The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.DOI:http://dx.doi.org/10.7554/eLife.21768.001 Cytoplasmic dynein-1 (hereafter referred to as dynein) is a 1.4 MDa, multi-subunit motor complex that is responsible for the vast majority of cargo transport towards the minus ends of microtubules in eukaryotic cells. Dynein’s cargoes include organelles, vesicles, aggregated proteins, mRNAs and pathogenic viruses . In addiDynein – like all cytoskeletal motors – functions within cells on a dynamic network of tracks. Whereas the microtubule minus ends are often stabilised in vivo, the plus ends alternate between phases of polymerisation (‘growth’) and depolymerisation (‘shrinkage’)—a process known as dynamic instability. Microtubule plus ends are stabilised during growth by a cap of tubulin-GTP, with depolymerisation triggered when the GTP bound to β-tubulin at the microtubule tip is hydrolysed . The dynDynein is detected along the lattice of the microtubule, but is enriched at the plus end in a wide variety of eukaryotic cells . AssociaGlued (hereafter p150), a subunit of the dynactin complex. Dynactin is a 1.1 MDa complex of 11 distinct components that co-precipitates with dynein and is critical for its plus-end targeting and cargo transport functions in vivo  protein is another key player in the regulation of dynein function. Reduced expression of LIS1 causes the neurodevelopmental disease type 1 lissencephaly in humans by perturbing neuronal proliferation and migration . LIS1 foIn addition to regulating association of dynein with microtubule plus ends, LIS1 is required for efficient transport of many dynein-associated cargoes . In vitrHere, we use total internal reflection fluorescence (TIRF)-microscopy to investigate in vitro the roles of EB1 and LIS1 in plus-end tracking of human dynein in the presence of the full dynactin complex. We also investigate the influence of these proteins on interactions of minus end-directed dynein complexes with dynamic microtubules by including dynactin and BICD2N in the assay system. Our approach elucidates how mammalian dynein function is spatially regulated on dynamic microtubules and reveals context-dependent effects of LIS1 that help clarify its in vivo functions.We developed assay conditions that are permissive for the interaction of non-processive and minus end-directed mammalian dynein complexes with microtubules exhibiting dynamic instability . The fulWe first asked whether, in conjunction with EB1, the native dynactin complex is sufficient to induce the association of dynein with the plus ends of growing microtubules . EB1 traAs described in the Introduction, LIS1 is required for association of dynein with plus ends of growing microtubules in mammalian cells . We therWe next investigated if LIS1 increases the frequency of plus-end tracking events by dynein at the growing plus end by promoting the initial landing of the motor complex at this site. In order to do this, we compared the incidence of dynein complexes landing on the growing plus end vs an equivalently-sized site on the GDP lattice. The presence of both EB1 and dynactin in the chamber made dynein approximately twice as likely to land at the plus-end site than at the lattice site . The addInterestingly, in the same assays in which LIS1 increased the duration of plus-end tracking events by dynein in the presence of EB1 and dynactin, there was no significant change in the duration of lattice binding events . Thus, LThe observation that LIS1 does not influence the dwell time of human dynein on the microtubule lattice was unexpected as yeast LIS1 strongly increases the dwell time of monomeric yeast dynein on stabilised microtubules in the presence of ATP . MoreoveThe experiments described above shed light on how dynein interacts with the plus ends of growing microtubules in the absence of activators of processive minus end-directed movement. In order to investigate how mammalian dynein complexes that undergo minus end-directed motion are targeted to dynamic microtubules, both dynactin and the N-terminal region of BICD2 (BICD2N) were included in the in vitro assay. This region of BICD2 is sufficient to associate with dynein and dynactin  and stimIt has been proposed that LIS1 regulates initiation of dynein-based cargo transport from microtubule plus ends in at least some contexts . We therCLIP-170 can contact EB1, LIS1 and p150 and plays an important role in enriching dynein at microtubule plus ends in vivo . We therIt has been hypothesised that tracking of dynein on the plus ends of growing microtubules in vivo produces a loading zone for cargo that is destined for minus end-directed transport , 2002. TPlus-end tracking events may not be converted into minus end-directed transport events because dynein complexes are in a state that cannot accommodate BICD2N. To test this possibility, we produced Alexa647-labelled BICD2N and analysed its localisation on microtubules in the presence of EB1, dynactin, LIS1 and TMR-labelled dynein. As expected, Alexa647-BICD2N was detected on almost all minus end-directed TMR-dynein complexes , includiOur earlier results showed that LIS1 does not regulate the site of initiation of minus end-directed dynein-dynactin-BICD2N movements on dynamic microtubules. We next asked if LIS1 has any influence on the motility of dynein along microtubules in the presence of dynactin and BICD2N. Interestingly, LIS1 increased the proportion of microtubule-associated dynein complexes that underwent minus end-directed motion from 45% to 70% . When thWe and others have demonstrated that LIS1 can promote the association of dynein with dynactin in cells or cell extracts , althougIn the presence of LIS1, the mean and maximum velocity of minus end-directed dynein-dynactin-BICD2N complexes on dynamic microtubules also increased markedly, regardless of whether EB1 was included in the assay . LIS1 diSome studies have proposed that the copy number of dyneins bound to a physiological cargo can regulate velocity in vivo, with higher motor numbers associated with higher speeds . We usedThe strong stimulation of dynein-dynactin-BICD2N velocity by LIS1 was surprising as previous in vitro studies have shown that LIS1 reduces the activity of isolated dynein complexes . For insIn vivo work in fungal systems has provided compelling evidence that LIS1 dissociates from dynein cargoes before transport begins . We therThe experiments described above were designed to shed light on how dynein complexes interact with microtubules during bouts of growth. However, we were also able to observe the behaviour of dynein on the plus end of microtubules when growth phases switched to shrinkage phases. Many dyneins that tracked the plus end during a growth phase did not dissociate from this site when a shrinkage event began and instead retreated with the depolymerising end . Dynein Our finding that EB1 is not needed for association of dynein with the plus end of shrinking microtubules is consistent with the observation that EB family members only track plus ends during growth phases . We confWe next asked whether LIS1 regulates association of dynein with the plus end of the microtubule during shrinkage. We first used fluorescently labelled proteins to determine if LIS1 is present on dynein that tracks shrinking ends. TMR-LIS1 was detected with Alexa647-dynein in 20 of 22 events analysed in which the motor remained associated with the plus end during shrinkage . FurtherWe next considered the possibility that dynein transitions to an active, minus end-directed stepping mechanism in order to remain attached to the plus end during shrinkage. To test this notion, we performed assays in the presence of ATP and vanadate. Vanadate leads to the formation of a stable complex (dynein-ADP·Vi), which mimics the ADP·Pi transition state and hence the pre-powerstroke conformation of the motor . The expWe have used an in vitro reconstitution approach to shed light on how mammalian dynein complexes interact with microtubules undergoing growth and shrinkage. These experiments include an assessment of how purified dynein complexes behave on dynamic microtubules in the presence of activators of minus end-directed transport. Our results reveal a minimal set of interactions that govern association of end tracking and minus end-directed motor complexes with dynamic microtubules in the presence of the full dynactin complex, and reveal several unexpected functions of LIS1 in regulating the behaviour of dynein complexes  (Cat. No. 11496015)) and purified as described previously , DIC2 , DLIC2 , Tctex , LC8  and Robl . The DHC gene was fused at its 5’ to sequences encoding a His-ZZ-LTLT tag (Staphylococcus aureus and the LTLT region includes a cleavage site for the tobacco etch virus (TEV) protease. The presence of each gene in the expression construct was verified by PCR using Quickload Taq 2x Master Mix ). The ZZ tag on DHC was used for affinity purification of the dynein complex with IgG Sepharose 6 FastFlow beads ) prior to a TEV protease-mediated elution step.The human dynein complex  was expressed in Sf9 cells ). EB1 was purified via a His tag as described . All purification steps for LIS1 and LIS1::SNAPf were performed at 4°C. Frozen pellets from 50 ml of Sf9 cells were thawed on ice and resuspended in 2 ml of Lysis Buffer 2, 1 mM EGTA, 10% (v/v) glycerol, 1 mM DTT, 2 mM PMSF). Cells were lysed with 15 strokes of a 1 ml dounce homogenizer . The lysate was cleared of cell debris by centrifugation at 4°C in an Eppendorf 5424 microfuge . The cleared lysate was incubated in 2-ml tubes with 400 μl IgG Sepharose 6 Fast Flow beads that had been pre-washed in Lysis Buffer. The lysate and beads were incubated for 2 hr at 4°C on a flat roller, followed by transfer into a 5 ml gravity flow polypropylene column ). The beads were washed three times with 5 ml TEV Buffer (Lysis Buffer without PMSF) and resuspended in 1 ml of TEV buffer, followed by transfer to a 1.5-ml tube. The beads were pelleted by centrifugation , resuspended in 300 μl TEV Buffer and transferred to a 0.5-ml tube. To fluorescently label the SNAPf tag on LIS1, the LIS1::SNAPf bound beads were incubated with SNAP-Cell TMR-Star (NEB) at this stage as described below. To elute LIS1 or LIS1::SNAPf from the beads, 25 μl of 4 mg/ml TEV protease was added and the tube filled with TEV Buffer. Tubes were incubated on a tube roller overnight at 4°C. The beads were then pelleted by centrifugation  and the supernatant used for size exclusion chromatography on a Superdex 200 Increase column on an ÄKTAmicro system  in TEV buffer. The fractions containing LIS1 were pooled, followed by dispensing of small aliquots and flash freezing.The pFastbac expression construct for human full-length LIS1 with an N-terminal His-ZZ-LTLT tag was kindly provided by A. Carter (MRC-LMB). To produce the LIS1::SNAPSDS–PAGE of purified proteins was performed using Novex 4–12% Bis–Tris precast gels using either MOPS or MES buffer ) and ECL Full-Range Rainbow Molecular Weight Markers . Gels were stained with the Coomassie-based