Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a ‘foothold’ that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus.DOI:http://dx.doi.org/10.7554/eLife.04489.001 Molecules can be transported around a cell by so-called motor proteins that move along a network of filaments called microtubules. Many motor proteins—including the kinesin family of these proteins—can only move in one direction along a microtubule. In most cells, kinesins tend to transport other molecules away from the center and towards the cell edge.Kinesins can have different structures, but most are made up of two subunits that are joined and work together to create a walking-like movement. Each subunit has a region called a motor domain  that can bind to the microtubule and to a molecule called ATP, which provides the energy required for the motor to step forward.Kinesins can be classed either as processive or non-processive motors. Processive motors can walk continuously along a microtubule for several hundred steps, whereas non-processive motors fall off after just a few steps. A motor protein called Kar3 belongs to a group of non-processive kinesins. Kar3 is unusual; unlike most of the motors in this group (which work together in pairs), Kar3 motor protein subunits each bind to and work with non-motor protein subunits, including one called Cik1. The head of the non-motor protein cannot bind to ATP, although it can bind to microtubules. This means that the non-motor protein subunits are not provided with the energy to make a stepping motion; this raises questions about how the Kar3 motor protein moves along the microtubule, and whether this affects the roles the motor performs.Mieck et al. studied how a molecular motor made up of Kar3 and Cik1 moves along microtubules using sensitive microscopy that allows single molecules to be observed. This revealed that, contrary to what is expected from a non-processive motor, Kar3–Cik1 moves long distances on microtubules without detaching from them. Further investigation showed that Cik1 provides a ‘foothold’ for the motor, binding it to the microtubule in such a way that allows it to move along the microtubule in the opposite direction to most kinesins. In addition, Mieck et al. found that the Kar3–Cik1 motor binds to and transports a protein complex that is crucial for separating chromosomes during cell division.A challenge for the future is to understand in even greater detail how the movement of such a motor occurs. If it doesn't ‘walk’ like other motors, then how can its motion be explained? The benefits for the cell that underlie why this mechanism evolved also remain to be discovered.DOI:http://dx.doi.org/10.7554/eLife.04489.002 Motors of the kinesin family are ubiquitous enzymes essential for intracellular transport along microtubules in eukaryotes. The mechanism by which kinesin motor proteins convert the chemical energy of ATP hydrolysis into coordinated, long-range directional movement has fascinated cell biologists, biochemists, and engineers for many decades. Biophysical studies of kinesins have focused on conventional Kinesin-1 and established the ‘hand-over-hand’ model for the processive walking behavior of this type of motor . In analDrosophila Ncd motor, are common examples for nonprocessive kinesins . Purification and labeling yielded a homogenous preparation containing a heterodimer of Cik1 and TMR-labeled Kar3  or removed a portion of the carboxyterminus (Cik11–521) had severe effects and prevented directional movement. The specific nature of the defect was most apparent for the shorter truncation Cik11–521–Kar3, which was able to bind microtubules under our standard conditions, but instead of smooth translocation it displayed erratic forward and backward displacements that did not lead to directional movement as revealed by kymographs  with short residence times (τ = 0.6 ± 0.1 s)  and in the presence of the non-hydrolyzable ATP analog AMP-PNP Kar3 motors bound with high affinity to the microtubule but displayed no movement . Binding± 0.1 s) .10.7554/1–360–Kar3)  and about 30-fold more diffusive in the AMPPNP state (D = 0.0036 ± 0.00011 μm2/s vs 0.00011 ± 0.00001 μm2/s)  . In contotubules . MSD datmovement . Further1 μm2/s) . Furtherr states . We addir states . Taken tHaving established that the non-catalytic partner is critical for achieving mechanically processive Kar3, we sought to gain further insights into the underlying mechanism by studying the alternative Vik1–Kar3 motor. The non-catalytic proteins Cik1 and Vik1 are paralogs that display about 45% sequence similarity between each other. We purified Vik1–Kar3 complexes from yeast extracts and imaged their interaction with microtubules under the same conditions as previously employed for Cik1–Kar3 . Interesbim1Δ strain, pheno-copying a cik1 deletion . Together with our finding that the Vik1head–Cik1tail chimera fully supports viability, this implies that the key function of Kar3 in vegetatively growing yeast cells requires motility—supported by any of the two non-catalytic heads—and the Cik1 tail domain, but does not involve spindle localization via Bim1. Based on previously characterized mutants and on our finding that the Ndc80 complex co-purifies with Cik1, this supports a key role for Kar3 at the kinetochore. Consistent with this notion, we found that 80% of Cik11–521 cells arrested as large budded cells upon shift to the restrictive temperature, indicative of mitotic checkpoint activation. Analysis of chromosome segregation at the semi-permissive temperature of 25°C by fluorescent labeling of chromosome V showed that 50% of large budded Cik1 mutant cells attempted nuclear division without proper bi-orientation of sister chromatids  full-length Kar3, Cik1, and Vik1 were amplified by PCR and cloned into the overexpression pESC-TRP  vector. The non-catalytic motor subunit was tagged C-terminally with 1xFLAG and the motor itself was fused to a HaloTag  at the 5′ end of the coding sequence, separated by a 13 amino acid linker. Mutated and truncated versions of Cik1 and Kar3 were generated by site-directed mutagenesis PCR . To determine the TMR-labeling efficiency, Halo-Kar3 was fused with enhanced green fluorescent protein (eGFP), creating Halo-eGFP-Kar3. All motor proteins were overexpressed in budding yeast using the pESC vectors (Agilent Technologies) following the manufacturer's instructions. In brief, yeast cells harboring the respective pESC plasmid were induced with 2% Galactose at OD600 = 1.0 for 7–9 hr, harvested by centrifugation, washed, and frozen as droplets in liquid nitrogen. Lysis was conducted in liquid nitrogen using a freezer mill (Biospec Inc.). The cell powder was resuspended in lysis buffer . The lysed cells were centrifuged twice, first at 43.146×g for 20 min and afterwards at 125.749×g for 1 hr. The resulting supernatant was incubated with M2 affinity agarose (Sigma–Aldrich) for 1 hr, gently rotating at 4°C. The agarose resin was washed five times with lysis buffer . Elution of the kinesin heterodimeric constructs from M2 agarose was achieved by applying one resin volume of 3xFLAG peptide at final 2 mg/ml in lysis buffer . If needed, the elution was loaded onto a cation exchange chromatography  in running buffer  to remove the FLAG peptide. Afterwards a linear salt gradient eluted a single peak, pure motor fraction at 250 mM salt. Elution fractions were supplemented with glycerol , snap-frozen in liquid nitrogen and stored at −80°C. The protein concentration was measured using the DC Assay kit (Bio-Rad). All proteins were pre-cleared by centrifugation using a 0.1-μm spin filter (Millipore) to remove aggregates before each experiment.The protein coding sequences of 2-terminus of Kar3 with the HaloTag TMR ligand or HaloTag Alexa488 ligand  was performed during the above-described purification before eluting the kinesin heterodimer from the M2 affinity agarose: the proteins were incubated with 10 μM HaloTag ligand for 3 hr. Extensive washing removed unbound TMR ligand and the kinesin was eluted as described before. In order to assess the labeling efficiency the Halo-eGFP-Kar3 construct was expressed, purified, and TMR-labeled. Observing the motor in our multi-color single-molecule imaging setup in the absence or presence of ATP revealed that >90% of eGFP-labeled kinesins also had a TMR-ligand covalently bound to the HaloTag.Labeling of the NHTo obtain a stoichiometric heterodimer of Cik1 and Kar3, the motor was subjected to analytical SEC. The purified motor was loaded onto a Superose 6 PC 3.2/30 column , and 100 μl fractions were collected and separated by SDS-PAGE. Proteins were stained with Coomassie Brilliant blue R250.Expression and purification of the full-length and mutant Ndc80 complex (Ndc80p-6xHis/Nuf2p-EGFP and Spc24p/6xHis/Spc25p) was performed as described previously . Bim1 waPeak fractions of motor from the gel filtration experiments were diluted to a final concentration of 60 μg/ml in spraying buffer, containing 100 mM ammonium acatate and 30% (vol/vol) glycerol, pH adjusted to 7.4. After dilution, the samples were sprayed onto freshly cleaved mica chips and immediately transferred into a Bal-Tec MED020 high vacuum evaporator equipped with electron guns. After drying in the vacuum, the rotating samples were coated with 0.6 nm Platinum at an elevation angle between 5° and 6°, followed by 9.5 nm Carbon at 90°. The produced replicas were floated off from the mica chips and picked up on 400 mesh Cu/Pd grids. The grids were inspected in an FEI Morgagni 268D TEM operated at 80 kV. Electron micrographs were acquired using an 11 megapixel Morada CCD camera from Olympus-SIS. Images were examined and analyzed using ImageJ.Single-step purified Cik1–Halo–Kar3 was loaded on top of a 4.4 ml 5–25% linear sucrose gradient and spun at 50,000 rpm for 16 hr using an Sorvall TH-660 rotor and a Sorvall Discovery 90SE centrifuge. Fractions (270 μl) were collected and analyzed by SDS-PAGE and Coomassie Brilliant Blue R250 staining. The sedimentation value for Cik1-Halo-Kar3 was defined by comaring the mobilities of the motor with linear plots of mobility standards.The single molecule motor assays were conceptually designed as described previously . Biotin-High temporal resolution recordings  to the flow chamber. After the excess of antibody was washed out with BRB80 supplemented with 0.5 mg/ml casein, motors were introduced to the chamber and incubated for 5 min. Finally a microtubule-containing solution supplemented with 5 mM ATP and oxygen scavenger mix was perfused into the chamber. Microtubules used for this experiment were assembled from porcine tubulin mixed together with HiLyte-647-labeled tubulin (Cytoskeleton Inc.) in the presence of 10 μM taxol. Time-lapse videos were recorded at 28°C in 3-s intervals between frames using a TIRF microsopy setup described previously . MetaMorThe tracking of the proteins was performed automatically using the Definiens Software Suite. Prior to the analysis, the image data were processed performing a shading correction and smoothing. This was done by applying an 11 × 11 pixel (1463 × 1463 nm) kernel median filter and dividing the original raw image by the filtered image data. The resulting