Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay.

Confirming the binding of a transcription factor with a particular DNA sequence may be important in characterizing interactions with a synthetic promoter. Electrophoretic mobility shift assay is a powerful approach to demonstrate the specific DNA sequence that is bound by a transcription factor and also to confirm the specific transcription factor involved in the interaction. In this chapter we describe a method we have successfully used to demonstrate interactions of endogenous transcription factors with sequences derived from endogenous and synthetic promoters

Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle- shaped viruses

The spindle-shaped virion morphology is common among archaeal viruses, where it is a defining characteristic of many viral families. However, structural heterogeneity intrinsic to spindle-shaped viruses has seriously hindered efforts to elucidate the molecular architecture of these lemon-shaped capsids. We have utilized a combination of cryo-electron microscopy and X-ray crystallography to study Acidianus tailed spindle virus (ATSV). These studies reveal the architectural principles that underlie assembly of a spindle-shaped virus. Cryo-electron tomography shows a smooth transition from the spindle-shaped capsid into the tubular-shaped tail and allows low-resolution structural modeling of individual virions. Remarkably, higher-dose 2D micrographs reveal a helical surface lattice in the spindle-shaped capsid. Consistent with this, crystallographic studies of the major capsid protein reveal a decorated four-helix bundle that packs within the crystal to form a four-start helical assembly with structural similarity to the tube-shaped tail structure of ATSV and other tailed, spindle-shaped viruses. Combined, this suggests that the spindle-shaped morphology of the ATSV capsid is formed by a multistart helical assembly with a smoothly varying radius and allows construction of a pseudoatomic model for the lemon-shaped capsid that extends into a tubular tail. The potential advantages that this novel architecture conveys to the life cycle of spindle-shaped viruses, including a role in DNA ejection, are discussed

Increased RV:LV ratio on chest CT-angiogram in COVID-19 is a marker of adverse outcomes

Background: Right ventricular (RV) dilation has been used to predict adverse outcomes in acute pulmonary conditions. It has been used to categorize the severity of novel coronavirus infection (COVID-19) infection. Our study aimed to use chest CT-angiogram (CTA) to assess if increased RV dilation, quantified as an increased RV:LV (left ventricle) ratio, is associated with adverse outcomes in the COVID-19 infection, and if it occurs out of proportion to lung parenchymal disease. Results: We reviewed clinical, laboratory, and chest CTA findings in COVID-19 patients (n = 100), and two control groups: normal subjects (n = 10) and subjects with organizing pneumonia (n = 10). On a chest CTA, we measured basal dimensions of the RV and LV in a focused 4-chamber view, and dimensions of pulmonary artery (PA) and aorta (AO) at the PA bifurcation level. Among the COVID-19 cohort, a higher RV:LV ratio was correlated with adverse outcomes, defined as ICU admission, intubation, or death. In patients with adverse outcomes, the RV:LV ratio was 1.06 ± 0.10, versus 0.95 ± 0.15 in patients without adverse outcomes. Among the adverse outcomes group, compared to the control subjects with organizing pneumonia, the lung parenchymal damage was lower (22.6 ± 9.0 vs. 32.7 ± 6.6), yet the RV:LV ratio was higher (1.06 ± 0.14 vs. 0.89 ± 0.07). In ROC analysis, RV:LV ratio had an AUC = 0.707 with an optimal cutoff of RV:LV ≥ 1.1 as a predictor of adverse outcomes. In a validation cohort (n = 25), an RV:LV ≥ 1.1 as a cutoff predicted adverse outcomes with an odds ratio of 76:1. Conclusions: In COVID-19 patients, RV:LV ratio ≥ 1.1 on CTA chest is correlated with adverse outcomes. RV dilation in COVID-19 is out of proportion to parenchymal lung damage, pointing toward a vascular and/or thrombotic injury in the lungs. © 2022, The Author(s).Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Prediction of signal peptides in archaea

Computational prediction of signal peptides (SPs) and their cleavage sites is of great importance in computational biology; however, currently there is no available method capable of predicting reliably the SPs of archaea, due to the limited amount of experimentally verified proteins with SPs. We performed an extensive literature search in order to identify archaeal proteins having experimentally verified SP and managed to find 69 such proteins, the largest number ever reported. A detailed analysis of these sequences revealed some unique features of the SPs of archaea, such as the unique amino acid composition of the hydrophobic region with a higher than expected occurrence of isoleucine, and a cleavage site resembling more the sequences of gram-positives with almost equal amounts of alanine and valine at the position-3 before the cleavage site and a dominant alanine at position-1, followed in abundance by serine and glycine. Using these proteins as a training set, we trained a hidden Markov model method that predicts the presence of the SPs and their cleavage sites and also discriminates such proteins from cytoplasmic and transmembrane ones. The method performs satisfactorily, yielding a 35-fold cross-validation procedure, a sensitivity of 100% and specificity 98.41% with the Matthews' correlation coefficient being equal to 0.964. This particular method is currently the only available method for the prediction of secretory SPs in archaea, and performs consistently and significantly better compared with other available predictors that were trained on sequences of eukaryotic or bacterial origin. Searching 48 completely sequenced archaeal genomes we identified 9437 putative SPs. The method, PRED-SIGNAL, and the results are freely available for academic users at http://bioinformatics.biol.uoa.gr/PRED-SIGNAL/ and we anticipate that it will be a valuable tool for the computational analysis of archaeal genomes