reagent Instant Blue ) or Imperial Protein Stain (ThermoFisher Scientific) according to the manufacturers’ instructions and imaged using a ChemiDoc XRS+ system with Image Lab 4.0 software ). Protein concentrations were measured using the Coomassie Protein Assay Kit (ThermoFisher Scientific) and an Eppendorf BioPhotometer Plus ).f::dynein complexes or LIS1::SNAPf were incubated at 4°C for 40 min with ~5 μM SNAP-Surface Alexa Fluor 647 or SNAP-Cell TMR-Star (NEB) for SNAPf::dynein, or ~40 μM SNAP-Cell TMR-Star for LIS1::SNAPf. Prior to TEV cleavage, excess dye was removed with three washes  in TEV Buffer, and beads resuspended in 300 μl TEV buffer. The labelling efficiency of dynein preparations was determined by spectrophotometric analysis of concentrated protein samples with a Nanodrop 1000 spectrophotometer ). The labelling efficiency was between 81% and 94% per dynein monomer, which equates to 96% to 99.6% of dimeric dynein complexes being labelled. The labelling efficiency of LIS1::SNAPf could not be evaluated by spectrophotometry as the protein could not be concentrated sufficiently without precipitating out of solution. However, the results of our co-localisation analysis of LIS1::SNAPf with dynein on microtubules . The microscope was equipped with a 100× objective  and the following lasers: 150 mW 488 nm, 150 mW 561 nm laser ), and 100 mW 641 nm (Coherent CUBE (Coherent Inc.)). Images were acquired with an EMCCD camera ). For multicolour experiments, images were captured sequentially by switching emission filters between GFP, Cy3, and Cy5 ). The size of each pixel was 105 nm × 105 nm. The data in Imaging was performed at 24 ± 1°C. The majority of data were acquired on a TIRF microscope system ) controlled with Micro-Manager software  for 2 hr 30 min at 37°C. Polymerised microtubules were sedimented by centrifugation at 18,400 x g in an Eppendorf 5424 centrifuge for 8 min at room temperature. The microtubule pellet was washed by resuspension in 1 ml of prewarmed BRB80 and sedimented again by centrifugation. Microtubules were then resuspended in BRB80 that had been prewarmed to 37°C and kept at room temperature for use within 1–2 hr.Unlabelled porcine tubulin, Hilyte488-tubulin and biotin-tubulin were supplied by Cytoskeleton Inc. . To polymerise fluorescent GMPCPP-stabilised microtubules, 1.66 μM unlabelled tubulin, 0.15 μM Hilyte488-tubulin and 0.4 μM biotin-tubulin were incubated together with 0.5 mM GMPCPP ) in BRB80  for 5 min, followed by two washes with BRB80. Exposed glass surfaces were then blocked with 5 mg/ml α-casein (Sigma) dissolved in BRB80 for 5 min. Fluorescent, biotinylated GMPCPP-stabilised microtubule seeds (polymerised as described above) were immobilised on the biotin-PEG-coated glass surface via streptavidin as described previously . For each imaging chamber, dynein was incubated for 5 min on ice with combinations of dynactin, BICD2N, CLIP-170 and LIS1 in Assay Buffer (‘Assembly Mix’). The concentration of dynein dimers in assembly mixes was 20 nM. In experiments evaluating the effects of LIS1, control protein mixes lacking LIS1 were supplemented with LIS1 storage buffer to ensure identical buffer conditions. Dynactin (1.1 MDa per complex), SNAPf::BICD2N dimers (134 kDa per complex), LIS1 dimers  and CLIP170 dimers (324 kDa per complex) were used in, respectively, a 2-fold, 10-fold, 20-fold and 25-fold molar excess to the full dynein complex (1.42 MDa per complex). Assembly mixes were diluted 1 in 2 to 1 in 10 to give a ‘chamber mix’ solution with a final concentration of 100 nM EB1 or mCherry::EB3 dimers, 26 μM unlabelled tubulin, 1.12 μM Hilyte488-tubulin, 2.5 mM ATP, 2 mM GTP, 0.2% (w/w) methyl cellulose, 10 mM KOAc, 10 mM KCl, 1250 nM glucose oxidase, 140 nM catalase, 71 mM 2-mercaptoethanol, 24.9 mM glucose, 80 mM PIPES pH 6.85, 2 mM MgCl2, 0.5 mM EGTA, 0.5 mg/ml BSA and 1 mM DTT . Varying the dilution factor for the assembly mixes was necessary at certain points in the study to give densities of complexes on microtubules that were conducive to the analysis of single binding events. The concentrations of dynein in the assembly mix and chamber mix were kept constant in any experiments that were compared to each other. In a subset of experiments, sodium orthovanadate (vanadate (NEB)) was added to a final concentration of 100 μM (i.e. 2.5 mM ATP +100 μM vanadate). In the experiment documented in 2, 0.5 mM EGTA, 0.5 mg/ml BSA and 1 mM DTT (i.e. as above but without ATP).Flow chambers were prepared as described above and passivated for 5 min with 1% (w/v) pluronic F-127 ) in dHeviously . Chamber2, 0.5 mM EGTA, 0.5 mg/ml BSA and 1 mM DTT. The dynein concentration in the assembly mix for these experiments was also 20 nM, except for Assessment of the behaviour of dynein or dynein-dynactin-BICD2N complexes on non-dynamic microtubules was assayed as described above, with two differences. First, microtubules were resuspended in BRB80 supplemented with 40 μM taxol (paclitaxel (Sigma)). Second, the final ‘chamber mix’ contained a dilution of the assembly mix in 2.5 mM ATP, 10 mM KOAc, 10 mM KCl, 1250 nM glucose oxidase, 140 nM catalase, 71 mM 2-mercaptoethanol, 24.9 mM glucose, 80 mM PIPES pH 6.85, 2 mM MgClSCR_002285) and analysed manually.Chamber mixes were introduced into flow chambers, which were subsequently sealed with nail polish. Flow chambers were then visualised by TIRF microscopy. Acquisition parameters were typically as follows: 100 ms exposures for one-colour imaging with a total acquisition time per frame of 0.137 s; 100 ms exposures per channel for two-colour imaging and a total acquisition time for both channels of 0.6 s; 50 ms exposure per channel for three-colour imaging and a total acquisition time for all three channels of 0.66 s. The exception was for the experiment in SCR_014990) was used to determine fluorescent intensity of minus end-directed Alexa647-dynein complexes on microtubules directly from the original image series. Fluorescence intensities of all unidirectional, minus end-directed dynein complexes were calculated as the volume under the Gaussian function. Velocities were also determined automatically with FIESTA for this analysis.In dynamic microtubule assays, the microtubule plus end was identified by its substantially faster growth rate compared to the minus end . We clasThe following procedure was performed to determine the ratio of initial dynein docking events on the growing plus end versus an equivalently sized lattice site. A diagonal line ~0.1 pixels wide was drawn on a kymograph in FIJI to demarcate the plus end of the growing microtubule. Docking events at the plus end were then scored manually as the initial appearance of a dynein signal (regardless of the dwell time) that overlaps with the diagonal line. Docking events on the GDP lattice were scored with the same procedure using a parallel line of the same thickness that was positioned randomly . The overlaps of dynein signals with diagonal lines drawn at growing plus end and randomly selected GDP lattice sites were also used to evaluate the initiation site of minus end-directed dynein movements.Throughout the study, each chamber utilised a unique assembly mix. In experiments used for statistical evaluations different experimental conditions within one series were typically assayed in an interleaved manner on the same day with at least two days of experimentation in total. Data were analysed to confirm that there was no significant day-to-day variability between results.2O, placed on an ice-cold metal block and washed with Assay Buffer. The following procedure was followed for all gliding assays except those documented in 2, 0.5 mM EGTA, 0.5 mg/ml BSA and 1 mM DTT was flowed into the chamber, which was promptly sealed with nail polish.Hilyte488-labelled microtubules were polymerised as described above, except that biotin-tubulin was not added to the polymerisation mix. Polymerised microtubules were resuspended in BRB80 containing 40 μM taxol that was prewarmed to 37°C. Flow chambers assembled from untreated glass surfaces were passivated for 5 min with 1% (w/v) pluronic F-127 in dHFor the experiments documented in In all gliding assays, microtubules were visualised by TIRF microscopy for no longer that 15 min (during which time three regions of the chamber were imaged), with a 100 ms exposure and a frame interval of 1 s. The time between addition of microtubules and the start of imaging was kept constant (2 min) within each experimental series in order to control for any effects of gradual dissociation of LIS1 from dynein. However, we observed no decrease in the inhibitory effect of LIS1 on microtubule gliding by dynein during the period of image acquisition. This finding indicates that sufficient LIS1 associated with dynein for a strong inhibitory effect for the duration of the gliding assay.Velocities of gliding microtubules were determined by manual analysis of kymographs with FIJI. In the gliding assays documented in SCR_002798) ) was used for data plotting and statistical analysis. Evaluations of statistical significance are described in the figure legends.Prism 6 Two preprints have recently been posted that also investigate the influence of LIS1 on mammalian dynein complexes in vitro  While EB1 and dynactin are required for plus end targeting of dynein, the addition of LIS1 increases the frequency and duration of dynein's interactions with growing microtubule plus-ends. 2) Dynein-dynactin-BICD2N complexes, which are capable of long distance processive motility, are more likely to begin a run at the microtubule plus end in the presence of EB1, but EB1 does not alter the complexes velocity or run length. 3) Dynein-dynactin-BICD2N complexes that track with the growing microtubule plus end are rarely competent to engage in minus-end-directed motility. 4) LIS1 co-migrates with moving minus-end-directed dynein-dynactin-BICD2N complexes and increases the velocity of these complexes. This is a surprising finding as previous studies on LIS1 suggested that LIS1 decreases velocity. 