Sense and simplicity in HADDOCK scoring: Lessons from CASP-CAPRI round 1.

Our information-driven docking approach HADDOCK is a consistent top predictor and scorer since the start of its participation in the CAPRI community-wide experiment. This sustained performance is due, in part, to its ability to integrate experimental data and/or bioinformatics information into the modelling process, and also to the overall robustness of the scoring function used to assess and rank the predictions. In the CASP-CAPRI Round 1 scoring experiment we successfully selected acceptable/medium quality models for 18/14 of the 25 targets - a top-ranking performance among all scorers. Considering that for only 20 targets acceptable models were generated by the community, our effective success rate reaches as high as 90% (18/20). This was achieved using the standard HADDOCK scoring function, which, thirteen years after its original publication, still consists of a simple linear combination of intermolecular van der Waals and Coulomb electrostatics energies and an empirically derived desolvation energy term. Despite its simplicity, this scoring function makes sense from a physico-chemical perspective, encoding key aspects of biomolecular recognition. In addition to its success in the scoring experiment, the HADDOCK server takes the first place in the server prediction category, with 16 successful predictions. Much like our scoring protocol, because of the limited time per target, the predictions relied mainly on either an ab initio center-of-mass and symmetry restrained protocol, or on a template-based approach whenever applicable. These results underline the success of our simple but sensible prediction and scoring scheme. Proteins 2017; 85:417-423. © 2016 Wiley Periodicals, Inc.

Besifloxacin ophthalmic suspension: emerging evidence of its therapeutic value in bacterial conjunctivitis.

OBJECTIVE: To outline the pharmacodynamics, efficacy and safety of besifloxacin ophthalmic suspension 0.6% in the treatment of bacterial conjunctivitis. QUALITY OF EVIDENCE: MEDLINE database was searched to review recent pharmacodynamic and clinical studies evaluating besifloxacin and comparing besifloxacin to other topical antibiotics for ophthalmic use. Findings were limited to full-text articles from clinical journals in the English language. MAIN MESSAGE: Bacterial resistance is a common source for treatment failure in bacterial conjunctivis. Besifloxacin, a novel fourth generation synthetic fluoroquinolone is likely to show lower resistance rates due to its mechanism of action and its short-term use for ocular infections only (decreased systemic exposure). Besifloxacin displays improved pharmacodynamic properties compared to other commonly used fluoroquinolones and has shown to be efficacious and safe in clinical studies. CONCLUSION: Besifloxacin ophthalmic suspension 0.6% provides safe and efficacious treatment for bacterial conjunctivitis. The factors leading to bacterial resistance are diminished, which allows besifloxacin to be a favorable treatment option.

The neural bases of momentary lapses in attention.

Momentary lapses in attention frequently impair goal-directed behavior, sometimes with serious consequences. Nevertheless, we lack an integrated view of the brain mechanisms underlying such lapses. By investigating trial-by-trial relationships between brain activity and response time in humans, we determined that attentional lapses begin with reduced prestimulus activity in anterior cingulate and right prefrontal regions involved in controlling attention. Less efficient stimulus processing during attentional lapses was also characterized by less deactivation of a 'default-mode' network, reduced stimulus-evoked sensory activity, and increased activity in widespread regions of frontal and parietal cortex. Finally, consistent with a mechanism for recovering from attentional lapses, increased stimulus-evoked activity in the right inferior frontal gyrus and the right temporal-parietal junction predicted better performance on the next trial. Our findings provide a new, system-wide understanding of the patterns of brain activity that are associated with brief attentional lapses, which informs both theoretical and clinical models of goal-directed behavior.

Force production by single kinesin motors.

Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis-Menten relationship.

Clinical and biochemical changes following 131I therapy for hyperthyroidism in patients not pretreated with antithyroid drugs.

