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Genetically determined disorders are highly heterogenous in clinical presentation and underlying molecular mechanism. The evidence underpinning these conditions in the peer-reviewed literature requires robust critical evaluation for diagnostic use. Here, we present a structured curation process for Gene2Phenotype (G2P). This draws on multiple lines of clinical, bioinformatic and functional evidence. The process utilises and extends existing terminologies, allows for precise definition of the molecular basis of disease, and confidence levels to be attributed to a given gene-disease assertion. In-depth disease curation using this process will prove useful in applications including in diagnostics, research and development of targeted therapeutics. G2P: www.ebi.ac.uk/gene2phenotype ., (© 2024. The Author(s).)

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

Background: Pediatric disorders include a range of highly penetrant, genetically heterogeneous conditions amenable to genomewide diagnostic approaches. Finding a molecular diagnosis is challenging but can have profound lifelong benefits., Methods: We conducted a large-scale sequencing study involving more than 13,500 families with probands with severe, probably monogenic, difficult-to-diagnose developmental disorders from 24 regional genetics services in the United Kingdom and Ireland. Standardized phenotypic data were collected, and exome sequencing and microarray analyses were performed to investigate novel genetic causes. We developed an iterative variant analysis pipeline and reported candidate variants to clinical teams for validation and diagnostic interpretation to inform communication with families. Multiple regression analyses were performed to evaluate factors affecting the probability of diagnosis., Results: A total of 13,449 probands were included in the analyses. On average, we reported 1.0 candidate variant per parent-offspring trio and 2.5 variants per singleton proband. Using clinical and computational approaches to variant classification, we made a diagnosis in approximately 41% of probands (5502 of 13,449). Of 3599 probands in trios who received a diagnosis by clinical assertion, approximately 76% had a pathogenic de novo variant. Another 22% of probands (2997 of 13,449) had variants of uncertain significance in genes that were strongly linked to monogenic developmental disorders. Recruitment in a parent-offspring trio had the largest effect on the probability of diagnosis (odds ratio, 4.70; 95% confidence interval [CI], 4.16 to 5.31). Probands were less likely to receive a diagnosis if they were born extremely prematurely (i.e., 22 to 27 weeks' gestation; odds ratio, 0.39; 95% CI, 0.22 to 0.68), had in utero exposure to antiepileptic medications (odds ratio, 0.44; 95% CI, 0.29 to 0.67), had mothers with diabetes (odds ratio, 0.52; 95% CI, 0.41 to 0.67), or were of African ancestry (odds ratio, 0.51; 95% CI, 0.31 to 0.78)., Conclusions: Among probands with severe, probably monogenic, difficult-to-diagnose developmental disorders, multimodal analysis of genomewide data had good diagnostic power, even after previous attempts at diagnosis. (Funded by the Health Innovation Challenge Fund and Wellcome Sanger Institute.)., (Copyright © 2023 Massachusetts Medical Society.)

Background: The process of identifying and discontinuing medicines in instances in which harms outweigh benefits (deprescribing) can mitigate the negative consequences of problematic polypharmacy. This process should be conducted with a focus on the patient and involve collaborative decision-making. Evidence is needed regarding patients' views on how deprescribing should be safely and routinely implemented in English primary care to improve its application. This study aimed to identify optimal methods of introducing and actioning deprescribing from the patient's perspective. Methods: Participants in England aged 65 and above who were taking five or more medicines and residing in their own homes were recruited through social media and service user groups. An interview guide was created from deprescribing literature and input from patients and the public, guided by the Normalisation Process Theory (NPT). The interviews were held online using Microsoft Teams® or via phone, recorded, and then transcribed. The data was analysed using the Framework analysis. Results: Twenty patients (mean age of 74.5, SD = 6.93), with 75% being female, were enrolled in the study. Three main themes emerged: (1) 'Why deprescribe now?' emphasised the significance of explaining the reasons behind deprescribing; (2) 'Monitoring and follow-up' underscored the necessity of safety measures during deprescribing and patients' willingness to self-monitor post-intervention; (3) 'Roles and relationships' explored patient perceptions of various healthcare professionals involved in deprescribing and the essential interpersonal skills for fostering therapeutic relationships. Conclusion: Optimal methods of introducing deprescribing included communicating a convincing rationale for stopping medicines and preparing patients for deprescribing conversations. Patients required support from a range of healthcare professionals with whom they had an existing therapeutic relationship. Whilst patients were motivated to self-monitor unwanted/unexpected effects post-deprescribing, timely support was required. The nature of such bolstered collective action and cognitive participation within NPT enhances the normalisation potential of deprescribing. These findings highlight the significance of considering the content and process of deprescribing consultations to enhance normalisation and tackle problematic polypharmacy. This provides a deeper understanding of patients' needs for implementing safe and routine deprescribing in primary care, which should be considered when designing medication review and deprescribing services. [ABSTRACT FROM AUTHOR]

