Printability and bio-functionality of a shear thinning methacrylated xanthan-gelatin composite bioink.

3D bioprinting is a recent technique that can create complex cell seeded scaffolds and therefore holds great promise to revolutionize the biomedical sector by combining materials and structures that more closely mimic the 3D cell environment in tissues. The most commonly used biomaterials for printing are hydrogels, however, many of the hydrogels used still present issues of printability, stability, or poor cell-material interactions. We propose that bioinks with intrinsic self-assembling and shear thinning properties, such as xanthan gum, can be methacrylated (XGMA) and combined with a bio-functional material such as gelatin methacryloyl (GelMa) to create a stable, cell-interactive bioink with improved properties for 3D bioprinting. These biomaterials have reduced viscosity under high shear and recover their viscosity rapidly after the shear is removed, retaining their shape, which translates to easier extrusion whilst maintaining accurate fidelity after printing. This was confirmed in printing studies, with measured normalized strand widths of 1.2 obtained for high gel concentrations (5+5 % XGMA-GelMA). Furthermore, the introduction of a secondary photo-cross-linking method allowed tuning of the mechanical properties of the hydrogel with stiffness between 15 and 30 kPa, as well as improving the stability of the hydrogel with retention of 75 % of its mass after 90 d. The hydrogel was shown to be biocompatible and bio-active with 97 % cell viability, and cell spreading after 7 d of culture for low gel concentrations (3+3 % XGMA-GelMA). Shear stresses were relatively low while printing (1 kPa) as a result of the shear thinning property of the material, which supported cell viability during extrusion. Finally, printed hydrogels retained high cell viability for lower gel concentrations, and showed improved cell viability for more concentrated hydrogels when compared to cells cultured in bulk hydrogels, presumably due to improved nutrient/oxygen diffusion and cell migration. In conclusion, stability and formulation of a XGMA-GelMA shear thinning composite hydrogel has been optimized to create a bio-functional bioink, with improved printability, andin vitroculture stabilityviasecondary photo-induced cross-linking, making this composite a promising bioink for 3D bioprinting.

In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells.

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Priorities for research in multiple conditions in later life (multi-morbidity): findings from a James Lind Alliance Priority Setting Partnership.

INTRODUCTION: multiple conditions in later life (multi-morbidity) is a major challenge for health and care systems worldwide, is of particular relevance for older people, but has not (until recently) received high priority as a topic for research. We have identified the top 10 research priorities from the perspective of older people, their carers, and health and social care professionals using the methods of a James Lind Alliance Priority Setting Partnership. METHODS: in total, 354 participants (162 older people and carers, 192 health professionals) completed a survey and 15 older people and carers were interviewed to produce 96 'unanswered questions'. These were further refined by survey and interviews to a shortlist of 21 topics, and a mix of people aged 80+ living with three or more conditions, carers and health and social care providers to prioritised the top 10. RESULTS: the key priorities were about the prevention of social isolation, the promotion of independence and physical and emotional well-being. In addition to these broad topics, the process also identified detailed priorities including the role of exercise therapy, the importance of falls (particularly fear of falling), the recognition and management of frailty and Comprehensive Geriatric Assessment. CONCLUSION: these topics provide a unique perspective on research priorities on multiple conditions in later life and complement existing UK and International recommendations about the optimisation of health and social care systems to deliver essential holistic models of care and the prevention and treatment of multiple co-existing conditions.

Polyethylene glycol-gelatin hydrogels with tuneable stiffness prepared by horseradish peroxidase-activated tetrazine-norbornene ligation.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

The duration of the orthostatic blood pressure drop is predictive of death.

