Differentiering av GnRH neuroner från humana pluripotenta stamceller

The onset of puberty and sexual development as well as normal reproductive function are dependent on pulsatile secretion of gonadotropin-releasing hormone (GnRH). GnRH is secreted from the GnRH neuron nerve terminals in the hypothalamic median eminence into the portal vessels that lead to the anterior pituitary. The pulsatile secretion of GnRH stimulates the release of gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which, in turn, regulate various gonadal functions. In rare occasions, the onset of puberty is delayed or completely absent. This can be caused by disrupted development, migration, or function of GnRH neurons, resulting in defects in sexual development and infertility. Congenital GnRH deficiency is termed congenital hypogonadotropic hypogonadism (CHH), and CHH combined with hyposmia or anosmia (reduced or absent sense of smell) is known as Kallmann syndrome (KS). CHH and KS are genetically heterogeneous diseases, with over 30 genes reported in association with KS and CHH to date. How mutations in these genes cause GnRH deficiency is not yet comprehensively understood, but several are postulated to affect GnRH neuron development. Human pluripotent stem cells (hPSCs) are the equivalent of undifferentiated cells in the early embryo, and able to give rise to all tissues and cell types in the human body. Thus, hPSCs have become a widely used tool for studying the differentiation of specialized cell types and the causes for human diseases in vitro. Developing methods for directed differentiation of hPSCs into GnRH neurons requires insight into the events which lead to the specification of GnRH neurons during embryonic development. GnRH neurons are born in the olfactory placodes in the nasal area of the developing embryo. After their delamination from the olfactory neuroepithelium, the differentiated postmitotic GnRH neurons take an upward migratory route along the axon fibers of the terminal nerve around the olfactory bulb, cross the cribriform plate to the forebrain, and finally make a ventral turn into to the preoptic area of the hypothalamus. The exact cell type within the olfactory placodes that gives rise to GnRH neurons is not entirely known. Its precursors have been proposed to be of both preplacodal ectoderm and neural crest origin. The aim of this work was to create a model in which to study the molecular mechanism of GnRH neuron differentiation and the mechanisms of CHH-associated genetic mutations on GnRH neurogenesis in humans. The literature review of this thesis addresses the relevant background in the field of GnRH neuron development; from neural crest and preplacodal development at gastrulation stages, to ontogeny, migration, and maturation of GnRH neurons at puberty. This thesis presents experimental validation of the methods for in vitro generation of GnRH neurons from human pluripotent stem cells, and the findings include the discovery of several genes and proteins expressed during GnRH neuron differentiation.Människans pubertetsutveckling samt fertilitet styrs av ett noggrannt reglerat system av hormonella signaler, dvs. hypotalamus-hypofys-gonad-axeln (HPG-axeln). Gonadotropinfrisättande hormon (eng. Gonadotropin-releasing hormone, GnRH), utsöndras av en grupp specialiserade nervceller i hypotalamus, GnRH neuronerna. Hormonet GnRH fungerar som en startsignal för utsöndring av follikelstimulerande och luteiniserande hormon (FSH och LH) från hypofysen, som i sin tur stimulerar tillverkningen av könshormon. Eftersom GnRH neuroner har en central roll i HPG-axeln, kan avbrott i deras utveckling eller funktion leda till pubertetsstörningar och infertilitet. Den sällsynta genetiska sjukdomen hypogonadotropisk hypogonadism (CHH) beror på nedsatt utsöndring av GnRH i hypotalamus och leder till försenad eller utebliven pubertet, begränsad könskaraktärsutveckling och infertilitet. Kallmann Syndrom är en form av CHH som förekommer i kombination med nedsatt luktsinne. Det finns ett påvisat samband mellan utvecklingen av luktsinnets celler och GnRH neuroner. Både olfaktoriska receptorceller och GnRH neuroner uppkommer i nasala plakoderna, i neuroepitelet som senare kommer att bilda i embryots luktepitel. Under veckorna 6-8 av fosterutvecklingen migrerar de nybildade GnRH neuronerna från näsan till hypotalamus i hjärnan tillsammans med olfaktoriska receptorcellers axonfibrer. Mekanismer som stör utvecklingen i nasala plakoderna, neuroners migration, eller cellernas mognad, kan därmed vara orsaker till Kallmann Syndrom. Mutationer i över 30 gener har påvisats vara förknippade med CHH och Kallmann Syndrom, men de molekylära mekanismer som orsakar avvikelser i GnRH neuroners fysiologi är till stor del okända. Humana pluripotenta stamceller (hPSC) är ospecialiserade celler som förekommer under den tidiga embryonalutvecklingen, och som genom differentiering ger upphov till alla specialiserade celltyper i kroppens vävnader och organ. Genom att odla hPSC celler i laboratoriemiljö är det möjligt att återskapa samt studera utvecklingen av specialiserade celler och utforska biologiska mekanismer som gynnar eller förhindrar cellernas uppkomst och funktion. Denna avhandling beskriver utvecklingen av metoder för differentiering av GnRH neuroner från hPSC in vitro, samt presenterar användningen av differentieringsmetoden som forskningsmodell i studier om GnRH neuroners utveckling. För övrigt diskuteras flera tidigare obeskrivna gener och protein som uttrycks under differentieringsprocessen, och deras potentiella roller i regleringen av humana GnRH neuroners uppkomst och funktion, och därmed även i regleringen av människans pubertetsutveckling och fertilitet

