Selection of bacteriophage λ integrases with altered recombination specificity by in vitro compartmentalization

In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage λ integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the λ integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications.     

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Divergent strategies for controlling the nuclear membrane satisfy geometric constraints during nuclear division

Eukaryotes segregate chromosomes in "open" or "closed" mitosis, depending on whether their nuclear envelopes (NEs) break down or remain intact. Here we show that the control of the nuclear surface area may determine the choice between these two modes. The dividing nucleus does not expand its surface in the fission yeast Schizosaccharomyces japonicus, confining the mitotic spindle and causing it to?buckle. The NE ruptures in anaphase, releasing the compressive stress and allowing chromosome segregation.?Blocking the NE expansion in the related species Schizosaccharomyces pombe that undergoes closed mitosis induces spindle buckling and collapse in the absence of an intrinsic NE rupture mechanism. We propose that scaling considerations could have shaped the evolution of eukaryotic mitosis by necessitating either nuclear surface expansion or the NE breakdown.     

Human papillomavirus infection and anxiety: analyses in women with low-grade cervical cytological abnormalities unaware of their infection status

Background

Women testing positive for human papillomavirus (HPV) infection experience increased levels of anxiety that have been attributed to fears of stigmatization and developing cervical cancer. The objective of this study was to investigate the association between HPV infection and anxiety in women who were unaware they had been tested specifically for HPV, to determine if any anxiety experienced by HPV-positive women could be due to causes other than learning of test results.

Methods

This study was nested within a randomised controlled trial of management of women with abnormal cervical cytology conducted in the United Kingdom with recruitment between 1999 and 2002. At baseline, prior to having a sample taken for HPV testing, the results of which were not disclosed, women were assessed for anxiety using the Hospital Anxiety and Depression Scale and asked about fears of developing cervical cancer (“cancer worries”); this assessment was repeated at 12, 18, 24, and 30 months of follow-up. Logistic regression and generalized estimating equations were used for the cross-sectional (baseline) and longitudinal analyses, respectively.

Results

Among the 2842 participants, there was no association between HPV status and anxiety among white women. Among non-white women, however, anxiety was less common among HPV-positive than HPV-negative women (adjusted odds ratio 0.41, 95% confidence interval 0.22 to 0.77). Among non-smokers, cancer worry was more common in HPV-positive than HPV-negative women; the opposite association was observed among ex-smokers.

Conclusions

Associations between HPV status and anxiety may be explained by factors other than learning of test results and may vary by ethnicity and lifestyle factors.     

Receivers limit the prevalence of deception in humans: evidence from diving behaviour in soccer players

Deception remains a hotly debated topic in evolutionary and behavioural research. Our understanding of what impedes or facilitates the use and detection of deceptive signals in humans is still largely limited to studies of verbal deception under laboratory conditions. Recent theoretical models of non-human behaviour have suggested that the potential outcome for deceivers and the ability of receivers to discriminate signals can effectively maintain their honesty. In this paper, we empirically test these predictions in a real-world case of human deception, simulation in soccer. In support of theoretical predictions in signalling theory, we show that cost-free deceit by soccer players decreases as the potential outcome for the signaller becomes more costly. We further show that the ability of receivers (referees) to detect deceptive signals may limit the prevalence of deception by soccer players. Our study provides empirical support to recent theoretical models in signalling theory, and identifies conditions that may facilitate human deception and hinder its detection.     

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration

BackgroundThe genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate.ConclusionGlobal surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.