PSI+]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [DOI:http://dx.doi.org/10.7554/eLife.04288.001 Proteins must fold into specific shapes to work inside cells, and the misfolding of proteins is associated with a growing number of diseases. For example, prions are misfolded proteins that form insoluble aggregates called amyloids. These aggregates are not easily destroyed and can cause other nearby proteins to misfold and join the amyloid. This process of amyloid assembly leads to progressive diseases such as mad cow disease, Huntington's disease, Alzheimer's disease, and Parkinson's disease, which are collectively known as amyloidoses.A series of biological pathways called the proteostasis network control protein integrity in a cell. Under normal conditions or even mildly stressful conditions—such as at slightly increased temperatures—the proteostasis network is able to prevent proteins from misfolding. However, if a cell is placed under lots of stress this network may become overwhelmed and misfolded proteins can accumulate. To date, the proteostasis network has not been linked to the clearance of amyloids.A protein called Sup35, which is found in budding yeast, can exist as two different prion forms. Previous studies have shown that briefly heating the yeast cells can ‘cure’ the so-called ‘weak’ form of the prion. The ‘strong’ prion form, however, was thought to be unaffected by elevated temperature. These previous studies had only tested yeast cells that had been dividing for a few generations; it was unknown if cells that had been dividing for longer might respond differently.Klaips et al. found that a protein called Hsp104—which helps to fold proteins properly—can break down the amyloid aggregates. This protein is normally only present in small amounts, but heating causes the levels of Hsp104 to rise. Klaips et al. found that the extra Hsp104 protein associated with the aggregates and led to their disassembly. When Hsp104 was prevented from associating with the prions, the aggregates were not cured even if high levels of Hsp104 were present in the cell.When budding yeast form new cells, a daughter cell ‘buds’ off from the mother cell. Klaips et al. found that mother cells exposed to heat retain most of the Hsp104 when the cell divides, and this retention allowed Hsp104 to accumulate to a level required for the breakdown of amyloid aggregates. Therefore, under normal conditions, amyloids persist because cell division keeps the amount of Hsp104 below this threshold.Previously it had been thought that the physical characteristics of amyloids accounted for their resilience in the face of the cell mechanisms designed to counteract protein misfolding. However, Klaips et al. show that the balance of the different mechanisms involved in proteostasis can be manipulated to create environments where amyloids are either created and maintained or destroyed. Targeting these mechanisms could therefore present new treatment options for amyloidosis.DOI:http://dx.doi.org/10.7554/eLife.04288.002 The proper folding of proteins is essential to cellular homeostasis, and an extensive collection of protein quality control (PQC) pathways, known as the proteostasis network, has evolved to protect nascent and metastable proteins from misfolding and to reactivate or remove proteins that have already misfolded . The PQCCaenorhabditis elegans, mice, Drosophila, yeast, and tissue culture-cell models of amyloidoses reduces proteotoxicity  at elevated temperature in comparison with [PSI+]Strong, whose propagation is unaltered under the same conditions  in the ADE1 gene. In [PSI+] strains, Sup35 is functionally compromised, leading to stop-codon read-through and the formation of white or pink colonies on rich medium, but in [psi−] strains, termination is faithful at the PTC, leading to the formation of red colonies on rich medium  to the non-amyloid [psi−] state (i.e. SDS-sensitive)  . In a consitive) , as expensitive) . Howevernsitive) , indicatnsitive) , and in d strain . Thus, HPSI+]Strong strain  or for a Sup35 disruption, which both reduce propagation efficiency   . These oPSI+]Weak curing, we took advantage of the fact that we could modulate the efficiency of curing with variations in temperature. For example, while exposure to 40°C induced quantitative [PSI+]Weak curing, pretreatment at 37°C prior to exposure to 40°C slightly reduced curing , and Sis1 (Hsp40) proteins, which have all been implicated in Sup35 amyloid fragmentation . By quanl stress  induces k curing , 1.5 genl stress    , a trans3% loss)  through 3% loss) . Our stuk curing . Rather,k curing . As a rek curing , and thik curing . Thus, cC. elegans and human homologs of these chaperones , the juxtanuclear quality control compartment (JUNQ), StiF-inducible foci (StiF), and Q-bodies, the latter of which form under the mild thermal stress conditions employed in our studies , which contains firefly luciferase as an Xba1/BamHI fragment and mCherry as a BamHI/XhoI fragment, separated by a three-repeat glycine–serine linker. The ORFs were amplified by PCR using primers 5XbaI firefly/3BamHI firefly and 5BamHIGS3mCherry/3XhoImCherry, respectively (Saccharomyces cerevisiae used in this study are derivatives of 74-D694 (PSI+]Weak diploid strain (SY945) was generated by mating SY2600 with SLL3252  into SY945 and selection on medium lacking leucine. Disruptions were verified by PCR and 2:2 marker segregation upon sporulation and dissection. SY957, a [PSI+]Strong diploid strain containing a heterozygous disruption of SUP35, was created by transforming a PCR-generated cassette using pFA6a-KanMX4 as a template with primers SD27 and SD28  was created by transforming a PCR-generated cassette using pFA6a-GFP(S65T)-KanMX6 as a template with primers HSP104-GFP F-A and HSP104-GFP R-A (PSI+]Strong strains and selection on medium containing 300 μg/ml G418. Integration was confirmed by PCR using primers Hsp104for/GFP-R and pFa6 test/Hsp104 3 flank R (PSI+]Weak variant (SY2126) was generated by mating SY2125 to a WT [PSI+]Weak strain (SLL2600) and sporulation. Tetrads were dissected to recover haploids, and HSP104-GFP isolates were verified by G418 resistance, fluorescence microscopy, and quantitative immunoblotting for Hsp104. The heat inducible GFP strain (SY2091) was generated by transformation of a WT [psi−] strain (SLL2119) with Bsu36I-digested SB849 (SSA1-GFP (SY2658) and SIS1-GFP (SY2447) [PSI+]Weak strains were created by transforming PCR-generated cassettes using pFA6a-GFP(S65T)-KanMX6 as a template with primers GFP-GS-Ssa1-F/GFP-Ssa1-R or Sis1-GFP-F GS/Sis1-GFP-R  were generated by transforming SY2658 or SY2447 with Bsu36I-digested SB503 (bni1 [PSI+]Weak strain (SY1888) was created by transforming a PCR-generated cassette using pFA6a-KanMX4 as a template with primers AD-BNI1-f and AD-BNI1-r  was created by transforming SY2126 with a PCR-generated cassette using pFA6a-hphMX4 as a template with primers AD-BNI1-f and AD-BNI1-r , sectored (part red and part white), or [PSI+] (completely white). Unless otherwise indicated, fully cured and sectored colonies were combined in the ‘cured’ category. For all colony counting assays, at least 150 colonies were counted for each experimental condition/timepoint. For the galactose-inducible Hsp104 experiments, cells were grown in rich YP medium containing 3% raffinose supplemented with 3% galactose during induction. α-factor and nocodazole arrests were performed in YPD liquid medium containing final concentrations of 5 μg/ml α-factor or 15 μg/ml nocodazole, respectively, for ∼2 hr. Following confirmation of arrest based on cell morphology by bright-field microscopy, cultures were washed three times with medium containing 1 mM DMSO followed by one wash in YPD before resuspension for indicated manipulation. GdnHCl treatment was performed at 3 mM final concentration in liquid YPD, and for experiments involving recovery, cultures were washed three times with medium before resuspension in YPD for indicated manipulation.Unless otherwise specified, yeast cultures were grown in rich YPD medium supplemented with 0.3 mM adenine. Cultures were maintained at an ODg and total protein content was quantified using the BioRad Bradford assay in triplicate. Lysates were subjected to 15,000×g centrifugation for 15 min, and pellets were washed with 10 mM sodium phosphate buffer (pH7.5) containing 2% NP-40 before being resuspended in 10 mM sodium phosphate buffer (pH7.5) and quantified again in triplicate using the Bradford assay. For the Hsp104 immunocapture, native lysates were prepared at 4°C in IP buffer (50 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and protease inhibitors , protease inhibitor cocktail ). Lysates were pre-cleared for 1 min at 500×g, and then incubated for 1 hr with Protein G magnetic beads  with nutation. Immunocapture was performed using Protein G magnetic beads and anti-GFP mouse monoclonal antibody . Beads were washed 4× with IP buffer and 1× with 50 mM HEPES–NaOH (pH 7.5), and protein was eluted by boiling in SDS sample buffer. Co-captured proteins were resolved by SDS-PAGE and analyzed by gel staining with Flamingo  and fluorescent scanning on a Typhoon imager  according to the manufacturer's instructions or by western blot for GFP.SDS-PAGE and quantitative immunoblotting were performed as previously described . Anti-SsStatic images were obtained on a Zeiss Axio Imager M2 fluorescence light microscope equipped with a 100× objective. Confocal images were obtained on a Zeiss LSM 510 Meta confocal microscope using a 100× objective. Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm). Imaging was performed in complete minimal medium supplemented with 2% glucose and 2.5 mM adenine. Fluorescence intensity was analyzed using the Zen software package .Flow cytometry and cell sorting was performed on a BD FACSAria fluorescence-activated cell sorter using a 488 nm laser and a FITC-A filter to measure GFP fluorescence intensity in single cells. Data were obtained at least in triplicate with representative spectra shown. Data were analyzed using the FlowJo software package . 