Model systems for regeneration: Xenopus.

Understanding how to promote organ and appendage regeneration is a key goal of regenerative medicine. The frog, Xenopus, can achieve both scar-free healing and tissue regeneration during its larval stages, although it predominantly loses these abilities during metamorphosis and adulthood. This transient regenerative capacity, alongside their close evolutionary relationship with humans, makes Xenopus an attractive model to uncover the mechanisms underlying functional regeneration. Here, we present an overview of Xenopus as a key model organism for regeneration research and highlight how studies of Xenopus have led to new insights into the mechanisms governing regeneration.

Stage-dependent cardiac regeneration in Xenopus is regulated by thyroid hormone availability.

Despite therapeutic advances, heart failure is the major cause of morbidity and mortality worldwide, but why cardiac regenerative capacity is lost in adult humans remains an enigma. Cardiac regenerative capacity widely varies across vertebrates. Zebrafish and newt hearts regenerate throughout life. In mice, this ability is lost in the first postnatal week, a period physiologically similar to thyroid hormone (TH)-regulated metamorphosis in anuran amphibians. We thus assessed heart regeneration in Xenopus laevis before, during, and after TH-dependent metamorphosis. We found that tadpoles display efficient cardiac regeneration, but this capacity is abrogated during the metamorphic larval-to-adult switch. Therefore, we examined the consequence of TH excess and deprivation on the efficiently regenerating tadpole heart. We found that either acute TH treatment or blocking TH production before resection significantly but differentially altered gene expression and kinetics of extracellular matrix components deposition, and negatively impacted myocardial wall closure, both resulting in an impeded regenerative process. However, neither treatment significantly influenced DNA synthesis or mitosis in cardiac tissue after amputation. Overall, our data highlight an unexplored role of TH availability in modulating the cardiac regenerative outcome, and present X. laevis as an alternative model to decipher the developmental switches underlying stage-dependent constraint on cardiac regeneration.

Predicting fracture risk in osteoporosis: the use of fracture prediction tools in an osteoporosis clinic population.

BACKGROUND: In the UK, the National Institute for Health and Care Excellence recommends either fracture risk assessment tool (FRAX) or QFracture to estimate the 10 year fracture risk of individuals. However, it is not known how these tools compare in determining risk and subsequent treatment using set intervention thresholds or guidelines. METHODS: The 10 year major osteoporotic (MO) and hip (HI) fracture risks were calculated for 100 women attending osteoporosis clinic in 2010 using FRAX and QFracture, and subsequent agreement to treatment between the tools was looked at using National Osteoporosis Foundation and National Bone Health Alliance thresholds (FRAX-20/3 and QFracture 20/3). We also looked at using these thresholds for QFracture and comparing them with the National Osteoporosis Guideline Group (NOGG) guidelines for FRAX (FRAX-NOGG). RESULTS: The 10 year risk for MO fracture for FRAX was 17.0% (IQR 10.8-24.0) and that of QFracture was 15.8% (IQR 9.5-27.7) (p=0.732). The 10 year risk for HI fracture for FRAX was 5.0% (IQR 2.1-8.9) and that of QFracture was 8.1% (IQR 2.5-21.6) (p<0.001). The agreement between FRAX-20/3 and QFracture-20/3 was greater than the agreement between FRAX-20/3 and FRAX-NOGG or QFracture-20/3 and FRAX-NOGG. CONCLUSIONS: The calculated 10 year risk for MO fracture between FRAX and QFracture was similar, whereas that of HI fracture was significantly different. The agreement to treatment between QFracture-20/3 and FRAX-NOGG was only 45%. Treatment decisions can differ depending on the fracture calculation tool used when coupled with certain intervention thresholds or guidelines.

Epidemiology of appendicular skeletal fractures: a cross-sectional analysis of data from the Nottingham Fracture Liaison Service.

