Inducing pluripotency in the domestic cat (Felis Catus)

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into derivatives of all three germ layers, therefore overcoming the limitations of primary differentiated cells and the ethical concerns of using embryonic stem cells. No studies however report the generation of iPSCs from domestic cats (fiPSCs). Feline adipose derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4, Sox2, Klf4, cMyc and Nanog. Isolated iPSC clones were expanded on mouse inactivated embryonic fibroblasts in the presence of feline leukaemia inhibitory factor (LIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required foetal bovine serum and feline LIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4, Sox2, Nanog and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by qPCR. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the generation of the first induced pluripotent cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies

Synthesis and formulation of four-arm PolyDMAEA-siRNA polyplex for transient downregulation of collagen type III gene expression in TGF-β1 stimulated tenocyte culture

The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-β1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-β1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL–1 TGF-β1 and 3-fold increase of COL3α1 expression at 48 h in cultured tenocytes. The PDMAEA-siRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries

Osteoblast differentiation of equine induced pluripotent stem cells.

Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including COL1A1, SPARC, SPP1, IBSP, RUNX2 and BGALP We also demonstrate that iPSC-osteoblasts are able to produce a mineralised matrix with both calcium and hydroxyapatite deposition. Alkaline phosphatase activity is also significantly increased during osteoblast differentiation. Although the genetic background of the iPSC donor animal affects the level of differentiation observed after 21 days of differentiation, less variation between lines of iPSCs derived from the same horse was observed. The successful, direct, differentiation of equine iPSCs into osteoblasts may provide a source of cells for future regenerative medicine strategies to improve fracture repair in horses undergoing surgery. iPSC-derived osteoblasts will also provide a potential tool to study equine bone development and disease.Anne Duchess of Cambridge Charitable Trust, Paul Mellon Foundation, Cambridge Turs

A Functional Single-Nucleotide Polymorphism Upstream of the Collagen Type III Gene Is Associated with Catastrophic Fracture Risk in Thoroughbred Horses

Fractures caused by bone overloading are a leading cause of euthanasia in Thoroughbred racehorses. The risk of fatal fracture has been shown to be influenced by both environmental and genetic factors but, to date, no specific genetic mechanisms underpinning fractures have been identified. In this study, we utilised a genome-wide polygenic risk score to establish an in vitro cell system to study bone gene regulation in horses at high and low genetic risk of fracture. Candidate gene expression analysis revealed differential expression of COL3A1 and STAT1 genes in osteoblasts derived from high- and low-risk horses. Whole-genome sequencing of two fracture cases and two control horses revealed a single-nucleotide polymorphism (SNP) upstream of COL3A1 that was confirmed in a larger cohort to be significantly associated with fractures. Bioinformatics tools predicted that this SNP may impact the binding of the transcription factor SOX11. Gene modulation demonstrated SOX11 is upstream of COL3A1, and the region binds to nuclear proteins. Furthermore, luciferase assays demonstrated that the region containing the SNP has promoter activity. However, the specific effect of the SNP depends on the broader genetic background of the cells and suggests other factors may also be involved in regulating COL3A1 expression. In conclusion, we have identified a novel SNP that is significantly associated with fracture risk and provide new insights into the regulation of the COL3A1 gene

The incidence of myocardial injury following post-operative Goal Directed Therapy.

BACKGROUND: Studies suggest that Goal Directed Therapy (GDT) results in improved outcome following major surgery. However, there is concern that pre-emptive use of inotropic therapy may lead to an increased incidence of myocardial ischaemia and infarction. METHODS: Post hoc analysis of data collected prospectively during a randomised controlled trial of the effects of post-operative GDT in high-risk general surgical patients. Serum troponin T concentrations were measured at baseline and on day 1 and day 2 following surgery. Continuous ECG monitoring was performed during the eight hour intervention period. Patients were followed up for predefined cardiac complications. A univariate analysis was performed to identify any associations between potential risk factors for myocardial injury and elevated troponin T concentrations. RESULTS: GDT was associated with fewer complications, and a reduced duration of hospital stay. Troponin T concentrations above 0.01 microg l-1 were identified in eight patients in the GDT group and six in the control group. Values increased above 0.05 microg l-1 in four patients in the GDT group and two patients in the control group. There were no overall differences in the incidence of elevated troponin T concentrations. The incidence of cardiovascular complications was also similar. None of the patients, in whom troponin T concentrations were elevated, developed ECG changes indicating myocardial ischaemia during the intervention period. The only factor to be associated with elevated troponin T concentrations following surgery was end-stage renal failure. CONCLUSION: The use of post-operative GDT does not result in an increased incidence of myocardial injury

Effects of noise exposure on young adults with normal audiograms II: Behavioral measures

An estimate of lifetime noise exposure was used as the primary predictor of performance on a range of behavioral tasks: frequency and intensity difference limens, amplitude modulation detection, interaural phase discrimination, the digit triplet speech test, the co-ordinate response speech measure, an auditory localization task, a musical consonance task and a subjective report of hearing ability. One hundred and thirty-eight participants (81 females) aged 18–36 years were tested, with a wide range of self-reported noise exposure. All had normal pure-tone audiograms up to 8 kHz. It was predicted that increased lifetime noise exposure, which we assume to be concordant with noise-induced cochlear synaptopathy, would elevate behavioral thresholds, in particular for stimuli with high levels in a high spectral region. However, the results showed little effect of noise exposure on performance. There were a number of weak relations with noise exposure across the test battery, although many of these were in the opposite direction to the predictions, and none were statistically significant after correction for multiple comparisons. There were also no strong correlations between electrophysiological measures of synaptopathy published previously and the behavioral measures reported here. Consistent with our previous electrophysiological results, the present results provide no evidence that noise exposure is related to significant perceptual deficits in young listeners with normal audiometric hearing. It is possible that the effects of noise-induced cochlear synaptopathy are only measurable in humans with extreme noise exposures, and that these effects always co-occur with a loss of audiometric sensitivity