BACKGROUND: Orthostatic hypotension (OH) affects 6% of community-dwelling older people. This increases to 60% when non-invasive, continuous blood pressure (BP) monitoring is used, due to identification of transient drops in BP which recover rapidly. AIM: To determine the clinical relevance of these transient orthostatic BP drops. DESIGN: Five-year clinical observational study. METHODS: One hundred three consecutive new patients attending a Falls and Syncope Clinic in the UK from 1 February 2009 underwent continuous BP monitoring during an active stand. BP profiles were analysed to quantify all reductions in BP, measuring the duration of any drop below diagnostic criteria. Five-year follow-up data were extracted from hospital clinical records to assess clinical outcomes. RESULTS: Systolic BP (sBP) dropped ≥20 mmHg in 76 (74%) individuals, with 65 (63%) having ≥10 mmHg drop in diastolic BP. However, only 22 (21%) cases were diagnosed clinically with OH. A sustained reduction in BP (≥30 s) had a sensitivity of 0.91 and specificity of 0.88 for a clinical diagnosis of OH, being more accurate than absolute BP reduction alone. A sustained reduction in sBP was associated with greater use of vasopressors (36%,P0.001) and an independent, significantly greater risk of death (45% at 5 years,P0.009). CONCLUSION: An orthostatic reduction in sBP lasting ≥30 s improves accuracy of diagnosis. Moreover, given the significant adverse outcomes with a sustained reduction, clinicians should consider this when diagnosing and treating patients, as transient OH does not appear to be clinically significant.

Effects of electric fields on human mesenchymal stem cell behaviour and morphology using a novel multichannel device.

The intrinsic piezoelectric nature of collagenous-rich tissues, such as bone and cartilage, can result in the production of small, endogenous electric fields (EFs) during applied mechanical stresses. In vivo, these EFs may influence cell migration, a vital component of wound healing. As a result, the application of small external EFs to bone fractures and cutaneous wounds is actively practiced clinically. Due to the significant regenerative potential of stem cells in bone and cartilage healing, and their potential role in the observed improved healing in vivo post applied EFs, using a novel medium throughput device, we investigated the impacts of physiological and aphysiological EFs on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for up to 15 hours. The applied EFs had significant impacts on hBM-MSC morphology and migration; cells displayed varying degrees of conversion to a highly elongated phenotype dependent on the EF strength, consistent perpendicular alignment to the EF vector, and definitive cathodal migration in response to EF strengths ≥0.5 V cm(-1), with the fastest migration speeds observed at between 1.7 and 3 V cm(-1). We observed variability in hBM-MSC donor-to-donor responses and overall tolerances to applied EFs. This study thus confirms hBM-MSCs are responsive to applied EFs, and their rate of migration towards the cathode is controllable depending on the EF strength, providing new insight into the physiology of hBM-MSCs and possibly a significant opportunity for the utilisation of EFs in directed scaffold colonisation in vitro for tissue engineering applications or in vivo post implantation.

A new method to prevent degradation of lithium-oxygen batteries: reduction of superoxide by viologen.

Lithium-oxygen battery development is hampered by degradation reactions initiated by superoxide, which is formed in the pathway of oxygen reduction to peroxide. This work demonstrates that the superoxide lifetime is drastically decreased upon addition of ethyl viologen, which catalyses the reduction of superoxide to peroxide.

Impaired blood pressure variability in chronic fatigue syndrome--a potential biomarker.

INTRODUCTION: Autonomic dysfunction is common in chronic fatigue syndrome (CFS). This study set out to derive an autonomic biomarker using a comprehensive assessment of heart rate and blood pressure variability. METHODS: Heart rate and non-invasive continuous blood pressure measurements (task force monitor) at rest and on standing were performed in CFS (Fukuda n = 68) and matched controls (n = 68) to derive high frequency (HF; parasympathetic) and low frequency (LF; sympathetic) heart rate variability (HRV), systolic (SBPV) and diastolic (DBPV) blood pressure variability. Variables of significance were combined using receiver operator curves to explore the diagnostic utility of parameters particularly at rest. RESULTS: At rest, LF-HRV (sympathetic) was significantly increased in CFS compared to controls, while parasympathetic markers were significantly reduced (P = 0.006). Total DBP spectral power was increased (P = 0.0003) across all domains, with a shift towards sympathetic and away from parasympathetic SBPV (P = 0.05). On standing, overall SBPV response was significantly reduced with reductions in both sympathetic and parasympathetic components of SBPV (all P < 0.0001). Change in LF-DBP and relative balance of LF/HF DBP on standing differed between CFS and controls (P < 0.0001). Using the 85% sensitivity levels, we determined a threshold for three chosen resting BPV parameters of LF DBP >3.185, rest HF DBP >0.86, rest total DBP >7.05. Achieving all of these differentiated between CFS and controls with 77% sensitivity and 53% specificity. CONCLUSION: This study has shown that there are objectively measured abnormalities of blood pressure variability in CFS and that these abnormalities have the potential to be a bedside diagnostic tool.