Background: In recent years, community pharmacists have seen their profession transition from a dispensing-focused role to a rapidly evolving clinically oriented practice. In Québec, Bill 31, adopted in 2020, increased the clinical opportunities for pharmacists with independent prescribing privileges in various defined clinical situations. As this expanded role can lead to different barriers, it is crucial to explore pharmacists' comfort levels with implementing such changes in their practice. Methods: A web-based survey was conducted from March 25 to May 28, 2021, among community pharmacists in Québec. We collected data with a questionnaire developed for this study. Questions were grouped into 4 domains: (1) characteristics of the respondents; (2) workload and work setting; (3) comfort level with, and barriers to, adjusting medications and following up pharmacologic treatments (86 clinical situations evaluated); and (4) general barriers and facilitators to implementation. Results: A total of 146 community pharmacists completed the questionnaire. Most were women (71.9%), younger than 50 years of age (86.2%), had a bachelor's degree (64.4%) as their highest academic level and had more than 10 years of experience as pharmacists (56.8%). Most of them worked exclusively in a community pharmacy (86.3%). Among the 86 clinical situations evaluated, there were 16 in which at least 80% of respondents felt comfortable. The main barriers identified were a lack of knowledge, experience and dedicated time and difficulties integrating these activities into the workflow; facilitators were having an adequate environment and resources. Conclusion: This study shows community pharmacists can confidently adjust pharmacotherapy for several conditions. However, they must have adequate time and resources. Also, the more complex the clinical situations were, the less comfortable community pharmacists felt adjusting pharmacotherapy. This study identified several areas where continuing education, training and mentoring could be offered and where the work environment and organization could be improved. [ABSTRACT FROM AUTHOR]

Background: Promotional videos are an effective means of marketing products or services in this era of social media. However, little is known if such videos can be a useful tool to promote pharmacy services. Objective: To assess the impact of promotional videos on public awareness of the health services provided by New Zealand pharmacists. Methods: This experimental study used an online questionnaire and promotional videos developed by the Pharmaceutical Society of New Zealand as an intervention to assess the public's perspectives about pharmacy services before and after the intervention. The survey was sent out to 1 127 randomly selected members of the public aged 18 and above from all over New Zealand. Data were analysed using descriptive statistics and thematic analysis. Results: A total of 313 participants completed the survey. The majority (95.8%) had not heard of the promotional videos before. However, 84.3% found the videos informative and nearly three-quarters (74.1%) reported a change in their perception of pharmacy services after watching the video. Similarly, 63.2% reported that they or a family member would be more likely to visit a pharmacy in the future after watching the promotional video. Less than half (40%) of the participants reported that the videos were too long and only 11.5% found them boring. Conclusion: The PSNZ videos were found to be an effective tool for educating New Zealanders on different pharmacy services and the pharmacy profession, however, there is a need to choose the advertising platform carefully so as to enhance the coverage. [ABSTRACT FROM AUTHOR]

Progressive neurodegenerative disorders such as Parkinson Disease (PD) lack curative or long-term treatments. At the same time, the increase of the worldwide elderly population and, consequently, the extension in the prevalence of age-related diseases have promoted research interest in neurodegenerative disorders. Caenorhabditis elegans is a free-living nematode widely used as an animal model in studies of human diseases. Here we evaluated cannabidiol (CBD) as a possible neuroprotective compound in PD using the C. elegans models exposed to reserpine. Our results demonstrated that CBD reversed the reserpine-induced locomotor alterations and this response was independent of the NPR-19 receptors, an orthologous receptor for central cannabinoid receptor type 1. Morphological alterations of cephalic sensilla (CEP) dopaminergic neurons indicated that CBD also protects neurons from reserpine-induced degeneration. That is, CBD attenuates the reserpine-induced increase of worms with shrunken soma and dendrites loss, increasing the number of worms with intact CEP neurons. Finally, we found that CBD also reduced ROS formation and α-syn protein accumulation in mutant worms. Our findings collectively provide new evidence that CBD acts as neuroprotector in dopaminergic neurons, reducing neurotoxicity and α-syn accumulation highlighting its potential in the treatment of PD. [ABSTRACT FROM AUTHOR]

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming a1 subunit and two auxiliary β and α2d subunits. Although auxiliary subunits are critical for channel function, whether they are required for a1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/a1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2d, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to validate the content of the specialized competency frameworks for pharmacists working in hospital settings (hospital and clinical pharmacists) and pilot the frameworks for practice assessment. Methods: This online cross-sectional study was carried out between March and October 2022 among a sample of 96 Lebanese pharmacists working in hospital settings. The frameworks were distributed to full-time hospital and clinical pharmacists, who filled them out according to their role in the hospital. Results: Overall, the competencies were distributed over five domains for hospital pharmacists (fundamental skills, safe and rational use of medicines, patient-centered care, professional skills, and preparedness for emergencies), while for clinical pharmacists, competencies were distributed over seven domains (quality improvement, clinical knowledge and skills, soft skills, ability to conduct clinical research, ability to provide effective education, use information technology to make decisions and reduce errors, and emergency preparedness). Moreover, Cronbach alpha values were appropriate, indicating sufficient to high internal consistency. Pharmacists were highly confident in most competencies, with some exceptions related to research in emergency settings (data evaluation, research, and reporting). Conclusions: This study could validate competency frameworks for clinical and hospital pharmacists, with the competencies and their respective behaviors showing an adequate construct analysis. It also identified the domains that require further development, i.e., soft skills and research in emergency settings. Both these domains are timely and needed to overcome the current practice challenges in Lebanon. [ABSTRACT FROM AUTHOR]

