Cell size and actin architecture determine force generation in optogenetically activated cells.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

3D-printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect.

The reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a minipig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D-printed scaffold made of clinical-grade polylactic acid (PLA), coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (computed tomography scans, microcomputed tomography scans, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20- to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. Three-dimensional printed PLA scaffolds loaded with reduced doses of BMP-2 may be a safe and simple solution for large bone defects faced in the clinic.

The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function.

Using DNA encoding the largest subunit of Drosophila melanogaster RNA polymerase II, we isolated the homologous hamster RPO21 gene. Nucleotide sequencing of both the hamster and D. melanogaster RPO21 DNAs confirmed that the RPO21 polypeptides of these two species, like the Saccharomyces cerevisiae RPO21 polypeptide, contain both an N-terminal region homologous to the Escherichia coli RNA polymerase subunit beta' and a unique polymerase II-specific C-terminal domain. This C-terminal domain, encoded by separate exons in the D. melanogaster and hamster genes, consists of a tandemly repeated heptapeptide sequence. By constructing a series of deletions in DNA encoding the 26 heptapeptide repeats normally present in the S. cerevisiae RPO21 polypeptide, we have established that a minimum of between 9 and 11 repeats is necessary for RPO21 function in yeast cells. Replacement of the yeast RPO21 heptapeptide repeats by the longer hamster repetitive domain resulted in viable yeast cells with no detectable mutant phenotype, while a similar replacement of the yeast repeats by the more divergent D. melanogaster repeats was a recessive lethal mutation. We suggest that this novel repetitive domain is essential for proper initiation of transcription by RNA polymerase II and that it may mediate the functions of TATA boxes, upstream activating sequences, and enhancers.

Gamma-heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175.

A novel neuregulin isoform, termed gamma-HRG, was cloned and characterized from the human breast cancer cell line, MDA-MB-175. As observed with other neuregulins, gamma-HRG, is a product of alternative mRNA splicing of the neuregulin gene. Gamma-HRG contains the EGF-like and immunoglobulin-like domains that are commonly found in other family members, but lacks a transmembrane and cytoplasmic region. The new isoform possesses a unique N-terminal region that includes a hydrophobic domain that may function as a secretion signal. A purified recombinant version of gamma-HRG competes for binding to soluble ErbB3- and ErbB4-IgG fusion proteins with affinities similar to those observed for rHRGbeta1(177-244). Gamma-HRG has a wide distribution in mesenchymal or neuronal tissues but in contrast to other neuregulins, it is not present in breast, lung, liver and small intestine. Expression of gamma-HRG with its cognate receptors, ErbB3 and ErbB2 suggested that the growth of the MDA-MB-175 cell line might be a result of the autocrine stimulation of a growth factor signaling pathway. Treatment of MDA-MB-175 cells with an anti-ErbB2 monoclonal antibody that interferes with the ligand-dependent formation of ErbB2-ErbB3 heterodimer complexes shows a strong growth inhibitory effect on this cell line. Moreover, incubation with a receptor-IgG fusion protein that neutralizes secreted gamma-HRG, also inhibits cell growth. These data suggest that the secretion of gamma-HRG by MDA-MB-175 cells leads to the formation of a constitutively active receptor complex and stimulates the growth of these cells in an autocrine manner.

Randomized, double-blind, placebo-controlled dose-ranging study of tirofiban (MK-383) platelet IIb/IIIa blockade in high risk patients undergoing coronary angioplasty.

OBJECTIVES: The objectives of this double-blind, placebo-controlled, randomized dose-ranging study were 1) to examine the safety and tolerability of tirofiban (MK-383), a new nonpeptide platelet IIb/IIIa receptor antagonist, on a background of intravenous heparin and aspirin therapy; 2) to study the pharmacodynamics and pharmacokinetics of tirofiban; and 3) to evaluate the incidence of adverse cardiac outcomes (urgent repeat revascularization, myocardial infarction and death) with tirofiban versus placebo in a high risk subset of patients undergoing coronary angioplasty. BACKGROUND: Abrupt vessel closure complicates 4% to 8% of angioplasty procedures. Recent data have suggested that agents that antagonize the platelet glycoprotein IIb/IIIa receptor may reduce the incidence of adverse ischemic outcomes after coronary angioplasty. METHODS: Seventy-three patients received tirofiban in three sequential dose panels and 20 patients received placebo. Patients within each panel were randomized to receive either tirofiban or placebo in a 3:1 randomization design. Bolus doses of 5, 10 and 10 microg/kg and continuous infusion (16 to 24 h) doses of 0.05, 0.10 and 0.15 microg/kg per min were administered in panels I, II and III, respectively. Patients received concomitant heparin and aspirin for the angioplasty procedure. Data on patients receiving placebo (heparin and aspirin only) were pooled across panels for comparisons. The pharmacodynamic effect of tirofiban on ex vivo platelet aggregation to 5 micromol/liter adenosine diphosphate (ADP) and bleeding times were measured. Clinical outcomes were assessed in all patients, but the power to detect clinically meaningful differences (a one-third reduction in clinical events) between groups was limited (5%). RESULTS: Tirofiban was associated with a dose-dependent inhibition of ex vivo ADP-mediated platelet aggregation that was sustained during intravenous infusion and resolved rapidly after drug cessation. Adverse bleeding events, largely related to vascular access site hemorrhage, were slightly increased at the highest dose. Adverse clinical outcomes were infrequent in all patients and were not different among the small number of patients within each group. CONCLUSIONS: This study establishes a rational and generally well tolerated dosing regimen for administration of tirofiban as adjunctive therapy in high risk angioplasty patients. The impact of tirofiban on adverse clinical outcomes after angioplasty awaits definition by a larger clinical trial.

