The burden of alcohol-related caring for others in Australia in the 2021 pandemic period.

OBJECTIVE: The magnitude of caring for others due to excessive alcohol use is unclear in Australia and internationally. This study explores the prevalence, sociodemographic predictors, and consequences of informal care for others due to alcohol use. METHOD: A survey on harm from others' drinking was conducted among 2,574 Australian adults in November 2021 from national random digit dial and "Life in Australia" panel samples to elicit representative data. Respondents who indicated they had "heavy drinker/s" in their lives (n= 1,585), were asked about their experience of caring for these drinkers and their dependents in the last 12 months. Weighted logistic and linear regressions examined a) sociodemographic factors associated with informal care due to others' drinking, and b) the impact of the caregiving burden on caregiver's financial status, overall health, and quality of life. RESULTS: Overall, 20% of participants reported caregiving responsibilities arising from others' drinking. Older age, unemployment, residing in capital cities and reporting birth in a non-English speaking background country were associated with a reduced likelihood of caregiving, whereas higher education and more frequent risky drinking were associated with an increased likelihood. Caregivers reported significantly higher financial disadvantage, a lower quality of life and poorer overall health. CONCLUSIONS: One in five adults reported caring for drinkers in the previous 12 months, with this associated with negative consequences for those providing care. Service providers, health promotion practitioners and policy makers should focus upon younger age groups, those who live in regional areas, and those born in Australia who are at greater risk of being burdened by caregiving due to others' alcohol use.

General wellbeing and work impacts among community pharmacists during crisis management.

BACKGROUND: Community pharmacists are highly accessible for advice, as most pharmacies are open long hours and no appointment is needed. Community pharmacists, as essential community health workers, play a critical role in the fight against coronavirus disease 2019 (COVID-19). This study aimed to determine the general wellbeing and work impacts of pharmacists and the factors important for adaptability and resilience during the COVID-19 pandemic. METHODS: This study adopted a cross-sectional design. Community pharmacists from various professional networks in Australia were invited through emails and social media posts to complete an anonymous online survey during the second wave of the COVID-19 pandemic in Victoria, Australia. RESULTS: Sixty-five community pharmacists completed the online survey. The respondents reported fair levels of general wellbeing during the COVID-19 pandemic, with a mean self-related health score of 33.57 (s.d.=13.19) out of a maximal of 96, despite relatively high levels of job stress and emotional labour. Lower levels of general wellbeing were correlated with higher levels of job stress (r =0.645, P <0.01) and emotional labour (r =0.513, P <0.01), and lower levels of occupational self-efficacy (r =-0.566, P <0.01). Leader member exchange was negatively correlated with job stress (r =-0.419, P <0.01) and positively correlated with psychological safety (r =0.693, P <0.01). The linear regression models showed that female pharmacists had lower occupational self-efficacy (ß =-0.286, P =0.024), but higher psychological safety (ß =0.234, P =0.042). Higher work ability was associated with lower job stress (ß =-0.529, P <0.001), higher occupational self-efficacy (ß =0.511, P =0.001), and poorer self-related health (ß =-0.659, P <0.001). CONCLUSIONS: The findings highlight the importance of a supportive work environment in helping community pharmacists to feel psychologically safe and reduce stress during a crisis.

Adaptation strategies in transnational education: a case study of an australian Master of Health Administration Course offered to chinese managers.

BACKGROUND: Management decisions in health influence patient care outcomes; however, health management development courses in China are rare. This study aims to document and evaluate a transnational Master of Health Administration (MHA) course launched in 2000 for the benefit of Chinese health managers. METHODS: A case study of the MHA program jointly run by an Australian university and a Chinese Medical University was conducted. We reviewed the development of the MHA course through a document analysis (key events recorded in achieves, minutes, and audits) followed by reflection (by two course coordinators), extracting key themes related to adaptative strategies. We then conducted a questionnaire survey of 139 graduates seeking their views on relevance, satisfaction and challenges associated with each subject within the course, the relevance of key management skills (as determined by the Australasian College of Health Service Management competency framework), and the impact of the course on their personal career trajectories. Chi-square tests identified differences in the responses by age, gender, pre-training position, and current workplace. RESULTS: The curriculum pedagogy followed the principles of practice-based reflective learning. Research findings and student feedback shaped the curriculum design and subject content, to enhance management practices of the students. Survey participants expressed high levels of satisfaction and confirmed the relevance of all study subjects. Two subjects, health economics and data management, were perceived as being the most challenging. Of the ten management skills we assessed, relatively low self-rated confidence was found in "strategic thinking" and "planning". Younger and less experienced graduates were more likely to report learning challenges (p < 0.05). Frontline managers were least likely to obtain promotion by changing employers (χ2 = 6.02, p < 0.05) or being seconded to another position (χ2 = 9.41, p < 0.01). CONCLUSIONS: This case study illustrates the suitability of cross-country partnerships in health management training, which offers opportunities for managers to systematically explore and acquire a comprehensive set of management skills applicable to their career needs. Opportunities for developing training aligned to career development opportunities are critical for attracting and developing a competent and well-prepared health service management workforce in China.

