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Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

Buffers↗

Spotting optimization for oligo microarrays on aldehyde-glass.

Low-density microarrays that utilize short oligos (<100 nt) for capture are highly attractive for use in diagnostic applications, yet these experiments require strict quality control and meticulous reproducibility. However, a survey of current literature indicates vast inconsistencies in the spotting and processing procedures. In this study, spotting and processing protocols were optimized for aldehyde-functionalized glass substrates. Figures of merit were developed for quantitative comparison of spot quality and reproducibility. Experimental variables examined included oligo concentration in the spotting buffer, composition of the spotting buffer, postspotting "curing" conditions, and postspotting wash conditions. Optimized conditions included the use of 3-4 microM oligo in a 3x standard saline citrate/0.05% sodium dodecyl sulfate/0.001% (3-[(3-cholamidopropyl) dimethylammonia]-1-propane sulfonate) spotting buffer, 24-h postspotting reaction at 100% relative humidity, and a four-step wash procedure. Evaluation of six types of aldehyde-functionalized glass substrates indicated that those manufactured by CEL Associates, Inc. yield the highest oligo coverage.

Aldehydes↗

Current status of pancreatic islet transplantation.

DM (diabetes mellitus) is a metabolic disorder of either absolute or relative insulin deficiency. Optimized insulin injections remain the mainstay life-sustaining therapy for patients with T1DM (Type I DM) in 2006; however, a small subset of patients with T1DM (approx. 10%) are exquisitely sensitive to insulin and lack counter-regulatory measures, putting them at higher risk of neuroglycopenia. One alternative strategy to injected insulin therapy is pancreatic islet transplantation. Islet transplantation came of age when Paul E. Lacy successfully reversed chemical diabetes in rodent models in 1972. In a landmark study published in 2000, Shapiro et al. [A. M. Shapiro, J. R. Lakey, E. A. Ryan, G. S. Korbutt, E. Toth, G. L. Warnock, N. M. Kneteman and R. V. Rajotte (2000) N. Engl. J. Med. 343, 230-238] reported seven consecutive patients treated with islet transplants under the Edmonton protocol, all of whom maintained insulin independence out to 1 year. Substantial progress has occurred in aspects of pancreas procurement, transportation (using the oxygenated two-layer method) and in islet isolation (with controlled enzymatic perfusion and subsequent digestion in the Ricordi chamber). Clinical protocols to optimize islet survival and function post-transplantation improved dramatically with the introduction of the Edmonton protocol, but it is clear that this approach still has potential limitations. Newer pharmacotherapies and interventions designed to promote islet survival, prevent apoptosis, to promote islet growth and to protect islets in the long run from immunological injury are rapidly approaching clinical trials, and it seems likely that clinical outcomes of islet transplantation will continue to improve at the current exponential pace.

Blood Glucose↗

Estimation of postmortem interval using the protein marker cardiac Troponin I.

The importance of determining the time since death is crucial to criminal, civil and forensic cases. A technique exploiting the degradation of a protein, cardiac Troponin I (cTnI), to estimate the postmortem interval (PMI) was investigated. Cardiac Troponin I is a basic regulatory protein found as part of a ternary complex responsible for calcium dependent muscle contraction. An efficient extraction protocol to analyze the banding pattern of cTnI in postmortem tissue was developed. The analysis involves extraction of the protein, separation by denaturing gel electrophoresis (SDS-PAGE) and visualization by Western blot using cTnI specific monoclonal antibodies. A bovine model was used to develop and optimize the protocol. The homology of cTnI amongst mammalian species allows for the cross-reaction of human anti-cTnI antibodies with bovine cTnI. The results indicate a characteristic banding pattern amongst human cadavers (n=6), a pseudo-linear relationship between percent cTnI degraded and the log of the time since death (r>0.95), and a qualitative degradation band pattern that in a simple comparative analysis with a standard human heart (known time since death) can be used to estimate the postmortem interval. The degradation-banding pattern of tissue cTnI is useful in the determination of the early postmortem (pm) interval (0-5 days). Overall, this technique offers advantages such a wide postmortem interval, measurable degradation pattern, a temporal semiquantitative relationship and manageable temperature dependence over direct temperature methods.

Animals↗

Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes.

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.

Antibodies↗

Theoretical considerations for optimizing intensity differences between primary musculoskeletal tumors and normal tissue with spin-echo magnetic resonance imaging.