The complexities of defining atopy in severe childhood asthma.

BACKGROUND: Defining atopy in children with severe, therapy-resistant asthma is complex. There is currently no gold standard test; both skin prick testing (SPT) and allergen-specific IgE (sIgE) are used. Furthermore, atopy is increasingly considered to be a spectrum, not an all-or-none phenomenon. HYPOTHESIS: SPTs and sIgE cannot be used interchangeably, and if both tests are not performed, opportunities for intervention will be missed. Furthermore, the severity of atopy will be defined differently by the two tests. METHODS: Cross-sectional study of 47 children with severe, therapy-resistant asthma, mean age 11.8 years, range 5.3-16.6 years, who underwent SPT, and measurement of total and sIgE as part of their clinical work-up. RESULTS: Overall, 42/47 (89%) were atopic (defined as either one positive SPT or sIgE). There was 98% concordance between the two tests in classifying atopy. When each allergen was considered individually, in 40/200 (20%), the SPT and sIgE results were discordant, most commonly in 25/200 (12.5%), the SPT was negative and the sIgE was positive. House dust mite and cat sensitization were more likely detected by sIgE, but dog sensitization by SPT. When atopy was quantified, the sum of sIgEs compared with the sum of SPT weal diameter showed a moderate correlation (r(2) =0.44, P<0.001). Total IgE increased with an increasing number of positive sIgEs (P=0.028), but not significantly with increasing numbers of positive SPTs. CONCLUSION AND CLINICAL RELEVANCE: SPT and sIgE identify group prevalence of atopy equally well; however, for individual allergens, concordance is poor, and when used to quantify atopy, SPTs and sIgE were only moderately correlated. In a clinical setting, if allergen avoidance is contemplated in children with severe, therapy-resistant asthma, both tests should be performed in order to detect sensitization.

Fatigue severity remains stable over time and independently associated with orthostatic symptoms in chronic fatigue syndrome: a longitudinal study.

OBJECTIVES: to examine fatigue variability over time in chronic fatigue syndrome (CFS) and the effect of other symptoms on its predictability. DESIGN: longitudinal cohort study of patients with CFS (Fukuda criteria). SETTING: specialist CFS clinical service. SUBJECTS: phase 1: 100 patients who participated in a study of CFS symptoms in 2005 were revisited in 2009. Phase 2: 25 patients completed fatigue diaries to address intra- and inter-day variability in perceived fatigue. MAIN OUTCOME MEASURES: phase 1: subjects completed fatigue impact scale (FIS), Epworth sleepiness scale (ESS), orthostatic grading scale (OGS) and hospital anxiety and depression scale (HADS). Changes in variables represented the differences between 2005 and 2009. Phase 2: subjects rated fatigue on a scale of 0 (no fatigue) to 10 (severe fatigue) four times a day for 5 weeks. RESULTS: symptom assessment tools were available in both 2005 and 2009 for 74% of patients. FIS and HADS depression (HAD-D) and anxiety (HAD-A) scores significantly improved during follow-up whereas ESS and OGS remained stable. FIS improved in 29/74 (39%) subjects, and by ≥ 10 points in 19 (26%). FIS worsened by ≥ 10 points in 33/74 (45%) subjects. On multivariate analysis, independent predictors of current fatigue (FIS in 2009) were FIS in 2005, HAD-D in 2009, OGS in 2009 and change in HAD-A. Reported fatigue was stable from week to week and from day to day. Patients reported higher fatigue in the morning (mean ± SD; 6.4 ± 2), becoming significantly lower at lunchtime (6.2 ± 2; P < 0.05) and increasing again to 7 ± 2 at bedtime. CONCLUSIONS: current fatigue is independently associated with current autonomic symptom burden, current depression and change in anxiety during follow-up. These findings have implications for targeted symptom management in CFS.

