AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein.The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and  Within the last decade there has been a renewed interest in androgen receptor (AR) signaling, as it pertains to normal prostatic function, prostate carcinogenesis, and metastatic progression. In the normal prostate, AR functions via a reciprocal paracrine interaction between the epithelial and stromal cells in vitroin vivoDuring prostate carcinogenesis, both AR-independent and AR-dependent signaling mechanisms contribute to the malignant transformation of epithelial cells The presence of AR expression, somatic AR mutations or amplification does not necessarily mean, however, that a prostate cancer cell is addicted to AR signaling. This statement is based on the present study, which documents an additional subtype of human prostate cancer cells. In this subtype, AR has undergone a dominant-negative, loss-of-function mutation even though AR is genetically amplified without the patient ever receiving androgen ablation therapy. Such a dominant-negative loss of AR function in malignant cells produces a selective growth advantage by eliminating the normal androgen-dependent AR signaling-induced growth suppression; however, while necessary, it is insufficient for these malignant cells acquiring an oncogenic addiction to AR-signaling.All animal studies were performed according to animal protocol MO09M434 approved by the Johns Hopkins Animal Care and Use Committee specifically for this study., and LNCaP C4-2B  cells were maintained in RPMI 1640 , and LAPC-4 cells in IMDM (Invitrogen) plus 1 nM R1881, both containing 10% FBS , 1× Pen/Strep, and L-glutamine. CWR22 xenograft tumor tissue was a kind gift from Thomas Pretlow . Charcoal/dextran stripped FBS (csFBS) was obtained from Hyclone and used to supplement phenol red-free RPMI (Invitrogen) for luciferase and chromatin immunoprecipitation assays. The synthetic androgen R1881 was purchased from Perkin-Elmer . PSA analysis was conducted by the JHMI Clinical Chemistry Laboratory. Cell growth was measured by a 3--2,5-diphenyltetrazolium bromide (MTT) assay ). Briefly, cells were plated in a 96-well format and allowed to adhere overnight. Cells were treated with vehicle or drug. At specific days, 15 µl of the MTT dye reagent was added to each well, plates incubated at 37°C for 4 hrs, 100 µl of Stop reagent was added to each well, and plates read by a Molecular Devices SpectraMAX plus plate reader at 570 and 650 nm. Absorbance readings were then plotted against a standard curve to determine cell numbers. Results are presented from day 4. Clonogenic survival was assayed as follows: 500 cells  were plated per well in 6-well plates, allowed to adhere and colonize for six days, then simultaneously fixed and stained in a 0.5% crystal violet/25% methanol solution and colonies were counted manually.E006AA Cytogenetic analysis was performed using standard methods. Chromosomal abnormalities were described according to ISCN guidelines In vivo growth assays were performed as previously described Immunostaining for AR , PSA , ΔNp63 , Nkx3.1 (UMBC), and CK5  was done on xenograft sections as previously described TM lentiviral shRNA transduction particles targeting either the AR alone or a non-silencing shRNA control . 1×104 E006AA or LNCaP cells were plated per well and allowed to adhere for 4 or 24 hrs, respectively. Cells were transduced at an MOI of 2 (2×104 particles/well) without polybrene. Antibiotic selection [1.5 µg/ml puromycin (Sigma)] was initiated 48 hrs later.Stable wild-type human AR cDNA expression and FACS sorting for GFP-positive cells was done as previously described Pfx DNA polymerase (Invitrogen) according to the manufacturer's specifications with the following overlapping primer sets: (1) 5′-AGAGAGGTAACTCCCTTTGGCT-3′ and 5′-ACGCTCTGGAACAGATTCTGG-3′ (740bp), (2) 5′-TCCCGCAAGTTTCCTTCTCT-3′ and 5′-GATACTGCTTCCTGCTGCTGTT-3′ (756bp), (3) 5′-TGAGGAACAGCAACCTTCACAG-3′ and 5′-ACAGGGTAGACGGCAGTTCAA-3′ (704bp), (4) 5′-GCCGAATGCAAAGGTTCTCT-3′ and 5′-TTGACACAAGTGGGACTGGGA-3′ (700bp), (5) 5′-CAGTTGTATGGACCGTGTGGT-3′ and 5′-CTACACCTGGCTCAATGGCT-3′ (723bp), (6) 5′-CGGAAGCTGAAGAAACTTGG-3′ and 5′-AGAAGCGTCTTGAGCAGGAT-3′ (685bp), (7) 5′-ATTCCAGTGGATGGGCTGAA-3′ and 5′-TAGCTCTCTAAACTTCCCGTGG-3′ (714bp), (8) 5′-CTTCCCATTGTGGCTCCTAT-3′ and 5′-ACCTTCTCGTCACTATTGGC-3′ (361bp); and for genomic DNA amplification of exon 3 of the AR gene: (9) 5′-TGTTTGGTGCCATACTCTGTCCAC-3′ and 5′-GCATCCTCACTCACCTTCTGTTGG-3′ (530bp). PCR products were column purified using the Qiagen QIAquick kit (Qiagen) and were sequenced at the Johns Hopkins University DNA Analysis Facility using both forward and reverse primers.RNA was harvested using the Qiagen miRNeasy Mini kit  and contaminating DNA was removed using the Ambion DNA-free kit . Superscript reverse transcriptase III  and 1 µg of