Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.

The relationship between the pH value of a hydration solution and the biomechanical properties of Crosado-embalmed human iliotibial bands.

Determining the biomechanical properties of human tissues commonly involves the immersion or spraying of the tissues to maintain them in a hydrated state. However, the influence of the pH value of these solutions on the biomechanical properties of the tissues is not well understood. This study investigated the effects of the pH value on the biomechanical properties of the collagen-rich human iliotibial band (ITB). A total of 124 samples were allocated to polyethylene glycol (PEG) solutions of pH values between 3 and 13 for 24 h, which is a frequently used immersion time prior to biomechanical tests. After this, the samples were biomechanically tested in a uniaxial tensile testing setup using an established testing routine. Similarly, 69 samples were allocated to pH groups of 6, 7 and 8 and biomechanically tested after 1, 2 and 3 weeks. The cross-sectional area of all samples was determined after immersion into the PEG solutions for the specified time frames. In the 24-h experiment, the elastic modulus (pH 12: p ≤ 0.045; pH 13: p ≤ 0.020) and the ultimate tensile strength (pH 12: p ≤ 0.031; pH 13: p ≤ 0.026) of the pH groups 12 and 13 were significantly lower and their cross-sectional areas were higher (pH 12: p ≤ 0.005; pH 13: p ≤ 0.003) compared to several groups of acidic to alkaline pH values. There was no difference in the maximum forces between the different groups within a 24-h immersion time (p > 0.999). In the 3-week-test, a decrease of the ultimate tensile strength was noted between the 24-h and 3 week values for the pH groups 7 (p = 0.034) and 8 (p = 0.029). It is concluded that pH-dependent tissue swelling influences the cross-sectional area-dependent biomechanical properties of the human ITB. Therefore, the pH value of storage and hydration solutions for the preparation of biomechanical tests should be recorded. From a biomechanical perspective, the collagen stability of the human ITB is largely unaltered in PEG solutions with pH values between 3 and 13 over 24 h.

Plant traits controlling growth change in response to a drier climate.

Plant traits are increasingly being used to improve prediction of plant function, including plant demography. However, the capability of plant traits to predict demographic rates remains uncertain, particularly in the context of trees experiencing a changing climate. Here we present data combining 17 plant traits associated with plant structure, metabolism and hydraulic status, with measurements of long-term mean, maximum and relative growth rates for 176 trees from the world's longest running tropical forest drought experiment. We demonstrate that plant traits can predict mean annual tree growth rates with moderate explanatory power. However, only combinations of traits associated more directly with plant functional processes, rather than more commonly employed traits like wood density or leaf mass per area, yield the power to predict growth. Critically, we observe a shift from growth being controlled by traits related to carbon cycling (assimilation and respiration) in well-watered trees, to traits relating to plant hydraulic stress in drought-stressed trees. We also demonstrate that even with a very comprehensive set of plant traits and growth data on large numbers of tropical trees, considerable uncertainty remains in directly interpreting the mechanisms through which traits influence performance in tropical forests.

Amazonia trees have limited capacity to acclimate plant hydraulic properties in response to long-term drought.

The fate of tropical forests under future climate change is dependent on the capacity of their trees to adjust to drier conditions. The capacity of trees to withstand drought is likely to be determined by traits associated with their hydraulic systems. However, data on whether tropical trees can adjust hydraulic traits when experiencing drought remain rare. We measured plant hydraulic traits (e.g. hydraulic conductivity and embolism resistance) and plant hydraulic system status (e.g. leaf water potential, native embolism and safety margin) on >150 trees from 12 genera (36 species) and spanning a stem size range from 14 to 68 cm diameter at breast height at the world's only long-running tropical forest drought experiment. Hydraulic traits showed no adjustment following 15 years of experimentally imposed moisture deficit. This failure to adjust resulted in these drought-stressed trees experiencing significantly lower leaf water potentials, and higher, but variable, levels of native embolism in the branches. This result suggests that hydraulic damage caused by elevated levels of embolism is likely to be one of the key drivers of drought-induced mortality following long-term soil moisture deficit. We demonstrate that some hydraulic traits changed with tree size, however, the direction and magnitude of the change was controlled by taxonomic identity. Our results suggest that Amazonian trees, both small and large, have limited capacity to acclimate their hydraulic systems to future droughts, potentially making them more at risk of drought-induced mortality.

