Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

<p>Abstract</p> <p>Background</p> <p>To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering.</p> <p>Results</p> <p>An interactive <b>Alignment Viewer </b>allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The <b>Construct Design Module </b>enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned <it>in silico </it>into defined expression vectors. The <b>Gene Design Module </b>uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies.</p> <p>Conclusion</p> <p>We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies.</p

Probing the inflaton: Small-scale power spectrum constraints from measurements of the CMB energy spectrum

In the early Universe, energy stored in small-scale density perturbations is quickly dissipated by Silk-damping, a process that inevitably generates mu- and y-type spectral distortions of the cosmic microwave background (CMB). These spectral distortions depend on the shape and amplitude of the primordial power spectrum at wavenumbers k < 10^4 Mpc^{-1}. Here we study constraints on the primordial power spectrum derived from COBE/FIRAS and forecasted for PIXIE. We show that measurements of mu and y impose strong bounds on the integrated small-scale power, and we demonstrate how to compute these constraints using k-space window functions that account for the effects of thermalization and dissipation physics. We show that COBE/FIRAS places a robust upper limit on the amplitude of the small-scale power spectrum. This limit is about three orders of magnitude stronger than the one derived from primordial black holes in the same scale range. Furthermore, this limit could be improved by another three orders of magnitude with PIXIE, potentially opening up a new window to early Universe physics. To illustrate the power of these constraints, we consider several generic models for the small-scale power spectrum predicted by different inflation scenarios, including running-mass inflation models and inflation scenarios with episodes of particle production. PIXIE could place very tight constraints on these scenarios, potentially even ruling out running-mass inflation models if no distortion is detected. We also show that inflation models with sub-Planckian field excursion that generate detectable tensor perturbations should simultaneously produce a large CMB spectral distortion, a link that could potentially be established by PIXIE.Comment: 13 pages, 10 figures, submitted to Ap

High Prevalence of Tuberculosis and Serious Bloodstream Infections in Ambulatory Individuals Presenting for Antiretroviral Therapy in Malawi

Background Tuberculosis (TB) and serious bloodstream infections (BSI) may contribute to the high early mortality observed among patients qualifying for antiretroviral therapy (ART) with unexplained weight loss, chronic fever or chronic diarrhea. Methods and Findings A prospective cohort study determined the prevalence of undiagnosed TB or BSI among ambulatory HIV-infected adults with unexplained weight loss and/or chronic fever, or diarrhea in two routine program settings in Malawi. Subjects with positive expectorated sputum smears for AFB were excluded. Investigations Bacterial and mycobacterial blood cultures, cryptococcal antigen test (CrAg), induced sputum (IS) for TB microscopy and solid culture, full blood count and CD4 lymphocyte count. Among 469 subjects, 52 (11%) had microbiological evidence of TB; 50 (11%) had a positive (non-TB) blood culture and/or positive CrAg. Sixty-five additional TB cases were diagnosed on clinical and radiological grounds. Nontyphoidal Salmonellae (NTS) were the most common blood culture pathogens (29 cases; 6% of participants and 52% of bloodstream isolates). Multivariate analysis of baseline clinical and hematological characteristics found significant independent associations between oral candidiasis or lymphadenopathy and TB, marked CD4 lymphopenia and NTS infection, and severe anemia and either infection, but low positive likelihood ratios (<2 for all combinations). Conclusions We observed a high prevalence of TB and serious BSI, particularly NTS, in a program cohort of chronically ill HIV-infected outpatients. Baseline clinical and hematological characteristics were inadequate predictors of infection. HIV clinics need better rapid screening tools for TB and BSI. Clinical trials to evaluate empiric TB or NTS treatment are required in similar populations

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes