Repository-based plasmid design

There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to over 300,000 among three of the largest repositories: iGEM, Addgene, and DNASU. A challenge in biodesign remains how to use these and other repository-based sequences effectively, correctly, and seamlessly. This work describes an approach to plasmid design where a plasmid is specified as simply a DNA sequence or list of features. The proposed software then finds the most cost-effective combination of synthetic and PCR-prepared repository fragments to build the plasmid via Gibson assembly®. It finds existing DNA sequences in both user-specified and public DNA databases: iGEM, Addgene, and DNASU. Such a software application is introduced and characterized against all post-2005 iGEM composite parts and all Addgene vectors submitted in 2018 and found to reduce costs by 34% versus a purely synthetic plasmid design approach. The described software will improve current plasmid assembly workflows by shortening design times, improving build quality, and reducing costs.Accepted manuscrip

Automated robotic liquid handling assembly of modular DNA devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

Effect of heavy-intensity 'priming' exercise on oxygen uptake and muscle deoxygenation kinetics during moderate-intensity step-transitions initiated from an elevated work rate

We examined the effect of heavy-intensity ‘priming’ exercise on the rate of adjustment of pulmonary O2 uptake (τ 2p) initiated from elevated intensities. Fourteen men (separated into two groups: τ 2p≤25s [Fast] or τ 2p>25s [Slow]) completed step-transitions from 20W-to- 45%lactate threshold (LT; lower-step, LS) and 45%-to-90%LT (upper-step, US) performed (i) without; and (ii) with US preceded by heavy-intensity exercise (HUS). Breath-by-breath 2p and near-infrared spectroscopy-derived muscle deoxygenation ([HHb+Mb]) were measured. Compared to LS, τ 2p was greater (p0.05) from LS or Fast group US. In Slow, τ[HHb+Mb] increased (p<0.05) in US relative to HUS; this finding coupled with a reduced τ 2p indicates a priming-induced improvement in matching of muscle O2 delivery-to-O2 utilization during transitions from elevated intensities in those with Slow but not Fast 2p kinetics

Multi-wavelength lens construction of a Planck and Herschel-detected star-bursting galaxy

We present a source-plane reconstruction of a Herschel and Planck-detected gravitationally lensed dusty star-forming galaxy (DSFG) at z = 1.68 using Hubble, Submillimeter Array (SMA), and Keck observations. The background submillimeter galaxy (SMG) is strongly lensed by a foreground galaxy cluster at z = 0.997 and appears as an arc with a length of ∼15″ in the optical images. The continuum dust emission, as seen by SMA, is limited to a single knot within this arc. We present a lens model with source-plane reconstructions at several wavelengths to show the difference in magnification between the stars and dust, and highlight the importance of multi-wavelength lens models for studies involving lensed DSFGs. We estimate the physical properties of the galaxy by fitting the flux densities to model spectral energy distributions leading to a magnification-corrected starformation rate (SFR) of 390 ± 60 M yr−1 and a stellar mass of 1.1 ± 0.4 10 x 11 M. These values are consistent with high-redshift massive galaxies that have formed most of their stars already. The estimated gas-to-baryon fraction, molecular gas surface density, and SFR surface density have values of 0.43 ± 0.13, 350 ± 200 M pc−2, and ~ 12 7 M yr−1 kpc−2, respectively. The ratio of SFR surface density to molecular gas surface density puts this among the most star-forming systems, similar to other measured SMGs and local ULIRGs

Increased Mitochondrial Calcium Sensitivity and Abnormal Expression of Innate Immunity Genes Precede Dopaminergic Defects in Pink1-Deficient Mice

BACKGROUND: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson\u27s disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice. METHODS AND FINDINGS: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting. CONCLUSIONS: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls

Tubulin's response to external electric fields by molecular dynamics simulations.

Tubulin heterodimers are the building blocks of microtubules and disruption of their dynamics is exploited in the treatment of cancer. Electric fields at certain frequencies and magnitudes are believed to do the same. Here, the tubulin dimer's response to external electric fields was determined by atomistic simulation. External fields from 50 to 750 kV/cm, applied for 10 ns, caused significant conformational rearrangements that were dependent upon the field's directionality. Charged and flexible regions, including the α:H1-B2 loop, β:M-loop, and C-termini, were susceptible. Closer inspection of the α:H1-B2 loop in lower strength fields revealed that these effects were consistent and proportional to field strength, and the findings indicate that external electric fields modulate the stability of microtubules through conformational changes to key loops involved in lateral contacts. We also find evidence that tubulin's curvature and elongation are affected, and external electric fields may bias tubulin towards depolymerization

Phase I study of low-dose metronomic temozolomide for recurrent malignant gliomas

Background: The treatment goal for recurrent malignant gliomas centers on disease stabilization while minimizing therapy-related side effects. Metronomic dosing of cytotoxic chemotherapy has emerged as a promising option to achieve this objective. Methods: This phase I study was performed using metronomic temozolomide (mTMZ) at 25 or 50 mg/m2/day continuously in 42-day cycles. Correlative studies were incorporated using arterial spin labeling MRI to assess tumor blood flow, analysis of matrix metalloproteinase-2 (MMP-2) and MMP-9 activities in the cerebrospinal fluid (CSF) as surrogates for tumor angiogenesis and invasion, as well as determination of CSF soluble interleukin-2 receptor alpha (sIL-2Rα) levels as a marker of immune modulation. Results: Nine subjects were enrolled and toxicity consisted of primarily grade 1 or 2 hematological and gastrointestinal side effects; only one patient had a grade 3 elevated liver enzyme level that was reversible. Tumor blood flow was variable across subjects and time, with two experiencing a transient increase before a decrease to below baseline level while one exhibited a gradual drop in blood flow over time. MMP-2 activity correlated with overall survival but not with progression free survival, while MMP-9 activity did not correlate with either outcome parameters. Baseline CSF sIL-2Rα level was inversely correlated with time from initial diagnosis to first progression, suggesting that subjects with higher sIL-2Rα may have more aggressive disease. But they lived longer when treated with mTMZ, probably due to drug-related changes in T-cell constituency. Conclusions: mTMZ possesses efficacy against recurrent malignant gliomas by altering blood flow, slowing invasion and modulating antitumor immune function