BACKGROUND:Idiopathic ventricular ectopy (VE) shows predilection to sites within the left ventricular (LV) base such as the outflow tract/aortic sinuses, LV summit, and areas adjacent to the aortomitral continuity. We characterize VE arising from the inferior septum of the LV base that was successfully managed by LV endocardial ablation from the inferoseptal recess of the LV.OBJECTIVE:The purpose of this study was to determine the incidence, electrocardiographic (ECG) findings, electrophysiological findings, and anatomical features associated with VE arising from the basal inferoseptal process of the LV (ISP-LV) ablated using an LV endocardial approach via the inferoseptal recess of the LV.METHODS:A total of 425 consecutive patients undergoing VE ablation between January 1, 2012 and December 31, 2016 at 3 centers were evaluated. Demographic characteristics, ECG findings, and procedural data were analyzed for patients with ISP-LV VEs.RESULTS:Seven (1.5%) had a site of origin from the ISP-LV. Common ECG findings were a right bundle branch block concordant pattern or an atypical left bundle branch block early transition pattern, suggestive of a basal origin with a left superior axis, a biphasic QRS complex in lead aVR, and a small s wave in lead V6. Earliest activation was seen in an area below the outflow tract accessed from the inferoseptal recess inferior to the His bundle. In 3 cases, transient junctional rhythm was seen during ablation. All cases were ablated successfully with no complications.CONCLUSION:VE arising from the ISP-LV represents a distinct subset of idiopathic arrhythmia and can be successfully treated by endocardial catheter ablation from the inferoseptal recess. They share common surface ECG and electrophysiological findings with special anatomical features that need recognition for successful catheter ablation.

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence micro- scope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromo- some-specific composite probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The tech- nique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2- phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facil- itate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.

Skin burns are a rare complication associated with radiofrequency catheter ablation of cardiac arrhythmias. Burns related to the indifferent electrode patch may be severe and result in significant comorbidity. We describe our experience of skin burns and discuss potential predisposing and possible causative factors.

Jessica Webb and David Ward investigated how long it takes for hospital doctors to speak to patients’ general practitioners by telephone

Percutaneous and surgical left atrial ablation has been widely used to treat paroxysmal and persistent atrial fibrillation. However, left atrial ablation may result in left atrial tachycardia due to an iatrogenic substrate created by the ablation lesion sets. Ablation of these iatrogenic arrhythmias can be technically challenging, requiring prolonged procedures and the use of three-dimensional electroanatomical mapping systems. In some cases, the atrial tachycardia may terminate during mapping, or may degenerate into atrial fibrillation during the procedure before adequate mapping. Some patients also have several arrhythmia circuits, each requiring separate mapping, which may be time consuming. The present article reports the cases of three patients in whom a large cryoballoon was used to empirically ablate the pulmonary vein antral region, which is important for the initiation and maintenance of these arrhythmias.

Left atrial isomerism (LAI) is characterized by the presence of two morphologically identical atria. It is commonly associated with conduction defects. We report a case of LAI presenting with highly symptomatic atrioventricular nodal reentrant tachycardia, which was cured by ablation. (PACE 2012; 35:e291–e292)

A 62-year-old man with uncorrected cyanotic congenital heart disease involving double inlet left ventricle with visceral and atrial situs solitus, L-looped ventricles, L-transposed great vessels, and pulmonary stenosis, presented with recurrent atrial tachycardia. Entrainment mapping revealed the arrhythmia mechanism to be an uncommon micro-reentrant cavotricuspid isthmus-dependent circuit (intra-isthmus reentry), which was amenable to radiofrequency ablation. This uncommon right atrial arrhythmia is yet to be reported in patients with complex congenital heart disease and was amenable to radiofrequency ablation.