image was smoothed, using a 3 × 3 (399 × 399 nm) kernel Gaussian filter. The processed image data were searched and segmented for fluorescent signal. The signal area was searched for local intensity maxima within a search range of 532 nm. Circular objects with a radius of 332 nm were created on the found maxima and used for tracking the identified proteins.The tracking of the movement of the TMR-, Alexa-488-, or GFP-labeled proteins was done through linking the proteins frame by frame by direct overlap, using the best fitting overlap.Saccharomyces cerevisiae (accession number NP_013925.1), Saccharomyces kudriavzevii (EJT42048.1), Saccharomyces arboricola (EJS44163.1) and Vik1 sequences from S. cerevisiae (NP_015070.1), S. kudriavzevii (EJT43871.1), S. arboricola (EJS41496.1) were retrieved from the National Center for Biotechnology Information (NCBI). The Saccharomyces bayanus protein sequences of Cik1 (WashU_Sbay_Contig651.30) and Vik1 (WashU_Sbay_Contig637.18) were retrieved from the Saccharomyces Genome Database (SGD). The sequences were aligned with MAFFT  and further processed with Jalview and colored according to ClustalW.Cik1 protein sequences of FL, GST-Bim10–185, GST-Bim1185–344) were immobilized on 30 μl glutathione sepharose  in 0.5 ml binding buffer . The binding partner was added at a constant concentration between 0.5 and 1 μM and incubation lasted for 1 hr at 4°C. Afterwards beads were washed three times with 0.5 ml binding buffer and analyzed by SDS-PAGE and Coomassie Brilliant blue R250 staining.Varying amounts (0.1–1 μM) of recombinant bait protein  plates. Plates were incubated at the indicated temperatures to 2–3 days.For the spot assay, the desired strains were grown overnight in YPD medium. The following day cells were diluted to ODImaging strains were grown in synthetic medium containing 2% glucose and imaged on concanavalin A-coated culture dishes (Matek) at ambient temperature. Eight z stacks with planes 0.3 μm apart were acquired at 30 s intervals on an Axiovert 200M microscope (Carl Zeiss) using an UPlanSApo 100× NA 1.40 oil immersion objective lens (Olympus) and a Coolsnap HQ CCD camera (Photometrics). Images were projected to two-dimensional images (SoftWoRx software) and further analyzed by MetaMorph (Molecular Devices). The linescans showing the fluorescence intensity for Kar3-3xGFP on spindles were plotted using ImageJ.600 = 1.2 in YPD, centrifuged, drop frozen in liquid nitrogen, and lysed by freezer mill treatment. 5 g of yeast powder was dissolved in 10 ml buffer A . The lysed cells were centrifuged twice, first at 43.146×g for 20 min and afterwards at 125.749×g for 1 hr. The resulting supernatant was incubated for 2–3 hr with 100 μl Dynabeads (Life Technologies) that were coupled to 50 μl anti-FLAG M2 antibody (Sigma Aldrich). Beads were washed three times with buffer A and four times with buffer B . Elution was achieved by using 1 beads volume 2 mg/ml 3xFLAG peptide in buffer B. The elution fractions were analyzed by SDS-PAGE and silver stain. For mass spectrometry analysis, an on-bead digest replaced the elution procedure: 500 ng LysC protease was added per 50 μl dynabeads in ammonium bicarbonate buffer and incubated at 37°C overnight. The supernatant was filtered and applied to mass spectrometry analysis, which was performed on three independent preparations.Desired strains were grown to OD review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and two reviewers.Thank you for sending your work entitled “Non-catalytic motor domains enable processive movement and functional diversification of the kinesin Kar3” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.eLife, but to make it so, the authors will need to carry out additional experiments and to reduce the strength of their claims, which both reviewers agreed were exaggerated (one reviewer even used the word “reckless”). The key result that the reviewers agreed makes the paper suitable for eLife is that the heterodimer walks processively toward the minus end of a MT.The reviewers agreed that in principle this paper would be suitable for publication in 1) The main conclusion of this manuscript is that Kar3 needs a second non-catalytic microtubule binding domain for processive translocation. If monomeric kinesin Kif1A can move along microtubules in vitro using a biased diffusion mechanism (2) Examples of exaggerated claims or incorrect information, all of which need to be fixed in a revision:Abstract:(i) “Whether this (hand-over-hand) model is universal, or if alternative mechanisms can support highly processive motion along microtubules, is an open question.” This statement is wrong. It is no longer an open question. Dewitt et al., Science, 2012 and Qiu et al., NSMB, 2012 showed that dynein walks in an uncoordinated manner.(ii) “The noncatalytic domain acts as a foothold that allows Kar3 to bias translocation towards the minus end via a diffusive ADP state.” The reported experiments do not prove this statement. That kinesins have lower MT affinity in the ADP state was already known. The authors did not demonstrate how this motor steps along MT. In order to make these claims, they must demonstrate that: a) Cik1/Vik1 biases translocation of Kar3 to the minus end, b) translocation of Kar3 occurs in the ADP bound state and c) diffusion of the Kar3 head to the next tubulin binding site is a critical step. The authors need to delete this statement and replace it with one that sticks to the facts.(iii) “This mechanism… allows transport of the Ndc80 kinetochore complex in vitro and is critical for Kar 3 function in vivo.” The authors show that Kar3 can bind and transport the isolated Ndc80 complex, not the Ndc80 complex as part of an assembled kinetochore. It has yet to be shown whether this in vitro transport reflects a property critical for in vivo function of Kar3. This latter statement is what they propose, but do not show, in this manuscript. The authors need to delete this statement and replace it with one that sticks to the facts.Introduction section:“In general motors equipped with only one catalytically active head fail to achieve long-range processive motility”. This is not true. The authors need to rewrite this paragraph citing: (i) the Okada et al. 2003 Kif1A paper showing processive single-headed motility; (ii) the Kaseda, Higuchi and Hirose  and Thoresen et al.  papers showed that processive motility of kinesin heterodimers with mutant motor domains that have lower catalytic activity; (iii) the The manuscript lacks evidence that their preps do not include aggregates. The authors could rule out aggregation by running gel filtration experiments, which are standard in the field.is also necessary.The authors do cite evidence in Figure 1–figure supplement 1, for mostly single step photobleaching of labeled motors, arguing that processive motility is a property of individual motors. While this may be true, the single step photobleaching is not definitive. First, the data clearly show that some motors show 2 or >2 steps of photobleaching, suggesting protein aggregation or transient interactions. Second, the labeling efficiency is not calculated. If it is low (e.g. 40%), the fact that 10-20% of motors have 2 or more steps of photobleaching would suggest most proteins are aggregates. Third, if the motors are aggregated (e.g. 10 motors in an aggregate) then almost all the aggregates will have TMR even if the coupling efficiency is low. Aggregation may be an even bigger problem with the delta K-loop construct, which is expected to be less soluble. So gel filtration on this construct In Third paragraph of the Results section:“We noticed that due to their relatively large velocity distribution…” This whole paragraph does not make sense. How do they know that the motors run into each other ? What is the evidence that motors travel together in teams? Are the proteins in contacts with each other (which cannot be seen by a fluorescence microscope due to diffraction limited resolution of the assay). In this section, they can also cite the Cin8 study by the Surrey lab .Eighth paragraph of the Results section:The interpretation of the K-tail mutant experiments is counterintuitive. One would expect that removing positively charged residues from the noncatalytic head would reduce its affinity for MTs . Since the authors wish to refer to the function of this head as “a foothold”, these mutations would be expected to alter motility by reducing processivity (because it is no longer a very strong MT tether), with increased or unchanged velocity . The description of these results and their interpretation need reconsideration and revision.The authors do not present direct evidence that Kar3 transports Ndc80 in vivo. Therefore, they need to change “We conclude that the Ndc80 complex is a direct cargo for Kar3 transport” to “We conclude that the Ndc80 complex can be transported by the Kar3/Cik1 motor.”Discussion:(i) In the first paragraph, the authors claim to have discovered that Kar3/Cik or Kar3/Vik1 heterodimers have a new mode of processive motility, and they assert that this is the first example of a motor that violates hand over hand motility. These statements are wrong (see comments above). Also note that without high resolution stepping studies their conclusions are too speculative.(ii) The authors claim that “Kar3's motile characteristics share similarities to Kif1A”. How so? Kif1A was claimed to be processive on its own and not to require a foothold. The effect of the positive charges is different. Note that processive motility of single-headed Kif1 has not been replicated in another laboratory. The authors should analyze the motility of a Kar3 monomer and compare their results with those published by Hirokawa on Kif1A.The model shown does not relate to the main story, and there is no evidence provided for it. It should be removed from the manuscript. The entire discussion of diffusive states, anisotropy and powerstroke are far too speculative. It would be more appropriate for some future review. The problem is that without high-resolution stepping data many of the conclusions are in fact speculations. The authors have to be very careful to distinguish fact from fiction.eLife, a revised manuscript will need to include at least the following:In summary, to be suitable for 1) More convincing and complete demonstration, by careful gel filtration experiments, that the motor proteins are not aggregated.2) More thorough analysis of the data in 3) Comparison of the behavior of a Kar3 monomer with the reported behavior of Kif1A.The revised manuscript must also avoid the exaggerated and overstated claims that mar the current version. In summary, to be suitable for eLife, a revised manuscript will need to include at least the following:1) More convincing and complete demonstration, by careful gel filtration experiments, that the motor proteins are not aggregated.2) More thorough analysis of the data in: is there directionality in the motion detected for the mutants?3) Comparison of the behavior of a Kar3 monomer with the reported behavior of Kif1A.The revised manuscript must also avoid the exaggerated and overstated claims that mar the current version.1) In