Covert observation in practice: lessons from the evaluation of the prohibition of smoking in public places in Scotland

Background: A ban on smoking in wholly or substantially enclosed public places has been in place in Scotland since 26th March 2006. The impact of this legislation is currently being evaluated in seven studies, three of which involve direct observation of smoking in bars and other enclosed public places. While the ethical issues around covert observation have been widely discussed there is little practical guidance on the conduct of such research. A workshop was therefore convened to identify practical lessons learned so far from the Scottish evaluation. Methods: We convened a workshop involving researchers from the three studies which used direct observation. In addition, one of the fieldwork managers collected written feedback on the fieldwork, identifying problems that arose in the field and some solutions. Results: There were four main themes identified: (i) the difficulty of achieving and maintaining concealment; (ii) the experience of being an observer; (iii) the risk of bias in the observations and (iv) issues around training and recruitment. These are discussed. Conclusion: Collecting covert observational data poses unique practical challenges, in particular in relation to the health and safety of the researcher. The findings and solutions presented in this paper will be of value to researchers designing similar studies

Effects of noise exposure on young adults with normal audiograms I: electrophysiology

Noise-induced cochlear synaptopathy has been demonstrated in numerous rodent studies. In these animal models, the disorder is characterized by a reduction in amplitude of wave I of the auditory brainstem response (ABR) to high-level stimuli, whereas the response at threshold is unaffected. The aim of the present study was to determine if this disorder is prevalent in young adult humans with normal audiometric hearing. One hundred and twenty six participants (75 females) aged 18-36 were tested. Participants had a wide range of lifetime noise exposures as estimated by a structured interview. Audiometric thresholds did not differ across noise exposures up to 8 kHz, although 16- kHz audiometric thresholds were elevated with increasing noise exposure for females but not for males. ABRs were measured in response to high-pass (1.5 kHz) filtered clicks of 80 and 100 dB peSPL. Frequency-following responses (FFRs) were measured to 80 dB SPL pure tones from 240- 285 Hz, and to 80 dB SPL 4 kHz pure tones amplitude modulated at frequencies from 240-285 Hz (transposed tones). The bandwidth of the ABR stimuli and the carrier frequency of the transposed tones were chosen to target the 3-6 kHz characteristic frequency region which is usually associated with noise damage in humans. The results indicate no relation between noise exposure and the amplitude of the ABR. In particular, wave I of the ABR did not decrease with increasing noise exposure as predicted. ABR wave V latency increased with increasing noise exposure for the 80 dB peSPL click. High carrier-frequency (envelope) FFR amplitudes decreased as a function of noise exposure in males but not females. However, these correlations were not significant after the effects of age were controlled. The results suggest either that noise-induced cochlear synaptopathy is not a significant problem in young, audiometrically normal adults, or that the ABR and FFR are relatively insensitive to this disorder in young humans, although it is possible that the effects become more pronounced with age

Improving higher education standards through reengineering in West African universities–A case study of Nigeria

This article examines the context of higher education (HE), policies and challenges in the West African context. A multi-level framework and analysis of reengineering, leading change in complexity, activity-based view of the University Business Model and Pedagogical Content Knowledge enable the development of deep connections between the macro- and meso-level and -micro challenges of Higher Education System (HES). These include elements of effective leadership, structures and curriculum and learning pedagogies. Drawing on the analyses of interviews from 25 overseas trained senior academics from Nigerian universities, a preliminary refinement of the philosophy of reengineering, re-thinking and revaluing the higher education system (HES) is offered. These have traditionally been addressed in a piecemeal perspective in HE policy and the academic literature; such a traditional approach has not been the systematic rethinking advocated in the philosophy of reengineering

Slow Solar Wind Connection Science during Solar Orbiter’s First Close Perihelion Passage

The Slow Solar Wind Connection Solar Orbiter Observing Plan (Slow Wind SOOP) was developed to utilize the extensive suite of remote-sensing and in situ instruments on board the ESA/NASA Solar Orbiter mission to answer significant outstanding questions regarding the origin and formation of the slow solar wind. The Slow Wind SOOP was designed to link remote-sensing and in situ measurements of slow wind originating at open–closed magnetic field boundaries. The SOOP ran just prior to Solar Orbiter’s first close perihelion passage during two remote-sensing windows (RSW1 and RSW2) between 2022 March 3–6 and 2022 March 17–22, while Solar Orbiter was at respective heliocentric distances of 0.55–0.51 and 0.38–0.34 au from the Sun. Coordinated observation campaigns were also conducted by Hinode and IRIS. The magnetic connectivity tool was used, along with low-latency in situ data and full-disk remote-sensing observations, to guide the target pointing of Solar Orbiter. Solar Orbiter targeted an active region complex during RSW1, the boundary of a coronal hole, and the periphery of a decayed active region during RSW2. Postobservation analysis using the magnetic connectivity tool, along with in situ measurements from MAG and SWA/PAS, showed that slow solar wind originating from two out of three of the target regions arrived at the spacecraft with velocities between ∼210 and 600 km s−1. The Slow Wind SOOP, despite presenting many challenges, was very successful, providing a blueprint for planning future observation campaigns that rely on the magnetic connectivity of Solar Orbiter