BACKGROUND:The multielectrode array (MEA) enables noncontact mapping of cardiac arrhythmias; our experience is reviewed and reported.HYPOTHESIS:The MEA has a role as first line therapy in multiple arrhythmias.METHODS:Retrospective and prospective analysis of all consecutive cases performed using the MEA over a 6 year period.RESULTS:Electrophysiological study and catheter ablation, 46% under general anaesthesia, using radiofrequency (RF), cryothermal and low energy direct current (DC) was performed in 66 procedures on 31 females and 29 males, average age 50.2 yrs (19.3-81.6); 8 patients underwent multiple procedures. 24 patients (36%) had undergone past ablation for the same arrhythmia. A wide variety of arrhythmias from all chambers were treated, majority right atrial (56%) and right ventricular (29%). Procedural success was complete in 77.4% and partial/indeterminate in 11.3%, highest in right atrial tachycardia, right ventricular outflow tract ectopy and typical atrial flutter (79, 82 and 100%). RF energy was most commonly used (67%) and DC carried 100% success. Ablation was delivered at 'early activation' and 'breakout' in focal arrhythmias. In re-entrant circuits linear ablation transecting path of activation extending to regions of functional/anatomic block was performed. Two of 7 complications were attributed to the MEA: groin haematomas. At mean 12.4 mo follow up 56% were arrhythmia free, 15% asymptomatic or minimally symptomatic and 12 patients had new arrhythmias.CONCLUSIONS:The MEA is effective, safe and suitable for first line therapy in multiple cardiac arrhythmias particularly in the right heart. Further study is warranted comparing it to other mapping techniques.

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.

Background: Existing guidelines for electrical cardioversion (ECV) of atrial arrhythmias suggest starting at a low energy setting on the grounds that shocks of high energy might damage the myocardium or trigger more serious arrhythmias. We hypothesised that more powerful shocks would exceed the upper limit of vulnerability for inducing ventricular fibrillation. The initial use of higher energy could therefore reduce arrhythmic complications.Methods: We collected data on the sequence of shocks delivered and the resulting changes in cardiac rhythm in 1896 patients who underwent transthoracic ECV. Rhythm strips derived from 200 consecutive ECV attempts were studied to verify the accuracy of the synchronisation of the shocks delivered.Results: In 2522 attempts at transthoracic ECV, 6398 shocks were delivered, 1243 in atrial flutter or atrial tachycardia, the others in AF. Ventricular fibrillation was significantly more common after shocks of < 200 J (5 of 2959 vs. 0 of 3439 shocks, p < 0.05, Fischer's exact test). Conversion of atrial flutter or atrial tachycardia to AF was also more common at < 200 J (20 of 930 shocks vs. 1 of 313 shocks at >= 200 J, p < 0.05, chi(2) test). Sinus bradycardia or sinus arrest complicated 0.95% of cardioversion attempts, but none required emergency pacing. The incidence of bradycardia was not related to the energy used.Conclusions: Shocks of > 200 J are associated with fewer tachyarrhythmic complications, and do not increase the risk of other serious complications. Bradycardia after cardioversion is very rarely of clinical importance. (c) 2007 Published by Elsevier Ireland Ltd.

OBJECTIVE:To explore the dimensional attributes of haploid and disomic X-bearing and Y-bearing spermatozoa.DESIGN:Morphometric evaluation of more than 2,000 X-bearing and Y-bearing spermatozoa after identification of the genotype with fluorescence in situ hybridization.SETTING:Academic clinical and research andrology laboratory.MAIN OUTCOME MEASURE(S):Sperm head area, perimeter, long axis, short axis, shape factor, elliptical form factor (long axis/short axis), and tail length.RESULT(S):We found no differences in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa, whether in the native or the decondensed state, or in oligozoospermic or normozoospermic men. There were inconsistent differences and a 70% overlap in the dimensions of haploid and disomic spermatozoa. The other 30% of sperm with disomic nuclei were either smaller or larger compared to haploid spermatozoa.CONCLUSION(S):There are no differences, or distinguishing characteristics, in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa. Dimensional attributes do not discriminate between dysomic and haploid spermatozoa.

BACKGROUND:LQTS may cause sudden cardiac death (SCD), but the mechanisms linking gene mutations to ventricular fibrillation (VF) are unclear. OBJECTIVE:To determine whether ventricular activation delays in congenital long QT syndrome (LQTS) are associated with VF and to describe these delays clinically by measuring activation through ventricular myocardium after a premature extrastimulus. METHODS:Forty-six patients with LQTS, including 16 with VF (LQTS VF) were investigated, and the results were compared with those from 24 patients with hypertrophic cardiomyopathy and VF (HCM VF). Electrograms in response to premature stimuli were analyzed for increases in electrogram duration (DeltaED) and the S1S2 coupling intervals at which electrogram latency starts to increase (S1S2(delay)). Two piecewise continuous straight line segments were fitted to the last electrogram deflection as a function of S1S2 interval in the LQTS and HCM VF populations, and the difference in their gradient (alpha) was taken as an index of the abruptness of the onset of this delay. RESULTS:Thirteen LQTS VF and six LQTS non-VF patients had values of DeltaED and S1S2(delay) comparable to those in HCM VF patients, while the remainder (three LQTS VF and 24 LQTS non-VF) had lower values (P<.001). There was only a weak correlation between delay and the corrected QT interval. The HCM and LQTS VF patients could be separated by the value of alpha (P<.01), with the LQTS patients having a more abrupt onset of delay. CONCLUSIONS:Large delays in ventricular activation after an extrastimulus occur in patients with the LQTS, especially those with VF. The change in delay is abrupt in the LQTS, indicating sudden block to activation creating a dynamic substrate for arrhythmogenesis.