total RNA were used to generate cDNAs with Oligo-dT primers. Genomic DNA was extracted using the Qiagen DNA Mini kit and incubated with RNase  at 37°C for 30 min to remove contaminating RNA. PCR amplification of cDNAs and genomic DNA was performed using Platinum Whole cell lysates were prepared on a per cell basis in lysis buffer  supplemented with PhosSTOP phosphatase inhibitor tablet and protease inhibitor tablet (Roche), and 1 mM dithiothreitol. Whole cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk (TBS +0.1% Tween-20) for 1 hr. The rabbit polyclonal AR antibody (SC-816), and mouse monoclonal CK8 (SC-53266) and mouse monoclonal CK18 (SC-32722) antibodies were purchased from Santa Cruz Biotechnology , and the mouse monoclonal ß-Actin antibody was purchased from Sigma (A5441) and were used in conjunction with an anti-rabbit-HRP antibody  or anti-mouse-HRP .Nuclear and cytoplasmic cell fractions were prepared using the Nuclear Complex Co-IP kit  according to manufacturer's instructions. In brief, cells were washed and then scraped in cold PBS supplemented with phosphatase inhibitors. Cell pellets were resuspended and lysed in 1× hypotonic solution plus detergent, centrifuged at 6,000×g for 5 min at 4°C, and cytoplasmic fractions removed. Nuclear pellets were resuspended in digestion buffer plus enzymatic shearing cocktail and incubated at 4°C for 90 min. 0.5 M EDTA was added to stop enzymatic shearing reactions and nuclear lysates centrifuged for 10 min at 4°C. All fractions were combined with equal volume 2× SDS-sample buffer and denatured at 95°C for 5 min. Samples were separated on 4–15% PAGE and blotted with either the anti-AR , the cytoplasmic marker Vinculin (Sigma), or the nuclear marker HDAC (Santa Cruz Biotechnologies).Cells were plated (per well) in white polystyrene, clear bottom, sterile 96-well tissue culture plates accordingly: LNCaP C4-2B ; PC-3 ; E006AA, E006AA LV-AR, E006AA LV-non-silencing-shRNA, E006AA LV-AR-shRNA . Cells with or without androgen (1.0 nM R1881) were plated in six replicates in RPMI (Invitrogen) supplemented with 10% csFBS (Hyclone) but without phenol red or pen/strep and allowed to adhere overnight. Cells were transfected using Fugene 6 (Roche) with 50 ng Probasin-PSA-firefly and 5 ng pRL-TK-renilla  Luciferase reporter constructs per well at a ratio of 3∶1  in serum-free media 7 cells were resuspended in 10 ml media, fixed by adding 275 µl of 37% formaldehyde and gently shaken at 22°C for 12 min. After crosslinking, 1 ml of glycine was added and cells gently shaken for 5 min at 22°C, washed twice in cold PBS, resuspended in 500 µl cell lysis buffer plus 2.5 µl protease inhibitors  and centrifuged for 5 min at 4°C. Cell pellets were resuspended in 500 µl cell lysis buffer plus 2.5 µl protease inhibitors, incubated 15 min on ice with brief vortex every 5 min, and centrifuged at 800×g for 5 min at 4°C. Nuclear pellets were resuspended in nuclear lysis buffer plus 2.5 µl protease inhibitors . Chromatin was sonicated to an average DNA length of 500–1200 bp using Fisher Scientific Sonic Dismembrator Model 100 (4×15 sec pulses at setting 3). Five µl was saved for control input and 50 µl aliquots were diluted in 450 µl dilution buffer . Four µg of antibody was added to respective tubes: 4 µl of 1 mg/ml rabbit IgG or 40 µl 100 µg/ml rabbit anti-AR  and rotated O/N at 4°C. Immune complexes were centrifuged 10 min at 10,000×g for 4°C to pellet any aggregates or non-specific complexes. The supernatant (desired immune complexes) was harvested and combined with 20 µl protein A Dynabeads  and gently stirred 2 hrs at 4°C. Tubes were placed in a Millipore magnetic rack and immune complexes sequentially washed 1 time with low salt buffer , high salt buffer , LiCl buffer, and TE, with 3 min stirring each wash step. To reverse the crosslinks, beads were resuspended in 100 µl of Chelex and boiled 10 min at 95°C, centrifuged 1 min at 12,000×g at 4°C, and supernatants collected. Beads were resuspended in 120 µl water, centrifuged again, and supernatants pooled with previous supernatant. The captured DNA was purified using the Qiagen PCR purification kit (#28104) according to manufacturer's instructions and analyzed by PCR and q-PCR.LNCaP C4-2B and E006AA cells grown in phenol red-free RPMI, supplemented with 10% csFBS, were harvested via trypsinization and chromatin prepared according to the MagnaChIP kit from Upstate, Temecula, CA. ∼1×105′-GCCAAGACATCTATTTCAGGAGC-3′, rev 5′-CCCACACCCAGAGCTGTGGAAGG-3′; KLK2 promoter (52°C) fwd 5′-CTCCAGACTGATCTAGTATG-3′, rev 5′-TTGGCACCTAGATGCTGACC-3′; SP1 site P45Skp2 promoter (53°C) fwd 5′-GATCCACGCTCAGAGACGAC-3′, rev 5′-TTCGAGATACCCACAACCCC-3′; Tcf4 binding element in c-Myc promoter (55°C) fwd 5′-GCTCTCCACTTGCCCCTTTTA-3′, rev 5′-GTTCCCAATTTCTCAGCC-3′.Using 5 Prime HotMasterMix (Eppendorf), PCR reactions were performed as follows: 94°C for 2 