Fabrication and analogue applications of nanoSQUIDs using Dayem bridge junctions

We report here recent work at the U.K. National Physical Laboratory on developing nanoscale SQUIDs using Dayem bridge Josephson junctions. The advantages are simplicity of fabrication, exceptional low-noise performance, toward the quantum limit, and a range of novel applications. Focused ion beam patterned Nb SQUID, possessing exceptionally low noise (∼200 nΦ0/Hz1/2 above 1 kHz), and operating above 4.2 K can be applied to measurement of nanoscale magnetic objects or coupled to nanoelectromechanical resonators, as well as single particle detection of photons, protons, and ions. The limited operating temperature range may be extended by exposing the Dayem bridges to carefully controlled ion beam implantation, leading to nonreversible changes in junction transition temperature.The work reported here was supported in part by the EMRP projects ‘MetNEMS’ NEW-08 and ‘BioQUART’SIB-06. The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union

Regulation of B cell fate by chronic activity of the IgE B cell receptor.

IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses

Transcranial magnetic stimulation for the treatment of epilepsy

© 2016 The Cochrane Collaboration.Background: Epilepsy is a highly prevalent neurological condition characterized by repeated unprovoked seizures with various etiologies. Although antiepileptic medications produce clinical improvement in most individuals, nearly a third of individuals have drug-resistant epilepsy that carries significant morbidity and mortality. There remains a need for non-invasive and more effective therapies for this population. Transcranial magnetic stimulation (TMS) uses electromagnetic coils to excite or inhibit neurons, with repetitive pulses at low-frequency producing an inhibitory effect that could conceivably reduce cortical excitability associated with epilepsy. Objectives: To assess the evidence for the use of TMS in individuals with drug-resistant epilepsy compared with other available treatments in reducing seizure frequency, improving quality of life, reducing epileptiform discharges, antiepileptic medication use, and side-effects. Search methods: We searched the Cochrane Epilepsy Group Specialized Register, the Cochrane Central Register of Controlled Trials (CENTRAL) via the Cochrane Register of Studies Online (CRSO), MEDLINE (Ovid 1946 to 10 March 2016), ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform (ICTRP) up to March 2016. We also searched SCOPUS (1823 to June 2014) as a substitute for Embase (but it is no longer necessary to search SCOPUS, because randomized controlled trials (RCTs) and quasi-RCTs in EMBASE are now included in CENTRAL). Selection criteria: Eligible studies were RCTs that were double-blinded, single-blinded or unblinded, and placebo, no treatment, or active controlled, which used repetitive transcranial magnetic stimulation (rTMS) without restriction of frequency, duration, intensity, or setup (focal or vertex treatment) on patients with drug-resistant epilepsy. The search revealed 274 records from the databases, that after selection provided seven full-text relevant studies for inclusion. Of the seven studies included, five were completed studies with published data and included randomized, blinded trials. The total number of participants in the seven trials was 230. Data collection and analysis: We extracted information from each trial including methodological data; participant demographics including baseline seizure frequency, type of epileptic drugs taken; intervention details and intervention groups for comparison; potential biases; and outcomes and time points, primarily change in seizure frequency or responder rates, as well as quality of life and epileptiform discharges, adverse effects, and changes in medication use. Main results: Two of the seven studies analyzed showed a statistically significant reduction in seizure rate from baseline (72% and 78.9% reduction of seizures per week from the baseline rate, respectively). The other five studies showed no statistically significant difference in seizure frequency following rTMS treatment compared with controls. We were not able to combine the results of the trials in analysis due to differences in the designs of the studies. Four studies evaluated our secondary endpoint of mean number of epileptic discharges, and three of the four showed a statistically significant reduction in discharges. Quality of life was not assessed in any of the studies. Adverse effects were uncommon among the studies and typically involved headache, dizziness, and tinnitus. No significant changes in medication use were found in the trials. Authors' conclusions: Overall, we judged the quality of evidence for the primary outcomes of this review to be low. There is evidence that rTMS is safe and not associated with any adverse events, but given the variability in technique and outcome reporting that prevented meta-analysis, the evidence for efficacy of rTMS for seizure reduction is still lacking despite reasonable evidence that it is effective at reducing epileptiform discharges