A functional ultrasound brain GPS for automatic vascular-based neuronavigation.

Recent advances in ultrasound imaging triggered by transmission of ultrafast plane waves have rendered functional ultrasound (fUS) imaging a valuable neuroimaging modality capable of mapping cerebral vascular networks, but also for the indirect capture of neuronal activity with high sensitivity thanks to the neurovascular coupling. However, the expansion of fUS imaging is still limited by the difficulty to identify cerebral structures during experiments based solely on the Doppler images and the shape of the vessels. In order to tackle this challenge, this study introduces the vascular brain positioning system (BPS), a GPS of the brain. The BPS is a whole-brain neuronavigation system based on the on-the-fly automatic alignment of ultrafast ultrasensitive transcranial Power Doppler volumic images to common templates such as the Allen Mouse Brain Common Coordinates Framework. This method relies on the online registration of the complex cerebral vascular fingerprint of the studied animal to a pre-aligned reference vascular atlas, thus allowing rapid matching and identification of brain structures. We quantified the accuracy of the automatic registration using super-resolution vascular images obtained at the microscopic scale using Ultrasound Localization Microscopy and found a positioning error of 44 µm and 96 µm for intra-animal and inter-animal vascular registration, respectively. The proposed BPS approach outperforms the manual vascular landmark recognition performed by expert neuroscientists (inter-annotator errors of 215 µm and 259 µm). Using the online BPS approach coupled with the Allen Atlas, we demonstrated the capability of the system to position itself automatically over chosen anatomical structures and to obtain corresponding functional activation maps even in complex oblique planes. Finally, we show that the system can be used to acquire and estimate functional connectivity matrices automatically. The proposed functional ultrasound on-the-fly neuronavigation approach allows automatic brain navigation and could become a key asset to ensure standardized experiments and protocols for non-expert and expert researchers.

Guidance to implementing evidence-based initiatives to manage pandemics in healthcare services: a discussion paper.

Planning and implementing surge capacity in healthcare services is challenging. The evolving nature of viral transmission makes it difficult to predict the potential impact on the health system and the readiness of healthcare managers and professional staff in responding to system transformation and consequential impacts. This is compounded by sudden increased demands in the types of work required. Guidance for implementing targeted strategies for health systems and services should include evidence that considers the feasibility, appropriateness, meaningfulness and effectiveness of healthcare practices at both the system and organization level. This is consistent with the Joanna Briggs Institute (JBI) model of evidence synthesis and translation. Evidence-based decision-making is a core management competency for health service leadership and the mechanism for improving the quality of management decisions, and hence better service delivery, effectiveness and efficiency. Human resources are the most important asset in crisis management. Health managers must create responsive surge capacity by quantifying existent human and material resources and the shortfall needs required to meet anticipated additional demand. We believe a multifaceted approach is the key to developing systemic surge capacity within the heath sector when faced with the management of crises such as pandemics. We explore and discuss the desirability of strategies based on the JBI model of evidence synthesis and implementation with a view to encouraging the creation of registries and follow-up systems. We believe this will encourage the effective use of human resources including the upskilling in how future crises may be addressed.

