A novel Dual-Branch Asymmetric Encoder-Decoder Segmentation Network for accurate colonic crypt segmentation.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with colonic crypts (CC) being crucial in its development. Accurate segmentation of CC is essential for decisions CRC and developing diagnostic strategies. However, colonic crypts' blurred boundaries and morphological diversity bring substantial challenges for automatic segmentation. To mitigate this problem, we proposed the Dual-Branch Asymmetric Encoder-Decoder Segmentation Network (DAUNet), a novel and efficient model tailored for confocal laser endomicroscopy (CLE) CC images. In DAUNet, we crafted a dual-branch feature extraction module (DFEM), employing Focus operations and dense depth-wise separable convolution (DDSC) to extract multiscale features, boosting semantic understanding and coping with the morphological diversity of CC. We also introduced the feature fusion guided module (FFGM) to adaptively combine features from both branches using cross-group spatial and channel attention to improve the model representation in focusing on specific lesion features. These modules are seamlessly integrated into the encoder for effective multiscale information extraction and fusion, and DDSC is further introduced in the decoder to provide rich representations for precise segmentation. Moreover, the local multi-layer perceptron (LMLP) module is designed to decouple and recalibrate features through a local linear transformation that filters out the noise and refines features to provide edge-enriched representation. Experimental evaluations on two datasets demonstrate that the proposed method achieves Intersection over Union (IoU) scores of 81.54% and 84.83%, respectively, which are on par with state-of-the-art methods, exhibiting its effectiveness for CC segmentation. The proposed method holds great potential in assisting physicians with precise lesion localization and region analysis, thereby improving the diagnostic accuracy of CRC.

Clustering-Guided Twin Contrastive Learning for Endomicroscopy Image Classification.

Learning better representations is essential in medical image analysis for computer-aided diagnosis. However, learning discriminative semantic features is a major challenge due to the lack of large-scale well-annotated datasets. Thus, how can we learn a well-structured categorizable embedding space in limited-scale and unlabeled datasets? In this paper, we proposed a novel clustering-guided twin-contrastive learning framework (CTCL) that learns the discriminative representations of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. Compared with traditional contrastive learning, in which only two randomly augmented views of the same instance are considered, the proposed CTCL aligns more semantically related and class-consistent samples by clustering, which improved intra-class tightness and inter-class variability to produce more informative representations. Furthermore, based on the inherent properties of CLE (geometric invariance and intrinsic noise), we proposed to regard CLE images with any angle rotation and CLE images with different noises as the same instance, respectively, for increased variability and diversity of samples. By optimizing CTCL in an end-to-end expectation-maximization framework, comprehensive experimental results demonstrated that CTCL-based visual representations achieved competitive performance on each downstream task as well as more robustness and transferability compared with existing state-of-the-art SSL and supervised methods. Notably, CTCL achieved 75.60%/78.45% and 64.12%/77.37% top-1 accuracy on the linear evaluation protocol and few-shot classification downstream tasks, respectively, which outperformed the previous best results by 1.27%/1.63% and 0.5%/3%, respectively. The proposed method holds great potential to assist pathologists in achieving an automated, fast, and high-precision diagnosis of GI tumors and accurately determining different stages of tumor development based on CLE images.

Context-aware dynamic filtering network for confocal laser endomicroscopy image denoising.

