Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

Increased Production of the Soluble Tumor-Associated Antigens CA19-9, CA125, and CA15-3 in Rheumatoid Arthritis

Some tumor-associated antigens (TAAs) are expressed on inflammatory cells. We previously detected carcinoembryonic antigen (CEA; CD66) in the rheumatoid (RA) synovium. The production of CEA, CA19-9, CA125, and CA15.3, may be increased in patients with RA, scleroderma, lupus, and SjÖgren's syndrome (SS). Some of these TAAs contain sialylated carbohydrate motifs and they are involved in tumor-associated cell adhesion and metastasis. We assessed levels of TAAs in the sera of RA patients and healthy subjects. Serum TAA levels were correlated with disease markers including serum rheumatoid factor (RF), C-reactive protein (CRP), and anti-CCP antibody levels, DAS28, age disease duration. TAAs including CEA, CA15-3, CA72-4, CA125, and CA19-9, and neuron-specific enolase (NSE) were assessed by immunoassay in the sera of 75 patients with RA and 50 age- and sex-matched healthy controls. Normal upper limits for these TAAs were 3.4 Μg/L, 25 kU/L, 6.9 kU/L, 35 kU/L, 34 kU/L, and 16.3 Μg/L, respectively. There were significantly more RA patients showing abnormally high levels of CA125 (10.8% versus 7.1%), CA19-9 (8.1% versus 0%), and CA15-3 (17.6% versus 14.3%) in comparison to controls ( P < 0.05). The mean absolute serum levels of CA125 (23.9 ± 1.8 versus 16.8 ± 2.2 kU/L) and CA19-9 (14.2 ± 1.2 versus 10.5 ± 1.6 kU/L) were also significantly higher in RA compared to controls ( P < 0.05). Among RA patients, serum CEA showed significant correlation with RF ( r = 0.270; P < 0.05). None of the assessed TAAs showed any correlation with CRP, anti-CCP, DAS28, age or disease duration. The concentration of some TAAs may be elevated in the sera of patients with established RA in comparison to healthy subjects. CEA, CA19-9, CA125, and CA15-3 contain carbohydrate motifs and thus they may be involved in synovitis-associated adhesive events. Furthermore, some TAAs, such as CEA, may also correlate with prognostic factors, such as serum RF levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73224/1/annals.1422.037.pd

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.Optical Imaging; Biological Services; Biophysic

Percutaneous electrical stimulation of lumbosacral roots in man.

High voltage percutaneous electrical stimulation over the lumbosacral spinal column was used to assess conduction in the cauda equina of 13 normal subjects. Electromyographic activity elicited by such stimulation was recorded from various muscles of the lower limbs. The stimulating cathode was placed over the spinous process of each vertebral body and the anode kept on the iliac crest contralateral to the studied limb. Shifting the cathode in a rostro-caudal direction shortened the response latency in quadriceps, tibialis anterior and extensor digitorum brevis muscles. At moderate intensities (60% maximum), this occurred abruptly when the cathode was placed at levels corresponding to the exit sites from the spinal canal of the roots innervating these muscles. At these intensities, the size of the response in each muscle was largest when the cathode was placed over the conus medullaris or at or below the exit of the motor roots from the spine. Latencies were always equal to or shorter than those obtained with F-wave measurements, suggesting that peripheral motor axons, rather than intraspinal structures were activated by the stimulus. Collision experiments demonstrated that activation occurred at two sites: near the spinal cord and at the root exit site in the vertebral foramina. Recordings made from soleus indicated that larger diameter proprioceptive afferent fibres also could be activated. This technique might have useful clinical applications in the study of both proximal and distal lesions of the cauda equina and provide a non-invasive method of localising such lesions electrophysiologically

Trap attributes influencing capture of Diabrotica speciosa (Coleoptera: Chrysomelidae) on common bean fields Atributos da armadilha influenciam captura de Diabrotica speciosa (Coleoptera: Chrysomelidae) em feijoeiro

Refinements in trap characteristics may improve ability to monitor and mass-trap beetles. Field assays were conducted in common bean fields to assess responses of Diabrotica speciosa (Germar) to some trap characteristics. Golden yellow plastic cups (750 mL) traps caught more D. speciosa females and males than did clear traps. Carrot slices in Petri dishes baited with Lagenaria vulagaris L. powder (cucurbitacin source - 0.28%) caught more beetles than did dishes with carrot alone. Dispensers for the floral volatile attractant 1,4-dimethoxybenze were also compared. Rubber septa dispenser attracted more beetles than did control (dental wicks saturated with acetone). Captures on dental wick, starch matrix and feminine pad dispensers were intermediate and did not differ from those on rubber septa and unbaited controls. Perforated bottle traps (2000 mL), when baited with the floral attractant, caught more beetles than did window bottle traps (both traps contained L. vulgaris powder) in most assessments done from two to ten days after trap placement in the field. Traps with the insecticide carbaryl captured more beetles than did traps without it, 2-4 and 8-10 days after trap placement in the field, but not in the remaining periods (0-2, 4-6 and 6-8 days). Traps baited with 1,4-dimethoxybenzene captured more beetles than did the unbaited ones in all assessments (each other day from two to ten days after trap placement in the field). Finally, similar amounts of beetles were captured using plastic bottle traps (2000 mL): perforated, window (both with cucurbitacin) and sticky (without cucurbitacin) traps, when were baited with the floral attractant.<br>Refinamentos em caraterísticas de armadilhas podem incrementar sua habilidade para monitorar e capturar em massa os insetos. Experimentos foram conduzidos em lavoura de feijoeiro para verificar as respostas de Diabrotica speciosa (Germar) a algumas características de armadilhas. Armadilhas de copos plástico (750 mL) amarelo ouro capturaram mais fêmeas e machos de D. speciosa do que transparentes. Placas de Petri com pedaços de cenoura, com pó seco de purungo, Lagenaria vulgaris L. (fonte de cucurbitacina - 0,28%) capturaram mais insetos do que placas com somente cenoura. Liberadores para o atraente floral 1,4-dimetoxibenzeno foram também comparados. Liberador de septo de borracha atraiu mais insetos do que o controle (flocos dentais saturados com acetona). Capturas nos tratamentos com liberadores de floco dental, matriz borato e absorvente higiênico foram intermediárias mas não diferiram da testemunha e septo. Modelo de armadilha de garrafa (2000 mL) furada capturou mais insetos do que armadilha vazada ("janelas") (ambas contendo pó seco de purungo) na maioria das avaliações, dos dois até os dez dias após a instalação das armadilhas no campo. Armadilhas com inseticida carbaryl capturaram mais insetos do que armadilhas sem, nos intervalos de 2-4 e 9-10 dias após a instalação das armadilhas no campo. Nos períodos restantes (0-2, 4-6- e 6-8 dias), diferenças não foram detectadas. Armadilhas com 1,4-dimetoxibenzeno capturaram mais insetos do que armadilhas sem o volátil (avaliações a cada dois dias dos dois aos dez dias após a instalação no campo). Finalmente, quantidades similares de insetos foram capturadas usando armadilhas de garrafa pet (2000 mL): vazadas ("janelas"), furadas (ambas com cucurbitacina) e adesivas quando estas foram iscadas com o atraente floral