Objective: To test a newly invented intracytoplasmic sperm injection (ICSI) sperm selection method based on sperm hyaluronic acid (HA) binding.Design: Comparison of chromosomal disomy and diploidy frequencies in sperm arising from semen and in HA-bound sperm.Setting: Academic andrology laboratory.Patient(s): Men presenting for semen analysis.Intervention(s): Washed sperm fractions of 32 semen samples were applied to Petri dishes or glass slides coated with immobilized HA. The unbound sperm were rinsed gently, and the HA-bound sperm were removed with an ICSI pipette. The control sperm population was the unselected sperm. Both HA-selected and unselected sperm were treated with fluorescence in situ hybridization with centromeric probes for the X, Y, and 17 chromosomes.Main Outcome Measure(s): Chromosomal disomy and diploidy frequencies.Result(s): In the HA-bound sperm (495-2,079 per man, 41,670 in all) compared with unselected sperm (4,770 per man, 162,210 in all), the chromosomal disomy frequencies were reduced to 0.16% from 0.52%, diploidy to 0.09% from 0.51%, and sex chromosome disomy to 0.05% from 0.27% (a 5.4-fold reduction vs. 4-fold respective increase in ICSI offspring).Conclusion(s): The HA sperm selection method for ICSI, which is based on a relationship between sperm receptors for zona pellucida and HA, will likely reduce the potential genetic complications and adverse public health effects of ICSI.

9508 Introduction: A major limitation in the treatment of ovarian cancer (OC) is the lack of early detection tumor markers. Early diagnosis would significantly decrease the morbidity and mortality rate from this disease. CA 125, the only available test is elevated in only 40–50% of women with early stage disease with a positive predictive value of only 10%. Sensitive and specific methods of early stage OC detection are urgently needed. Here we report a new blood test, based on four analytes, which can discriminate between disease-free, and OC patients, including Stage I/II patients. Methods: 169 proteins were measured in serum samples (28 healthy controls and 36 OC patients) using a microarray immunoassay (Nat. Biotech (2000) 20:359). Ten proteins showing the greatest concentration differences between patients and controls were evaluated by independent ELISA assays and four were found to correctly classify all serum samples in this training set. The four biomarkers then were validated in a blind study of serum samples collected from 106 healthy females and 100 OC patients (24% Stage I/II disease and 76% Stage III/IV). The results of the selected analytes then were compared using three different classifiers: support vector machines, K-nearest neighbor classifiers and classification trees. Results: None of the 169 analytes used individually could correctly classify cancer patients and healthy controls. However, in a blind study with analysis of 4 biomarkers, 100 of the 106 women (94%) in the normal (disease-free) group had no more than one marker out of the normal range, while 99 of 100 OC patients (99%) had two or more markers out of normal range. The sensitivity of the test is 96%, specificity 97%, PPV 97% and NPV 96%. All 24 Stage I/II sera were correctly identified. Conclusion: We describe a multiplex serum protein screening assay that more accurately discriminates between normal women and OC patients and has the ability to detect early stage I and II OCs. No significant financial relationships to disclose.

Early diagnosis of epithelial ovarian cancer (EOC) would significantly decrease the morbidity and mortality from this disease but is difficult in the absence of physical symptoms. Here, we report a blood test, based on the simultaneous quantization of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II), that can discriminate between disease-free and EOC patients, including patients diagnosed with stage I and II disease, with high efficiency (95%). Microarray analysis was used initially to determine the levels of 169 proteins in serum from 28 healthy women, 18 women newly diagnosed with EOC, and 40 women with recurrent disease. Evaluation of proteins that showed significant differences in expression between controls and cancer patients by ELISA assays yielded the four analytes. These four proteins then were evaluated in a blind cross-validation study by using an additional 106 healthy females and 100 patients with EOC(24 stage I/II and 76 stage III/IV). Upon sample decoding, the results were analyzed by using three different classification algorithms and a binary code methodology. The four-analyte test was further validated in a blind binary code study by using 40 additional serum samples from normal and EOC cancer patients. No single protein could completely distinguish the cancer group from the healthy controls. However, the combination of the four analytes exhibited the following: sensitivity 95%, positive predictive value (PPV) 95%, specificity 95%, and negative predictive value (NPV) 94%, a considerable improvement on current methodology.

