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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]

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Background: Complexin binds the SNARE complex at synapses and regulates exocytosis, but genetic studies indicate contradictory roles: in flies it predominantly inhibits synaptic vesicle fusion, whereas in mice it promotes evoked responses., Results: Here we characterize the complexin mutant in the nematode Caenorhabditis elegans and reveal bipolar functions in neurotransmission: complexin inhibits spontaneous fusion of synaptic vesicles but is also essential for evoked responses. Complexin mutants exhibit a doubling of vesicle fusion in the absence of extracellular calcium. Even more profoundly, mutants exhibit an almost complete loss of evoked responses, and current amplitudes are reduced by 94%. One possible interpretation is that complexin is required for the stabilization of docked vesicles and that, in its absence, vesicles may fuse or undock from the plasma membrane. Consistent with this hypothesis, docked synaptic vesicles are reduced by 70% in complexin-1 mutants., Conclusion: These data suggest that the main function of complexin is to maintain the docked state both by inhibiting fusion and by promoting priming., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

Background: Little is known about patients' opinions upon the development of non-medical prescribing (NMP)., Objective: To explore the opinions of patients on the development of NMP., Methods: In-depth interviews using qualitative methodology (Interpretative Phenomological Analysis). Eighteen interviews were undertaken in Bristol (Sites 1 and 3), Swindon (Site 2) and Brighton (Site 4). [Site 1 = primary care, GP prescriber (n = 5), Site 2 = secondary care, consultant prescriber (n = 5), Site 3 = primary care (n = 5) and Site 4 = secondary care (n = 3) (both pharmacist supplementary prescribers.] Participants (n = 18) were randomly sampled from patients under the care of the participating prescriber. Participants were aged between 42 and 81 years of age (n = 11 male and n = 7 female). Interviews took place between January and August 2006., Results: Participants expressed concerns about clinical governance, privacy and whether sufficient space were available to provide the service in community pharmacies. Participants acknowledged the expert drug knowledge of pharmacists and their accessibility. These factors enhanced acceptability of this role for pharmacists. Nurses were highly regarded, accepted and preferred as prescribers with few concerns., Conclusions: The results indicate support for pharmacists and nurses as prescribers, which aid successful implementation. Further research may be needed to evaluate the level of understanding that the public has of NMP and their views of the service once NMP is more widely established. Stakeholders should be mindful that the public may be hesitant regarding the professionalism, quality and clinical governance standards of clinics in community pharmacies in particular.

Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT(6)-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.

Serotonin (5-HT) regulates key processes in both vertebrates and invertebrates. Previously, four 5-HT receptors that contributed to the 5-HT modulation of egg laying were identified in Caenorhabditis elegans. Therefore, to assess potential receptor interactions, we generated animals containing combinations of null alleles for each receptor, especially animals expressing only individual 5-HT receptors. 5-HT-stimulated egg laying and egg retention correlated well with different combinations of predicted excitatory and inhibitory serotonergic inputs. For example, 5-HT did not stimulate egg laying in ser-1, ser-7, or ser-7 ser-1 null animals, and ser-7 ser-1 animals retained more eggs than wild-type animals. In contrast, 5-HT-stimulated egg laying in ser-4;mod-1 animals was greater than in wild-type animals, and ser-4;mod-1 animals retained fewer eggs than wild-type animals. Surprisingly, ser-4;mod-1;ser-7 ser-1 animals retained the same number of eggs as wild-type animals and exhibited significant 5-HT-stimulated egg laying that was dependent on a previously uncharacterized receptor, SER-5. 5-HT-stimulated egg laying was absent in ser-5;ser-4;mod-1;ser-7 ser-1 animals, and these animals retained more eggs than either wild-type or ser-4;mod-1;ser-7 ser-1 animals. The 5-HT sensitivity of egg laying could be restored by ser-5 muscle expression. Together, these results highlight the dual excitatory/inhibitory serotonergic inputs that combine to modulate egg laying.

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]

Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.

Unlabelled: OBJECTIVE (OF THE STUDY): To provide data on the views of chief pharmacists (CPs) and primary care trust pharmacists (PCTPs) on the risks and concerns surrounding supplementary prescribing., Setting: Secondary and primary care within England., Method: Postal questionnaire surveys of chief pharmacists and primary care trust pharmacists., Main Outcome Measure: Significance of the association between the extracted factors., Results: The response rate was 68% for both the primary care (183/271) and secondary care surveys (97/143). The survey tool was subjected to factor analysis and reliability testing. For both sectors, the three factors that were extracted described concerns over the training model for supplementary prescribing, concerns about the professional competency/responsibility of the supplementary prescribers once trained, and positivity about the implementation of supplementary prescribing. For both sectors, as trusts have more experience of supplementary prescribing by nurses, the respondents had less concerns about the supplementary prescribing training model. For secondary care, as the total number of pharmacists employed within the trust increases, the respondents had less concerns over the limitations of the supplementary prescribing training model., Conclusion: Although both sectors have concerns over the training model for supplementary prescribing and also professional competence and responsibility once trainees qualify, there is overall a positive attitude towards supplementary prescribing and there is a belief that pharmacists wish to take this role on.

