RESUMEN
BACKGROUND: Zebrafish visual function depends on quality optics. An F3 screen for developmental mutations in the Zebrafish nervous system was conducted in wild-type (wt) AB Zebrafish exposed to 3 mM of N-ethyl-N-nitrosourea (ENU). RESULTS: Mutant offspring, identified in an F3 screen, were characterized by a small pupil, resulting from retinal hypertrophy or hyperplasia and a small lens. Deficits in visual function made feeding difficult after hatching at approximately 5-6 days postfertilization (dpf). Special feeding conditions were necessary for survival of the occhiolino (occ) mutants after 6 dpf. Optokinetic response (OKR) tests measured defects in visual function in the occ mutant, although electroretinograms (ERGs) were normal in the mutant and wt. Consistent with the ERGs, histology found normal retinal structure in the occ mutant and wt Zebrafish. However, lens development was abnormal. Multiphoton imaging of the developmental stages of live embryos confirmed the formation of a secondary mass of lens cells in the developing eye of the mutant Zebrafish at 3-4 dpf, and laminin immunohistochemistry indicated the lens capsule was thin and disorganized in the mutant Zebrafish. CONCLUSIONS: The occ Zebrafish is a novel disease model for visual defects associated with abnormal lens development. Developmental Dynamics 246:915-924, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Cristalino/crecimiento & desarrollo , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Embrión no Mamífero , Anomalías del Ojo/genética , Inmunohistoquímica , Laminina , Cápsula del Cristalino/anatomía & histología , Cápsula del Cristalino/patología , Cristalino/embriología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Aims In March 2020 the World Health Organisation (WHO) declared the COVID-19 virus a global pandemic. The United Kingdom's National Health Service (NHS) was placed under unprecedented pressure and hospitals were forced to adapt their working practices to continue offering world-leading healthcare. This project aims to highlight the lessons learnt within hand surgical departments throughout Wales. Using this knowledge, we can consider how these lessons can be implemented in both emergency and elective hand practice. Methods A qualitative questionnaire was distributed to hand consultants working across Health Boards within Wales during the pandemic. The questionnaire encompasses the impact of the pandemic on usual practices and what local departmental changes have been implemented in response to patient needs. Results Across the Welsh Health Boards, we received 12 of 19 consultant responses achieving a 63% response rate and captured data from five of seven (71%) major health boards. The questionnaire revealed that 100% of respondents changed their routine management of elective cases whilst 83% changed their management of hand trauma. 50% reported the need to issue updated management guidelines to junior doctors. The major highlighted lessons were the importance of a dedicated hand fracture clinic, coupled with a ring-fenced day-surgical unit (offering regional anaesthetic support) to manage trauma and elective patients independently from general trauma. Conclusion This qualitative research demonstrates that the pandemic drove the restructuring of many hand departments enabling us to find new, efficient ways of working. We must take these lessons forward to tackle the ever-growing waiting list, increased patient expectations and increasingly complex workloads.
RESUMEN
Vertebrates rely on rod photoreceptors for vision in low-light conditions. The specialized downstream circuit for rod signalling, called the primary rod pathway, is well characterized in mammals, but circuitry for rod signalling in non-mammals is largely unknown. Here we demonstrate that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA sequencing, we identified two bipolar cell types in zebrafish that are related to mammalian rod bipolar cell (RBCs), the only bipolar type that directly carries rod signals from the outer to the inner retina in the primary rod pathway. By combining electrophysiology, histology and ultrastructural reconstruction of the zebrafish RBCs, we found that, similar to mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells postsynaptic to one RBC type is strikingly similar to that of mammalian RBCs and their amacrine partners, suggesting that the cell types and circuit design of the primary rod pathway emerged before the divergence of teleost fish and mammals. The second RBC type, which forms separate pathways, was either lost in mammals or emerged in fish.
Asunto(s)
Células Bipolares de la Retina , Células Fotorreceptoras Retinianas Bastones , Pez Cebra , Animales , Pez Cebra/fisiología , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Evolución Biológica , Retina/fisiología , Retina/citología , MamíferosRESUMEN
Vertebrates rely on rod photoreceptors for vision in low-light conditions. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage. Thus, it has been long assumed that the primary rod pathway evolved in mammals. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs, suggesting that the cell types and circuit design of the primary rod pathway have emerged before the divergence of teleost fish and amniotes. The second RBC type, which forms separate pathways, is either lost in mammals or emerged in fish.
RESUMEN
Vertebrates rely on rod photoreceptors for vision in low-light conditions1. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage2-6. Thus, it has been long assumed that the primary rod pathway evolved in mammals3,5-7. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs8, both zebrafish RBC types connect with all rods and red-cones in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs. This suggests that the cell types and circuit design of the primary rod pathway may have emerged before the divergence of teleost fish and amniotes (mammals, bird, reptiles). The second RBC type in zebrafish, which forms separate pathways from the first RBC type, is either lost in mammals or emerged in fish to serve yet unknown roles.