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 , Jeffery W Kelly (Reviewing editor), and 3 reviewers.Thank you for sending your work entitled “Spatial Quality Control Bypasses Cell-Based Limitations on Proteostasis To Promote Prion Curing” 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.PSI+]Weak cells induces curing through the disassembly of existing Sup35 amyloid by Hsp104. Serio et al. showed that Hsp104 must engage heat-induced protein aggregates for a period that exceeds the time to the next cell division. As a result, Hsp104 is asymmetrically localized to the cells that experienced the thermal stress  It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation? How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations?2) A broader context here would be useful as a number of laboratories have made observations on Q-bodies , iPOD, J3) The use of Sup35 as a client and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. Some brief comments addressing this issue will allow the reader to comprehend the bigger picture.Reviewer #1:Protein aggregation is linked to many degenerative diseases, either through loss of function of the aggregated protein, or through a novel gain of toxic function of the aggregate. It is therefore of high importance to identify pathways that can prevent or reverse protein aggregation. The submitted manuscript investigates the relationship between protein aggregation and the aggregate-remodeling factor Hsp104.°C strongly reduces aggregation of the model substrate Sup35 in its prion form [PSI+]Weak. Heat shock treatment reduces the size of the aggregates and increases the amount of soluble Sup35 after a recovery period. This process is dependent on the activity of Hsp104. The authors convincingly demonstrate that heat-induced aggregates of proteins other than Sup35 asymmetrically retain Hsp104 during cell division, in a process that is dependent of the enzymatic activity of Hsp104, and thereby increase Hsp104 concentration in mother cells to a level that allows Sup35 disaggregation and prion curing during recovery from heat stress.The authors report the seemingly paradoxical observation that incubation of cells at an elevated temperature of 40The concept that the (temporary) sequestration of important proteostasis factors into an aggregate can be beneficial instead of detrimental is novel and exciting, and provides insight into an unanticipated role of asymmetric cell division in protein aggregate management. The paper is clearly written and the main conclusions are generally well supported by data.There are however a few points the authors should address:1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation?2) The use of nocodazole in 3) Some of the colors in Reviewer #2:Klaips et. al. (Serio) have explored the mechanism of curing of Sup35 prion aggregates upon exposure of yeast to transient thermal stress to test a hypothesis that the cellular environment sets limits on protein quality control (PQC). The authors show that exposure of yeast (S. cerevisiae) at specific stages of the cell cycle to short periods of thermal stress leads to the disassembly of Sup35 amyloid, and propose that this is due to the spatial sequestration of Hsp104 in the mother cell rather than the result of overall elevated levels of Hsp104 or other chaperones. The sequestration of Hsp104 is correlated with the accumulation of heat-induced, non-prion aggregates upon thermal stress and the data suggest that the disassembly of Sup35 is more a function of amplification efficiency than thermodynamic stability. FACS experiments coupled with the use of two different cell-cycle inhibitors demonstrated that prion curing was highly dependent upon the cell-cycle stage.Overall, the experiments are well done, the results are intriguing and the paper is nicely presented. Some of the conclusions are correlative rather than causal, thus limiting the overall enthusiasm. Also, the use of Sup35 as a probe and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations? An argument is made that it is the asymmetrical partitioning of Hsp104 by its association with thermal aggregates that leads to a sufficiently high concentration of Hsp104 to resolve Sup35 amyloids. What is this concentration, what is the nature and basis of the interaction? Will elevated levels of Hsp104 achieved using conditional systems or shield-based metastable DHFR achieve the same outcome? Finally, the effects of different temperature conditions are intriguing. What is the nature of the biophysical transformation of Sup35 in 30C cells compared to those exposed to 40C beyond that rather vague terminology of shift from SDS-resistant to SDS-sensitive? What is special about 40C? Is this a useful tool to understand more completely the cellular environment?A broader context here would be useful as a number of laboratories have made observations on Q-bodies , iPOD, JAdditional comments:PSI+]Weak and a ∼50% decrease in curing efficiency was observed in a heterozygous deletion of Hsp104 (1) Evidence for involvement of Hsp104 in Sup35 amyloid disassembly comes both from the literature and new data presented here, that (i) the GdnHCl curing of  at elevated temperatures in yeast. Interestingly, this curing process depends upon an unprecedented spatiotemporal mechanism. Thus, asymmetric Hsp104 retention by heat induced, non-prion aggregates in late cell cycle-stage cells, leads to high enough Hsp104 levels to promote dissolution of Sup35 prions encoding weak [PSI+]. Upon recognizing this potential mechanism, the authors then cleverly tweak the system to enable curing of Sup35 prions that encode strong [PSI+]. Although Sup35 prion dissolution by Hsp104 is not very surprising per se, the spatiotemporal mechanism revealed by these studies is entirely novel and should interest a broad audience. Indeed, this paper will raise awareness that spatiotemporal mechanisms can have a profound impact on proteostasis and amyloid resolution. In my view, the study is high quality and is carefully conducted and controlled, and I do not have any issues with the experiments or data as presented. A limitation is that several conclusions are based upon correlative evidence, but it has already been shown in vitro that Hsp104 can rapidly dissolve Sup35 prions. My only minor objections concern several statements in the Introduction that are incorrect or misleading:1) 'but a direct demonstration of amyloid resolubilization in vivo has yet to be reported in any system'. Serio and colleagues have themselves already convincingly demonstrated amyloid resolublization by Hsp104 in vivo in their 2011 NSMB paper . Park et2) 'Rather, the effects of chaperone overexpression have been shown in some cases to be independent of their catalytic function '. This sC. elegans and mammalian tissues and cell lines similarly promote amyloid solubilization .' This statement should also be revised since Murray et al. subsequently published :1531-41.) that 'our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity.'3) 'Extracts from  1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation?PSI+]Weak curing, which is increased at 40°C relative to 37°C , which denatures upon thermal stress and is reactivated in an Hsp104-dependent manner . Importa to 37°C  and is i to 37°C , providiHow does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations?PSI+]Weak curing. To summarize here, curing correlates only with the highest levels of Hsp104 accumulation . In add reduced , indicatPSI+]Strong  A broader context here would be useful as a number of laboratories have made observations on Q-bodies , iPOD, JUNQ , aggresomes , and spatial sequestration and symmetrical inheritance in yeast  have demonstrated that aggregates have spatial restrictions and numerous papers have shown that multiple chaperones are associated with these aggregate structures using a wide range of proteomic and co-localization methods, often with substantial consequences on cellular activity. Some of these would seem to have opposing outcomes, which then poses questions how the mother-daughter cell relationship of yeast relates to metazoan cell division, and broader relevance to neurons being post-mitotic. Integrating these observations into this work without dramatically expanding the Discussion would be useful.We thank the reviewers for these helpful suggestions. We have reworked the Discussion to more clearly link our studies to the literature available on spatial quality control and to address the distinctions between our observations of enhanced proteostasis capacity and those in the literature demonstrating reduced proteostasis capacity. We believe that these distinct outcomes arise from imbalance between chaperones and substrates created by significant perturbations of the cellular environment beyond what can be achieved naturally.While the limitations on proteostasis capacity imposed by chaperone partitioning are clearly not applicable to post-mitotic neurons, we believe that the issues raised by a comparison of our work with those previous studies highlight the importance of system balance, particularly the role of chaperone-substrate dynamics in determining proteostasis capacity. These latter points are likely to be independent of cell-type and are clarified in the revised Discussion.3) The use of Sup35 as a client and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. Some brief comments addressing this issue will allow the reader to comprehend the bigger picture.The reviewer raises an important point that we failed to address in the original submission. We have now