting in a practical product for in-field use and commercialization.

Analysis of change in gait in the ovine stifle: normal, injured, and anterior cruciate ligament reconstructed.

BACKGROUND: Many patients who undergo anterior cruciate ligament (ACL) reconstructive surgery develop post-traumatic osteoarthritis (PTOA). ACL reconstructive surgery may not fully restore pre-injury joint biomechanics, thereby resulting in further joint damage and contributing to the development of PTOA. In an ovine model of idealized ACL reconstruction (ACL-R), it has been shown that signs of PTOA develop within surgical joints by 20 weeks post-surgery. The aim of the present study was to investigate whether altered kinematics contribute to early PTOA development within ACL-R joints of the ovine injury model by comparing the gait of these surgical animals to the gait of a stable normal control group, and an unstable injury group in which the ACL and medial collateral ligament (MCL) had been transected. METHODS: Fifteen skeletally mature female sheep were allocated evenly into 3 treatment groups: normal control, ACL-R, and ACL/MCL Tx (each group n = 5). Each animal's gait was recorded at baseline, 4 weeks post injury, and 20 weeks post injury. Principal component analysis (PCA) was used to identify the kinematic patterns that may be discriminant between treatment groups. Results from previous studies were referenced to present the amount of gross PTOA-like changes that occurred in the joints. RESULTS: ACL-R and ACL/MCL transected (Tx) animals developed a similar amount of early PTOA-like changes within the surgical joints, but differed significantly in the amount of kinematic change present at 20 weeks post-surgery. We showed that the stifle joint kinematics of ACL/MCL Tx differed significantly from those of CTRL and the majority of ACL-R animals, while no significant differences in joint kinematic changes were found between ACL-R and CTRL animals. CONCLUSIONS: These results suggest that the early PTOA-like changes reported in the ACL-R model cannot be attributed exclusively to post-surgical kinematic changes, and therefore biologic components in the post-injury environment must be contributing significantly to PTOA development.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Principal angles separate subject illumination spaces in YDB and CMU-PIE.

The theory of illumination subspaces is well developed and has been tested extensively on the Yale Face Database B (YDB) and CMU-PIE (PIE) data sets. This paper shows that if face recognition under varying illumination is cast as a problem of matching sets of images to sets of images, then the minimal principal angle between subspaces is sufficient to perfectly separate matching pairs of image sets from nonmatching pairs of image sets sampled from YDB and PIE. This is true even for subspaces estimated from as few as six images and when one of the subspaces is estimated from as few as three images if the second subspace is estimated from a larger set (10 or more). This suggests that variation under illumination may be thought of as useful discriminating information rather than unwanted noise.

Pulsed electric field treatment as a potential method for microbial inactivation in scaffold materials for tissue engineering: the inactivation of bacteria in collagen gel.

AIMS: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels. METHODS AND RESULTS: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm(-1). For low levels of E. coli contamination (10(3) CFU ml(-1)), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (>or=10(4) CFU ml(-1)) did not achieve complete inactivation of E. coli. CONCLUSIONS: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log(10) CFU ml(-1). Complete inactivation was hindered at high cell densities by the tailing effect observed. SIGNIFICANCE AND IMPACT OF THE STUDY: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.

Accelerated image processing on FPGAs.

The Cameron project has developed a language called single assignment C (SA-C), and a compiler for mapping image-based applications written in SA-C to field programmable gate arrays (FPGAs). The paper tests this technology by implementing several applications in SA-C and compiling them to an Annapolis Microsystems (AMS) WildStar board with a Xilinx XV2000E FPGA. The performance of these applications on the FPGA is compared to the performance of the same applications written in assembly code or C for an 800 MHz Pentium III. (Although no comparison across processors is perfect, these chips were the first of their respective classes fabricated at 0.18 microns, and are therefore of comparable ages.) We find that applications written in SA-C and compiled to FPGAs are between 8 and 800 times faster than the equivalent program run on the Pentium III.

Precise matching of 3-D target models to multisensor data.

This paper presents a three-dimensional (3-D) model-based ATR algorithm that operates simultaneously on imagery from three heterogeneous, approximately boresight aligned sensors. An iterative search matches models to range and optical imagery by repeatedly predicting detectable features, measuring support for these features in the imagery, and adjusting the transformations relating the target to the sensors in order to improve the match. The result is a locally optimal and globally consistent set of 3-D transformations that precisely relate the best matching target features to combined range, IR, and color images. Results show the multisensor algorithm recovers 3-D target pose more accurately than does a traditional single-sensor algorithm. Errors in registration between images are also corrected during matching. The intended application is imaging from semiautonomous military scout vehicles.

