Real-time closed-loop electrophysiology: towards new frontiers in in vitro investigations in the neurosciences

Reflected at any level of organization of the central nervous system, most of the processes ranging from ion channels to neuronal networks occur in a closed loop, where the input to the system depends on its output. In contrast, most in vitro preparations and experimental protocols operate autonomously, and do not depend on the output of the studied system. Thanks to the progress in digital signal processing and real-time computing, it is now possible to artificially close the loop and investigate biophysical processes and mechanisms under increased realism. In this contribution, we review some of the most relevant examples of a new trend in in vitro electrophysiology, ranging from the use of dynamic-clamp to multi-electrode distributed feedback stimulation. We are convinced these represents the beginning of new frontiers for the in vitro investigation of the brain, promising to open the still existing borders between theoretical and experimental approaches while taking advantage of cutting edge technologies.

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

A two-microphone noise reduction system for cochlear implant users with nearby microphones. Part I: Signal processing algorithm design and development

Users of cochlear implant systems, that is, of auditory aids which stimulate the auditory nerve at the cochlea electrically, often complain about poor speech understanding in noisy environments. Despite the proven advantages of multimicrophone directional noise reduction systems for conventional hearing aids, only one major manufacturer has so far implemented such a system in a product, presumably because of the added power consumption and size. We present a physically small (intermicrophone distance 7 mm) and computationally inexpensive adaptive noise reduction system suitable for behind-the-ear cochlear implant speech processors. Supporting algorithms, which allow the adjustment of the opening angle and the maximum noise suppression, are proposed and evaluated. A portable real-time device for test in real acoustic environments is presented.

A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

BACKGROUND: The Fip1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. METHODS: In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. RESULTS: Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. CONCLUSIONS: We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias.

Conserved leucine residue in the head region of morbillivirus fusion protein regulates the large conformational change during fusion activity

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.

Agency and ownership are independent components of 'sensing the self' in the auditory-verbal domain

'Sensing the self' relies on the ability to distinguish self-generated from external stimuli. It requires functioning mechanisms to establish feelings of agency and ownership. Agency is defined causally, where the subjects action is followed by an effect. Ownership is defined by the features of the effect, independent from the action. In our study, we manipulated these qualities separately. 13 right-handed healthy individuals performed the experiment while 76-channel EEG was recorded. Stimuli consisted of visually presented words, read aloud by the subject. The experiment consisted of six conditions: (a) subjects saw a word, read it aloud, heard it in their own voice; (b) like a, but the word was heard in an unfamiliar voice; (c) subject heard a word in his/her own voice without speaking; (d) like c, but the word was heard in an unfamiliar voice; (e) like a, but subjects heard the word with a delay; (f) subjects read without hearing. ERPs and difference maps were computed for all conditions. Effects were analysed topographically. The N100 (86-172 ms) displayed significant main effects of agency and ownership. The topographies of the two effects shared little common variance, suggesting independent effects. Later effects (174-400 ms) of agency and ownership were topographically similar, suggesting common mechanisms. Replicating earlier studies, significant N100 suppression was observed, with a topography resembling the agency effect. 'Sensing the self' appears to recruit from at least two very distinct processes: an agency assessment that represents causality and an ownership assessment that compares stimulus features with memory content.

A mutation in the SUV39H2 gene in Labrador Retrievers with hereditary nasal parakeratosis (HNPK) provides insights into the epigenetics of keratinocyte differentiation.

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (p(raw) = 4.4×10⁻¹⁴). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis.

A re-examination of petrogenesis and 40Ar/39Ar systematics in the the Chain of Ponds K-feldspar: "diffusion domain" archetype versus polyphase hygrochronology