BACKGROUND: Radioiodine therapy (131I) for the treatment of hyperthyroidism has been shown to be effective and safe. Despite the extensive experience with radioiodine therapy, the necessity for pretreatment with antithyroid drugs is controversial. Pretreatment is partly based on the concept that antithyroid drugs deplete the thyroidal hormonal stores, thereby reducing the risk of a radioiodine-induced aggravation of hyperthyroidism or thyroid storm. Few data are available on the frequency of clinically significant exacerbations of hyperthyroidism following 131I therapy without prior treatment with antithyroid drugs. The aim of the present study was to determine prospectively the early clinical and biochemical changes after 131I therapy in patients who were not pretreated with antithyroid drugs. METHODS: Patients with Graves' disease (n = 21), toxic multinodular goiter (n = 11) or toxic adenoma (n = 2) were studied before and after 131I therapy. Clinical and biochemical parameters of thyroid function were investigated before and 1, 2, 8, 11, 18 and 25 days after 131I treatment. Patients were given no antithyroid drugs prior to 131I therapy, all patients received beta-blocking agents for symptomatic relief. RESULTS: In 19 of 34 patients, a transient increase in thyroid hormone levels was observed, predominantly in the first week following 131I therapy. None of these patients experienced worsening of thyrotoxic symptoms. This transient increase in thyroid hormone levels was demonstrated in all patients with toxic multinodular goiter, whereas it was found in only six of 21 patients with Graves' disease. This difference could not readily be explained by differences in pretreatment thyroid hormone levels, administered dose or effectively absorbed dose of 131I. CONCLUSIONS: 131I treatment of hyperthyroidism without pretreatment with antithyroid drugs may cause a transient increase in thyroid hormone levels. Clinically significant exacerbations of hyperthyroidism were, however, not observed in our study population. Increased hormone levels following 131I therapy were more often seen in patients with toxic multinodular goiter than in patients with Graves' disease.

Single kinesin molecules studied with a molecular force clamp.

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

Enhanced rates of subpicosecond energy transfer in blue-shifted light harvesting LH2 mutants of Rhodobacter sphaeroides.

Energy transfer within various LH2 antenna complexes of the photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas acidophila has been studied at 77 K using tunable femtosecond and subpicosecond infrared pulses. The complexes examined include the wild-type B800-850 as well as three different specifically mutated complexes. The site-directed mutant strains were altered at positions 44 and 45 near the C-terminus of the alpha-subunit, which introduces a spectral blue-shift of the 850-nm absorption band. In addition to a constant band at 800 nm, the mutations alpha Tyr44,Tyr45-->Phe,Tyr; -->Tyr,Phe; and -->Phe,Leu have absorption peaks at 838, 838, and 826 nm, respectively. As the spectral overlap between the B800 and the variable bands increases, the rate of energy transfer as measured by the lifetime of the B800 excited state also increases from 2.4 +/- 0.2 to 1.8 +/- 0.2, 1.6 +/- 0.2, and 0.8 +/- 0.1 ps. This correlation between energy-transfer rate and spectral blue-shift of the B850 absorption band is in qualitative agreement with the trend predicted from Förster spectral overlap calculations, although the variation of the experimentally determined rate through the series of mutants is somewhat wider than what is predicted by simulations. In addition to the decay time constants relate to the B800-->B850 energy transfer, the B800 excited state is seen to decay with a faster 150-500-fs component due to energy transfer between spectrally inhomogeneous B800 molecules and possibly also vibrational relaxation and cooling in the bacteriochlorophyll excited state.

Activity of urea amidohydrolase immobilized within poly[di(methoxyethoxyethoxy)phosphazene] hydrogels.

Urea amidohydrolase (urease) was immobilized within poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels. This was accomplished by mixing an aqueous solution (pH 7) of the soluble polymer with the enzyme. Films of the conjugate were cast and the solvent removed to yield an MEEP/enzyme composite. The conjugate films were dried in a vacuum and were then cross-linked by exposure to 0.2 or 0.5 Mrad of 60Co gamma-radiation to give an MEEP network with the enzyme entrapped within its matrix. The cross-linked films were sectioned into strips and were washed with pH 7 buffer to remove enzyme adhering to the surface. The films were then allowed to swell to form a hydrogel in pH 7 buffer to which was added a 1.0 M aqueous urea solution. The increase in pH from the conversion of urea to ammonia was monitored over a 24 h period. The immobilized enzyme could be recycled at least five times without significant loss of activity. Several control experiments were also performed by monitoring the pH of buffer solutions that contained hydrogels devoid of entrapped urease, and by monitoring the pH of solutions of the free, non-irradiated and free, irradiated urease after the addition of the urea solution. The polymer-free, irradiated urease lost little to no activity compared with its non-irradiated counterpart. The MEEP gel-immobilized enzyme retained approximately 80% of the activity of the non-irradiated, polymer-free urease.

Energy transfer in the inhomogeneously broadened core antenna of purple bacteria: a simultaneous fit of low-intensity picosecond absorption and fluorescence kinetics.