Drought stress and tree size determine stem CO2 efflux in a tropical forest.

CO2 efflux from stems (CO2_stem ) accounts for a substantial fraction of tropical forest gross primary productivity, but the climate sensitivity of this flux remains poorly understood. We present a study of tropical forest CO2_stem from 215 trees across wet and dry seasons, at the world's longest running tropical forest drought experiment site. We show a 27% increase in wet season CO2_stem in the droughted forest relative to a control forest. This was driven by increasing CO2_stem in trees 10-40 cm diameter. Furthermore, we show that drought increases the proportion of maintenance to growth respiration in trees > 20 cm diameter, including large increases in maintenance respiration in the largest droughted trees, > 40 cm diameter. However, we found no clear taxonomic influence on CO2_stem and were unable to accurately predict how drought sensitivity altered ecosystem scale CO2_stem , due to substantial uncertainty introduced by contrasting methods previously employed to scale CO2_stem fluxes. Our findings indicate that under future scenarios of elevated drought, increases in CO2_stem may augment carbon losses, weakening or potentially reversing the tropical forest carbon sink. However, due to substantial uncertainties in scaling CO2_stem fluxes, stand-scale future estimates of changes in stem CO2 emissions remain highly uncertain.

Stand dynamics modulate water cycling and mortality risk in droughted tropical forest.

Transpiration from the Amazon rainforest generates an essential water source at a global and local scale. However, changes in rainforest function with climate change can disrupt this process, causing significant reductions in precipitation across Amazonia, and potentially at a global scale. We report the only study of forest transpiration following a long-term (>10 year) experimental drought treatment in Amazonian forest. After 15 years of receiving half the normal rainfall, drought-related tree mortality caused total forest transpiration to decrease by 30%. However, the surviving droughted trees maintained or increased transpiration because of reduced competition for water and increased light availability, which is consistent with increased growth rates. Consequently, the amount of water supplied as rainfall reaching the soil and directly recycled as transpiration increased to 100%. This value was 25% greater than for adjacent nondroughted forest. If these drought conditions were accompanied by a modest increase in temperature (e.g., 1.5°C), water demand would exceed supply, making the forest more prone to increased tree mortality.

Malocclusion model of temporomandibular joint osteoarthritis in mice with and without receptor for advanced glycation end products.

OBJECTIVE: This study has two aims: 1. Validate a non-invasive malocclusion model of mouse temporomandibular joint (TMJ) osteoarthritis (OA) that we developed and 2. Confirm role of inflammation in TMJ OA by comparing the disease in the presence and absence of the receptor for advanced glycation end products (RAGE). DESIGN: The malocclusion procedure was performed on eight week old mice, either wild type (WT) or without RAGE. RESULTS: We observed TMJ OA at two weeks post-misalignment/malocclusion. The modified Mankin score used for the semi-quantitative assessment of OA showed an overall significantly higher score in mice with malocclusion compared to control mice at all times points (2, 4, 6 and 8 weeks). Mice with malocclusion showed a decrease in body weight by the first week after misalignment but returned to normal weight for their ages during the following weeks. The RAGE knock out (KO) mice had statistically lower modified Mankin scores compared to WT mice of the same age. The RAGE KO mice had statistically lower levels of Mmp-13 and HtrA1 but higher Tgf-ß1, as measured by immunohistochemistry, compared to WT mice at eight weeks post malocclusion. CONCLUSIONS: We demonstrate an inexpensive, efficient, highly reproducible and non-invasive model of mouse TMJ OA. The mechanical nature of the malocclusion resembles the natural development of TMJ OA in humans, making this an ideal model in future studies that aim to elucidate the pathogenesis of the disease leading to the discovery of a treatment. The RAGE plays a role in mouse TMJ OA.