Primary biliary cirrhosis is associated with falls and significant fall related injury.

BACKGROUND: Osteoporosis and autonomic dysfunction are prevalent in the autoimmune liver disease primary biliary cirrhosis (PBC). Postural hypotension is one consequence of autonomic dysfunction and is a recognized risk factor for falls, which, alongside osteoporosis could lead to significant injury and fractures. AIM: To determine the prevalence and sequelae of falls in PBC and to identify modifiable risk factors. DESIGN: Cross-sectional, geographical, population census of PBC and two control groups: primary sclerosing cholangitis and a community dwelling population. Multidisciplinary falls assessment of a representative group of PBC. METHODS: Symptom assessment tools, completed by the three cohorts, determined the prevalence of falls, injuries and associated symptoms. Multidisciplinary assessments, adhering to NICE guidelines, identified modifiable fall associations. RESULTS: Significantly more of the PBC population had fallen (72% P < 0.001) than both control groups. Fifty-five percent had fallen in the last year (P < 0.001), and 22% more than once in the last year (P < 0.01). Seventy percent of PBC fallers were injured, 27% fractured a bone and 19% were admitted to hospital, all significantly more common than controls. Postural dizziness was significantly worse in fallers (P < 0.001), as were balance (P < 0.001) and lower limb strength (P = 0.002). Lower limb strength was independently associated with number of falls in previous year (beta = 0.184, P < 0.001). CONCLUSION: Falls and resultant injury are prevalent in PBC and more common than previously recognized. Addressing postural dizziness, poor balance and lower limb weakness using a multidisciplinary approach has the potential to reduce falls, morbidity and mortality and as a result improve quality of life.

The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis.

OBJECTIVE: The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma-dichloromethylene) triphosphate (AppCCl2p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed. METHODS: High-performance liquid chroma-tography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl2p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of RaplA, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals. RESULTS: AppCCl2p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl2p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl2p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl2p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase RaplA in osteoclasts or macrophages in vivo. CONCLUSION: These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl2p.

Cellular and molecular mechanisms of action of bisphosphonates.

BACKGROUND: Bisphosphonates currently are the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia, Paget's disease, and osteoporosis. These compounds have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. METHODS: This article reviews the pharmacology of bisphosphonates and the relation between the chemical structure of bisphosphonates and antiresorptive potency, and describes recent new discoveries of their molecular mechanisms of action in osteoclasts. RESULTS: Bisphosphonates can be grouped into two pharmacologic classes with distinct molecular mechanisms of action. Nitrogen-containing bisphosphonates (the most potent class) act by inhibiting the mevalonate pathway in osteoclasts, thereby preventing prenylation of small GTPase signaling proteins required for osteoclast function. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that may involve the formation of cytotoxic metabolites in osteoclasts or inhibition of protein tyrosine phosphatases. CONCLUSIONS: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts. After more than 30 years of clinical use, their molecular mechanisms of action are only just becoming clear.

The use and perceived value of diabetes clinical management guidelines in general practice.

OBJECTIVE: To assess attitudes to and use of NSW Health Department (NSWHD) guidelines for the clinical management of diabetes mellitus by divisions of general practice, general practitioners, patients and allied health professionals. METHOD: A mail cross sectional survey of divisions of general practice and a series of focus groups of GPs, patients and allied health professionals, on extent of use and perceived usefulness of the NSWHD guidelines. RESULTS: Forty divisions had or recently concluded a diabetes shared care project. Thirteen out of the 31 diabetes projects with guidelines reported using the NSWHD guidelines. Nineteen divisions were aware of the NSWHD guidelines. While the general response to the guidelines in both the survey and the focus groups was positive, there were suggestions for modifications. Many respondents were not aware of the guidelines. CONCLUSION: Optimal use of the guidelines in general practice requires improved dissemination and an implementation strategy based not only on GP education but also systems to reduce barriers to implementation and to support better quality of GP care.