included a paragraph on mammalian disaggregase activity in the Discussion, including the similarities and distinctions with the yeast system.Reviewer #1:1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation?Please see the response to point 1 under “Response to points common to at least two reviews” above.2) The use of nocodazole inseems problematic as it may influence several other pathways that are involved in the formation and degradation of aggregates. Since the cell cycle analysis is not contributing critically to the overall model, it might be better to remove this section, or move it to the supplement.PSI+]Weak curing. Both reviewer 2 and reviewer 3 concur with the importance of this point in their reviews, and we have therefore retained the figure in the main figures of the revised manuscript.We were also concerned about off-pathway effects of nocodazole treatment, which prompted us to assess both Hsp104 expression and localization in the presence of this treatment , and to 3) Some of the colors inare hard to distinguish  and are not explained in the panels.We have changed the colors in panel Reviewer #2:Some of the conclusions are correlative rather than causal, thus limiting the overall enthusiasm.We have addressed the specific comments of this reviewer below.Also, the use of Sup35 as a probe and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment.Please see the response to point 4 under “Response to points common to at least two reviews” above.How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations?Please see the response to point 2 under “Response to points common to at least two reviews” above.An argument is made that it is the asymmetrical partitioning of Hsp104 by its association with thermal aggregates that leads to a sufficiently high concentration of Hsp104 to resolve Sup35 amyloids. What is this concentration, what is the nature and basis of the interaction?PSI+]Weak occurs when Hsp104 levels are increased ∼3.5-fold by thermal stress, relative to 30°C  Evidence for involvement of Hsp104 in Sup35 amyloid disassembly comes both from the literature and new data presented here, that (i) the GdnHCl curing of [PSI+]weak and a ∼50% decrease in curing efficiency was observed in a heterozygous deletion of Hsp104 , (ii) Hsp104 was asymmetric localized upon thermal stress  and (iii) no significant changes were observed for Hsp104, Ssa1 and Sis1 chaperone expression levels upon heat stress . This establishes necessity, but not sufficiency and therefore does not rule out other 'factor(s)' induced upon heat stress that could contribute to amyloid disassembly.These arguments can be strengthened by: (a) demonstrating whether the expression of other chaperone candidates  are affected by these thermal stress conditions. For example, there could be small changes in the expression of a number of chaperones that affects the cellular environment and enables amyloid resolubilization. Examples of candidates to test include chaperones examined in studies by;;and, and (b) showing the over-expression data, currently not shown (p.18) that establishes that the o/e of Hsp104 alone, to the same level achieved in this study through its asymmetric retention, suffices to induce curing.PSI+]Weak curing. We believe that establishing these points represents a significant advance in the field because up until this point, the engagement of chaperones with substrates has been correlated with reduced, not enhanced, activity. As we address in the Discussion, we believe that our system, which retains all factors within their native balance, is a major reason for this distinction.We completely agree that our studies demonstrate necessity of Hsp104 asymmetric localization and activity for Strong and [PSI+]Weak variants (With regard to the studies of Park variants . We have2) 'Rather, the effects of chaperone overexpression have been shown in some cases to be independent of their catalytic function '. This statement is a little misleading as in several cases the effect has been shown to depend on catalytic function (e.g.PLoS Genet.)Again, we apologize for the confusion. Our statement was meant to only refer to the citations listed (hence the “some”), but we can see how this narrow discussion of the literature can lead to confusion. Rather than expand to include the Cushman-Nick study, which incidentally shows that overexpression of Hsp104 promotes amyloid formation by MJD rather than clearance of existing amyloid, we have deleted this sentence.3) 'Extracts from C. elegans and mammalian tissues and cell lines similarly promote amyloid solubilization .' This statement should also be revised since Murray et al. subsequently published :1531-41.) that 'our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity.'We thank the reviewer for pointing us to the Murray et al. 2013 manuscript. We have removed the previous references to disaggregase activity in metazoan extracts.


PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs.Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( Preservation of genetic diversity of extant populations that can be reintroduced at later times to avoid population bottlenecks is central to controlled flock management. This is especially important for the biosecurity of poultry production systems, which are at risk of emerging disease pandemics such as avian influenza , UK. These White Leghorn lines were back crossed and selected to express a single MHC haplotype and vary in their susceptibility to many avian viral and bacterial pathogens The lines used in this study are the inbred line 6 (varying in their susceptibility to Marek's virus) the partially inbred line O lacking endogenous avian leucosis viruses (ALVs), Cornell partially inbred lines N and P (differing in resistance to MD), and the Wellcome inbred line W;  Cole, . These cHere we describe the establishment of a biobank of frozen primordial germ cells from 5 chicken lines of research interest. Each archived line comprises between 15 to 32 individuals in total, and at least 5 individuals of each sex.Avian PGC culture medium contained 1 × B-27 supplement, 2.0 mM GlutaMax, 1 × NEAA, 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM pyruvate, 0.2% ovalbumin (Sigma), 0.2% sodium heparin (Sigma) 5 μg/mL in avian DMEM, a custom basal medium . The following growth factors were added before use: human Activin A, 25 ng/mL (Peprotech); human FGF2, 4 ng/mL (R&D Biosystems); 0.2% chicken serum (Biosera). All reagents were purchased from ThermoFisher Scientific unless otherwise specified.Chicken flocks of several partially inbred White Leghorn lines were produced and maintained at IAH, Compton and are now housed at the NARF, UK. Line 0, 6 and N birds originate from the Poultry Research Laboratory, East Lansing, MI. Lines N and P line birds from Cornell University, USA and Wellcome B14 line (W line) from Wellcome Research Laboratories, Beckenham, UK.5, total volume of medium was changed every 2 d and cells were propagated at 2 to 4 × 105 cells/mL medium. Cells were frozen in Avian DMEM containing 4% DMSO/5% chicken serum and stored at –150°C. The donor embryo was isolated and sex was determined as published  embryos in 300 μL medium in a 48-well plate. One-third of the medium was changed every 2 d. When total cell number reached 1.0 × 10To investigate the efficiency of germline transmission, one male and one female line 6 cell line were expanded in culture in FAcs medium and cryopreserved for 1 wk. Cells were thawed, cultured for several wk and counted. Eggs were incubated until stage 16 HH and windowed through the pointy end. 5,000 to 6,000 cells total of male or female cells were injected into the dorsal aorta, the egg was resealed with parafilm and incubated with the pointy end down until hatching ) and 1 μL of blood was aspirated from the dorsal aorta and transferred to culture medium. The blood sample was cultured in suspension for 2 to 3 wk. During this period the blood cells present in the sample lysed and PGCs present in the well proliferated as single dispersed cells  or too old (older than stage 17 HH) for blood sampling.The data in Table For 203 cultures initiated from single embryos, 133 independent cell lines (genotypes) were expanded and frozen in a total of 478 vials. The overall derivation rate was 67%, which is consistent with past cell line derivation results (Whyte et al., To avoid inbreeding depression, it has been estimated that a minimum population containing 13 breeding pairs is needed FAO, . We haveTo test that the cryopreserved PGC lines were germline competent and as a first step to demonstrate the reconstitution of a chicken breed from frozen propagated PGC lines, we wanted to verify that the cryopreserved PGCs could produce viable gametes when transplanted into an outbred commercial layer line. A male and a female line 6 PGC cryovial was thawed and expanded in culture for several wk. Five thousand to 6,000 male or female PGCs were injected into the dorsal aorta of stage 16 host ISA brown embryos. The embryos were incubated until hatching, hatched, and the hatched chicks were sexed by PCR. The hosts containing correctly sex-matched donor PGCs were raised to sexual maturity. Three hens and 3 cockerels containing donor line 6 PGCs were produced. An initial examination of the cockerel semen using microsatellite analysis tentatively identified the male containing the highest contribution of donor PGCs in the semen (used in Table The storage and faithful recovery of breeds of poultry from stored germplasm is needed for the long-term safeguarding and management of poultry genetic resources (Whyte et al., The reconstitution of a chicken breed using cryopreserved chicken semen and backcrossing is predicted to take 4 crosses to re-establish 97% of the genome of the original breed (Blesbois et al.,


Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. Schwannomatosis (SWN), a rare form of neurofibromatosis characterized by the development of multiple benign schwannomas. Schwannomatosis is estimated to affect 1 in 40,000 people. However, given the increasing understanding of the phenotypic heterogeneity of this disorder, its true incidence is unknown. SWN differs from Neurofibromatosis Type 2 in that patients do not develop vestibular tumors. Schwannomatosis patients also do not harbor germline mutations in the merlin gene, NF2. , althougThus far there has been very limited research focused on schwannomatosis, in part because it is considered a rare disease, in part because there have been limited resources dedicated to the syndrome and most importantly because a relevant cell line model of SWN has been lacking. No phenotypically & physiologically relevant screening systems for drug discovery or drug re-purposing are currently available for the schwannomatosis research community. Currently, surgical resection persists as the standard of care for schwannomatosis. It is therefore critical to develop research tools to elucidate the genetic basis of schwannoma tumorigenesis and to identify novel therapeutic agents. With no commercially available schwannomatosis cell lines, the need has arisen to generate a method to dissociate and produce high-purity Schwann cell cultures from patient tumor specimens in order to advance peripheral nerve sheath tumor treatment options.Schwann cells dissociated from the sciatic nerve of SMARCB1/INI1 knockout mice have been used as an in vitro model of schwannomatosis. Given the complex genetics and supporting cell types that make up a schwannoma, however, this may not be an accurate disease model. Schwannomatosis tumors have been difficult to grow in culture as they are benign cells that do not proliferate rapidly and survive only a few passages before senescence. Our lab has extensive experience in establishing primary Schwann cell cultures, from rat, mouse and human. We have established immortalized human Schwann cell lines using hTERT and SV40 large T antigen, which retained phenotype after immortalization . Here weWe obtained schwannoma tissue specimens from patients with well-characterized clinical cases of schwannomatosis from surgical resection performed in the Comprehensive Neurofibromatosis (NF) Center at Johns Hopkins Hospital. We established 2 cells lines from separate schwannomatosis (SWN) patients. One tumor caused the patient great pain (Hp-SWN-14F) and the other was removed due to neurological deficit but was not associated with pain (Hnp-SWN-14O). The cell lines were established in our laboratory using standard cell culturing techniques, with minor modifications. In brief, tumor specimens were collected directly in the operating room in a vial of ice cold saline. The tissue was immediately processed by manual dissociation by mincing with a scalpel. The tissue was kept in cold saline while processing. The tissue was then incubated in L-15 media with collagenase and dispase for 3 hours to dissociate the cells. The cells were filtered through a 70um filter and seeded on plates coated with laminin. The cells were grown to confluency in T-25 flasks. After passaging the cells were examined by immunocytochemistry for the Schwann cell marker S100B, and the fibroblast marker Thy-1. The SWN cells were S100B positive. Fibroblast contamination was ruled out by a lack of Thy-1 immunostaining . At passAs of the submission of this manuscript, the SWN cells have been passaged 11 times since immortalization with SV40. Total passaging since initial dissociation is 14. Primary Human Schwann cells, and schwannomatosis cell lines (Hp-SWN-14F and Hnp-SWN-14O) all positively stained for S100B after multiple passages and freeze-thaw cycles. . A potenWe also compared gene expression in the immortalized SWN cell lines (passage 6) and their parent tumors. The Illumina HT-12 microarray was used for gene expression analysis. This microarray platform contains probes for 47,000 transcripts and splice variants, with 31,000 annotated genes represented. This assay revealed some differences in gene expression between parent tumor cells and their respective immortalized cell lines. 509/31,000 (1.6%) of genes showed a 2 fold difference in the immortalized cell line when compared to the parent tissue. . Using DWe have created human immortalized schwannomatosis cell lines derived from surgical resections of tumors from schwannomatosis patients. Previously reported cell culturing methods rely upon relatively harsh conditions to dissociate Schwann cells from the schwannoma tumor specimens. Frequently, trypsin is utilized as the predominant enzyme to break apart the tumor sample into its cellular components . Some olIt has been of utmost importance that our immortalized SWN cell lines retain phenotypic and genotypic characteristics of the tumor tissues from which they were derived. To this end we have fully characterized gene expression of both cell lines and their parent tumor. The non-painful SWN cell line (Hnp-SWN-14O) was derived from a patient harboring a mutation in SMARCB1. The expression level of SMARCB1 in both the tumor tissue and cell line was significantly downregulated when compared to non-SWN related schwannomas. The SWN tumor tissue and corresponding cell line exhibit similar SMARCB1 expression levels. Therefore the SWN cell line reflects the parental tumor regarding the expression of the key gene linked to schwannomatosis. Upon examination of gene expression in cell lines and their parent tumor by microarray analysis, we discovered some differences . The genes downregulated in the SWN cell lines were related to immune response, and myelination. This was not surprising as the tumor tissue is vascularized, and the SWN cell lines are not myelinating cultures. Schwann cells in culture initiate a myelination program when co-cultured with neurons under precise myelination media conditions . The uprThe immortalized human SWN cell lines and normal primary human Schwann cells showed a similar rate of growth. SV40 immortalization, a widely accepted method for creating cell lines has recently raised a few concerns among the scientific community. SV40 uses blockage of the p53 and the retinoblastoma gene (RB1B) pathways to overcome cell cycle arrest. There is a concern that SV40 immortalization can create cells that are not representative of the parent tissue and are increasingly tumorigenic. Many genes have been shown to be affected by SV40 . In our In conclusion, we have created the first immortalized Schwannomatosis cell lines that retain essential genotypes, phenotypes, and cell growth patterns. These cell lines will be invaluable tools for advancing research in schwannomatosis.3) was finely minced. The minced tumor was placed in into a new sterile 15mL conical tube containing 4ml L-15 media, and 10mg/ml collagenase/dispase. The tumor was incubated for 3 hours at 37°C to dissociate the cells. The cells were collected by centrifugation at 1,000 rpm for 2 min and the cell pellet was washed twice with 5ml of L-15 media. After the final wash the cell pellet was resuspended in in 1 mL of L15 and DNase solution (10mg/ml). The suspended cells were passed through a 70 uM cell strainer, to remove unwanted debris. The strainer was washed with an additional 3ml of L-15/DNase solution to increase cell yield. The collected cells were centrifuged at 1,000 rpm for 10 minutes. The cell pellet was suspended in 1mL of the human Schwann cell medium (Sciencell). The cells were plated in a poly-l-lysine coated T25 flask. 2–3 days after initial plating the media on the cells was changed to high glucose DMEM/pen strep, plus 10% fetal bovine serum, 2μM forskolin, and 20ng/ml neuregulin. Cultures were maintained in this media until the reached 80% confluence, at which time the cells were split into three T-25 flasks or frozen (in DMEM +20% FBS+ 10% DMSO) for later use.Schwannomatosis tumors were collected from surgical cases from December 2012 through October 2014, occurring at Johns Hopkins School of Medicine. Schwannoma diagnosis was confirmed by Pathology. This study was approved by the Institutional Review Board of the Johns Hopkins School of Medicine. After collection, the tumor specimen was placed in saline for transport to the lab. In a laminar flow hood, a section of the tumor  SV40 Forward Primer Sequence 5’ ACTGAGGGGCCTGAAATGA; SV40 Reverse Primer Sequence 5’ GACTCAGGGCATGAAACAGG. The product size is 61 bp.Cells were immortalized using pLenti-SV40 from ABM  according to manufacturer’s instructions. In brief, cells at passage 3 were infected with 2 ml/well of viral supernatant in the presence of 5μg/ml Polybrene, in the morning. 6–8 hours later, the viral supernatant from first infection was removed from the wells and the cells were re-infected with 2 ml/well of fresh supernatant/ polybrene. The next day, viral supernatant was removed and replaced with complete growth medium. The cells were incubated at 37°C for ~72 hours incubation and subcultured. After subculturing the cells were screened for the SV40 transgene by qRT-PCR as per manufacturer’s instructions . RNA DNA and protein were extracted using the Allprep extraction kit (Qiagen). RNA from cells in culture was extracted using Trizol, followed by clean up with RNeasy RNA extraction kit (Qiagen).1ug of total RNA was converted to cDNA using the Quantitect Reverse Transcription kit (Qiagen).B2M, GAPDH, SMARCB1, p75/NGFR, NGF, S100B, CCNA2, CCNB1, CCNB2, CCND1, CCNF, and CDC20 were purchased from Qiagen. Amplified bands were analyzed for correct size and sequenced by the Johns Hopkins Sequencing core to assure correct targets.t) as 2−Δ(ΔCt), where ΔCt = Ct,GENE − Ct housekeeping genes and Δ(ΔCt) = ΔCt,C− ΔCt,pt One microliter of each tumor, cell line, and normal primary human Schwann cell cDNA, was used for real-time qRT-PCR using QuantiTect SYBR Green PCR kit (Qiagen). Amplifications were carried out in 96 well plates in a Roche Lightcycler. All reactions were performed in triplicate. Dissociation curve analysis was performed to rule out experimental PCR artifacts or non-specific amplification. Expression of genes relative to Beta-2-Microglobulin (B2M) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated based on the threshold cycle  method following the guidelines of Zhang and Ruschhaupt 2013 S1 FigAuthentication of the cell line using Short Tandem Repeat (STR) profiling was employed to ensure the proper identity of the subsequent passages.