After more than a decade of soil moisture deficit, tropical rainforest trees maintain photosynthetic capacity, despite increased leaf respiration.

Determining climate change feedbacks from tropical rainforests requires an understanding of how carbon gain through photosynthesis and loss through respiration will be altered. One of the key changes that tropical rainforests may experience under future climate change scenarios is reduced soil moisture availability. In this study we examine if and how both leaf photosynthesis and leaf dark respiration acclimate following more than 12 years of experimental soil moisture deficit, via a through-fall exclusion experiment (TFE) in an eastern Amazonian rainforest. We find that experimentally drought-stressed trees and taxa maintain the same maximum leaf photosynthetic capacity as trees in corresponding control forest, independent of their susceptibility to drought-induced mortality. We hypothesize that photosynthetic capacity is maintained across all treatments and taxa to take advantage of short-lived periods of high moisture availability, when stomatal conductance (gs ) and photosynthesis can increase rapidly, potentially compensating for reduced assimilate supply at other times. Average leaf dark respiration (Rd ) was elevated in the TFE-treated forest trees relative to the control by 28.2 ± 2.8% (mean ± one standard error). This mean Rd value was dominated by a 48.5 ± 3.6% increase in the Rd of drought-sensitive taxa, and likely reflects the need for additional metabolic support required for stress-related repair, and hydraulic or osmotic maintenance processes. Following soil moisture deficit that is maintained for several years, our data suggest that changes in respiration drive greater shifts in the canopy carbon balance, than changes in photosynthetic capacity.

'Sink or swim': an evaluation of the clinical characteristics of individuals with high bone mass.

SUMMARY: High bone mineral density on routine dual energy X-ray absorptiometry (DXA) may indicate an underlying skeletal dysplasia. Two hundred fifty-eight individuals with unexplained high bone mass (HBM), 236 relatives (41% with HBM) and 58 spouses were studied. Cases could not float, had mandible enlargement, extra bone, broad frames, larger shoe sizes and increased body mass index (BMI). HBM cases may harbour an underlying genetic disorder. INTRODUCTION: High bone mineral density is a sporadic incidental finding on routine DXA scanning of apparently asymptomatic individuals. Such individuals may have an underlying skeletal dysplasia, as seen in LRP5 mutations. We aimed to characterize unexplained HBM and determine the potential for an underlying skeletal dysplasia. METHODS: Two hundred fifty-eight individuals with unexplained HBM (defined as L1 Z-score ≥ +3.2 plus total hip Z-score ≥ +1.2, or total hip Z-score ≥ +3.2) were recruited from 15 UK centres, by screening 335,115 DXA scans. Unexplained HBM affected 0.181% of DXA scans. Next 236 relatives were recruited of whom 94 (41%) had HBM (defined as L1 Z-score + total hip Z-score ≥ +3.2). Fifty-eight spouses were also recruited together with the unaffected relatives as controls. Phenotypes of cases and controls, obtained from clinical assessment, were compared using random-effects linear and logistic regression models, clustered by family, adjusted for confounders, including age and sex. RESULTS: Individuals with unexplained HBM had an excess of sinking when swimming (7.11 [3.65, 13.84], p < 0.001; adjusted odds ratio with 95% confidence interval shown), mandible enlargement (4.16 [2.34, 7.39], p < 0.001), extra bone at tendon/ligament insertions (2.07 [1.13, 3.78], p = 0.018) and broad frame (3.55 [2.12, 5.95], p < 0.001). HBM cases also had a larger shoe size (mean difference 0.4 [0.1, 0.7] UK sizes, p = 0.009) and increased BMI (mean difference 2.2 [1.3, 3.1] kg/m(2), p < 0.001). CONCLUSION: Individuals with unexplained HBM have an excess of clinical characteristics associated with skeletal dysplasia and their relatives are commonly affected, suggesting many may harbour an underlying genetic disorder affecting bone mass.

