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BACKGROUND: The laparoscopic approach to liver resection may reduce morbidity and hospital stay. However, uptake has been slow due to concerns about patient safety and oncological radicality. Image guidance systems may improve patient safety by enabling 3D visualisation of critical intra- and extrahepatic structures. Current systems suffer from non-intuitive visualisation and a complicated setup process. A novel image guidance system (SmartLiver), offering augmented reality visualisation and semi-automatic registration has been developed to address these issues. A clinical feasibility study evaluated the performance and usability of SmartLiver with either manual or semi-automatic registration. METHODS: Intraoperative image guidance data were recorded and analysed in patients undergoing laparoscopic liver resection or cancer staging. Stereoscopic surface reconstruction and iterative closest point matching facilitated semi-automatic registration. The primary endpoint was defined as successful registration as determined by the operating surgeon. Secondary endpoints were system usability as assessed by a surgeon questionnaire and comparison of manual vs. semi-automatic registration accuracy. Since SmartLiver is still in development no attempt was made to evaluate its impact on perioperative outcomes. RESULTS: The primary endpoint was achieved in 16 out of 18 patients. Initially semi-automatic registration failed because the IGS could not distinguish the liver surface from surrounding structures. Implementation of a deep learning algorithm enabled the IGS to overcome this issue and facilitate semi-automatic registration. Mean registration accuracy was 10.9 ± 4.2 mm (manual) vs. 13.9 ± 4.4 mm (semi-automatic) (Mean difference - 3 mm; p = 0.158). Surgeon feedback was positive about IGS handling and improved intraoperative orientation but also highlighted the need for a simpler setup process and better integration with laparoscopic ultrasound. CONCLUSION: The technical feasibility of using SmartLiver intraoperatively has been demonstrated. With further improvements semi-automatic registration may enhance user friendliness and workflow of SmartLiver. Manual and semi-automatic registration accuracy were comparable but evaluation on a larger patient cohort is required to confirm these findings.
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Realidad Aumentada , Hígado/cirugía , Cirugía Asistida por Computador/métodos , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We report the development and characterisation of highly miniaturised fibre-optic sensors for simultaneous pressure and temperature measurement, and a compact interrogation system with a high sampling rate. The sensors, which have a maximum diameter of 250 µm, are based on multiple low-finesse optical cavities formed from polydimethylsiloxane (PDMS), positioned at the distal ends of optical fibres, and interrogated using phase-resolved low-coherence interferometry. At acquisition rates of 250 Hz, temperature and pressure changes of 0.0021 °C and 0.22 mmHg are detectable. An in vivo experiment demonstrated that the sensors had sufficient speed and sensitivity for monitoring dynamic physiological pressure waveforms. These sensors are ideally suited to various applications in minimally invasive surgery, where diminutive lateral dimensions, high sensitivity and low manufacturing complexities are particularly valuable.
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Tecnología de Fibra Óptica/instrumentación , Interferometría/métodos , Presión , Temperatura , Diseño de Equipo , Fibras Ópticas , TransductoresRESUMEN
A versatile and scalable fabrication method for laser-generated focused ultrasound transducers is proposed. The method is based on stamping a coated negative mold onto polydimethylsiloxane, and it can be adapted to include different optical absorbers that are directly transferred or synthesized in situ. Transducers with a range of sizes down to 3 mm in diameter are presented, incorporating two carbonaceous (multiwalled carbon nanoparticles and candle soot nanoparticles) and one plasmonic (gold nanoparticles) optically absorbing component. The fabricated transducers operate at central frequencies in the vicinity of 10 MHz with bandwidths in the range of 15-20 MHz. A transducer with a diameter of 5 mm was found to generate a positive peak pressure greater than 35 MPa in the focal zone with a tight focal spot of 150 µm in lateral width. Ultrasound cavitation on the tip of an optical fiber was demonstrated in water for a transducer with a diameter as small as 3 mm.