Bacterial whole genome-based phylogeny: construction of a new benchmarking dataset and assessment of some existing methods

BackgroundWhole genome sequencing (WGS) is increasingly used in diagnostics and surveillance of infectious diseases. A major application for WGS is to use the data for identifying outbreak clusters, and there is therefore a need for methods that can accurately and efficiently infer phylogenies from sequencing reads. In the present study we describe a new dataset that we have created for the purpose of benchmarking such WGS-based methods for epidemiological data, and also present an analysis where we use the data to compare the performance of some current methods.ResultsOur aim was to create a benchmark data set that mimics sequencing data of the sort that might be collected during an outbreak of an infectious disease. This was achieved by letting an E. coli hypermutator strain grow in the lab for 8 consecutive days, each day splitting the culture in two while also collecting samples for sequencing. The result is a data set consisting of 101 whole genome sequences with known phylogenetic relationship. Among the sequenced samples 51 correspond to internal nodes in the phylogeny because they are ancestral, while the remaining 50 correspond to leaves.We also used the newly created data set to compare three different online available methods that infer phylogenies from whole-genome sequencing reads: NDtree, CSI Phylogeny and REALPHY. One complication when comparing the output of these methods with the known phylogeny is that phylogenetic methods typically build trees where all observed sequences are placed as leafs, even though some of them are in fact ancestral. We therefore devised a method for post processing the inferred trees by collapsing short branches (thus relocating some leafs to internal nodes), and also present two new measures of tree similarity that takes into account the identity of both internal and leaf nodes.ConclusionsBased on this analysis we find that, among the investigated methods, CSI Phylogeny had the best performance, correctly identifying 73% of all branches in the tree and 71% of all clades.We have made all data from this experiment (raw sequencing reads, consensus whole-genome sequences, as well as descriptions of the known phylogeny in a variety of formats) publicly available, with the hope that other groups may find this data useful for benchmarking and exploring the performance of epidemiological methods. All data is freely available at: https://cge.cbs.dtu.dk/services/evolution_data.php

Pre-surgical radiographic and clinical features as predictors for temporomandibular joint discectomy prognosis

Objectives This study aimed to identify potential clinical and radiological predictors associated with the outcome of discectomies. Methods In this retrospective observational study, the material comprised preoperative CBCT images and medical records of 62 patients with disc derangement disorders, who had undergone discectomy because of disc displacement with reduction (DDwR), disc displacement without reduction (DDwoR), systemic arthritis (SA), or joint hypermobility. Clinical and radiographic variables were analysed in relation to success rate determined by subjective, objective and combined outcomes. Results The success odds ratio was 11 times higher in patients with painful DDwR versus that of SA (p = 0.03), and even 25.9 times higher when considering solely objective outcome (p = 0.03). In the absence of subchondral pseudocyst, there were 5.2 times higher odds to have a successful subjective outcome (p = 0.04). Extensive bone apposition on the temporal joint component indicated a 9.3 times higher likelihood of a failed objective outcome (p = 0.04). Conclusions There is a significant higher risk for combined outcome failure for the diagnosis SA involving the TMJ compared with DDwR. Predictors of importance based on CBCT findings related to the objective outcome failure were extensive bone apposition on the temporal joint component and condylar subchondral pseudocysts for the subjective outcome failure.publishedVersio

