A functioning artificial secretory cell

We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane

Model-based relationship between the molecular bacterial load assay and time-to-positivity in liquid culture

The molecular bacterial load (MBL) assay is a new tuberculosis biomarker which provides results in ∼4 hours. The relationship between MBL and time-to-positivity (TTP) has not been thoroughly studied and predictive models do not exist. We aimed to develop a model for MBL and identify the MBL-TTP relationship in patients. The model was developed on data from 105 tuberculosis patients from Malawi, Mozambique and Tanzania with joint MBL and TTP observations quantified from patient sputum collected for 12 weeks. MBL was quantified using polymerase chain reaction (PCR) of mycobacterial RNA and TTP using the Mycobacterial Growth Indicator Tube (MGIT) 960 system. Treatment consisted of isoniazid, pyrazinamide and ethambutol in standard doses together with rifampicin 10 or 35 mg/kg. The developed MBL-TTP model included several linked sub-models; a component describing decline of bacterial load in sputum, another component describing growth in liquid culture and a hazard model translating bacterial growth into a TTP signal. Additional components for contaminated and negative TTP samples were included. Visual predictive checks performed using the developed model gave good description of the observed data. The model predicted greater total sample loss for TTP than MBL due to contamination and negative samples. The model detected an increase in bacterial killing for 35 versus 10 mg/kg rifampicin (p=0.002). In conclusion, a combined model for MBL and TTP was developed that described the MBL-TTP relationship. The full MBL-TTP model or each sub-model used separately. Secondly, the model can be used to predict biomarker response for MBL given TTP data or vice versa in historical or future trials.PostprintPeer reviewe

Phosphorylated Nucleolin Interacts with Translationally Controlled Tumor Protein during Mitosis and with Oct4 during Interphase in ES Cells

BACKGROUND: Reprogramming of somatic cells for derivation of either embryonic stem (ES) cells, by somatic cell nuclear transfer (SCNT), or ES-like cells, by induced pluripotent stem (iPS) cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl) is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. METHODOLOGY: Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA), colocalization and immunoprecipitation (IP) in ES cells. PRINCIPAL FINDINGS: We found that phosphorylated Ncl (Ncl-P) interacted with translationally controlled tumor protein (Tpt1) in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s) of mammalian development. CONCLUSIONS: Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells

The disruptive positions in human G-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts

Specific guanine-rich sequence motifs in the human genome have considerable potential to form four-stranded structures known as G-quadruplexes or G4 DNA. The enrichment of these motifs in key chromosomal regions has suggested a functional role for the G-quadruplex structure in genomic regulation. In this work, we have examined the spectrum of nucleotide substitutions in G4 motifs, and related this spectrum to G4 prevalence. Data collected from the large repository of human SNPs indicates that the core feature of G-quadruplex motifs, 5′-GGG-3′, exhibits specific mutational patterns that preserve the potential for G4 formation. In particular, we find a genome-wide pattern in which sites that disrupt the guanine triplets are more conserved and less polymorphic than their neutral counterparts. This also holds when considering non-CpG sites only. However, the low level of polymorphisms in guanine tracts is not only confined to G4 motifs. A complete mapping of DNA three-mers at guanine polymorphisms indicated that short guanine tracts are the most under-represented sequence context at polymorphic sites. Furthermore, we provide evidence for a strand bias upstream of human genes. Here, a significantly lower rate of G4-disruptive SNPs on the non-template strand supports a higher relative influence of G4 formation on this strand during transcription

The disruptive positions in human G-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts

Specific guanine-rich sequence motifs in the human genome have considerable potential to form four-stranded structures known as G-quadruplexes or G4 DNA. The enrichment of these motifs in key chromosomal regions has suggested a functional role for the G-quadruplex structure in genomic regulation. In this work, we have examined the spectrum of nucleotide substitutions in G4 motifs, and related this spectrum to G4 prevalence. Data collected from the large repository of human SNPs indicates that the core feature of G-quadruplex motifs, 5′-GGG-3′, exhibits specific mutational patterns that preserve the potential for G4 formation. In particular, we find a genome-wide pattern in which sites that disrupt the guanine triplets are more conserved and less polymorphic than their neutral counterparts. This also holds when considering non-CpG sites only. However, the low level of polymorphisms in guanine tracts is not only confined to G4 motifs. A complete mapping of DNA three-mers at guanine polymorphisms indicated that short guanine tracts are the most under-represented sequence context at polymorphic sites. Furthermore, we provide evidence for a strand bias upstream of human genes. Here, a significantly lower rate of G4-disruptive SNPs on the non-template strand supports a higher relative influence of G4 formation on this strand during transcription