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In this paper we present a system aimed at demonstrating the photoacoustic (PA) effect for educational purposes. PA imaging is a hybrid imaging modality that requires no contrast agent and has a great potential for spine and brain lesion characterisation, breast cancer and blood flow monitoring notably in the context of fetal surgery. It relies on combining light excitation with ultrasound reception. Our brief was to present and explain PA imaging in a public-friendly way suitable for a variety of ages and backgrounds. We developed a simple, accessible demonstration unit using readily available materials. We used a modulated light emitting diode (LED) torch and an electronic stethoscope. The output of a music player was used for light modulation and the chest piece of the stethoscope covered by a black tape was used as an absorbing target and an enclosed chamber. This demonstration unit was presented to the public at the Bloomsbury Festival On Light in October 2015. Our stall was visited by over 100 people of varying ages. Twenty families returned in-depth evaluation questionnaires, which show that our explanations of the photoacoustic effect were well understood. Their interest in biomedical engineering was increased.
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BACKGROUND: Bevacizumab improves outcome for most recurrent glioblastoma patients, but the duration of benefit is limited and survival after initial bevacizumab progression is poor. We evaluated bevacizumab continuation beyond initial progression among recurrent glioblastoma patients as it is a common, yet unsupported practice in some countries. METHODS: We analysed outcome among all patients (n=99) who received subsequent therapy after progression on one of five consecutive, single-arm, phase II clinical trials evaluating bevacizumab regimens for recurrent glioblastoma. Of note, the five trials contained similar eligibility, treatment and assessment criteria, and achieved comparable outcome. RESULTS: The median overall survival (OS) and OS at 6 months for patients who continued bevacizumab therapy (n=55) were 5.9 months (95% confidence interval (CI): 4.4, 7.6) and 49.2% (95% CI: 35.2, 61.8), compared with 4.0 months (95% CI: 2.1, 5.4) and 29.5% (95% CI: 17.0, 43.2) for patients treated with a non-bevacizumab regimen (n=44; P=0.014). Bevacizumab continuation was an independent predictor of improved OS (hazard ratio=0.64; P=0.04). CONCLUSION: The results of our retrospective pooled analysis suggest that bevacizumab continuation beyond initial progression modestly improves survival compared with available non-bevacizumab therapy for recurrent glioblastoma patients require evaluation in an appropriately randomised, prospective trial.
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Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Anciano , Inhibidores de la Angiogénesis/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Bevacizumab , Progresión de la Enfermedad , Esquema de Medicación , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Chronic disseminated candidosis, often referred to as hepatosplenic candidosis (HSC), is an infection due to Candida spp. that mainly involves the liver and spleen. HSC occurs mostly in patients after profound and prolonged neutropenia, which is more often seen in patients with acute haematological malignancies. The incidence of HSC ranges from 3% to 29% in patients suffering from Acute Leukaemia. However, it is now seen less frequently with the widespread use of antifungal agents as prophylaxis or as preemptive therapy. Early and adequate diagnosis and treatment of HSC are crucial, as treatment delays can negatively affect the prognosis of the underlying condition. The pathogenesis is not well understood, but it is believed that it may be due to an unbalanced adaptive immune response that leads to an exacerbated inflammatory reaction, resulting in an Immune Reconstitution Inflammatory Syndrome. In this context, new therapeutic approaches such as the use of adjuvant high-dose corticosteroids have been shown beneficial. This article will focus on the clinical, diagnostic and therapeutic aspects of HSC and provide an accurate review of recent pathophysiological data.
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Candida/fisiología , Candidiasis/microbiología , Hepatopatías/microbiología , Enfermedades del Bazo/microbiología , Animales , Antifúngicos/uso terapéutico , Candida/aislamiento & purificación , Candidiasis/tratamiento farmacológico , Candidiasis/inmunología , Humanos , Hepatopatías/tratamiento farmacológico , Hepatopatías/inmunología , Enfermedades del Bazo/tratamiento farmacológico , Enfermedades del Bazo/inmunologíaRESUMEN
OBJECTIVE: To report a case of primary intracerebral Hodgkin lymphoma with disease recurrence. METHODS: Case report and review of the literature. RESULTS: A 58-year-old immunocompetent male presented with aphasia. Neuroimaging revealed a left temporal lobe lesion. A craniotomy and resection were performed, and the diagnosis of classical Hodgkin lymphoma was made. Systemic work-up for lymphoma was negative. Postoperatively, the patient was treated with whole brain irradiation. 14 months later, the patient developed an enhancing lesion in his pons and received combination chemotherapy and radiation therapy. Repeat imaging demonstrated leptomeningeal enhancement and multiple lesions throughout the cerebral hemispheres, cerebellum and brainstem. COMMENT: We report what appears to be the first case of a patient with aggressive primary intracerebral Hodgkin lymphoma with disease recurrence.