Context: Xanthates are organic compounds that present great interest for coordination chemistry, because they can bond in different ways to the metal ion. Thus, these compounds have several applications, being best known for their environmental application. In fact, xanthates are recognized for their application as heavy metal collector agents in aqueous environments. In view of this application, this study is aimed at showing the thermochemical and electronic parameters obtained for the reactions of substitution water molecules in the aqua zinc complexes, by xanthate ligands (n-propyl, n-butyl, and n-pentyl xanthates). In addition to their environmental application, xanthates have shown biological properties, such as anti-bacterial and anti-cancer. In recent years, xanthates have also been used in the technological area, where it participates as a precursor of sulfides for the manufacture of thin films. Our results showed complexes with distorted octahedral geometries and with negative values of enthalpy and Gibbs free energy, indicating exothermic and spontaneous processes. For all the complexes, it was observed that Zn2+ complexes have both an ionic and covalent character. However, the monosubstituted complexes showed a predominance of the ionic character. In addition, high donor-acceptor interaction energies were obtained, indicating a good superposition between the s and p orbitals involved in the Zn-S bond. Methods: This work consists in theoretical studies of Zn2+ complexes with alkyl xanthate ligands, with different structures, where optimization and normal modes calculations were performed at different DFT levels: M06L, M06-2X, wB97XD, and B3LYP/6-311++G**+LANL2TZ, with Gaussian09 program. The process of substitution of two aqua by two xanthate ligands was analyzed in stages, forming cationic and neutral complexes, in the first and second stages, respectively. In addition, electronic energy decomposition (EDA) and natural bond orbital (NBO) analysis were performed at level M06L/6-311++G**+LANL2TZ with Gamess program. [ABSTRACT FROM AUTHOR]

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gaq-coupled SER-1 and Gas-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gaq or SER-7/Gas alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gaq and Gas in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gaq and Gas signaling to promote muscle activity and egg laying. [ABSTRACT FROM AUTHOR]

Neurodevelopmental disorders (NDDs) are a clinically and genetically heterogeneous group of early-onset pediatric disorders that affect the structure and/or function of the central or peripheral nervous system. Achieving a precise molecular diagnosis for NDDs may be challenging due to the diverse genetic underpinnings and clinical variability. In the current study, we investigated the underlying genetic cause(s) of NDDs in four unrelated Pakistani families. Using exome sequencing (ES) as a diagnostic approach, we identified disease-causing variants in established NDD-associated genes in all families, including one hitherto unreported variant in RELN and three recurrent variants in VPS13B, DEGS1, and SPG11. Overall, our study highlights the potential of ES as a tool for clinical diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Context: Xanthates are organic compounds of great interest in coordination chemistry due to their different basic sites, which allow them to form complexes with different coordination modes and geometries. These compounds are relevant in the environment and act as heavy metal collectors in aqueous environments. In this theoretical-experimental work, electronic spectroscopy studies of n-propyl xanthate complexes with group 12 metals were performed. This study verified structural differences in these systems, depending on the environment in which they are inserted. In addition, structural differences were observed when the solid was changed to an n-hexane solution. Thus, it was observed that the complexes assume a mononuclear structure in solution, while they present a polymeric form in the solid phase. The electronic spectra obtained through TD-DFT calculations were compared to those of the previously synthesized complexes. In the final theoretical analysis, the main orbitals involved in these transitions were assigned using population analysis calculations. The synthesis of the complexes was confirmed through infrared (MID and FAR), UV‒Vis, Raman, and NMR- 1 H spectroscopic analyses., Methods: The structures of the mononuclear and polymeric complexes were optimized in vacuum and n-hexane. Under vacuum, DFT levels M06L/6-311 + + G** + LANL2TZ and M06L/def2-TZVP were used for the mononuclear complexes, and M06L/LANL2DZ + LANL2 were used for the polymer complexes. For the calculations of the mononuclear complexes in n-hexane, the same level of theory was used for the solid state. TD-DFT calculations for 300 excited states were performed with the same levels of theory and used the optimized structures of the complexes. Furthermore, population analysis was carried out on all the systems studied. Gaussian 09 software was used for the structure optimization, TD-DFT, and population analysis calculations. GaussSum software was used to evaluate the molecular orbitals and electronic spectra., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Many genotype-phenotype databases and bioinformatic software tools utilize phenotype-genotype matching following Human Phenotype Ontology (HPO) standardized vocabulary and this has become the global standard for the International Rare Diseases Research Consortium (IRDiRC)[39]. Increasingly, high-resolution, rapid-sequencing MRI is used to enhance the diagnosis of congenital structural anomalies in the fetus[37], and, in approximately 15% of cases, incidental findings within the fetus are identified prospectively, which may make an important contribution to delineating the fetal phenotype and establishing the risk of genetic disease[38]. 43 Meier N, Bruder E, Lapaire O, Hoesli I, Kang A, Hench J, Hoeller S, De Geyter J, Miny P, Heinimann K, Chaoui R, Tercanli S, Filges I. Exome sequencing of fetal anomaly syndromes: novel phenotype-genotype discoveries. Increasingly, prenatal phenotype descriptions and heterogeneity will be recorded in clinical databases, and it is vitally important that fetal-medicine specialists, who utilize fetal imaging modalities, work within a multidisciplinary team in order to maximize their understanding of fetal phenotypes associated with genetic disease and, in particular, prenatal findings associated with significant long-term morbidity. [Extracted from the article]