The effect of prior bisphosphonate therapy on the subsequent therapeutic effects of strontium ranelate over 2 years.

UNLABELLED: Many osteoporotic women prescribed strontium ranelate have previously received bisphosphonates. Prior bisphosphonate use blunted the spinal bone mineral density (BMD) response for 6 months. Hip BMD was blunted to a degree for 2 years, although there was an overall increase in hip BMD in contrast to the heel where BMD did not increase. INTRODUCTION: Many osteoporotic women commenced on strontium ranelate have already received treatment with bisphosphonates. This study investigates whether prior bisphosphonate use impairs the subsequent therapeutic response to strontium ranelate. METHODS: Women were recruited who were either bisphosphonate naïve or currently receiving a bisphosphonate. All women received strontium ranelate and were followed up for 2 years. RESULTS: One hundred and twenty women were recruited. After 2 years, the bisphosphonate-naïve group had significant BMD increases of 8.9%, 6.0% and 6.4% at the spine, hip and heel, respectively. In the prior bisphosphonate group, BMD increased significantly at the spine (4.0%) and hip (2.5%) but not at the heel. At all time points at all sites, the BMD increase was greater in the bisphosphonate-naïve group. BMD at the spine did not increase during the first 6 months in the prior bisphosphonate group but then increased in parallel with the bisphosphonate-naïve group. In contrast, the difference between the two groups in hip BMD continued to increase throughout the 2 years. P1NP was suppressed in the prior bisphosphonate group for the first 6 months. CONCLUSIONS: After bisphosphonate exposure, the BMD response to strontium ranelate is blunted for only 6 months at the spine. At the hip, a degree of blunting was observed over 2 years, although there was an overall increase in hip BMD in contrast to the heel where no increase in BMD was observed.

Obstetric and perinatal outcomes in pregnancies complicated by Type 1 and Type 2 diabetes: influences of glycaemic control, obesity and social disadvantage.

AIMS: To compare obstetric and perinatal outcomes in women with Type 1 and Type 2 diabetes and relate these to maternal risk factors. METHODS: Prospective cohort study of 682 consecutive diabetic pregnancies in East Anglia during 2006-2009. Relationships between congenital malformation, perinatal mortality and perinatal morbidity (large for gestational age, preterm delivery, neonatal care) with maternal age, parity, ethnicity, glycaemic control, obesity and social disadvantage were examined using bivariable and multivariate models. RESULTS: There were 408 (59.8%) Type 1 and 274 (40.2%) Type 2 diabetes pregnancies. Women with Type 2 diabetes were older (P < 0.001), heavier (P < 0.0001), more frequently multiparous (P < 0.001), more ethnically diverse (p < 0.0001) and more socially disadvantaged (P = 0.0004). Although women with Type 2 diabetes had shorter duration of diabetes (P < 0.0001) and better pre-conception glycaemic control [HbA(1c) 52 mmol/mol (6.9%) Type 2 diabetes vs. 63 mmol/l (7.9%) Type 1 diabetes; p < 0.0001), rates of congenital malformation and perinatal mortality were comparable. Women with Type 2 diabetes had fewer large-for-gestational-age infants (37.6 vs. 52.9%, P < 0.0008), fewer preterm deliveries (17.5 vs. 37.1%, P < 0.0001) and their offspring had fewer neonatal care admissions (29.8 vs. 43.2%, P = 0.001). Third trimester HbA(1c) (OR 1.35, 95% CI 1.09-1.67, P = 0.006) and social disadvantage (OR 0.80, 95% CI 0.67-0.98; P = 0.03) were risk factors for large for gestational age. CONCLUSIONS: Despite increased age, parity, obesity and social disadvantage, women with Type 2 diabetes had better glycaemic control, fewer large-for-gestational-age infants, fewer preterm deliveries and fewer neonatal care admissions. Better tools are needed to improve glycaemic control and reduce the rates of large for gestational age, particularly in Type 1 diabetes.