Functional EpoR pathway utilization is not detected in primary tumor cells isolated from human breast, non-small cell lung, colorectal, and ovarian tumor tissues.

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

Combinatorial gene expression using multiple episomal vectors.

Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.

Formation of a high affinity heregulin binding site using the soluble extracellular domains of ErbB2 with ErbB3 or ErbB4.

ErbB2 functions as a shared signal transducing component for other ErbB receptor family members. Two of these receptors, ErbB3 and ErbB4, bind the heregulin (HRG) or neuregulin family of polypeptide growth factors. Cells expressing ErbB3 alone display a single class of low affinity HRG binding sites, whereas both high and low affinity binding sites can be measured on cells that co-express both ErbB3 and ErbB2. To assess the interaction of the extracellular domains of ErbB receptors, a series of soluble homodimeric and heterodimeric IgG fusion proteins were constructed. Heregulin binding analysis revealed that a heterodimer composed of either ErbB3 or ErbB4 with ErbB2 is sufficient for the formation of a high affinity binding state. In contrast, heterodimeric ErbB3/4-IgG, as well as homodimeric ErbB3-IgG or ErbB4-IgG, contained only low affinity HRG binding sites. Further evidence for the unique specificity of ErbB2 in generating this high affinity binding site was determined by inhibiting HRG binding with an ErbB2 monoclonal antibody.

Trityl monitoring of automated DNA synthesizer operation by conductivity: a new method of real-time analysis.

AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide after each base addition and is a useful measure of synthesis efficiency. The traditional absorbance method of collecting each trityl effluent with a fraction collector, followed by dilution with an acid solution and careful quantitation by UV/VIS spectroscopy is costly, tedious and prone to error. The absorbance method for trityl cation analysis must usually wait until the synthesis is complete. Interruption of a failed operation, for a variety of reasons, such as an empty reagent reservoir, is thus not possible. Taking advantage of the conductive properties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream during the detritylation step after each nucleoside addition in DNA synthesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the current average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthesizer will interrupt, preserving reagents. AutoAnalysis allows trityl monitoring with complete automation on the Applied Biosystems Models 392 and 394 DNA/RNA Synthesizers.

Losartan potassium as initial therapy in patients with severe hypertension.

This 12-week, open-label study was conducted to gain experience with losartan potassium, an angiotensin II receptor antagonist, in patients with severe hypertension. Patients were either untreated or withdrawn from current therapy for at least 48 h before initiation of losartan 50 mg once daily. Patients were titrated to 100 mg as needed to achieve a goal of sitting diastolic blood pressure (SiDBP) 90 or 95 mm Hg. Hydrochlorothiazide (12.5 mg once daily titrated to 25 mg) was added and followed by either a dihydropyridine calcium channel blocker (CCB) and/or atenolol, if BP was not controlled. A total of 179 patients with a pretreatment mean baseline BP of 172 +/- 17/112 +/- 18 mm Hg enrolled in the trial and BP was recorded 24 h after dosing at baseline and weeks 2, 4, 8 and the final week (10-12 weeks). The mean reductions in SiDBP from baseline were 7.3, 9.3, 15.9 and 18.9 mm Hg, respectively, and these changes from baseline were statistically significant, P < 0.001. At the end of the trial, 22% of patients remained on losartan monotherapy, 30% required the addition of hydrochlorothiazide (HCTZ) and 31% required both HCTZ and a CCB; 11% required HCTZ and atenolol while 4% required HCTZ, a CCB and atenolol; 2% of patients were on regimens not specified by the protocol. SiDBP < 90 mm Hg was achieved in 68 patients by the final visit; 24% of these patients were treated with losartan monotherapy (50 or 100 mg), 41% achieved control with the addition of HCTZ (12.5 or 25 mg) and 24% required triple therapy which included losartan, HCTZ and a CCB. As assessed by the investigator, 25% of the patients in the study had drug-related clinical adverse experiences. Headache was the most frequently reported clinical adverse event (26% of patients). No clinically significant changes in laboratory parameters were observed. It is concluded that losartan potassium can be used as initial therapy for patients with severe hypertension and can be administered concurrently with hydrochlorothiazide, calcium channel blockers and atenolol.