Objectives: Non-medical prescribers, including pharmacists, have been found to achieve comparable clinical outcomes with doctors for certain health conditions. Legislation supporting pharmacist prescribing (PP) has been implemented in the United Kingdom (UK), Canada and New Zealand (NZ); however, to date, Australian pharmacists have not been extended prescribing rights. The purpose of this review was to describe the barriers to PP found in the literature from the UK, Canada, NZ and Australia, and examine the implications of these for the development of PP in Australia., Methods: We conducted a scoping review, which included peer-reviewed and grey literature, and consultation with stakeholders. Sources - Scopus, PubMed and CINAHL; Google Scholar, OpenGrey and organisational websites from January 2003 to March 2018 in the UK, Canada, NZ and Australia. Inclusion criteria - articles published in English, related to implementation of PP and articulated barriers to PP., Key Findings: Of 863 unique records, 120 were reviewed and 64 articles were eligible for inclusion. Three key themes emerged: (1) Socio-political context, (2) Resourcing issues and (3) Prescriber competence. The most common barriers were inadequate training regarding diagnostic knowledge and skills, inadequate support from authorities and stakeholders, and insufficient funding/reimbursement., Conclusions: If implementation of PP is to occur, attention needs to be focused on addressing identified barriers to PP implementation, including fostering a favourable socio-political context and prescriber competence. As such, a concerted effort is required to develop clear policy pathways, including targeted training courses, raising stakeholder recognition of PP and identifying specific funding, infrastructure and resourcing needs to ensure the smooth integration of pharmacist prescribers within interprofessional clinical teams., (© 2019 Royal Pharmaceutical Society.)

In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.

The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin‐1, SNAP‐25 and synaptobrevin. This complex is disassembled by N‐ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18‐1 and Munc13s organize SNARE complex assembly in an NSF‐SNAP‐resistant manner. Synaptotagmin‐1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane‐binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re‐interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses. [ABSTRACT FROM AUTHOR]

Photocatalysis is considered to be one of the possible routes to solve energy crises at a low cost. Graphitic-C3N4 combined with other semiconductors at the nanoscale has demonstrated a promising potential in photocatalysis application owing to the heterojunction configuration. In this study, Ag/g-C3N4/CuNb2O6-4 (mass ratio of CuNb2O6 to g-C3N4 = 1:4) composites were prepared by photo-deposition of Ag particles on the hydrothermally synthesized g-C3N4/CuNb2O6-4 composites. The microstructure, morphology, and light absorption property of the photocatalysts were characterized by X-ray diffractometry, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and UV–Vis diffuse reflectance spectroscopy. The photocatalytic properties of photocatalysts and active species responsible for the degradation of RhB dye under visible light were investigated. After loading Ag particles on the g-C3N4/CuNb2O6-4 composites, the resulting Ag/g-C3N4/CuNb2O6-4 composites show a bandgap of ~ 2.31 eV and a significant increase in photocatalytic efficiency. In particular, the photocatalytic activity of 11% Ag/g-C3N4/CuNb2O6-4 composite (Ag mass fraction = 11%) is nearly seven times that of orthorhombic CuNb2O6 and 3.8 times that of g-C3N4/CNO-4 composite. The Z-scheme heterojunction has been successfully formed in the Ag/g-C3N4/CuNb2O6-4 composites and it enhances the photocatalytic performance significantly with the best degradation efficiency of 88% after 2 h. [ABSTRACT FROM AUTHOR]

Correlative light and electron microscopy (CLEM) is a powerful imaging approach that allows the direct correlation of information obtained on a light and an electron microscope. There is a growing interest in the application of CLEM in biology, mainly attributable to technical advances in field of fluorescence microscopy in the past two decades. In this review, we summarize the important developments in CLEM for biological applications, focusing on the combination of fluorescence microscopy and electron microscopy. We first provide a brief overview of the early days of fluorescence CLEM usage starting with the initial rise in the late 1970s and the subsequent optimization of CLEM workflows during the following two decades. Next, we describe how the engineering of fluorescent proteins and the development of super‐resolution fluorescence microscopy have significantly renewed the interest in CLEM resulting in the present application of fluorescence CLEM in many different areas of cellular and molecular biology. Lastly, we present the promises and challenges for the future of fluorescence CLEM discussing novel workflows, probe development and quantification possibilities. [ABSTRACT FROM AUTHOR]

Herein, a flexible light weight lead-free piezoelectric nanogenerator has been fabricated using electrospun web based on polyacrylonitrile (PAN) and copper oxide (CuO) nanorods. The influence of CuO nanorods on the piezoelectric performance of PAN nanofibrous structure (in the form of a nanocomposite) has been investigated critically. Structural analysis of the electrospun nanocomposite has been done by FTIR and XRD techniques. Morphology of the developed material has been evaluated by scanning electron microscopy (SEM), whereas thermal behaviour has been investigated by thermogravimetric analyser (TGA) and differential scanning calorimetry (DSC). Finally, piezoelectric performance of the device has been measured by a digital oscilloscope. The piezoelectric nanogenerator (PAN/0.5% CuO) has shown an output voltage of ~ 5 V, current ~ 0.172 µA and power density ~ 0.215 µW/cm2 (when pressure is applied by finger tapping). The same nanogenerator has shown ~ 1.25 V and ~ 118 nA output voltage and short circuit current, respectively by using an automatic force-imparting machine. The fabricated nanogenerator is a potent device in lieu of the conventional battery powered devices. [ABSTRACT FROM AUTHOR]

