Transient peak-strain matching partially recovers the age-impaired mechanoadaptive cortical bone response

Mechanoadaptation maintains bone mass and architecture; its failure underlies age-related decline in bone strength. It is unclear whether this is due to failure of osteocytes to sense strain, osteoblasts to form bone or insufficient mechanical stimulus. Mechanoadaptation can be restored to aged bone by surgical neurectomy, suggesting that changes in loading history can rescue mechanoadaptation. We use non-biased, whole-bone tibial analyses, along with characterisation of surface strains and ensuing mechanoadaptive responses in mice at a range of ages, to explore whether sufficient load magnitude can activate mechanoadaptation in aged bone. We find that younger mice adapt when imposed strains are lower than in mature and aged bone. Intriguingly, imposition of short-term, high magnitude loading effectively primes cortical but not trabecular bone of aged mice to respond. This response was regionally-matched to highest strains measured by digital image correlation and to osteocytic mechanoactivation. These data indicate that aged bone’s loading response can be partially recovered, non-invasively by transient, focal high strain regions. Our results indicate that old murine bone does respond to load when the loading is of sufficient magnitude, and bones’ age-related adaptation failure may be due to insufficient mechanical stimulus to trigger mechanoadaptation

Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion

The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells

Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (KD = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7'-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7'-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7'-13 and LigBCen7'-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction

Medication Use Patterns among Urban Youth Participating in School-Based Asthma Education

Although pharmaceutical management is an integral part of asthma control, few community-based analyses have focused on this aspect of disease management. The primary goal of this analysis was to assess whether participation in the school-based Kickin’ Asthma program improved appropriate asthma medication use among middle school students. A secondary goal was to determine whether improvements in medication use were associated with subsequent improvements in asthma-related symptoms among participating students. Students completed an in-class case-identification questionnaire to determine asthma status. Eligible students were invited to enroll in a school-based asthma curriculum delivered over four sessions by an asthma health educator. Students completed a pre-survey and a 3-month follow-up post-survey that compared symptom frequency and medication use. From 2004 to 2007, 579 participating students completed pre- and post-surveys. Program participation resulted in improvements in appropriate use across all three medication use categories: 20.0% of students initiated appropriate reliever use when “feeling symptoms” (p < 0.001), 41.6% of students reporting inappropriate medication use “before exercise” initiated reliever use (p < 0.001), and 26.5% of students reporting inappropriate medication use when “feeling fine” initiated controller use (p < 0.02). More than half (61.6%) of participants reported fewer symptoms at post-survey. Symptom reduction was not positively associated with improvements in medication use in unadjusted and adjusted analysis, controlling for sex, asthma symptom classification, class attendance, season, and length of follow-up. Participation in a school-based asthma education program significantly improved reliever medication use for symptom relief and prior-to-exercise and controller medication use for maintenance. However, given that symptom reduction was not positively associated with improvement in medication use, pharmaceutical education must be just one part of a comprehensive asthma management agenda that addresses the multifactorial nature of asthma-related morbidity

Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics

Relatively Low HIV Infection Rates in Rural Uganda, but with High Potential for a Rise: A Cohort Study in Kayunga District, Uganda

BACKGROUND: Few studies have been conducted in Uganda to identify and quantify the determinants of HIV-1 infection. We report results from a community-based cohort study, whose primary objectives were to determine HIV-1 prevalence, incidence, and determinants of these infections, among other objectives. METHODOLOGY: Consenting volunteers from the rural district of Kayunga in Uganda aged 15-49 years were enrolled between March and July 2006. Participants were evaluated every six months. A questionnaire that collected information on behavioral and other HIV-1 risk factors was administered, and a blood sample obtained for laboratory analysis at each study visit. PRINCIPAL FINDINGS: HIV-1 prevalence among the 2025 participants was 9.9% (95% CI = 8.6%-11.2%). By the end of 12 months of follow-up, 1689.7 person-years had been accumulated, with a median follow-up time of 11.97 months. Thirteen HIV-1 incident cases were detected giving an annual HIV-1 incidence of 0.77% (95% CI = 0.35-1.19). Prevalence of HSV-2 infection was 57% and was strongly associated with prevalent HIV-1 infection (adjusted Odds Ratio = 3.9, 95% CI = 2.50-6.17); as well as incident HIV-1 infection (adjusted Rate Ratio (RR) = 8.7, 95% CI = 1.11-67.2). The single most important behavioral characteristic associated with incident HIV infection was the number of times in the past 6 months, a participant had sex with person(s) they suspected/knew were having sex with others; attaining statistical significance at 10 times and higher (adjusted RR = 6.3, 95% CI = 1.73-23.1). By the end of 12 months of follow-up, 259 participants (13%) were lost to follow-up, 13 (0.6%) had died, and 2 (0.1%) had withdrawn consent. CONCLUSIONS: Despite relatively low HIV-1 incidence observed in this community, prevalence remains relatively high. In the presence of high prevalence of HSV-2 infection and the behavioral characteristic of having sex with more than one partner, there is potential for increase in HIV-1 incidence