5) LIS1, in conjunction with EB1 and dynactin, promotes dynein association with shrinking microtubules. In summary, this study defines novel ways in which Lis1 can impact on dynein motor behaviour.Essential revisions:1) The authors report a surprising ability of LIS1 to activate both the frequency and velocity of dynein-dependent runs. This observation appears to directly contrast with previous studies showing that LIS1 leads to dynein inhibition during gliding assays, and that the binding of LIS1 enhances the development of an ATPase insensitive state in which dynein remains bound to the microtubule. To the credit of the investigators, they repeat the gliding assay inhibition, which suggests that the difference is not due to differential activities of the purified dyneins used in the various labs. However, they provide no significant insights into the dramatic differences between their single molecule and gliding data, nor any insights into how their new observations can be reconciled with the many previous single molecule studies on LIS1 from the Reck-Peterson, Vallee, and Gross labs. Some explanation of the discrepancy between the single molecule and gliding assays should be provided. The authors should also provide much better controls and description for both the single molecule and gliding assays:A) The authors should investigate whether Lis1 has an effect on human dynein microtubule interactions at the single molecule level, in the absence of other regulators . The focus of this study is on more complex reconstitutions, but these data are fundamental and it seems very likely the authors already have them. B) A major experimental concern relates to the microtubule gliding assay , which tFirst, did the presence of Lis1 (up to ~2.7 mg/ml) reduce the amount of dynein that non-specifically adsorbed to the surface? Lis1 is a sticky protein, so this seems plausible, especially at the 200-fold Lis1 molar excess. The presence of microtubules on the surface does not rule this out, as microtubules adsorb to many surfaces, including those lacking motors. Either the consistency of dynein surface density in the different conditions needs to be confirmed (e.g. using fluorescent dynein) or, to avoid this danger entirely, the authors could specifically immobilize dynein, then subsequently add the desired concentration of Lis1.Second, washing the chamber in a solution lacking Lis1 means that the dynein-Lis1 binding reaction will not have been at the desired equilibrium during the experiment: Lis1 will have been gradually dissociating in the time post wash out, and thus the fraction of dynein molecules with Lis1 bound will have depended critically on the time that elapsed between the washout and the start of the data acquisition. Was this elapsed time kept constant? It would be much safer to include Lis1 at the desired concentration in the final assay mixture.2) The experiments with the dynein tail construct are somewhat confusing. Is the tail alone sufficient to track growing and shrinking plus ends? What is the minimum set of proteins required to allow the tail to track growing and shrinking ends? Can this tracking be seen with purified dynein, or is it limited to recombinant forms? Does Lis1 influence the frequency/duration of tracking events for the dynein tail, or is the motor domain required? The authors propose the model that the different effects of Lis1 may arise from different binding sites, invoking interactions reported between Lis1 and various fragments of dynein and dynactin. However, interactions reported from the cited yeast 2-hybrid and pull-down experiments have not all been reproduced with intact proteins, so it is necessary to exercise caution before setting the field off down this path. Importantly, it seems that the authors may already have the data to test this model in their end-tracking experiments with the dynein tail construct. i.e. if Lis1 influences the frequency/duration of tracking events for the dynein tail, this would provide strong evidence for a functional binding site outside the characterised one in the dynein motor domain.3) It is possible that dynein-dynactin-BICDN complexes that track the plus end contain a "dead" motor , or a dy4) Despite the general high quality of the experimental data, the apparent number of experimental repeats is low. The authors state that the values shown represent 4 chambers per condition, with 15 microtubules analysed per chamber. But this doesn't clarify how many independent experiments were performed , and they do not specify the number of total events measured for each experiment.5) Throughout the manuscript, protein levels are reported as molar ratios rather than absolute concentrations. However, a 1:10 dynein:Lis1 ratio can result in essentially all the dynein having Lis1 bound (if the concentrations are well above the Kd) or essentially no dynein having Lis1 bound (if the concentrations are well below the Kd). Therefore, it is essential to state the absolute final concentration of all species in the reactions in order to interpret them. This is especially important when comparing single-molecule and microtubule-gliding assays, in which the concentrations are presumably quite different.6) Binding to the plus end is expressed per growth phase, rather than per unit time. This seems problematic for experiments +/- EB1, as EB1 substantially shortens the duration of the growth phase as shown in 7) Please include additional, or higher quality kymographs of dynein and BICD2 moving towards the minus end of dynamic microtubules .8) eLife, 2014; Duellberg, et al. Nat. Cell. Biol., 2014). The authors have purified and functional Clip170 in hand (9) It is well established that Clip170 interacts with LIS1 and the p150 subunit of dynactin  and that Clip170 plays an important role in dynein localization to microtubule plus ends "Dynein complexes that track the plus ends of microtubules are unlikely to act directly as a source of motors for minus end-directed transport." This statement has the potential to mislead. While it may be true under these assay conditions (i.e. in the presence of a 10-fold molar excess of a constitutively active cargo adaptor fragment), the situation in the cell (where cargoes with bound adaptors are likely to be limiting) might be very different indeed. This should be acknowledged.11) The discussion of the literature is selective, with the potential to mislead:Nudel/NudE are not mentioned. However, these proteins are known to be critical for Lis1 functions in vivo, and relieve Lis1's inhibitory effect on mammalian dynein in vitro, and must be discussed.The major of models of McKenney et al. (2010) and (2011) are not mentioned i.e. that Lis1 and NudE induce a persistent force-generating state in mammalian dynein, and that this is mutually exclusive with dynactin regulation.Treatment of the yeast literature seems selective, with the inhibitory effects of Lis1/Pac1 in vitro played up but the substantial body of work elucidating Lis1/Pac1's role in vivo (consistent with a targeting/inhibitory function) played down. Notably, Lis1/Pac1 is absent from dynein's site of action in yeast, and evidence suggests it must be displaced in order to activate yeast dynein."It is unclear how [Lis1's] activities can account for the reduction in dynein-based processes that is generally observed when LIS1 function is inhibited in cells." This seems an oversimplification, as incorrect targeting/inhibitory regulation can perturb motor processes as much as loss of an activator."However, LIS1's specific function in plus end tracking of dynein was also not addressed in previous in vitro reconstitution assays." This is true for mammalian proteins, but Lis1/Pac1's role has been addressed in reconstitutions with yeast proteins."However, this [search-and-capture] mechanism has not been visualised directly." Plus-end capture and minus-end directed motility of melanophores has been visualized directly . Transfer of yeast dynein from the plus end to the cell cortex has been visualized directly .There are other factors that could make plus end localization more complex in vivo as well. There are also additional mechanisms of dynein recruitment to the plus end and many other plus end binding proteins. Additional discussion of this complexity would strengthen the paper. Essential revisions:1) The authors report a surprising ability of LIS1 to activate both the frequency and velocity of dynein-dependent runs. This observation appears to directly contrast with previous studies showing that LIS1 leads to dynein inhibition during gliding assays, and that the binding of LIS1 enhances the development of an ATPase insensitive state in which dynein remains bound to the microtubule. To the credit of the investigators, they repeat the gliding assay inhibition, which suggests that the difference is not due to differential activities of the purified dyneins used in the various labs. However, they provide no significant insights into the dramatic differences between their single molecule and gliding data, nor any insights into how their new observations can be reconciled with the many previous single molecule studies on LIS1 from the Reck-Peterson, Vallee, and Gross labs. Some explanation of the discrepancy between the single molecule and gliding assays should be provided. The authors should also provide much better controls and description for both the single molecule and gliding assays:We regret that we did not state explicitly that our results do not contradict the findings of previous studies in the literature, which were generated with very different experimental systems. For example, the work of McKenney, Gross, Vallee and colleagues assayed dynein behaviour in the absence of processivity activators and predominantly under load. Huang, Roberts, Reck-Peterson and colleagues studied yeast dynein, which has features distinct from mammalian dynein . We have now stated that our findings do not contradict those in the literature . We believe that our improved coverage of the existing literature on LIS1’s regulation of dynein, which was requested by the reviewers, also helps clarify the relationship between our findings and those of other groups.Most importantly, we have now performed two types of experiments that clarify the different effects of LIS1 in different assays. One set of experiments provides evidence that LIS1 stimulates the assembly of the dynein-dynactin-BICD2N complex new . This exThe mechanism by which mammalian LIS1 increases the velocity of dynein-dynactin-BICD2N complexes will require a separate in depth study, although our new data demonstrating that LIS1 can influence the interaction of dynein with dynactin and BICD2N suggests an exciting route for investigation. The second set of new experiments that address the effects of LIS1 in different types of assays is described in response to point 1A. Collectively, our results are consistent with LIS1 exerting different effects on the activity of mammalian dynein in the presence and absence of dynactin and BICD2N.A) The authors should investigate whether Lis1 has an effect on human dynein microtubule interactions at the single molecule level, in the absence of other regulators . The focus of this study is on more complex reconstitutions, but these data are fundamental and it seems very likely the authors already have them. We thank the reviewers for this excellent suggestion. We have performed a set of new experiments investigating the effects of LIS1 on the single molecule interactions of human dynein complexes with microtubules new . These eThe published in vitro studies of the influence of LIS1 on interactions of yeast dynein with microtubules seemingly used a large molar excess of LIS1. Our new experiments therefore not only used the relative concentrations of LIS1 and dynein used in the rest of our single molecule experiments, but also a 15-fold further excess of LIS1 to dynein. There was no significant influence of the lower concentration of LIS1 on the interactions of dynein with the microtubule lattice. This finding is