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Cryothermal energy ablation of cardiac arrhythmias 2005: state of the art.

At the time of antiarrhythmic surgery, cryothermal energy application by a hand-held probe was used to complement dissections and resections and permanently abolish the arrhythmogenic substrate. Over the last decade, significant engineering advances allowed percutaneous cryoablation based on catheters, apparently not very different from standard radiofrequency ablation catheters. Cryothermal energy has peculiar characteristics. In fact, it allows testing in a reversible way the effects of energy application at higher temperature, before producing a permanent lesion at -75 degrees C. Moreover, slow formation of the lesion allows timely discontinuation of the application, as soon as inadvertent modifications of normal atrioventricular conduction are observed during ablation in the proximity of atrioventricular node and His bundle, avoiding its permanent damage. Over the last years, percutaneous cryothermal ablation has been widely used for a variety of cardiac arrhythmias. From the data gathered, it is unlikely that cryoablation will replace standard ablation in unselected cases. Nevertheless, for the above mentioned peculiarities, cryothermal ablation has proved very effective and safe for ablation of arrhythmogenic substrates close to the normal conduction pathways, becoming the first choice method to ablate anteroseptal and midseptal accessory pathways. It can be also the best treatment for ablation of the slow pathway to abolish atrioventricular node reentrant tachycardia in pediatrics or when particular anatomy of the Koch's triangle is observed. Cryothermal ablation of the pulmonary veins for atrial fibrillation, although longer than radiofrequency ablation, is not associated with pulmonary vein stenosis and is expected to be less thrombogenic; new catheter designs for cryothermal ablation of this challenging arrhythmia are to be tested to assess their efficacy and clinical usefulness.

Journal Article↗

Regulated production of mature insulin in rat hepatoma cells: insulin production is up-regulated by dexamethasone and down-regulated by insulin.

We engineered an artificial beta cell line that produces an up-regulation of insulin in response to dexamethasone, and a down-regulation in response to insulin. A regulatory secretion system was devised by placing proinsulin cDNA containing genetically engineered furin endoprotease cleavage sites and a regulatory promoter for phosphoenolpyruvate carboxykinase (PEPCK), and an insulin expressing retrovirus vector (pN-PEPCK-mINS) was constructed and transfected into Hepa1-6 cells. The levels of insulin in culture medium and expression of insulin gene was estimated by radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The clone (Hepa1-6/INS21), which secreted the highest level of insulin (10.79 microIU/106 cells per day), was selected for the regulation experiment. Compared with the non-treated Hepa1-6/INS21 cells, insulin production was augmented 3.6-fold by the addition of 10-7 M of dexamethasone. Addition of exogenous insulin to the culture medium decreased insulin mRNA expression remarkably on RT-PCR results, while dexamethasone increased insulin gene expression at the transcriptional level. The data indicated that genetically engineered Hepa1-6 cells could synthesize process and secrete insulin in a physiological manner.

Animals↗

[Structures and functions of ribonucleases].

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.

Amino Acid Sequence↗

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Crystallography, X-Ray↗

Functional evaluation of bioartificial liver using RT-PCR.

In order to evaluate functions of a bioartificial liver (BAL), containing porcine hepatocytes in a hollow fiber cartridge, some chemical loading tests have been employed. However the kinds of functions that can be evaluated by chemical loading tests are limited. We desire versatile methods to estimate various BAL functions. The purpose of this report is to propose a method using the reverse transcription polymerase chain reaction (RT-PCR) for functional evaluation of a BAL. In vitro perfusion culture of a BAL cartridge was carried out using 20% of human whole blood. At pre-determined periods of perfusion culture, hepatocytes were taken from the cartridge and mRNA was extracted from the hepatocytes. The mRNA expression levels of albumin and cytochrome P450 (CYP3A29) were determined by RT-PCR method. In order to quasi-quantitatively determine the time courses of mRNA expression levels during the perfusion culture, PCR of target DNA was carried out by co-amplification with its competitor DNA that was constructed by means of the partial deletion of target DNA. The results showed that the amounts of both albumin and cytochrome P450 mRNA rapidly decreased during the initial few days' perfusion culture and remained at a constant level for the following week. This fact corresponded with the analysis of lidocaine metabolic functions via pharmacokinetics in our previous study. Additionally, the RT-PCR is so sensitive that we can measure much more minute quantity of various proteins. Consequently, this method is useful for comprehensive evaluation of BAL functions.

Albumins↗

Use of an internal standard in a TaqMan nested reverse transcription-polymerase chain reaction for the detection of bovine viral diarrhoea virus.

