A Single Antibody based ELISA for the N-terminal sequence of BAG-75, a New Biomarker for Bone Formation [abstract]

Biomedical Tissue Engineering, Biomaterials, & Medical Devices Poster SessionBone acidic glycoprotein-75 (BAG-75) is a secreted product of osteoblastic cells localized predominantly to areas of new bone formation. We have identified the N-terminal sequence of BAG-75 as LPVARYQNTEEEE and shown that anti-peptide antibodies against residues #3-13 only recognize the 75 kDa precursor and apparent 50 kDa fragment in serum and in osteoblastic cultures. Formation of the 50 kDa fragment is blocked by AEBSF, a serine protease inhibitor which we also showed blocks mineralization in osteoblastic cultures. Measurement of BAG-75 and its fragment concentration in serum represents a new method to estimate the rate of new bone formation in vivo. Our purpose was to establish an anti-VARYQNTEEEE peptide antibody based ELISA test to measure cross-reactive proteins released from bone into blood. Western blotting was performed using young rat serum from different ages, rats subjected to ovariectomy (OVX) or sham surgery, and normal human serum. Immunoreactive 50 kDa fragment peaked at 18 days after birth which parallels bone formation. Ovariectomized rats displayed a peak of 50 kDa immunoreactivty at 21 days after surgery which corresponds to a spike in bone formation in this model (~2.5-fold above controls). Comparable assays for osteocalcin showed only a 39% increase. Also, human serum contains a 50 kDa protein which cross-reacts with anti-VARYQNTEEEE antibodies. We then established a competitive 96-well ELISA using anti-peptide antibody and new sera at 21 days from ovariectomized or sham rats, a model for stimulated bone formation. VARYQNTEEEE peptide conjugated to keyhole limpet hemocyanin (KLH) was used as the bound antigen. KLH-peptide amount, primary antibody concentration, secondary antibody concentration, and blocking agent were optimized in a series of experiments. Optimal conditions were determined to be 2 µg input KLH-peptide per well, 1/5,000 dilution of primary anti-VARYQNTEEEE antibody, 1/10,000 dilution of secondary antibody, and gelatin as a blocking agent. Sera from OVX rats and sham-operated controls were compared to the standard curve (r = 0.9923) created with free KLH-peptide as competitor to determine the equivalent amount of KLH-peptide present. OVX sera (n=3) contained an average 2.6 x 10-4 (+/- 1.4 x 10-4) µg peptide equivalent versus 1.05 x 10-4 (+/- 0.68 x 10-4) µg for sham sera (n=3). The difference was not significant (t-test, p=0.157), however, doubling the sample size is predicted to yield significance. Conclusions: A. Cross-reactive 75 kDa and 50 kDa proteins are present in human and rat serum and increase in concentration when bone formation is stimulated. B. A new, single antibody based ELISA assay was established to quantitate antigen released from bone into blood. C. In contrast to other commercial bone formation assays (collagen peptides and osteocalcin), the size of cross-reactive protein (>50 kDa) should preclude kidney filtration and facilitate measurement. D. This serum biomarker undergoes a 2-3 fold average increase within 3 weeks after simulation of bone. This test may be useful to monitor the early response to stimulatory therapy in osteoporosis patients or to repressive glucocorticoid therapy in sarcoidosis patients. Currently, a 1% change in bone mineral density requires 12-18 months to detect by x-ray methods

Insect Eggs Can Enhance Wound Response in Plants: A Study System of Tomato Solanum lycopersicum L. and Helicoverpa zea Boddie