A checklist for streamlining organ donation after cardiac death.

There is currently a shortage of organ donors to meet the demands of transplantation waiting lists. In recent years there has been renewed interest in donation after cardiac death in order to increase the pool of potential donors. The Organ and Tissue Authority has recently developed a national policy for donation after cardiac death. We describe here a checklist that is used by our hospital-based staff for organ donation which outlines important steps in the donation after cardiac death process.

Personal experiences of women with diabetes who do not attend pre-pregnancy care.

AIMS: To explore the views of women who did not attend pre-pregnancy care (PPC), in particular their accounts of contraception, previous pregnancies and the influence of healthcare advice. METHODS: We conducted semi-structured interviews with 29 pregnant women (21 with Type 1 diabetes, eight with Type 2 diabetes) at three UK specialist diabetes antenatal clinics. Interviews explored women's journeys to becoming pregnant, including use of contraception, their views regarding diabetes and pregnancy and the factors which encouraged and discouraged them from attending PPC. RESULTS: All women had some understanding of the issues concerning diabetes during pregnancy, predominantly regarding the benefits of PPC (90%) and optimal glycaemic control (80%) and risks of malformation (48%) and macrosomia (35%). Most were not regularly using contraception (70%), having stopped deliberately (45%), become unintentionally less rigorous (28%) or experienced side effects/contraindications (14%). Knowledge concerning the risks of pregnancy (90%) and past pre-conception counselling (38%) did not encourage women to attend PPC, and neither did personal experience of miscarriage, malformation or stillbirth in women with previous poor pregnancy outcome (41%). Barriers included conceiving faster than anticipated (45%), fertility concerns (31%), negative experiences with health professionals (21%), desire for a 'normal' pregnancy (17%) and the logistics of attending (10%). CONCLUSIONS: More integrated diabetes and reproductive health/contraceptive advice, increased awareness of the potentially short time between stopping contraception and conception and more intensive support between pregnancies are required, particularly for women with previously poor outcomes. Research is also needed into how communication between health professionals and women with diabetes can be improved.

Altered resource availability and the population dynamics of tree species in Amazonian secondary forests.

Despite research demonstrating that water and nutrient availability exert strong effects on multiple ecosystem processes in tropical forests, little is known about the effect of these factors on the demography and population dynamics of tropical trees. Over the course of 5 years, we monitored two common Amazonian secondary forest species-Lacistema pubescens and Myrcia sylvatica-in dry-season irrigation, litter-removal and control plots. We then evaluated the effects of altered water and nutrient availability on population demography and dynamics using matrix models and life table response experiments. Our results show that despite prolonged experimental manipulation of water and nutrient availability, there were nearly no consistent and unidirectional treatment effects on the demography of either species. The patterns and significance of observed treatment effects were largely dependent on cross-year variability not related to rainfall patterns, and disappeared once we pooled data across years. Furthermore, most of these transient treatment effects had little effect on population growth rates. Our results suggest that despite major experimental manipulations of water and nutrient availability-factors considered critical to the ecology of tropical pioneer tree species-autogenic light limitation appears to be the primary regulator of tree demography at early/mid successional stages. Indeed, the effects of light availability may completely override those of other factors thought to influence the successional development of Amazonian secondary forests.

The Alara Metriscan phalangeal densitometer: evaluation and triage thresholds.