Reducing uncertainty in health-care resource allocation

A key task for health policymakers is to optimise the outcome of health care interventions. The pricing of a new generation of cancer drugs, in combination with limited health care resources, has highlighted the need for improved methodology to estimate outcomes of different treatment options. Here we introduce new general methodology, which for the first time employs continuous hazard functions for analysis of survival data. Access to continuous hazard functions allows more precise estimations of survival outcomes for different treatment options. We illustrate the methodology by calculating outcomes for adjuvant treatment of gastrointestinal stromal tumours with imatinib mesylate, which selectively inhibits the activity of a cancer-causing enzyme and is a hallmark representative for the new generation of cancer drugs. The calculations reveal that optimal drug pricing can generate all win situations that improve drug availability to patients, make the most of public expenditure on drugs and increase pharmaceutical company gross profits. The use of continuous hazard functions for analysis of survival data may reduce uncertainty in health care resource allocation, and the methodology can be used for drug price negotiations and to investigate health care intervention thresholds. Health policy makers, pharmaceutical industry, reimbursement authorities and insurance companies, as well as clinicians and patient organisations, should find the methodology useful

Greater early bactericidal activity at higher rifampicin doses revealed by modeling and clinical trial simulations

This work was supported by the Swedish Research Council (grant 521-2011-3442 to R. J. S. and U. S. H. S.), the Innovative Medicines Initiative Joint Undertaking (award 115337, with contribution from the European Union’s Seventh Framework Programme [FP7/2007–2013] and the European Federation of Pharmaceutical Industries and Associations [in-kind contribution]), the European and Developing Countries Clinical Trials Partnership (awards IP.2007.32011.011, IP.2007.32011.012, and IP.2007.32011.013), the Netherlands-African Partnership for Capacity Development and Clinical Interventions Against Poverty-Related Diseases, and the Bill and Melinda Gates Foundation.Background The currently recommended rifampicin dose (10 mg/kg) for treating tuberculosis is suboptimal. The PanACEA HIGHRIF1 trial evaluated the pharmacokinetics and early bactericidal activity of rifampicin doses of up to 40 mg/kg. Conventional statistical analyses revealed no significant exposure-response relationship. Our objectives were to explore the exposure-response relationship for high-dose rifampicin by using pharmacokinetic-pharmacodynamic modeling and to predict the early bactericidal activity of 50 mg/kg rifampicin. Methods Data included time to Mycobacterium tuberculosis positivity of liquid cultures of sputum specimens from 83 patients with tuberculosis who were treated with 10 mg/kg rifampicin (n = 8; reference arm) or 20, 25, 30, 35, or 40 mg/kg rifampicin (n = 15/arm) for 7 days. We used a semimechanistic time-to-event approach to model the time-to-positivity data. Rifampicin exposure and baseline time to culture positivity were explored as covariates. Results The baseline time to culture positivity was a significant covariate on the predicted initial bacterial load, and rifampicin exposure was a significant covariate on the bacterial kill rate in sputum resulting in increased early bactericidal activity. The 90% prediction interval for the predicted median day 7 increase in time to positivity for 50 mg/kg rifampicin was 7.25–10.3 days. Conclusions A significant exposure-response relationship was found between rifampicin exposure and early bactericidal activity. Clinical trial simulations showed greater early bactericidal activity for 50 mg/kg rifampicin.PostprintPeer reviewe

Anticipatory governance for social-ecological resilience

Anticipation is increasingly central to urgent contemporary debates, from climate change to the global economic crisis. Anticipatory practices are coming to the forefront of political, organizational, and citizens’ society. Research into anticipation, however, has not kept pace with public demand for insights into anticipatory practices, their risks and uses. Where research exists, it is deeply fragmented. This paper seeks to identify how anticipation is defined and understood in the literature and to explore the role of anticipatory practice to address individual, social, and global challenges. We use a resilience lens to examine these questions. We illustrate how varying forms of anticipatory governance are enhanced by multi-scale regional networks and technologies and by the agency of individuals, drawing from an empirical case study on regional water governance of Malaren, Sweden. Finally, we discuss how an anticipatory approach can inform adaptive institutions, decision making, strategy formation, and societal resilience