Objective.As an emerging diagnosis technology for gastrointestinal diseases, confocal laser endomicroscopy (CLE) is limited by the physical structure of the fiber bundle, leading to the inevitable production of various forms of noise during the imaging process. However, existing denoising methods based on hand-crafted features inefficiently deal with realistic noise in CLE images. To alleviate this challenge, we proposed context-aware kernel estimation and multi-scale dynamic fusion modules to remove realistic noise in CLE images, including multiplicative and additive white noise.Approach.Specifically, a realistic noise statistics model with random noise specific to CLE data is constructed and further used to develop a self-supervised denoised model without the participation of clean images. Secondly, context-aware kernel estimation, which improves the representation of features by similar learnable region weights, addresses the problem of the non-uniform distribution of noises in CLE images and proposes a lightweight denoised model (CLENet). Thirdly, we have developed a multi-scale dynamic fusion module that decouples and recalibrates features, providing a precise and contextually enriched representation of features. Finally, we integrated two developed modules into a U-shaped backbone to build an efficient denoising network named U-CLENet.Main Results.Both proposed methods achieve comparable or better performance with low computational complexity on two gastrointestinal disease CLE image datasets using the same training benchmark.Significance.The proposed approaches improve the visual quality of unclear CLE images for various stages of tumor development, helping to reduce the rate of misdiagnosis in clinical decision-making and achieve computer graphics-assisted diagnosis.

SEVERE BURN-INDUCED MITOCHONDRIAL RECRUITMENT OF CALPAIN CAUSES ABERRANT MITOCHONDRIAL DYNAMICS AND HEART DYSFUNCTION.

ABSTRACT: Mitochondrial damage is an important cause of heart dysfunction after severe burn injury. However, the pathophysiological process remains unclear. This study aims to examine the mitochondrial dynamics in the heart and the role of µ-calpain, a cysteine protease, in this scenario. Rats were subjected to severe burn injury treatment, and the calpain inhibitor MDL28170 was administered intravenously 1 h before or after burn injury. Rats in the burn group displayed weakened heart performance and decreased mean arterial pressure, which was accompanied by a diminishment of mitochondrial function. The animals also exhibited higher levels of calpain in mitochondria, as reflected by immunofluorescence staining and activity tests. In contrast, treatment with MDL28170 before any severe burn diminished these responses to a severe burn. Burn injury decreased the abundance of mitochondria and resulted in a lower percentage of small mitochondria and a higher percentage of large mitochondria. Furthermore, burn injury caused an increase in the fission protein DRP1 in the mitochondria and a decrease in the inner membrane fusion protein OPA1. Similarly, these alterations were also blocked by MDL28170. Of note, inhibition of calpain yielded the emergence of more elongated mitochondria along with membrane invagination in the middle of the longitude, which is an indicator of the fission process. Finally, MDL28170, administered 1 h after burn injury, preserved mitochondrial function and heart performance, and increased the survival rate. Overall, these results provided the first evidence that mitochondrial recruitment of calpain confers heart dysfunction after severe burn injury, which involves aberrant mitochondrial dynamics.

Boosting few-shot confocal endomicroscopy image recognition with feature-level MixSiam.

As an emerging early diagnostic technology for gastrointestinal diseases, confocal laser endomicroscopy lacks large-scale perfect annotated data, leading to a major challenge in learning discriminative semantic features. So, how should we learn representations without labels or a few labels? In this paper, we proposed a feature-level MixSiam method based on the traditional Siamese network that learns the discriminative features of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. The proposed method is divided into two stages: self-supervised learning (SSL) and few-shot learning (FS). First, in the self-supervised learning stage, the novel feature-level-based feature mixing approach introduced more task-relevant information via regularization, facilitating the traditional Siamese structure can adapt to the large intra-class variance of the pCLE dataset. Then, in the few-shot learning stage, we adopted the pre-trained model obtained through self-supervised learning as the base learner in the few-shot learning pipeline, enabling the feature extractor to learn richer and more transferable visual representations for rapid generalization to other pCLE classification tasks when labeled data are limited. On two disjoint pCLE gastrointestinal image datasets, the proposed method is evaluated. With the linear evaluation protocol, feature-level MixSiam outperforms the baseline by 6% (Top-1) and the supervised model by 2% (Top1), which demonstrates the effectiveness of the proposed feature-level-based feature mixing method. Furthermore, the proposed method outperforms the previous baseline method for the few-shot classification task, which can help improve the classification of pCLE images lacking large-scale annotated data for different stages of tumor development.

