A simple and accurate ratiometric sensor for determination of dopamine based on dual-emission carbon dots.

Dopamine (DA) is a neuromodulatory molecule that plays critical roles in many biological processes. The dysfunctions of the DA system are closely associated with several nervous system diseases. Therefore, it is urgent to establish a simple and accurate method for DA analysis. In this study, an economic and accurate DA ratiometric sensor was established using dual-emission carbon dots (DE-CDs). DE-CDs were first synthesized by the one-step solvothermal method and two separate fluorescence emission peaks at 340 and 500 nm were observed under the excitation of 310 nm. In the presence of Hg2+, the fluorescence signal at 340 nm was significantly quenched, while the signal at 500 nm keeps stable. Upon adding DA, the quenched signal at 340 nm was significantly recovered, whereas the signal at 500 nm remains stable. Therefore, a novel ratiometric sensor for DA analysis was established. This method shows a good linear range from 500 nM to 100 µM, and the detection limit was calculated to be 80 nM. Moreover, this established method shows excellent specificity and could be applied in real sample analysis, showing great potential for application in clinical research.

A Review of Dual-Emission Carbon Dots and Their Applications.

Carbon dots (CDs), as a rising star among fluorescent nanomaterials with excellent optical properties and fascinating dual-emission characteristics, have attracted increasing attention in sensing, bio-imaging, drug delivery, and so on. The synthesis of dual-emission CDs (DE-CDs) and the establishment of ratiometric fluorescence sensors can effectively diminish background interference and provide more accurate results than single-emission CDs. Although DE-CDs have generated increased attention in many fields, the review articles about DE-CDs are still insufficient. Therefore, we summarized the latest results and prepared this review. This review first provides an overview of the primary synthesis route and commonly used precursors in DE-CDs synthesis. Then, the photoluminescence mechanism behind the dual-emission phenomenon was discussed. Thirdly, the application of DE-CDs in metal cation detection, food safety analysis, biosensing, cell imaging, and optoelectronic devices has been extensively discussed. Finally, the main challenges and prospects for further development are presented. This review presents the latest research progress of DE-CDs synthesis and its application in ratiometric sensing; hopefully, it can help and encourage researchers to overcome existing challenges and broaden the area of DE-CDs research.

Mucin Glycans: A Target for Cancer Therapy.

Mucin glycans are an important component of the mucus barrier and a vital defence against physical and chemical damage as well as pathogens. There are 20 mucins in the human body, which can be classified into secreted mucins and transmembrane mucins according to their distributions. The major difference between them is that secreted mucins do not have transmembrane structural domains, and the expression of each mucin is organ and cell-specific. Under physiological conditions, mucin glycans are involved in the composition of the mucus barrier and thus protect the body from infection and injury. However, abnormal expression of mucin glycans can lead to the occurrence of diseases, especially cancer, through various mechanisms. Therefore, targeting mucin glycans for the diagnosis and treatment of cancer has always been a promising research direction. Here, we first summarize the main types of glycosylation (O-GalNAc glycosylation and N-glycosylation) on mucins and the mechanisms by which abnormal mucin glycans occur. Next, how abnormal mucin glycans contribute to cancer development is described. Finally, we summarize MUC1-based antibodies, vaccines, radio-pharmaceuticals, and CAR-T therapies using the best characterized MUC1 as an example. In this section, we specifically elaborate on the recent new cancer therapy CAR-M, which may bring new hope to cancer patients.

miR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A.