The aim of this study was to improve molecular methods for the detection of bovine viral diarrhoea virus (BVDV). A single-tube nested reverse-transcriptase polymerase chain reaction (nRT-PCR) employing the 5'-3'-exonuclease assay (TaqMan) system was optimised for use with bulk milk, semen and whole blood samples. An artificial template (mimic) was engineered to provide in-tube validation of negative samples by demonstrating the absence of substances inhibitory to RT or PCR. This mimic was constructed by disrupting the BVDV amplicon at the TaqMan probe site by inserting a 295bp fragment of human genomic DNA. The mimic amplicon was discriminated from the BVDV RT-PCR products using a second TaqMan probe, with a different fluorochrome specific for the inserted DNA. This new method was more sensitive than BVDV antigen ELISA methods and the existing RT-PCR method used in the laboratory for detection of BVDV in bulk milk. Furthermore, RNA extracted by robotic methods has proved suitable for use in this assay. This TaqMan nRT-PCR will be a valuable method for the detection of BVDV in a variety of biological matrices including milk and semen.

Animals↗

Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and "cell conditioning" for improved recombinant protein yield.

The Escherichia coli stress gene transcription profile and response to recombinant protein overexpression were substantially altered at high cell density when compared with low cell density. Reverse trascription-polymerase chain reaction RT-PCR-amplified mRNA from low (4 g[DCW]/L) and high-cell-density (43.5 g [DCW]/L) conditions were hybridized with a DNA microarray of Kohara clones encompassing 16% of the E. coli genome, and differentially displayed genes were identified. Transcript-specific RNA dot blots indicated that molecular chaperones (groEL, ibpA, degP), proteases (degP, ftsH), the lysis gene mltB, and DNA damage/bacteriophage-associated gene transcript levels (ftsH, recA, alpA, uvrB) increased 10- to 43-fold at high cell density. In addition, overexpression of recombinant green fluorescent protein (GFP(uv))/chloramphenicol acetyltransferase (CAT) fusion protein did not change the rates of cell growth or cell lysis. The stress gene transcription profile at high cell density was used to evaluate "cell conditioning" strategies to alter the levels of chaperones, proteases, and other intracellular proteins prior to recombinant protein overexpression. Interestingly, the addition of 1 g/L dithiothreitol (DTT) 20 min prior to induction of a GFP(uv)/CAT fusion protein resulted in a twofold increase in CAT activity when compared with the unconditioned controls. In addition, RNA dot blots of five stress genes confirmed that cell conditioning strategies significantly altered the dynamic stress gene response to foreign protein overexpression.

Bacterial Proteins↗

A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.

Global gene regulation throughout the Escherichia coli stress response to overexpression of each of five recombinant proteins was evaluated. Reverse-transcriptase polymerase chain reaction-amplified mRNA from induced and control cells were hybridized with a DNA array of Kohara clones representing 16% (700 genes) of the E. coli genome. Subsequently, Northern analysis was performed for quantification of specific gene dynamics and statistically significant overlap in the regulation of 11 stress-related genes was found using correlation analysis. The results reported here establish that there are dramatic changes in the transcription rates of a broad range of stress genes (representing multiple regulons) after induction of recombinant protein. Specifically, the responses included significantly increased upregulation of heat shock (ftsH, clpP, lon, ompT, degP, groEL, aceA, ibpA), SOS/DNA damage (recA, lon, IS5 transposase), stationary phase (rpoS, aceA), and bacteriophage life cycle (ftsH, recA) genes. Importantly, similarities at the microscopic (gene) level were not clearly reflected at the macroscopic (growth rate, lysis) level. The use of such dynamic data is critical to the design of gene-based sensors, the engineering of metabolic pathways, and the determination of parameters (harvest and induction times) needed for successful recombinant E. coli fermentations.

Bacteriophage lambda↗

Genetic and dopaminergic modulation of reversal learning in a touchscreen-based operant procedure for mice.