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Neoplasias Encefálicas/patología , Enfermedad de Hodgkin/patología , Recurrencia Local de Neoplasia/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirugía , Irradiación Craneana , Resultado Fatal , Enfermedad de Hodgkin/radioterapia , Enfermedad de Hodgkin/cirugía , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/radioterapia , Procedimientos NeuroquirúrgicosRESUMEN
BACKGROUND AND PURPOSE: Despite high interest in machine-learning algorithms for automated segmentation of MRIs of patients with brain tumors, there are few reports on the variability of segmentation results. The purpose of this study was to obtain benchmark measures of repeatability for a widely accessible software program, BraTumIA (Versions 1.2 and 2.0), which uses a machine-learning algorithm to segment tumor features on contrast-enhanced brain MR imaging. MATERIALS AND METHODS: Automatic segmentation of enhancing tumor, tumor edema, nonenhancing tumor, and necrosis was performed on repeat MR imaging scans obtained approximately 2 days apart in 20 patients with recurrent glioblastoma. Measures of repeatability and spatial overlap, including repeatability and Dice coefficients, are reported. RESULTS: Larger volumes of enhancing tumor were obtained on later compared with earlier scans (mean, 26.3 versus 24.2 mL for BraTumIA 1.2; P < .05; and 24.9 versus 22.9 mL for BraTumIA 2.0, P < .01). In terms of percentage change, repeatability coefficients ranged from 31% to 46% for enhancing tumor and edema components and from 87% to 116% for nonenhancing tumor and necrosis. Dice coefficients were highest (>0.7) for enhancing tumor and edema components, intermediate for necrosis, and lowest for nonenhancing tumor and did not differ between software versions. Enhancing tumor and tumor edema were smaller, and necrotic tumor larger using BraTumIA 2.0 rather than 1.2. CONCLUSIONS: Repeatability and overlap metrics varied by segmentation type, with better performance for segmentations of enhancing tumor and tumor edema compared with other components. Incomplete washout of gadolinium contrast agents could account for increasing enhancing tumor volumes on later scans.
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Neoplasias Encefálicas , Glioblastoma , Algoritmos , Neoplasias Encefálicas/diagnóstico por imagen , Glioblastoma/diagnóstico por imagen , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Carga TumoralRESUMEN
BACKGROUND: We evaluated bevacizumab with metronomic etoposide among recurrent malignant glioma patients in a phase 2, open-label trial. METHODS: A total of 59 patients, including 27 with glioblastoma (GBM) and 32 with grade 3 malignant glioma, received 10 mg kg(-1) bevacizumab biweekly and 50 mg m(-2) etoposide daily for 21 consecutive days each month. The primary end point was a 6-month progression-free survival, and secondary end points included safety and overall survival. Vascular endothelial growth factor (VEGF), VEGFR-2, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor-2alpha (HIF-2alpha) were assessed semiquantitatively in archival tumours using immunohistochemistry and were correlated with outcome. RESULTS: Among grade 3 and GBM patients, the 6-month progression-free survivals were 40.6% and 44.4%, the radiographic response rates were 22% and 37% and the median survivals were 63.1 and 44.4 weeks, respectively. Hypertension predicted better outcome among both grade 3 and GBM patients, whereas high CA9 and low VEGF were associated with poorer progression-free survival (PFS) among those with GBM. The most common grade > or = 3 adverse events included neutropaenia (24%), thrombosis (12%), infection (8%) and hypertension (3%). Two patients had asymptomatic, grade 1 intracranial haemorrhage and one on-study death occurred because of pulmonary embolism. CONCLUSION: Bevacizumab with metronomic etoposide has increased toxicity compared with previous reports of bevacizumab monotherapy. Its anti-tumour activity is similar to that of bevacizumab monotherapy or bevacizumab plus irinotecan. (ClinicalTrials.gov: NCT00612430).