Structural and functional diversity of connexin genes in the mouse and human genome

Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.

Connexin genes in the mouse and human genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

SMARTDIAB: a communication and information technology approach for the intelligent monitoring, management and follow-up of type 1 diabetes patients

SMARTDIAB is a platform designed to support the monitoring, management, and treatment of patients with type 1 diabetes mellitus (T1DM), by combining state-of-the-art approaches in the fields of database (DB) technologies, communications, simulation algorithms, and data mining. SMARTDIAB consists mainly of two units: 1) the patient unit (PU); and 2) the patient management unit (PMU), which communicate with each other for data exchange. The PMU can be accessed by the PU through the internet using devices, such as PCs/laptops with direct internet access or mobile phones via a Wi-Fi/General Packet Radio Service access network. The PU consists of an insulin pump for subcutaneous insulin infusion to the patient and a continuous glucose measurement system. The aforementioned devices running a user-friendly application gather patient's related information and transmit it to the PMU. The PMU consists of a diabetes data management system (DDMS), a decision support system (DSS) that provides risk assessment for long-term diabetes complications, and an insulin infusion advisory system (IIAS), which reside on a Web server. The DDMS can be accessed from both medical personnel and patients, with appropriate security access rights and front-end interfaces. The DDMS, apart from being used for data storage/retrieval, provides also advanced tools for the intelligent processing of the patient's data, supporting the physician in decision making, regarding the patient's treatment. The IIAS is used to close the loop between the insulin pump and the continuous glucose monitoring system, by providing the pump with the appropriate insulin infusion rate in order to keep the patient's glucose levels within predefined limits. The pilot version of the SMARTDIAB has already been implemented, while the platform's evaluation in clinical environment is being in progress.

Design of a semi-implantable hearing device for direct acoustic cochlear stimulation

A new hearing therapy based on direct acoustic cochlear stimulation was developed for the treatment of severe to profound mixed hearing loss. The device efficacy was validated in an initial clinical trial with four patients. This semi-implantable investigational device consists of an externally worn audio processor, a percutaneous connector, and an implantable microactuator. The actuator is placed in the mastoid bone, right behind the external auditory canal. It generates vibrations that are directly coupled to the inner ear fluids and that, therefore, bypass the external and the middle ear. The system is able to provide an equivalent sound pressure level of 125 dB over the frequency range between 125 and 8000 Hz. The hermetically sealed actuator is designed to provide maximal output power by keeping its dimensions small enough to enable implantation. A network model is used to simulate the dynamic characteristics of the actuator to adjust its transfer function to the characteristics of the middle ear. The geometry of the different actuator components is optimized using finite-element modeling.

Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study

Basal dendrites receive the majority of synapses that contact neocortical pyramidal neurons, yet our knowledge of synaptic processing in these dendrites has been hampered by their inaccessibility for electrical recordings. A new approach to patch-clamp recordings enabled us to characterize the integrative properties of these cells. Despite the short physical length of rat basal dendrites, synaptic inputs were electrotonically remote from the soma (>30-fold excitatory postsynaptic potential (EPSP) attenuation) and back-propagating action potentials were significantly attenuated. Unitary EPSPs were location dependent, reaching large amplitudes distally (>8 mV), yet their somatic contribution was relatively location independent. Basal dendrites support sodium and NMDA spikes, but not calcium spikes, for 75% of their length. This suggests that basal dendrites, despite their proximity to the site of action potential initiation, do not form a single basal-somatic region but rather should be considered as a separate integrative compartment favoring two integration modes: subthreshold, location-independent summation versus local amplification of incoming spatiotemporally clustered information.

Effects of butorphanol on the withdrawal reflex using threshold, suprathreshold and repeated subthreshold electrical stimuli in conscious horses

OBJECTIVE: To assess the effects of a single intravenous dose of butorphanol (0.1 mg kg(-1)) on the nociceptive withdrawal reflex (NWR) using threshold, suprathreshold and repeated subthreshold electrical stimuli in conscious horses. STUDY DESIGN: 'Unblinded', prospective experimental study. ANIMALS: Ten adult horses, five geldings and five mares, mean body mass 517 kg (range 487-569 kg). METHODS: The NWR was elicited using single transcutaneous electrical stimulation of the palmar digital nerve. Repeated stimulations were applied to evoke temporal summation. Surface electromyography was performed to record and quantify the responses of the common digital extensor muscle to stimulation and behavioural reactions were scored. Before butorphanol administration and at fixed time points up to 2 hours after injection, baseline threshold intensities for NWR and temporal summation were defined and single suprathreshold stimulations applied. Friedman repeated-measures analysis of variance on ranks and Wilcoxon signed-rank test were used with the Student-Newman-Keul's method applied post-hoc. The level of significance (alpha) was set at 0.05. RESULTS: Butorphanol did not modify either the thresholds for NWR and temporal summation or the reaction scores, but the difference between suprathreshold and threshold reflex amplitudes was reduced when single stimulation was applied. Upon repeated stimulation after butorphanol administration, a significant decrease in the relative amplitude was calculated for both the 30-80 and the 80-200 millisecond intervals after each stimulus, and for the whole post-stimulation interval in the right thoracic limb. In the left thoracic limb a decrease in the relative amplitude was found only in the 30-80 millisecond epoch. CONCLUSION: Butorphanol at 0.1 mg kg(-1) has no direct action on spinal Adelta nociceptive activity but may have some supraspinal effects that reduce the gain of the nociceptive system. CLINICAL RELEVANCE: Butorphanol has minimal effect on sharp immediate Adelta-mediated pain but may alter spinal processing and decrease the delayed sensations of pain.