A phase I randomized study of subcutaneous adjuvant IL-2 in combination with an autologous tumor vaccine in patients with advanced renal cell carcinoma.

We performed a prospective, randomized study to determine whether subcutaneous administration of interleukin-2 (IL-2) in combination with an autologous renal cell vaccine is feasible and can potentiate antitumor immunity. Seventeen patients with metastatic renal cell carcinoma underwent surgical resection with preparation of an autologous tumor cell vaccine. Patients were vaccinated intradermally twice at weakly intervals with 10(7) irradiated tumor cells plus bacillus Calmette-Guérin, and once with 10(7) tumor cells alone. Patients were randomized to one of three groups: no adjuvant IL-2, low-dose IL-2 (1.2 x 10(6) IU/m2), or high-dose IL-2 (1.2 x 10(7) IU/m2). IL-2 was administered subcutaneously on the day of vaccination and the subsequent 4 days. Immune response was monitored by delayed-type hypersensitivity (DTH) response to tumor cells as compared with normal autologous renal cells. Sixteen of 17 patients received vaccine therapy. Four patients developed cellular immunity specific for autologous tumor cells as measured by DTH responses; two had received no IL-2 and two had received high-dose IL-2. There were two partial responses (PR) noted, both in patients who received high-dose IL-2. One responding patient was DTH(+) and one was negative. A third patient who was DTH(+) after vaccination with no IL-2 had a dramatic PR after receiving IL-2 subcutaneously in a subsequent protocol. Prospective testing of response to recall antigens indicated that only 5 of 12 tested patients were positive, including both clinical responders. These data suggest that subcutaneously administered adjuvant IL-2 does not dramatically augment the immunologic response to autologous renal cell vaccines as determined by the development of tumor-specific DTH response.

Intensive therapy with cisplatin, interleukin-2 and interferon-alpha-2a in patients with metastatic melanoma. A phase II Study.

OBJECTIVE: Based upon their individual clinical activity and combined effects in animal models or in vitro, we wished to evaluate a regimen of cisplatin, interferon-alpha, and IL-2 in patients with metastatic melanoma. DESIGN: Phase II pilot study. SETTING: Referral-based US Government clinical research unit. PATIENTS: Nine patients with metastatic malignant melanoma. INTERVENTION: Cisplatin 75-100 mg/m2 was administered intravenously over 30 minutes on days 1 and 8. Interferon-alpha 2a 5 Mu/m2 body surface area (BSA) was given subcutaneously for 4 days beginning 1 day before each dose of cisplatin. Beginning on day 15 and day 22, IL-2 was administered by intravenous continuous infusion at 3 Mu/m2 BSA/d for 96 hours and by daily intravenous bolus concurrent with daily subcutaneous doses of interferon-alpha 2a. MAIN OUTCOME MEASURES: Antitumor response and toxicities. RESULTS: The study was stopped due to renal and hematopoietic toxicity and severe, delayed nausea and vomiting associated with the cisplatin-interferon treatment. Three of 9 patients achieved a partial response (duration 2.5, 4, 14+ months), and an additional patient had a 50% reduction in measurable tumor volume before undergoing resection of residual disease. Overall response rate was 45%. CONCLUSION: This regimen was associated with excessive toxicity, and the lack of complete responses in a patient cohort with favorable characteristics for response (good performance status, predominance of skin and lymph node metastatic sites) suggests that it had no advantage over less toxic treatment regimens. REGISTRATION: National Cancer Institute/Cancer Therapy Evaluation Program T89-0137.

Prolonged, continuous treatment of hairy cell leukemia patients with recombinant interferon-alpha 2a.

Interferons are not curative in hairy cell leukemia (HCL), and retreatment is necessary in most patients whose therapy is stopped. In an attempt to maintain or improve responses, we administered recombinant interferon-alpha 2a (rIFN-alpha 2a) continuously to patients with HCL who initially responded to this therapy. Of 53 evaluable patients enrolled in this study, 32 have received rIFN-alpha 2a continuously for a median of 5 years. Patients received 3 million units of rIFN-alpha 2a subcutaneously (SC) daily for 6 months, followed, in responding patients, by the same dose three times weekly. Twenty-one patients (40%) discontinued IFN after a median of 29 months, seven of whom developed resistant disease in association with anti-IFN antibodies. Treatment produced high response rates: complete response plus partial response (CR + PR) = 40 of 53 (76%), CR + PR + minor response (MR) = 43 of 53 (82%), with no differences in response rates between patients with and without splenectomy. Sixteen patients who had MR at 18 months had PR with prolonged treatment, nine of whom had a significant further reduction in the hairy cell infiltrate in the bone marrow (BM). The median granulocyte and platelet counts have continued to increase and the median serum soluble interleukin-2 receptor (sIL-2R) level has continued to decrease with prolonged treatment. Two patients developed erythrocytosis that may be treatment related, but no other new toxicities were noted with prolonged treatment. We conclude that prolonged, continuous rIFN-alpha 2a treatment has acceptable toxicity, is not associated with late development of IFN resistance, and results in continued hematologic improvement with time on treatment.