Analysis of an adenine nucleotide-containing metabolite of clodronate using ion pair high-performance liquid chromatography-electrospray ionisation mass spectrometry.

Clodronate belongs to the family of bisphosphonates, which are synthetic analogues of pyrophosphate. Bisphosphonates are widely used in the treatment of metabolic bone diseases. Some bisphosphonates, including clodronate, can be metabolized in cells into non-hydrolysable nucleotide analogues. In this paper, we describe a new method for extraction and quantitation of the clodronate metabolite in cell lysates by using ion-pairing HPLC method that is compatible with negative ion electrospray ionization mass spectrometry (ESI-MS). The method was used for detection of the metabolite of clodronate in extracts from RAW 264 macrophage cells after treatment with clodronate.

Is it MS? Presenting symptoms and diagnosis of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is the most common chronic neurological disease in our community. OBJECTIVE: To discuss the presenting symptoms that suggest the diagnosis of MS and consider how to confirm the diagnosis and evaluate the differential diagnosis. DISCUSSION: There are protean presenting symptoms of MS but because of the distribution of pathology predominantly in the peri-ventricular region of the brain, certain symptom complexes are more common. With the recent availability of treatment for MS which has been demonstrated to slow the progress of the disease, the need for early diagnosis has become even more important.

The pharmacology of bisphosphonates and new insights into their mechanisms of action.

Bisphosphonates are chemically stable analogs of inorganic pyrophosphate, which are resistant to breakdown by enzymatic hydrolysis. The biological effects of bisphosphonates on calcium metabolism were originally ascribed to their physico-chemical effects on hydroxyapatite crystals. Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds. Remarkable progress has been made in increasing the potency of bisphosphonates as inhibitors of bone resorption, and the most potent compounds in current use are characterized by the presence of a nitrogen atom at critical positions in the side chain which, together with the bisphosphonate moiety itself, seems to be essential for maximal activity. As a class the bisphosphonates offer a very effective means of treating Paget's disease.

Farnesol and geranylgeraniol prevent activation of caspases by aminobisphosphonates: biochemical evidence for two distinct pharmacological classes of bisphosphonate drugs.

Recently, advances have been made in understanding the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Studies with the macrophage-like cell line J774 have suggested that alendronate, an amino-containing bisphosphonate, causes apoptosis by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. However, clodronate, a nonaminobisphosphonate, does not inhibit protein isoprenylation but can be metabolized intracellularly to a cytotoxic, beta-gamma-methylene (AppCp-type) analog of ATP. These observations raise the possibility that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R2 side chain. We addressed this question by directly comparing the ability of three aminobisphosphonates (alendronate, ibandronate, and pamidronate) and three nonaminobisphosphonates (clodronate, etidronate, and tiludronate) to inhibit protein isoprenylation and activate caspase-3-like proteases or to be metabolized to AppCp-type nucleotides by J774 cells. All three aminobisphosphonates inhibited protein isoprenylation and activated caspase-3-like proteases. Apoptosis and caspase activation after 24-h treatment with the aminobisphosphonates could be prevented by addition of farnesol or geranylgeraniol, confirming that these bisphosphonates inhibit the metabolic mevalonate pathway. No AppCp-type metabolites of the aminobisphosphonates could be detected by mass spectrometry. The three nonaminobisphosphonates did not inhibit protein isoprenylation or cause activation of caspase-3-like proteases, but were incorporated into AppCp-type nucleotides. Taken together, these observations clearly demonstrate that bisphosphonate drugs can be divided into two pharmacological classes: the aminobisphosphonates, which act by inhibiting protein isoprenylation, and the less potent nonaminobisphosphonates, which act through the intracellular accumulation of AppCp-type metabolites.