Contextual fear memory impairment in Angelman syndrome model mice is associated with altered transcriptional responses.

Angelman syndrome (AS) is a rare neurogenetic disorder caused by UBE3A deficiency and characterized by severe developmental delay, cognitive impairment, and motor dysfunction. In the present study, we performed RNA-seq on hippocampal samples from both wildtype (WT) and AS male mice, with or without contextual fear memory recall. There were 281 recall-associated differentially expressed genes (DEGs) in WT mice and 268 DEGs in AS mice, with 129 shared by the two genotypes. Gene ontology analysis showed that extracellular matrix and stimulation-induced response genes were prominently enriched in recall-associated DEGs in WT mice, while nuclear acid metabolism and tissue development genes were highly enriched in those from AS mice. Further analyses showed that the 129 shared DEGs belonged to nuclear acid metabolism and tissue development genes. Unique recall DEGs in WT mice were enriched in biological processes critical for synaptic plasticity and learning and memory, including the extracellular matrix network clustered around fibronectin 1 and collagens. In contrast, AS-specific DEGs were not enriched in any known pathways. These results suggest that memory recall in AS mice, while altering the transcriptome, fails to recruit memory-associated transcriptional programs, which could be responsible for the memory impairment in AS mice.

A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health.

KIF1A is a critical cargo transport motor within neurons. More than 100 known mutations result in KIF1A-associated neurological disorder (KAND), a degenerative condition for which there is no cure. A missense mutation, P305L, was identified in children diagnosed with KAND, but the molecular basis for the disease is unknown. We find that this conserved residue is part of an unusual 310 helix immediately adjacent to the family-specific K-loop, which facilitates a high microtubule-association rate. We find that the mutation negatively affects several biophysical parameters of the motor. However, the microtubule-association rate of the motor is most markedly affected, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that the 310 helix facilitates a specific K-loop conformation that is critical for its function. We find that the function of this proline is conserved in kinesin-1, revealing a fundamental principle of the kinesin motor mechanism.

Net emission reductions from electric cars and heat pumps in 59 world regions over time.

Electrification of passenger road transport and household heating features prominently in current and planned policy frameworks to achieve greenhouse gas emissions reduction targets. However, since electricity generation involves using fossil fuels, it is not established where and when the replacement of fossil fuel-based technologies by electric cars and heat pumps can effectively reduce overall emissions. Could electrification policy backfire by promoting their diffusion before electricity is decarbonised? Here, we analyse current and future emissions trade-offs in 59 world regions with heterogeneous households, by combining forward-looking integrated assessment model simulations with bottom-up life-cycle assessment. We show that already under current carbon intensities of electricity generation, electric cars and heat pumps are less emission-intensive than fossil fuel-based alternatives in 53 world regions, representing 95% of global transport and heating demand. Even if future end-use electrification is not matched by rapid power sector decarbonisation, it likely avoids emissions in almost all world regions.

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.

Synthesis and Biological Evaluation of Fentanyl Analogues Modified at Phenyl Groups with Alkyls.

A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce ß-arrestin2 recruitment. Compound 12 is a potent µ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak ß-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak ß-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals.

The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight the difficulty in adapting in vitro methods to high-throughput format for screening the skin sensitization potential of large numbers of chemicals whilst ensuring that the data produced are both accurate and reproducible.

In vitro methods for hazard assessment of industrial chemicals - opportunities and challenges.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the 'method of choice' for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals.

Novel polymeric bioerodable microparticles for prolonged-release intrathecal delivery of analgesic agents for relief of intractable cancer-related pain.

Intractable cancer-related pain complicated by a neuropathic component due to nerve impingement is poorly alleviated even by escalating doses of a strong opioid analgesic. To address this unmet medical need, we developed sustained-release, bioerodable, hydromorphone (potent strong opioid)- and ketamine (analgesic adjuvant)-loaded microparticles for intrathecal (i.t.) coadministration. Drug-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared using a water-in-oil-in-water method with evaporation. Encapsulation efficiency of hydromorphone and ketamine in PLGA (50:50) microparticles was 26% and 56%, respectively. Microparticles had the desired size range (20-60 µm) and in vitro release was prolonged at ≥28 days. Microparticles were stable for ≥6 months when stored refrigerated protected from light in a desiccator. Desirably, i.t. injected fluorescent dye-labeled PLGA microparticles in rats remained in the lumbar region for ≥7 days. In a rat model of neuropathic pain, i.t. coinjection of hydromorphone- and ketamine-loaded microparticles (each 1 mg) produced analgesia for 8 h only. Possible explanations include inadequate release of ketamine and/or hydromorphone into the spinal fluid, and/or insufficient ketamine loading to prevent development of analgesic tolerance to the released hydromorphone. As sub-analgesic doses of i.t. ketamine at 24-48 h intervals restored analgesia on each occasion, insufficient ketamine loading appears problematic. We will investigate these issues in future work.

Endomorphin analogues with mixed µ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking ß-arrestin2 recruitment activity.

Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high µ-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Kiδ-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of ß-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.