the revised manuscript we have included a biochemical characterization of Cik1-Kar3 motors by gel filtration and velocity gradient centrifugation analysis new . To go f2>= a·tn, indicating random diffusion with no directional component. In contrast, data for mean squared displacement of the wild-type motor is fitted with n=2, indicating directed movement.2) As suggested we have more carefully analyzed the movement of wild-type and mutant motors in ATP in the new 3) We have constructed a monomeric, Halo-tagged Kar3 construct and imaged it in the TIRF assay. In the presence of ATP and under low nanomolar motor concentrations, only very short-lived binding events can be detected on microtubules new . Thus, tWe believe that taken together these three points have greatly strengthened the manuscript. The biochemical and EM characterization shows that well-behaved, non-aggregated motors were purified. The additional experiments show that indeed the non-catalytic head is strictly required for processive movement, as no directional movement of the heterodimer can be detected after its removal and in the reciprocal experiment; the Kar3 monomeric head alone does not display processive movement.The reviewers agreed that in principle this paper would be suitable for publication in eLife, but to make it so, the authors will need to carry out additional experiments and to reduce the strength of their claims, which both reviewers agreed were exaggerated (one reviewer even used the word “reckless”). The key result that the reviewers agreed makes the paper suitable for eLife is that the heterodimer walks processively toward the minus end of a MT.1) The main conclusion of this manuscript is that Kar3 needs a second non-catalytic microtubule binding domain for processive translocation. If monomeric kinesin Kif1A can move along microtubules in vitro using a biased diffusion mechanism , Kar3 might do the same, obviating (or at least reducing) the requirement for a second motor head. A key question is whether the Cik1(1-360)-Kar3 mutant (or the Cik1(1-521)-Kar3 mutant) moves directionally in the presence of ATP. Now it is clear fromthat the velocity is substantially lower, but the authors need to analyze these data in more detail. If Cik1(1-360)-Kar3 and Cik1(1-521)-Kar3 do have significant directionality, then the authors might need to modify their description of the “foot-hold” mechanism.Please see our response to this point above. In addition to the analysis for the Cik1 (1-360)-Kar3 mutant presented in the new 2) Examples of exaggerated claims or incorrect information, all of which need to be fixed in a revision:Abstract:(i) “Whether this (hand-over-hand) model is universal, or if alternative mechanisms can support highly processive motion along microtubules, is an open question.”This statement is wrong. It is no longer an open question. Dewitt et al., Science, 2012 and Qiu et al., NSMB, 2012 showed that dynein walks in an uncoordinated manner.As suggested we have rewritten the Abstract. We would like to point out, however, that we explicitly referred to kinesin motors in the original Abstract. Indeed, Dynein movement does not require head-to-head coordination and we have cited the above mentioned papers in our Discussion.(ii) “The noncatalytic domain acts as a foothold that allows Kar3 to bias translocation towards the minus end via a diffusive ADP state.”The reported experiments do not prove this statement. That kinesins have lower MT affinity in the ADP state was already known. The authors did not demonstrate how this motor steps along MT. In order to make these claims, they must demonstrate that: a) Cik1/Vik1 biases translocation of Kar3 to the minus end, b) translocation of Kar3 occurs in the ADP bound state and c) diffusion of the Kar3 head to the next tubulin binding site is a critical step. The authors need to delete this statement and replace it with one that sticks to the facts.We have deleted and replaced the corresponding statements in the revised Abstract.(iii) “This mechanism… allows transport of the Ndc80 kinetochore complex in vitro and is critical for Kar 3 function in vivo.” The authors show that Kar3 can bind and transport the isolated Ndc80 complex, not the Ndc80 complex as part of an assembled kinetochore. It has yet to be shown whether this in vitro transport reflects a property critical for in vivo function of Kar3. This latter statement is what they propose, but do not show, in this manuscript. The authors need to delete this statement and replace it with one that sticks to the facts.We have replaced the statement with “… can support transport of the Ndc80 complex in vitro…”.Introduction section:“In general motors equipped with only one catalytically active head fail to achieve long-range processive motility”. This is not true. The authors need to rewrite this paragraph citing: (i) the Okada et al. 2003 Kif1A paper showing processive single-headed motility; (ii) the Kaseda, Higuchi and Hirose  and Thoresen et al.  papers showed that processive motility of kinesin heterodimers with mutant motor domains that have lower catalytic activity; (iii) thepaper on cytoplasmic dynein showing that processive motility can occur when one of the heads in the dimer is replaced by a non-motor domain; and (iv) reports of processive motility with axonemal dynein. In other words, the authors need to remove overblown claims, discuss earlier results in a balanced way, and clearly and accurately indicate how their results advance the field.We have re-written the Introduction as suggested. In general we have put less emphasis on a comparison of a conventional hand-over-hand mechanism with the observed motility of Kar3 heterodimers and instead have focused on a comparison of Kar3 to other Kinesin-14 family members such as Ncd.The manuscript lacks evidence that their preps do not include aggregates. The authors could rule out aggregation by running gel filtration experiments, which are standard in the field.We have included these experiments in the revised manuscript new , please The authors do cite evidence in Figure 1–figure supplement 1, for mostly single step photobleaching of labeled motors, arguing that processive motility is a property of individual motors. While this may be true, the single step photobleaching is not definitive. First, the data clearly show that some motors show 2 or >2 steps of photobleaching, suggesting protein aggregation or transient interactions. Second, the labeling efficiency is not calculated. If it is low (e.g. 40%), the fact that 10-20% of motors have 2 or more steps of photobleaching would suggest most proteins are aggregates. Third, if the motors are aggregated (e.g. 10 motors in an aggregate) then almost all the aggregates will have TMR even if the coupling efficiency is low. Aggregation may be an even bigger problem with the delta K-loop construct, which is expected to be less soluble. So gel filtration on this construct is also necessary.We have estimated the TMR labeling efficiency using a construct that in addition to the Halo tag contains an eGFP fusion. By comparing eGFP to TMR fluorescence at low motor concentrations we estimate the labeling efficiency to exceed 90% ? What is the evidence that motors travel together in teams? Are the proteins in contacts with each other (which cannot be seen by a fluorescence microscope due to diffraction limited resolution of the assay). In this section, they can also cite the Cin8 study by the Surrey lab .We have re-worded this section to state that we are simply comparing properties of motors with different brightness, i.e. heterodimers compared to teams that consist of two or more heterodimersEighth paragraph of the Results section:The interpretation of the K-tail mutant experiments is counterintuitive. One would expect that removing positively charged residues from the noncatalytic head would reduce its affinity for MTs . Since the authors wish to refer to the function of this head as “a foothold”, these mutations would be expected to alter motility by reducing processivity (because it is no longer a very strong MT tether), with increased or unchanged velocity . The description of these results and their interpretation need reconsideration and revision.While our experiments establish that the non-catalytic head in combination with Kar3 determines the velocity of the motor complex, we indeed at this point cannot give a mechanistic explanation for the role of the “K-tail”. In additional experiments we determined that simply adding the “K-tail” to the carboxyterminus of Cik1-Kar3 is not sufficient to alter the velocity of this motor. Thus, the K-tail’s role in the mechanochemical cycle might be more complex than simply altering the affinity for microtubules. This is in line with recent work from Yoshi et al., JBC, 2013. We have decided to remove the figure panels describing the K-tail experiments from the revised manuscript, as these points require further experimentation and do not contribute to the main message of the paper.The authors do not present direct evidence that Kar3 transports Ndc80 in vivo. Therefore, they need to change “We conclude that the Ndc80 complex is a direct cargo for Kar3 transport” to “We conclude that the Ndc80 complex can be transported by the Kar3/Cik1 motor.”We agree and have changed the wording accordingly.Discussion:(i) In the first paragraph, the authors claim to have discovered that Kar3/Cik or Kar3/Vik1 heterodimers have a new mode of processive motility, and they assert that this is the first example of a motor that violates hand over hand motility. These statements are wrong (see comments above). Also note that without high resolution stepping studies their conclusions are too speculative.(ii) The authors claim that “Kar3's motile characteristics share similarities to Kif1A”. How so? Kif1A was claimed to be processive on its own and not to require a foothold. The effect of the positive charges is different. Note that processive motility of single-headed Kif1 has not been replicated in another laboratory. The authors should analyze the motility of a Kar3 monomer and compare their results with those published by Hirokawa on Kif1A.As suggested, we have tested the motility of a monomeric Kar3 head construct . We have found no evidence for processive movement under conditions that allow robust motility of the heterodimer new . We agreThe model shown does not relate to the main story, and there is no evidence provided for it. It should be removed from the manuscript. The entire discussion of diffusive states, anisotropy and powerstroke are far too speculative. It would be more appropriate for some future review. The problem is that without high-resolution stepping data many of the conclusions are in fact speculations. The authors have to be very careful to distinguish fact from fiction.As suggested, we have re-written the discussion section to remove the more speculative statements and focus on the experimentally supported insights. We have removed the original We agree that further biophysical experiments are required to establish a mechanism and we have greatly tightened the discussion to avoid too much speculation about the mechanism.


The RecQ family DNA helicases Werner syndrome protein (WRN) and Bloom syndrome protein (BLM) play a key role in protecting the genome against deleterious changes. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. WRN and BLM are distinguished from other helicases by possessing signature tandem domains toward the C terminus, referred to as the RecQ C-terminal (RQC) and helicase-and-ribonuclease D-C-terminal (HRDC) domains. Although the precise function of the HRDC domain remains unclear, the previous crystal structure of a WRN RQC-DNA complex visualized a central role for the RQC domain in recognizing, binding and unwinding DNA at branch points. In particular, a prominent hairpin structure (the β-wing) within the RQC winged-helix motif acts as a scalpel to induce the unpairing of a Watson–Crick base pair at the DNA duplex terminus. A similar RQC-DNA interaction was also observed in the recent crystal structure of a BLM-DNA complex. I review the latest structures of WRN and BLM, and then provide a docking simulation of BLM with a Holliday junction. The model offers an explanation for the efficient branch migration activity of the RecQ family toward recombination and repair intermediates. RecQ helicases, a family of DNA unwinding enzymes that belong to the SF2 superfamily helicases, play crucial roles at multiple steps in DNA recombination, replication and repair. Whereas the genomes of bacteria typically encode a single recQ gene, the human genome contains five recQ genes that encode Werner syndrome protein (WRN), Bloom syndrome protein (BLM), RECQ1, RECQ4, and RECQ5. Mutations in WRN and BLM are associated with the rare genetic diseases Werner and Bloom syndromes, respectively. These two diseases are characterized by a high frequency of cancer predisposition, illustrating the primary importance of WRN and BLM in preventing tumorigenesis. Indeed, cells derived from aﬄicted patients show pronounced genomic instabilities such as sister chromatid exchange and telomere shortening.The Werner and Bloom syndromes, however, are also characterized by many distinct clinical symptoms: Werner patients display features of accelerated aging including the early onset of osteoporosis, atherosclerosis, arteriosclerosis, type II diabetes and cataracts , while BTo date, a number of reviews on the biological functions of WRN and BLM have been published, including the latest ones that discuss the diverse genome-maintenance mechanisms of the RecQ family . In thisFurthermore, I present a novel docking simulation of BLM with a Holliday junction (HJ), using the recently determined crystal structure of a BLM-DNA complex . The modFigure 1. WRN and BLM are multi-domain helicases composed of 1,432 and 1,417 amino acids (a.a.), respectively. The two proteins share the structured ATPase, RQC, and HRDC domains, while an exonuclease domain is present only at the N terminus of WRN. Previous crystal structures of the exonuclease domain from human  of the ATPase domain with a short linker, is unique to the RecQ family of proteins. This region folds into a winged-helix motif, a subset of the helix-turn-helix superfamily . Helix-tFigure 2 shows the co-crystal structure of the WRN RQC domain bound to a DNA duplex  was determined  was determined . First, aromatic and non-polar residues at the tip of the β-wing, key elements that WRN uses for DNA strand separation, are each replaced by polar and acidic residues in BLM. A detailed discussion of this feature is given below. Second, a BLM-specific 14-a.a. insertion (referred to as the BLM insertion) between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Third, the C-terminal residues of BLM RQC adopt a novel extended structure (referred to as the C-term extended loop) by being tightly packed against the domain core. These unique structures in BLM RQC may be associated with the preferential activity of BLM toward HJs  and BLM (B) RQC domains with those of the conventional winged-helix domains of the transcription factors ETS  of WRN RQC (A) and BLM RQC (B) is located more than 4 Å away from the bound DNA and is not involved in the direct interaction with DNA. Instead, the positively charged loop between helices α2 and α3 (the α2–α3 loop) serves as the prominent DNA binding site by interfacing with the major groove of the DNA, and the β-wing  exhibits a unique interaction with the terminus of the duplex. Considering that the fundamental role of the recognition helix is to promote sequence-specific DNA recognition, its exceptional lack of use in RQC seems essential for realizing sequence-independent helicase reactions . The RQCThe protruding β-wing within the RQC domain is also essential for WRN and BLM to prevent non-specific binding to DNA, since the β-wing exhibits steric hindrance with linear paired bases . Due to Figure 2) and BLM  forms a hydrogen bond with a backbone phosphate of the DNAs  is capped by polar (Asn1164) and acidic (Asp1165) residues  completely lack HRDC sequences . The exiFigure 6 shows the crystal structure of the WRN HRDC domain , whereas the BLM HRDC surface is largely electronegative with many acidic residues exposed to the solvent (D). The isoelectric point (pI) of BLM HRDC (5.1) is also much lower than that of WRN HRDC  is highly electronegative, and is therefore unlikely to be involved in direct DNA interaction. Consistent with these observations, neither of the purified WRN , which is six times longer than that of BLM . The long linker region of WRN is probably unstructured  that is tightly packed against the upper surface of the folding core. This extra structure increases the surface area of WRN HRDC, and assists in the formation of WRN-specific structures such as a hydrophobic pocket at the back of the domain . This hydrophobic pocket is a candidate for interaction with other proteins . The domain is constituted of two RecA-like subdomains (1A and 2A) that are characteristic of a wide variety of DNA and RNA helicases  preferentially act on DNA structures that resemble recombination and repair intermediates . Such stFigures 7A,B I present a new binding model of a BLM dimer with a HJ. This model has been constructed in silico by superimposing the BLM 640–1291 structure  were manually tilted by 21° rotation from their ideal vertical positions, to avoid steric conflicts with the two ATPase domains. The obtained model shows that the two molecules of BLM can simultaneously bind to the single HJ with no significant steric hindrance to each other. Each RQC domain binds the east or west  arm of the HJ with twofold symmetry, while the ATPase domains interact with the north and south arms. Binding of a BLM dimer to a single HJ is also suggested by a recent single-molecule visualization study , a line of basic regions (encircled by a dashed line) that traverses subdomains 1A, 2A, and Zn of the ATPase domain is observed. The lower part of these regions binds to the 3′-overhang ssDNA, suggesting a role for these regions as a DNA translocation route for the helicase reactions. In the present BLM-HJ model , the basic regions in each ATPase domain run precisely in parallel with the north or south duplexes, representing the best operational arrangement for the translocations of the two arms.In the electrostatic surface potentials of BLM 640–1291  offers several plausible explanations for the branch migration reactions undertaken by BLM. First, co-insertion of the two acidic β-wings into a small hole of the HJ enables the BLMs to catalyze simultaneous unpairing of the east and west arms of the HJ. This is reminiscent of the acidic hairpin of bacterial RuvA, a protein that also catalyzes branch migration of HJs , of each Zn subdomain is extended in parallel with the yellow or pink strand at the intersection, exposing the basic residue Arg1003 toward the unpaired 3′-nucleotides. This residue probably forms a salt bridge with the DNA backbone phosphate. Therefore, the two Zn subdomains located around the central hole seem likely to act as a guiderail for the unpaired 3′-nucleotides to move smoothly into the north-south duplexes, helping them to rapidly anneal with new partners. At the same time, the Zn subdomain would also act as a joint to adjust the relative orientation of the ATPase domain against the RQC domain.Third, the two ATPase domains would pull the north and south duplexes in opposite directions, by the conventional inch-worm mechanism . HydrolyIn summary, although a genuine BLM-HJ co-crystal structure is still lacking, the proposed binding model yields a possible HJ branch migration mechanism for BLM, in which DNA unwinding by the RQC domains and DNA annealing/translocation by the ATPase domains are effectively coordinated.Figure 8B) and archaeal Hel308  also possess a conserved β-hairpin to act as an unwinding element. However, these hairpins are located directly within the ATPase domains  and cannot be inserted into the narrow hole of the HJ, since severe steric conflicts would occur between their ATPase domains and DNA strands . For the RecQ family, separation of the unwinding module (the RQC domain) from the translocation module (the ATPase domain) is likely to be crucial to process multi-stranded DNAs such as the HJs.In contrast, WRN and BLM prepare the unwinding element in the compact RQC domain  also showed that the BLM HRDC domain is situated ∼28 Å apart from the DNA substrate and binds to the ATPase domain within the same molecule  and open planar conformations , the two HRDC domains are located in the empty spaces between the north–west and east–south arms of the HJ. Considering the electronegativity of the BLM HRDC domain surface, the HRDC domain may act as a wedge to open the HJ arms by electrostatic repulsion  bound by WRN (or BLM). Since the structure of the D-loop is comparable to that of the right half of the HJ  DNA . The G4-Escherichia coli RecQ (Deinococcus radiodurans RecQ (E. coli RecQ without DNA (Recent advances in the structural studies of WRN and BLM, in particular the discovery of the “DNA zip-slider” function of the RQC domain to catalyze strand separation, have greatly improved our understanding of WRN and BLM in terms of their preferential activities toward recombination and repair intermediates. In this paper, I have focused on the structures of WRN and BLM, but it should be mentioned that other important RecQ structures that could not be discussed here are also available, including those of oli RecQ , Deinocoans RecQ  and humaans RecQ . The strhout DNA  gave us hout DNA . The β-whout DNA , in conthout DNA .Besides their biological importance in the prevention of tumorigenesis and accelerated aging, WRN and BLM are also new targets for cancer chemotherapy . Recent The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro, Aspirin synergized with Metformin in inducing apoptosis of triple negative and endocrine-sensitive BC cells, and in activating AMPK in BC and in white adipose tissue (WAT) progenitors known to cooperate to BC progression. Both Aspirin and Atenolol added to the inhibitory effect of Metformin against complex I of the respiratory chain. In both immune-deficient and immune-competent preclinical models, Atenolol increased Metformin activity against angiogenesis, local and metastatic growth of HER2+ and triple negative BC. Aspirin increased the activity of Metformin only in immune-competent HER2+ BC models. Both Aspirin and Atenolol, when added to Metformin, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of Atenolol but not by the addition of Aspirin. Our data indicate that the addition of Aspirin or of Atenolol to Metformin might be beneficial for BC control, and that this activity is likely due to effects on both BC and microenvironment cells.Metformin can induce breast cancer (BC) cell apoptosis and reduce BC local and metastatic growth in preclinical models. Since Metformin is frequently used along with Aspirin or beta-blockers, we investigated the effect of Metformin, Aspirin and the beta-blocker Atenolol in several BC models.  Here we report about the combinatory effect of these three drugs over BC cells (triple negative and endocrine-responsive) and WAT progenitor cells in vitro and in vivo.Aspirin (Asp) and beta blockers are currently used together with Met by millions of patients with diabetes and cardiovascular diseases (CVD)1213in vitro studies18in vivo effects of Met, Asp and the beta blocker Atenolol (At) in orthotopic local and metastatic models of triple negative and HER2+ BC. Considering the known effect of Met and of Asp over immune cells20in vivo both immune-competent and immune-deficient BC models.The AMPK/mTOR pathway and the complex I of the respiratory chain are currently considered the most relevant possible targets of Met activity in BC, and both were investigated in our in vitro and in vivo12in vitro and in vivo.Our studies on Met activity towards BC and WAT progenitors, found that another biguanide, Phenformin (Phe), was significantly more active than Met in vitro, the BC cell lines MDA-MB-436 and ZR-75-1 were treated with different drug combinations including 10 mM Asp, 500 uM At, 10 mM Met, and 2 mM Phe for 48 and 72 hours. Results of preliminary studies investigating the in vitro activity of different concentrations of Met and Asp are reported in in vitro under these culture conditions. Cell proliferation data are reported in To evaluate the inhibitory effect of Met along with Asp or At on human BC cells We observed an increased level of p-AMPK over total AMPK after 24 h culture in both MDA-MB-436  and ZR-7We treated WAT progenitor cells with 20 mM Met and 0.5 mM Phe for 16 h and analyzed both AMPK and mTOR activation. The combination of Met with At or Asp further activates AMPK as compared to Met alone, and Met+Asp treatment showed the highest phosphorylation status of the protein . In ordeWe also analyzed the activation of the downstream mediators of mTOR pathway, 4E-BP1 and p70S6K. As shown in One of the primary cellular targets of Met and Phe is the mitochondrionin vivo to the activity of Met against local and metastatic BC growth in both HER2+ and triple negative models. In the same immune-deficient NGS mice, the addition of At significantly increased the activity of Met against local and metastatic HER2+ .in vivo preclinical studies are higher than those used in human diabetic patients, the current dosage is in the range that was previously observed to prolong lifespan in miceMet, Asp and beta blockers are currently used worldwide by millions of patients with diabetes and cardiovascular diseases (CVD). In the past few years, the use of Met has been associated in preclinical models and in clinical retrospective studies in diabetic patients with a reduced incidence and severity of several types of cancer, including BC123456789101112131415et al.Three large observational studies have shown a survival benefit in BC patients using Asp or other non steroidal anti inflammatory drugs NSAIDs,2627. Sim26282930The choice to study together Met and Asp was also due to the increasing evidence that molecular targets of Met and Asp are multiple and not only restricted to AMPK. Also, cancer and microenvironment cells are differently targeted (in multiple pathways) by Met and Asp.in vitro and reduces angiogenesis, local and metastatic BC progression in vivo by targeting both cancer and microenvironment WAT cells17in vitro and in preclinical local and metastatic BC models the association of Met with Asp and of Met with a beta blocker. To minimize the use of animals, in preclinical studies we focused on models of the most aggressive types of breast neoplasia, namely triple negative and HER2+ diseases. At was chosen as a beta blocker for its safety profile in long-term administration along with Met. To the best of our knowledge, this is the first report investigating these drug combinations in BC cells in vitro and in orthotopic local and metastatic BC models of triple negative and HER2+ disease  was significantly more active than Met, both in vitro and in vivo. However, the safety profile of Phe renders its possible future development as an anticancer drug unclear. Data obtained with Phe are reported here as a reference of how the addition of Asp or At can increase the effect of Met in vitro and in vivo.In our study on Met activity against BC AMPK, which is activated by Met and by Asp in neoplastic cells34in vitro in BC and in WAT-derived progenitor cells. Adding Asp to Met increased the inhibition of complex I of the respiratory chain both in triple negative and in endocrine-responsive BC cells. On the other hand, the addition of At to Met had no effect. At variance, in vitro studies on WAT progenitor cells, the addition of At to Met increased the inhibition of complex I at levels similar to those observed when Asp was added to Met.As recent data underlines that also the mitochondrion might be among of the primary cellular targets of Met and Phe18in vitro results showing an additive effect of Asp to Met, in immune-deficient mice Asp did not further increase to the activity of Met against angiogenesis, local and metastatic BC growth in both HER2+ and triple negative models. In contrast, in the same immune-deficient models At significantly increased the activity of Met against local and metastatic BC, both HER2+ and triple negative preclinical models. In immune-competent models, the addition of Asp increased the activity of Met against local and metastatic HER2+ BC growth, as did the addition of At. Notably, the addition of At to Met led to an inhibition of local and metastatic BC growth similar to the data observed after the administration of the more toxic biguanide, Phe.We then investigated whether Asp or At might add to the activity of Met against angiogenesis, local and metastatic BC growth in triple negative and in HER2+ preclinical models. Despite the in vitro studies . WAT EPC and ASC purity was always greater than 96% with a recovery of 70-80%.Samples were continuously cooled to 4 °C and a forward scatter pulse height and side scatter analyses were performed to exclude cell clusters and doublets. A two way cell sorting procedure was performed with a 140 um nozzle with a 5.5 PSI pressure, and with an events rate of 1,000-1,500 events per second, using a sort pure mode. Samples were collected into sterile polypropylene tubes containing 20% FBS, and used for Human MDA-MB-436 (triple negative), and ZR-75-1 (ER+ PR+ HER2-), BC cells were purchased from the American Type Culture Collection , tested and authenticated by Stem cell Elite ID (Promega) in January 2014 and cultured as previously described352 humidified incubator (n = 5 per study arm). Cells were collected and analyzed by flow cytometry for apoptosis 48 and 72 hours for BC cells and for 16 hours for WAT after drug supplementation. The nuclear staining Syto16 was used to discriminate between DNA containing cells and cell debris, which were excluded by gating. 7-Amino-actinomycin D (7-AAD) was used to determine the viability status of the cells1736Cell lines and sorted WAT progenitors were plated 24 and 16 hours respectively before culture in the presence of vehicle or of different concentrations and combinations of Met, Phe, Asp or At (Sigma) in a 37 °C, 5% COAMPK and phospho-AMPK, as well as phospho-mTOR and mTOR protein expression were examined in BC cells and purified WAT progenitors maintained under different treatment conditions. Briefly, MDA-MB-436 and ZR-75-1 human BC cells and purified human WAT progenitors were treated, respectively, with 10 mM Met or 2 mM Phe for 24 h (BC cells) and 20 mM Met or 0.5 mM Phe for 16 h (WAT) alone or in combination with 10 mM Asp or 500 μM At. Cells were lysed in ripa buffer containing phos-STOP solution (Roche LifeScience). Lysates were then centrifuged at 12,000 rpm at 4 °C for 20 minutes to remove any cell debris. Protein concentrations were determined using Bradford assay . Total tissue lysate were then heated at 90 °C and resolved on a 4–20% gradient Mini-Protean TGX gels (Bio-Rad). Proteins were subjected to PAGE, electrotransferred to nitrocellulose membranes (Amersham Biosciences), incubated in blocking buffer (5% Non-fat dry milk in PBS/0.1% Tween) followed by overnight incubation with the indicated antibodies in PBS-Tween containing 5% (w/v) BSA for 16 h at 4 °C. Antibodies used for western blotting were anti-phospho-AMPK, anti-AMPKa1, anti-phospho-mTOR, anti-mTOR, anti-phospho-4E-BP1, anti-4E-BP1, anti-phospho-p70S6K and anti-p70S6K  and anti-actin . Detection was performed using anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody and chemiluminescence with ECL reagent (Amersham Biosciences).As NAD+/NADH is another possible target of biguanides activity, NAD(H) was analyzed both in CD45-CD34+ WAT-derived progenitor cells and BC cell lines using the fluorescent NAD/NADH detection kit  following the manufacturer’s protocol.Experiments were carried out on immune-deficient NOD SCID IL2RG null (NSG) and immune-competent FVB female mice, 6 to 9-weeks-old. NSG mouse strain were bred and housed under pathogen-free conditions in our animal facilities at the European Institute of Oncology–Italian Foundation for Cancer Research (FIRC) Institute of Molecular Oncology . Mice were expanded from breeding pairs kindly donated by Dr. Leonard Shultz, The Jackson Laboratory, Bar Harbor, ME. FVB mice were purchased from Harlan Laboratories .Experiments involving animals have been done in accordance with the approved guidelines. In particular, studies were carried out in accordance with the Italian Laws (D.L.vo 116/92 and following additions), which enforce EU 86/609 Directive . Mice have been housed accordingly to the guidelines set out in Commission Recommendation 2007/526/EC - June 18, 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes.Until March 28, 2014, the Italian legislation did not require a specific ethical review process for all the experiments involving animals. A central (Government) review was required only for particular species  or for experiments done without anesthesia or that will or may cause severe pain. In the other cases, only a notification of the experiments to the Ministry of Health was required. Accordingly, the project was notified to the Ministry of Health.6 cells/13 μL. Briefly, after mechanical dissociation with gentle MACS Dissociator , tumors were placed in culture medium  containing collagenase (Sigma) and hyaluronidase (Sigma), and digested for 2 h at 37 °C. After lysis of the red blood cells, a single cell suspension was obtained by sequential dissociation of the fragments by gentle pipetting for 3 min in 0.25% trypsin (EuroClone), and then 3 min in 5 mg/ml dispase (StemCell technologies) plus 0.1 mg/ml DNase I  followed by filtration through a 100-mm mesh. Prior to injection in female FVB recipient mice, tumor cells were mixed with 5 μL of Matrigel (BD) and 2 μL of trypan blue solution (Sigma) and maintained on ice until injection. Surgical procedure was performed in aseptic conditions under a laminar flow hood. Mice were anesthetized with 2.5% 2-2-2-tribromoethanol , laid on their backs, and injected with 20 μL of cell suspension in Matrigel directly in the fourth mammary fad pad through the nipple with a Hamilton syringe. Tumor growth was monitored weekly using digital calliper, and tumor volume was calculated according to the formula: L X W2/2 = mm3, where W represents the width and L the length of the tumor mass.To produce orthotopic primary human tumors in NSG mice, MDA-MB-436 BC cells were injected in the mammary fat pad as we previously described36Tumor resection (mastectomy) was done as previously describedad libitum through their drinking water, starting the day after tumor injection. Daily fluid intake of fluids was monitored and fresh drugs were administered twice a week. Drug dosage in drinking water  was based on literature data and was associated with no toxicity and no significant changes in mouse weight, and circulating levels of glucose, cholesterol and triglycerides. In mice treated with Phe, drinking water was supplemented with 5% sucrose to increase palatability.Mice (n = 5 per study arm) were treated with vehicle (water) or different drugs Formalin-fixed paraffin-embedded samples were sectioned 5 μm thick on poly-L-lysine slides . After deparaffination and re-hydratation, antigen retrieval was obtained by heating the sections at 95 °C in 10 mM Sodium Citrate Buffer added with 0.05% Tween 20  for 30 minutes. Sections were then permeabilized with 1% Triton X-100 (Sigma) in PBS for 30 min at room temperature. A blocking step was performed for 30 minutes using PBS/10% BSA (Sigma). Overnight incubation at 4 °C was performed with primary antibodies diluted in PBS/BSA 10%: rabbit polyclonal antibody anti-mouse CD31  and mouse monoclonal antibody anti-α-SMA . Sections were then incubated with secondary antibodies for 1 hour at room temperature . Slides were stained with 4′, 6-diamino-2phenylindole  and mounted with Fluorashield (Sigma). Negative controls without primary antibodies were conducted for all reactions.2 (about 228 × 228 μm2), whereas the z-step between two images within a stack is 0.8 μm, resulting in a voxel size of 0.223 × 0.223 × 0.8 μm3. The acquisition parameters, the microscope, and all the detectors were controlled by means of LAS-AF software (Leica Microsystems). Quantification of blood vessel density and mean area of lumina was obtained with ImageJ analysis software.A preliminary scan of sections was done using a Leica SP5 II confocal microscope  by means of a 20× oil objective in “tile scan acquisition mode”, such that all the section surface was imaged. The optical configuration was set-up as described below. For detailed confocal imaging, sequential z-stacks were collected by the Leica confocal microscope using a 40× oil immersion objective with a numerical aperture of 1.3. Fluorochromes were excited using a 405-nm diode laser for DAPI, an argon 488 laser for Alexa Fluor 488 and a 561 diode laser for Alexa Fluor 555. Detector slits were configured to minimize the cross-talk between channels and to maximize the fluorescent signal arising from the sample: PMT1, 407 to 485 nm (DAPI); PMT2, 492 to 556 nm (Alexa Fluor 488); PMT3, 579 to 684 nm (Alexa Fluor 555); the cross-talk is also limited by the fact that the stacks were collected in “between frame mode”: the blue and red fluorescence  were collected simultaneously while the green signal was obtained alone. Every single image of the collected z-stacks is 1024 × 1024 pixelU test of Mann–Whitney. All reported P values were 2 sided.The Shapiro–Wilk test was used to assess for normality. Considering that the very large majority of data were not normally distributed, statistical comparisons were carried out using the nonparametric How to cite this article: Talarico, G. et al. Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells. Sci. Rep.6, 18673; doi: 10.1038/srep18673 (2016).


Purpose. Since recent reports suggest that Hashimoto thyroiditis (HT) may be associated with IgG4-related disease, we aimed to find out whether the measurement of serum IgG4 allows for the identification of distinct types of HT, with different clinical, sonographic, and serologic characteristics. Methods. The group studied consisted of 53 patients with HT and 28 healthy individuals who underwent thyroid ultrasonography and body composition analysis. Serum concentrations of IgG4, TSH, anti-peroxidase antibodies (TPOAb), anti-TSH receptor antibodies, TNF-α, TGF-β1, Fas Ligand, TRAIL, and chemokines  were measured by ELISA or radioimmunoassay. Results. The group with IgG4 level >135 IU/ml accounted for 32.5% of the patients. The signs of fibrosis were present in 27.0% of the high-IgG4 patients and in 9.1% of the normal-IgG4 group. The patients with elevated IgG4 required higher doses of L-thyroxine and had significantly lower level of TPOAb (P=0.02) than the non-IgG4-HT individuals and higher TNF-α level in comparison with the controls (P=0.01). Conclusions. Our results suggest that the measurement of serum IgG4 allows for an identification of patients with more rapid progression of HT, requiring higher doses of L-thyroxine. Low TPOAb level and the absence of coexisting autoimmune diseases may suggest distinct pathomechanism of this type of thyroiditis. Hashimoto's thyroiditis —an autoimmune disease described for the first time more than a hundred years ago , 2—is reTherefore, in the present study, we aimed to find out whether the measurement of serum IgG4 allows for an identification of distinct types of HT, with different clinical and serologic characteristics. To test this hypothesis, we compared thyroid function, ultrasonographic findings, and the levels of anti-thyroid antibodies, proinflammatory cytokines, and apoptotic markers in patients with HT and low/normal and elevated levels of IgG4.The study comprised 81 subjects, including 53 patients with diagnosed HT  and 28 healthy individuals , who had never been treated for any autoimmune or malignant diseases, matched for age and gender. None of the participants was suspected/diagnosed of having any IgG4-RD (except IgG4-HT). All patients and controls gave their informed consent to participate in the study before enrolment. The protocol was approved by the local ethics committee .All the patients underwent thyroid ultrasonography to establish thyroid volume, echogenicity, vascularity (with Power Doppler method), signs of fibrosis, and calcification.Biospace, Korea), which allows measurement of body mass, total body water (TBW), fat and free fat mass, skeletal muscle mass (SMM), BMI, percentage of body fat (PBF), and basic metabolic rate (BMR). The reliability of an estimated BMR was confirmed by the calorimetric method performed in 5 patients with HT. Differences between these results were smaller than 5%. The waist to hip ratio (WHR) was calculated as waist circumference in centimeters divided by hip circumference in centimeters.A body composition analysis was performed using the medical body analyzer INBODY 220 (α) levels were also determined by commercial immunoassays . The concentration of anti-TSH receptor antibodies (TRAb) was measured by a commercial radioimmunoassay . IgG4 was estimated using Human IgG4 Platinum ELISA . The following chemokines and apoptotic markers were also measured: Human Fas Ligand/TNFSF6, Human TRAIL/TNFSF10, Human TGF-β1, and chemokines CXCL9/I-TAC, CXCL10/IP-10, and CXCL11/I-TAC .Blood samples were collected from the antecubital vein between 7:30 and 8:30 am, after an overnight fast, in order to avoid diurnal variations. Serum TSH concentration was measured using an enzyme-linked immunoassay . Anti-peroxidase antibodies (TPOAb) and tumor necrosis factor-alpha  and IBM SPSS Statistics 21.0  Software were used for the statistical analysis. Prior to the analysis, data were tested for normality of distribution using Kołmogorow-Smirnow test with Lilliefors correction and Shapiro-Wilk test. Differences between the groups were compared by Mann-WhitneyP < 0.001). Furthermore, the IgG4-HT patients required higher L-thyroxine dose in order to reach euthyreosis in comparison with the non-IgG4-HT group (The HT group was divided into IgG4-HT (IgG4 > 135 IU/mL) and non-IgG4-HT (IgG4 < 135 IU/mL), depending on the patient's IgG4 level. The group with IgG4 >135 IU/mL accounted for 32.5% of all HT patients. The percentage of male patients was 7.5% in the non-IgG4-HT and 18.2% in the group with a high level of IgG4. Mean HT duration was 4.9 ± 4.6 years in non-IgG4-HT group and 2.5 ± 1.96 in IgG4-HT group  (P = 0.001) in comparison with the control group (P = 0.001), whereas TSH values did not differ markedly between the groups studied , as well as a comparable percentage of thyroid calcifications (9.1% versus 13.9%). The signs of fibrosis were present in 27.0% of IgG4-HT patients in comparison with 9.1% of the non-IgG4-HT group, but the difference was not significant. The non-IgG4-HT patients had a lower number of thyroid nodules compared with IgG4-HT patients ( = 0.02) , and 66. studied .P = 0.02) than the non-IgG4-HT individuals (P = 0.02), whereas TRAb concentrations were similar in both groups with HT , BMI (P = 0.02), WHR (P = 0.01), and fat mass (P = 0.02) in comparison with the healthy subjects.As shown in P = 0.001) and in the control group; however, the difference between the IgG4-HT and healthy individuals was not significant. Serum TNF-α level was markedly higher in the IgG4-HT in comparison with the controls (P = 0.01). There was also a trend towards higher TGF-β1 concentration in the IgG4-HT, as compared with the non-IgG4-HT group (P = 0.05), whereas the levels of other chemokines and apoptotic markers (Fas Ligand and TRAIL) were similar in all groups studied  proteins are expressed in normal thyrocytes, but their concentrations are insufficient to initiate apoptosis –37. ChanThe main limitation of our study is the fact that the division into the IgG4-TH and non-IgG4-HT groups was made only on the basis of serum IgG4 level, without immunohistochemical analysis. However, we aimed to find out whether a single measurement of IgG4 concentration may help us to identify patients with a distinct form of thyroiditis.In conclusion, our results suggest that the measurement of serum IgG4 allows for an early identification of patients with more rapid progressing and destructive form of HT, who require higher doses of L-thyroxine. A relatively low TPOAb level and the absence of coexisting autoimmune diseases may suggest a distinct pathomechanism of this type of thyroiditis. It should be mentioned that measuring serum IgG4 level may be a clinically useful marker for differentiating two distinct immunophenotypes of the disease. Furthermore, the identification of HT patients with high IgG4 levels might provide new therapeutic possibilities since there is good evidence that the use of oral glucocorticoids in patients with IgG4-RD results in a considerable clinical improvement.


Clostridium difficile infection, the negative impact on microbiota, and undertreatment risks. In this paper, the principle of and strategies for paediatric antimicrobial stewardship (AS) programs, the effects of AS interventions and the common barriers to development and implementation of AS programs are discussed.Antibiotics are among the drugs most commonly prescribed to children in hospitals and communities. Unfortunately, a great number of these prescriptions are unnecessary or inappropriate. Antibiotic abuse and misuse have several negative consequences, including drug-related adverse events, the emergence of multidrug resistant bacterial pathogens, the development of Over the last few years, there have been significant shortages in the development and availability of new antibiotics; therefore, the implementation of strategies to preserve the activity of existing antimicrobial agents has become an urgent public health priority. AS is one such approach. The need for formal AS programs in paediatrics was officially recognized only recently, considering the widespread use of antibiotics in children and the different antimicrobial resistance patterns that these subjects exhibit in comparison to adult and elderly patients. However, not all problems related to the implementation of AS programs among paediatric patients are solved. The most important remaining problems involve educating paediatricians, creating a multidisciplinary interprofessional AS team able to prepare guidelines, monitoring antibiotic prescriptions and defining corrective measures, and the availability of administrative consensuses with adequate financial support. Additionally, the problem of optimizing the duration of AS programs remains unsolved. Further studies are needed to solve the above mentioned problems.In paediatric patients, as in adults, the successful implementation of AS strategies has had a significant impact on reducing targeted- and nontargeted-antimicrobial use by improving the quality of care for hospitalized patients and preventing the emergence of resistance. Considering that rationalization of antibiotic misuse and abuse is the basis for reducing emergence of bacterial resistance and several clinical problems, all efforts must be made to develop multidisciplinary paediatric AS programs in hospital and community settings. Antibiotics are among the drugs most commonly prescribed to children in hospital and community settings –3. It haClostridium difficile infection [Antibiotic abuse and misuse have several negative consequences. The incidence of drug-related adverse events is significantly increased in instances of improper antibiotic use , 16. Mornfection , 18 and nfection , 20. Inanfection .In the last 15 years, significant deficiencies have occurred in the development and availability of new antibiotics able to combat instances of emerging resistance ; therefoFor several years, AS efforts were focused on adult populations. A very successful and evidence-based AS program in adults was the Transatlantic Taskforce on Antimicrobial Resistance, established by American President Barack Obama and President of the European Union (EU) Fredrik Reinfeldt from Sweden . RecommeThe need for formal AS programs in paediatrics was officially recognized only recently, considering the widespread use of antibiotics in children and the different antimicrobial resistance patterns that these subjects exhibit in comparison to adult and elderly patients . In the From a theoretical point of view, to be maximally effective in rationing antibiotic prescriptions, AS strategies and protocols must account for several elements strictly related to the antibiotic prescription but should also consider the need for continuous access to expertise in clinical pharmacology and infectious diseases and for transparent monitoring of antibiotic use Table . PracticWith regards to the problem of selection of the appropriate antibiotic regimen for each child, it must be considered that for many paediatric infectious diseases, one of a limited number of bacterial pathogens is usually the etiologic cause and that, lacking direct microbiological evidence, the antibiotic of choice to treat each disease is the one active against the most probable infectious agent. To solve the problem of the delay with which culture results indicate the etiology of a given disease, the use of rapid diagnostic tests must be considered . MoreoveThe questions of the appropriate dosage and timing of antibiotic administration can be resolved by restricting formulary for empiric treatment to 48–72 h to permit a re-evaluation of the prescribed therapy and a decision as to whether the therapy should be continued, modified or suspended. However, in planning AS programs, particular attention must be paid to drugs that have been found to be predictive of receiving paediatric AS recommendations. In their study, Goldman et al. reported that third-generation cephalosporins and clindamycin were the antimicrobials with the highest predictive probability of warranting an AS program recommendation, whereas linezolid had the lowest probability . MoreoveDevelopment of local AS teams including experts from different fields is considered essential to ensure adequate development of AS programs and continuously updated information useful for avoiding antibiotic misuse and abuse . InfectiImplementation of paediatric AS programs is easier in the hospital setting, where technical and human resources are more plentiful and easily available than in the community. Hyun et al. have listed potential strategies to promote AS programs in community-based settings, highlighting the need for identification of local physician champions and potential resources  as well as the great difficulties in developing common guidelines for antibiotic use and control .Although AS programs should account for all the aspects previously discussed, in most previously implemented programs, the development and implementation were only partial. A relevant example is provided by the study published by Bryant on behalf of the Australasian Stewardship of Antimicrobials in Paediatrics group . FourteeSeveral evaluations of paediatric AS programs have shown favourable outcomes, including a reduction in antibiotic prescriptions and lower costs Table . Most ofAgwu et al. evaluated a World Wide Web-based antimicrobial restriction program at a 175-bed, tertiary care paediatric teaching hospital . The proMetjian et al. performed a prospective observational study in which data were collected on clinician's requests for targeted antibiotics and the interventions made by an AS program . During Di Pentima et al. reported the results of an AS program carried out in a paediatric teaching hospital in Tennessee, USA . An autoHowever, all of these studies were single-centre studies and were carried out over a limited period of time. For these reasons, they can be criticized because they are subject to multiple biases, including publication bias, a failure to consider secular trends in antibiotic use and a lack of comparisons with antibiotic prescriptions in similar hospitals without AS programs. The study by Hersh et al. overcame these limits, confirming the effectiveness of AS programs in children and the need for their systematic use in all paediatric hospitals . These aOutpatient AS interventions are uncommon. However, the available data seem to indicate that AS can be effective in reducing antibiotic misuse in community settings as well, although the results are less impressive than those obtained in hospitals. From 1998–2003 Finkelstein et al. conducted a controlled, community-level, cluster-randomized trial in 16 non-overlapping Massachusetts communities . During More recently, Gerber et al. performed an outpatient AS interventional trial that involved 162 clinicians and consisted of a 1-h on-site clinician education session followed by one year of personalized quarterly audits of and feedback on prescriptions for acute respiratory tract infections or usual practice . Broad-sOne of the most successful interventions to reduce antibiotic prescriptions among children in outpatient settings has been performed in Sweden . Since 1Despite a growing evidence base supporting the value of paediatric AS programs, a number of well-recognized barriers hinder their development and growth. A lack of knowledge regarding the fundamental rules on which antibiotic prescriptions are based remains one of the most important problems in the implementation of AS programs. A recent study by Bowes et al. highlighted several challenges that paediatric practitioners face with respect to the knowledge of and approaches to prescribing antimicrobials . In partAll of these findings provide evidence that educational programs specifically devoted to illustrate the rules for rational use of antibiotics are needed before and during AS program implementation. Guidelines and protocols can be useful in this regard, but to be most effective, these should be shared between all specialists directly or indirectly involved in the prescription of antibiotics, including pharmacists, microbiologists, and infectious disease specialists. Shared suggestions for antibiotic administration can permit a facility to overcome the risk of any new intervention being perceived by front-line physicians as interfering with their routine practice and being questioned. Unfortunately, creation of a team specifically dedicated to educate providers and monitor antibiotic use to correct possible mistakes is not always possible because it requires substantial financial support. A lack of funds can cause difficulties in the implementation of an AS program. Additionally, a lack of technical resources may further limit the implementation and effectiveness of AS programs. Daily surveillance of antibiotic prescriptions and control of the concordance between actual and suggested uses, including evaluations of dosing and length of administration, are largely facilitated by the availability of computerized programs. Availability of hardware and software needed for monitoring first requires financial support and administrative consensus, which is not easily obtained, even though cost saving is routinely achieved by reducing unnecessary antibiotic use and decreases in the pharmacy budget usually cover the expenses for AS program implementation within a few months .As recently indicated by the updated IDSA guidelines for implementing an AS program , passiveAmong adult patients, the successful implementation of AS strategies have had a significant impact on reducing targeted- and nontargeted-antimicrobial use, improving quality of care of hospitalized patients and preventing the emergence of resistance. Similar results have also been reported in paediatric patients, although there has been significantly less widespread implementation of AS programs among children. However, not all of the problems related to implementation of AS programs in paediatric settings are solved. The most important remaining problems involve educating paediatricians, creating a multidisciplinary interprofessional AS team able to prepare guidelines, monitoring antibiotic prescriptions and defining corrective measures, and the availability of administrative consensuses with adequate financial support. Additionally, the problem of optimizing the duration of AS programs remains unsolved. It is not clear whether such programs must be maintained continuously or can instead be implemented until rationalization of antibiotic use is obtained and re-initiated when significant variations in antibiotic use are needed. Some reports, including the recent experience of Gerber et al. in a community setting , seem to


For each SL pair, a confidence score was calculated by integrating individual scores derived from different evidence sources. We also developed a statistical analysis module to estimate the druggability and sensitivity of cancer cells upon drug treatments targeting human SL partners, based on large-scale genomic data, gene expression profiles and drug sensitivity profiles on more than 1000 cancer cell lines. To help users access and mine the wealth of the data, we developed other practical functionalities, such as search and filtering, orthology search, gene set enrichment analysis. Furthermore, a user-friendly web interface has been implemented to facilitate data analysis and interpretation. With the integrated data sets and analytics functionalities, SynLethDB would be a useful resource for biomedical research community and pharmaceutical industry.Synthetic lethality (SL) is a type of genetic interaction between two genes such that simultaneous perturbations of the two genes result in cell death or a dramatic decrease of cell viability, while a perturbation of either gene alone is not lethal. SL reflects the biologically endogenous difference between cancer cells and normal cells, and thus the inhibition of SL partners of genes with cancer-specific mutations could selectively kill cancer cells but spare normal cells. Therefore, SL is emerging as a promising anticancer strategy that could potentially overcome the drawbacks of traditional chemotherapies by reducing severe side effects. Researchers have developed experimental technologies and computational prediction methods to identify SL gene pairs on human and a few model species. However, there has not been a comprehensive database dedicated to collecting SL pairs and related knowledge. In this paper, we propose a comprehensive database, SynLethDB ( Two genes are said to be in a synthetic lethality (SL) relationship if a perturbation of either gene alone is not lethal but perturbations of both genes lead to cell death or a dramatic decrease in cell viability . For exaDrosophila melanogaster models , species, diseases, references to PubMed and other relevant information, so that users can access the detailed information to explore the SL gene pairs. Furthermore, to prioritize SL pairs according to their reliability, we developed a scoring scheme to compute an integrative confidence score for each SL pair based on the annotations, as described in the following section.In summary, the current version of SynLethDB contains 34 089 SL pairs that comprise 19 952 of The SL pairs in our database were collected from different types of sources, including biochemical assays, other related databases, computational predictions and text mining results. Furthermore, biochemical assays were based on different experimental technologies and platforms, such as genetic mutation and transfection, RNA interference and drug inhibition. As multiple types of evidence contribute to the identification of a specific SL, an integrative confidence score combining scores from all these evidence sources can give an overall estimation of the reliability of an SL interaction. In principle, we assume that (i) experimental evidence contributes more significantly to the confidence score than evidence derived from predictive algorithms or text mining, and (ii) the SL pairs supported by more evidence sources should be given higher confidence scores than those supported by less evidence sources.quantification and integration, to compute the confidence scores. A large number of SL pairs collected from wet-lab experiments and other related databases have only qualitative annotation evidence (such as ‘high-throughput’ or ‘low-throughput’), or technological descriptions about the wet-lab experiments (such as ‘shRNA screening’ or ‘mutation’), hence the quantification step is necessary to assign quantitative scores to those SL pairs before the calculation of integrative scores. Similar to the scoring scheme for protein–protein interactions (PPI) proposed by Cao et al. (1 − 0.5) = 0.875. Note that the probability disjunction formula has been frequently used to calculate combined scores in the case that multiple pieces of evidence exist, such as in STITCH  supporting a specific SL pair, we adopted the probability disjunction formula to combine the individual scores as follows:n STITCH  and ComPn STITCH .integration step, we introduced weight factors to reflect the importance of different types of evidence. To obtain a normalized score between 0 and 1, such that a score closer to 1 represents higher confidence, we computed the normalized weighted sum as:S represents the integrative confidence score; m, d, p and t are the weight factors of biochemical experiment, other related databases, computational prediction and text mining-based evidence; sm, sd, sp and st are corresponding individual scores. Following the convention that evidence from biochemical experiments is the most reliable, followed by other related databases and in silico predictions, and text mining-based evidence is the least reliable, we set the weight factors m, d, p and t to 0.8, 0.5, 0.3 and 0.2, respectively.In the Although a perturbation of an SL pair via genetic mutation or RNAi inhibition can induce cell death with a high probability, it is likely that only low sensitivity or even no lethal response upon drug treatments can be observed. A reason may be that the proteins encoded by the SL parters are not accessible to drug molecules (i.e. lack of druggability), or their biological functions are not completely blocked by small drug molecules (i.e. low efficacy). Insufficient response to drug treatments could hinder the practical application of the SL concept to anticancer drug design.A and B, a Wilcoxon rank sum test can be conducted to examine if inhibiting gene B by drugs yields significant drug sensitivity levels in samples in which gene A is inactive (or overactive) than in the rest of the samples. It is worth noting that such a statistical test was also used by the DAISY method to detect SL pairs from somatic copy number alterations and shRNA essentiality screening data  , HomoloGstatistical analysis of drug sensitivity functional module to test the druggability and efficacy to drugs targeting SL partners based on the collected large-scale drug sensitivity data sets. For each SL pair, one click can launch the statistical analysis procedure and the statistical significance  will be calculated. To facilitate data interpretation, graphical representations with interactive features, such as scatter plots, statistical boxplots and scatter plots, are employed. In these graphical plots, drug names, sensitivity values and cancer cell lines are interactively displayed. Also, the drugs targeting the SL partners of interest can be viewed via the drug-SL partner interaction query functionality. All displayed drugs are linked to the PubChem database  is helpful for understanding the molecular mechanisms of SL interactions, we carried out gene set enrichment analysis to find statistically significant pathways and GO (gene ontology) functional annotation terms, based on the subset of genes constituting SL relationships with each specific gene. For the identified pathways and GO terms, links to external databases, such as KEGG (Furthermore, as  as KEGG , Reactom as KEGG  and Gene as KEGG , are proIn this paper, we proposed SynLethDB, a comprehensive database of synthetic lethality. SL pairs were collected from multiple sources, including biochemical assays, other related databases, computational predictions and text-mining outputs for five species. To extend the coverage of SL gene pairs, we adopted text mining tools to analyze the PubMed literature related to synthetic lethality. To facilitate the data interpretation and evaluation, we developed useful functional modules such as orthology search, query and filtering, statistical analysis on drug sensitivity and gene set enrichment analysis, etc. As the first comprehensive database dedicated to synthetic lethality, which is an emerging anticancer strategy promising to be selective and sensitive, SynLethDB can be a valuable resource to facilitate the discovery of new anticancer drug targets.In future, we will expand the coverage of data types and species, on the basis of a rapidly increasing numbers of studies focused on SL screening and sensitivity analysis of cancer cells to drugs. We will continuously increase the number of manually curated SL pairs to ensure the reliability of data, and build a gold standard for human SL, which would be very helpful for biomedical research community in validating and evaluating results produced with both experimental and computational approaches. In addition, we will incorporate new SL pairs from other sources, such as more computational predictions and text mining results, to complement the manual curations.Furthermore, it has been realized that the cellular response of cancer cells to drug treatments depends strongly on the genetic context, such as spectrum of mutations, copy number alterations and epigenetic modifications . We will


It has come to the publisher’s attention that in the original article , Table 3In addition, the authors noticed errors in certain reference citations within the discussion section following the publication of 1]. The section of text affected has now been amended in the original article [. The secThe correct reference for ‘Yirgalam’ is: Markos E, Worku A, Davey G. Adherence to ART in PLWHA at Yirgalem hospital, South Ethiopia. Ethiop J Health Dev. 2008;22(2):174–9.The correct references for ‘Jimma’ are: Amberbir A, Woldemichael K, Getachew S, Girma B, Deribe K. Predictors of adherence to antiretroviral therapy among HIV-infected persons: a prospective study in Southwest Ethiopia. BMC Public Health. 2008;8:265; and Tiyou A, Belachew T, Alemseged F, Biadgilign S. Predictors of adherence to antiretroviral therapy among people living with HIV/AIDS in resource-limited setting of southwest Ethiopia. AIDS Res Ther. 2010;7:39.The correct references for ‘Nigeria’ are: Bello SI. HIV/AIDS patients’ adherence to Antiretroviral therapy in Sobi specialist hospital, Ilorin Nigeria. J Adv Sci Res. 2011;2(3):52–7; and Erah P, Arute J. Adherence of HIV/AIDS patients to antiretroviral therapy in a tertiary health facility in Benin City, Nigeria. Afr J Pharm Pharmacol. 2007;2(7):145–52.http://ir-library.ku.ac.ke/handle/123456789/1725.The correct reference for ‘Kenya’ is: Anthony N. Factors that influence non-adherence to antiretroviral therapy among HIV and AIDs patients in central province, Kenya, 2011.