Automatic Design of Synthetic Gene Circuits through Mixed Integer Non-linear Programming

Automatic design of synthetic gene circuits poses a significant challenge to synthetic biology, primarily due to the complexity of biological systems, and the lack of rigorous optimization methods that can cope with the combinatorial explosion as the number of biological parts increases. Current optimization methods for synthetic gene design rely on heuristic algorithms that are usually not deterministic, deliver sub-optimal solutions, and provide no guaranties on convergence or error bounds. Here, we introduce an optimization framework for the problem of part selection in synthetic gene circuits that is based on mixed integer non-linear programming (MINLP), which is a deterministic method that finds the globally optimal solution and guarantees convergence in finite time. Given a synthetic gene circuit, a library of characterized parts, and user-defined constraints, our method can find the optimal selection of parts that satisfy the constraints and best approximates the objective function given by the user. We evaluated the proposed method in the design of three synthetic circuits (a toggle switch, a transcriptional cascade, and a band detector), with both experimentally constructed and synthetic promoter libraries. Scalability and robustness analysis shows that the proposed framework scales well with the library size and the solution space. The work described here is a step towards a unifying, realistic framework for the automated design of biological circuits

A phase I/II study of oxaliplatin when added to 5-fluorouracil and leucovorin and pelvic radiation in locally advanced rectal cancer: a Colorectal Clinical Oncology Group (CCOG) study

The purpose of this study was to evaluate the maximum tolerated dose (MTD) and recommended dose of oxaliplatin given synchronously with 5-fluorouracil (5FU), leucovorin (LV) and preoperative pelvic radiation for primary unresectable, locally advanced, rectal cancer. Preoperative pelvic radiotherapy using a three- or four-field technique and megavoltage photons comprised 45 Gy given in 25 fractions, 1.8 Gy per fraction, and delivered with escalating doses of oxaliplatin in combination with low-dose LV and 5FU. Chemotherapy was given synchronously with radiotherapy in weeks 1 and 5. Escalating doses of oxaliplatin (85, 130 and 150 mg m−2) were given on days 2 and 30, followed by low-dose LV (20 mg m−2) and 5FU (350 mg m−2), both given on days 1–5 and 29–33. Surgery was performed 6–10 weeks later. The MTD was determined as the dose causing more than a third of patients to have a dose-limiting toxicity (DLT). Once the MTD was reached, a further 14 patients were treated at the dose level below the MTD. In all, 32 patients received oxaliplatin at the three dose levels, median age 60 years (range 31–79), 24 males and eight females. The MTD was reached at 150 mg m−2 when four out of six patients experienced DLT. Dose-limiting grade 3 or 4 diarrhoea was reported in two out of six patients at 85 mg m−2, 5 out of 20 at 130 mg m−2 and four out of 6 at 150 mg m−2. Grade 3 neuropathy was reported at 130 mg m−2 (1 out of 20) and at 150 mg m−2 (two out of six), and serious haematological toxicity was minimal; one grade 3 anaemia at 150 mg m−2. In all, 28 out of 32 patients completed all treatments as planned; three had radiotherapy interrupted and three a chemotherapy dose reduction. Four patients did not proceed to surgery due to the presence of metastatic disease (two), unfitness (one) or patient refusal (one). Also, 28 patients underwent surgical resection. Histopathology demonstrated histopathological complete response (pCR) 2 out of 27 (7%), Tmic 3 out of 27 (11%), pCR+Tmic 5 out of 27 (19%), pT0–2 6 out of 27 (22%) and histologically confirmed clear circumferential resection margins in 22 out of 27 (81%). Dose-limiting toxicity with oxaliplatin is 150 mg m−2 given days 2 and 30 when added to the described 5FU LV and 45 Gy radiation preoperatively. The acceptable toxicity and compliance at 130 mg m−2 recommend testing this dose in future phase II studies. The tumour downstaging and complete resection rates are encouragingly high for this very locally advanced group

Diversity in sound pressure levels and estimated active space of resident killer whale vocalizations