In the radiographic assessment of primary musculoskeletal tumors, it is important for therapy planning to accurately define the extent of a tumor. Using a double spin-echo pulse sequence, the T1 and T2 relaxation times and relative hydrogen densities of several neoplastic tissues and of several normal tissues in four patients were measured. Neoplasms measured included one fibrosarcoma, two osteosarcomas, and one giant cell tumor. Normal tissues measured included normal muscle, fat, and bone marrow. Using a mathematical model of the double spin-echo pulse sequence, the intensity difference between each tumor and each normal tissue for multiple values of TR and TE was calculated. These calculated intensity differences were then used to plot isodifference contour curves for each tissue pair. These plots enabled us to pick combinations of TR and TE that optimized the signal difference between tumor and normal tissue. When comparing tumor with predominantly fatty tissue such as marrow or subcutaneous fat, optimal signal difference in our imager occurred at a TR of 600 to 800 msec and a very short TE. When comparing tumor with muscle, optimal signal difference occurred with very long TR times, and TE times ranging from 30 to 90 msec. These preliminary results suggest that an optimal scanning protocol for primary musculoskeletal tumors should contain at least two different pulse sequences with widely separated TR values (500 and 2000 msec in our instrument), and short to intermediate values of TE (28 and 56 msec in our instrument). It is believed that analysis of isodifference contour plots is a useful method for optimizing intensity differences between any two tissue types.

Bone Neoplasms↗

Short implants in the severely resorbed maxilla: a 2-year retrospective clinical study.

BACKGROUND: Although the predictability of endosseous dental implants is well documented, the restoration of the posterior region of the maxilla remains a challenge. The placement of short implants is one therapeutic option that reduces the need for augmentation therapy. PURPOSE: The purpose of this retrospective study was to assess the survival rates of 6 to 8.5 mm-long implants in the severely resorbed maxilla following a surgical protocol for optimized initial implant stability. MATERIALS AND METHODS: The study included 85 patients with 96 short (6-8.5 mm) implants (Brånemark System, Nobel Biocare AB, Göteborg, Sweden) supporting single-tooth and partial reconstructions. The implants had a machined (54) or an oxidized (TiUnite, Nobel Biocare AB) (42) surface. A one-stage surgical protocol with delayed loading was used. The patients were followed for at least 2 years after loading (average follow-up period 37.6 months). The marginal bone resorption was assessed by radiographic readings. RESULTS: Five implants were lost during the first 9 months, and four implants were lost to follow-up. The cumulative survival rate was 94.6%. Four of the failed implants had a machined surface, and one had an oxidized surface. The mean marginal bone resorption after 2 years in function was 0.44 +/- 0.52 mm. CONCLUSION: This study demonstrates that the use of short implants may be considered for prosthetic rehabilitation of the severely resorbed maxilla as an alternative to more complicated surgical techniques.

Alveolar Bone Loss↗

Management of patients with acute asthma: what do we know? What do we need to know?

Management of patients with acute, severe asthma mandates the use of comprehensive monitoring and aggressive therapeutic drug regimens. Global clinical and objective assessment criteria can help determine the severity of the acute episode and will also help determine optimal management protocols. The response to aggressive therapy, rather than the initial severity of the attack, predicts outcome. Beta 2 sympathomimetic agents are the treatment of choice for acute asthmatic episodes. Aminophylline and parasympatholytic agents may be useful adjuvants, but more data are needed before firm conclusions can be reached regarding risk-benefit ratios. The optimal timing of steroid therapy is not yet known, but even this form of treatment is slow in producing results.

Acute Disease↗

An analytical study on the thermal effects of cryosurgery on selective cell destruction.