Generation of an induced pluripotent stem cell line (ZJULLi003-A) from a hypertrophic cardiomyopathy patient carrying MYH7/c.4384G > A mutation.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited cardiovascular disease characterized by left ventricular hypertrophy and cardiomyocyte disarray. In this study, a skin biopsy was obtained from a HCM patient, who carried a missense mutation (c.4384G > A; p.E1462K) in the myosin heavy chain 7 (MYH7) gene. The skin fibroblasts were subsequently reprogrammed with a non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical morphology and normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Establishment of an induced pluripotent stem cell line (ZJULLi004-A) from a hypertrophic cardiomyopathy patient carrying MYBPC3/c.3764C>A mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease characterized by left ventricular hypertrophy and a high risk of sudden death. In this study, a skin biopsy was obtained from a HCM patient harboring a heterozygous missense mutation (c.3764C>A; p.A1225D) in the myosin binding protein C3 (MYBPC3) gene. The isolated fibroblasts were reprogrammed using non-integrated Sendai viral method to establish the patient-specific induced pluripotent stem cell (iPSC) line. The established iPSC line displayed normal morphology and karyotype, expressed pluripotency markers, and can differentiate into three germ layers in vivo.

Generation of an induced pluripotent stem cell line from a long QT syndrome patient carrying KCNH2/1956C > A mutation.

Long QT syndrome (LQT) is an inherited primary arrhythmic disorder characterized by prolonged QT interval on the surface electrocardiogram and life-threatening arrhythmia. In this study, a skin biopsy was obtained from an LQT type 2 (LQT2) patient, who carried a nonsense mutation (c.1956C > A; p.Y652X) in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. The skin fibroblasts were reprogrammed by non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical embryonic stem cell-like morphology, exhibited normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Impairment of µ-calpain activation by rhTNFR:Fc reduces severe burn-induced membrane disruption in the heart.

Stress cardiomyopathy is a major clinical complication after severe burn. Multiple upstream initiators have been identified; however, the downstream targets are not fully understood. This study assessed the role of the plasma membrane in this process and its relationship with the protease µ-calpain and tumor necrosis factor-alpha (TNF-α). Here, third-degree burn injury of approximately 40% of the total body surface area was established in rats. Plasma levels of LDH and cTnI and cardiac cell apoptosis increased at 0.5 h post burn, reached a peak at 6 h, and gradually declined at 24 h. This effect correlated well with not only the disruption of cytoskeletal proteins, including dystrophin and ankyrin-B, but also with the activation of µ-calpain, as indicated by the cleaved fragments of α-spectrin and membrane recruitment of the catalytic subunit CAPN1. More importantly, these alterations were diminished by blocking calpain activity with MDL28170. Burn injury markedly increased the cellular uptake of Evans blue, indicating membrane integrity disruption, and this effect was also reversed by MDL28170. Compared with those in the control group, cardiac cells in the burn plasma-treated group were more prone to damage, as indicated by a marked decrease in cell viability and increases in LDH release and apoptosis. Of note, these alterations were mitigated by CAPN1 siRNA. Moreover, after neutralizing TNF-α with rhTNFR:Fc, calpain activity was blocked, and heart function was improved. In conclusion, we identified µ-calpain as a trigger for severe burn-induced membrane disruption in the heart and provided evidence for the application of rhTNFR:Fc to inhibit calpain for cardioprotection.

CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts.