The excited state decay kinetics of chromatophores of the purple photosynthetic bacterium Rhodospirillum rubrum have been recorded at 77 K using picosecond absorption difference spectroscopy under strict annihilation free conditions. The kinetics are shown to be strongly detection wavelength dependent. A simultaneous kinetic modeling of these experiments together with earlier fluorescence kinetics by numerical integration of the appropriate master equation is performed. This model, which accounts for the spectral inhomogeneity of the core light-harvesting antenna of photosynthetic purple bacteria, reveals three qualitatively distinct stages of excitation transfer with different time scales. At first a fast transfer to a local energy minimum takes place (approximately 1 ps). This is followed by a much slower transfer between different energy minima (10-30 ps). The third component corresponds to the excitation transfer to the reaction center, which depends on its state (60 and 200 ps for open and closed, respectively) and seems also to be the bottleneck in the overall trapping time. An acceptable correspondence between theoretical and experimental decay kinetics is achieved at 77 K and at room temperature by assuming that the width of the inhomogeneous broadening is 10-15 nm and the mean residence time of the excitation in the antenna lattice site is 2-3 ps.

Direct energy transfer from the peripheral LH2 antenna to the reaction center in a mutant of Rhodobacter sphaeroides that lacks the core LH1 antenna.

The light-harvesting apparatus of the photosynthetic bacterium Rhodobacter sphaeroides is composed of a peripheral LH2 complex which directs excitation energy to the LH1/reaction center core. The puf BA genes encoding the LH1 polypeptides have been deleted, producing a photosynthetically-competent strain which contains LH2 and reaction centers. Time-resolved absorption and fluorescence measurements demonstrate that energy is efficiently transferred from LH2 to the reaction center, despite the absence of LH1. Energy trapping takes place in 55 +/- 5 ps at room temperature, compared to the result for the wild-type strain of 60 +/- 5 ps. At 77 K, the results for the mutant and wild type are 75 +/- 5 and approximately 35 ps, respectively; the slower time in the mutant is attributed to the small differences in antenna/reaction center contacts and relative distances that are bound to exist as a consequence of LH1 and LH2 being assembled from different alpha- and beta-polypeptides. Measurements with closed reaction centers provided new information on the nature of fast energy transfer within the B850 pigments of LH2. We conclude that the absorption band is inhomogeneously broadened, and the fast (approximately 10 ps) lifetime observed in the 847-857-nm region is interpreted as very rapid (1-5 ps) hopping of the excitation energy from high-energy to low-energy pigments within the B850 absorption band. Time-resolved anisotropy studies demonstrate that energy-transfer events within B850 occur on a subpicosecond to picosecond time scale.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of single yeast cells by optical trapping.

Individual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light. The cells can be selected on morphological criteria by high resolution microscopy. The isolation device is constructed from two coverslips separated by spacers, in which selected cells are transferred to a plastic capillary, using the optical trap. To test the procedure, selection experiments were done with a mixture of two Saccharomyces cerevisiae strains, distinguishable both in fluorescence microscopy and on agar plates. These experiments showed that only selected cells were isolated, and close to 100% of the isolated stationary-phase cells formed colonies on agar plates, indicating a high recovery. A lower recovery was obtained with exponential-phase cells, possibly because of a higher sensitivity to laser irradiation. Applications for this method may include the isolation of mutants with altered morphology and the isolation of subpopulations of yeast cultures, for their separate investigation or for the initiation of pure cultures.

Micromanipulation by "multiple" optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope.

We have developed a novel micromanipulator consisting of multiple optical traps created by scanning one single beam trap along a variable number of positions. Among other things, this enables the orientation of irregularly shaped and relatively large structures which could not be oriented by just one trap as is demonstrated on long Escherichia coli bacteria filaments. We expect that the multiple trap manipulator will broaden the field of applications of optical trapping as a micromanipulation technique. For example, it facilitates the study of mechanical properties of extended structures as illustrated by a "bending"-experiment using E. coli bacterium filaments. A special application of the multiple trap manipulator is the "indirect trapping" of objects which we did by keeping them held between other optically trapped particles. Indirect trapping makes it possible to trap particles which either cannot be trapped directly due to their optical properties (refractive index) or for which exposure to the laser radiation is undesirable. The multiple optical trap manipulator is controlled interactively by a UNIX workstation coupled to a VME instrumentation bus. This provides great flexibility in the control of the position and the orientation of the optical traps. Micromanipulation makes it desirable to have real time 3D microscopy for imaging and guidance of the optical traps. Therefore we integrated optical micromanipulation and a specially developed real-time confocal microscope. This so called bilateral confocal scanning laser microscope (bilateral CSLM) [Brakenhoff and Visscher, J Microsc 165:139-146, 1992] produces images at video rate.