Insect oviposition on plants frequently precedes herbivory. Accumulating evidence indicates that plants recognize insect oviposition and elicit direct or indirect defenses to reduce the pressure of future herbivory. Most of the oviposition-triggered plant defenses described thus far remove eggs or keep them away from the host plant or their desirable feeding sites. Here, we report induction of antiherbivore defense by insect oviposition which targets newly hatched larvae, not the eggs, in the system of tomato Solanum lycopersicum L., and tomato fruitworm moth Helicoverpa zea Boddie. When tomato plants were oviposited by H. zea moths, pin2, a highly inducible gene encoding protease inhibitor2, which is a representative defense protein against herbivorous arthropods, was expressed at significantly higher level at the oviposition site than surrounding tissues, and expression decreased with distance away from the site of oviposition. Moreover, more eggs resulted in higher pin2 expression in leaves, and both fertilized and unfertilized eggs induced pin2 expression. Notably, when quantified daily following deposition of eggs, pin2 expression at the oviposition site was highest just before the emergence of larvae. Furthermore, H. zea oviposition primed the wound-induced increase of pin2 transcription and a burst of jasmonic acid (JA); tomato plants previously exposed to H. zea oviposition showed significantly stronger induction of pin2 and higher production of JA upon subsequent simulated herbivory than without oviposition. Our results suggest that tomato plants recognize H. zea oviposition as a signal of impending future herbivory and induce defenses to prepare for this herbivory by newly hatched neonate larvae

Design, baseline characteristics, and retention of African American light smokers into a randomized trial involving biological data

<p>Abstract</p> <p>Background</p> <p>African Americans experience significant tobacco-related health disparities despite the fact that over half of African American smokers are light smokers (use ≤10 cigarettes per day). African Americans have been under-represented in smoking cessation research, and few studies have evaluated treatment for light smokers. This paper describes the study design, measures, and baseline characteristics from <it>Kick It at Swope III </it>(KIS-III), the first treatment study of bupropion for African American light smokers.</p> <p>Methods</p> <p>Five hundred forty African American light smokers were randomly assigned to receive bupropion (150mg bid) (n = 270) or placebo (n = 270) for 7 weeks. All participants received written materials and health education counseling. Participants responded to survey items and provided blood samples for evaluation of phenotype and genotype of CYP2A6 and CYP2B6 enzymes involved in nicotine and bupropion metabolism. Primary outcome was cotinine-verified 7-day point prevalence smoking abstinence at Week 26 follow-up.</p> <p>Results</p> <p>Of 2,628 individuals screened, 540 were eligible, consented, and randomized to treatment. Participants had a mean age of 46.5 years and 66.1% were women. Participants smoked an average of 8.0 cigarettes per day, had a mean exhaled carbon monoxide of 16.4ppm (range 1-55) and a mean serum cotinine of 275.8ng/ml. The mean Fagerström Test for Nicotine Dependence was 3.2, and 72.2% of participants smoked within 30 minutes of waking. The average number of quit attempts in the past year was 3.7 and 24.2% reported using pharmacotherapy in their most recent quit attempt. Motivation and confidence to quit were high.</p> <p>Conclusion</p> <p>KIS-III is the first study designed to examine both nicotine and bupropion metabolism, evaluating CYP2A6 and CYP2B6 phenotype and genotype in conjunction with psychosocial factors, in the context of treatment of African American light smokers. Of 1629 smokers screened for study participation, only 18 (1.1%) were ineligible to participate in the study because they refused blood draws, demonstrating the feasibility of recruiting and enrolling African American light smokers into a clinical treatment trial involving biological data collection and genetic analyses. Future evaluation of individual factors associated with treatment outcome will contribute to advancing tailored tobacco use treatment with the goal of enhancing treatment and reducing health disparities for African American light smokers.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="URL">NCT00666978</a></p

Business mathematics

xix, 682 p. ; 27 cm

Business mathematics. Thirteenth edition

Harlow Essex, England776 p.: app., index; 28 c

Novel Oral Anticoagulants for Venous Thromboembolism with Special Emphasis on Risk of Hemorrhagic Complications and Reversal Agents

