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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.

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.

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.