Traditional medicines used by Q'eqchi' Maya to treat diabetic symptoms and their antiglycation potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Because of the recent increase in type 2 diabetes and the need for complementary treatments in remote communities in many parts of the world, we undertook a study of treatments for diabetic symptoms used by traditional Q'eqchi' Maya healers of Belize. We used quantitative ethnobotany to rank culturally important taxa and subsequent pharmacological and phytochemical studies to assess bioactivity. MATERIALS AND METHODS: Antidiabetic plants identified in field interviews with traditional healers were ranked by syndromic importance value (SIV) based on 15 symptoms of diabetes. Species ranked with high SIV were tested in an assay relevant to many diabetes complications, the advanced glycation endproduct (AGE) inhibition assay. Active principles were identified by phytochemical analysis and bioassay. RESULTS: We collected over 70 plant species having a promising SIV score. The plants represented a broad range of neotropical taxa. Selected Q'eqchi' antidiabetic plants with high SIV were collected in bulk and tested in the advanced glycation endproduct (AGE) inhibition assay. All plant extracts showed AGE inhibition and the half maximal inhibitory concentration (IC50) ranged from 40.8 to 733 µg/mL, while the most active species was Tynanthus guatemalensis Donn (Bignoniaceae). A linear regression showed a significant relationship between 1/ IC50 and SIV. Phytochemical analysis revealed the presence of verbascoside, as a major component and active principle of the T guatemalensis which had an IC50 = 5.1 µg/mL, comparable to the positive control quercetin. CONCLUSION: The results reveal a rich botanical tradition of antidiabetic symptom treatments among the Q'eqchi'. Study of highly ranked plants revealed their activity in AGE inhibition correlated with SIV. T. guatemalensis was identified as a promising species for further evaluation and local use.

Organizational barriers associated with the implementation of national essential medicines policy: A cross-sectional study of township hospitals in China.

This study identifies potential organizational barriers associated with the implementation of the Chinese National Essential Medicines Policy (NEMP) in rural primary health care institutions. We used a multistage sampling strategy to select 90 township hospitals from six provinces, two from each of eastern, middle, and western China. Data relating to eight core NEMP indicators and institutional characteristics were collected from January to September 2011, using a questionnaire. Prescription-associated indicators were calculated from 9000 outpatient prescriptions selected at random. We categorized the eight NEMP indicators using an exploratory factor analysis, and performed linear regressions to determine the association between the factor scores and institution-level characteristics. The results identified three main factors. Overall, low levels of expenditure of medicines (F1) and poor performance in rational use of medicines (F2) were evident. The availability of medicines (F3) varied significantly across both hospitals and regions. Factor scores had no significant relationship with hospital size (in terms of number of beds and health workers); however, they were associated with revenue and structure of the hospital, patient service load, and support for health workers. Regression analyses showed that public finance per health worker was negatively associated with the availability of medicines (p < 0.05), remuneration of prescribers was positively associated with higher performance in the rational use of medicines (p < 0.05), and drug sales were negatively associated with higher levels of drug expenditure (p < 0.01). In conclusion, irrational use of medicines remains a serious issue, although the financial barriers for gaining access to essential medicines may be less for prescribers and consumers. Limited public finance from local governments may reduce medicine stock lines of township hospitals and lead them to seek alternative sources of income, jeopardizing their capacity to meet the needs of local consumers.

Urban-rural inequality regarding drug prescriptions in primary care facilities - a pre-post comparison of the National Essential Medicines Scheme of China.

OBJECTIVE: To assess the impact of the National Essential Medicines Scheme (NEMS) with respect to urban-rural inequalities regarding drug prescriptions in primary care facilities. METHODS: A stratified two-stage random sampling strategy was used to sample 23,040 prescriptions from 192 primary care facilities from 2009 to 2010. Difference-in-Difference (DID) analyses were performed to test the association between NEMS and urban-rural gaps in prescription patterns. Between-Group Variance and Theil Index were calculated to measure urban-rural absolute and relative disparities in drug prescriptions. RESULTS: The use of the Essential Medicines List (EML) achieved a compliance rate of up to 90% in both urban and rural facilities. An overall reduction of average prescription cost improved economic access to drugs for patients in both areas. However, we observed an increased urban-rural disparity in average expenditure per prescription. The rate of antibiotics and glucocorticoids prescription remained high, despite a reduced disparity between urban and rural facilities. The average incidence of antibiotic prescription increased slightly in urban facilities (62 to 63%) and reduced in rural facilities (67% to 66%). The urban-rural disparity in the use of parenteral administration (injections and infusions) increased, albeit at a high level in both areas (44%-52%). CONCLUSION: NEMS interventions are effective in reducing the overall average prescription costs. Despite the increased use of the EML, indicator performances with respect to rational drug prescribing and use remain poor and exceed the WHO/INRUD recommended cutoff values and worldwide benchmarks. There is an increased gap between urban and rural areas in the use of parenteral administration and expenditure per prescription.