consistent with our observations with dynamic microtubules. However, the higher concentration of LIS1 significantly increased the landing rate of dynein on microtubules without a detectable effect on the dwell time of the motor. Our finding that LIS1 can increase the landing rate of dynein is compatible with previous observations that LIS1 increases the association of other mammalian dyneins with microtubules in co-sedimentation assays . We point out that our results with human proteins differ from those of the study with yeast proteins , which found that LIS1 increases the dwell time of monomeric dynein on microtubules. This finding suggests that LIS1 can exert different effects on dynein’s interactions with microtubules in yeast and mammals, a potentially significant finding when seeking to reconcile results from these systems.Although further mechanistic studies need to be performed in the future, we believe our new experiments also offer a possible explanation for the effects of LIS1 on mammalian dynein in different assays. We speculate that the ability of high relative concentrations of LIS1 to increase the association of human dynein with microtubules contributes to the inhibitory effect of LIS1 on microtubule translocation . At lower relative concentrations of LIS1, such as those used in our other single molecule assays, there is no effect on the interaction of dynein with microtubules unless co-factors, such as plus end-associated dynactin complexes, are also present. We also point out that the ability of LIS1 to prolong the interaction of mammalian dynein with microtubules under load  may also contribute to the inhibition of microtubule gliding by LIS1.We have now added representative kymographs of dynamic microtubules incubated with human dynein alone and in the presence of LIS1 to B) A major experimental concern relates to the microtubule gliding assay , which tFirst, did the presence of Lis1 (up to ~2.7 mg/ml) reduce the amount of dynein that non-specifically adsorbed to the surface? Lis1 is a sticky protein, so this seems plausible, especially at the 200-fold Lis1 molar excess. The presence of microtubules on the surface does not rule this out, as microtubules adsorb to many surfaces, including those lacking motors. Either the consistency of dynein surface density in the different conditions needs to be confirmed (e.g. using fluorescent dynein) or, to avoid this danger entirely, the authors could specifically immobilize dynein, then subsequently add the desired concentration of Lis1.We mixed dynein with LIS1 prior to washing off unbound motor from the glass surface as we thought that this was the best way to combine the proteins in a defined molar ratio. Controlling molar ratios is not practical when LIS1 is added to chambers with prebound dynein because the amount of motor complex bound to the surface cannot be readily quantified. Nonetheless, the reviewers’ comments made us appreciate that additional controls are needed to support our interpretations of the gliding assays.In the assay conditions employed, microtubules do not associate with the glass surface when dynein is omitted. We now cite a recent publication from our group that includes this control . This finding suggests that LIS1 did not inhibit gliding by causing strong dissociation of the motor complex from the glass, as this would inhibit microtubule association. As suggested by the reviewers, we have now investigated directly the effects of LIS1 on the association of dynein with the surface using the fluorophore on the heavy chain. We show that in conditions in which LIS1 strongly inhibits microtubule gliding there is no reduction in the amount of motor complex on the glass surface . In thesWe also performed an additional control showing that LIS1 alone does not cause microtubules to adhere to the glass surface in our assay buffer . This fiOne of the reviewers’ comments led us to check the concentrations of dynein and LIS1 in our previous experiments. There was an unfortunate miscalculation when compiling the previous Materials and methods; the concentration of dynein in the solution incubated with the glass surface was 30 nM, and the concentration of LIS1 was 0.6 or 6 μM. This error has been corrected and we have confirmed that protein concentrations in all other assays are stated correctly.Second, washing the chamber in a solution lacking Lis1 means that the dynein-Lis1 binding reaction will not have been at the desired equilibrium during the experiment: Lis1 will have been gradually dissociating in the time post wash out, and thus the fraction of dynein molecules with Lis1 bound will have depended critically on the time that elapsed between the washout and the start of the data acquisition. Was this elapsed time kept constant? It would be much safer to include Lis1 at the desired concentration in the final assay mixture.We kept the time between addition of proteins and imaging constant in our previous image series and observed no diminution over time of the inhibitory effect of LIS1 on microtubule gliding. This was also the case in our new experimental series. We illustrate this point in Thus, there is sufficient LIS1 associated with dynein to exert a strong inhibitory effect for the duration of the gliding assay. Including LIS1 with the microtubules in the final assay mixture is therefore not necessary . We have now made it clear in the Materials and methods that the time between protein addition and image acquisition was kept constant and that there was no reduction in LIS1’s inhibitory effect over the time course of the experiment.2) The experiments with the dynein tail construct are somewhat confusing. Is the tail alone sufficient to track growing and shrinking plus ends? What is the minimum set of proteins required to allow the tail to track growing and shrinking ends? Can this tracking be seen with purified dynein, or is it limited to recombinant forms? Does Lis1 influence the frequency/duration of tracking events for the dynein tail, or is the motor domain required? The authors propose the model that the different effects of Lis1 may arise from different binding sites, invoking interactions reported between Lis1 and various fragments of dynein and dynactin. However, interactions reported from the cited yeast 2-hybrid and pull-down experiments have not all been reproduced with intact proteins, so it is necessary to exercise caution before setting the field off down this path. Importantly, it seems that the authors may already have the data to test this model in their end-tracking experiments with the dynein tail construct. i.e. if Lis1 influences the frequency/duration of tracking events for the dynein tail, this would provide strong evidence for a functional binding site outside the characterised one in the dynein motor domain.The observation with the tail was a minor part of the study and was not emphasised in the Discussion because of several open questions, which were also picked up on by the reviewers. We could not previously address the minimal requirements for tracking of the dynein tail as the lack of a fluorescently labelled tail construct meant that the behaviour of the tail had to be visualised by fluorescent BICD2N, which only associates with the tail in the presence of dynactin. In this assay configuration it was also not possible to determine if LIS1 promotes end tracking of the isolated dynein tail; altered end tracking behaviour of labelled BICD2N could also result from an affect of LIS1 on the association of the dynein tail with BICD2N and dynactin. We also could not compare the efficiency of tracking by the dynein tail versus the full complex using labelled BICD2N as the results could be confounded by any differences in the efficiency of dynactin-dependent binding of BICD2N to the tail compared to the full dynein complex. We agree that the experiments with the dynein tail construct were therefore confusing and unsatisfactory. We were unable to convincingly address the questions raised above in the time available for revision, and have therefore decided to remove the two panels in the old We have retained some discussion in the Perspective about the possibility of LIS1 having more than one binding site on the dynein-dynactin complex as we think this is an attractive hypothesis for how LIS1 exerts context-dependent (and concentration-dependent) effects. We have now stated that the earlier evidence for LIS1 interactions with the tail and p50 was from yeast two hybrid and pulldown methods, which should allow the reader to exercise appropriate caution when reading this section. We now make it clear that this section is speculation and not a proposed model. Incidentally, we would not have been able to address the reviewers’ question about the tracking behaviour of the native dynein tail, as we can only produce the tail in a recombinant form.3) It is possible that dynein-dynactin-BICDN complexes that track the plus end contain a "dead" motor , or a dyWe had also considered this possibility but not looked into it further. We have now followed the reviewers’ helpful suggestion and quantified the percentage of all static dynein motors on the lattice that co-localise with BICD2N (and therefore dynactin). Quantification was performed using our previous kymographs of dynamic microtubules, so that the results could be compared directly to our analysis of BICD2N and dynein co-localisation on plus end tracking complexes in the presence of dynactin. Approximately 60% of static dynein complexes on the lattice had a BICD2N signal , which i4) Despite the general high quality of the experimental data, the apparent number of experimental repeats is low. The authors state that the values shown represent 4 chambers per condition, with 15 microtubules analysed per chamber. But this doesn't clarify how many independent experiments were performed , and they do not specify the number of total events measured for each experiment.We apologise for not including this information in the first submission. Each chamber is derived from a unique assembly mix, which we operationally define as an independent experiment. For each chamber, movies of three different regions were acquired. Different experimental conditions in the same series where typically performed in an interleaved manner on the same day. The data set for each experimental condition comprises chambers from at least two different days, and all the data were analysed carefully to check that there is no significant day-to-day variability between results. This information is now included in the Materials and methods. Information on the number of events analysed per condition is now included in each figure legend. A very large number of events was analysed throughout the study, and all of this was done manually. Consistent effects in the presence and absence of LIS1 were also seen in independent experimental series .5) Throughout the manuscript, protein levels are reported as molar ratios rather than absolute concentrations. However, a 1:10 dynein:Lis1 ratio can result in essentially all the dynein having Lis1 bound (if the concentrations are well above the Kd) or essentially no dynein having Lis1 bound (if the concentrations are well below the Kd). Therefore, it is essential to state the absolute final concentration of all species in the reactions in order to interpret them. This is especially important when comparing single-molecule and microtubule-gliding assays, in which the concentrations are presumably quite different.We have now added information to the Materials and methods and figure legends on the concentration of dynein in the assembly mixes.6) Binding to the plus end is expressed per growth phase, rather than per unit time. This seems problematic for experiments +/- EB1, as EB1 substantially shortens the duration of the growth phase as shown in We confirmed that the duration of growth phases is not significantly different between conditions in key experiments comparing the frequency of dynein binding events per growth phase . We did 7) Please include additional, or higher quality kymographs of dynein and BICD2 moving towards the minus end of dynamic microtubules .We have now included an additional kymograph of dynein and BICD2N complexes moving towards the minus end of dynamic microtubules . Please 8) We now see that the inclusion of the shrinking end analysis with the labelled BICD2N and the dynein tail in the same figure with the results of the experiments with labelled full-length dynein was confusing. This is no longer an issue as the tail experiments have been removed from the manuscript for the reasons described above. To further simplify the figure, we have moved the data for experiments without dynactin to the supplement .9) It is well established that Clip170 interacts with LIS1 and the p150 subunit of dynactin  and that Clip170 plays an important role in dynein localization to microtubule plus ends . The authors have purified and functional Clip170 in hand . Have th in vitro reconstitutions? In principle, it would be nice to know whether Clip170 affects the number and duration of dynein plus end tracking events in the presence of LIS1, dynactin, and EB1? If the authors have reasons not to include these experiments, they should at least include some discussion on this issue.We had performed experiments with CLIP-170 but had not analysed the experiments as we planned to focus on the effects of this protein in a future study. The reviewer’s comment made us appreciate that readers would be interested in the effects of CLIP-170 in the current study. We have now analysed the experiments and added the data to the manuscript. We show that, in the presence of LIS1, CLIP-170 has no detectable effect on the ability of EB1 to target the initiation of processive transport events to dynein-dynactin-BICD2N complexes to growing plus ends, or on the frequency and duration of plus end tracking events . This fi10)"Dynein complexes that track the plus ends of microtubules are unlikely to act directly as a source of motors for minus end-directed transport." This statement has the potential to mislead. While it may be true under these assay conditions (i.e. in the presence of a 10-fold molar excess of a constitutively active cargo adaptor fragment), the situation in the cell (where cargoes with bound adaptors are likely to be limiting) might be very different indeed. This should be acknowledged.This is another very good point. We now make it explicit that we are referring to the findings in our assay conditions. We agree with the reviewer that several other factors could influence the initiation of processive movement from the plus end in vivo. We discuss the possibility that the cargo or microtubule modifications could be involved in a physiological setting . In response to the reviewers’ comments we now also include experiments assaying the effects of CLIP-170 (see above), and mention the importance of assessing the effects of NudE proteins in the future (subsection “Perspective”).11) The discussion of the literature is selective, with the potential to mislead:We thank the reviewers for these suggestions for improving the discussion of the literature. We have made several changes.Nudel/NudE are not mentioned. However, these proteins are known to be critical for Lis1 functions in vivo, and relieve Lis1's inhibitory effect on mammalian dynein in vitro, and must be discussed.We now mention prominently in the Perspective section that addressing the role of NudE/NudEL is a priority for future experiments (see above).The major of models of McKenney et al. (2010) and (2011) are not mentioned i.e. that Lis1 and NudE induce a persistent force-generating state in mammalian dynein, and that this is mutually exclusive with dynactin regulation.We had cited the McKenney et al. (2010) paper but realise that we should have introduced the model explicitly. We now do so in the Introduction (seventh paragraph) and Discussion . The 2011 McKenney et al. paper focuses on the competition between NudE and dynactin. In the absence of NudE experiments in our study, we had not cited this paper in our first submission. The one experiment with LIS1 in the 2011 study shows that it promotes the ability of the CC1 domain of p150 to release dynein from NudE, which suggests a complex set of interactions between these factors. The revised manuscript introduces the need to study NudE/NudEL in our reconstitution assays in the future and we now cite the McKenney et al. 2011 paper in this context.Treatment of the yeast literature seems selective, with the inhibitory effects of Lis1/Pac1 in vitro played up but the substantial body of work elucidating Lis1/Pac1's role in vivo (consistent with a targeting/inhibitory function) played down. Notably, Lis1/Pac1 is absent from dynein's site of action in yeast, and evidence suggests it must be displaced in order to activate yeast dynein.We now emphasise in the Introduction that there is substantial evidence for a targeting/inhibitory function of Lis1/Pac1 from different studies. This section includes citations to papers that elucidate the effect of Lis1/Pac1 on dynein in yeast. These citations are also used in the Results and Discussion. We expand on the findings of the Roberts et al. study from 2014 to state that the LIS1 and CLIP170 orthologues from yeast can couple dynein to a plus end-directed motor, Kip2."It is unclear how [Lis1's] activities can account for the reduction in dynein-based processes that is generally observed when LIS1 function is inhibited in cells." This seems an oversimplification, as incorrect targeting/inhibitory regulation can perturb motor processes as much as loss of an activator.We include more discussion of other models for how LIS1 can promote dynein cargo transport in vivo . We have been careful not to give the impression that our results contradict these models. However, they do offer an additional explanation for how LIS1 promotes cargo transport in the presence of processivity activators, which we find exciting. We believe our new evidence that LIS1 can promote the assembly of the dynein-dynactin-BICD2N complex strengthens this aspect of the manuscript significantly."However, LIS1's specific function in plus end tracking of dynein was also not addressed in previous in vitro reconstitution assays." This is true for mammalian proteins, but Lis1/Pac1's role has been addressed in reconstitutions with yeast proteins.We now refer specifically to mammalian proteins in this section. We also mention in the Discussion specific findings of the reconstitution studies with yeast proteins."However, this [search-and-capture] mechanism has not been visualised directly." Plus-end capture and minus-end directed motility of melanophores has been visualized directly . Transfer of yeast dynein from the plus end to the cell cortex has been visualized directly .There are other factors that could make plus end localization more complex in vivo as well. There are also additional mechanisms of dynein recruitment to the plus end and many other plus end binding proteins. Additional discussion of this complexity would strengthen the paper.The restructuring of the Introduction to give more emphasis to existing models of how LIS1 regulates dynein (in response to the points above) has resulted in the section on visualisation of search-and-capture by plus end-associated dynein in vivo being removed. We now make it more explicit in the Discussion and the Perspective that additional factors could be involved in regulating transport initiation and plus end localisation in vivo. The Lomakin et al. and Markus et al. studies are cited elsewhere in our manuscript.


Although uncomplicated urinary tract infections (UTIs) are often self-limiting, most patients will be prescribed antibiotic treatment. We assessed whether treatment with ibuprofen was non-inferior to pivmecillinam in achieving symptomatic resolution by day 4, with a non-inferiority margin of 10%.n = 194, 181 analyzed) or pivmecillinam . By day 4, 38.7% of the patients in the ibuprofen group felt cured versus 73.6% in the pivmecillinam group. The adjusted risk difference with 90% confidence interval was 35% (27% to 43%) in favor of pivmecillinam, which crossed the prespecified non-inferiority margin. Secondary endpoints were generally in favor of pivmecillinam. After 4 weeks’ follow-up, 53% of patients in the ibuprofen group recovered without antibiotic treatment. Seven cases of pyelonephritis occurred, all in the ibuprofen group, giving a number needed to harm of 26 (95% CI 13 to 103). Five of these patients were hospitalized and classified as having serious adverse events; 2 recovered as outpatients. A limitation of the study was the extensive list of exclusion criteria, eliminating almost half of the patients screened. We did not register symptoms in the screening process; hence, we do not know the symptom burden for those who declined to participate. This might make our results less generalizable.This was a randomized, controlled, double-blind non-inferiority trial. We recruited patients from 16 sites in a general practice setting in Norway, Sweden, and Denmark. Non-pregnant women aged 18–60 years presenting with symptoms of uncomplicated UTI were screened for eligibility from 11 April 2013 to 22 April 2016. Patients with informed consent were randomized (1:1 ratio) to treatment with either 600 mg ibuprofen or 200 mg pivmecillinam 3 times a day for 3 days. The patient, treating physician, and study personnel were blinded to treatment allocation. The primary outcome was the proportion of patients who felt cured by day 4, as assessed from a patient diary. Secondary outcomes included the proportion of patients in need of secondary treatment with antibiotics and cases of pyelonephritis. A total of 383 women were randomly assigned to treatment with either ibuprofen , EudraCT Number  In a multicenter trial, Ingvild Vik and colleagues studied a non-antimicrobial treatment for urinary tract infections in women. Most uncomplicated urinary tract infections (UTIs) are self-limiting, but they are commonly treated with