min, then 32 cycles 30 sec denaturation at 94°C, 30 sec of annealing (as indicated below for each primer pair), and 30 sec of elongation at 65°C, then a 2 min final elongation at 65°C. PCR products were separated on 2% Agarose gels and stained with ethidium bromide. Primers for amplification of DNA fragments are as follows: PSA promoter ARE (52°C) fwd 5′- CCTAGATGAAGTCTCCATGAGCTACA-3′, rev 5′-GGGAGGGAGAGCTAGCACTTG-3′; ARE III fwd 5′-TGGGACAACTTGCAAACCTG-3′, rev 5′-CCAGAGTAGGTCTGTTTTCAATCCA-3′AR ChIP – Ctinput)x100. Student's t-test was used to determine p-value.Using BioRad IQ SYBER Green Supermix , PCR reactions were performed with the BioRad ICycler as follows: 95°C for 3 min, then 40 cycles 30 sec denaturation at 95°C, 30 sec of annealing at 55°C, and 45 sec of elongation at 72°C (data were collected during extension), followed by a 110cycle melt curve from 45–100°C to ensure primer efficiency. Previously designed primers for quantitative amplification of DNA fragments containing a putative ARE in the PSA gene promoter and an ARE III in the PSA enhancer region are: ARE fwd cnull triple deficient mice  were initiated 3 by six weeks, in 100% of the mice. After six weeks, however, all of the E006AA tumors in intact male nude mice stopped growing and regressed over a second period of 5–10 weeks  or nude mice (n = 5). Similar to our previous observations, transplanted E006AA tumors underwent delayed regression in intact male nude mice while forming continuously growing tumors in NOG-SCID hosts . These cin vivo, by inoculating these cells into both intact (n = 10) and castrated (n = 8) male NOG-SCID hosts. Tumors developed equally well in both hosts and there was no difference in the growth rate or tumor volume in the castrated vs. intact NOG-SCID animals  was performed with probes specific for the AR gene [Xq12]  and the X centromere   . Interphth position (AGC to GGC). This single base change results in an amino acid substitution from serine to glycine at position 599  . Sequenc9 S599G, . The S59AR gene within E006AA cells all harbor the same mutation. These data document that the AR gene mutation in E006AA was a somatic mutation that likely occurred prior to gene amplification and X-chromosome duplication.To validate this single nucleotide change, each E006AA cDNA PCR product, generated from multiple cell passages, was sequenced using both sense and anti-sense primers. Additionally, genomic DNA was obtained from E006AA as well as from the patient-matched S006AA cells; PCR amplification using an intronic forward primer and an exonic reverse primer, designed to amplify only the mutation-containing region in exon 3, verified that E006AA cancer cells harbor the S599G mutation while the matching stromal S006AA cells do not. All sequencing reactions demonstrated an unambiguous signal at the mutation site, indicating that amplified copies of the Quantitative PCR analysis of AR mRNA in E006AA compared to LNCaP cells found that E006AA cells express approximately 23-fold lower levels of AR transcript than LNCaP cells , but wheAR is mutated and amplified in E006AA cells, the in vitro growth of these cells, as determined by a cell growth and viability MTT assay normalized for cell counts, is not affected by the addition of an increasing dose of synthetic androgen , or anti-androgen   . Also, Eanalysis  and, as To begin resolving whether the lack of AR signaling is due to the mutant AR, wild-type AR (wt-AR) was introduced into E006AA cells via lentiviral transduction, to co-express ectopic wt-AR, and cells isolated via fluorescence activated cell sorting (FACS) for GFP-positive cells in vivo castration studies, these data demonstrate that the growth of E006AA cells is not dependent on AR protein expression and function.These results raise the issue of whether AR signaling is needed at all for the growth of E006AA cells. To address this, AR protein expression was suppressed using infection of anti-AR shRNA lentiviral particles. Western blotting confirmed a stable >95% knockdown of AR protein in these E006AA LV-AR-shRNA transduced cells . Such ARThe inability of both wt-AR expression and targeted knockdown of the endogenous mutant AR to affect the growth of E006AA cells implies that the AR signaling machinery is deficient in E006AA cells. This could involve, among many possible mechanisms, impaired AR nuclear translocation or a loss-of-function due to the S599G mutation. To test this, the nuclear translocation and transcriptional activity of both the mutant and exogenously expressed wt-AR were analyzed. It has been well documented that, in the absence of ligand, AR is predominantly localized in the cytoplasm; however, upon binding ligand they localize to the nucleus where they bind DNA and activate target genes Skp2 promoter Knowing that endogenous, mutant AR in E006AA cells translocates to the nucleus upon stimulation