Major recent advances and previous data have led to a plausible model of how key proteins mediate neurotransmitter release. In this model, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin form tight complexes that bring the membranes together and are crucial for membrane fusion. NSF and SNAPs disassemble SNARE complexes and ensure that fusion occurs through an exquisitely regulated pathway that starts with Munc18-1 bound to a closed conformation of syntaxin-1. Munc18-1 also binds to synaptobrevin, forming a template to assemble the SNARE complex when Munc13-1 opens syntaxin-1 while bridging the vesicle and plasma membranes. Synaptotagmin-1 and complexin bind to partially assembled SNARE complexes, likely stabilizing them and preventing fusion until Ca2+ binding to synaptotagmin-1 causes dissociation from the SNARE complex and induces interactions with phospholipids that help trigger release. Although fundamental questions remain about the mechanism of membrane fusion, these advances provide a framework to investigate the mechanisms underlying presynaptic plasticity. [ABSTRACT FROM AUTHOR]

Cadmium selenide quantum dots (QDs) are fluorescent and electron-dense nanoparticles. When used as reporter of immunolabeling, this dual visibility is essential for direct comparison of its fluorescent signals on light microscopy (LM) and their ultrastructrual counterparts on electron microscopy (EM) as correlative light and electron microscopy (CLEM). To facilitate EM recognition, QDs on EM grid were gold enhanced, which increased their size and electron density. On histological sections as well, gold-enhanced QDs, used as a reporter of immunolabeling, were easily recognized on EM. Because target structures are visible on bright field microscopy, gold enhancement facilitated trimming the target structures into final EM sections. Furthermore, gold enhancement of rod-shaped QD655 on EM grid was accentuated on their tips while spherical QD565 was gold-enhanced as sphere in contrast. This EM distinction was evident on histological sections where QD565 (green fluorescence) and QD655 (red fluorescence) were used as a reporter pair for double immunolabeling. Double-labeled immuno-fluorescent images, initially captured before EM processing, are now compared with their respective immuno EM counterparts. Specific labeling of each epitope was corroborated by mutual comparison between LM and EM. Although fluoronanogold may be a candidate reporter partner with QDs for gold-enhanced, double-labeling CLEM, its limited penetration into fixed tissue hampers universal use for thick histological sections. Gold-enhancement of QD immunolabeling, now expanded to double-labeling CLEM for human brain samples, will pave the way to translate molecular events into ultrastructural morphopathogenesis in situ. [ABSTRACT FROM AUTHOR]

Background Studies have demonstrated the potential for patient feedback to inform quality care as well as a direct relationship between patient experience and clinical outcomes. Over recent years, there has been increasing use of online patient feedback platforms, however, there has been little study of the content of patient feedback relating to pharmacy and pharmacy services. Objective This study explores the content of online feedback provided by patients from across the UK in relation to their experiences of their interaction with pharmacy staff and pharmacy services. Main outcome measure Content of online patient feedback relating to pharmacy. Method Patient stories published on Care Opinion, a national online patient feedback platform, for a one-year period were searched for all content relating to patients' pharmacy experiences. A thematic and sentiment analysis was conducted on 237 patient stories. Results Patient stories related to supply, staff attitudes, services, accessibility, systems, and errors. Patient sentiment depended on pharmacy setting, but staff attitudes, services, and accessibility were generally positive across all settings. Waiting time was the most common complaint in both hospital and community pharmacies with stories relaying experiences of slow discharge, stock shortages and poor communication and collaboration between pharmacies and GP surgeries. Conclusions Online patient feedback highlighted factors important to patients when interacting with pharmacies and their staff. Medication supply was the primary topic of patient stories with waiting times and stock shortages being clear areas for improvement; however, accessibility, pharmacy services and advice were key strengths of the profession. Further research is needed to understand how online patient feedback can be used effectively to inform improvements in pharmacy services. [ABSTRACT FROM AUTHOR]

Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention. [ABSTRACT FROM AUTHOR]

Attenuated intracellular survival of Mycobacterium tuberculosis (Mtb) secretory gene mutants exemplifies their role as virulence factors. Mtb peptidyl prolyl isomerase A (PPiA) assists in protein folding through cis/trans isomerization of prolyl bonds. Here, we show that PPiA abets Mtb survival and aids in disease progression by exploiting host-associated factors. While the deletion of PPiA has no discernable effect on bacillary survival in a murine infection model, it compromises the formation of granuloma-like lesions and promotes host cell death through ferroptosis. Overexpression of PPiA enhances the bacillary load and exacerbates pathology in mice lungs. Importantly, PPiA interacts with the integrin α5β1 receptor through a conserved surface-exposed RGD motif. The secretion of PPiA as well as interaction with integrin contributes to disease progression by upregulating multiple host matrix metalloproteinases. Collectively, we identified a novel nonchaperone role of PPiA that is critical in facilitating host-pathogen interaction and ensuing disease progression. [ABSTRACT FROM AUTHOR]