The impact of the National Essential Medicines Policy on prescribing behaviours in primary care facilities in Hubei province of China.

AIM: To assess the impact of the National Essential Medicines Policy (NEMP) on the use of medicines in government-owned primary care institutions in Hubei province of China. STUDY DESIGN: Quasi-experimental design and time-trend analysis. METHODS: A systematic random sampling strategy was employed to select 55,800 prescriptions from 18 primary care organizations who progressively implemented the NEMP from January 2009 to July 2011. We examined the change of patterns of prescriptions. The facilities that implemented the NEMP at a later stage served as control. RESULTS: An immediate increased uptake of essential medicines of all drugs prescribed which ultimately neared 95%. In total, 38,151 prescriptions (68%) involved antibiotics, and we found no evidence of reduction after the NEMP interventions. A high percentage (59-66%) of prescription drugs were administered through parenteral routes and no reduction was found after the NEMP interventions. Although the average number of medicines per prescription remained unchanged (nearly four), the average cost per prescription declined significantly after the NEMP interventions (¥ 44.67 vs ¥ 26.67 CNY, P < 0.03). CONCLUSIONS: The NEMP interventions reduced the average cost per prescription; however, the irrational use of antibiotics and unnecessary parenteral administration remains prevalent. The goals of the NEMP are partially achieved; we therefore recommend a strategic approach involving all stakeholders to comprehensively achieve all aspirations.

Using historical data for bioprocess optimization: modeling wine characteristics using artificial neural networks and archived process information.

Optimization of fermentation processes is a difficult task that relies on an understanding of the complex effects of processing inputs on productivity and quality outputs. Because of the complexity of these biological systems, traditional optimization methods utilizing mathematical models and statistically designed experiments are less effective, especially on a production scale. At the same time, information is being collected on a regular basis during the course of normal manufacturing and process development that is rarely fully utilized. We are developing an optimization method in which historical process data is used to train an artificial neural network for correlation of processing inputs and outputs. Subsequently, an optimization routine is used in conjunction with the trained neural network to find optimal processing conditions given the desired product characteristics and any constraints on inputs. Wine processing is being used as a case study for this work. Using data from wine produced in our pilot winery over the past 3 years, we have demonstrated that trained neural networks can be used successfully to predict the yeast-fermentation kinetics, as well as chemical and sensory properties of the finished wine, based solely on the properties of the grapes and the intended processing. To accomplish this, a hybrid neural network training method, Stop Training with Validation (STV), has been developed to find the most desirable neural network architecture and training level. As industrial historical data will not be evenly spaced over the entire possible search space, we have also investigated the ability of the trained neural networks to interpolate and extrapolate with data not used during training. Because a company will utilize its own existing process data for this method, the result of this work will be a general fermentation optimization method that can be applied to fermentation processes to improve quality and productivity.

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.

Electrically measuring viscoelastic parameters of adherent cell layers under controlled magnetic forces.

Electrical cell-substrate impedance sensing (ECIS) was used to measure the time-dependence and frequency-dependence of impedance for current flowing underneath and between cells. Osteosarcoma cells with a topology similar to a short cylinder (coin-like) surmounted by a dome were used in this study. Application of a small step increase in net vertical stress to the cells (4 and 7 dyn/cm2), via magnetic beads bound to the dorsal (upper) surface, causes an increase in cell body height and an increase in cell-cell separation, as well as stretching of the cell-substrate adhesion bonds. This results in a fast drop in measured resistance (less than 2 s), followed by a slower change with a time constant of 60-150 s. This time constant is about 1.5 times longer at 22 degrees C than that at 37 degrees C; it also increases with applied stress. Our frequency scan data, as well as our data for the time course of resistance and capacitance, show that the fast change is associated with both the under-the-cells and between-the-cells resistance. The slower change in resistance mainly reflects the between-the-cells resistance. To obtain viscoelastic parameters from our data we use a simple viscoelastic model comprising viscous and elastic elements (i.e., dashpot and two springs) for the cell body, and an elastic element (a spring) for the cell-substrate adhesion system. Our results show that the spring constants and the viscosity of the cell body components of this viscoelastic model decrease as the temperature increases, whereas the elastic modulus of cell-substrate adhesion increases with temperature. At 37 degrees C, for the cell body we obtain a value of about 10(5) P for the viscous element of the viscoelastic model, and a spring constant expressed in units of an elastic modulus of about 10(4) dyn/cm2 for the spring in series with the viscous element, with another spring with a modulus of about 2 x 10(3) dyn/cm2 in parallel with these. In comparable units, we have a modulus for the cell-substrate adhesion system of about 3 x 10(3) dyn/cm2.