RESUMEN
This case report aims to highlight that not all shoulder dislocations are simple to treat and that early recognition of complications is key in managing these injuries successfully. We report the case of a 68-year-old gentleman who presented to Accident and Emergency (A&E) following a fall and sustaining an anterior dislocation of his right shoulder. This was reduced under sedation; however, the patient had an ongoing feeling that his shoulder "was not right." The subsequent investigation demonstrated persistent anterior subluxation of the humeral head with rotator cuff interposition in the glenohumeral joint. This case appears to be the first of its kind to be reported in which the supraspinatus, subscapularis, and long head of biceps were collectively interposed. This was treated operatively with open reduction and rotator cuff repair, even though the procedure was technically difficult due to tissue fibrosis and the formation of adhesions. The patient progressed well and had a good clinical outcome. This case highlights that rotator cuff interposition following shoulder dislocation is a rare but debilitating complication and is often neglected in initial care. We must recognise that patients are their own experts, and if they report something is "not right," further investigation and prompt treatment are required.
RESUMEN
In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant (pde6c(w59)) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone death in vivo in pde6c(w59) fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild-type (WT) and pde6c(w59) cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones.
Asunto(s)
Mutación/fisiología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Animales , Efecto Espectador/fisiología , Muerte Celular , Degeneración Retiniana/fisiopatología , Pez Cebra , Proteínas de Pez Cebra/fisiologíaRESUMEN
Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuits in vivo by simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia cells (MGs). We conducted in vivo time-lapse multiphoton imaging of the rapidly developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MGs are absent. We found that removal of MGs had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.
Asunto(s)
Neuroglía/fisiología , Neuronas/fisiología , Retina/citología , Sinapsis/fisiología , Aminoácidos , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Imagenología Tridimensional/métodos , Proteínas Luminiscentes/genética , Microscopía Confocal/métodos , Microscopía Electrónica de Transmisión/métodos , Neuroglía/ultraestructura , Neuronas/clasificación , Neuronas/ultraestructura , Fotoblanqueo , Receptores de Glutamato/metabolismo , Sinapsis/ultraestructura , Factores de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/genética , Cromosomas Humanos X/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Ubiquitina-Proteína Ligasas/genética , Secuencia de Bases , Western Blotting , Análisis Mutacional de ADN , ADN Complementario/genética , Dosificación de Gen/genética , Duplicación de Gen , Humanos , Hibridación Fluorescente in Situ , Análisis por Micromatrices , Datos de Secuencia Molecular , Mutación/genética , Linaje , Proteínas Supresoras de TumorRESUMEN
Age-related hearing loss (ARHL), also known as presbycusis, is a widespread and debilitating condition impacting many older adults. Conventionally, researchers utilize mammalian model systems or human cadaveric tissue to study ARHL pathology. Recently, the zebrafish has become an effective and tractable model system for a wide variety of genetic and environmental auditory insults, but little is known about the incidence or extent of ARHL in zebrafish and other non-mammalian models. Here, we evaluated whether zebrafish exhibit age-related loss in auditory sensitivity. The auditory sensitivity of adult wild-type zebrafish (AB/WIK strain) from three adult age subgroups (13-month, 20-month, and 37-month) was characterized using the auditory evoked potential (AEP) recording technique. AEPs were elicited using pure tone stimuli (115-4500 Hz) presented via an underwater loudspeaker and recorded using shielded subdermal metal electrodes. Based on measures of sound pressure and particle acceleration, the mean AEP thresholds of 37-month-old fish [mean sound pressure level (SPL) = 122.2 dB ± 2.2 dB SE re: 1 µPa; mean particle acceleration level (PAL) = -27.5 ± 2.3 dB SE re: 1 ms-2] were approximately 9 dB higher than that of 20-month-old fish [(mean SPL = 113.1 ± 2.7 dB SE re: 1 µPa; mean PAL = -37.2 ± 2.8 dB re: 1 ms-2; p = 0.007)] and 6 dB higher than that of 13-month-old fish [(mean SPL = 116.3 ± 2.5 dB SE re: 1 µPa; mean PAL = -34.1 ± 2.6 dB SE re: 1 ms-2; p = 0.052)]. Lowest AEP thresholds for all three age groups were generally between 800 Hz and 1850 Hz, with no evidence for frequency-specific age-related loss. Our results suggest that zebrafish undergo age-related loss in auditory sensitivity, but the form and magnitude of loss is markedly different than in mammals, including humans. Future work is needed to further describe the incidence and extent of ARHL across vertebrate groups and to determine which, if any, ARHL mechanisms may be conserved across vertebrates to support meaningful comparative/translational studies.