BACKGROUND: The aim of this study is to describe the population-based age and gender appendicular skeletal fracture incidence in Nottinghamshire, a medium-size health county in the UK. METHOD: Data were analysed from the Nottingham Fracture Liaison Service Database over 4 years (2008-2011) on patients over 50 years of age. ICD-9 codes were used to classify fracture sites. Population figures from the Office of National Statistics were used to calculate fracture incidence rates per 10,000 population. Those aged 75 years and older were used to define the elderly population. RESULTS: A total of 7,767 patients with 7,806 fractures presented over this time period. The overall incidence in males was 24/10,000 and in females 73/10,000. The incidence of fracture increased with age in females (R (2) = 0.90) compared to males (R (2) = 0.06). The over 75 population is more likely to fracture their wrist (OR 1.36, 95% CI 1.24-1.50, p < 0.01), humerus (OR 1.87, 95% CI 1.65-2.12, p < 0.01) and clavicle (OR 1.34, 95% CI 1.07-1.69, p = 0.011) compared to other fractures. There was linearity with radius/ulna and humerus fractures with age (R (2) = 0.92, R (2) = 0.97, respectively). CONCLUSION: The incidence of appendicular skeletal fractures is high especially in the elderly female population with fractures of the upper limb more common. With an ageing population these numbers are projected to increase and therefore effective strategies to prevent falls and osteoporosis are clearly necessary.

Seasonality of adult fragility fractures and association with weather: 12-year experience of a UK Fracture Liaison Service.

PURPOSE: To investigate the monthly and seasonal variation in adult osteoporotic fragility fractures and the association with weather. METHODS: 12-year observational study of a UK Fracture Liaison Service (outpatient secondary care setting). Database analyses of the records of adult outpatients aged 50 years and older with fragility fractures. Weather data were obtained from the UK's national Meteorological Office. In the seasonality analyses, we tested for the association between months and seasons (determinants), respectively, and outpatient attendances, by analysis of variance (ANOVA) and Tukey's test. In the meteorological analyses, the determinants were mean temperature, mean daily maximum and minimum temperature, number of days of rain, total rainfall and number of days of frost, per month, respectively. We explored the association of each meteorological variable with outpatient attendances, by regression models. RESULTS: The Fracture Liaison Service recorded 25,454 fragility fractures. We found significant monthly and seasonal variation in attendances for fractures of the: radius or ulna; humerus; ankle, foot, tibia or fibula (ANOVA, all p-values <0.05). Fractures of the radius or ulna and humerus peaked in December and winter. Fractures of the ankle, foot, tibia or fibula peaked in July, August and summer. U-shaped associations were showed between each temperature parameter and fractures. Days of frost were directly associated with fractures of the radius or ulna (p-value <0.001) and humerus (p-value 0.002). CONCLUSION: Different types of fragility fractures present different seasonal patterns. Weather may modulate their seasonality and consequent healthcare utilisation.

The impact of lockdown during the COVID-19 pandemic on osteoporotic fragility fractures: an observational study.

We investigated whether osteoporotic fractures declined during lockdown, among adults aged 50 years and older. We showed that fewer outpatients attended the Fracture Clinic, for non-hip fractures, during lockdown; in contrast, no change in admissions for hip fractures was observed. This could be due to fewer outdoors falls, during lockdown. PURPOSE: Many countries implemented a lockdown to control the spread of the COVID-19 pandemic. We explored whether outpatient attendances to the Fracture Clinic for non-hip fragility fracture and inpatient admissions for hip fracture declined during lockdown, among adults aged 50 years and older, in a large secondary care hospital. METHODS: In our observational study, we analysed the records of 6681 outpatients attending the Fracture Clinic, for non-hip fragility fractures, and those of 1752 inpatients, admitted for hip fracture, during the time frames of interest. These were weeks 1st to 12th in 2020 ("prior to lockdown"), weeks 13th to 19th in 2020 ("lockdown") and corresponding periods over 2015 to 2019. We tested for differences in mean numbers (standard deviation (SD)) of outpatients and inpatients, respectively, per week, during the time frames of interest, across the years. RESULTS: Prior to lockdown, in 2020, 63.1 (SD 12.6) outpatients per week attended the Fracture Clinic, similar to previous years (p value 0.338). During lockdown, 26.0 (SD 7.3) outpatients per week attended the Fracture Clinic, fewer than previous years (p value < 0.001); similar findings were observed in both sexes and age groups (all p values < 0.001). During lockdown, 16.1 (SD 5.6) inpatients per week were admitted for hip fracture, similar to previous years (p value 0.776). CONCLUSION: During lockdown, fewer outpatients attended the Fracture Clinic, for non-hip fragility fractures, while no change in inpatient admissions for hip fracture was observed. This could reflect fewer non-hip fractures and may inform allocation of resources during pandemic.