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Administración Oral , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Bevacizumab , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/mortalidad , Etopósido/administración & dosificación , Etopósido/efectos adversos , Femenino , Glioma/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Strongly directional ultrasound sources are desirable for many minimally invasive applications, as they enable high-quality imaging in the presence of positioning uncertainty. All-optical ultrasound is an emerging paradigm that exhibits high frequencies, large bandwidths, and a strong miniaturisation potential. Here, we report the design, modelling, and fabrication of a highly directional fibre-optic ultrasound transmitter that uses a composite of reduced graphene oxide and polydimethylsiloxane as the optical ultrasound generator. The ultrasound transmitter, which had an outer diameter of just 630 µm, generated ultrasound with a pressure exceeding 0.4 MPa for axial distances up to 16 mm, at a large bandwidth of 24.3 MHz. The ultrasound beam exhibited low divergence, with a beam diameter ranging between 0.6 and 2.1 mm for distances between 0 and 16 mm. The presented directional optical ultrasound source is hence well-suited to high-resolution interventional imaging.
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Polarization sensitive optical coherence tomography (PS-OCT) provides a cross-sectional image of birefringence in biological samples that is complementary in many applications to the standard reflectance-based image. Recent ex vivo studies have demonstrated that birefringence mapping enables the characterization of collagen and smooth muscle concentration and distribution in vascular tissues. Instruments capable of applying these measurements percutaneously in vivo may provide new insights into coronary atherosclerosis and acute myocardial infarction. We have developed a polarization sensitive optical frequency domain imaging (PS-OFDI) system that enables high-speed intravascular birefringence imaging through a fiber-optic catheter. The novel design of this system utilizes frequency multiplexing to simultaneously measure reflectance of two incident polarization states, overcoming concerns regarding temporal variations of the catheter fiber birefringence and spatial variations in the birefringence of the sample. We demonstrate circular cross-sectional birefringence imaging of a human coronary artery ex vivo through a flexible fiber-optic catheter with an A-line rate of 62 kHz and a ranging depth of 6.2 mm.
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Vasos Coronarios/ultraestructura , Tecnología de Fibra Óptica/instrumentación , Aumento de la Imagen/instrumentación , Microscopía de Polarización/instrumentación , Tomografía de Coherencia Óptica/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Tecnología de Fibra Óptica/métodos , Humanos , Aumento de la Imagen/métodos , Microscopía de Polarización/métodos , Fibras Ópticas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tomografía de Coherencia Óptica/métodosRESUMEN
RATIONALE: Second-line chemotherapy is disappointing in recurrent high-grade gliomas. Dramatic responses in recurrent high-grade gliomas have been reported in a recent monocentric trial with a novel association combining bevacizumab (anti-VEGF monoclonal antibody agent) and irinitecan. OBJECTIVE: To report the experience of the ANOCEF group (French speaking neuro-oncology association) using the bevacizumab-irinotecan combination in recurrent high-grade gliomas. METHODS: Eight centers were involved in this retrospective multicenter study. Bevacizumab-irinotecan was delivered as previously described in a compassional setting to non-selected patients suffering from a high-grade glioma (WHO grade III and IV). Response rate at two months of the onset of the treatment was analyzed using the Macdonald criteria. The toxicity profile of the treatment was also investigated. RESULTS: From 2006 to 2007, 77 patients were treated (median age: 52 years; median Karnofsky score: 70) for a recurrent high-grade glioma (49 grade IV, 28 grade III). At two months, the response rates were objective response=36% (54% in grade III and 27% in grade IV); stable disease=39%; progressive disease=13%; patients not evaluable because of a rapid fatal clinical deterioration=12%. Improvement was noted in 49% of patients. Among the main toxicities, we noted; intratumoral hemorrage (n=5 with spontaneous regression in three) and thromboembolic complications including venous thrombophlebitis (n=4), pulmonary embolism (n=2), myocardial infarction (n=1), grade III-IV hematotoxicity (n=2), reversible leukoencephalopathy (n=1). CONCLUSION: This retrospective multicenter study adds further arguments in favor of the promising results of this new combination and its potential rapidity of action in recurrent high-grade gliomas. Antiangiogenic agents expose the patients to a well-known risk of thromboembolic and hemorragic complications, necessitating careful follow-up and patient selection in light of the cardiovascular contraindications.