Immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis B virus proteins

Viruses employ various means to evade immune detection. Reduction of CD8(+) T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8(+) T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.     

Separase biosensor reveals that cohesin cleavage timing depends on phosphatase PP2A(Cdc55) regulation

In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1?min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.     

Structural basis for the preference of UTP over ATP in human deoxycytidine kinase: illuminating the role of main-chain reorganization

Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65-79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240-247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK.     

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k_cat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.     

WIS‐neuromath enables versatile high throughput analyses of neuronal processes

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS‐NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS‐NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS‐NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole‐mount sections. WIS‐NeuroMath is freely available to academic users, providing a versatile and cost‐effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Evidence for fractal correlation properties in variations of peripheral arterial tone during REM sleep

Previous studies utilizing detrended fluctuation analysis (DFA) of heart rate variability during sleep revealed a higher fractal exponent during rapid eye movement (REM) sleep than non-REM sleep. The aim of this study was to determine whether the same difference exists in the variations of peripheral arterial tone (PAT). Finger pulse wave measured by a novel plethysmographic technique was monitored during sleep in 12 chronic heart failure patients, 8 heavy snorers, and 12 healthy volunteers. For each subject, at least two 15-min time series were constructed from the interpulse intervals and from pulse wave amplitudes during REM and non-REM sleep. Fractal scaling exponents of both types of time series were significantly higher for REM than non-REM sleep in all groups. In each of the groups and in both sleep stages, the fractal scaling exponents based on pulse wave amplitude were significantly higher than those based on pulse rate variability. A repeat of the analysis for short-, intermediate-, and long-term intervals revealed that the fractal-like exponents were evident only in the short- and intermediate-term intervals. Because PAT is a surrogate of sympathetic activation, our results indicate that variations in sympathetic activation during REM sleep have a fractal-like behavior.     

Ultradian rhythms in renal excretion in dogs

Urine flow, urinary osmolality, concentrations of sodium and potassium were measured every 10 min for 24 hours in 6 female dogs, whose sleep-wake stages were monitored electrographically. Experiments were conducted under hydration and progressive dehydration conditions. Visual examination and spectral analysis revealed significant diurnal ultradian rhythms of about 200 min/cycle in urine flow, which were out of phase with similar rhythms in osmolality in different hydratory conditions. Diurnal rhythms with similar characteristics were found in urinary potassium only in the hydration condition. Sodium excretions did not show consistent rhythmic patterns. No consistent rhythms were found in nocturnal urine parameters in different hydratory conditions. The variations in renal activities were related to the dogs' diurnal ultradian sleep-wake cycle, urine volume increased and osmolality decreased during diurnal waking episodes, and conversely volume decreased and osmolality increased during the diurnal sleep episodes.