Rotating shaft avulsion amputations of the thumb.

In a three-year period twenty-nine patients were treated for rotating shaft avulsion amputations of the thumb. Twenty-three thumbs were considered suitable for replantation. A staged approach, consisting of primary replantation and secondary nerve grafting and tendon reconstruction, was used because of concern that survival rates would be low. Survival was achieved in nineteen of twenty-three replantations. At mean follow-up of 20.5 months grip strength was 94.6% of the unaffected side and key pinch was 77.1%. Five patients achieved a two-point discrimination less than five millimeters. Success was better anticipated and now a full reconstruction is carried out in a single-stage procedure.

Use of exogenous electric current in the treatment of delayed lesions in peripheral nerves.

A commonly observed clinical problem following nerve injury is the incomplete recovery of function associated with the formation of a neuroma in continuity. In the present study, exogenous direct electric current was tested for its ability to promote growth of axons through a neuroma-like lesion. Neuroma-like structures were created by crushing rat sciatic nerves at two sites 4 mm apart and applying phenol to the intermediate region. A bulbous axonally impenetrable structure is formed 3 weeks later. At that time, silicone cuffs were sewn onto the nerve proximal to the phenol application site and 7 mm distally. In experimental groups, cuffs were attached to wires leading to a subcutaneously implanted Traxon power source, the distal cuff being cathodal. In control groups, cuffs were not electrically connected. In electrically active groups, substantial numbers of myelinated axons were seen distal to the cathode 3 weeks after implantation. Four times fewer fibers were observed in control groups. Footprint patterns from electrically active animals revealed a significant improvement over control neuroma preparations, as quantitated using the Sciatic Functional Index.

Anatomic basis of a fascio-cutaneous flap supplied by the perforating branch of the peroneal artery.

The length, caliber and course of the perforating branch of the peroneal artery are described following examination of both feet of 20 cadavers. This terminal branch of the peroneal artery has a constant, predictable course and may normally be sacrificed without any vascular compromise in the foot. It has great surgical potential at it is strategically situated so that it can act as a vascular pedicle for a large cutaneous flap situated on the lower lateral leg, with an axis of rotation centered at the midtarsal joint. This has been used by the senior author (AC Masquelet) as a pedicle flap and as an island flap for reconstruction of the foot and ankle with satisfying results.

The lateral supramalleolar flap.

An anatomic study (40 fresh dissected specimens) and clinical experience (14 patients) have shown the reliability of a skin flap designed on the lower third of the lateral aspect of the leg. It is supplied by a cutaneous branch from the perforating branch of the peroneal artery. This perforating branch continues distally deep to the fascia along the anterior ankle and into the foot. This can be used as a reversed pedicle, giving the flap an arc of rotation that allows coverage of the dorsal, lateral, and plantar aspects of the foot, the posterior heel, and the lower medial portion of the leg.

Palliation in cyanotic congenital heart disease. Fifteen years' experience of various shunt procedures.

During the past 15 years, 143 systemic pulmonary shunt procedures have been performed in 117 patients. These have been evaluated for their clinical effectiveness, the need for a repeat operation and the mortality; particular attention was paid to the Teflon shunt. Variations were found in shunt performance, depending on the primary defect, the type of shunt that was employed and the year of operation. The overall shunt patency after three years was 77% (85% with the Teflon shunt). Although, in our total experience, mortality at 30 days was 12%, with 16% late deaths, "modified Blalock" (Teflon) shunts had only a 5% hospital mortality and a 5% late mortality within three years. Pulmonary atresia, without a ventricular septal defect, is often insufficiently palliated by a shunt alone. Ten of 82 patients with variations of the tetralogy complex died within 30 days of operation, and a further 11 died in the late follow-up period. Six of these 21 shunts were patent at autopsy. Less common defects, such as univentricular heart, transposition and double-outlet right ventricular connections, that are associated with pulmonary stenosis had no early mortality but led to four late deaths among 27 patients. Two of the four patients had patent shunts. Results in the early part of this experience were less than acceptable owing to inferior shunting techniques, postoperative management errors and, particularly, inadequate follow-up surveillance. With correction of these factors we find that the modified Blalock shunt provides very good early and late mortality results, with excellent clinical palliation and patency rates.