The aim of cryosurgery is to kill cells within a closely defined region maintained at a predetermined low temperature. To effectively kill cells, it is important to be able to predict and control the cooling rate over some critical range of temperatures and freezing states in order to regulate the spatial extent of injury during any freeze-thaw protocol. The objective of manipulating the freezing parameters is to maximize the destruction of cancer cells within a defined spatial domain while minimizing cryoinjury to the surrounding healthy tissue. An analytical model has been developed to study the rate of cell destruction within a liver tumor undergoing a freeze-thaw cryosurgical process. Temperature transients in the tumor undergoing cryosurgery have been quantitatively investigated. The simulation is based on solving the transient bioheat equation using the finite volume scheme for a single or multiple-probe geometry. Simulated results show good agreement with experimental data obtained from in vivo clinical study. The calibrated model has been employed to study the effects of different freezing rates, freeze-thaw cycle(s), and multi-probe freezing on cell damage in a liver tumor. The effectiveness of each treatment protocol is estimated by generating the cell survival-volume signature and comparing the percentage of cell damaged within the ice-ball. Results from the model show that employing freeze-thaw cycles has the potential to enhance cell destruction within the cancerous tissue. Results from this study provide the basis for designing an optimized cryosurgical protocol which incorporates thermal effects and the extent of cell destruction within tumors.

Biophysical Phenomena↗

Outpatient parenteral inotropic therapy for advanced heart failure.

BACKGROUND: Patients with advanced heart failure generally have hemodynamic perturbation characterized by low cardiac output and high ventricular filling pressures. This creates a clinical milieu with profound symptomatology that includes weakness, fatigue, and fluid-retention states causing peripheral edema, mesenteric congestion, and dyspnea syndromes. Great morbidity including hospital admissions and readmissions as well as high mortality rates ensue. Though medication and/or surgical intervention often attenuate heart failure symptomatology, morbidity, and mortality, some patients reach more advanced stages despite aggressive maneuvers. Indeed, patients presenting with acute decompensation of chronic congestive heart failure frequently receive parenteral inotropic drugs during their hospitalization with clinical improvement. Because these agents generally increase cardiac output and reduce pre-load and afterload, they can be lifesaving. Some patients, however, have symptomatic and hemodynamic rebound to worsened heart failure states during or shortly after inotrope weaning. METHODS: It was, then, a logical step to segue from acute inpatient inotrope infusion to long-term administration of these drugs in the outpatient setting when patients were dependent on these agents. Dopamine, dobutamine, and milrinone are all generally available inotropes that have been used singly or in combination in a chronic outpatient infusion setting. CONCLUSIONS: Data from a few small clinical trials and anecdotal case experience suggest that these drugs result in both hemodynamic and clinical improvement that is generally sustained during chronic administration, and even noted long after discontinuation of infusions in some patients. Some reports have suggested that intermittent infusion therapy in outpatients (so-called pulsed therapy) is effective in attenuating congestive heart failure symptoms long term, with more data supporting chronic infusion of these agents. Though questions regarding safety of these agents have been raised, a reasonable compendium of data published to date supports the contention that inotropic drugs used in this fashion ameliorate symptoms. Legitimate concern may be raised regarding exacerbation of arrhythmias with subsequent sudden cardiac death syndrome; however, in severely symptomatic heart failure patients, the trade-off between symptomatic amelioration and the chance of sudden cardiac death may be worthwhile. Unfortunately, precise guidance regarding the best drug, dose, optimal administration technique, weaning protocol, and actual risk/benefit ratio are not well characterized. Practice as been guided, in large part, by anecdotal experience. However, it appears that chronic or pulsed outpatient parenteral inotropic infusion therapy is frequently prescribed and that this treatment option is an effective alternative for carefully selected patients with severely symptomatic and advanced heart failure. Formulating optimal protocols for home inotropic drug infusion therapy by conducting properly designed clinical trials will be an essential endeavor.

Adrenergic beta-Agonists↗

Investigation of laser annealing parameters for optimal laser-damage performance in deuterated potassium dihydrogen phosphate.

Laser annealing via preexposure to laser pulses at sub-damage-threshold fluences is known to improve the resistance of KDP crystals to laser-induced damage. Using a specific damage-testing method, we investigate the laser annealing process as a function of fluence and number of preexposure pulses (at 355 nm, 2.5 ns). Our aim is to reveal the key laser parameters in order to devise a practical and efficient protocol for optimizing performance of the material for operation in laser systems in the near UV. Results suggest that a near twofold improvement to the laser-damage performance can be achieved with a limited number of preexposure pulses.

Journal Article↗

Cryoloop vitrification in assisted reproduction: analysis of survival rates in > 1000 human oocytes after ultra-rapid cooling with polymer augmented cryoprotectants.