Integration of electrical neuroimaging with other functional imaging methods

Integrating evidence from different imaging modalities is important to overcome specific limitations of any given imaging method, such as insensitivity of the EEG to unsynchronized neural events, or the lack of fMRI sensitivity to events of low metabolic demand. Processes that are visible in one modality may be related in a nontrivial way to other processes visible in another modality and insight may only be obtained by integrating both methods through a common analysis. For example, brain activity at rest seems to be at least partly determined by an interaction of cortical rhythms (visible to EEG but not to fMRI) with sub-cortical activity (visible to fMRI, but usually not to EEG without averaging). A combination of EEG and fMRI data during rest may thus be more informative than the sum of two separate analyses in both modalities. Integration is also an important source of converging evidence about specific aspects and general principles of neural functions and their dysfunctions in certain pathologies. This is because not only electrical, but also energetic, biochemical, hemodynamic and metabolic processes characterize neural states and functions, and because brain structure provides crucial constraints upon neural functions. Focusing on multimodal integration of functional data should not distract from the privileged status of the electric field as the primary direct, noninvasive real-time measure of neural transmission. The preceding chapters illustrate how electrical neuroimaging has turned scalp EEG into an imaging modality which directly captures the full temporal dynamics of neural activity in the brain.

Informative State-Based Video Communication

We study state-based video communication where a client simultaneously informs the server about the presence status of various packets in its buffer. In sender-driven transmission, the client periodically sends to the server a single acknowledgement packet that provides information about all packets that have arrived at the client by the time the acknowledgment is sent. In receiver-driven streaming, the client periodically sends to the server a single request packet that comprises a transmission schedule for sending missing data to the client over a horizon of time. We develop a comprehensive optimization framework that enables computing packet transmission decisions that maximize the end-to-end video quality for the given bandwidth resources, in both prospective scenarios. The core step of the optimization comprises computing the probability that a single packet will be communicated in error as a function of the expected transmission redundancy (or cost) used to communicate the packet. Through comprehensive simulation experiments, we carefully examine the performance advances that our framework enables relative to state-of-the-art scheduling systems that employ regular acknowledgement or request packets. Consistent gains in video quality of up to 2B are demonstrated across a variety of content types. We show that there is a direct analogy between the error-cost efficiency of streaming a single packet and the overall rate-distortion performance of streaming the whole content. In the case of sender-driven transmission, we develop an effective modeling approach that accurately characterizes the end-to-end performance as a function of the packet loss rate on the backward channel and the source encoding characteristics.

Assessing distractors and teamwork during surgery: developing an event-based method for direct observation

OBJECTIVE: To develop a behavioural observation method to simultaneously assess distractors and communication/teamwork during surgical procedures through direct, on-site observations; to establish the reliability of the method for long (>3 h) procedures. METHODS: Observational categories for an event-based coding system were developed based on expert interviews, observations and a literature review. Using Cohen's κ and the intraclass correlation coefficient, interobserver agreement was assessed for 29 procedures. Agreement was calculated for the entire surgery, and for the 1st hour. In addition, interobserver agreement was assessed between two tired observers and between a tired and a non-tired observer after 3 h of surgery. RESULTS: The observational system has five codes for distractors (door openings, noise distractors, technical distractors, side conversations and interruptions), eight codes for communication/teamwork (case-relevant communication, teaching, leadership, problem solving, case-irrelevant communication, laughter, tension and communication with external visitors) and five contextual codes (incision, last stitch, personnel changes in the sterile team, location changes around the table and incidents). Based on 5-min intervals, Cohen's κ was good to excellent for distractors (0.74-0.98) and for communication/teamwork (0.70-1). Based on frequency counts, intraclass correlation coefficient was excellent for distractors (0.86-0.99) and good to excellent for communication/teamwork (0.45-0.99). After 3 h of surgery, Cohen's κ was 0.78-0.93 for distractors, and 0.79-1 for communication/teamwork. DISCUSSION: The observational method developed allows a single observer to simultaneously assess distractors and communication/teamwork. Even for long procedures, high interobserver agreement can be achieved. Data collected with this method allow for investigating separate or combined effects of distractions and communication/teamwork on surgical performance and patient outcomes.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.