with androgen, the next step was to evaluate whether the S599G mutant AR in E006AA cells had the capacity to bind DNA. Using an anti-AR antibody, chromatin was immunoprecipitated from E006AA and LNCaP C4-2B cells grown in reduced serum conditions (csFBS + phenol red-free RPMI) in the presence or absence of androgen. The captured DNA was subjected to semi-quantitative PCR amplification using primers designed to amplify putative androgen response elements (ARE) located in the PSA promoter. Positive amplification is detected in AR-immunoprecipitated chromatin from both E006AA and C4-2B cells, demonstrating that the endogenous, mutant AR expressed in E006AA cells binds DNA, regardless of the presence of ligand . To testTo test whether endogenous AR in E006AA cells possesses the capacity for AR-mediated gene transcription, transient transfection assays were performed using a Probasin-PSA-Firefly Luciferase vector, which is optimized for measuring AR transcriptional activity The earlier demonstration that S599G mutant AR in E006AA cells translocates to the nucleus upon androgenic stimulation, where it effectively binds ARE-containing DNA but does not stimulate transcription of AR target genes, raises the issue of whether the S599G mutation results in a loss-of-function. To evaluate this possibility, the S599G mutant AR was cloned and placed in a Lentiviral vector to create a LV-AR-S599G construct. We then assessed Luciferase activity in the positive control LNCaP C4-2B cells transiently co-transfected with the LV-AR-S599G and Probasin-PSA-Firefly Luciferase vectors. In LNCaP C4-2B cells transiently expressing S599G AR, Luciferase activity was reduced 63%, in multiple experiments, compared to parental LNCaP C4-2B cells . These cProstate cancer cells can be subdivided based upon AR protein status and response to castration. In one subtype, AR protein is not expressed  and thus cell survival and proliferation is completely independent of AR signaling. These AR negative cells are thus castration resistant. In another subtype of prostate cancer cells , AR is expressed and has acquired a gain-of-function such that it no longer inhibits but now stimulates proliferation when activated by physiological levels of androgen AR gene mutations database for AIS [http://androgendb.mcgill.ca], but has not been previously linked with prostate cancer. This mutation, however, does not disrupt normal androgen binding kinetics E006AA cells represent a third new subtype of AR-expressing prostate cancer cells, which exhibit castrate-resistant growth characteristics even though these cells were derived from a hormonally naïve patient. E006AA cells possess many of the same molecular characteristic changes commonly observed in prostate cancer metastases and cell lines derived from patients who were androgen ablated. These include AR gene amplification and mutation. The significance of the S599G mutation is its precise location within the DBD, and more specifically in the dimerization interface region known as the D box The defining characteristic of E006AA cells is the complete absence of AR growth-regulatory function, even though these cells express AR protein. This raises the issue of why such molecular changes in AR  would have a selective advantage. One explanation is that while these changes prevent AR signaling from promoting either survival or proliferation of malignant prostate cells, the lack of AR signaling eliminates the growth suppression normally induced by AR signaling in non-transformed prostate epithelial cells in a host with normal physiological levels of androgen. This is consistent with the loss of AR-mediated growth suppression providing a selective growth advantage, even without the simultaneous acquisition of a gain-of-function ability of AR signaling to stimulate survival and proliferation. This suggests that the biochemical mechanism for loss of AR signaling-induced growth suppression is not identical, and not always coupled, to that associated with the gain of AR signaling-induced growth stimulation in prostate cancer cells. The inability to express AR regulated genes, such as PSA, Nkx3.1, and KLK2, could also be due to alterations in the AR transcriptional machinery in E006AA cells, in addition to possible epigenetic changes that have altered the chromatin configuration found in E006AA cells. These possibilities raise important questions for future studies and defining these distinguishable mechanisms is a focus of our ongoing experiments, for which E006AA cells are an important reagent.Regardless of the mechanism, the realization that loss of growth suppression and gain of survival and proliferation stimulation by AR signaling are distinct processes has significant clinical implications. For example, as many as 50% of patients with castrate-resistant metastatic prostate cancer have circulating cancer cells that have substantial amplification of the AR locus, which has been interpreted to predict that, in such patients, androgen signaling continues to play an important growth stimulatory role