(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Background. Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. Materials and Methods. To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. Results. Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. Conclusion. Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma. A. nidulans cells from entering mitosis. Thus, “NIM” stands for “never in mitosis” [ Drosophila, Xenopus, and mouse [Nek2 is a serine/threonine protein kinase, belonging to the Nek family of cell cycle regulators . The firmitosis” . Modern nd mouse –6. Therend mouse –10. Becand mouse –13, a fend mouse –16. ex vivo and in vitro models of multiple myeloma [While the effectiveness of proteasome inhibition in breast cancer continues to be evaluated, bortezomib continues to be a mainstay treatment for relapsed refractory MM , 18. In  myeloma . in vitro and mitigated bortezomib resistance induced by Nek2 overexpression. Taken together, the data suggest that Nek2 plays an important role in the uncontrolled proliferation of MM cells and introduces Nek2 as a therapeutic target in relapsed refractory MM cells resistant to bortezomib.Although several groups have tried to validate Nek2 as a therapeutic target using both small molecules and siRNA, few of them actually achieved efficient inhibition of Nek2 by small molecules , 22–25.  NEK2 gene into the pCMV6-GFP vector (OriGene). The correct sequence of pCMV6-NEK2-GFP was verified by sequencing. Plasmid was generated in Top 10 cells (Invitrogen) and the plasmid was purified using the Small Scale Plasmid DNA Purification Kit (QIAGEN). Purified pCMV6-NEK2-GFP was used to transfect HeLa cells in 6-well plates, using Lipofectamine 2000 (Invitrogen). We chose to transfect HeLa cells with the pCMV6-NEK2-GFP plasmid because a previous report indicated the successful transfection of plasmids into NT2/D1 and HeLa cells using Lipofectamine 2000 without visible toxicity [μg/mL and the cell density was 8 × 105 cells per well. G418 (Invitrogen) was added to DMEM medium with final concentration of 1 mg/mL. HeLa cells were cultured in G418 containing medium for one month. Clones were then isolated and continuously cultured as stable Nek2 OE HeLa cells. The same process was conducted using the pCMV6-GFP vector to establish stable GFP OE HeLa cells.The Nek2 coding sequence was purchased and subcloned from a pCMV6-Entry vector (OriGene). Restriction enzymes AsiSI and XhoI were used to ligate thetoxicity . The finμg/mL and the cell density was 8 × 105 cells per well. Three additional clones of the ARP-1 cell line, Nek2-OE, Nek2-knockdown (KD), and bortezomib-resistant lines were generated as described in our previous report [Other Nek2 OE multiple myeloma cell lines, including ARP1, H929, and KMS28PE cells, were developed as described in our previous report . As withs report .Following the indicated treatments, cells were washed by cold 1 X PBS buffer and were lysed using NP-40 cell lysis buffer (Life Technology). Whole-cell lysates were prepared and subjected to Western blot analysis as described in our previous report . Brieflyμg/mL.The 26S proteasome was isolated from whole-cell lysates by ultracentrifugation as previously described . ProteasμM, 30 μM, 10 μM, 3 μM, 1 μM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, and 0.3 nM.Compounds were incubated with human Nek2 kinase (Invitrogen) and then kinase activity was examined by the Kinase-Glo Luminescence Kinase Assay (Promega). The assay was performed according to the manufacturer's protocol in 384-well plates' format using 60 mM Nek2. Twelve different concentrations were set for each compound: 100 Cell viability was determined using the ATPlite 1Step Kit (PerkinElmer) in 96-well plates. The assay was performed according to the vendor's protocol. Cell viability was assessed by measuring live cell ATP activity.μg/mL propidium iodide, 20 μg/mL RNase A, and 0.5% NP-40). Flow cytometry was conducted on a FACScan cytometer . Collected data was analyzed by FlowJo 6.0b software .Cell cycle analysis was performed as described . HeLa cet-tests using the Graph-Pad InStat Software. Data was considered statically significant when P < 0.05.Data was tested for statistical significance by unpaired α, a known substrate of Nek2 [We previously reported that bortezomib resistance is accompanied with Nek2 upregulation in MM patients . To conf of Nek2 , was alsThe two most viable HeLa Nek2-OE clones and HeLa GFP-OE clones were selected for the following experiments. Bortezomib was used to treat these HeLa cells in a 96-well plate under different concentrations  with 0.1% DMSO as control. After 72 hours, cell viability was examined by the ATP lite assay. At every concentration of bortezomib, Nek2-OE clones yielded higher cell viability than GFP clones . These dBecause bortezomib is able to target cancer cells by proteasome inhibition , we hypoIn vitro proteasome activity from the isolated proteasome was tested by the Proteasome-Glo Trypsin-Like Assay. For all the studied cell lines, the proteasome activity of the Nek2-OE cells was significantly higher than the control . Bortezomib resistant ARP-1 cells exhibited the highest proteasome activity  which was tested by the Kinase-Glo assay with the drug preincubated with Nek2. The Nek2 inhibitory activity of HCI-2389 was significantly increased after preincubation times as short as 0.5 hours  with our Nek2 inhibitors. These 36 cell lines were either responsive  to both HCI-2184 and HCI-2389 or nonresponsive to either of the two inhibitors selected for the proteasome activity assay. Data analysis showed that although there was not a strict proportional relation between proteasome activity and Nek2 inhibitor responsiveness, the average proteasome activity of the sensitive cell lines was significantly higher than that of the nonsensitive cell lines  of protein degradation. Ubiquitination regulates multiple cell cycle aspects including checkpoints control and cell growth progression , 43, 44.rocesses . Cdk1 anrocesses , 46. Therocesses . In addirocesses , 46. Sturocesses , 47, 48.rocesses , 42. in vitro proteasome activity is inhibited by our Nek2 inhibitors HCI-2184 and HCI-2389 which rescue drug resistance of Nek2-OE HeLa cells. However, the mechanism of how Nek2 regulates proteasome activity is still unknown and needs further investigation.Previous reports suggested that Nek2 primarily played a role in regulating centrosome separation –10. OverTogether with the Polo and Aurora kinase families, the NIMA-related protein kinases (Neks) have been called the third family of mitotic kinases . PreviouBy examining the proteasome activity of multiple cancer cell lines, we have identified Nek2 upregulation as a potential mechanism for bortezomib resistance related to proteasome activity elevation. However, for ARP1 cells, the proteasome activity of the bortezomib resistant clone was higher than that of the Nek2 OE clone . TherefoAlthough we have synthesized several potent Nek2 inhibitors with demonstrated activity , these iIn summary, we have discovered that high levels of Nek2 expression are at least partly responsible for elevated proteasome activity and subsequent bortezomib resistance in human MM treatment. More excitingly, we have shown that Nek2 inhibition results in proteasome activity suppression and cell cycle arrest. This research provides important knowledge for future studies of Nek2's biological function and provides potential solutions for bortezomib resistance in MM therapy. in vitro. It is clear that there is an urgent need for exploring the mechanism linking the Nek2 kinase to drug resistance and the development of novel Nek2 inhibitors. To our knowledge, this study represents the first link connecting Nek2's biological function of regulating proteasome activity as the mechanism of bortezomib resistance in multiple myeloma. It is also the first to establish highly effective Nek2 inhibitors that successfully inhibit proteasome activity in cancer cell lines.Although progress in the treatment of MM has been made in the past decade , 41, myeThe Supplementary Material provides further detail about the small molecule Nek2 inhibitors used in the studies. The first Supplementary Figure depicts the structural binding mode of HCI-2389 in the ATP-binding pocket of Nek2 and rationalizes the irreversible bond between the small molecule and Cys22 on Nek2. The second Supplementary Figure shows the selectivity of HCI-2184 and HCI-2389 against representative panel of 39 kinases. Percent inhibition is shown on the graph at a single screening concentration (200 nM).


The structures were elucidated by extensive HRESIMS and NMR spectroscopic analyses. All compounds were tested for their cytotoxicities against five human cancer cell lines by MTT method, among which compound 1 showed moderate inhibitory effects on these cell lines. This was the first report of azaphilones isolated from Diaporthe sp. Three new azaphilone compounds, isochromophilones X–XII (Supplementary material is available for this article at 10.1007/s13659-012-0023-2 and is accessible for authorized users. Supplementary material, approximately 819 KB.