Background: The introduction of new health professional roles, such as that of the nurse practitioner and pharmacist prescriber in primary health care can lead to changes in health service delivery. Consumers, having used these roles, often report high satisfaction. However, there is limited knowledge of how these individuals position nurse practitioner and pharmacist prescriber roles within existing practice structures.Methods: Semi-structured interviews were conducted with 21 individuals receiving services from these practitioners in New Zealand primary health care. Interviews were recorded and transcribed verbatim for thematic analysis.Results: Participant views reflect established practice hierarchies, placing advanced practitioners 'below' general practitioners. Participants are unable to articulate what it was about these practitioners that meant they operated at lower tiers and often considered practitioners to act as 'their doctor'. They also highlight structural barriers impairing the ability of these providers to operate within their full scope of practice.Conclusions: While seeing value in the services they receive, consumers are often unable to position nurse practitioner and pharmacist prescriber roles within health system contexts or to articulate how they value their practitioner's skills. Embedded structural barriers may be more visible to consumers than their interactions with the health system suggest. This may influence peoples' ability to receive intended or optimal health services. Consumer 'health professional literacy' around the functions of distinct health practitioners should be supported so that they may make informed service provision choices. [ABSTRACT FROM AUTHOR]

Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity. Pyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. A new 3D correlative light-electron microscopy (3D-CLEM) approach enables the cartography of synaptic ultrastructure with cell type-, dendrite type- and synapse type-specific resolution in brain tissues, revealing unique properties of spines that receive dual innervation. [ABSTRACT FROM AUTHOR]

Organismal functionality and reproduction depend on metabolic rewiring and balanced energy resources. However, the crosstalk between organismal homeostasis and fecundity and the associated paracrine signaling mechanisms are still poorly understood. Using Caenorhabditis elegans, we discovered that large extracellular vesicles (known as exophers) previously found to remove damaged subcellular elements in neurons and cardiomyocytes are released by body wall muscles (BWM) to support embryonic growth. Exopher formation (exopheresis) by BWM is sex‐specific and a non‐cell autonomous process regulated by developing embryos in the uterus. Embryo‐derived factors induce the production of exophers that transport yolk proteins produced in the BWM and ultimately deliver them to newly formed oocytes. Consequently, offspring of mothers with a high number of muscle‐derived exophers grew faster. We propose that the primary role of muscular exopheresis is to stimulate reproductive capacity, thereby influencing the adaptation of worm populations to the current environmental conditions. SYNOPSIS: Caenorhabditis elegans body wall muscles release vesicles called exophers that transport muscle‐synthesized yolk proteins to nourish the next generation. Exopher formation is a bulky event in muscle cells.Muscular exopheresis is a non‐cell autonomous process regulated by in utero developing embryos.Exophers represents a transgenerational metabolic/resource management system that supports embryonic growth. [ABSTRACT FROM AUTHOR]

Defects in mitochondrial function activate compensatory responses in the cell. Mitochondrial stress that is caused by unfolded proteins inside the organelle induces a transcriptional response (termed the "mitochondrial unfolded protein response" [UPRmt]) that is mediated by activating transcription factor associated with stress 1 (ATFS-1). The UPRmt increases mitochondrial protein quality control. Mitochondrial dysfunction frequently causes defects in the import of proteins, resulting in the accumulation of mitochondrial proteins outside the organelle. In yeast, cells respond to mistargeted mitochondrial proteins by increasing activity of the proteasome in the cytosol (termed the "unfolded protein response activated by mistargeting of proteins" [UPRam]). The presence and relevance of this response in higher eukaryotes is unclear. Here, we demonstrate that defects in mitochondrial protein import in Caenorhabditis elegans lead to proteasome activation and life span extension. Both proteasome activation and life span prolongation partially depend on ATFS-1, despite its lack of influence on proteasomal gene transcription. Importantly, life span prolongation depends on the fully assembled proteasome. Our data provide a link between mitochondrial dysfunction and proteasomal activity and demonstrate its direct relevance to mechanisms that promote longevity. Mitochondrial import stress is a challenge for cellular protein homeostasis but at low level can be beneficial by activating the proteasome and increasing protein degradation. This study shows that activation of proteostasis compensatory mechanisms can lead to extension of lifespan in the nematode Caenorhabditis elegans. [ABSTRACT FROM AUTHOR]

Successful execution of behavior requires coordinated activity and communication between multiple cell types. Studies using the relatively simple neural circuits of invertebrates have helped to uncover how conserved molecular and cellular signaling events shape animal behavior. To understand the mechanisms underlying neural circuit activity and behavior, we have been studying a simple circuit that drives egg-laying behavior in the nematode worm Caenorhabditis elegans. Here we show that the sex-specific, ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to serotonin. Ca2+ imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca2+ activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. We also find the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca2+ activity and requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other. Together, our results demonstrate that the VC neurons facilitate efficient execution of egg-laying behavior by coordinating postsynaptic muscle contractility in response to serotonin and mechanosensory feedback. [ABSTRACT FROM AUTHOR]