Author Posting. © The Author, 2005. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology 192 (2006): 449-459, doi:10.1007/s00359-005-0085-2.Signal source intensity and detection range, which integrates source intensity with propagation loss, background noise and receiver hearing abilities, are important characteristics of communication signals. Apparent source levels were calculated for 819 pulsed calls and 24 whistles produced by free-ranging resident killer whales by triangulating the angles-of-arrival of sounds on two beamforming arrays towed in series. Levels in the 1-20 kHz band ranged from 131-168 dB re 1μPa @1m, with differences in the means of different sound classes (whistles: 140.2 ± 4.1 dB; variable calls: 146.6 ± 6.6 dB; stereotyped calls: 152.6 ± 5.9 dB), and among stereotyped call types. Repertoire diversity carried through to estimates of active space, with “long-range” stereotyped calls all containing overlapping, independently-modulated high-frequency components (mean estimated active space of 10-16km in sea state zero) and “short-range” sounds (5-9 km) included all stereotyped calls without a high-frequency component, whistles, and variable calls. Short-range sounds are reported to be more common during social and resting behaviors, while long-range stereotyped calls predominate in dispersed travel and foraging behaviors. These results suggest that variability in sound pressure levels may reflect diverse social and ecological functions of the acoustic repertoire of killer whales.Funding was provided by WHOI’s Ocean Ventures Fund and Rinehart Coastal Research Center and a Royal Society fellowship

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition

Polycomb repressive complex 2 (PRC2) is a key regulator of epigenetic states catalyzing histone H3 lysine 27 trimethylation (H3K27me3), a repressive chromatin mark. PRC2 composition is conserved from humans to plants, but the function of PRC2 during the early stage of plant life is unclear beyond the fact that it is required for the development of endosperm, a nutritive tissue that supports embryo growth. Circumventing the requirement of PRC2 in endosperm allowed us to generate viable homozygous null mutants for FERTILIZATION INDEPENDENT ENDOSPERM (FIE), which is the single Arabidopsis homolog of Extra Sex Combs, an indispensable component of Drosophila and mammalian PRC2. Here we show that H3K27me3 deposition is abolished genome-wide in fie mutants demonstrating the essential function of PRC2 in placing this mark in plants as in animals. In contrast to animals, we find that PRC2 function is not required for initial body plan formation in Arabidopsis. Rather, our results show that fie mutant seeds exhibit enhanced dormancy and germination defects, indicating a deficiency in terminating the embryonic phase. After germination, fie mutant seedlings switch to generative development that is not sustained, giving rise to neoplastic, callus-like structures. Further genome-wide studies showed that only a fraction of PRC2 targets are transcriptionally activated in fie seedlings and that this activation is accompanied in only a few cases with deposition of H3K4me3, a mark associated with gene activity and considered to act antagonistically to H3K27me3. Up-regulated PRC2 target genes were found to act at different hierarchical levels from transcriptional master regulators to a wide range of downstream targets. Collectively, our findings demonstrate that PRC2-mediated regulation represents a robust system controlling developmental phase transitions, not only from vegetative phase to flowering but also especially from embryonic phase to the seedling stage

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.This is the final published version. It was originally published by PLOS in PLOS Genetics here: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1004417

Health-related quality of life dynamics of HIV-positive South African women up to ART initiation : evidence from the CAPRISA 002 acute infection cohort study.

CAPRISA, 2014.Few studies have investigated the long-term dynamics in health-related quality of life (HRQoL) among HIV-positive persons from acute infection. From 2004, 160 women were enrolled into the CAPRISA 002 Acute Infection study at two sites in the province of KwaZulu-Natal and underwent 3–6 monthly HRQoL assessments using the functional assessment of HIV infection (FAHI) instrument. Overall and 5 sub-scale FAHI scores [physical well-being (PWB), emotional well-being (EWB), functional and global well-being (FGWB), social well-being (SWB) and cognitive functioning (CF)] were calculated up to antiretroviral therapy (ART) initiation and scores at enrollment were compared to the acute, early and established infection phases. Mixed-effects regression models adjusting for behavioral and clinical factors were applied to assess HRQoL trends and the proportion of women meeting minimally important differences was calculated. Our analyses revealed that overall/sub-scale scores improved over time, except from PWB and CF. A higher educational status, contraceptive use and a higher BMI were the strongest predictors of higher overall/sub-scale FAHI scores. CD4 count and HIV viral load were strongly associated with PWB and CF, but not overall FAHI and other sub-scales. Women newly diagnosed with acute HIV infection face profound HRQoL challenges. While early ART delivery may be important for PWB and CF, factors such as education, contraception provision and good nutritional status should be promoted to maximize HRQoL in HIV positive individuals