The overall objectives of this project were (1) to develop allosteric ribozymes capable of acting as molecular switches for monitoring the levels of both wild-type and mutant mRNA species in living cells and whole animals and (2) to develop highly efficient reagents to deliver nucleic acid molecular switches into living cells, tissues and animals with the ultimate goal of expression profiling specific mRNAs of diagnostic or prognostic value within tumors in animals. During the past year, we have moved our laboratory to Nevada and in the moving process we have lost electronic and paper copies of prior progress reports concerning the construction and biological properties of the molecular switches. Since there was minimal progress during the last year on molecular switches, we are relying on past project reports to provide a summary of our data on this facet of the grant. Here we are summarizing the work done on the delivery reagents and their application to inducing mutations in living cells, which will include work done during the no cost extension.

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic −794 CATT5–8 repeat and −173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

ObjectiveWe have adapted the method of haplotyping by MDA (multiple displacement amplification; PNAS, 2002 and 2003) to detect single nucleotide polymorphism in DNA of individual sperm cells, for future application in the study of genetic aspects of male infertility.DesignThis study combines three different methods: (a) MDA for whole genome amplification, (b) PCR (polymerase chain reaction) for amplification of specific DNA regions, and (c) single nucleotide polymorphism genotyping with optical thin-film biosensor chips that facilitate the detection of positive hybridization/ligation reactions via color changes at defined regions of the chip surface.Materials and methodsFrom washed semen samples, groups of 60–100 sperm were isolated, and from the same sperm suspensions single sperm were also selected with a micromanipulator pipette. The cells were lysed and DNA amplification was carried out directly on the multiple or single cell samples using MDA (Ø29 DNA polymerase, Amersham), generating microgram amounts of DNA with high fidelity locus representation. The MDA amplified DNA was used as a template for PCR. In the present work, we are focusing on chromosome 17; thus we used 20 different chromosome 17 PCR primer pairs. Each individual PCR product was tested using allele-specific single nucleotide polymorphism probes arrayed on the biosensor chips. The target DNA was denatured and incubated on the array in a hybridization/ligation reaction, using a thermostable DNA ligase. All non-ligated molecules were removed by an alkaline wash step. Individual single nucleotide polymorphism were detected as gold to blue/purple color changes on the chip surface, signaling positive ligation events.ResultsSperm from a total of 6 patients were analyzed. The pattern of homozygous and heterozygous alleles for the 20 markers of chromosome 17 were examined by the MDA, PCR and chip analysis of the 60–100 sperm lysates. For each locus exhibiting heterozygous alleles in the multiple sperm cell lysates, testing of single sperm DNA samples showed one or the other allele, but not both alleles in individual spermatozoa.ConclusionWith the adaptation of the MDA technology, we have demonstrated the feasibility of genetic haplotyping in single spermatozoa. The haplotype analysis of specific sperm regions may allow the detection of single nucleotide, or more extensive, genomic changes in the Y and other chromosomes that may be associated with diminished spermatogenesis or sperm function in infertile men. ObjectiveWe have adapted the method of haplotyping by MDA (multiple displacement amplification; PNAS, 2002 and 2003) to detect single nucleotide polymorphism in DNA of individual sperm cells, for future application in the study of genetic aspects of male infertility. We have adapted the method of haplotyping by MDA (multiple displacement amplification; PNAS, 2002 and 2003) to detect single nucleotide polymorphism in DNA of individual sperm cells, for future application in the study of genetic aspects of male infertility. DesignThis study combines three different methods: (a) MDA for whole genome amplification, (b) PCR (polymerase chain reaction) for amplification of specific DNA regions, and (c) single nucleotide polymorphism genotyping with optical thin-film biosensor chips that facilitate the detection of positive hybridization/ligation reactions via color changes at defined regions of the chip surface. This study combines three different methods: (a) MDA for whole genome amplification, (b) PCR (polymerase chain reaction) for amplification of specific DNA regions, and (c) single nucleotide polymorphism genotyping with optical thin-film biosensor chips that facilitate the detection of positive hybridization/ligation reactions via color