Central and peripheral circadian clocks and their role in Alzheimer's disease

Molecular and cellular oscillations constitute an internal clock that tracks the time of day and permits organisms to optimize their behaviour and metabolism to suit the daily demands they face. The workings of this internal clock become impaired with age. In this review, we discuss whether such age-related impairments in the circadian clock interact with age-related neurodegenerative disorders, such as Alzheimer's disease. Findings from mouse and fly models of Alzheimer's disease have accelerated our understanding of the interaction between neurodegeneration and circadian biology. These models show that neurodegeneration likely impairs circadian rhythms either by damaging the central clock or by blocking its communication with other brain areas and with peripheral tissues. The consequent sleep and metabolic deficits could enhance the susceptibility of the brain to further degenerative processes. Thus, circadian dysfunction might be both a cause and an effect of neurodegeneration. We also discuss the primary role of light in the entrainment of the central clock and describe important, alternative time signals, such as food, that play a role in entraining central and peripheral circadian clocks. Finally, we propose how these recent insights could inform efforts to develop novel therapeutic approaches to re-entrain arrhythmic individuals with neurodegenerative disease

Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.

Circadian rhythms are essential for health and are frequently disturbed in disease. A full understanding of the causal relationships between behavioural and molecular circadian rhythms requires simultaneous longitudinal observations over time in individual organisms. Current experimental paradigms require the measurement of each rhythm separately across distinct populations of experimental organisms, rendering the comparability of the resulting datasets uncertain. We therefore developed FLYGLOW, an assay using clock gene controlled luciferase expression detected by exquisitely sensitive EM-CCD imaging, to enable simultaneous quantification of parameters including locomotor, sleep consolidation and molecular rhythms in single flies over days/weeks. FLYGLOW combines all the strengths of existing techniques, and also allows powerful multiparametric paired statistics. We found the age-related transition from rhythmicity to arrhythmicity for each parameter occurs unpredictably, with some flies showing loss of one or more rhythms during middle-age. Using single-fly correlation analysis of rhythm robustness and period we demonstrated the independence of the peripheral clock from circadian behaviours in wild type flies as well as in an Alzheimer's model. FLYGLOW is a useful tool for investigating the deterioration of behavioural and molecular rhythms in ageing and neurodegeneration. This approach may be applied more broadly within behavioural neurogenetics research

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation

Molecular Structure of the Human CFTR Ion Channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function. © 2017 Elsevier Inc

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Background The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.published_or_final_versio

Comprehensive Characterization of the Transmitted/Founder env Genes From a Single MSM Cohort in China

Background: The men having sex with men (MSM) population has become one of the major risk groups for HIV-1 infection in China. However, the epidemiological patterns, function of the env genes, and autologous and heterologous neutralization activity in the same MSM population have not been systematically characterized. Methods: The env gene sequences were obtained by the single genome amplification. The time to the most recent common ancestor was estimated for each genotype using the Bayesian Markov Chain Monte Carlo approach. Coreceptor usage was determined in NP-2 cells. Neutralization was analyzed using Env pseudoviruses in TZM-bl cells. Results: We have obtained 547 full-length env gene sequences by single genome amplification from 30 acute/early HIV-1–infected individuals in the Beijing MSM cohort. Three genotypes (subtype B, CRF01_AE, and CRF07_BC) were identified and 20% of the individuals were infected with multiple transmitted/founder (T/F) viruses. The tight clusters of the MSM sequences regardless of geographic origins indicated nearly exclusive transmission within the MSM population and limited number of introductions. The time to the most recent common ancestor for each genotype was 10–15 years after each was first introduced in China. Disparate preferences for coreceptor usages among 3 genotypes might lead to the changes in percentage of different genotypes in the MSM population over time. The genotype-matched and genotype-mismatched neutralization activity varied among the 3 genotypes. Conclusions: The identification of unique characteristics for transmission, coreceptor usage, neutralization profile, and epidemic patterns of HIV-1 is critical for the better understanding of transmission mechanisms, development of preventive strategies, and evaluation of vaccine efficacy in the MSM population in China