The incidence of physical disability in the community resulting from neurological dysfunction is predicted to increase in the coming years. The impetus for immediate and critical evaluation of physical neurorehabilitation strategies stems from the largely incomplete recovery following neurological damage, questionable efficacy of individual rehabilitation techniques, and the progressive acceptance of evidence-based medicine. The emergent technologies of non-invasive brain stimulation (NBS) and rehabilitation robotics enable a better understanding of the recovery process, as well as the mechanisms and effectiveness of intervention. With a more precise grasp of the relationship between dysfunctional and treatment-related plasticity, we can anticipate a move toward highly controlled and individualised prescription of rehabilitation. Both robotics and NBS can also be used to enhance motor control and learning in patients with neurological dysfunction. The merit of these contemporary methods as investigative and rehabilitation tools requires clarification and discussion. In this thematic series, five cohesive and eloquent papers address this issue from leading clinicians and scientists in the fields of robotics, NBS, plasticity and motor learning. There is a pressing need to improve physical neurorehabilitation strategies. This might be accomplished by learning from observations of accurately defined physiological and behavioural aspects of motor recovery. Technologies such as non-invasive brain stimulation (NBS) and robotics can provide sensitive and reliable measures to achieve this, and might additionally be incorporated as therapeutic tools to augment rehabilitation strategies. While these techniques are still developing, over a decade of scientific work is uncovering their mechanisms and efficacy. To the rehabilitation practitioner, this literature may seem esoteric and far removed from daily practice. This thematic series brings together leaders in the field of robotics, NBS and plasticity, to provide an up-to-date overview for scientists and clinicians interested in physical neurorehabilitation.st century may be an important time in the advancement of the medical field, where practice not based on credible scientific evidence is increasingly questioned. As well, this time might be characterized by rapid and global dissemination of research findings, and the emergence of technologies that may translate into significant improvements in preventative medicine and acute care. Yet, according to the World Health Report 2008, we can anticipate an unprecedented impact of diseases resulting from our progressively ageing population [The early 21pulation ). Physicspecific'. But there was debate amongst rehabilitation professionals about the most effective type and amount of therapy. Presently, the superiority of the various techniques still remains questionable when held to scientific scrutiny.Historically, the management of stroke survivors involved bed-rest and convalescence, reminiscent of the familiar quote attributed to Voltaire (1694–1778); "the role of the physician is to entertain his patient while nature takes it course". The concept of physical therapies developed through the early to mid 1900s, resulting largely from efforts to help world-war and polio survivors. The branch of medicine devoted to active rehabilitation was formed, and a shift from 'do nothing' to 'do something' occurred. This was based on the understanding of neurological dysfunction at the time, and the observation that patients appeared to tolerate and benefit from physical interventions. Observations that patients had better outcomes if exposed to 'enriched environments' versus bed-rest were increasingly reported. However the critical aspects of the enriched experience remained an enigma. Distinct approaches to rehabilitation practice emerged in parallel to increasing understanding of the human nervous system and the response to physical interventions. These approaches had different characteristics which might be loosely summarized as focusing on the following outcomes: impairment reduction, suppression of muscle tone and restoration of normal movement, or repetitive training to improve function. As hospitals and care facilities world-wide increasingly incorporated physical therapies as routine management for neurological patients, there was a move from 'do something', to 'do something anything'? This is of course impudent since informed and experienced practitioners intelligently prescribe safe and appropriate therapies leading to clinically meaningful improvements. But what makes therapies effective? Was Voltaire right? Do we serve to 'entertain' or perhaps more appropriately 'encourage' or 'motivate'? There is clearly something about the psychosocial nature of regular interaction with health professionals that is beneficial, but also that active, motivated engagement in activity leads to higher levels of function. If the benefit is to extend beyond orthopaedic, and impact recovery of motor control, we know that therapies cannot be passive and patients must be engaged. Perhaps part of the 'magic' in the hands of the individual therapist might be the ability to engage the patient and direct appropriate attention to task.A recent review of randomized controlled trials in physical therapy treatment alternatives (in over 1000 stroke patients), indicates that physical therapies provide improvement in function when compared to no treatment or control . Yet impIt is apparent then, that focused guided rehabilitation efforts, performed repetitively, result in improved motor function together with the decreased risk of secondary health problems. Yet this is labour intensive. Furthermore, patients often find the training uninteresting, tiring and lacking meaning for them. Robotic or Virtual Reality technology may overcome these barriers by making physical rehabilitation interesting, challenging and even addictive, all perhaps necessary ingredients for relearning motor skills after neurological damage. There is clearly a need for optimising rehabilitation strategies to provide cost efficient, fun, effective methods that maximize long-term health and function. The scientific challenge is to build on existing technologies and strategies to achieve this important outcome.clearly-described physical rehabilitation methods, at a specific time for specific patients. Rehabilitation robotics may represent the most sophisticated method available today for reliably and precisely driving therapeutic engagement and measuring precise outcomes. Robotic technology can be used to quantify and track motor behaviour for individual patients. Kinematic and kinetic data can be obtained during therapy sessions or during a separate evaluation, thus making rehabilitation robots an ideal tool to provide motor control measures – more sensitive and reliable than standard clinical scales. In addition to helping us understand motor control in relation to specific lesion and individual characteristics, robotics can help us understand the effectiveness of motor learning paradigms. Robotics can be used to investigate if patients recovering from neurological lesion might acquire motor skills similar to healthy adults. Huang and Krakauer present a paper titled 'Robotic Neurorehabilitation: A Computational Motor Learning Perspective' which provides a comprehensive and lucid discussion of what is currently known about return of motor function following neurological damage, and what we can take from computational motor learning literature to set up training paradigms using robotic devices that might optimally promote development and retention of motor skills and translate into long-term reduction in disability.In order to progress from current rehabilitation strategies, we need to have a solid and precise understanding of specific therapies. There is a need for research into the effectiveness of Also supporting a quantitative scientific understanding of mechanisms of post-stroke recovery, Krebs, Volpe, and Hogan present a paper titled 'A Working Model of Stroke Recovery from Rehabilitation Robotics Practitioners'. Their perspective is based on experience with the implementation of rehabiltitation robotics in a large number of stroke patients, and they present data supporting a model of motor recovery post-stroke, resulting from their experiences. They propose that coordination training may be most the beneficial form of intervention and may lead to motor relearning, while resistive training is only beneficial in some cases, and passive training resulted in no motor learning.training paradigm in relation to a given stimulation paradigm. The authors suggest that the combination of NBS with functional therapies has the potential to drive plastic changes in brain-damaged patients, only if guided by a careful consideration of underlying mechanisms. This is important, since combined therapies may not necessarily be complementary.One of the contemporary methods under investigation as a tool to promote neuroplasticity is non-invasive brain stimulation. The concept of treating neurological disorders with electricity is not new, and despite an interesting history dating back to ancient times, was considered to be first seriously attempted in the mid 1700s; perhaps the start of medical electrotherapy . At thisOne premise of NBS as a potential adjuvant for therapy is that it induces plasticity and that plasticity is important and helpful. While evidence exists that NBS in humans can be beneficial, the