Pharmacists' job satisfaction and perceived utilization of skills.

The relationship between job satisfaction and perceived utilization of skills among pharmacists practicing in institutional and ambulatory care settings in Arizona was studied, and factors thought to influence pharmacists' perceived utilization of skills were evaluated. Questionnaires on job satisfaction and perceived utilization of skills were mailed to a random sample of 600 pharmacists. Information on workplace factors such as hours worked, practice setting, and job title was collected. A 4-item measure of general job satisfaction and a 10-item measure of perceived utilization of skills were used. Responses were measured on a five-point Likert scale ranging from "strongly disagree" to "strongly agree." The response rate was 35%. There was a significant positive relationship between job satisfaction and perceived utilization of skills and between job satisfaction and adequate staffing, where "staffing" referred to factors such as competence of coworkers and workload. Pharmacists with training beyond a B.S. degree in pharmacy were more satisfied with their job than those whose highest degree was a B.S. in pharmacy. Pharmacists practicing in institutional settings, pharmacists with management titles, and older pharmacists perceived that they were utilizing their skills to a greater extent than did pharmacists practicing in ambulatory care settings, pharmacists with a general staff title, and younger pharmacists. Among a sample of Arizona pharmacists in institutional and ambulatory care settings, job satisfaction was influenced by perceived utilization of skills, staffing, and education; practice setting, job title, and age were significantly related to perceived utilization of skills.

The sole source: the Library of Congress National Library Service for the Blind and Physically Handicapped.

The Library of Congress National Library Service for the Blind and Physically Handicapped leads a co-operative network of specialized libraries and ancillary agencies that are delivering a successful programme of reading and informational services to blind, visually impaired and handicapped users. Its unique position within the Library of Congress and the benefits which emanate from this relationship are described. This current review outlines and discusses the development, role and functioning of the National Library Service.

6agonist selectivity determinants in somatostatin receptor subtypes I and II.

The biological activities of the peptide hormone somatostatin are mediated through a recently identified family of G-protein-linked receptors. A number of somatostatin analogs have been characterized with selective affinities for particular somatostatin receptor subtypes. Using one such molecule (MK-678), we have delineated receptor regions that determine analog selectivity in the murine Type 1 and Type 2 somatostatin receptors. We find that the regions about the second and third extracellular loops of these two receptors contain the determinants for MK-678 selectivity and affinity.

The Drosophila fushi tarazu polypeptide is a DNA-binding transcriptional activator in yeast cells.

Many of the regulatory genes controlling the developmental pattern of segmentation during embryonic development in Drosophila melanogaster encode nuclear proteins containing either homoeobox or 'zinc-finger' domains with putative or demonstrated sequence-specific DNA-binding properties. One of these Drosophila homoeobox-containing proteins is encoded by the fushi tarazu (ftz) gene. The expression of ftz is spatially restricted during embryogenesis and the ftz polypeptide has an important role in different stages of development. To determine whether the ftz polypeptide is a sequence-specific DNA-binding activator of transcription, we expressed portions of ftz as fusions with the yeast transcription factor GAL4 in yeast cells. Chimaeric GAL4/ftz proteins, like GAL4 itself, activated the transcription of a GAL4-dependent reporter gene. With reporter constructs containing Drosophila-derived chromosomal DNA sequences as transcriptional elements, the ftz polypeptide acted as a sequence-specific DNA-binding transcriptional activator. In Drosophila, the ftz product may therefore be a positive regulator of transcription.

The effect of familiar size at familiar distances.

The effect of familial size as a distance cue was tested with familiar objects at familiar distances. Experiment 1 showed that there were no uncontrolled distance cues available and that in their absence the retinal image did not affect depth or size perception. Under these conditions, size and distance judgments were essentially indeterminate and independent of each other. In experiment 2 a paradigm was employed which allowed a direct determination of whether equivalent changes either in size of a familiar object or in its true distance produced equivalent changes in its perceived distance. The results showed that there were no uncontrolled distance cues, and that subjects perceived the familiar object as having its familiar size. Moreover, changing the retinal image of the objects had almost exactly the same effect on their perceived distance as did changing their true distance. Hence, familial size does effectively govern the perception of distance when there are no competing cues.