Osnovni cilj istraživanja završnog rada bio je ispitati empatiju, prosocijalno ponašanje i samoregulaciju studentica koje se osposobljavaju za rad s djecom predškolske dobi. To može biti pokazatelj u kojoj su mjeri kod njih razvijene kompetencije empatiziranja s osobama u njihovom okruženju, prepoznavanja i razumijevanja tuđih emocija, moralno rasuđivanje i postupci te regulacija vlastitih emocija i ponašanja. Istraživanje je provedeno na uzorku od 58 studentica predškolskog odgoja. Iako ne u svim dimenzijama, studentice su kod većine pokazale viši stupanj razvoja empatije te srednje razvijeno prosocijalno ponašanje i samoregulaciju. Također, pokazala se i značajna pozitivna povezanost među nekim dimenzijama ovih kompetencija. Biti odgojiteljica izrazito je izazovan profesionalni zadatak. Odgojiteljica koja se trudi biti uspješna u svojem zadatku, osim što treba biti usmjerena na odgojno-obrazovni aspekt za poticanje razvoja djeteta, također treba biti vrlo svjesna sebe, svojih emocionalnih stanja i kapaciteta, svojih komunikacijskih obrazaca, kako s djecom, tako i s roditeljima i kolegicama i kolegama na poslu te načina postavljanja granica u ponašanju djece. To nužno uvjetuje stalno profesionalno usavršavanje i stalni rad na sebi. Radom na sebi i povećanjem razvoja vlastitih sposobnosti utjecat ćemo na pozitivan rast i razvoj socioemocionalnih kompetencija djece.The main purpose in this research was to explore empathy, prosocial behaviour and self-regulation of students who are trained to work in nursery and kindergarten. It can be a predictor of how well they have developed competencies of empathizing with people in their environment, recognizing and understanding other people’s emotions, making moral judgments and actions and regulation of their own emotions and behaviour. The research was conducted on a sample of 58 university students. Although not in all dimensions, students showed higher developed empathy, and moderately developed prosocial behaviour and self-regulation. There is, also, a significant positive correlation between certain dimensions of these competences. Being a nursery and kindergarten teacher is a very challenging profession. The teachers that are trying to be successful in their work, in addition to being focused on the educational aspect of child development, also need to be very aware of themselves, their emotional states and capacities, communication skills with children, parents and colleagues at work and the ways to set boundaries in children's behaviour. This necessarily requires constant professional development and constant work on themselves. By working on themselves and enhancing the development of the own abilities, they will influence the positive growth and development of children's socio-emotional competences

Spectrum of disease caused by Rhodococcus equi in human immunodeficiency virus infection: Report of a case and review of the literature

Since the first report of Rhodococcus equi infection in an acquired immune deficiency syndrome patient in 1986, seven additional cases have been described. A patient is described in whom the diagnosis was delayed due to misidentification of the organism as an atypical mycobacterial species. The literature regarding R equi infection in persons infected with the human immunodeficiency virus is reviewed. The most common presentation is one of a chronic, indolent pulmonary infiltrative disease (78%). Fever (78%), cough (67%), and hemoptysis (44%) are frequently present. Coexistent opportunistic illnesses are common (67%). In the laboratory identification of this organism, it is important to communicate the clinical setting to the microbiologist and to recognize the potential for the organism to be overlooked as normal flora or a contaminant, or misidentified as an organism with similar phenotypic characteristics (Nocardia species or a rapidly growing mycobacterium). Based on experience in foals, therapy with erythromycin and rifampin is suggested

Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time

Viruses may play an important role in the evolution of human microbial communities. Clustered regularly interspaced short palindromic repeats (CRISPRs) provide bacteria and archaea with adaptive immunity to previously encountered viruses. Little is known about CRISPR composition in members of human microbial communities, the relative rate of CRISPR locus change, or how CRISPR loci differ between the microbiota of different individuals. We collected saliva from four periodontally healthy human subjects over an 11- to 17-mo time period and analyzed CRISPR sequences with corresponding streptococcal repeats in order to improve our understanding of the predominant features of oral streptococcal adaptive immune repertoires. We analyzed a total of 6859 CRISPR bearing reads and 427,917 bacterial 16S rRNA gene sequences. We found a core (ranging from 7% to 22%) of shared CRISPR spacers that remained stable over time within each subject, but nearly a third of CRISPR spacers varied between time points. We document high spacer diversity within each subject, suggesting constant addition of new CRISPR spacers. No greater than 2% of CRISPR spacers were shared between subjects, suggesting that each individual was exposed to different virus populations. We detect changes in CRISPR spacer sequence diversity over time that may be attributable to locus diversification or to changes in streptococcal population structure, yet the composition of the populations within subjects remained relatively stable. The individual-specific and traceable character of CRISPR spacer complements could potentially open the way for expansion of the domain of personalized medicine to the oral microbiome, where lineages may be tracked as a function of health and other factors