While human oocytes have been successfully cryopreserved using traditional slow-rate freezing protocols, inconsistent results post-thaw have limited the routine clinical application of oocyte cryopreservation. Despite interest in the potential benefits of vitrification as an alternative laboratory approach to long-term oocyte preservation in assisted reproduction, there is little agreement on how best to configure such cryopreservation protocols to optimize oocyte viability. To comparepost-thaw oocyte survival rates,we performed cryoloop vitrification of human oocytes utilizing two different cryoprotectant mixtures that included polymer macromolecules. Human oocytes (n = 1120) were obtained from consenting patients undergoing in vitro fertilization, but only failed-matured (uninseminated) or failed-fertilized (inseminated but without 2pn development) were included in this investigation. Protocol A consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.4 M sucrose and 20% synthetic serum substitute. Protocol B consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.65 M sucrose, 1 mg/ml polyethylene glycol, 10 mg/ml Ficoll and 20% synthetic serum substitute. Following cryostorage for 10-14 d at -196 degrees C, the survival rate for oocytes vitrified with protocol A was 80.9%, whereas the post-thaw viability among protocol B oocytes was 80.6% (p > 0.005). Our results indicate that an ethylene glycol + dimethyl sulphoxide mixture (with or without polymer macromolecules) can be an effective cryoprotectant strategy for human oocyte vitrification; either approach can be employed without any observed compromise in post-warming survival and/or morphology.

Cell Survival↗

Cryopreservation of in vitro cultured mouse preimplantation embryos.

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.

Animals↗

Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.

This study explored the biologic response of chondrocytes and mesenchymal stem cells (MSCs) to a dynamic mechanical loading regime. We developed a time-efficient methodology for monitoring regional changes in extracellular matrix gene transcription using reporter promoter constructs. Specifically, transfected cells were homogenously distributed throughout agarose hydrogel constructs, and spatial and temporal gene expression and the ability to form functional ECM were analyzed in response to dynamic mechanical stimuli. Theoretical analyses were used to predict the physical signals generated within the gel in response to these loading regimes. Using a custom compression bioreactor system, changes in aggrecan and type II collagen promoter activity in transfected chondrocyte-laden cylindrical constructs were evaluated in response to a range of loading frequencies and durations. In general, aggrecan promoter activity increased with increasing duration of loading, particularly in the outer annulus region. Interestingly, type II collagen promoter activity decreased in this annular region under identical loading conditions. In addition, we explored the role of mechanical compression in directing chondrogenic differentiation of MSCs by monitoring short-term aggrecan promoter activity. As an example of long-term utility, a specific loading protocol was applied to MSC-laden constructs for 5 days, and the resultant changes in glycosaminoglycan (GAG) production were evaluated over a 4-week period. This dynamic loading regime increased not only short-term aggrecan transcriptional activity but also GAG deposition in long-term culture. These results demonstrate the utility of a new reporter promoter system for optimizing loading protocols to improve the outcome of engineered chondrocyte- and MSC-laden cartilaginous constructs.

Aggrecans↗

Determination of half-dose depth in skin for soft x-rays.

Unlike superficial x-rays, the soft x-rays normally used in dermatologic practice spare unaffected underlying organs during treatment of cutaneous malignancies. However, since the dose with depth from soft x-rays varies markedly, it is important to know this relationship for optimal therapeutic results. The peak kilovoltage, and thus the energy of the beam, is generally selected so that the dose to the base of the lesion is one-half the surface dose. An absorbed dose of 3,400 rads to the surface and a dose of about one-half this amount to the base of most malignant lesions is one standard protocol for optimal therapeutic results. An accurate value of half-depth dose in skin is therefore necessary and is readily obtained from ordinary half-value layer measurements using the technic described.

Aluminum↗

Detection of etiological agent for cholera by PCR protocol.

BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report. We report the detection of Vibrio species in drinking water samples in a duplex PCR reaction. The target loci used in the study were ctxA and tcpA. The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, wherein monitoring of target organisms is very crucial. MATERIAL AND METHODS: Single step thermocycling programs have been reported for amplification of specific target loci. We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR. The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatible for PCR. The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples. RESULTS: The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100 cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specific template. The developed protocol was successfully extended to environmental samples. CONCLUSIONS: The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.

Bacterial Outer Membrane Proteins↗

[Stress and stress tolerance in chronic heart failure].