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Bevacizumab , Neoplasias Encefálicas/patología , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Preescolar , Femenino , Glioma/patología , Humanos , Irinotecán , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Estudios RetrospectivosRESUMEN
We present a novel method for rapidly acquiring optical coherence tomography (OCT) images at multiple backscattering angles. By angularly compounding these images, high levels of speckle reduction were achieved. Signal-to-noise ratio (SNR) improvements of 3.4 dB were obtained from a homogeneous tissue phantom, which was in good agreement with the predictions of a statistical model of speckle that incorporated the optical parameters of the imaging system. In addition, the fast acquisition rate of the system (10 kHz A-line repetition rate) allowed angular compounding to be performed in vivo without significant motion artifacts. Speckle-reduced OCT images of human dermis show greatly improved delineation of tissue microstructure.
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As the 20th century ended, Fusarium mycotoxicology entered the age of genomics. With complete genomes of Fusarium graminearum and F. verticillioides and several Fusarium gene expression sequence databases on hand, researchers worldwide are working at a rapid pace to identify mycotoxin biosynthetic and regulatory genes. Seven classes of mycotoxin biosynthetic genes or gene clusters have been identified in Fusarium to date; four are polyketide synthase gene clusters for equisetin, fumonisins, fusarins, and zearalenones. Other Fusarium mycotoxin biosynthetic genes include a terpene cyclase gene cluster for trichothecenes, a cyclic peptide synthetase for enniatins, and a cytochrome P450 for butenolide. From the perspective of the United States Department of Agriculture, the ultimate goal of research on Fusarium molecular biology is to reduce mycotoxins in cereal grains. With this goal in mind, efforts have focused on identifying aspects of mycotoxin biosynthesis and regulation that can be exploited for mycotoxin control. New information on fungal and plant genomes and gene expression will continue to provide information on genes important for fungal-plant interactions and to facilitate the development of targeted approaches for breeding and engineering crops for resistance to Fusarium infection and mycotoxin contamination.
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Fusarium/genética , Regulación de la Expresión Génica , Genes Fúngicos , Micotoxinas/genética , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Fusarium/metabolismo , Familia de Multigenes , Micotoxinas/biosíntesisRESUMEN
Speckle noise significantly limits the information content provided by coherent optical imaging methods such as optical coherence tomography and its recent derivative, optical frequency-domain imaging (OFDI). In this paper, we demonstrate a novel OFDI system that simultaneously acquires hundreds of angularly resolved images, which can be compounded to reduce speckle noise. The system comprises an InGaAs line-scan camera and an interferometer, configured so that the elements of the detector array simultaneously capture light spanning a backscattering angular range of 32 degrees. On successive read-outs of the array, the wavelength of the laser source was stepped through a range of 130 nm centered at 1295 nm to concurrently generate 400 angle-resolved OFDI images. A theory of angle-resolved OFDI and the design equations of the system are presented. Incoherent averaging of the angle-resolved data is shown to yield substantial speckle reduction (as high as an 8 dB SNR improvement) in images of a tissue phantom and esophageal tissue ex vivo.