changes at defined regions of the chip surface. Materials and methodsFrom washed semen samples, groups of 60–100 sperm were isolated, and from the same sperm suspensions single sperm were also selected with a micromanipulator pipette. The cells were lysed and DNA amplification was carried out directly on the multiple or single cell samples using MDA (Ø29 DNA polymerase, Amersham), generating microgram amounts of DNA with high fidelity locus representation. The MDA amplified DNA was used as a template for PCR. In the present work, we are focusing on chromosome 17; thus we used 20 different chromosome 17 PCR primer pairs. Each individual PCR product was tested using allele-specific single nucleotide polymorphism probes arrayed on the biosensor chips. The target DNA was denatured and incubated on the array in a hybridization/ligation reaction, using a thermostable DNA ligase. All non-ligated molecules were removed by an alkaline wash step. Individual single nucleotide polymorphism were detected as gold to blue/purple color changes on the chip surface, signaling positive ligation events. From washed semen samples, groups of 60–100 sperm were isolated, and from the same sperm suspensions single sperm were also selected with a micromanipulator pipette. The cells were lysed and DNA amplification was carried out directly on the multiple or single cell samples using MDA (Ø29 DNA polymerase, Amersham), generating microgram amounts of DNA with high fidelity locus representation. The MDA amplified DNA was used as a template for PCR. In the present work, we are focusing on chromosome 17; thus we used 20 different chromosome 17 PCR primer pairs. Each individual PCR product was tested using allele-specific single nucleotide polymorphism probes arrayed on the biosensor chips. The target DNA was denatured and incubated on the array in a hybridization/ligation reaction, using a thermostable DNA ligase. All non-ligated molecules were removed by an alkaline wash step. Individual single nucleotide polymorphism were detected as gold to blue/purple color changes on the chip surface, signaling positive ligation events. ResultsSperm from a total of 6 patients were analyzed. The pattern of homozygous and heterozygous alleles for the 20 markers of chromosome 17 were examined by the MDA, PCR and chip analysis of the 60–100 sperm lysates. For each locus exhibiting heterozygous alleles in the multiple sperm cell lysates, testing of single sperm DNA samples showed one or the other allele, but not both alleles in individual spermatozoa. Sperm from a total of 6 patients were analyzed. The pattern of homozygous and heterozygous alleles for the 20 markers of chromosome 17 were examined by the MDA, PCR and chip analysis of the 60–100 sperm lysates. For each locus exhibiting heterozygous alleles in the multiple sperm cell lysates, testing of single sperm DNA samples showed one or the other allele, but not both alleles in individual spermatozoa. ConclusionWith the adaptation of the MDA technology, we have demonstrated the feasibility of genetic haplotyping in single spermatozoa. The haplotype analysis of specific sperm regions may allow the detection of single nucleotide, or more extensive, genomic changes in the Y and other chromosomes that may be associated with diminished spermatogenesis or sperm function in infertile men. With the adaptation of the MDA technology, we have demonstrated the feasibility of genetic haplotyping in single spermatozoa. The haplotype analysis of specific sperm regions may allow the detection of single nucleotide, or more extensive, genomic changes in the Y and other chromosomes that may be associated with diminished spermatogenesis or sperm function in infertile men.

Placental site trophoblastic tumour (PSTT) is a neoplastic proliferation of the implantation intermediate trophoblast. Although clinicopathological studies are not uncommon in case reports or small series, molecular and genetic studies are quite limited. Four archived cases of PSTT were successfully analysed by comparative genomic hybridization (CGH) in this study. Regional chromosomal gains were observed in two cases. One case showed chromosomal gains in the regions of 19p13.2, 21q11–21 and 22q12. The second case demonstrated a single regional chromosomal gain involving 21q21. No chromosomal loss is observed. The remaining two cases showed a balanced CGH profile without detectable chromosomal gain or loss. In summary, although chromosomal alterations detectable by CGH are not common, rare chromosomal gains do occur in PSTT. The recurrent chromosomal gain involving chromosomal 21q observed in two of our cases deserves additional studies to ascertain whether it carries any pathobiological significance.

Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO-peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with 'naked' oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO-peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.