Mice are uniquely suited as experimental subjects for various approaches to the study of the molecular and genetic basis of behavior, and there has been a corresponding explosion in the use of mice in behavioral neuroscience. Rats and monkeys, however, remain the preferred species for high-order cognitive models largely due to the unavailability of valid, reliable and translatable endpoint measures of behavior in the mouse. Here we present further development and validation of a touchscreen-based operant method for measuring cognition that is comparable to methods used in other species and human patients. C57BL/6J mice were found to show good performance on visual discrimination and reversal learning using this method. Demonstrating the sensitivity of the paradigm to genetic factors, C57BL/6J and DBA/2J mice exhibited marked differences in discrimination and reversal learning. Systemic treatment with the selective D1-like agonist, SKF81297, produced an impairment in the early phase of reversal learning, but did not alter visual discrimination, in C57BL/6J mice. The same treatment impaired spatial working memory on the T-maze delayed alternation task, but did not alter control measures of behavior including motivation and locomotor activity. These data demonstrate the sensitivity of visual discrimination and reversal learning measured by this method to genetic factors and pharmacological challenge, and thereby provide an extension and further validation of the method for measuring cognition in mice. When combined with emerging molecular techniques uniquely suited to this species such as genetic engineering and RNA modification this paradigm could provide a powerful new tool for behavioral neuroscience.

Analysis of Variance↗

Protein Language Model Decoys for Target Decoy Competition in Proteomics: Quality Assessment and Benchmarks.

Large-scale proteomics relies heavily on target-decoy competition for false discovery rate estimation in peptide identification, and the performance of this strategy depends strongly on the design of the decoy database. Classical generators such as reversal and shuffling remain widely used. Here, we introduce the first protein language model-based (PLM) decoy generation for peptide identification and benchmark it against classical strategies. We evaluate these approaches using three complementary quality-control layers: sequence-based separability, search-engine-agnostic spectral-space diagnostics, and end-to-end mass spectrometry benchmarks, including pipelines with rescoring. Across these analyses, PLM-based decoys are harder for sequence-only neural networks to distinguish than most classical generators, suggesting fewer obvious sequence-level artifacts. However, this signal is only weakly informative for search performance. Spectral diagnostics further show that short peptides occupy a particularly crowded target-decoy space and are therefore especially prone to local collisions across all generators. In full search pipelines, reverse decoys remain a strong baseline, and current PLM-based generators do not yet provide a clear overall advantage. We therefore view PLM-based decoys not as universal replacements for reverse decoys but as tunable tools for benchmarking, diagnostics, stress testing, and future adaptive decoy optimization, with increasing value as search models become more expressive.

Proteomics↗

Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Base Sequence↗

Genetic engineering of Periplaneta fuliginosa densovirus as an improved biopesticide.

The smoky-brown cockroach (Periplaneta fuliginosa) densovirus (PfDNV) has previously shown potential in urban pest control. To improve its efficacy as a biopesticide, the genome of PfDNV was engineered by inserting the insect-specific toxin gene BmKIT1 in the open reading frame encoding the major structural proteins. A green fluorescent protein (GFP) marker was tagged to the BmKIT1 at its C-terminus for in vivo imaging using Confocal laser scanning microscopy (CFSM). Using a virion rescue strategy, the genomes of recombinant and wild-type (wt) PfDNV were then cotransfected in P. fuliginosa nymphs. Reverse transcription PCR (RT-PCR) showed that the inserted BmkIT1 genes were expressed in the P. fuliginosa nymphs 48 h after cotransfection. CFSM and transmission electron microscopy also confirmed the generation of virus particles and expression of BmKIT1-GFP fusion protein in the cotransfected nymphs. The recombinant viruses remained infective to P. fuliginosa nymphs in feeding tests. Using the LT(50) bioassay method, the coninfection of the recombinant and wt PfDNV killed the P. fuliginosa nymphs approximate 32% faster than wt PfDNV only. This is the first report showing the improvement of engineered densovirus for the potential application of biopesticide.

Animals↗

Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery.

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanateinduced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/n49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the His127Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

Dimerization↗

In vivo Cre/loxP mediated recombination in mouse Clara cells.

In small airways, Clara cells are the main epithelial cell type and play an important physiological role in surfactant production, protection against environmental agents, regulation of inflammatory and immune responses in the respiratory system. Thus, Clara cells are involved in lung homeostasis and pathologies like asthma, Chronic Obstructive Pulmonary Diseases (COPD) or cancers. To date, Clara cells implication in these pathological processes remains largely enigmatic. The engineering of a transgenic strain mouse allowing specific gene invalidation in Clara cells may be of interest to improve our knowledge about the genes involved in these diseases. By using the Cre/loxP strategy we report the engineering of a transgenic mouse strain with expression of Cre recombinase under the control of the Clara Cell Secretory Protein (CCSP) promoter. Specific staining and immuno-histochemistry performed after breeding with reporter mice revealed that CCSP drives a functional Cre expression specifically in Clara cells. This mouse strain is a powerful tool for Cre-loxP-mediated conditional recombination in the lung and represents a new tool to study Clara cell physiology.