BACKGROUND: Exercise intolerance in patients with chronic heart failure (CHF) shows no correlation to the degree of left ventricular dysfunction. This surprising finding has directed attention to peripheral changes in CHF. During the last years several different peripheral factors as determinants of exercise intolerance have been defined, i.e. abnormalities in ventilation, reduced endothelium-dependent vasodilatation of peripheral conduit and resistance vessels, and altered skeletal muscle metabolism. Skeletal muscle alterations are characterized by a reduced oxidative capacity, a catabolic state with reduced local IGF-I expression and muscle atrophy, chronic inflammation with local expression of the inducible isoform of nitric oxide synthase (iNOS) and an accelerated rate of programmed cell death (apoptosis). EFFECTS OF PHYSICAL EXERCISE: Physical exercise training has evolved as an important therapeutic approach to influence these non-cardiac causes of exercise intolerance. After the first studies documenting the effect of aerobic training on the peripheral causes of exercise intolerance in CHF the question was asked: Should we treat the heart or the periphery to improve exercise intolerance in CHF? Today, we have come closer to the answer: It is now clear that these two systems are not mutually exclusive. Exercise training in CHF has been shown to improve skeletal muscle metabolism and function, to avert muscle catabolism, to reduce neurohumoral overactivation, to reverse endothelial dysfunction and to contribute to the prevention of pathologic left ventricular remodeling. After 6 months of regular exercise training oxidative capacity of the working skeletal muscle increases by approximately 40%. Regular exercise training leads to a significant improvement of endothelium-dependent vasodilatory capacity of peripheral resistance vessels, thereby reducting peripheral resistance in particular during exercise. These beneficial training effects result in a small, but significant improvement of stroke volume and reduction in cardiomegaly. CONCLUSION: Although several questions regarding patient selection, optimal training protocol and training intensity remain unanswered, exercise training can been seen as an established adjunct to pharmacotherapy in CHF. We may soon reach the conclusion that by treating the periphery with exercise programs we are in fact treating the heart, as well. All exercise-induced adaptations converge to increase peak oxygen uptake by up to 2 ml/kg.min. For patients in stable CHF on optimal cardiac medication a combination of in-hospital and home-based aerobic endurance training in combination with local muscle strength training seems most promising. Although exercise training offers no causal treatment of CHF, it has great potentials as an adjunct therapy directed at improving exercise tolerance and expanding the physical limits of CHF patients.

Chronic Disease↗

Population plasma pharmacokinetics of 11C-flumazenil at tracer concentrations.

OBJECTIVE: The objectives of the study were to develop a population pharmacokinetic model for (11)C-flumazenil at tracer concentrations, to assess the effects of patient-related covariates and to derive an optimal sampling protocol for clinical use. METHODS: A population pharmacokinetic model was developed using nonlinear mixed effects modelling (NONMEM) with data obtained from 51 patients with either depression or epilepsy. Each patient received approximately 370 MBq (1-4 microg) of (11)C-flumazenil. The effects of selected covariates (gender, weight, type of disease and age) were investigated. The model was validated using a bootstrap method. Finally, an optimal sampling design was established. RESULTS: The population pharmacokinetics of tracer quantities of (11)C-flumazenil were best described by a two compartment model. Type of disease and weight were identified as significant covariates (P < 0.002). Mean population pharmacokinetic parameters (percent coefficient of variation) were: CL 1530 mL min(-1) (6.6%), V(1) 24.8 x 10(3) mL (3.8%), V(2) 27.3 x 10(3) mL (5.4%), and Q 2510 mL min(-1) (6.5%). CL was 20% lower in patients with epilepsy, and the influence of weight on V(1) was 0.55% kg(-1). For the prediction of the AUC, a combination of two time points at t = 30 and 60 min post injection was considered optimal (bias -0.7% (95% CI -2.2 to 0.8%), precision 5.7% (95% CI 4.5-6.9%)). The optimal sampling strategy was cross-validated (observed AUC = 296 MBql(-1) min(-1) (95% CI 102-490), predicted AUC = 288 MBql(-1) min(-1) (95% CI 70-506)). CONCLUSIONS: The population pharmacokinetics of tracer quantities of (11)C-flumazenil are well described by a two-compartment model. Inclusion of weight and type of disease as covariates significantly improved the model. Furthermore, an optimal sampling procedure may increase the feasibility and applicability of (11)C-flumazenil PET.

Adult↗