Is adult cardiac regeneration absent in Xenopus laevis yet present in Xenopus tropicalis?

We recently used an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis, obtaining the result that the adult Xenopus heart is unable to regenerate. At 11 months post-amputation, cellular and biological marks of scarring persisted. We thus concluded that, contrary to urodeles and teleosts, adult anurans share a cardiac injury outcome similar to adult mammals. However, in their work published in this journal on the 13 December 2017, Liao et al. showed that the adult Xenopus tropicalis heart is capable of efficient, almost scar free regeneration, a result at odds with our previous observation. These findings contrast with and challenge the outcome of adult heart repair following injury in Xenopus species. Here we discuss the question of the intrinsic cardiac regenerative properties of an adult heart in anuran amphibians.

Probing the origin of matching functional jaws: roles of Dlx5/6 in cranial neural crest cells.

Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories.

In Vivo Transfection of Naked DNA into Xenopus Tadpole Tail Muscle.

In vivo gene transfer systems are important to study foreign gene expression and promoter regulation in an organism, with the benefit of exploring this in an integrated environment. Direct injection of plasmids encoding exogenous promoters and genes into muscle has numerous advantages: the protocol is easy, efficient, and shows time-persistent plasmid expression in transfected muscular cells. After injecting naked-DNA plasmids into tadpole tail muscle, transgene expression is strong, reproducible, and correlates with the amount of DNA injected. Moreover, expression is stable as long as the tadpoles remain, or are maintained, in premetamorphic stages. By directly expressing genes and regulated promoters in Xenopus tadpole muscle in vivo, one can exploit the powerful experimental advantages of gene transfer systems in an intact, physiologically normal animal.

Persistent fibrosis, hypertrophy and sarcomere disorganisation after endoscopy-guided heart resection in adult Xenopus.

Models of cardiac repair are needed to understand mechanisms underlying failure to regenerate in human cardiac tissue. Such studies are currently dominated by the use of zebrafish and mice. Remarkably, it is between these two evolutionary separated species that the adult cardiac regenerative capacity is thought to be lost, but causes of this difference remain largely unknown. Amphibians, evolutionary positioned between these two models, are of particular interest to help fill this lack of knowledge. We thus developed an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis. This method allowed in situ live heart observation, standardised tissue amputation size and reproducibility. During the first week following amputation, gene expression of cell proliferation markers remained unchanged, whereas those relating to sarcomere organisation decreased and markers of inflammation, fibrosis and hypertrophy increased. One-month post-amputation, fibrosis and hypertrophy were evident at the injury site, persisting through 11 months. Moreover, cardiomyocyte sarcomere organisation deteriorated early following amputation, and was not completely recovered as far as 11 months later. We conclude that the adult Xenopus heart is unable to regenerate, displaying cellular and molecular marks of scarring. Our work suggests that, contrary to urodeles and teleosts, with the exception of medaka, adult anurans share a cardiac injury outcome similar to adult mammals. This observation is at odds with current hypotheses that link loss of cardiac regenerative capacity with acquisition of homeothermy.