Animals↗

The effect of cholecyst-derived extracellular matrix on the phenotypic behaviour of valvular endothelial and valvular interstitial cells.

Cholecyst-derived extracellular matrix (CEM) is a novel, proteinaceous biomaterial, derived from the porcine cholecyst, which may have potential applications as a scaffold in the area of heart valve tissue engineering. In this study the potential of CEM to support the proliferation of valvular endothelial cells (VECs) and valvular interstitial cells (VICs), while maintaining their phenotypic mRNA synthesis, protein expression and morphology was assessed by biochemical assays, electron microscopy, immunostaining and reverse-transcriptase polymerase chain reaction. VICs and VECs were isolated from the porcine aortic valve and techniques were developed for the isolation of CEM for cell culture. VECs and VICs cultured on CEM adhered and proliferated, maintaining their phenotypic morphology. VECs synthesised von Willebrand factor mRNA and endothelial nitric oxide synthase (eNOS) mRNA and expressed eNOS and VICs synthesised alpha-smooth muscle actin (alphaSMA) mRNA and expressed alphaSMA. Cellular area fraction of VICs expressing alphaSMA was 87.7+/-6.8% and cellular area fraction of VECs expressing eNOS was 93.8+/-9.3%. Findings of this study support the hypothesis that CEM is a potential biomaterial for tissue engineered heart valve scaffold design.

Animals↗

DNA vaccination with an insulin construct and a chimeric protein binding to both CTLA4 and CD40 ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D), a T-cell-mediated autoimmune disease, could be attributed to many defects in nonobese diabetic (NOD) mice, including deficient expressions of costimulatory molecules that impair antigen presentation. Thus, this deficient antigen presentation may result in a reduced ability to induce a tolerogenic response through negative selection/regulation of autoreactive T cells. Improperly activated T cells seem to be able to induce autoimmune responses causing diabetes. To re-establish tolerance to autoantigens by modulating costimulation, we constructed and tested a new type of DNA vaccine encoding a membrane-bound preproinsulin (mbPPI) and a chimeric gene vector encoding mutant B7.1/CD40L (mB7.1/CD40L) fusion protein. This mutant B7.1 binds CTLA4 but not CD28. We report that young NOD mice immunized with mbPPI along with mB7.1/CD40L DNA vectors significantly reduced diabetes incidence while treatment with CTLA4/IgG1 exacerbated diabetes. In conclusion, the combination of mbPPI and mB7.1/CD40L was able to protect against autoimmunity and diabetes in NOD mice possibly by promoting a more efficient presentation of autoantigen PPI and inducing specific tolerance to PPI by negatively regulating autoreactive T cells.

Animals↗

Production and characterization of recombinant rat brain-derived neurotrophic factor and neurotrophin-3 from insect cells.

Rat brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were engineered for expression in a baculovirus-infected Spodoptera frugiperda insect cell system. The BDNF and NT-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. The purification procedure yielded approximately 2 mg of pure rat BDNF or NT-3 per liter of culture supernatant. A single N-terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell-produced rat BDNF and NT-3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT-3, the BDNF and NT-3 recovered after purification were > 95% fully processed, mature protein. Mature recombinant rat BDNF and NT-3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT-3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT-3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.

Amino Acid Sequence↗

Tobacco use as drug addiction: the scientific foundation.

Tobacco use is strongly driven by the pharmacological actions of nicotine in the central nervous system. This review will summarize some of the seminal research findings relating to nicotine dependence and will highlight fundamental questions that must yet be answered. The evidence that nicotine is an addictive drug was summarized in the 1988 Report of the Surgeon General which concluded that nicotine fulfills the criteria for a dependence-producing drug. More recently, research has further characterized the pharmacological effects of nicotine in the brain and elucidated the basic pathophysiology of nicotine addition. Moreover, research shows that nicotine replacement therapy, such as nicotine patch or gum, can prevent or reverse withdrawal symptoms. It is also clear that the form of nicotine delivery is a major determinant of addiction potential and that cigarettes and smokeless tobacco products are both highly engineered drug delivery devices that act not only to provide users with controllable doses of nicotine, but also to maximize the addictive effects of nicotine. Along with the understanding of the dependence process has come a rapidly expanding arsenal of treatment for the disorder. There are many major questions about the nature and course of nicotine addiction that remain unanswered and must be addressed if we are to continue to improve our ability to prevent tobacco dependence as well as to provide more effective and acceptable options for treatment and disease prevention in those who are already addicted.

Animals↗