A novel synthetic method for hybridoma cell encapsulation.

We report here what we believe is the first example of the encapsulation of hybridoma cells within a synthetic polymer by a simple gelation with dissolved cations in water, and at room temperature. Two lines of hybridoma cells were encapsulated within calcium cross-linked polyphosphazene gel microbeads without affecting their viability or their capability to produce antibodies. Interaction of these gel beads with the positively-charged polyelectrolyte, poly(L-lysine), of 102-kD molecular weight, produced a semipermeable membrane that was capable of retaining the cell-secreted antibodies inside the beads. Cell density increased 3.5-fold within 13 days concomitant with a 6.4-fold increase in antibody production. These synthetic membranes have the potential to aid in protein recovery schemes.

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature dependence of energy transfer from the long wavelength antenna BChl-896 to the reaction center in Rhodospirillum rubrum, Rhodobacter sphaeroides (w.t. and M21 mutant) from 77 to 177K, studied by picosecond absorption spectroscopy.

Decay of the bacteriochlorophyll excited state was measured in membranes of the purple bacteria Rhodospirillum (R.) rubrum, Rhodobacter (Rb.) sphaeroides wild type and Rb. sphaeroides mutant M21 using low intensity picosecond absorption spectroscopy. The excitation and probing pulses were chosen in the far red wing of the long wavelength absorption band, such that predominantly the minor antenna species B896 was excited. The decay of B896 was studied between 77 and 177K under conditions that the traps were active. In all species the B896 excited state decay is almost temperature independent between 100 and 177K, and probably between 100 and 300 K. In this temperature range the decay rates for the various species are very similar and close to 40 ps. Below 100 K this rate remains temperature independent in Rb. sphaeroides w. t. and M21, while in R. rubrum a steep decrease sets in. An analysis of this data with the theory of nuclear tunneling indicates an activation energy for the final transfer step from B896 to the special pair of 70cm(-1) for R. rubrum and 30cm(-1) or less for Rb. sphaeroides.

Kinetics and mechanism of electron transfer between purines and pyrimidines, their dinucleotides and polynucleotides after reaction with hydrated electrons; a pulse radiolysis study.

The radical spectra of mixtures of thymidine 5'-monophosphate (TMP) or uridine 5'-monophosphate (UMP) with adenine 5'-monophosphate (AMP) after hydrated electron attack, measured from 5 to 3000 microsecond after pulse radiolysis, can only be described in terms of the radical spectra of the nucleotides if an electron transfer is taken into account from the purine radical anion to the pyrimidine, resulting in the formation of a pyrimidine radical anion. From analysis of the spectra of the dinucleoside phosphates ApU, dApT and dCpdA after eaq- attack it follows that the electron-donating species is the purine radical anion (A-.) rather than the protonated purine radical. The electron transfer competes with the fast protonation of the purine radical anion: A-. + py----A + py.- and A-. + H2O in equilibrium AH. respectively. The electron transfer is found to have a diffusion-controlled reaction rate constant of approximately 1.2 X 10(10) for TMP and 3.5 X 10(9) dm3 mol-1 s-1 for UMP.

Four-parameter white blood cell differential counting based on light scattering measurements.

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinophilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0 degrees and 2.6 degrees and angles between 3.0 degrees and 11.0 degrees. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.

Fast protonation of adenosine and of its radical anion formed by hydrated electron attack; a nanosecond optical and dc-conductivity pulse radiolysis study.

The study of the reaction of the hydrated electron with adenosine by optical and dc-conductivity pulse radiolysis on nano- and microsecond timescales has been carried out in an attempt to answer the question whether the electron adduct radical becomes protonated or not. The following conclusions have been reached: (1) the reaction of the hydrated electron with adenosine is followed by a water-mediated protonation, which must be complete with 5 ns; (2) no spectral indication of a further protonation of the protonated electron adduct of adenosine of 2'-deoxyadenosine has been found between 40 and 5000 ns; (3) the equilibrium reaction between radiation produced H3O+ and adenosine with a pKa of 3.5 plays an important role in the kinetics of the conductivity transients.