In recent years, increasing evidence indicates that, not only lymphocytes but also glial cells, in particular astrocytes, play a role in the pathophysiology of HAM/TSP. In this study we used a model of co-culture between human HTLV-1-infected (CIB and C91PL) and non-infected (CEM) T lymphocyte cell lines and astrocyte (U251 and U87) cell lines to mimic the We first observed that CIB and C91PL adhere strongly to cultured astrocytes cell lines, and that co-cultures of HTLV-1 infected and astrocyte cell lines cells resulted in rapid syncytium formation, accompanied by severe morphological alterations and increased apoptotic cell death of astrocyte cells. Additionally, cultures of astrocyte cell lines in presence of supernatants harvested from HTLV-1-infected T cell cultures resulted in significant increase in the mRNA of CCL2, CXCL1, CXCL2, CXCL3, CXCL10, IL-13, IL-8, NFKB1, TLR4, TNF, MMP8 and VCAM1, as compared with the values obtained when we applied supernatants of non-infected T- cell lines. Lastly, soluble factors secreted by cultured astrocytic cell lines primed through 1-h interaction with infected T cell lines, further enhanced migratory responses, as compared to the effect seen when supernatants from astrocytic cell lines were primed with non-infected T cell lines.Collectively, our results show that HTLV-1 infected T lymphocyte cell lines interact strongly with astrocyte cell lines, leading to astrocyte damage and increased secretion of attracting cytokines, which in turn may participate in the further attraction of HTLV-1-infected T cells into central nervous system (CNS), thus amplifying and prolonging the immune damage of CNS.The online version of this article (doi:10.1186/s12985-015-0398-x) contains supplementary material, which is available to authorized users. Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic and slowly progressive neurodegenerative disease of the central nervous system (CNS)  and AdulHistopathological findings of CNS revealed that HAM/TSP affects mostly the lower and middle thoracic spinal cord, with marked degeneration of the corticospinal tracts and demyelination, accompanied by diffuse and symmetrical degeneration of the anterolateral and inner portion of the posterior columns . These fpost mortem tissues revealed that astrocytes from HAM/TSP lesions bear an activated phenotype and produce high amounts of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and chemokines [in vitro studies demonstrated that interactions with HTLV-1-infected lymphocytes resulted in morphological changes of astrocytes similarly to those found in post mortem [To date, the precise mechanisms by which HTLV-1 promotes these lesions remain poorly understood. Nevertheless, available data indicate that progression to HAM/TSP is characterized by presence of an exaggerated and chronic immune response , accompaemokines , 29, 30.t mortem , 32, beit mortem , 34.in vitro model of T cell-astrocyte cell lines interaction to approach the potential the impact of HTLV-1-infected T cell lines in the integrity and gene expressing profile of migration-related genes of astrocytic cell lines. We also analyzed the migratory response of HTLV-1-T lymphocyte cell lines under the stimulation of astrocytic cell lines primed with supernatants derived from HTLV-1+ T cell lines. Our results indicate that under transient interactions with HTLV-1-infected T cell line cells, astrocytic cell lines undergo major morphological changes, together with modulation in the expression of a variety of cell-migration genes. In turn, such reactive astrocytic cell lines increase migratory responses of HTLV-1-infected lymphocytes, thus suggesting a role of these glial elements in the recruitment of additional T cells into CNS.However the participation of astrocytes in the pathophysiology of HAM/TSP remains poorly understood, particularly their role in the recruitment and trafficking of peripheral T cells into CNS. In this context, we conducted a study to investigate the morphological and functional alterations exerted by HTLV-1-infected T cell lines upon astrocytoma-derived cell lines. In particular, we used an In the first set of experiments, we investigated the adhesion of HTLV-1-infected (CIB and C91PL) and non-infected (CEM) T cell lines to astrocytoma monolayers (U251). The adhesion assay was performed during 30 min, after which non-adherent lymphocytes cell lines were removed and adherent lymphocytes cell lines counted after Giemsa staining. We found that after 30 min in co-cultures, the adhesion degree of HTLV-1 infected T cell lines,  were co-cultured with the astrocytoma cell lines (U251 and U87) for up to 20 h, after which, non-adherent cells were removed and the alterations observed after Giemsa staining. We observed heterocellular cell fusion, and syncytium formation between HTLV-1 infected T cell lines and astrocytoma cell lines as early as 6 h post co-culture. These changes were equally seen when the CIB Fig.  and C91PIn the following set of experiments, we were interested to investigate the long term impact of HTLV-1 infected T cells on astrocytes cell lines, including the morphological and ultra structural alterations and induction of apoptosis. For this purpose, we co-cultured astrocytoma cells (U251 and U87) with infected (CIB) or non-infected T cell lines (CEM). After 3 h interaction, lymphocyte cell lines were removed from the co-cultures through vigorous cold PBS washings. Complete removal of T cell lines was confirmed in paired co-cultures stained by Giemsa and observed by light microscopy. The astrocytoma cells were then kept in culture for up to 10 days with daily microscopic observation. Figure These changes were further characterized by transmission electron microscopy Fig. . AstrocyNo morphological changes were observed in the mock control  in astrocytoma cell lines after short-term interaction with each T cell line. Figure Subsequent experiments were performed in order to investigate if the cytopathic effect described above involved increased apoptosis. We thus performed annexin V + T cell lines could affect the expression of cell adhesion and migration-related genes on astrocytoma cell lines. Indeed, transient exposure (30 min) to fractionated supernatants from the HTLV-1+ cell lines resulted in statistically significant increase in the mRNA expression of various cytokines/chemokines and adhesion molecules, including the pro-inflammatory cytokine TNF-α, the TNF-α-regulated chemokines CXCL1, CXCL2, CXCL3, CXCL8, CCL2, IL13 and CXCL10, as well as V-CAM-1 and MMP-8, as compared to the values obtained when the astrocytoma cells were subjected to supernatants from non-infected T cell line cultures  previously primed with a transient exposure with the HTLV-1 infected (CIB) or non-infected (CEM) T cell lines, as migratory stimuli for the migration of further HTLV-1+ T cells. Migratory response of these cells to supernatant of astrocytoma cell lines primed with infected cell lines was significantly enhanced, as compared to supernatants from untreated astrocytoma cell lines, or those co-cultured with the non-infected T cell line , a T cell line originally infected by HTLV-1 in vitro (C91PL) and two astrocytoma cell lines (U251 and U87).The mechanisms underlying the pathogenesis of HAM/TSP are still poorly understood. Although previous evidence strongly suggests that astrocytes take part in this process through several mechanisms –32, theiWe first demonstrated that HTLV-1-infected T cell lines adhered significantly higher to the astrocytoma monolayers as compared to the non-infected T cell line. This finding suggests that astrocytes may represent a critical target of HTLV-1 infected lymphocytes. Previous studies also demonstrated that HTLV-1 infected cell lines adhered stronger to endothelial cell lines . We also+ T cell line [bcl-2 [In a second set of experiments, we observed several and extensive morphological changes and cytopathic effect in 5-day culture of astrocytoma cell cultures that were transiently exposed to HTLV-1-infected T cell lines, but not to non-infected counterparts. The cytopathic effect was characterized by loss of cell-cell contact, cell shrinkage, accumulation of lysosomal vesicles and cell death. Notably, similar morphological alterations have been observed in endothelial cells after transient interaction with the MT-2 HTLV-1ell line . These aell line –38. Sevee [bcl-2 , 37 and + T cells line, but not with the non-infected T cell line. Infection of astrocytes with HTLV-1 has been reported in previous studies [Of note, we observed the presence of HTLV-1 viral particles in the cytoplasm of astrocytoma cells after transient interaction with HTLV-1 studies –41, and + T cell lines on cultured astrocyte cell lines. We used qPCR arrays to quantitatively evaluate the expression levels of a large numbers of cell migration-related genes, including cytokines/chemokines and extracellular matrix/cell adhesion proteins. A short-term (1 h) exposure of astrocytoma cell lines to fractionated HTLV-1-infected T cell lines-derived supernatants, resulted in statistically significant increase in the mRNA of TNF-α, various chemokines, as well as VCAM-1 and MMP-8, as compared with supernatants derived from the non-infected T cell line, as shown in Fig. in vivo, when T-cells migrate into the CNS. Our results are also in line with previous histopathological findings from post-mortem tissues, in which it has been found that astrocytes from HAM/TSP lesions exhibit higher contents of proinflammatory cytokines, MMP and chemokines [We also investigated the role played by the soluble factors secreted by HTLV-1emokines , 29, 30.Moreover, our data corroborate those described by Akaoka (2011) and Szymocha (2000), who demonstrated that infiltrating HTLV-1-infected T lymphocytes in the CNS continuously induce significant changes in astrocytes, not only through cell-cell interaction , but alsConjointly, these findings strongly point to an important paracrine effect of HTLV-1 infected lymphocytes on astrocytes and highlight that the functional impairment of astrocytes occurring in HAM/TSP, would be much more complex than ever described.Our findings suggest that the functional impairment of astrocytes caused by interactions with HTLV-1-infected T cells contributes to the perpetuation and amplification of the CNS damage. Considering that we applied a 30–50 kDa supernatant fraction to stimulate the astrocytoma cells, it is conceivable that Tax1 protein is involved in these effects. Yet, additional studies with blocking antibodies are required to confirm this hypothesis.in vitro derived HTLV-1 infected T cell line (C91PL) was used as a control only in the first set of experiments when we observed that the effects were similar when using both cell lines.Since astrocytes primed with HTVL-1-infectd T cells expressed higher levels of various cell adhesion and migration-related mRNA molecules, it seemed plausible to hypothesize that supernatants from such primary cultures could enhance