circumstances under which it works most effectively and the mechanisms are not fully understood. Huerta and Volpe address potential mechanisms in their paper 'Transcranial magnetic stimulation, synaptic plasticity and network oscillations'. The paper provides insight from basic sciences literature about cellular mechanisms of plasticity, including an apposite discussion of processes leading to potentiation and depression of synaptic strength. The similarity of human NBS protocols to long-term-potentiation (LTP) and long-term-depression (LTD) protocols in animal studies is described, as well as implications for interpreting studies of NBS in human motor cortex. Successful therapeutic application of NBS techniques in humans additionally requires understanding the relationship of functional and dysfunctional plasticity across different neurological conditions. This is true even within a given condition, such as stroke, which itself represents a diverse mix of injury type, clinical presentation and recovery prognosis. Thickbroom and Mastaglia outline important considerations for advancing NBS as a potential therapeutic tool in disorders of the brain and spinal cord in a paper titled 'Plasticity in neurological disorders and challenges for noninvasive brain stimulation'. The paper describes how plasticity is implicated in neurological disorders, and discusses current NBS plasticity protocols applied in clinical research.This thematic series emphasizes the understanding and development of modern neurorehabilitation methods, and outlines some ways to quantify plasticity and recovery of function. However, we are only beginning to understand the complex interactions affecting recovery from acute brain injury (for review see ). The muThe rapid advancement in technology, together with more rigid clinical experimental protocols, will enable a better understanding of physical rehabilitation therapies. Coupled with an increasing knowledge of plasticity in neurological disorders, we can expect that rehabilitation practices and treatments will continue to evolve in parallel. The type and schedule of rehabilitation intervention may ultimately be prescribed based on detailed characteristics of the individual, and the time since lesion or state of the disease. I refer readers to the individual papers in this thematic series of JNER for an insightful and thorough account of the basis for these evolving practices.


Twelve patients with newly diagnosed non-Hodgkin's lymphoma (n = 9) or Hodgkin's disease (n = 3) participated in this study before start of oncologic treatment. Each patient underwent two [F18]FDG PET studies within 1 week after overnight fast: once during high fasting serum FFA concentrations and once after reduction of serum FFA by administration of acipimox. Acipimox is a nicotinic acid derivative that inhibits lipolysis in peripheral tissues and induces a striking reduction in circulating FFA concentration. In all cases, dynamic PET imaging over the tumour area was performed for 60 min after injection of [F18]FDG. Both graphical analysis (rMRFDG) and single scan approach (SUV) were used to compare tumour uptake of [F18]FDG under high fasting FFA concentrations and after pharmacologically decreased FFA concentrations. Serum FFA concentrations were reduced significantly from 0.92 ± 0.42 mmol l−1 at baseline to 0.26 ± 0.31 mmol l−1 after acipimox administration (P = 0.0003). Plasma glucose, serum insulin and lactate concentrations were similar during both approaches. The retention of glucose analogue [F18]FDG in tumour was similar between baseline and acipimox studies. Median rMRFDG of a total of 12 involved lymph nodes in 12 patients was 21.9 μmol 100 g−1 min−1 (range 8.7–82.5) at baseline and 20.1 μmol 100 g−1 min−1 (range 10.7–81.7) after acipimox. The respective values for median SUV were 7.8 (range 3.6–18.6) and 6.0 (range 4.1–20.2). As expected, [F18]FDG uptake in myocardium was clearly enhanced by acipimox due to reduction of circulating FFAs. In conclusion, blood fatty acids appear to have minor significance for [F18]FDG uptake in lymphoma. This suggests that glucose utilization is uncoupled of FFA metabolism and indicates that glucose-free fatty acid cycle does not operate in lymphomatous tissue. Glucose appears to be the preferred substrate for energy metabolism in tumours, in spite of the high supply of FFAs in the fasting state. Although acipimox and other anti-lipolytic drugs have potential for treatment of catabolic state induced by cancer, they are not likely to interfere with tumour energy metabolism which is fuelled by glucose. © 1999 Cancer Research CampaignIncreased use of glucose through glycolysis is characteristic for neoplastic growth while the significance of serum-free fatty acids for regulation of energy metabolism in cancer is poorly understood. We studied whether serum-free fatty acids (FFA) interfere with glycolytic metabolism of lymphoproliferative neoplasms as assessed with 2-F


