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PubMed2006

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA.

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.

DNA↗

PubMed2006

Elective liver transplantation for the treatment of classical maple syrup urine disease.

An 8.5-year-old girl with classical maple syrup urine disease (MSUD) required liver transplantation for hypervitaminosis A and was effectively cured of MSUD over an 8-year clinical follow-up period. We developed a collaborative multidisciplinary effort to evaluate the effects of elective liver transplantation in 10 additional children (age range 1.9-20.5 years) with classical MSUD. Patients were transplanted with whole cadaveric livers under a protocol designed to optimize safe pre- and post-transplant management of MSUD. All patients are alive and well with normal allograft function after 106 months of follow-up in the index patient and a median follow-up period of 14 months (range 4-18 months) in the 10 remaining patients. Leucine, isoleucine and valine levels stabilized within 6 hours post-transplant and remained so on an unrestricted protein intake in all patients. Metabolic cure was documented as a sustained increase in weight-adjusted leucine tolerance, normalization of plasma concentration relationships among branched-chain and other essential and nonessential amino acids, and metabolic and clinical stability during protein loading and intercurrent illnesses. Costs and risks associated with surgery and immune suppression were similar to other pediatric liver transplant populations.

Adolescent↗

PubMed1999

Present and future perspectives on children with type 1 diabetes.

This review presents current achievements in paediatric diabetology and discusses potential new approaches. Recent multinational investigations have demonstrated that in spite of multidisciplinary diabetes care only one-third of young patients with diabetes have an HbA1c below 8%, which is associated with a lower risk for development of secondary complications. Since there is no standard protocol for the optimal treatment of diabetes in children and adolescents, the therapy has to be tailored to the individual child's needs, involving not only paediatricians, but also dieticians, diabetes educators, psychologists and social workers. To improve the current situation a major goal for the next few years appears to be the establishment of standards for structure, process and outcome quality in paediatric diabetology. Future perspectives include possibilities for a cure via islet- or pancreas transplantation or improved pharmacological therapy following the introduction of new types of insulin or other beta cell-related peptides as adjunctive therapy not currently used.

Adolescent↗

PubMed1997

Treatment planning 2000: an esthetically oriented revision of the original implant protocol.

Soft and hard tissue ridge deformities are prevalent in areas of tooth loss and trauma and can compromise esthetic outcomes. Only a full understanding of the severity of the dimensional defects, the reconstructive techniques available, and the functional and esthetic needs of the final implant restoration will allow the design of a treatment approach that will achieve the desired outcome. This article emphasizes the synergistic relation of the osseous, gingival, and restorative triad in treatment planning. In addition, a systematic approach to the enhancement of this triad, in the partially edentulous patient, is outlined as part of a revised protocol for achieving optimal esthetics in implant therapy.

Dental Implantation↗

PubMed2026

Magnetic nanoparticle-mediated genetic transformation and gene editing system in loquat (Eriobotrya japonica).

Loquat (Eriobotrya japonica Lindl.) is a valuable subtropical fruit tree whose genetic improvement has been significantly constrained by the absence of an efficient genetic transformation system. Although Agrobacterium-mediated transformation is the most widely used method, it proves ineffective in loquat due to the species' recalcitrance to in vitro regeneration. Pollen-based transformation offers a promising alternative by bypassing the need for tissue culture. However, the pollen wall poses a major physical barrier to the uptake of exogenous DNA. In this study, we investigated magnetic nanoparticle (MNP)-mediated transformation as a novel strategy for loquat. We confirmed that loquat pollen contains tricolporate apertures with diameters ranging from 3.0 to 5.0 μm, which are structurally suitable for the entry of MNPs-DNA. Based on this finding, we developed and optimized a transformation protocol using polyethyleneimine-coated Fe3O4 nanoparticles to deliver genetic material into loquat pollen grains. Using this approach, we successfully generated stable transgenic loquat lines, including both overexpression and gene-edited mutants. To our knowledge, this is the first report of successful MNP-mediated pollen transformation in a woody plant species. This work establishes a robust and efficient genetic transformation platform for loquat, providing a valuable tool for functional genomics and molecular breeding, as well as a potentially applicable strategy for other recalcitrant woody plants.

Eriobotrya↗

PubMed2004

Chloromethane-dependent expression of the cmu gene cluster of Hyphomicrobium chloromethanicum.

The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH(3)Cl) as the sole carbon and energy source. Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH(3)Cl. These CH(3)Cl-specific genes were subsequently detected in H. chloromethanicum. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH(3)Cl metabolism in H. chloromethanicum. New developments in genetic manipulation of Hyphomicrobium are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H. chloromethanicum to generate stable CH(3)Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium. A reliable and reproducible selection procedure for screening of CH(3)Cl utilization-negative mutants has also been developed. Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH(3)Cl or to express the CH(3)Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H. chloromethanicum. This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5') of cmuA. The detrimental effect of mutation in hutI on the upstream (5')-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed. Expression of the cmuBCA-metF transcript was also shown to be strictly CH(3)Cl inducible and was not repressed by the alternative C(1) substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3' of the paaE gene in H. chloromethanicum. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M. chloromethanicum. Mutational and transcriptional analysis data indicated that, in H. chloromethanicum, CH(3)Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system.