Context: Xanthates are organic compounds that present great interest for coordination chemistry, because they can bond in different ways to the metal ion. Thus, these compounds have several applications, being best known for their environmental application. In fact, xanthates are recognized for their application as heavy metal collector agents in aqueous environments. In view of this application, this study is aimed at showing the thermochemical and electronic parameters obtained for the reactions of substitution water molecules in the aqua zinc complexes, by xanthate ligands (n-propyl, n-butyl, and n-pentyl xanthates). In addition to their environmental application, xanthates have shown biological properties, such as anti-bacterial and anti-cancer. In recent years, xanthates have also been used in the technological area, where it participates as a precursor of sulfides for the manufacture of thin films. Our results showed complexes with distorted octahedral geometries and with negative values of enthalpy and Gibbs free energy, indicating exothermic and spontaneous processes. For all the complexes, it was observed that Zn 2+ complexes have both an ionic and covalent character. However, the monosubstituted complexes showed a predominance of the ionic character. In addition, high donor-acceptor interaction energies were obtained, indicating a good superposition between the s and p orbitals involved in the Zn-S bond., Methods: This work consists in theoretical studies of Zn 2+ complexes with alkyl xanthate ligands, with different structures, where optimization and normal modes calculations were performed at different DFT levels: M06L, M06-2X, wB97XD, and B3LYP/6-311++G**+LANL2TZ, with Gaussian09 program. The process of substitution of two aqua by two xanthate ligands was analyzed in stages, forming cationic and neutral complexes, in the first and second stages, respectively. In addition, electronic energy decomposition (EDA) and natural bond orbital (NBO) analysis were performed at level M06L/6-311++G**+LANL2TZ with Gamess program., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Advanced practitioner services, such as those nurse practitioners and pharmacist prescribers provide, are an opportunity to improve health care delivery. In New Zealand, these practitioners remain underutilised, despite research suggesting they offer safe and effective care, and considerable international literature recording patient satisfaction with these roles. This study aimed to explore factors underlying consumer satisfaction with primary health care nurse practitioner and pharmacist prescriber services.Methods: As part of a larger realist evaluation, 21 individuals receiving advanced practitioner services participated in semi-structured interviews. These interviews were transcribed and coded against context-mechanism-outcome configurations tested and refined throughout the research.Results: Study findings emphasise the importance of consumer confidence in the provider as a mechanism for establishing advanced practitioner roles. Underlying this confidence is a recognition that these practitioners work in a more accessible manner, engage at the individual's 'level', and operate with passion.Conclusions: This research offers learnings to re-engineer service delivery within primary health care to make best use of the entire health care team by including consumers in the design and introduction of new roles. [ABSTRACT FROM AUTHOR]

Metazoans use protein homeostasis (proteostasis) pathways to respond to adverse physiological conditions, changing environment, and aging. The nervous system regulates proteostasis in different tissues, but the mechanism is not understood. Here, we show that Caenorhabditis elegans employs biogenic amine neurotransmitters to regulate ubiquitin proteasome system (UPS) proteostasis in epithelia. Mutants for biogenic amine synthesis show decreased poly‐ubiquitination and turnover of a GFP‐based UPS substrate. Using RNA‐seq and mass spectrometry, we found that biogenic amines promote eicosanoid production from poly‐unsaturated fats (PUFAs) by regulating expression of cytochrome P450 monooxygenases. Mutants for one of these P450s share the same UPS phenotype observed in biogenic amine mutants. The production of n‐6 eicosanoids is required for UPS substrate turnover, whereas accumulation of n‐6 eicosanoids accelerates turnover. Our results suggest that sensory neurons secrete biogenic amines to modulate lipid signaling, which in turn activates stress response pathways to maintain UPS proteostasis. SYNOPSIS: Protein homeostasis or proteostasis must respond to adverse physiological or environmental conditions. The Caenorhabditis elegans nervous system uses a signaling axis of biogenic amine neurotransmitters and eicosanoids to promote the ubiquitin proteasome proteostasis pathway in epithelia. The C. elegans nervous system releases biogenic amine neurohormones into the body.Biogenic amines promote the expression of cytochrome P450 monooxygenases.These P450 enzymes synthesize omega‐6 eicosanoids from poly‐unsaturated fatty acids.Omega‐6 eicosanoids promote the turnover of metastable proteins in epithelia by the ubiquitin proteasome system. [ABSTRACT FROM AUTHOR]

Background: Uptake of non‐medical prescribing by pharmacists working in primary care has been slow. This is despite benefits such as quicker and more efficient access to medicines for patients, a reduction in doctor workload, and enhanced professional satisfaction. This systematic review explores the views, opinions, and attitudes of pharmacists and graduates towards non‐medical prescribing. Methods: Medline, ScienceDirect, Embase, and the University of Reading Summon Service were searched to identify qualitative and mixed methods papers that examined the views, opinions, and attitudes of pharmacists and graduates towards non‐medical prescribing. Papers published between January 2003 and September 2017 were included. Studies were quality assessed using the CASP checklist and then analysed using thematic synthesis. Results: After 85 full‐text articles were assessed, a final 14 studies were eligible for inclusion. The included studies assessed pharmacists who currently prescribe, and other pharmacists and graduates with familiarity of non‐medical prescribing. Thematic synthesis identified two themes: (1) practice environment, and (2) pharmacist's role. Non‐medical prescribing was considered a natural extension to the role of a pharmacist despite difficulties in completing the required training. The ability to then prescribe was dependent on funding and access to medical records, time, and support staff. Pharmacists experienced professional rivalry with both support and resistance from members of the primary care team. The provision of training was frequently referred to as unsatisfactory. Pharmacists were motivated to prescribe, deriving increased job satisfaction and a sense of professionalism; however, they often felt underprepared for the reality of unsupervised practice. Furthermore, pharmacists reported a cautious approach with a fear of making errors frequently discussed. Conclusions: This review has identified themes and subsequent barriers and facilitators to non‐medical prescribing. Many of the barriers are more perceived than real and are diminishing. Consideration of these will assist and advance pharmacist prescribing in primary care, leading to positive outcomes for both patient care and the pharmacy profession. [ABSTRACT FROM AUTHOR]