Disruption of murine alpha-enolase by a retroviral gene trap results in early embryonic lethality.

Gene trapping with the retroviral ROSA beta geo vector was used to generate lines of mice carrying disrupted genes. Both cDNA and genomic flanks have been cloned from a number of these lines. One mutation has been shown to disrupt the alpha-enolase gene by insertion of the splice-trap vector into the first intron. In adult mice, lacZ expression was detected only in testes. Embryonic expression was detected from 10.5-day postcoitum embryos and was seen as a diffuse staining pattern over much of the embryo, consistent with the housekeeping gene function of alpha-enolase. This mutation results in an early recessive embryonic lethality. Mice heterozygous for the mutation have no obvious phenotype. Mutations of this gene in humans are reported to be associated with rare autosomal-dominant, non-spherocytic haemolytic anaemia. This phenotype is not reproduced in mice heterozygous for this mutation.

Cell-substrate separation: effect of applied force and temperature.

We measure the change in cell-substrate separation in response to an upward force by combining two relatively new techniques, Electric Cell-substrate Impedance Sensing (ECIS) to measure average cell-substrate separation, and collagen-coated magnetic beads to apply force to the top (dorsal) surface of cells. The collagen-coated ferric oxide beads attach to integrin receptors in the dorsal surfaces of osteoblastlike ROS 17/2.8 cells. Magnetic force is controlled by the position and the number of permanent magnets, applying an average 320 or 560 pN per cell. Comparing model calculations with experimental impedance data, the junctional resistivity of the cell layer and the average distance between the lower (ventral) cell surface and substrate can be determined. The ECIS analysis shows that these forces produce an increase in the distance between the ventral cell surface and the substrate that is in the range of 10 to 25%. At temperatures of 4 degrees, 22 degrees and 37 degrees C, the measured cell surface-substrate distances without magnetic beads are 84 +/- 4, 45 +/- 2 and 38 +/- 2 nm. The force-induced changes at 22 degrees are 11 +/- 3 and 21 +/- 3 nm for 320 and 560 pN, and at 37 degrees they are 5 +/- 2 and 9 +/- 2 nm. The resulting cell-substrate spring constants at 22 degrees and 37 degrees are thus about 28 and 63 pN nm-1 (dyne cm-1). Using a reasonable range for the number for individual integrin-ligand adhesion bonds gives a range for the spring constant of the individual adhesion bond of from about 10(-3) to 10(-1) pN nm-1. These data also provide evidence that the number of adhesion bonds per cell increases with temperature.

A new method for application of force to cells via ferric oxide beads.

We describe a new method that uses straightforward physics to apply force to substrate-attached cells. In this method, collagen-coated magnetic ferric oxide beads attach to the dorsal surface of cells via receptors of the integrin family, and a magnetic field gradient is applied to produce a force. In this paper we present a complete characterization of the method in a configuration that is easy to use, in which a permanent magnet provides a fairly uniform gradient over a relatively large area. This allows a fairly uniform average force that can be controlled in magnitude, direction, and duration to be applied to a large number of cells. We show how to determine the applied force per cell by measuring the force per unit volume of magnetic bead, the distribution of bead diameters, and the distribution of beads per cell. We also show how to calculate the force per unit volume of bead in a three-dimensional region near the permanent magnet on the basis of field measurements, and present results for three of the magnets. An upward force applied to fibroblasts by this method produces a measurable time-dependent increase in attachment of cytoskeletal actin filaments to the force application points, and an increase in actin cross-linking. This is accompanied by an actin-dependent retraction of the force-induced upward movement of the dorsal surface of the cells.

Diffuse nasal polyposis: postoperative long-term results after endoscopic sinus surgery and frontal irrigation.