Background: The ambiguity of renal cell carcinoma (RCC) symptoms hinders early diagnosis, thereby contributing to high mortality rates. By attaching to the 3'-untranslated region (UTR) of the target gene, microRNAs (miRNAs) exert significant control over the expression of genes. Objectives: To investigate the influence of miR-30c-2-3p and DNA topoisomerase II alpha (TOP2A) on RCC growth and the mechanisms underlying the regulation of its expression. Methods: The expression of miRNA-30c-2-3p and TOP2A in RCC cells was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MiR-30c-2-3p mimics, its inhibitors, and controls, as well as TOP2A short hairpin RNA (shRNA) and controls, were used to transfect the human RCC cell lines 786-O, Caki-1, and ACHN. Additionally, the roles of miRNA-30c-2-3p and TOP2A in the growth of RCC were evaluated using the cell counting kit (CCK)-8 test, colony formation assay, apoptosis analysis, and Western blotting. Meanwhile, binding of miRNA-30c-2-3p and TOP2A was verified using dual-luciferase reporter assays and Western blotting. Results: miR-30c-2-p is underexpressed in RCC cells. Overexpression of miR-30c-2-p promotes apoptosis and inhibits proliferation of ACHN, Caki-1, and 786-O cells. miR-30c-2-3p targets TOP2A, which is elevated in RCC tissues and cells, whereas TOP2A silencing inhibits the proliferation ability of RCC cells. The miRNA-30c-2-3p inhibitor compromises TOP2A shRNA-induced apoptosis of RCC. RCC cells cotransfected with miRNA-30c-2-3p inhibitors and TOP2A shRNAs have a higher proliferation rate than those transfected with only TOP2A shRNAs. Conclusions: Collectively, our results verify that miRNA-30c-2-3p has a tumor suppressor property. miRNA-30c-2-3p inhibits the proliferation of RCC through regulation of TOP2A. The data provide a viable therapeutic target for RCC.

A Sweet Warning: Mucin-Type O-Glycans in Cancer.

Glycosylation is a common post-translational modification process of proteins. Mucin-type O-glycosylation is an O-glycosylation that starts from protein serine/threonine residues. Normally, it is involved in the normal development and differentiation of cells and tissues, abnormal glycosylation can lead to a variety of diseases, especially cancer. This paper reviews the normal biosynthesis of mucin-type O-glycans and their role in the maintenance of body health, followed by the mechanisms of abnormal mucin-type O-glycosylation in the development of diseases, especially tumors, including the effects of Tn, STn, T antigen, and different glycosyltransferases, with special emphasis on their role in the development of gastric cancer. Finally, tumor immunotherapy targeting mucin-type O-glycans was discussed.

Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling.

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells.

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins.

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid-binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galß1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Display of the human mucinome with defined O-glycans by gene engineered cells.

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.

Probing the binding specificities of human Siglecs by cell-based glycan arrays.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells.

Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics.

Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis.

BACKGROUND: Sialylation of glycoproteins and glycolipids is important for biological processes such as cellular communication, cell migration and protein function. Biosynthesis of CMP-sialic acid, the essential substrate, comprises five enzymatic steps, involving ManNAc and sialic acid and their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood. METHODS AND RESULTS: We aimed to study the mechanisms of sialic acid metabolism in knockouts (KO) of the sialic acid pathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), NANS (sialic acid synthase) and CMAS (N-acylneuraminate cytidylyltransferase) genes, but was largely unaffected in NANP (N-acylneuraminate-9-phosphatase) KO, as studied by MAA and PNA lectin binding. NANP is the third enzyme in sialic acid biosynthesis and dephosphorylates sialic acid 9-phosphate to free sialic acid. LC-MS analysis of sialic acid metabolites showed that CMP-sialic acid was dramatically reduced in GNE and NANS KO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP-sialic acid levels in NANP KO were comparable to WT cells, even though sialic acid 9-phosphate, the substrate of NANP accumulated. Metabolic flux analysis with 13C6-labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid. CONCLUSIONS: Our data provide evidence that NANP activity is not essential for de novo sialic acid production and point towards an alternative phosphatase activity, bypassing NANP. GENERAL SIGNIFICANCE: This report contributes to a better understanding of sialic acid biosynthesis in humans.

Glycoengineering design options for IgG1 in CHO cells using precise gene editing.

Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.

Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads.

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs' population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Lab on a single microbead: an ultrasensitive detection strategy enabling microRNA analysis at the single-molecule level.

Detection of a single nucleic acid molecule is of great significance for both fundamental biochemistry studies and clinical diagnostics. By using microRNA (miRNA) as a model target, herein, we have developed a single-microbead-based sensing (SMBS) platform, which simply enables the detection of miRNA at the single-molecule level. In this strategy, an isothermal exponential amplification reaction (EXPAR) is rationally designed towards specific miRNAs and all products of the EXPAR are integrated onto a single microbead for signal amplification and fluorescence enrichment. This pushes the detection of miRNAs down to 1 aM in a 5 µL sample, corresponding to 3 copies of the miRNA molecule. This new strategy also affords high selectivity and it is capable of distinguishing among homologous miRNA family members even with a single-base difference. Due to its ultrahigh sensitivity and selectivity, the proposed SMBS platform has been successfully applied to the detection of miRNA extracted from a single cell.

