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At chemical synapses, voltage-gated Ca 2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca 2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABA A or GABA B receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 β-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation., Competing Interests: The authors declare no competing financial interest., (Copyright © 2024 the authors.)

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

Brain development is orchestrated by both innate and experience-dependent mechanisms, but their relative contributions are difficult to disentangle. Here we asked if and how central visual areas are altered in a vertebrate brain depleted of any and all signals from retinal ganglion cells throughout development. We transcriptionally profiled neurons in pretectum, thalamus and other retinorecipient areas of larval zebrafish and searched for changes in lakritz mutants that lack all retinal connections. Although individual genes are dysregulated, the complete set of 77 neuronal types develops in apparently normal proportions, at normal locations, and along normal differentiation trajectories. Strikingly, the cell-cycle exits of proliferating progenitors in these areas are delayed, and a greater fraction of early postmitotic precursors remain uncommitted or are diverted to a pre-glial fate. Optogenetic stimulation targeting groups of neurons normally involved in processing visual information evokes behaviors indistinguishable from wildtype. In conclusion, we show that signals emitted by retinal axons influence the pace of neurogenesis in visual brain areas, but do not detectably affect the specification or wiring of downstream neurons., (© 2023. The Author(s).)

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli., (Copyright © 2019 Woldemariam et al.)

Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Computerized neurocognitive tests (CNTs) are widely used at all competitive levels of sport to assess sport concussion (SC). Whereas there are multiple CNTs available, little is known about how some of the most popular platforms compare. The purpose of this study was to investigate the test-retest reliability of the Automated Neuropsychological Assessment Metrics (ANAM), Concussion Vital Signs (CVS) and the Immediate Postconcussion and Cognitive Testing battery (ImPACT) using clinically relevant time points in healthy college-age participants. Participants were healthy college-age students (N=128) randomly assigned into one of three groups which were administered ANAM, CVS, or ImPACT at Days 1, 45 and 50. Intraclass correlation coefficients (ICCs) and Pearson correlations were used to assess reliability of the various CNT scores and subtest scores between time points. Participants were tested approximately 47.1±2.75days after time point 1 and approximately 7.0±2.45days after time point 2. ICC values ranged from 0.18 (Procedural Reaction Time) to 0.53 (Mathematical Processing and Simple Reaction Time 1) for ANAM, 0.14 (Continuous Performance Test) to 0.85 (Reaction Time) for CVS, and 0.19 (Verbal Memory) to 0.89 (Visual Motor Speed) for ImPACT. Significant improvements (p<0.05) across time were observed for (7/10) CNS Vital Signs composite scores, but no additional significant changes in performance were observed for the remaining CNTs. Overall, weak to strong reliability coefficients for ANAM, CVS, and ImPACT were observed when using clinically relevant time points of repeated administration., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Organic cages were identified as highly potent affinity materials for the tracing of γ-butyrolactone. The selectivity over ethanol and water is based on the interior functional groups which allow preferential hydrogen bonding to the target analyte.

Porosity makes powerful affinity materials for quartz crystal microbalances. The shape-persistent organic cages and pores create superior affinity systems to existing ones for direct tracing of aromatic solvent vapors. A shape and size selectivity for the analytes is observed. These organic cages can be processed to thin films with highly reproducible sensing properties., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The one-pot synthesis of an exo-functionalized [4 + 6] imine cage compound is introduced. The material derived from this compound is highly porous in its amorphous state with a specific surface area of 1037 m(2) g(-1) as determined by nitrogen sorption at 77 K.

A new tripodal ligand has been designed by connecting pyridine-based coordination units to a rigid triptycene moiety. Its reaction with europium(III) provides three-dimensional tetranuclear edifices, whose structural and photophysical characteristics as well as host-guest interactions are discussed in this contribution.

The synthesis of various periphery-substituted shape-persistent cage compounds by twelve-fold condensation reactions of four triptycene triamines and six salicyldialdehydes is described, where the substituents systematically vary in bulkiness. The resulting cage compounds were studied as permanent porous material by nitrogen sorption measurements. When the material is amorphous, the steric demand of the cages exterior does not strongly influence the gas uptake, resulting in BET surface areas of approximately 700 m(2)  g(-1) for all cage compounds 3 c-e, independently of the substituents bulkiness. In the crystalline state, materials of the same compounds show a strong interconnection between steric demand of the peripheral substituent and the resulting BET surface area. With increasing bulkiness, the overall BET surface area decreases, for example 1291 m(2)  g(-1) (for cage compound 3 c with methyl substituents), 309 m(2)  g(-1) (for cage compound 3 d with 2-(2-ethyl-pentyl) substituents) and 22 m(2)  g(-1) (for cage compound 3 e with trityl substituents). Furthermore, we found that two different crystalline polymorphs of the cage compound 3 a (with tert-butyl substituents) differ also in nitrogen sorption, resulting in a BET surface area of 1377 m(2) g(-1), when synthesized from THF and 2071 m(2) g(-1), when recrystallized from DMSO., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: To understand the features of child abuse/neglect (CA/N) allegations in cases with emotional maltreatment (EMT) allegations, as well as the features of the EMT allegations themselves, and to describe any associations of EMT with distinct impairments of children's behavior, emotion and functioning., Method: The sample consisted of 806 high-risk children, 545 with one or more maltreatment reports to CPS. The Maltreatment Classification System was used to record the number and severity levels of maltreatment allegations, which compared cases with and without EMT. Multiple regression analyses were conducted using 10 outcome scales from the Child Behavior Checklist, Vineland Screener, and Trauma Symptom Checklist. Successive blocks of predictor variables included demographics, maltreatment classification variables, maternal and family characteristics, and study site., Results: When there were allegations of EMT as well as CA/N in a CPS case-record (by age 8), the CA/N allegations tended to be either more frequent or less severe than those kinds of allegations in cases without EMT. When neglect was alleged to occur with EMT, neglect allegations outnumbered allegations of EMT. However, when sexual abuse allegations were accompanied by EMT allegations, there were more EMT allegations than sexual abuse allegations in the cases. Higher severity ratings for EMT allegations than for physical abuse occurred when cases included any abuse. Distinctive effects of EMT subtypes were found between problems of safety/restriction and self-reported anger symptoms, and between problems of self-esteem/autonomy and posttraumatic stress., Conclusion: Differences exist between the CA/N allegations in cases with and without EMT. Having few cases containing only EMT allegations made it difficult to assess distinctive harm associated with EMT. Certain types of EMT allegations were associated with increases in children's anger and posttraumatic stress.