Previous studies indicated that Ca2+/calmodulin-dependent kinase II (CaMKII), a kinase involved in the modulation of ryanodine receptor activity, activates Ca2+-regulated protease µ-calpain to promote myocardial ischemia/reperfusion injury. This study was performed to explore the underlying mechanisms in CaMKII-induced calpain activation to better understand heart injury. To examine the Ca2+ paradox and ischemia/reperfusion injury, isolated rat hearts were subjected to a Ca2+-free solution for 3 min, or left coronary artery occlusion for 40 min, prior to restoration of normal perfusion. Blockade of trans-sarcoplasmic reticulum Ca2+ flux using ryanodine and thapsigargin failed to prevent Ca2+ paradox-induced heart injury. In contrast, the Ca2+ paradox increased CaMKII auto-phosphorylation at Thr287, while the CaMKII inhibitor KN-62 and the Na+/Ca2+ exchanger inhibitor KB-R7943 alleviated heart injury and calpain activity. Intriguingly, the binding of µ-calpain large subunit calpain-1 (CAPN1) to phospho-CaMKII was blunted by both inhibitors. Thus, a Ca2+ leak via the ryanodine receptor is not an essential element in CaMKII-elicited calpain activation. In hearts receiving vector injection, ischemia/reperfusion caused elevated calpain activity and α-fodrin degradation, along with membrane integrity damage, similar to the effects noted in control hearts. Importantly, all these alterations were diminished with delivery of adeno-associated virus expressing mutant CaMKIIδC T287A. Ischemia/reperfusion increased CaMKII auto-phosphorylation and binding of CAPN1 to phospho-CaMKII, and facilitated the translocation of phospho-CaMKII and CAPN1 to the plasma membrane, all of which were reversed by injecting CaMKII mutant. Furthermore, the relocation capacity and the interaction of CaMKII with CAPN1 appeared to be dependent upon CaMKII autophosphorylation, as its mutant delivery increased the level of CaMKII, but did not increase membrane content of CaMKII and CAPN1, or their interactions. Together, CaMKII/calpain interaction represents a new avenue for mediating myocardial ischemia/reperfusion injury, and CaMKII likely serves as both a kinase and a carrier, thereby promoting calpain membrane translocation and activation.

Human induced pluripotent stem cell-derived cardiomyocytes reveal abnormal TGFß signaling in type 2 diabetes mellitus.

Diabetes mellitus is a serious metabolic condition associated with a multitude of cardiovascular complications. Moreover, the prevalence of diabetes in heart failure populations is higher than that in control populations. However, the role of cardiomyocyte alterations in type 2 diabetes mellitus (T2DM) has not been well characterized and the underlying mechanisms remain elusive. In this study, two patients who were diagnosed as T2DM were recruited and patient-specific induced pluripotent stem cells (iPSCs) were generated from urine epithelial cells using nonintegrated Sendai virus. The iPSC lines derived from five healthy subjects were used as controls. All iPSCs were differentiated into cardiomyocytes (iPSC-CMs) using the monolayer-based differentiation protocol. T2DM iPSC-CMs exhibited various disease phenotypes, including cellular hypertrophy and lipid accumulation. Moreover, T2DM iPSC-CMs exhibited higher susceptibility to high-glucose/high-lipid challenge than control iPSC-CMs, manifesting an increase in apoptosis. RNA-Sequencing analysis revealed a differential transcriptome profile and abnormal activation of TGFß signaling pathway in T2DM iPSC-CMs. We went on to show that inhibition of TGFß significantly rescued the hypertrophic phenotype in T2DM iPSC-CMs. In conclusion, we demonstrate that the iPSC-CM model is able to recapitulate cellular phenotype of T2DM. Our results indicate that iPSC-CMs can therefore serve as a suitable model for investigating molecular mechanisms underlying diabetic cardiomyopathies and for screening therapeutic drugs.

Single nucleotide polymorphisms of PIN1 promoter region and cancer risk: evidence from a meta-analysis.

BACKGROUND: Peptidylprolyl cis/trans isomerase NIMA-interacting 1 (PIN1) is involved in the process of tumorigenesis. The two single nucleotide polymorphisms (-677T>C, -842G>C) in the PIN1 promoter region have been suspected of being associated with cancer risk for years, but the conclusion is still inconclusive. METHODS: Eligible case-control studies were retrieved by searching databases and references of related reviews and studies. Genotype distribution data, adjusted odds ratios (ORs) and 95% confidence (CIs) intervals were extracted to calculate pooled ORs. RESULTS: A total of 4619 cancer cases and 4661 controls were included in this meta-analysis. Overall, the PIN1 -667T>C polymorphism was not associated with cancer risk, while the -842C allele was significantly associated with reduced cancer risk (CC+GC vs. GG, OR = 0.725, 95% CI: 0.607-0.865; P(heterogeneity) = 0.012 and GC vs. GG: OR = 0.721, 95% CI: 0.591-0.880; P(heterogeneity) = 0.003). Results from genotype distribution data were in agreement with those calculated with adjusted ORs and 95% CIs. No publication bias was detected. CONCLUSIONS: Results of this meta-analysis suggest that the PIN1 -842G>C polymorphism is associated with decreased cancer risk, but that the -667T>C polymorphism is not.