Plant parasitic nematodes are microscopic pathogens that invade plant roots and cause extensive damage to crops. We have used a chemical biology approach to define mechanisms underpinning their parasitic behaviour: We discovered that reserpine, a plant alkaloid that inhibits the vesicular monoamine transporter (VMAT), potently impairs the ability of the potato cyst nematode Globodera pallida to enter the host plant root. We show this is due to an inhibition of serotonergic signalling that is essential for activation of the stylet which is used to access the host root. Prompted by this we identified core molecular components of G. pallida serotonin signalling encompassing the target of reserpine, VMAT; the synthetic enzyme for serotonin, tryptophan hydroxylase; the G protein coupled receptor SER-7 and the serotonin-gated chloride channel MOD-1. We cloned each of these molecular components and confirmed their functional identity by complementation of the corresponding C. elegans mutant thus mapping out serotonergic signalling in G. pallida. Complementary approaches testing the effect of chemical inhibitors of each of these signalling elements on discrete sub-behaviours required for parasitism and root invasion reinforce the critical role of serotonin. Thus, targeting the serotonin signalling pathway presents a promising new route to control plant parasitic nematodes. Author summary: Indian snakeroot is an herbal medicine prepared from the roots of the shrub Rauwolfia serpentine that has been used for centuries for its calming action. We have found that the major active constituent of snakeroot, reserpine, is a potentially powerful crop protectant as it protects the roots of plants from attack by microscopic plant parasitic nematodes. The pharmacology of reserpine is well known: it works by depleting a specific class of mood regulating chemical in the nervous system, the biogenic amines. We show that in the nematode, reserpine disables biogenic amine signalling and that loss of the amine serotonin halts the parasitic life cycle. We identified key components of the serotonin signalling pathway in the potato cyst nematode Globodera pallida and show that chemicals that target these sites also inhibit the ability of the nematode to invade its host plant. Therefore, the naturally occurring plant root alkaloid reserpine has provided insight into new targets for crop protection. [ABSTRACT FROM AUTHOR]

Maps of the synapses made and neurotransmitters released by all neurons in model systems, such as Caenorhabditis elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter GPCRs of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard laboratory conditions, suggesting that the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits. [ABSTRACT FROM AUTHOR]

The serotonergic modulation of feeding behaviour has been intensively studied in several invertebrate groups, including Arthropoda, Annelida, Nematoda and Mollusca. These studies offer comparative information on feeding regulation across divergent phyla and also provide general insights into the neural control of feeding. Specifically, model invertebrates are ideal for parsing feeding behaviour into component parts and examining the underlying mechanisms at the levels of biochemical pathways, single cells and identified neural circuitry. Research has found that serotonin is crucial during certain phases of feeding behaviour, especially movements directly underlying food intake, but inessential during other phases. In addition, while the serotonin system can be manipulated systemically in many animals, invertebrate model organisms also allow manipulations at the level of single cells and molecules, revealing limited and precise serotonergic actions. The latter highlight the importance of local versus global modulatory effects of serotonin, a potentially significant consideration for drug and pesticide design. [ABSTRACT FROM AUTHOR]

Cells, basic units of living structures and functions, build up a complicated small world, and their order and complexity resemble a small universe. The detailed understanding and elucidation of the matter transport and energy conversion mechanisms within a single cell will expand our knowledge about the origin and evolution of life, the disease mechanisms and much more. In past decades, single-cell analysis has been rapidly and significantly improved and various methodologies have been developed to reveal the complexity of mass, energy, and information within single cells. In this review, we focused on the methods developed in recent years for single-cell analysis, including electrochemical method, optical method, and mass spectrometry method. We reviewed the recent advances and representative studies in this research field, and also discussed the strengths and limitations of each method. Finally, we presented the existing technical challenges and further directions for single-cell analysis. [ABSTRACT FROM AUTHOR]

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca 2+ -triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca 2+ -triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive "open" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Ryanodine receptors (RyRs) are Ca 2+ release channels located in the endoplasmic reticulum membrane. Presynaptic RyRs play important roles in neurotransmitter release and synaptic plasticity. Recent studies suggest that the proper function of presynaptic RyRs relies on several regulatory proteins, including aryl hydrocarbon receptor-interacting protein, calstabins, and presenilins. Dysfunctions of these regulatory proteins can greatly impact neurotransmitter release and synaptic plasticity by altering the function or expression of RyRs. This chapter aims to describe the interaction between these proteins and RyRs, elucidating their crucial role in regulating synaptic function., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Cryo-correlative light and electron microscopy (Cryo-CLEM) is materializing as a widespread approach amalgamating the advantages of both fluorescence light microscopy (FLM) as well as three dimensional (3D) cryo-electron tomography (cryo-ET) to reveal the ultrastructure of significant target molecules with specific cellular functions. Cryo-CLEM allows imaging of cells by means of fluorescence microscopy exhibiting the location of the destined molecule at high temporal and spatial resolution while cryo-ET is employed to analyze the 3D structure at a molecular resolution in close-to-physiological condition. Present review focuses upon the practical strategies for Cryo-CLEM and recent technical developments that will assist the broad implementation of this technique to investigate and answer questions pertaining to various biological events occurring in the cell. [ABSTRACT FROM AUTHOR]