Asunto(s)
Presbiacusia , Pez Cebra , Estimulación Acústica , Animales , Umbral Auditivo , Potenciales Evocados Auditivos , SonidoRESUMEN
Study Design Retrospective study. Objectives To assess the fatty atrophy of the lumbar paraspinal muscles (LPMs) as determined using magnetic resonance imaging in patients with lumbar degenerative disk disease (DDD) and focal disk herniation and to determine if fatty atrophy is associated with patient-reported outcome measures (PROMS). Methods One hundred sixty-five patients with lumbar DDD were identified from a PROMS database of >1,500 patients. These patients were divided into two study groups: DDD alone (n = 58) and DDD with disk herniation (n = 107). A grid was randomly applied to the axial scans at the L3-L4, L4-L5, and L5-S1 levels. The muscle-to-fat ratio of the LPMs was recorded and compared with PROMS data. Subcutaneous fat thickness at each level was also measured. Results This study found no difference in the muscle-to-fat ratio between the DDD and disk herniation groups. There was no association between the muscle-to-fat ratio and PROMS data in either group. There was significantly more subcutaneous fat at all levels in the DDD group as compared with the disk prolapse group. In DDD and disk prolapses, subcutaneous fat was thicker in women (p = 0.013 and 0.001). In patients with DDD, more subcutaneous fat was associated with disability (p < 0.001). Muscle content of erector spinae and multifidus negatively correlated with increasing age in both groups at the L3-L4 level. Conclusions Muscle fat content in the LPM does not appear to relate to PROMS. Muscle content decreases with age. Those with low back pain (DDD) have greater subcutaneous fat thickness.
RESUMEN
The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein-protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.
Asunto(s)
Proteínas 14-3-3/metabolismo , Anexina A2/metabolismo , Biomarcadores de Tumor/metabolismo , Epidermis/metabolismo , Exorribonucleasas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas 14-3-3/genética , Biomarcadores de Tumor/genética , Células Cultivadas , Cristalización , Células Epidérmicas , Exorribonucleasas/genética , Proteínas Filagrina , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Unión Proteica , Transporte de Proteínas/fisiología , Proteínas S100/metabolismo , Sensibilidad y Especificidad , Espectrometría de FluorescenciaRESUMEN
The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.
Asunto(s)
Epidermis/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células Epidérmicas , Proteínas Filagrina , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Filamentos Intermediarios/genética , Queratina-10/metabolismo , Queratinocitos/citología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Dominios y Motivos de Interacción de Proteínas/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
Caspase-14 is a cysteine endoproteinase that is expressed in the epidermis and a limited number of other tissues. It is activated during keratinocyte differentiation by zymogen processing, but its precise function is unknown. To obtain caspase-14 for functional studies, we engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in Escherichia coli and cultured mammalian cells. Rev-hC14 required no proteolytic processing for activity, showed strong activity against the caspase substrate WEHD, and was inhibited by the pan-caspase inhibitor zVAD-fmk. Mammalian cells that expressed active caspase-14 showed normal cell adherence and morphology. Using positional scanning of synthetic tetrapeptide libraries, we determined the substrate preference of human caspase-14 to be W (or Y)-X-X-D. These studies affirm that caspase-14 has a substrate specificity similar to the group I caspases, and demonstrate that it functions in a distinct manner from executioner caspases to carry out specific proteolytic events during keratinocyte differentiation.
Asunto(s)
Caspasas/biosíntesis , Clorometilcetonas de Aminoácidos/farmacología , Animales , Células COS , Caspasa 14 , Inhibidores de Caspasas , Caspasas/metabolismo , Diferenciación Celular/fisiología , Chlorocebus aethiops , Clonación Molecular , Escherichia coli/enzimología , Humanos , Queratinocitos/citología , Oligopéptidos/metabolismo , Ratas , Especificidad por SustratoRESUMEN
An infant feeding survey was carried out in three parishes in Cornwall County, Jamaica, from March to August, 1984, in an attempt to determine the feeding practices of infants and beliefs of mothers in Western Jamaica. Forty-one enumeration districts (EDs) were randomly selected from two strata, urban and rural, in proportion to the population distribution. Six hundred and fourteen households with children under thirty-six months of age were selected from 41 EDs. The results showed that the prevalence of breastfeeding was 98 percent. Despite the high frequency of breastfeeding, infant formula was introduced very early, and only about half the mothers introduced semi-solids and solids within one month of the recommended time. The most popular foods, identified as good foods, were also popular weaning foods. It is concluded that there is need for adequate training of both primary and secondary health-care staff, and reinforcement of the effectiveness of nutrition education programmes is also important (AU)