Protective effects of adenosine on the diabetic myocardium against ischemia-reperfusion injury: role of calpain.

Myocardial ischemia/reperfusion injury (I/RI) is the principal cause of mortality and morbidity in diabetic patients undergoing cardiac surgery. However, there is no specific measure available to protect diabetic hearts in this clinical setting. Our clinical studies showed that adenosine pre-treatment or post-treatment and adding adenosine to cardioplegia solution had significant myocardial protective effects in patients undergoing cardiac surgery. However, the specific protective effects and mechanisms of adenosine in diabetic myocardial I/RI are not clear. Calpain is an important proteolytic enzyme in the myocardium. Studies show that the activation of calpain is an injury factor in not only the diabetic myocardium but also myocardial I/RI progression. We therefore hypothesize that adenosine play a protective role in diabetic myocardial I/RI through the inhibition of calpain.

The effects of curcumin post-treatment against myocardial ischemia and reperfusion by activation of the JAK2/STAT3 signaling pathway.

In this study, we evaluated the effect of curcumin (Cur) post-treatment on isolated perfused rat hearts that had been subjected to a protocol of ischemia and reperfusion injury. We also examined whether the Janus kinase 2 and signal transducer and activator 3 of transcription (JAK2/STAT3) signaling pathway plays a role in the cardioprotective effects of Cur post-treatment. Isolated perfused rat hearts were subjected to 60 min of ischemia, followed by 60 min of reperfusion. The hearts were exposed to 1-µM Cur during the first 10 min of reperfusion in the absence or presence of the JAK kinase-specific inhibitor AG490 (AG, 1 µM). The Cur treatment conferred a cardioprotective effect, and the treated hearts demonstrated an improved post-ischemic cardiac functional recovery, a decreased myocardial infarct size and decreased lactate dehydrogenase release in the coronary flow, a reduced number of apoptotic cardiomyocytes, up-regulation of the anti-apoptotic protein Bcl2 and down-regulation of the pro-apoptotic protein Caspase3. AG blocked the Cur-mediated cardioprotection by inhibiting the JAK2/STAT3 signaling pathway, as reflected by the abrogation of the Cur-induced up-regulation of Bcl2 and down-regulation of Caspase3. The results suggest that Cur post-treatment can attenuate IR injury through the activation of the JAK2/STAT3 signaling pathway, which transmits a survival signal to the myocardium.

Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.

Low extracellular K+ increases intracellular Ca2+ oscillation and injury by activating the reverse mode Na+-Ca2+ exchanger and inhibiting the Na+, K+ ATPase in rat cardiomyocytes.

BACKGROUND: The effects of low K(+) on post-ischemic reperfused heart cells are not clearly understood. Calcium overload is one of the major causes for myocardial reperfusion injury, the present study was to investigate the role of intracellular calcium oscillations in the effects of reperfusion with low K(+) on rat myocytes. METHODS: Ischemic myocytes were reperfused with Tyrode solution containing K(+) at 5.4 (control) or 3.0 mM (low K(+)) for 10 min. the changes of intracellular calcium was recorded by spectrofluorometry. The exclusion of trypan blue by myocytes served as indices of viability. Measurements of cell length, reverse-mode Na(+)-Ca(2+) exchanger (NCX) and Na(+), K(+) ATPase activity were performed. RESULTS: Compared to control, myocytes reperfused with low K(+) had greater number of calcium oscillations and reverse-mode NCX activity, which were accompanied with decreased cell length recovery and cell viability. Reperfusion with KB-R7943, an inhibitor of reverse-mode NCX, attenuated the effects of low K(+) on all the parameters. Inhibition of Na(+), K(+) ATPase with Ouabain increased the susceptibility to calcium oscillations in myocytes reperfused with low K(+), which was accompanied with cell length shortening and decreased cell viability. Reperfusion with K(+) at 9.0 mM, which activated Na(+), K(+) ATPase, attenuated calcium oscillations, protected cell length recovery, and increased cell viability. CONCLUSIONS: These results suggest that increased calcium oscillations mediate the exacerbating reperfusion injury with low K(+) on myocytes, and inhibition of Na(+), K(+) ATPase activity and increase of reverse-mode NCX activity contribute to these effects.