Base Sequence↗

PubMed2002

Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR.

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.

Bacteria↗

PubMed2026

Complete genome sequence of the Anaplasma phagocytophilum clinical isolate NCH-1.

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacterium and etiologic agent of human granulocytic anaplasmosis. A. phagocytophilum genomic sequencing has historically been performed via short-read platforms. Our optimized bacterial isolation protocol combined with Nanopore sequencing produced a single, closed 1,481,805 bp circular A. phagocytophilum strain NCH-1 chromosome.

Anaplasma phagocytophilum↗

PubMed1978

An evaluation of current diagnostic radiologic modalities in the investigation of acoustic neurilemmomas.

The radiologic investigation of patients with suspected acoustic neurilemmomas may include specialized plain films, thin-section tomography, CT scanning with intravenous contrast enhancement, Pantopaque cisternography, radionuclide scanning, angiography, and pneumoencephalography. Recently we have used intrathecally enhanced (Amipaque)2 CT cisternography tostudy the cerebellopontine angles and fourth ventricle. An assessment is made of the differential diagnostic value and limitations of radiologic modalities both in tumor detection and in accurate localization. A protocol designed to optimize use of the available radiologic modalities is projected from 30 patients with acoustic neurilemmomas.

Angiography↗

PubMed1996

CT of the stomach: spectrum of disease.

In evaluation of gastric disease, computed tomography (CT) has proved to be a valuable adjunct to barium studies and endoscopy. CT clearly demonstrates the primary pathologic condition and shows extension of disease to adjacent or distant structures. Useful in staging gastric cancer, CT has also proved valuable in detecting and defining the extent of other gastric neoplasms such as lymphoma, leiomyosarcoma, and metastasis to the stomach. Recent advances in CT technology such as spiral CT-coupled with air contrast gastric studies and a better understanding of the need to optimize CT protocols-suggest that the value of CT in these applications will increase. CT has also been shown to be valuable in detection and differentiation of other gastric conditions such as benign tumors, Helicobacter pylori and other infections, various forms of gastritis (radiation, eosinophilic, and emphysematous), ulcer disease, Ménétrier disease, and varices. Adequate gastric distention is essential for successful gastric CT.

Adult↗

PubMed1994

Neural crest tumors: I-123 MIBG imaging in children.

PURPOSE: To determine 24- and 48-hour distribution of iodine-123 metaiodobenzylguanidine (MIBG) and to establish an optimized pediatric imaging protocol. MATERIALS AND METHODS: Seventy-seven I-123 MIBG scans obtained in 33 children undergoing evaluation for neural crest tumor were reviewed. RESULTS: Activity in the salivary glands, myocardium, liver, gut, and bladder was demonstrated on almost all scans. Activity was noted in the neck muscles, thyroid gland, and uninvolved adrenal gland in fewer studies. In 20 studies, abnormal uptake was present in the chest or abdomen at 24 and 48 hours, and lesion identification was improved at 48 hours in 12 studies. Detection of skeletal lesions on 14 scans was similar at 24 and 48 hours. Foci of tumor were first detected at I-123 MIBG imaging in nine studies, and in only two patients was tumor involvement initially missed on I-123 MIBG studies. CONCLUSION: Distribution of I-123 MIBG on images in children differs from that in earlier descriptions in adults. Images of the torso at 48 hours after injection are a useful adjunct in detection of lesions.

3-Iodobenzylguanidine↗

PubMedDate not supplied

Nontraumatic emergent abdominal vascular conditions: advantages of multi-detector row CT and three-dimensional imaging.

In the past decade, great strides have been made in the development of helical computed tomography (CT) that have led to shorter scanning time and higher spatial resolution. A wide range of traumatic and nontraumatic emergent conditions may be quickly and accurately diagnosed with multi-detector row CT. Multi-detector row CT angiography is the preferred method for imaging in emergent abdominal vascular conditions because it enables the acquisition of high-spatial-resolution volumetric image data during a single breath hold. Unlike catheter angiography, multi-detector row CT angiography not only depicts the vessels but also allows assessment of perfusion in adjacent organs. To make the most effective diagnostic use of multi-detector row CT angiography and three-dimensional image postprocessing, radiologists must be familiar with the optimal CT angiographic protocols and with the typical CT findings in various emergent vascular conditions. This article describes the protocols used in 11 patients with conditions including ruptured abdominal aortic aneurysm, secondary aortoduodenal fistula, splanchnic segmental arterial mediolysis, and Wegener-type vasculitis with visceral involvement. All of the diagnoses in these 11 cases were made in the emergency department, and the delay between imaging and diagnosis was decreased considerably by avoiding the transfer of patients for catheter angiography.

Abdomen, Acute↗

PubMedDate not supplied

Pearls and pitfalls in interpretation of abdominal and pelvic PET-CT.