Both overlapping and distinct signaling pathways for somatostatin receptor subtypes SSTR1 and SSTR2 in pituitary cells.

To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.

Homeodomain-independent activity of the fushi tarazu polypeptide in Drosophila embryos.

The Drosophila segmentation gene fushi tarazu (ftz) encodes a homeodomain-containing protein, ftz, that can act as a DNA-binding activator of transcription. In the developing embryo, ftz is expressed in seven stripes which correspond to the even-numbered parasegments. These parasegments are missing in ftz- embryos. When ftz is expressed throughout blastoderm embryos under the control of a heat-shock promoter, the odd-numbered parasegments are lost. This 'anti-ftz' phenotype has been attributed to autoactivation of the endogenous ftz gene by the ectopically expressed protein. Here we show that the same phenotype is induced by ectopic expression of a ftz polypeptide containing a deletion in the homeodomain. Thus, ftz can alter gene expression without binding directly to DNA.

Estimating low-density lipoprotein cholesterol by the Friedewald equation is adequate for classifying patients on the basis of nationally recommended cutpoints.

We compared low-density lipoprotein cholesterol (LDL) values obtained by the Friedewald formula--i.e., total cholesterol minus high-density lipoprotein (HDL) cholesterol minus very-low-density lipoprotein (VLDL) cholesterol (estimated as triglyceride divided by 5)--with those obtained by lipoprotein fractionation, using 4736 specimens. When triglycerides were less than 2.0 g/L, greater than 90% of estimated LDL cholesterol values were acceptable, within +/- 10% of measured values. At triglyceride concentrations of 2.0-4.0 g/L and 4.0-6.0 g/L, only 72% and 39%, respectively, of the estimates were acceptable. LDL values derived from an alternative formula, estimating VLDL as triglycerides divided by 6, were even less accurate. Nevertheless, the use of estimated LDL for risk classification based on the National Cholesterol Education Program Adult Treatment Panel cutpoints of 1.30 and 1.60 g/L was considered acceptable. At triglyceride concentrations less than or equal to 5.0 g/L, 88% of classifications based on estimated LDL (using triglycerides divided by 5) were concordant with those by measured LDL. Eleven percent of classifications were shifted across one cutpoint, evenly distributed between high and low. Fewer than 1% of classifications, all with Type III hyperlipoproteinemia, were misclassified two cutpoints high. Refinements in the estimation model did not substantially improve LDL estimation or concordance of risk classification.

Climbing-specific finger endurance: a comparative study of intermediate rock climbers, rowers and aerobically trained individuals.

The aim of this study was to compare the climbing-specific finger endurance of climbers, rowers and aerobically leg trained athletes. Twenty-seven males aged 21.2 +/- 2.2 years (mean +/- s) volunteered for the study. The participants were intermediate rock climbers (n = 9), rowers (n = 9) and leg trained athletes (n = 9). Maximal voluntary contraction (MVC) was determined on climbing-specific finger apparatus. Endurance isometric exercise was performed at 40% MVC in three tests performed in a random order: (1) sustained exercise; (2) 6 s exercise, 4 s rest; and (3) 18 s exercise, 12 s rest. Pre- and post-exercise blood pressure and blood lactate concentration, together with post-exercise pain perception, were measured. The climbers had a significantly greater MVC (383 +/- 35.6 N) than the rowers (321 +/- 49.5 N, P = 0.007) and aerobically leg trained athletes (288 +/- 60.6 N, P = 0.001). There were no significant differences between the groups in terms of endurance times for any of the tests. In the test with 18 s exercise and 12 s rest, the climbers showed a significantly higher increase in blood lactate concentration, on average, than the rowers by 0.01-0.89 mmol x l(-1) (P = 0.006); there were no significant differences, on average, in the comparisons of climbers and the leg trained athletes and rowers and the leg trained athletes. There were no significant differences in the average changes in blood pressure from rest to post-exercise between any of the groups. Although the climbers had greater MVC on average than the other two groups, there were no significant differences in average endurance times amongthe groups. These findings suggest that training for rock climbing and participation in rock climbing may result in some specific adaptations. However, we acknowledge that this study is descriptive and there is the possibility that differences between groups could be attributed to self-selection.

Social work in pediatric oncology: a family needs assessment.

When a child is diagnosed with cancer, the family is confronted with meeting both the physical and psychosocial needs of the child and maintaining normal family functioning. This study assessed the perceived psychosocial needs of 77 families who have a child diagnosed with cancer. Preliminary results suggest practical application for social work interventions in specific areas such as the development of an informal support network, enhancement of communication within families concerning the disease, the need for adequate information at various stages of the disease, and continued supportive services for the family.