K-feldspar (Kfs) from the Chain of Ponds Pluton (CPP) is the archetypal reference material, on which thermochronological modeling of Ar diffusion in discrete “domains” was founded. We re-examine the CPP Kfs using cathodoluminescence and back-scattered electron imaging, transmission electron microscopy, and electron probe microanalysis. 40Ar/39Ar stepwise heating experiments on different sieve fractions, and on handpicked and unpicked aliquots, are compared. Our results reproduce the staircase-shaped age spectrum and the Arrhenius trajectory of the literature sample, confirming that samples collected from the same locality have an identical Ar isotope record. Even the most pristine-looking Kfs from the CPP contains successive generations of secondary, metasomatic/retrograde mineral replacements that post-date magmatic crystallization. These chemically and chronologically distinct phases are responsible for its staircase-shaped age spectra, which are modified by handpicking. While genuine within-grain diffusion gradients are not ruled out by these data, this study demonstrates that the most important control on staircase-shaped age spectra is the simultaneous presence of heterochemical, diachronous post-magmatic mineral growth. At least five distinct mineral species were identified in the Kfs separate, three of which can be traced to external fluids interacting with the CPP in a chemically open system. Sieve fractions have size-shifted Arrhenius trajectories, negating the existence of the smallest “diffusion domains”. Heterochemical phases also play an important role in producing non-linear trajectories. In vacuo degassing rates recovered from Arrhenius plots are neither related to true Fick’s Law diffusion nor to the staircase shape of the age spectra. The CPP Kfs used to define the "diffusion domain" model demonstrates the predominance of metasomatic alteration by hydrothermal fluids and recrystallization in establishing the natural Ar distribution amongst different coexisting phases that gives rise to the staircase-shaped age spectrum. Microbeam imaging of textures is as essential for 40Ar-39Ar hygrochronology as it is for U-Pb geochronology.

Three Shades of Embeddedness, State Capitalism as the Informal Economy, Emic Notions of the Anti-Market, and Counterfeit Garments in the Mauritian Export Processing Zone

Purpose This paper furthers the analysis of patterns regulating capitalist accumulation based on a historical anthropology of economic activities revolving around and within the Mauritian Export Processing Zone (EPZ). Design/methodology/approach This paper uses fieldwork in Mauritius to interrogate and critique two important concepts in contemporary social theory – “embeddedness” and “the informal economy.” These are viewed in the wider frame of social anthropology’s engagement with (neoliberal) capitalism. Findings A process-oriented revision of Polanyi’s work on embeddedness and the “double movement” is proposed to help us situate EPZs within ongoing power struggles found throughout the history of capitalism. This helps us to challenge the notion of economic informality as supplied by Hart and others. Social implications Scholars and policymakers have tended to see economic informality as a force from below, able to disrupt the legal-rational nature of capitalism as practiced from on high. Similarly, there is a view that a precapitalist embeddedness, a “human economy,” has many good things to offer. However, this paper shows that the practices of the state and multinational capitalism, in EPZs and elsewhere, exactly match the practices that are envisioned as the cure to the pitfalls of capitalism. Value of the paper Setting aside the formal-informal distinction in favor of a process-oriented analysis of embeddedness allows us better to understand the shifting struggles among the state, capital, and labor.

Assessing the Environmental Hazard of Using Seawater for Ore Processing at the Lasail Mine Site in the Sultanate of Oman

The Lasail mining area (Sultanate of Oman) was contaminated by acid mine drainage during the exploitation and processing of local and imported copper ore and the subsequent deposition of sulphide-bearing waste material into an unsealed tailings dump. In this arid environment, the use of seawater in the initial stages of ore processing caused saline contamination of the fresh groundwater downstream of the tailings dump. After detection of the contamination in the 1980s, different source-controlled remediation activities were conducted including a seepage water collection system and, in 2005, surface sealing of the tailings dump using an HDPE-liner to prevent further infiltration of meteoric water. We have been assessing the benefits of the remediation actions undertaken so far. We present chemical and isotopic (δ18O, δ 2H, 3H) groundwater data from a long-term survey (8–16 years) of the Wadi Suq aquifer along a 28 km profile from the tailings dump to the Gulf of Oman. Over this period, most metal concentrations in the Wadi Suq groundwater decreased below detection limits. In addition, in the first boreholes downstream of the tailings pond, the salinity contamination has decreased by 30 % since 2005. This decrease appears to be related to the surface coverage of the tailings pond, which reduces flushing of the tailings by the sporadic, but commonly heavy, precipitation events. Despite generally low metal concentrations and the decreased salinity, groundwater quality still does not meet the WHO drinking water guidelines in more than 90 % of the Wadi Suq aquifer area. The observations show that under arid conditions, use of seawater for ore processing or any other industrial activity has the potential to contaminate aquifers for decades.