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Antisense therapy for the treatment of bacterial infections is a very attractive alternative to overcome drug resistance problems. However, the penetration of antisense oligonucleotides into bacterial cells is a major huddle that has delayed research and application in this field. In the first part of this study, we defined efficient conditions to encapsulate plasmid DNA and antisense oligonucleotides in a fluid negatively charged liposome. Subsequently, we evaluated the potential of liposome-encapsulated antisense oligonucleotides to penetrate the bacterial outer membrane and to inhibit gene expression in bacteria. It was found that 48.9+/-12% and 43.5+/-4% of the purified plasmid DNA and antisense oligonucleotides were respectively encapsulated in the liposomes. Using fluorescence-activated cell sorting analysis, it was shown, after subtraction of the fluorescence values due to the aggregation phenomenon measured at 4 degrees C, that about 57% of bacterial cells had integrated the encapsulated antisense oligonucleotides whereas values for free antisenses were negligible. The uptake of the encapsulated anti-lacZ antisense oligonucleotides resulted in a 42% reduction of beta-galactosidase compared to 9% and 6% for the encapsulated mismatch antisense oligonucleotides and the free antisense oligonucleotides respectively. This work shows that it is possible to encapsulate relatively large quantities of negatively charged molecules in negative fluid liposomes and suggests that fluid liposomes could be used to deliver nucleic acids in bacteria to inhibit essential bacterial genes.
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ADN , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Liposomas , Oligonucleótidos Antisentido/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Citometría de Flujo , Operón Lac/efectos de los fármacos , Soluciones , beta-Galactosidasa/análisis , beta-Galactosidasa/antagonistas & inhibidores , beta-Galactosidasa/biosíntesisRESUMEN
Monte-Carlo simulation is an important tool in the field of biomedical optics, but suffers from significant computational expense. In this paper, we present the multicanonical Monte-Carlo (MMC) method for improving the efficiency of classical Monte Carlo simulations of light propagation in biological media. The MMC is an adaptive importance sampling technique that iteratively equilibrates at the optimal importance distribution with little (if any) a priori knowledge of how to choose and bias the importance proposal distribution. We illustrate the efficiency of this method by evaluating the probability density function (pdf) for the radial distance of photons exiting from a semi-infinite homogeneous tissue as well as the pdf for the maximum penetration depth of photons propagating in an inhomogeneous tissue. The results agree very well with diffusion theory as well as classical Monte-Carlo simulations. A six to sevenfold improvement in computational time is achieved by the MMC algorithm in calculating pdf values as low as 10(-8). This result suggests that the MMC method can be useful in efficiently studying numerous applications of light propagation in complex biological media where the remitted photon yield is low.
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Antibodies against the 19 amino acid encephalitogenic peptide )residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88-Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgG1, IgG2, and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.
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Idiotipos de Inmunoglobulinas/inmunología , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Cromatografía de Afinidad , Reacciones Cruzadas , Femenino , Cobayas , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Idiotipos de Inmunoglobulinas/aislamiento & purificación , Focalización Isoeléctrica , Ratas , Ratas Endogámicas LewRESUMEN
The trichodiene synthase gene (Tox5) was isolated from Gibberella pulicaris, and its nucleotide sequence was determined. Tox5 was disrupted through transformation with a plasmid carrying a doubly truncated copy of the coding region and a selectable marker for resistance to hygromycin B (Hygr). Analysis of 82 transformants for their ability to produce the trichothecene, 4,15-diacetoxyscirpenol (DAS), resulted in the identification of five DAS- strains. Southern hybridization analysis of DAS- Hygr transformants indicated that the plasmid integrated at the Tox5 locus. The disrupted Tox5 gene was shown to be mitotically stable. Analysis of nine tetrads revealed either the cosegregation of the disrupter plasmid and the DAS- phenotype or the loss of the disrupter plasmid. These results demonstrate the feasibility of using gene disruption in G. pulicaris and suggest a general method for obtaining Tox5- mutants in other trichothecene-producing fungi.
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Liasas de Carbono-Carbono , Genes Fúngicos , Gibberella/genética , Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN de Hongos , Gibberella/enzimología , Liasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Mapeo Restrictivo , Alineación de Secuencia , Transformación GenéticaRESUMEN
We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.