Purpose: The implementation of supplementary prescribing by pharmacists within primary care trusts (PCTs) and secondary care trusts (SCTs) in England was studied., Methods: A survey was developed and sent to pharmacists in PCTs and SCTs in England who would oversee the implementation of supplementary prescribing by pharmacists., Results: The response rate was 68% for both surveys. The majority of SCTs and PCTs intended to implement supplementary prescribing by pharmacists by the end of 2005 (57% and 56%, respectively). The majority of SCT respondents did not believe that it would be more difficult to recruit designated medical practitioners to supervise supplementary prescribing training for pharmacists as opposed to nurses (67%, n = 43), whereas the largest group of PCT pharmacists believed it would be (47%, n = 86). Within secondary care, the clinical areas in which pharmacists were intending to work as supplementary prescribers were those where they already had established roles. Within primary care, the main clinical areas for pharmacists were influenced by those areas in the new General Medical Services contract Quality and Outcomes Framework for general practitioners., Conclusion: A survey investigating the implementation of supplementary prescribing by pharmacists in England found that there were significantly more barriers to its establishment within primary care than secondary care settings. Within primary care, supplementary prescribing is being implemented to develop new services. Within secondary care, the supplementary prescribing model is more often used to legitimize services already being provided.

Serotonin (5-HT) stimulates both pharyngeal pumping and egg laying in Caenorhabditis elegans. Four distinct 5-HT receptors have been partially characterized, but little is known about their function in vivo. SER-7 exhibits most sequence identity to the mammalian 5-HT7 receptors and couples to a stimulation of adenyl cyclase when expressed in COS-7 cells. However, many 5-HT7-specific agonists have low affinity for SER-7. 5-HT fails to stimulate pharyngeal pumping and the firing of the MC motorneurons in animals containing the putative ser-7(tm1325) and ser-7(tm1728) null alleles. In addition, although pumping on bacteria is upregulated in ser-7(tm1325) animals, pumping is more irregular. A similar failure to maintain "fast pumping" on bacteria also was observed in ser-1(ok345) and tph-1(mg280) animals that contain putative null alleles of a 5-HT2-like receptor and tryptophan hydroxylase, respectively, suggesting that serotonergic signaling, although not essential for the upregulation of pumping on bacteria, "fine tunes" the process. 5-HT also fails to stimulate egg laying in ser-7(tm1325), ser-1(ok345), and ser-7(tm1325) ser-1(ok345) animals, but only the ser-7 ser-1 double mutants exhibit an Egl phenotype. All of the SER-7 mutant phenotypes are rescued by the expression of full-length ser-7gfp translational fusions. ser-7gfp is expressed in several pharyngeal neurons, including the MC, M2, M3, M4, and M5, and in vulval muscle. Interestingly, 5-HT inhibits egg laying and pharyngeal pumping in ser-7 null mutants and the 5-HT inhibition of egg laying, but not pumping, is abolished in ser-7(tm1325);ser-4(ok512) double mutants. Taken together, these results suggest that SER-7 is essential for the 5-HT stimulation of both egg laying and pharyngeal pumping, but that other signaling pathways can probably fulfill similar roles in vivo.

Background: In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system., Methods: The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP., Results: The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression., Conclusions: We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

The biogenic amines, serotonin, octopamine, tyramine and dopamine regulate many essential processes in parasitic nematodes, such as pharyngeal pumping, muscle contraction, and egg-laying, as well as more complex behaviors, such as mechanosensation and foraging, making biogenic amine receptors excellent targets for drug discovery. This review is designed to summarize our knowledge of nematode biogenic amine signaling and preliminarily identify some of the key receptors involved in the regulation of biogenic amine-dependent behaviors through an analysis of the free-living nematode, Caenorhabditis elegans.

Serotonin plays a key role in the regulation of pharyngeal pumping in nematodes. We have isolated a Caenorhabditis elegans cDNA (C09B7.1b, ser-7b) with greatest identity to the 5-HT7 receptor family. Membranes from COS-7 cells expressing SER-7b exhibit saturable [3H]-LSD binding (Kd = 45 nm) that is inhibited by serotonin (5-HT) and tryptamine, but not by other physiological biogenic amines. Expression of SER-7b in COS-7 cells results in dramatic increase in basal cAMP levels over untransfected cells that is dependent on expression level. 5-HT further elevates cAMP levels in a dose-dependent manner (pEC50 = 7.5 +/- 0.5). Mammalian 5-HT7 receptor inverse agonists reduce constitutive activity, with methiothepin the most potent (pIC50 = 7.8 +/- 0.1). Ser-7::GFP transcriptional fusions reveal that SER-7b appears to be expressed solely in the M4 pharyngeal motorneuron after hatching. This is the first report of a Galphas coupled biogenic amine receptor in nematodes and the localization of SER-7b in the M4 pharyngeal motorneuron suggests that SER-7b may play a role in the regulation of pharyngeal pumping.

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.

The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette-Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in M. tuberculosis during macrophage infection, by real-time RT-PCR. Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.

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]