Growth standard charts for monitoring bodyweight in dogs of different sizes

Limited information is available on what constitutes optimal growth in dogs. The primary aim of this study was to develop evidence-based growth standards for dogs, using retrospective analysis of bodyweight and age data from >6 million young dogs attending a large corporate network of primary care veterinary hospitals across the USA. Electronic medical records were used to generate bodyweight data from immature client-owned dogs, that were healthy and had remained in ideal body condition throughout the first 3 years of life. Growth centile curves were constructed using Generalised Additive Models for Location, Shape and Scale. Curves were displayed graphically as centile charts covering the age range 12 weeks to 2 years. Over 100 growth charts were modelled, specific to different combinations of breed, sex and neuter status. Neutering before 37 weeks was associated with a slight upward shift in growth trajectory, whilst neutering after 37 weeks was associated with a slight downward shift in growth trajectory. However, these shifts were small in comparison to inter-individual variability amongst dogs, suggesting that separate curves for neutered dogs were not needed. Five bodyweight categories were created to cover breeds up to 40kg, using both visual assessment and hierarchical cluster analysis of breed-specific growth curves. For 20/24 of the individual breed centile curves, agreement with curves for the corresponding bodyweight categories was good. For the remaining 4 breed curves, occasional deviation across centile lines was observed, but overall agreement was acceptable. This suggested that growth could be described using size categories rather than requiring curves for specific breeds. In the current study, a series of evidence-based growth standards have been developed to facilitate charting of bodyweight in healthy dogs. Additional studies are required to validate these standards and create a clinical tool for growth monitoring in pet dogs

Healthcare Challenges and Future Solutions in Dental Practice: Assessing Oral Antibiotic Resistances by Contemporary Point-Of-Care Approaches

Antibiotic resistance poses a global threat, which is being acknowledged at several levels, including research, clinical implementation, regulation, as well as by the World Health Organization. In the field of oral health, however, the issue of antibiotic resistances, as well as of accurate diagnosis, is underrepresented. Oral diseases in general were ranked third in terms of expenditures among the EU-28 member states in 2015. Yet, the diagnosis and patient management of oral infections, in particular, still depend primarily on empiric means. On the contrary, on the global scale, the field of medical infections has more readily adopted the integration of molecular-based systems in the diagnostic, patient management, and antibiotic stewardship workflows. In this perspective review, we emphasize the clinical significance of supporting in the future antibiotic resistance screening in dental practice with novel integrated and point-of-care operating tools that can greatly support the rapid, accurate, and efficient administration of oral antibioticspublishedVersio

MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1 -expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.Peer reviewe

Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.Peer reviewe

Association between life span and body condition in neutered client‐owned dogs

Background There is an association between overweight status and life span in kenneled dogs, but a similar association has not been reported for pet dogs. Objectives To examine the effects of being overweight in middle age on the life span of neutered client‐owned dogs. Animals Fifty‐thousand seven‐hundred eighty seven middle‐aged neutered client‐owned dogs attending a network of approximately 900 veterinary hospitals across North America. Methods Retrospective case‐control study. For each of 12 breeds, groups of dogs aged between 6.5 and 8.5 years were identified as being in “overweight” or “normal” body condition. Within each breed and sex, differences in life span between dogs in normal body condition and overweight body condition in the 2 groups were then analyzed by Cox proportional hazards models. Results For all breeds, instantaneous risk of death for dogs in overweight body condition was greater than those in normal body condition throughout the age range studied, with hazard ratios ranging from 1.35 (99.79% confidence interval [CI] 1.05‐1.73) for German Shepherd dog to 2.86 (99.79% CI 2.14‐3.83) for Yorkshire Terrier. In all breeds, median life span was shorter in overweight compared with normal weight dogs, with the difference being greatest in Yorkshire Terriers (overweight: 13.7 years, 99.79% CI 13.3‐14.2; normal: 16.2 years, 99.79% CI 15.7‐16.5) and least in German Shepherd dogs (overweight: 12.1 years, 99.79% CI 11.8‐12.4; normal: 12.5 years, 99.79% CI 12.2‐12.9). Conclusions and Clinical Importance Veterinary professionals should consider promoting healthy body condition for dogs, particularly from midlife onward