The Metriscan (Alara Inc, CA) is a compact digital radiographic absorptiometry device capable of determining phalangeal bone mineral density in arbitrary units (BMD(au)) from the second phalanges of the middle three digits. We have examined the utility and the in vitro and in vivo performances of the Metriscan, and established triage thresholds based on the UK's National Osteoporosis Society guidelines on peripheral densitometry. 170 white female participants (70 osteoporotic and 100 non-osteoporotic at the hip or spine) aged between 55 years and 70 years were recruited from patients attending for routine dual X-ray absorptiometry (DXA) examination. All participants underwent two scans of the non-dominant hand (with repositioning) and one of the dominant hand. An additional 10 participants were excluded owing to finger or hand deformities. Radiation exposure to the patient per scan was <0.1 microSv, and a controlled area of 1 m was established around the device. Phantom-based in vitro short-term precision (%CV) was 0.17% without, and 0.22% with, repositioning. Long-term in vitro precision was 0.31% over a 6-month period. In vivo short-term precision was 1.42% for the group as a whole, and 1.30% and 2.23% for the non-osteoporotic and osteoporotic groups, respectively. Triage thresholds for reassurance/referral or referral/treatment were 54.30 BMD(au) and 46.89 BMD(au), respectively, for the non-dominant hand, and 55.02 BMD(au) and 48.73 BMD(au) for the dominant hand. The dominant side proved superior for triage purposes, with a triage referral rate of 44%, compared with 48% for the non-dominant hand. The Metriscan is suitable for use on post-menopausal women in a community-based setting preferably in a triage role as an adjunct to axial BMD.

Routine versus targeted vertebral fracture assessment for the detection of vertebral fractures.

UNLABELLED: Fracture risk is underestimated in women with unknown vertebral fractures. Using VFA, we compared two screening methods: targeted (6,388 women) and routine (2,176 women). Routine screening detected fractures in 20%. Targeted screening only required 5% attending for DXA to undergo VFA but only detected 9.6% of women with fractures. INTRODUCTION: BMD alone underestimates fracture risk in women with unknown vertebral fractures. We report the results of routine vertebral fracture assessment (VFA) screening and compare with targeted screening. METHOD: Our centre initially targeted VFA at women with reasons to suspect a vertebral fracture. Later we changed to routine VFA screening for all women over 65. We retrospectively compare each screening method's ability to detect vertebral fractures. RESULTS: Six thousand three hundred and eighty-eight women over 65 underwent DXA during the period of targeted VFA and 2,176 during routine VFA. Routine VFA detected 420 (20.0%) women with fracture. Most vertebral fractures (56.2%) occurred in women with osteopenia. Routine VFA would be expected to alter the management of 1 in 6 osteopenic women. Targeted VFA was performed in 332 (5.2%) women detecting 122 (1.9%) women with fractures. It was estimated that targeted VFA only detected 9.6% of women with a vertebral fracture. Targeted VFA failed to detect fractures in 18.1% of the population attending for DXA and in 29% of those with osteoporosis. CONCLUSION: Routine VFA detects vertebral fractures in 20% of women over 65. Targeted VFA greatly reduces the number of VFAs performed but only detects a minority of the women with vertebral fractures.

The effects of short-term hormone replacement therapy on long-term bone mineral density.

INTRODUCTION: Short-term hormone replacement therapy (HRT) relieves menopausal symptoms and increases bone mineral density (BMD), but bone loss reoccurs upon discontinuation. This study assesses whether short-term HRT provides long-term BMD benefits. METHOD: This was a prospective study of women aged 50-54 years followed up for 9 years. Women were categorized into three groups according to the treatment they received: No-HRT (n = 340), Short-term HRT (2-4 years, n = 60), and Long-term HRT (9 years, n = 187). RESULTS: BMD increased significantly at the hip (2.4%, p < 0.001) and spine (8.0%, p < 0.001) over 9 years in the Long-term HRT group. Women without treatment lost BMD at the hip (-4.2%, p < 0.001) and spine (-3.5%, p < 0.001). Women in the Short-term HRT group had no significant loss of BMD at the hip (-1.6%, p = 0.08) or spine (-1.4%, p = 0.18) over 9 years. BMD in the Short-term HRT group was significantly higher at 9 years than in the No-HRT group at both spine (difference 0.023 g/cm(2), p = 0.048) and hip (difference 0.016 g/cm(2), p = 0.042). CONCLUSION: After 9 years, women who had taken short-term HRT had no significant loss of BMD and were better off in terms of BMD than those left untreated. Short-term HRT in the early postmenopausal period provides long-term BMD benefits.