Phylogenetic representativeness: a new method for evaluating taxon sampling in evolutionary studies

<p>Abstract</p> <p>Background</p> <p>Taxon sampling is a major concern in phylogenetic studies. Incomplete, biased, or improper taxon sampling can lead to misleading results in reconstructing evolutionary relationships. Several theoretical methods are available to optimize taxon choice in phylogenetic analyses. However, most involve some knowledge about the genetic relationships of the group of interest (i.e., the ingroup), or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications.</p> <p>Results</p> <p>We propose a new method to assess taxon sampling developing Clarke and Warwick statistics. This method aims to measure the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, our method also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses we describe in this paper.</p> <p>Conclusions</p> <p>We show that this method is sensitive and allows direct discrimination between representative and unrepresentative samples. It is also informative about the addition of taxa to improve taxonomic coverage of the ingroup. Provided that the investigators' expertise is mandatory in this field, phylogenetic representativeness makes up an objective touchstone in planning phylogenetic studies.</p

Population Genetics of Duplicated Alternatively Spliced Exons of the Dscam Gene in Daphnia and Drosophila

In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- And Rab8-Dependent and recycling endosome-independent

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the m1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells. © 2014 Bertuccio et al

Cost-effectiveness of tuberculosis evaluation and treatment of newly-arrived immigrants

BACKGROUND: Immigrants to the U.S. are required to undergo overseas screening for tuberculosis (TB), but the value of evaluation and treatment following entry to the U.S. is not well understood. We determined the cost-effectiveness of domestic follow-up of immigrants identified as tuberculosis suspects through overseas screening. METHODS: Using a stochastic simulation for tuberculosis reactivation, transmission, and follow-up for a hypothetical cohort of 1000 individuals, we calculated the incremental cost-effectiveness of follow-up and evaluation interventions. We utilized published literature, California Reports of Verified Cases of Tuberculosis (RVCTs), demographic estimates from the California Department of Finance, Medicare reimbursement, and Medi-Cal reimbursement rates. Our target population was legal immigrants to the United States, our time horizon is twenty years, and our perspective was that of all domestic health-care payers. We examined the intervention to offer latent tuberculosis therapy to infected individuals, to increase the yield of domestic evaluation, and to increase the starting and completion rates of LTBI therapy with INH (isoniazid). Our outcome measures were the number of cases averted, the number of deaths averted, the incremental dollar cost (year 2004), and the number of quality-adjusted life-years saved. RESULTS: Domestic follow-up of B-notification patients, including LTBI treatment for latently infected individuals, is highly cost-effective, and at times, cost-saving. B-notification follow-up in California would reduce the number of new tuberculosis cases by about 6–26 per year (out of a total of approximately 3000). Sensitivity analysis revealed that domestic follow-up remains cost-effective when the hepatitis rates due to INH therapy are over fifteen times our best estimates, when at least 0.4 percent of patients have active disease and when hospitalization of cases detected through domestic follow-up is no less likely than hospitalization of passively detected cases. CONCLUSION: While the current immigration screening program is unlikely to result in a large change in case rates, domestic follow-up of B-notification patients, including LTBI treatment, is highly cost-effective. If as many as three percent of screened individuals have active TB, and early detection reduces the rate of hospitalization, net savings may be expected