antibiotics as this gives quicker symptom relief.In 2010 a German study showed that two-thirds of women who received initial treatment with ibuprofen recovered without taking antibiotics.Antibiotic resistance is a serious public health problem, mainly caused by overuse of antibiotics. We performed a trial to explore whether treatment with ibuprofen could be a safe and efficient alternative to antibiotics in women with uncomplicated UTIs.Between April 2013 and June 2016 we recruited 383 women with symptoms of uncomplicated cystitis, who were randomized to receive a 3-day treatment of either ibuprofen or pivmecillinam (an antibiotic commonly used to treat UTIs in Scandinavia).It took 3 days longer for the patients in the ibuprofen group to get well.In the ibuprofen group, 46% of the patients returned to the doctor within 4 weeks with persisting, worsening, or new symptoms; 10% of the patients in the pivmecillinam group came back for a second consultation.Twelve patients (6.6%) developed a febrile UTI; all were initially treated with ibuprofen. In 7 of these patients (3.9%), the infection spread to the kidneys (pyelonephritis). All patients recovered with antibiotic treatment.More than half of the patients initially treated with ibuprofen got well without taking antibiotics. Looking at only the patients who got well without needing a subsequent prescription for antibiotics, the women in the ibuprofen group had symptoms for a median of 4 days, 1 day longer than the patients initially treated with antibiotics.Antibiotic treatment is the best treatment we have for uncomplicated UTI.Until we can identify the patients who need antibiotic treatment to avoid complications, we cannot recommend ibuprofen alone to women with uncomplicated UTI. More than half of all women will experience an uncomplicated urinary tract infection (UTI) during life , and 20%UTIs are the second most common cause for antibiotic prescribing in general practice . With anA small randomized pilot study from Germany was published in 2010 by Bleidorn et al. . Their rIn this paper we report the results from our trial comparing ibuprofen to pivmecillinam for uncomplicated UTI. The main objective of this study was to assess whether ibuprofen was non-inferior to antibiotics (pivmecillinam) in achieving symptomatic cure by day 4 in uncomplicated UTI. A 10% non-inferiority margin was established based on clinical expertise. We also wanted to assess the occurrence of complications.The study was a double-blind, randomized, parallel group, multicenter non-inferiority trial. Patients were randomized in a 1:1 ratio to treatment with either ibuprofen or pivmecillinam.All patients received oral and written information about the trial and signed an informed consent form. The trial was conducted in compliance with the International Conference on Harmonisation guidelines for good clinical practice (GCP) and the Declaration of Helsinki. The trial was approved by the Regional Ethical Committee in Norway (REK) and the Norwegian Medicines Agency (SLV) (2012/1569 C), the Committee on Biomedical Research Ethics for the Capital Region of Denmark (H-4-2013-145), the Regional Ethical Committee in Lund, Sweden (Dnr 2014/28), and the Medical Products Agency, Uppsala, Sweden (Dnr 5.1-2014-12072). Safety data were monitored throughout the trial by a certified monitor in each country. There was no independent data monitoring committee for the trial. All study personnel were trained in GCP. The study was conducted and analyzed according to the protocol  and the We recruited non-pregnant women aged 18–60 years with symptoms of an uncomplicated UTI. Inclusion criteria were dysuria combined with either increased urinary frequency or urinary urgency or both, with or without visible hematuria. The exclusion criteria were duration of symptoms for more than 7 days; allergies/adverse reactions to penicillin or ibuprofen; breastfeeding a child under 1 month of age; any sign of an upper UTI ; vaginal irritation/discharge; severe abdominal pain; diabetes; kidney disease; genetic aciduria; severe gastritis; ulcerative colitis; Crohn's disease; low platelets; use of probenecid, steroids, immunosuppressant drugs, or blood-thinning drugs; previous pyelonephritis; use of urinary catheter or symptoms of a UTI within the last 4 weeks; or use of antibiotics within the last 2 weeks . AbilityPatients were recruited from the accident and emergency outpatient clinics (AEOCs) in Oslo and Bergen, Norway, 7 general practices in Denmark, and 7 general practices in Sweden. The AEOCs in Norway are part of the primary healthcare system and are mainly staffed with GPs or doctors training to become GPs; we therefore consider them to be a general practice setting. Women presenting with symptoms of an uncomplicated UTI were consecutively screened for eligibility by a doctor or a study nurse using a questionnaire . The queA pilot study was performed at each site to test logistics: 30 patients were enrolled at the AEOC in Oslo, 10 patients at each of the other sites. We did not make any major changes to the protocol after these pilots; only 2 exclusion criteria were added (Crohn's disease and ulcerative colitis). The exclusion criteria were added when a patient with Crohn's disease wanted to be enrolled in the study. We had not thought of this patient group when writing the protocol, but when the issue emerged, we decided to exclude patents with inflammatory bowel disease. The patients from the pilot studies were included in the main study.A Norwegian pharmaceutical preparation company, Kragerø Tablettproduksjon, over-encapsulated the medicine used in the study. They used gelatin capsules where red iron oxide was used for color and titanium oxide as an opacifier. The final product was tested before the trial started to ensure that the capsules had the same look, feel, weight, and taste. The study medicine was packed in 2 different kits, one with 9 capsules containing 200 mg pivmecillinam each, the other with 9 capsules containing 600 mg ibuprofen each. Each kit was labeled with a study number. The labeling was done following a computer-generated randomization list created by an independent statistician using randomized block sizes of 2, 4, 6, or 8, stratified by country . DiffereFollowing the randomization list, Kragerø Tablettproduksjon distributed the study medicine to each study site, where it was kept in a locked facility. At inclusion, each patient received a kit labeled only with the study number. Neither the research nurse/doctor nor the patient knew which active substance was given, thus resulting in blinding for both the nurse/doctor and patient.The patients were consecutively allocated to active treatment with either pivmecillinam 200 mg or ibuprofen 600 mg 3 times a day for 3 days. They received a kit labeled with a study number; the kits were retrieved in chronological order from a locked facility at each study site. They were all given routine advice to stay hydrated alongside information on proper self-care. They received a patient diary for recording daily symptoms and on which day they felt completely cured. The diary was based on a previously validated version ,18. The Except for the drugs listed in the exclusion criteria, there are no known significant drug interactions for the study medications. The patients were directed to continue taking all prescription drugs as usual throughout the trial and to record all prescription and non-prescription drugs in the patient diary. They were not allowed to take any additional NSAIDs during the trial. They were allowed to take paracetamol if they needed additional pain relief. When the patients had completed the diary, they were asked to return it to the study site.A research nurse/doctor contacted the patients for a telephone follow-up after 14 and 28 days. In the first follow-up, the patients were asked if they felt cured, and if so, how many days it had taken; they were asked this question again in the second follow-up if they answered “no” in the first follow-up. In both follow-ups, they were asked if they had experienced a relapse of symptoms, if they had seen a doctor again, and if they had received antibiotic treatment.The monitors visited the study sites at the start of the pilot and after the pilot, and made 1 or 2 visits during the study and a final visit when all patient follow-up was done.The results of a urine dipstick  were recorded at inclusion. The dipstick results were not part of the inclusion criteria, but were recorded to provide baseline information for later predictor analysis. A urine sample was sent to the local microbiology laboratory for culturing. In Norway and Denmark, the urine sample was transported in a plastic container with boric acid. In Sweden, study personnel used plastic containers without any added chemicals; this was due to short delivery time to the laboratory (<6 hours).The patients received equipment to take a second urine sample at home and send it to the laboratory for a new culture after 2 weeks. They were instructed to collect a midstream urine sample after spreading of the labia, preferably morning urine. They could send the urine sample by mail in a freepost envelope or deliver it to the study site.3 /ml for primary pathogens, ≥104/ml for secondary pathogens, and ≥105/ml for doubtful pathogens [The uropathogens were quantified in colony-forming units per milliliter. Significant bacteriuria was defined according to current European guidelines for patients with symptoms of UTI as ≥10athogens . Clinicaathogens .If a patient experienced getting worse or not getting better, she was instructed to contact either the study doctor for advice or the study site for a second medical consultation. Contact information and routines were described in the consent form. There were written routines at each study site for health personnel to follow when a study patient came back for a second medical consultation.Patients who received additional treatment, had an allergic reaction, or were admitted to the hospital remained in the study unless they expressed a wish to withdraw their consent. Their data were included in the analyses according to the principle of intention to treat.In case of immediate need for unblinding, sealed, opaque envelopes for each study number were kept at each study site. Each envelope contained the name of the active substance given to the patient with the corresponding study number. If necessary, the envelope would be opened, and the patient and the doctor treating the patient would be informed which active substance the patient had received.If more than 10 patients within the first 100 patients included were hospitalized, this would be a cause for unblinding of the hospitalized patients. If it turned out that significantly more patients in the ibuprofen group were hospitalized, we would consider terminating the study.The trial by Gagyor et al. was published in December 2015 . We realAll outcomes are similar to outcomes chosen in previous studies demonstrating efficacy of antibiotic treatment for uncomplicated