Study of odontoclast dysregulation in feline tooth resorption

Feline tooth resorption (TR) is a common and painful disease characterised by loss of mineralised tissues of the tooth. Due to the progressive nature of the disease the only available treatment is to extract affected teeth. Odontoclasts are specialised cells that resorb teeth and they share many similarities with osteoclasts, the bone resorbing cells. The main physiological role of these cells is to resorb deciduous teeth to allow for the permanent tooth replacement. In some cats these cells become dysregulated and attack the permanent teeth later in life. However, the aetiology of tooth resorption is still unclear. The aim of this PhD project was to identify transcriptomic changes between TR negative and TR positive teeth, set up a TR model to study odontoclast dysregulation in vitro and to use this system to test a novel therapeutic target for the treatment of TR. In this study, a successful RNA isolation procedure for feline teeth collected in a clinical setting and at post-mortem was developed. To elucidate the role of inflammation and vitamin D involvement in TR, quantitative PCR was performed but there was no statistically significant difference in the expression of any inflammatory cytokines, vitamin D receptor or receptor activator of nuclear factor kappa-B ligand (RANKL) between TR negative and TR positive teeth. Feline osteoclasts were successfully generated from feline bone marrow in vitro. I also found that primary feline periodontal ligament cells could induce osteoclast formation. Osteoclast precursors from TR positive cats formed higher numbers of osteoclasts when co-cultured with periodontal ligament cells compared to TR negative cats. Transcriptome analysis by RNA sequencing identified many differentially expressed genes between TR negative and TR positive teeth from the same cat. I also found that up-regulated genes in TR positive teeth, from the literature, are known to be associated with osteoclast differentiation and calcium signalling pathways. Apart from the genes related to osteoclast biology, I also identified up-regulated genes involved in muscle and tooth development, which suggests TR positive teeth undergo repair or bone formation in response to tooth resorption. Based on RNA sequencing data and quantitative PCR, I was able to identify a list of genes which may be involved in odontoclast dysregulation in TR. Among those, matrix metalloproteinase 9 (MMP9) was of great interest due to its potential role in osteoclast biology. High MMP9 expressing odontoclasts were found in tooth sections undergoing tooth resorption by immunohistochemistry. In functional studies, MMP9 inhibitor reduced both osteoclast formation and resorption activity in vitro. Furthermore, feline MMP9 siRNA inhibited osteoclast differentiation but showed little effect on dentine resorption activity. These results confirm the hypothesis that the transcriptome of TR positive teeth was locally different from TR negative teeth. The results revealed an upregulation of genes in pathways associated with osteoclast formation in TR positive teeth. Further testing of candidate genes as potential therapeutic targets in feline TR could be performed using the feline tooth resorption model system developed in this study

Use of Omics Data in Fracture Prediction; a Scoping and Systematic Review in Horses and Humans

Despite many recent advances in imaging and epidemiological data analysis, musculoskeletal injuries continue to be a welfare issue in racehorses. Peptide biomarker studies have failed to consistently predict bone injury. Molecular profiling studies provide an opportunity to study equine musculoskeletal disease. A systematic review of the literature was performed using preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P) guidelines to assess the use of miRNA profiling studies in equine and human musculoskeletal injuries. Data were extracted from 40 papers between 2008 and 2020. Three miRNA studies profiling equine musculoskeletal disease were identified, none of which related to equine stress fractures. Eleven papers studied miRNA profiles in osteoporotic human patients with fractures, but differentially expressed miRNAs were not consistent between studies. MicroRNA target prediction programmes also produced conflicting results between studies. Exercise affected miRNA profiles in both horse and human studies (e.g., miR-21 was upregulated by endurance exercise and miR-125b was downregulated by exercise). MicroRNA profiling studies in horses continue to emerge, but as yet, no miRNA profile can reliably predict the occurrence of fractures. It is very important that future studies are well designed to mitigate the effects of variation in sample size, exercise and normalisation methods

DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature neurons.

Neuronal morphology and circuitry established during early development must often be maintained over the entirety of animal lifespans. Compared with neuronal development, the mechanisms that maintain mature neuronal structures and architecture are little understood. The conserved disco-interacting protein 2 (DIP2) consists of a DMAP1-binding domain and two adenylate-forming domains (AFDs). We show that the Caenorhabditis elegans DIP-2 maintains morphology of mature neurons. dip-2 loss-of-function mutants display a progressive increase in ectopic neurite sprouting and branching during late larval and adult life. In adults, dip-2 also inhibits initial stages of axon regeneration cell autonomously and acts in parallel to DLK-1 MAP kinase and EFA-6 pathways. The function of DIP-2 in maintenance of neuron morphology and in axon regrowth requires its AFD domains and is independent of its DMAP1-binding domain. Our findings reveal a new conserved regulator of neuronal morphology maintenance and axon regrowth after injury

Safety and efficacy study of laparoscopic or robotic radical surgery using an endoscopic stapler for inhibiting tumour spillage of cervical malignant neoplasms evaluating survival (SOLUTION): a multi-centre, open-label, single-arm, phase II trial protocol

The Laparoscopic Approach to Cervical Cancer trial and Surveillance, Epidemiology, and End Results program database study demonstrated that minimally invasive radical hysterectomy was inferior to abdominal radical hysterectomy in terms of disease recurrence and survival. Among risk factors related to poor prognosis after minimally invasive surgery (MIS), tumour spillage during intracorporeal colpotomy became a significant issue. Thus, we designed this trial to evaluate the efficacy and safety of minimally invasive radical hysterectomy using an endoscopic stapler for early-stage cervical cancer. This trial is a prospective, multi-centre, open-label, single-arm, non-inferiority phase II study. The nine organisations will participate in this trial after the approval of the institutional review board. Major eligibility criteria include women aged 20 years or older with cervical cancer stage IB1 squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma according to the revised 2009 FIGO staging system who will undergo type B2 or C hysterectomy by MIS. The primary endpoint is the 4.5-year disease-free survival (DFS) rate between abdominal radical hysterectomy and MIS using an endoscopic stapler. For calculating the sample size, we hypothesised that the 4.5-year DFS rate after MIS using an endoscopic stapler is assumed to be the same after abdominal radical hysterectomy at 90.9%, and the non-inferiority margin was 7.2%. When we consider a three-year accrual and 4.5-year follow-up, at least 13 events must happen, requiring a total of 111 patients assuming a statistical power of 80% and the one-tailed test of 5% significance. A total of 124 patients is needed, considering a drop-out rate of 10%. We expect intracorporeal colpotomy using an endoscopic stapler may prevent tumour spillage during MIS for stage IB1 cervical cancer, showing a comparable prognosis with abdominal radical surgery.This study was supported by Johnson & Johnson. The funder has no role in study design, writing of the manuscript and the decision to submit the report for publication