Using intramyocardial electrograms combined with other noninvasive methods for monitoring acute rejection following human heart transplantation.

BACKGROUND: Acute allograft rejection in heart transplantation remains as one of the major complications. Obligatory graft surveillance is still achieved with the invasive and expensive endomyocardial biopsy (EMB). Our study aimed to study the use of intramyocardial electrograms combined with other noninvasive methods for the monitoring of acute rejection after human heart transplantation. METHODS: Permanent pacemakers were implanted in 58 patients undergoing heart transplantations. Intramyocardial electrograms (IMEG) were recorded periodically and the results were compared with those from EMBs. The R wave amplitude of the IMEG was used as the index value, the average R wave amplitude at the third week following transplantation was considered as the baseline, and a reduction of > 20% compared with the baseline was regarded as a positive result. EMB was performed in cases of positive IMEG results and also at other times. Other noninvasive methods were used to help the diagnosis. Acute rejection (AR) was defined as International Society of Heart-Lung Transplantation grade IIIA or higher. RESULTS: We obtained 1231 IMEG records and 127 EMBs. Of the total 127 EMBs, 53 were positive, in which there were 42 IMEG positive results and 11 negative, while in the rest 74 negative EMBs, there were 9 IMEG positive results and 65 negative. The sensitivity of IMEG for the diagnosis of AR was 79.2%, and the specificity was 87.8%. The positive predictive value was 82.4% and the negative predictive value was 85.5%. Of the total of 1231 IMEG records, 51 were positive and 1180 were negative. Excluding 11 proved by EMB to be false negative, if the other 1169 were considered as no evidence of rejection, through the other noninvasive methods, AR diagnosed by this noninvasive monitoring strategy, the sensitivity was 79.2%, and the specificity was 99.2%. The positive predictive value was 82.4% and the negative predictive value was 99.1%. CONCLUSIONS: IMEG can be used as a noninvasive method for monitoring AR following heart transplantation. It is a continuous, safe and inexpensive method, and could reduce the need for EMB combined with other noninvasive methods, without reducing the detection of rejection.

Minimally invasive surgical pulmonary vein isolation alone for persistent atrial fibrillation: preliminary results of epicardial atrial electrogram analysis.

BACKGROUND: Minimally invasive surgical pulmonary vein isolation has become an alterative therapy for lone atrial fibrillation. This study evaluated the effect of the procedure on persistent atrial fibrillation by epicardial atrial electrography. METHODS: Five consecutive patients with lone persistent atrial fibrillation were enrolled. Intraoperative electrophysiology tests were performed before and after minimally invasive surgical pulmonary vein isolation. Morphology of the recordings and atrial fibrillation cycle length were analyzed. RESULTS: Sixty sites were recorded in 5 patients. Three types of bipolar electrogram were recorded at these sites. After ablation, all electrograms changed into type I in pulmonary veins and proximal antra, and remained unchanged in all proximal left atria. Atrial fibrillation cycle length at the proximal left atrium was shorter than that at the pulmonary veins. Atrial fibrillation cycle length recorded at proximal left atrium sites correlated with atrial diameter. The atrial fibrillation cycle length of the left atrium increased from 143 +/- 11 to 170 +/- 12 ms after pulmonary vein isolation. All 5 patients had atrial fibrillation immediately after the procedure and were treated with direct-current cardioversion and received amiodarone postoperatively. Freedom from atrial fibrillation was 100% at discharge and 60% at 6 months' follow-up. CONCLUSIONS: Ectopic foci outside the pulmonary veins play an important role in persistent atrial fibrillation. Minimally invasive surgical pulmonary vein isolation might not be sufficient for persistent atrial fibrillation termination. The pulmonary vein isolation procedure, however, slows atrial fibrillation and makes supplemental pharmacologic cardioversion effective.