UTI ,14,21.The main outcome measure of this trial was the proportion of patients who felt cured by day 4 as recorded in the patient diary. If we did not have information from the diary, we used the number of days until cure reported by the patient during the telephone follow-up.Secondary outcome measures included duration of symptoms and the patients’ symptom load for specific symptoms. We calculated a daily symptom sum score for each patient from the patient diary. The sum score consisted of the symptoms dysuria, urinary urgency, and urinary frequency. Each symptom was graded on a scale from 0 to 6 where 0 was “normal/not affected” and 6 was “as bad as it could be”, so the maximum potential sum score each day was 18.Further secondary outcomes were the proportion of patients (i) with positive second urine culture (significant bacteriuria), (ii) in need of a medical consultation within the 4 weeks of follow-up, and (iii) who received antibiotic treatment during this period. Regarding safety, we were specifically interested in how many patients developed an upper UTI (pyelonephritis/febrile UTI) and how many patients experienced adverse events and serious adverse events (SAEs) . Pyelonephritis was defined as symptoms of UTI with flank pain, self-reported fever, and C-reactive protein (CRP) ≥ 40 mg/l. If the patient had flank pain and self-reported fever but CRP < 40 mg/l, she was characterized as having a febrile UTI. Pyelonephritis is sometimes also classified as a febrile UTI , but in The main outcome was tested according to non-inferiority, the secondary outcomes according to superiority.Assuming no difference between the treatment groups in the proportion of patients feeling cured after 4 days, we calculated that 316 patients (158 in each arm) were required in the primary full analysis set (FAS) analyses to be 80% confident that the 1-sided 95% confidence limit would exclude a difference in favor of pivmecillinam of more than 10%. The anticipated rate of feeling cured after 4 days was 85% in both groups. Assuming a dropout rate of approximately 20%, we aimed to randomize 400 patients.The null hypothesis of this study was that treatment with ibuprofen would be inferior to pivmecillinam regarding the proportion of patients feeling cured after 4 days by a 10% margin. The alternative hypothesis was that ibuprofen would be non-inferior regarding the proportion of patients feeling cured after 4 days by at most 10%.The primary efficacy analyses were performed in the FAS, consisting of all randomized patients with at least 1 efficacy assessment after randomization. Secondary efficacy analyses were performed in the per protocol set, consisting of patients in the FAS with a treatment compliance rate of at least 80%.The primary and secondary dichotomous endpoints were analyzed using logistic regression (single measurement) or mixed effects logistic regression (repeated measurements) with treatment as a fixed effect, adjusted for randomization stratum . Efficacy measures were adjusted risk difference and adjusted relative risk using the delta method.Continuous endpoints were analyzed using linear mixed models with patient-specific random intercept and treatment, time (randomization to day of assessment), treatment–time interaction, baseline value, and randomization stratum as fixed factors. Time to event endpoints were analyzed using a Cox regression model adjusted for randomization stratum and are presented using Kaplan–Meier plots.The polytomous complications variable was analyzed using Fisher’s exact methods for contingency tables and Newcombe’s hybrid score method for calculation of risk difference confidence intervals. These analyses were decided upon after unblinding of the data, which revealed the low cell count in the pivmecillinam group. Thus, these analyses should be regarded as exploratory.Subgroup analyses compared the patients with positive baseline urine cultures to those with negative cultures within the 2 treatment groups, both with regards to symptom load and duration of symptoms. Within the patients with positive urine cultures, we also compared those with cultures susceptible to pivmecillinam to those with cultures resistant to pivmecillinam with regards to symptom burden.The null hypothesis was tested on the 1-sided 5% significance level as predefined in the SAP. Consequently, the primary efficacy estimate is reported with a 90% confidence interval. Efficacy estimates of secondary endpoints are presented with 95% confidence intervals.There were no missing data for the primary endpoint in the FAS. Missing dichotomous endpoints were imputed with the worse outcome, except for febrile UTI, pyelonephritis, and SAEs. If we had no information on these endpoints, they were interpreted as not present (best case imputation). We did not impute missing outcomes for continuous and time to event endpoints. Missing data for these endpoints were handled by the linear mixed model and the Cox regression model, assuming missing at random and non-informative censoring at the last point of contact. Further statistical details are described in the SAP . StatistPatients were assessed for eligibility from 11 April 2013 to 22 April 2016, and the last follow-up was made on 7 June 2016. A total of 2,942 women were screened: 1,290 patients met 1 or more exclusion criteria, 1,269 patients were eligible, and 383 patients were enrolled in the trial . The larp-value of >0.99. The corresponding number needed to treat was 3 (90% CI 2 to 4). Adjusted relative risk was 1.9 (90% CI 1.6 to 2.2). For secondary outcomes the patients in the pivmecillinam group generally felt cured sooner than the patients in the ibuprofen group  felt cured by day 4 (the primary outcome) versus 131 patients (73.6%) in the pivmecillinam group . Adjusteen group . Where wThroughout the first week of follow-up, the ibuprofen group had a higher symptom burden than the pivmecillinam group. At day 6, the mean symptom sum score was 2.3 for the ibuprofen group and 0.7 for the pivmecillinam group, giving an estimated difference of 1.6 (95% CI 0.8 to 2.4) . ClinicaWithin 2 weeks, 41.4% of the patients in the ibuprofen group had a second consultation and were prescribed antibiotic treatment versus 9.6% in the pivmecillinam group. In the ibuprofen group, 47.0% had 1 or more secondary treatments with antibiotics within 4 weeks, versus 11.2% in the pivmecillinam group. For the purpose of these analyses, where we did not have information on whether the patient had a second consultation, we assumed she had received secondary treatment with antibiotics. Robustness analyses assuming no secondary treatment with antibiotics where we had missing data gave highly similar results with a treatment difference in the FAS of −0.36 (95% CI −0.45 to −0.28).p < 0.001)  of the cultures in the ibuprofen group and 113 (63.8%) in the pivmecillinam group. After treatment, the numbers were 43 (27.9%) in the ibuprofen group and 16 (10.4%) in the pivmecillinam group (< 0.001) . In the E. coli and 53% of the patients with baseline growth of S. saprophyticus returned and received antibiotic treatment within 4 weeks. In the pivmecillinam group, the corresponding numbers were 13% for E. coli and 25% for S. saprophyticus   .Comparing the subgroup with recurrent UTIs  to the subgroup with 0–2 UTIs within the last 12 months, the highest symptom burden over 6 days was observed in the recurrent UTI patients treated with ibuprofen . The difE. coli in the baseline urine culture to those with significant growth of any other uropathogen. The difference in symptom burden between the treatment groups was bigger in the other uropathogen subgroup, but significant in both   .In this study, we were unable to show that ibuprofen was non-inferior to pivmecillinam for treating uncomplicated UTI. Reducing the use of antibiotics came at the cost of stronger symptom burden, longer duration of symptoms, and more complications.In the pivmecillinam group, there were no significant differences in symptom burden and time to resolution of symptoms between those with a positive and negative urine culture.E. coli and 2 S. saprophyticus. This indicates that patients with a positive culture are more likely to benefit from antibiotic treatment and that treating these patients could prevent unwanted complications.In the ibuprofen group, the patients with a positive culture had a higher symptom burden and it took longer to achieve symptom resolution than for those with a negative culture. Furthermore, 10 of the 12 patients with complications had a positive urine culture at baseline showing growth of a primary pathogen: 8 However, nearly half of the patients in the ibuprofen group with a positive urine culture at baseline recovered without antibiotics . About oThe strengths of our study were that the blinding was well performed, we reached our calculated sample size, and the dropout rate was less than 10%. Our participants were recruited from unselected patients presenting with UTI symptoms. We recruited patients from both general practices and outpatient clinics; thus, we have a quite diverse population.A weakness was the extensive list of exclusion criteria, eliminating almost half of the patients presenting with symptoms of an uncomplicated UTI. Many eligible patients were not asked to participate, mainly due to the heavy work load at the AEOCs. If the pressure of incoming patients was too high, the nurses did not feel they could prioritize enrolling patients for the trial, and sometimes the nurses had not yet received the proper training to enroll patients. Furthermore, we did not include symptom burden in the questionnaire at inclusion; hence, we do not know the degree of symptoms for those who declined to participate. This might make our results less generalizable. A few patients lost the diary and equipment to send the second urine culture; hence, we had to send new diaries and equipment by mail, and in some instances this led to a delay of up to several weeks in collecting data. Filling out the diary retrospectively might provide less reliable data.As the distinction between febrile UTI and pyelonephritis was not defined in the protocol, we went through the medical records and made the classification based on the doctors’ description of the patients’ symptoms and CRP value. The classification was done prior to unblinding the treatment allocation and thus should not have introduced bias.All patients were allowed to take paracetamol as additional pain medication if needed. The patients did not report this, so we do not know how many actually took additional paracetamol. If the patients in the ibuprofen group took additional paracetamol, it could possibly have masked symptomatic progression of upper UTI.E. coli, a low resistance-driving effect, and limited side effects [We chose to use ibuprofen over other NSAIDs because of its relatively low rate of severe adverse effects. The dosage used was lower than the maximum recommended daily intake. We chose to use an NSAID instead of paracetamol because of its greater anti-inflammatory effect, which led us to believe it would provide better pain relief. Using a placebo as a second comparator was discussed, but given the number of placebo trials proving inferiority to antibiotics, we felt this would be unethical. We chose pivmecillinam because it is a narrow spectrum antibiotic and one of the first choice antibiotics for treating uncomplicated UTI in Scandinavia . It has  effects ,25. We bIn our study, using worst case imputation for missing data, 53% of the patients in the ibuprofen group recovered without antibiotics, compared to 67% in the study conducted by Gagyor et al.  and 38% The pivmecillinam group in our study recovered more quickly and had fewer complications than the fosfomycin group in the trial by Gagyor et al. This could indicate that a 3-day treatment with pivmecillinam is more effective than a single dose of fosfomycin. In the trial by Kronenberg et al., they defined symptom resolution as a symptom score of less than 2, whereas in our trial and the trial by Gagyor et al., it was defined as a symptom score of 0. If we compare the number of patients with a symptom score of 0 by day 4, the pivmecillinam group recovered more quickly than the patients treated with norfloxacin. Even though the 3 study populations may differ somewhat, they have very similar inclusion and exclusion criteria and the study drugs are similar. Despite the discrepancies, we believe it is relevant to compare the different treatment groups from the 3 trials for the purpose of discussion.Our ibuprofen group had a higher symptom burden, longer duration of symptoms, and more complications than the ibuprofen group in the trial by Gagyor et al. It could be questioned whether it is safer to treat patients presenting with an uncomplicated UTI with 400 mg rather than 600 mg of ibuprofen. The trial by Gagyor et al. had 5 cases of pyelonephritis in the ibuprofen group and 1 in the fosfomycin group. The trial by Kronenberg et al. had 6 cases (5%) of pyelonephritis, all among the patients initially treated with diclofenac. We had 7 cases of pyelonephritis, all in the ibuprofen group. The rate of pyelonephritis in the ibuprofen group was 3.9%, and even though the number of patients was quite small, the percentage is high compared to what was found in a meta-analysis of randomized controlled trials comparing placebo with antibiotics, where the incidence of pyelonephritis in the placebo group ranged from 0.4% to 2.6% . Based oThe German group identified a moderate to severe degree of urinary urgency/frequency and a positive urinary dipstick for erythrocytes, leucocytes, and nitrite as predictors for needing subsequent antibiotic treatment . In the In our study, more than half of the women treated with ibuprofen achieved symptomatic cure without subsequent antibiotic treatment. This is in line with the findings of Little et al., who showed that a wait-and-see strategy for women with uncomplicated UTI reduced the use of antibiotics in a safe manner . With shMore than half of the women treated with ibuprofen achieved symptom resolution without any additional treatment. Initial treatment with ibuprofen could reduce unnecessary use of antibiotics in this group. However, until we can identify those women in need of antibiotic treatment to prevent complications, we cannot recommend ibuprofen alone to women with uncomplicated UTIs.S1 Appendix(PDF)Click here for additional data file.S1 CONSORT Checklist(DOC)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 TableFigures are number of women (percentage) unless stated otherwise.(DOCX)Click here for additional data file.S2 TableFigures are proportions (percentage) unless stated otherwise.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


International statistical classification of diseases and related health problems (ICD-11).To examine the empirical basis for including the diagnostic category of “a single episode of harmful substance use” in the 11th revision of the We used data on patients admitted to emergency departments in 21 countries with alcohol-related injuries (i.e. with drinking within the preceding six hours) who had no sign of alcohol intoxication or withdrawal, no alcohol in blood and no sign of alcohol dependence or harmful drinking as described in the ICD-10. We obtained data on alcohol-related injuries, the patient’s causal attribution of injury to drinking, the alcohol amount consumed, blood alcohol concentration and usual drinking pattern. Patients with and without alcohol dependence or harmful drinking were compared.We included a representative sample of 18 369 patients. After adjustment for unequal sampling, 18.8% reported drinking in the six hours before injury and 47.1% of these attributed their injury to drinking; 16.3% of those reporting drinking and 10.3% of those attributing their injury to drinking were not alcohol dependent or harmful drinkers. The majority of these last two groups reported never having had five or more drinks on one occasion during the last year and had a blood alcohol concentration less than 0.05%.Some individuals attending emergency departments had alcohol-attributable injuries due to a single episode of drinking but had no history of harmful use or dependence. These findings highlight the public health relevance of including the new diagnostic category in the ICD-11. The graduated-frequency approach was used to determine how frequently they consumed 5 to 11 drinks or 12 or more drinks on one occasion,Diagnostic and Statistical Manual of Mental Disorders, fourth edition.––,,To assess alcohol dependence, interviewers asked patients the four questions in the Rapid Alcohol Problems Screen (hereafter referred to as RAPS4):We divided patients who reported drinking in the six hours before injury and those who causally attributed their injury to drinking into three groups: (i) those who had a positive result on the RAPS4 (i.e. RAPS4-positive); (ii) those who had a negative result on the RAPS4 (i.e. RAPS4-negative) but positive responses to both the quantity and frequency questions (i.e. QF-positive); and (iii) those who were RAPS4-negative and had a negative response to one or both of the quantity and frequency questions (i.e. RAPS4-QF-negative). We compared these three groups to identify differences in the amount of alcohol consumed before injury, blood alcohol concentrations, usual drinking patterns, sex and age . In four studies, researchers oversampled patients at some times of the day for logistical reasons associated with shift patterns. Consequently, we weighted the data to adjust for unequal probability sampling .After adjustment for unequal sampling, it was estimated that 18.8% of the 18 369 patients reported drinking in the six hours before the event that caused injury and therefore had an alcohol-related injury. Overall, 16.3% of patients who reported drinking before injury showed no evidence of alcohol dependence or harmful drinking. In this group, 82.8% reported never having had five or more drinks on one occasion during the last year and 42.9% reported two or fewer drinks before the injury . In addiThe main diagnostic categories covering alcohol-related harm in the ICD-10 include alcohol dependence, harmful drinking, alcohol intoxication and alcohol withdrawal; no classification is available for individuals not covered by one of these four categories who may have experienced harm due to a single episode of drinking. Health professional might therefore not identify these individuals as eligible for alcohol-focused interventions.A single episode of use of alcohol that has caused damage to a person’s physical or mental health or has resulted in behaviour leading to harm to the health of others. The episode of harmful use of alcohol typically involves acute harm to health, which is not limited to symptoms of acute intoxication or withdrawal and may include substance-induced mental disorders. This diagnosis should not be made if the harm is attributed to a known pattern of alcohol use.This definition excludes three diagnostic categories already in effect: harmful pattern of use of alcohol (6B81), alcohol withdrawal (6B83) and alcohol dependence (6B82).Here we show that a substantial proportion of patients  were admitted to emergency departments with alcohol-related injuries after drinking relatively small amounts and had no sign of acute alcohol intoxication. These patients also had no history of heavy episodic drinking or of any other pattern of alcohol use that would qualify as an alcohol use disorder. Hence, these patients do not fall into one of the four ICD-10 diagnostic categories but who would have met diagnostic requirements for a single episode of harmful use of alcohol as described in the draft ICD-11. This draft includes a proposed diagnostic category for a single episode of harmful use of alcohol, with the definition:Our analysis did not take into account harm to others resulting from the patient’s drinking before injury, as included in the proposed definition. A previous analysis of data from emergency departments in 14 countries found that the number of patients who believed an injury due to violence could be attributed to alcohol increased by 62% if they were able to include the other person’s drinking when attributing the cause.Our findings underscore the importance of including the new diagnostic category of a single episode of harmful drinking in the ICD-11. Its inclusion would help identify patients who could benefit from a brief intervention for harmful drinking in the emergency department and would provide support for alcohol policies aimed at reducing alcohol-related harm. At present, interventions focused on alcohol use are not routinely implemented in emergency departments despite the expectation that an alcohol-related emergency admission could provide an effective learning moment for the patient to consider reducing his drinking or stopping altogether.One strength of our study is the inclusion of data from 21 countries that used identical protocols in representative samples of emergency department patients. There are some caveats, however. Patients themselves provided information on drinking before injury, on causal attribution of the injury and on usual alcohol consumption. However, previous emergency department studies found that only a very small percentage (ranging from 0.5% to 3.3%) of patients denied drinking but had a substantial blood alcohol concentration.In conclusion, current ICD-10 codes do not cover all alcohol-related injury, although the harmful consequence of alcohol use affects the global burden of disease. Here we show that some individuals attending emergency departments had alcohol-attributable injuries but no history of harmful use or dependence. This is just one setting in which a single episode of harmful alcohol use can be identified and recognizing such episodes can have clinical and public health implications. The findings highlight the importance of including the new diagnostic category of a single episode of the harmful use of alcohol in the ICD-11.