Research suggests that fathers' involvement in their children's lives is associated with enhanced child functioning. The current study examined (a) whether presence of a father was associated with better child functioning, (b) whether children's perceptions of fathers' support was associated with better functioning, and (c) whether the above association was moderated by the father's relationship to the child, the child's race, and the child's gender. Participants included 855 six-year-old children and their caregivers. Father presence was associated with better cognitive development and greater perceived competence by the children. For children with a father figure, those who described greater father support had a stronger sense of social competence and fewer depressive symptoms. The associations did not differ by child's gender, race, or relationship to the father figure. These findings support the value of fathers' presence and support to their children's functioning. Priorities for future research include clarifying what motivates fathers to be positively involved in their children's lives and finding strategies to achieve this.

The responses of isolated guinea pig tracheal spirals and parenchymal strips to histamine and carbachol were compared. The parenchymal strip, a 1.5 x 1.5 x 20-mm strip cut from the periphery of the lung, constricted at a lower dose and had a larger maximal response to histamine than to carbachol. In contrast, the response of the tracheal spiral to equimolar doses of histamine or carbachol was the same. The responsiveness of both muscle strips to histamine was decreased by treatment with the H1 receptor antagonist mepyramine (0.1 micrometer), and the response to carbachol was blocked by treatment with atropine (0.1 micrometer). Indomethacin (3 micrometer), cimetidine (1 micrometer), propranolol (10 micrometer), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (4 micrometer) did not alter the differential response of the two strips to histamine and carbachol. The differential response of parenchymal strips with many, few, or no conducting airways and blood vessels was identical, suggesting that the contractile element is alveolar duct smooth muscle or alveolar contractile elements. This differential pharmacologic response in vitro is consistent with the in vivo observation that histamine causes more peripheral airway constriction than does acetylcholine.

Effects of H2-receptor antagonism on the response to histamine was studied in the guinea pig in vivo and in vitro. The H2-receptor antagonist, metiamide (100 micro M), resulted in an enhanced histamine response in eight of eight parenchymal strips and in four of eight tracheal spirals. On the average the parenchymal strips were 20-fold more sensitive to histamine (P less than 0.001), whereas the tracheal spirals demonstrated an insignificant, 20%, increase in sensitivity after metiamide treatment. These results are consistent with the hypothesis that there are inhibitory H2-receptors in guinea pig airways and they predominate in the periphery. When we determined the effects of H2-antagonism on the histamine response in vivo we found that the histamine response was enhanced only in animals that had been treated with the beta-receptor antagonist propranolol. In these animals there was a mean 2.2-fold increase in histamine sensitivity. These results suggest that although there are inhibitory H2-receptors in the guinea pig lung, their role in modulating the in vivo response is much less than beta-adrenergic mechanisms.

The bronchodilator activity of the H2-recptor agonist, dimaprit, was assessed in vitro and in vivo. In vitro dimaprit relaxed guinea pig tracheal spirals and parenchymal strips that were constricted by the H1 receptor agonist, 2-PEA, or by carbachol. The H2-receptor antagonist, metiamide, inhibited this effect of dimaprit in vitro on both tissues constricted by 2-PEA but not on the carbachol constricted tracheal spiral. Intravenous infusion of dimaprit in the intact guinea pig resulted in reversal of bronchoconstriction induced by subcutaneous injection of 2-PEA. In vivo pretreatment with the H2-recptor antagonist, metiamide, resulted in a diminished sensitivity to the bronchodilating effects of intravenous dimaprit.

We compared the pharmacologic effects of a number of oxidative products of arachidonic acid metabolism, including prostaglandin (PG) F2 alpha, PGD2, PGI2, PGE2 6-keto-PGF1 alpha, and the 9 alpha, 11 alpha and 11 alpha, 9 alpha cyclic ether endoperoxide analogues, with that of histamine on guinea pig tracheal spirals and lung parenchymal strips. These agents demonstrated different profiles of activity on the tracheal (central) and parenchymal (peripheral) airway tissues. None of the metabolites studied exceeded the ability of histamine to constrict the tracheal spirals, whereas the cyclic ether endoperoxide analogues and PGD2 were as good as, or better, constrictors of the parenchymal strips than histamine. This suggests that these mediators, which are formed during hypersensitivity reactions, may play an important role in the peripheral airway constriction that is a part of this syndrome.