Diffuse nasal polyposis remains a challenge despite recent improvements in endonasal surgery. The purpose of this study is to evaluate the results after a radical complete sphenoethmoidectomy with peroperative and postoperative frontal irrigation in cases of diffuse nasal polyposis. In this prospective study, we include 50 consecutive patients with diffuse nasal polyposis suffering from nasal obstruction, anosmia, and other symptoms of chronic sinusitis. All patients were refractory to medical therapy. In each patient an endoscopic complete sphenoethmoidectomy including total excision of all diseased ethmoid mucosa was performed. Preoperative and postoperative frontal irrigation was performed systematically. The patients were followed closely with serial endoscopic examination, and CT scanning was performed between 2 and 3 years after surgery. There were no complications. Thirty-nine of the 50 patients regained satisfactory olfaction. Partial nasal obstruction persisted in four of the 50 patients. Endoscopically, polyp recurrence was noted in 3% of posterior ethmoids, 23% of anterior ethmoids, and 50% of frontal recesses. We conclude that in cases of refractory and extensive nasal polyposis, a total sphenoethmoidectomy with perioperative frontal irrigation followed by long-term postoperative topical steroid therapy provides excellent improvement or cure with safety and reliability.

Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro.

Osteopontin (OPN) and bone sialoprotein (BSP) are phosphorylated glycoproteins that, together with osteonectin/secreted protein, acidic, rich in cysteine (SPARC) and osteocalcin, comprise the major non-collagen proteins of bone. Although phosphorylation of OPN and BSP, which is known to influence the biological properties of these proteins, has been shown to occur intracellularly, recent studies have demonstrated ectokinase activity in bone cell populations [Mikuni-Takagaki, Kakai, Satoyoshi, Kawano, Suzuki, Kawase and Saito (1995) J. Bone Miner. Res. 10, 231-241]. To determine whether OPN and BSP are phosphorylated by ectokinase activity we have used [gamma-32P]ATP and [gamma-32P]GTP as cell-impenetrable phosphate donors to analyse for ectokinase activity in osteoblastic UMR106.06 cells and fetal rat calvarial cells (FRCCs). By pulse-labelling confluent cells with radiolabelled nucleotides, the phosphorylation of endogenous and exogenously added OPN and BSP was demonstrated together with the labelling of a number of cell surface proteins. These phosphorylation reactions were inhibited by a cell-impermeable ectokinase inhibitor, K252b, and cell surface phosphorylation was also inhibited by exogenously added OPN and BSP substrates, indicating competition for the ectokinase enzyme. However, phosphorylation of OPN and BSP, both of which can mediate cell attachment through Arg-Gly-Asp (RGD) motifs, was not inhibited by an RGD peptide, suggesting that binding of OPN and BSP to cell surface integrins is not required. In similar experiments, ectokinase-mediated phosphorylation of OPN and BSP was demonstrated during mineralized tissue formation by FRCCs in vitro. These studies demonstrate that OPN and BSP secreted by bone cells are phosphorylated by a casein kinase II-like ectokinase present on the surface of osteoblastic cells.

Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching.

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.

Differential purinergic receptor signalling in osteoclasts and osteoblastic cells.

There are differences between osteoclasts and osteoblastic cells in their cytosolic calcium responses to purinergic receptor activation. Application of 50 or 100 microM extracellular ATP inhibits the calcium response to a second application of ATP in osteoblastic rat osteosarcoma UMR 106 cells, but not in rabbit osteoclasts. This shows that there is adaptation to the extracellular, ATP in osteoclasts, but not in the UMR 106 cells. Extracellular washing of the UMR 106 cells restores the calcium response to ATP partially but not completely, indicating that there is a purinergic receptor activation-induced desensitisation of the receptor or its linked signalling pathways. In contrast to these results, if extracellular UTP is applied first, application of ATP produces no calcium response in osteoclasts, with or without washing, while in the UMR 106 cells there is some response to the ATP, which is greatly enhanced by washing. This indicates that UTP induces a complete desensitisation of the purinergic receptor/calcium signalling system in osteoclasts, but not in the osteoblastic cells, in which there is simply competition between UTP and ATP for the same receptors. Taken together, these results demonstrate that ATP and UTP could differentially regulate osteoblasts and osteoclasts.