T cell migration. Actually, this was the case and supernatants from HTLV-1 primed astrocytoma cell lines were able increase the migration of HTLV-1 infected T cell lines. Conceptually, these data suggest that activated astrocytes would enhance the recruitment of additional T cells into the CNS. Since main focus of this study was to understand the impact of a HAM/TSP derived T cell line (CIB), the Although we have not performed immune staining to confirm the presence of HTLV-1 inside the cells, we believe that the viral particle detection by electron microscopy is a convincing evidence of astrocyte infection. Similarly, we also think that the enhanced T cell migration after being primed by supernatants from infected astrocytes suggests an increased concentration of chemotactic factors, as indicated by the elevated expression of the corresponding mRNAs by HTLV-1-infected astrocytes.in vitro derived HTLV-1-infected T cell lines, such as MT-2 or C91PL [Of remark, our results are of utmost importance, since most of the previous work that investigated HTLV-1-induced changes on astrocytes used or C91PL –34, 45. or C91PL .In conclusion, our results show that HAM/TSP-derived T lymphocyte cell lines exert a pleiotropic effects upon astrocyte cell lines, leading to astrocyte damage and increased secretion of attracting cytokines, which in turn may participate in the further attraction of HTLV-1-infected T cells into central nervous system (CNS), thus amplifying and prolonging the immune damage of CNS.2. Hela cells, an epithelial cell line derived from cervical cancer cells [2. Cultures of the HTLV-1-infected T cell lines were further supplemented with 100 U/ml of interleukin 2 (IL-2) .The human malignant astrocytoma cell lines U251 and U87, both derived from malignant gliobastoma multiforme  , were grer cells , were user cells . The HTLer cells   were fixed in absolute ethanol, stained with Giemsa and counted to determine the adhesion index (AI), as previously described [For adhesion assays, astrocytoma and Hela cells were first plated in 25 cmescribed  and valiescribed , using t2 flasks until sub-confluence. HTLV-1-infected T lymphocytes (CIB or C91PL) or the non-infected T cell line (CEM) were added to the monolayer of astrocytoma at astrocyte/lymphocyte ratio of 1:4 and left to adhere for up to 20 h in culture conditions similar to those described above. Supernatant was then discarded, washed briefly with PBS, fixed in absolute ethanol, stained with Giemsa and observed by light microscopy. Similar assays were performed using a non-astrocytic cell line (Hela), to assess whether changes were specific for astrocytes.To assess syncytium formation, astrocytoma cells were initially grown in 25 cm2 flasks until sub-confluence. Transient co-cultures of astrocytes with either HTLV-1-infected T lymphocytes (CIB or C91PL) or the non-infected T cell line (CEM) were performed by replacing the medium in the astrocytoma cultures with those from cultures of HTLV-1-infected or non-infected T cell lines in the absence of IL-2 at 37 °C, 5 % CO2, at astrocyte/lymphocyte ratio of 1:4. After 3 h, lymphocytes were completely removed from the cultures by vigorous washing using cold PBS. Duration of co-culture was set at 3 h, since the study conducted by Mor-Vaknin et al., [Astrocytoma cells were initially grown in 25 cm et al., , demonstHela cells were used as controls for nonspecific interactions between astrocytes and HTLV-1-infected cells. For Mock controls, astrocytes were culture alone with RPMI.After lymphocyte removal by vigorous washings with cold PBS, astrocytoma and Hela cells were kept in culture for up to 5 days, replenishing fresh medium every two days. At days 5 post co-culture, astrocytes were processed for optical or transmission electron microscopy, as previously described , using cThe ability of HTLV-1 or non-infected T cells to induce apoptosis on the astrocytoma cells was measured by cytofluorometry using FITC-coupled Annexin V Apoptosis Detection Kit I . After transient co-cultures during 3 h, followed by lymphocyte removal by vigorous washings with cold PBS, astrocytes were kept in culture for additional 2 h, trypsinized and stained with FITC-Annexin V and Propidium Iodide according to the manufacturer’s instructions. Events were acquired using a FACSCalibur Flow Cytometer .2 culture flasks at an initial concentration of 1x107 cells/mL during 24hs. Supernatants were then collected and concentrated using the centripep YM-50 and YM-30 filters , according to the manufacturer’s instructions. The fraction was added to the astrocyte monolayer for 1 h at 37 °C and then, cells were then trypsinized and washed. As a mock control, growing astrocytes were treated with RPMI alone.We further evaluated the effect exerted by HTLV-1-infected cells on the expression of cell adhesion and cell migration-related genes in astrocytoma cells. Astrocytes were primed using supernatants from HTLV-1-infected T cell cultures, using as control counterpart supernatants from non-infected T cell cultures. For this purpose, lymphocytes were grown in 75 cm260/A230 ≈ 2.0.Total RNA was extracted using RNeasy Micro Kit , according to the manufacturers instructions, including DNAse treatment. Total RNA was quantified by spectrophotometry using Nanodrop 2000 . RNA integrity was assessed for the presence of ribosomal RNA 28S and 18S, using denaturating agarose gel electrophoresis as previously described . RNA was2 First Strand” , according to the manufacturers instructions. Then, RT2 SYBR Green/ROX PCR Master Mix (SABioscience) and’ nuclease-free water were added to the cDNA. Twenty-five μL of this mixture were added to 96 microplates containing primers for each of the 84 target genes for ECM proteins/adhesion molecules using corresponding qPCR arrays , as seen in supplementary data in S1 Table and S2 Table. Amplification signals were captured using the ABI Prism 7000 PCR device . Melting curve analysis was performed for each sample at the end of the PCR.Gene expressing profiles for cytokines/chemokines and extracellular matrix (ECM) proteins/adhesion molecules were ascertained by quantitative PCR. cDNAs were prepared from 1 μg RNA of each sample using “RTtranswell chambers , as described elsewhere [(3 h co-culture) were harvested and added into the lower chamber, whereas 106 CIB cells/100 μl (of RPMI/1 % BSA) were added into the upper chambers. After 4 h incubation at 37 °C in 5 % CO2, we counted the cells that migrated into the lower chambers using trypan blue cell viability analysis.Cell migration studies were conducted in lsewhere , using tt-test and the One Way ANOVA test were used to compare the differences among groups. Differences were considered statistically significant when the p values were ≤ 0.05.All statistical analyses were performed using the GraphPad Prism 5.01 software package. Quantitative data were expressed as the mean ± standard error. The Student’s


Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells.Listeriolysin O (LLO) is the main virulence factor of A purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells.in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.LLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated  Most bacterial pathogens produce toxins, which are important virulence factors. Some toxins act on cytoplasmic membranes, whereas others are receptor-targeted toxins or membrane damaging toxins. The latter toxins are referred to as cytolysins, and are produced by numerous Gram-positive and Gram-negative bacteria. Some of these toxins display enzymatic activity, while others have cytolytic capacity without enzymatic activity. Occasionally those two mechanisms may act together. For example some phospholipases facilitate the action of pore-forming cytolysins by hydrolysing membrane lipids .Listeria monocytogenes is an intracellular bacterial pathogen whose important virulence determinants are secreted toxins, one of which is listeriolysin O (LLO), a cytolysin encoded by the hly gene belonging to the CDC family. This protein is crucial for pathogen survival within the cytoplasm of the infected cell [Pore-forming cytolysins are a class of membrane-damaging toxins without enzymatic activity. They act by the insertion of their hydrophobic regions into the cell membrane phospholipid bilayer, effectively disrupting the target cells. The most homogenous and numerous group of membrane pore-forming cytolysins are Cholesterol Dependent Cytolysins (CDC). CDCs are produced mostly by Gram-positive bacteria and share similar amino acid sequences and biochemical properties . Listerited cell ,4.Escherichia coli strains [in vitro experiments with human T leukaemia Jurkat cells [in vitro experiments to determine the haemolytic and cytotoxic activity of purified LLO on peripheral blood leukocytes. The concentration-dependent activity of purified LLO was tested on a human T cell leukaemia cell line (Jurkat) and on normal peripheral blood mononuclear cells (PBMC). This study might provide useful information for future in vivo testing of LLO.There have been several attempts to develop effective LLO purification methods using recombinant  strains -9. Previat cells . Here weE. coli harbouring a pET29b-hly plasmid was optimized with a set of buffers with the following gradient: buffer pH ranging from 5 to 8 and NaCl concentration from 0 to 0.5 M. The concentration of imidazole ranged from 0 to 100 mM (for the column buffer) and from 0.25 to 1 M (for the elution buffer). Final protocol and optimal buffer composition are described in the Methods. Notably, lowering the pH of the elution buffer from 8 to 6 allowed a four-fold reduction of the imidazole concentration without a detectable loss of efficiency as compared to the original elution buffer . The analysis of electrophoretically separated E. coli lysates and purified LLO preparation was performed. The results of SDS-PAGE for LLO purified fractions and western blotting results with anti-LLO antibodies are presented in Figure E. coli sonicate (lane 1), purified LLO preparation (lane 2), and western blotting of purified LLO preparation with anti-LLO antibodies (right B 1 and 2 lanes) suggested the presence of a highly uniform protein preparation.The presence of a single protein of approximately 58 kDa, equivalent to the LLO molecular mass was observed. The cytotoxic potential of the purified LLO preparation, prior to application on a Jurkat cell line, was tested on SRBC (sheep red blood cells) for haemolytic activity assay. For most experiments preparations were standardized as follows: the LLO concentration was set at 1.5 μg/ml and samples (3000 HU/ml) were stored at −70°C. To facilitate its application, conditions for longer storage were optimized. The following compounds at various concentrations were tested: glycerol (from 0 to 20%), buffer pH (from 5 to 8), cysteine (from 0 to 10 mM), EDTA (0 to 20 mM) and AEBSF - fluoro 4-(2-aminoethylo)-benzenesulphonyl.HCl (0 to 2 mM). The results for the effects of glycerol and pH on LLO stability are presented in Figures The synthesis and affinity purification of His-tagged LLO from  samples 000 HU/mlThe most potent concentration of glycerol (15%) and pH value (6) were used for all further experiments. For the remaining reagents tested, the differences in their ability to preserve LLO activity were less distinguishable (data not shown). However, the presence of protease inhibitors was necessary to preserve optimum LLO activity. We concluded that the following conditions for LLO preparation storage were optimal for the purified LLO preparation: 1 mM AEBSF, 10 mM EDTA, 15% glycerol, 5 mM cysteine-HCl, pH 6 and a temperature of −70°C. We observed that even after several months of storage, LLO samples retained significant activity.The cytotoxicity of the purified LLO preparation was tested on a Jurkat cell line at 22°C and 37°C Figure . The cytThe cholesterol dependency of LLO was tested. The cytotoxicity of the LLO preparation was tested on Jurkat cell line in NCS  medium containing cholesterol (390 μM) Figure . The resThe cholesterol concentration dependence on LLO was compared with RPMI1640, and RPMI1640 containing 26 μM cholesterol or 5 mM of MβCD. Statistically significant (p < 0.004) differences were noted between cholesterol or MβCD samples and the controls. Interestingly, in medium containing 5% LLO and cholesterol, up to 54% of Jurkat cells survived. A similar percentage of cell survival (51.5%) was observed in the presence of MβCD in the medium as compared with control medium where only 2% of Jurkat cells survived. When both factors were present almost all cells survived, and the cytotoxic activity of LLO was completely blocked. Here the MβCD molar concentration was about 200 times higher than the cholesterol concentration.6 cells/ml, incubated in RPMI 1640-10% NCS with protease inhibitors and their survival rate tested after incubation with the purified LLO preparation. Concentration dependence survival was observed  with one exception [In this study, an xception , here wexception , the purxception ,4. The sListeria monocytogenes protein. Listeriolysin O is crucial for pathogen survival within the cytoplasm of infected cells [L. monocytogenes is better adopted to infect humans than mice and might explain its high activity towards leukocytes that normally threaten L. monocytogenes survival in human cells.It was shown that purified LLO demonstrates the properties of an authentic ed cells ,4. Less ed cells ,12. Whiled cells . Possibled cells . L. monoin vivo conditions, the presence of cholesterol is expected in peripheral blood plasma; however, it may also be present within the tissue matrix [in vitro by the additional application of cholesterol or MβCD. This might also have practical significance in vivo although the safety of such a treatment might be an issue. However, it might be possible since cyclodextrins are already in clinical use [Pseudomonas aeruginosa and diphtheria toxin from Corynebacterium diphtheriae[The cytotoxic activity of purified LLO was observed when applied to human leukocytes in the presence of a known concentration of cholesterol and/or MβCD in the medium, which removed cholesterol from cell membranes. We concluded that the presence of cholesterol in cell membranes and in the culture medium modified the cytotoxic activity of purified LLO. During e matrix . The LLOical use . The LLOphtheriae,18. Althphtheriae. The cytin vitro activity towards leukaemia cells. LLO activity was easily regulated in vitro and possibly in vivo, and this information might facilitate its future clinical application.In this study, we purified LLO by affinity chromatography demonstrating the usefulness of acidic buffer to remove LLO from Ni-NTA resin. The satisfactory storage conditions of the purified LLO preparation were also demonstrated to be an important step before planning use of the toxin. This study underscores the role of cryopreservative glycerol and a thiol-reducing agent, cysteine, for LLO stability and activity. The storage additives in the doses used were not toxic to eukaryotic cells, while LLO displayed potent E. coli strain BL21(DE3)  with plasmid pET29B (Novagen) carrying cloned hly gene, kindly provided by Dr. Higgins [The source of purified LLO was  Higgins . The bacEukaryotic cells were: SRBC - sheep red blood cells , human acute T cell leukaemia cell line , and PBMC isolated from humans or mice.The target Jurkat cells were grown in RPMI 1640 culture medium  with 10% of inactivated NCS containing 100 U of penicillin and 0.1 mg of streptomycin per 1 ml or in DMEM medium (Sigma) with 10% of inactivated NCS. Serum contained 150 mg/dl cholesterol, and the final cholesterol concentration in the culture medium was 15 μg/ml (39 μM).in vitro for 2–7 days before a cytotoxicity experiment. For human peripheral blood experiments in vitro, patients gave their personal consent and the procedure was accepted by the Bioethical Commission at the Medical Center of Postgraduate Education, Warsaw .PBMC were cultured, after isolation, g. The interlayer of PBMCs consisting of lymphocytes and monocytes was separated and diluted with 10 ml of culture medium, centrifuged for 5 min at 200 × g and the washed cells suspended in fresh culture medium were counted in a Burker camera. The concentration of the cell suspension was corrected to a final value of 2 × 106/ml before culturing. The cultures were performed in Falcon vessels (dishes) or in 6-well flat bottom plates in RPMI 1640 with 10% NCS and antibiotics, and half of the medium volume was changed twice weekly.Isolation of PBMC was performed by an overlayer of peripheral blood diluted with PBS (1:1), on 5 ml of Histopaque 1077 (Sigma) preparation in a test tube and centrifugation for 30 min at 540 × For some experiments cell cultures were supplemented with the following compounds to modify the LLO activity: cholesterol (Sigma), NCS containing 150 μg/ml cholesterol (390 μM), and MβCD (Sigma).E. coli suspension (approximately 108/ml) grown in the presence of 1 mM IPTG was centrifuged at 4400 × g for 10 min at 4°C, and the supernatant was removed. The pelleted cells were resuspended in 1 ml of sterile column buffer  and were ruptured by sonication in 3 pulses of 30 s each. Then, the bacterial fragments were removed by centrifugation . The supernatant was collected, diluted with column buffer and applied onto the affinity column  previously equilibrated with the column buffer. The column was washed with the same buffer, and then LLO was desorbed with the elution buffer . The collected fraction was subsequently dialysed to remove imidazole . Protease inhibitors: fluoro 4-(2-aminoethylo)-benzenesulphonyl.HCl , EDTA (Sigma), and glycerol (Merck) were added to a final concentration of 1 mM, 10 mM, and 15% (v/v), respectively. Protein concentrations were assayed with NanoOrange® Protein Quantitation Kit (Molecular Probes) using an infinite M200 PRO reader .Ten ml of Electrophoretically resolved proteins (SDS-PAGE) were transferred onto a nitrocellulose membrane using the BIO-RAD Trans-Blot system, according to a protocol recommended by the manufacturer. The membrane was blocked using 5% skimmed milk in Tris-buffered saline buffer (TBS), pH 7.6. Primary rabbit polyclonal anti-LLO antibody  and secondary goat polyclonal antibody (Abcam) conjugated with alkaline phosphatase were used at 1:1500 and 1:10,000 dilutions, respectively. The membrane was developed using the NBT/BCIP reagent (Merck).g. The released haemoglobin was measured spectrophotometrically at λ = 410 nm. Haemolytic Units (HU) were calculated after setting 0 HU as the activity of a negative control, and 100 HU for total haemolysis, observed in samples of erythrocytes lysed with 0.01% SDS.Haemolytic activity was assayed using SRBC. The erythrocytes were washed three times with PBS and suspended to a final concentration of 20% (v/v) in PBS (pH 7.4). The LLO preparation was diluted by adding 10 μl of its preparation to a final volume of 1 ml of 2% SRBC suspension in PBS. The prepared solution was incubated for 30 min at 37°C and then centrifuged for 3 min at 150 × Jurkat cells were resuspended in fresh RPMI medium without any additives (control), supplemented with 26 μM cholesterol, with 5 mM MβCD or with both these compounds. The cells were incubated for 1 h at 37°C, washed with PBS three times, counted and disrupted by sonication. Membrane cholesterol was extracted with isopropanol and quantified by a cholesterol assay kit .6/ml) of 20 μl, was mixed with 30 μl of LLO preparation diluted with physiological salt solution at different proportions, and immediately incubated for 30 min at room temperature (20–22°C). The time of the incubation was assumed to be the same as for the haemolytic assays. The final cell concentration was 0.4 × 106/ml. The effect of the LLO preparation was titrated in an assay volume of 50 μl containing cell suspension (20 μl) and 30 μl of physiological salt solution containing the LLO preparation. The LLO preparation content investigated was: 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, and 50% of the original LLO concentration (1.5 ng/μl) with activity set at 3 HU/μl. After cell sample incubation, physiological saline containing PI (1 μg/ml) was added before acquisition by a cytometer. PI penetrates the cell membrane of damaged cells and binds to the cell nucleus. In the fluorescence quadrant readings of forward scatter (FSC)/PI fluorescence, the PI unstained events indicate the percentage of living cells. In the FACSCalibur BD cytometer, 5000 events were collected from each sample and analysed by Cell Quest software . Experiments were repeated at least three times.The activities of the LLO preparations were tested on Jurkat cells or PBMC. The Jurkat cell suspension in a culture medium  after logit transformation of values. The post hoc test of Tukey was used. Results of haemolytic assays were presented as mean values ± standard deviation (SD). Analysis of variance was performed by ANOVA. Statistically significant values of p < 0.05 were assumed. Statistical software SAS 9.2 was used.The flow cytometry results were presented as median values and percentile values PThe authors declare no competing interests.RS contributed to experimental strategy, analysed data, conducted experiments, wrote and submitted the manuscript. MŁ performed cytometric assays of eukaryotic cells and drafted the manuscript. MG managed all aspects of the protein purification and optimised the method. KR conducted SDS-PAGE and western-blot analysis. TJ contributed to development of the LLO purification method and edited the manuscript. JB supervised the experimental design and execution and guided manuscript preparation. BB isolated PBMC from patients and cultured the cells. GH isolated and cultured mouse PBMC and managed tissue cultures. JK conceptualized the experimental strategy, supervised cytometric assays and significantly contributed to the writing of the manuscript. All authors read and approved the final manuscript.