A novel photochemical cross-linking technology to improve luminal gain, vessel compliance, and buckling post-angioplasty in porcine arteries.

UNLABELLED: Development of substituted 1,8-naphthalimides for photochemical cross-linking of biomolecules is the focus of this research. This study describes limited cross-linking of collagen in the artery wall to control recoil and buckling in arteries following balloon angioplasty. Isolated porcine arteries were overstretched (25%) with balloon angioplasty (BA) +/- light-activated naphthalimide treatment (NVS). Lumen size and recoil were measured as retention of stretch after angioplasty. Cross-sectional compliance and distensibility coefficients were measured as slope of cross-sectional area versus increasing hydrostatic pressure. Buckling was measured, with 30% axial pre-stretch and 200 mmHg, as deviation from the center line. Electron microscopy evaluation of collagen fibers was conducted. RESULTS: Uninjured arteries have low compliance and low levels of buckling, whereas the BA-injured arteries demonstrated much greater compliance and buckling behavior. Treatment of the injured artery with NVS reduced buckling and demonstrated compliance midway between the two groups while retaining the increased luminal diameter imparted by angioplasty compared to untreated vessels. In summary, limited collagen cross-linking with NVS treatment resulted in lumen retention, as well as improved compliance without the accompanying rigidity and stiffness of conventional stent therapy or current cross-linking materials. This treatment shows great promise for dilation, repair and strengthening of arteries damaged by injury or vascular disease.

A United Kingdom perspective on the relationship between body mass index (BMI) and bone health: a cross sectional analysis of data from the Nottingham Fracture Liaison Service.

OBJECTIVES: This study examines the relationship between high BMI, a diagnosis of osteoporosis and low trauma fractures. METHOD: This is a cross sectional analysis using data collected from the Nottingham Fracture Liaison Service. A total of 4288 participants with a low trauma fracture from 1 January 2007 to 31 August 2012 were analysed. Logistic regression adjusted for potential confounders was used investigate osteoporosis and BMI. Fracture types were compared between those who were obese and non-obese. RESULTS: A total of 30% (1285) were obese. Prevalence of osteoporosis was 13.4%, 24.9%, and 40.4% in the obese, overweight and normal category respectively. Being obese has an odds ratio of 0.23 (95% CI 0.19-0.28, p<0.01) of having osteoporosis compared to a normal BMI category. When variable BMI cut offs were used (BMI 25, 30 and 35) to calculate the positive predictive value of patients not having osteoporosis, it was 80.5%, 86.3% and 88.3%. Examining fracture types, obese patients when compared with the non-obese category, were more likely to fracture their ankle (OR 1.48, p<0.01) and upper arm (OR 1.48, p<0.001), but were less likely to fracture their wrist (OR 0.65, p<0.001). In the elderly (>70years), obesity no longer influenced ankle or wrist fractures but there is an increased risk of upper arm fractures (OR 1.46, p=0.005). CONCLUSION: Higher BMD in obesity is not protective against fractures as there are a significant number of fractures in this group which may be due to body habitus, mechanism of injury and the effect of adiposity on bone. A low trauma osteoporotic fracture will need to be redefined in light of these findings.

Differential effects of non-nicotine tobacco constituent compounds on nicotine self-administration in rats.