The interpretation of images obtained in the abdomen and pelvis can be challenging, and the coregistration of positron emission tomographic (PET) and computed tomographic (CT) scans may be especially valuable in the evaluation of these anatomic areas. PET-CT represents a major technologic advance, consisting of generally complementary modalities whose combined strength tends to overcome their respective weaknesses. However, this combined functional-structural imaging approach raises a number of controversial questions and presents some unique interpretative challenges. Accurate PET-CT scan interpretation requires awareness of the various pitfalls associated with the imaging components, both individually and in combination. The results of recent PET-CT studies have been very encouraging, but larger prospective studies will be needed to establish optimal hybrid scanning protocols. Applying sound imaging principles, paying attention to detail, and staying abreast of advances in this exciting new modality are necessary for harnessing the full diagnostic power of abdominopelvic PET-CT.

Abdomen↗

PubMed1998

Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species.

A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the alpha- and beta-isoforms of cardiac myosin heavy chain (MHC-alpha and MHC-beta) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-alpha and MHC-beta achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 microgram. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-alpha and MHC-beta in myocardial samples from different species. The effect of sample load on MHC-alpha and MHC-beta band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.

Animals↗

PubMed2006

Dynamic imaging of emerging resistance during cancer therapy.

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1omega) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2omega). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols.

Animals↗

PubMed2004

Classification of human breast cancer using gene expression profiling as a component of the survival predictor algorithm.

PURPOSE: Selection of treatment options with the highest likelihood of successful outcome for individual breast cancer patients is based to a large degree on accurate classification into subgroups with poor and good prognosis reflecting a different probability of disease recurrence and survival after therapy. Here we propose a breast cancer classification algorithm taking into account three main prognostic features determined at the time of diagnosis: estrogen receptor (ER) status; lymph node (LN) status; and gene expression signatures associated with distinct therapy outcome. EXPERIMENTAL DESIGN: Using microarray expression profiling and quantitative reverse transcription-PCR analyses, we compared expression profiles of the 70-gene breast cancer survival signature in established breast cancer cell lines and primary breast carcinomas from cancer patients. We classified 295 breast cancer patients using 14-, 13-, 6-, and 4-gene survival predictor signatures into subgroups having statistically distinct probability of therapy failure (P < 0.0001). We evaluated the prognostic power of breast cancer survival predictor signatures alone and in combination with ER and LN status using Kaplan-Meier analysis. RESULTS: The breast cancer survival predictor algorithm allowed highly accurate classification into subgroups with dramatically distinct 5- and 10-year survival after therapy of a large cohort of 295 breast cancer patients with either ER+ or ER- tumors as well as LN+ or LN- disease (P < 0.0001, log-rank test). CONCLUSIONS: Our data imply that quantitative laboratory tests measuring expression profiles of a limited set of identified small gene clusters may be useful in stratification of breast cancer patients at the time of diagnosis into subgroups with statistically distinct probability of positive outcome after therapy and assisting in selection of optimal treatment strategies. The estimated increase in survival due to the optimization of treatment protocols may reach many thousands of breast cancer survivors every year at the 10-year follow-up check point.

Algorithms↗

PubMed2006

Fragile histidine triad expression in oral squamous cell carcinoma and precursor lesions.

PURPOSE: Fragile histidine triad (FHIT) expression in precursor oral lesions (POL) and oral squamous cell carcinomas (OSCC) was studied with regard to (a) the frequency of loss of FHIT expression, (b) whether loss of FHIT expression correlates with degree of dysplasia in POLs, (c) whether FHIT loss predicts high-risk POLs that are more likely to transform, and (d) whether FHIT loss in OSCCs correlates with survival. EXPERIMENTAL DESIGN: Ninety-four POLs and 86 OSCCs were immunostained for FHIT. Survival analysis was done for cases with validated clinical outcomes. RESULTS: By optimizing the immunostaining protocol, we found that FHIT is expressed in a distinctive strong nuclear and weak cytoplasmic pattern in oral tissues. Loss of FHIT expression was found in 42 of 94 (45%) POLs and in 66 of 86 (77%) OSCCs. We observed a statistically significant positive correlation between frequency of FHIT loss and increasing grade of dysplasia (chi2=13.8; degrees of freedom=4; P=0.008). Loss of FHIT expression in POLs that progressed to malignancy was more frequent than in those that did not [17 of 25 (68%) versus 12 of 29 (41.4%), respectively]. This difference was statistically significant (chi2=3.8; degrees of freedom=1; P=0.046). In OSCCs, loss of FHIT staining indicated a worse prognosis (survival rate, 36.2%) than when positive FHIT staining was observed (survival rate, 50%), but the difference was not statistically significant (P=0.546, Kaplan-Meier, log-rank). CONCLUSIONS: FHIT seems to localize to both nuclear and cytoplasmic domains. FHIT inactivation occurs early in oral carcinogenesis and may be useful molecular marker for progressive dysplastic oral lesions.

Acid Anhydride Hydrolases↗

PubMed2006

Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells.

Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcumin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at approximately IC(50) concentrations has been seen. At IC(50), a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC(50), thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways.

Amino Acid Sequence↗