Myocardial protective effect of urethane on isolated rat hearts in prolonged hypothermic preservation.

BACKGROUND: One of the most important factors restricting heart transplantation is the limited myocardial ischemia time. This study investigated the effects of urethane on the hypothermic preservation of donor rat hearts. MATERIALS AND METHODS: Hearts isolated from rats were divided into 2 groups (n = 8), a control group with histidine-tryptophan-ketoglutarate (HTK) solution alone and an experimental group with HTK solution plus 30 mM urethane. Hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function; the hearts were then arrested and stored in one of the 2 solutions for 6 hours and 18 hours at 4 degrees C. After preservation, the hearts were reperfused, and cardiac function was evaluated. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) content, cardiomyocyte apoptosis, and myocardial ultrastructure were examined. RESULTS: Compared with the control group, the experimental group showed a significantly higher recovery of cardiac function for both 6 hours and 18 hours of preservation and demonstrated a lower rate of cardiomyocyte apoptosis (8.5% + or - 1.2% versus 12.2% + or - 1.8% for 6 hours; 14.1% + or - 2.1% versus 31.4% + or - 2.7% for 18 hours). ATP content was significantly higher in the experimental group than in the control group after 18 hours of preservation (229.4 + or - 29.7 microg/g versus 153.2 + or - 21.1 microg/g). The experimental group also showed lower levels of LDH release after 18 hours of preservation. Electron microscopy studies demonstrated better cardiomyocyte structure in the experimental group for both 6 hours and 18 hours of preservation. CONCLUSIONS: Use of urethane improved cardiac functional recovery and led to significant protective effects on rat hearts placed in a hypothermic preservation solution for a prolonged period.

Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.

Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.

The myocardial protective effects of a moderate-potassium adenosine-lidocaine cardioplegia in pediatric cardiac surgery.

OBJECTIVES: We sought to evaluate a moderate-potassium cardioplegic solution using adenosine and lidocaine as the arresting and protecting cardioprotective combination in pediatric cardiac surgery. METHODS: One hundred thirty-four patients with congenital heart disease were randomly allocated to one of 3 groups according to the cardioplegia formula used: the high-potassium (HP) group (K(+), 20 mmol/L), 46 patients; the high-potassium adenosine-lidocaine (HPAL) group (K(+), 20 mmol/L; adenosine, 0.7 mmol/L; and lidocaine, 0.7 mmol/L), 44 patients; and the moderate-potassium adenosine-lidocaine (MPAL) group (K(+), 10 mmol/L; adenosine, 0.7 mmol/L; and lidocaine, 0.7 mmol/L), 44 patients. Hemodynamic data during the operation and postoperative data were recorded. Serum cardiac troponin I concentrations were examined at the time points of before cardiopulmonary bypass and 1, 3, 6, 12, and 24 hours after aortic crossclamp removal. RESULTS: At the end of cardiopulmonary bypass and modified ultrafiltration, the systolic and pulse pressures of the MPAL group were significantly increased compared with the respective values of the HP group. At the time points of 1 to 12 hours after reperfusion, the levels of serum cardiac troponin I were significantly decreased in the MPAL group compared with those in the HP and HPAL groups. CONCLUSIONS: The MPAL cardioplegia formula was associated with better myocardial protective effects.

Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.

We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.