Tobacco smoking has been shown to be quite addictive in people. However, nicotine itself is a weak reinforcer compared to other commonly abused drugs, leading speculation that other factors contribute to the high prevalence of tobacco addiction in the human population. In addition to nicotine, there are over 5000 chemical compounds that have been identified in tobacco smoke, and more work is needed to ascertain their potential contributions to tobacco's highly addictive properties, or as potential candidates for smoking cessation treatment. In this study, we examined seven non-nicotine tobacco constituent compounds (anabasine, anatabine, nornicotine, myosmine, harmane, norharmane, and tyramine) for their effects on nicotine self-administration behavior in rats. Young adult female Sprague-Dawley rats were allowed to self-administer nicotine (0.03 mg/kg/50 µl infusion) under a fixed ratio-1 schedule of reinforcement. Each self-administration session lasted 45 min. Doses of each tobacco constituent compound were administered subcutaneously 10 min prior to the start of each session in a repeated measures, counterbalanced order two times. Anabasine displayed a biphasic dose-effect function. Pretreatment with 0.02 mg/kg anabasine resulted in a 25% increase in nicotine self-administration, while 2.0mg/kg of anabasine reduced nicotine infusions per session by over 50%. Pretreatment with 2.0mg/kg anatabine also significantly reduced nicotine self-administration by nearly half. These results suggest that some non-nicotine tobacco constituents may enhance or reduce nicotine's reinforcing properties. Also, depending upon the appropriate dose, some of these compounds may also serve as potential smoking cessation agents.

Safety net hospital performance on national quality of care process measures.

Several studies have found poor or mixed performance by safety net hospitals on national measures of quality. The study's purposes were to determine whether safety net hospital performance is similar to the average U.S. hospital, both currently and during earlier reporting periods, and to summarize features commonly used to assess performance, including definition of safety net and patient characteristics. This study reviewed quality performance data for the Joint Commission's accountability measures for hospitals that are members of the National Association of Public Hospitals and Health Systems (NAPH)-safety net hospitals that serve a large proportion of Medicaid and uninsured patients. Analyses of quality performance on the earliest data show that on average there was no statistically significant difference in performance between NAPH members and other hospitals on 6 of 15 measures. According to the most recent data, NAPH hospitals on average had no statistically significant differences as other hospitals on 13 of 18 measures and had statistically significantly better scores on two measures. These results are an important addition to the literature regarding safety net hospitals that serve a high proportion of Medicaid, low income, and uninsured patients, and support the case that quality of care at safety net hospitals is equivalent to that of non-safety net hospitals.

Lipid-lowering effect of berberine in human subjects and rats.

Due to serious adverse effects and the limited effectiveness of currently available pharmacological therapies for obesity, many research efforts have focused on the development of drugs from natural products. Our previous studies demonstrated that berberine, an alkaloid originally isolated from traditional Chinese herbs, prevented fat accumulation in vitro and in vivo. In this pilot study, obese human subjects (Caucasian) were given 500 mg berberine orally three times a day for twelve weeks. The efficacy and safety of berberine treatment was determined by measurements of body weight, comprehensive metabolic panel, blood lipid and hormone levels, expression levels of inflammatory factors, complete blood count, and electrocardiograph. A Sprague-Dawley rat experiment was also performed to identify the anti-obesity effects of berberine treatment. The results demonstrate that berberine treatment produced a mild weight loss (average 5 lb/subject) in obese human subjects. But more interestingly, the treatment significantly reduced blood lipid levels (23% decrease of triglyceride and 12.2% decrease of cholesterol levels) in human subjects. The lipid-lowering effect of berberine treatment has also been replicated in the rat experiment (34.7% decrease of triglyceride and 9% decrease of cholesterol level). Cortisol, calcitriol, ACTH, TSH, FT4, and SHBG levels were not significantly changed following 12 weeks of berberine treatment. However, there was interestingly, an increase in calcitriol levels seen in all human subjects following berberine treatment (mean 59.5% increase, p=0.11). Blood inflammatory factors (CRP, IL-6, TNFα, COX-2) and erythrocyte sedimentation rate (ESR) were not significantly affected by treatment with berberine. Tests of hematological, cardiovascular, liver, and kidney function following berberine treatment showed no detrimental side effects to this natural compound. Collectively, this study demonstrates that berberine is a potent lipid-lowering compound with a moderate weight loss effect, and may have a possible potential role in osteoporosis treatment/prevention.