Hyperglycemia in premature infants is associated with increased morbidity and mortality, but data on long-term outcome are limited. We investigated the effects of neonatal hyperglycemia  on growth and neurobehavioral outcome at 2 years of age.Retrospective follow-up study at 2 years of age among 859 infants ≤32 weeks of gestation admitted to a tertiary neonatal center between January 2002 and December 2006. Thirty-three survivors treated with insulin for hyperglycemia and 63 matched controls without hyperglycemia were evaluated at a corrected age of 2 years. Outcome measures consisted of growth  and neurological and behavioural development.66/859 (8%) infants ≤ 32 weeks of gestation developed hyperglycemia. Mortality during admission was 27/66 (41%) in the hyperglycemia group versus 62/793 (8%) in those without hyperglycemia (p < 0.001). Mortality was higher in infants with hyperglycemia with a birth weight ≤1,000 gram (p = 0.005) and/or gestational age of 24-28 weeks (p = 0.009) than in control infants without hyperglycemia. Sepsis was more prominent in infants with hyperglycemia and a birth weight of >1,000 gram (p = 0.002) and/or gestational age of 29-32 weeks (p = 0.009) than in control infants without hyperglycemia. Growth at 2 years of age was similar, but neurological and behavioural development was more frequently abnormal among those with neonatal hyperglycemia (p = 0.036 and 0.021 respectively).Mortality was higher in very preterm infants with hyperglycemia treated with insulin during the neonatal period. At 2 years of age survivors showed normal growth, but a higher incidence of neurological and behavioural problems. Better strategies to manage hyperglycemia may improve outcome of very preterm infants. Hyperglycemia is a common problem in very preterm infants. Among extremely low birth weight infants the incidence of neonatal hyperglycemia is estimated to be between 45% and 80%-3. This Prematurity is an important risk factor in the complex pathogenesis of hyperglycemia. Insulin resistance develops due to a high circulating level of inflammatory markers, cytokines and catecholamines. Consequently, glucose production in the liver is not inhibited. The pancreas needs to produce insulin to compensate, but is probably unable to do so due to immature beta-cells, leading to relative insulin deficiency,9,10.Since twenty-five years continuous insulin infusion is used, nevertheless in many centers restriction of glucose intake is still the first step in treatment of hyperglycemia.Comparison of these interventions has demonstrated the efficacy of continuous insulin infusion in glucose control. Infants treated with insulin had a higher glucose intake, a higher weight gain, less sepsis and an increased endogenous insulin production,12. The Whereas the short term effects of neonatal hyperglycemia in very preterm infants have been reported, less is known about the effects of hyperglycemia on growth and neurobehavioural outcome. The aim of this retrospective follow-up study was to determine the possible effects of hyperglycemia on growth and neurobehavioral development by comparing very preterm infants with and without a history of neonatal hyperglycemia at the corrected age of two years.The study population consisted of 859 very preterm infants (≤ 32 weeks) admitted to the Neonatal Intensive Care Unit (NICU) of the Leiden University Medical Center between January 2002 and December 2006. Neonatal data and data on growth and neurobehavioural outcome at the corrected age of 2 years were collected from the charts in the tertiary neonatal center and the regional hospitals where these infants were regularly seen for follow-up. The Medical Ethics Committee of the Leiden University Medical Center did not require approval of this study because it consisted of retrospective chart review, nor did the medical ethics committee require written consent by the parents for their infant's information to be stored in the hospital database and used for research.Hyperglycemia was defined as at least 2 blood glucose levels of ≥10.0 mmol/L (180 mg/dL) during a 12-hour period. Insulin treatment was started at 0.05 U/kg/h  when hypBlood glucose levels were measured in whole blood using the glucose oxidase method. . Frequency of glucose level measurements was based on clinical and laboratory findings. During the first week after birth blood glucose levels were measured at least 6 times a day, thereafter glucose levels were measured at least 3 times a day. In the acute phase of hyperglycemia, glucose levels were measured regularly with intervals of approximately 1-2 hours.Data for demographic and perinatal characteristics as well as postnatal clinical conditions of all infants were collected from the charts and included birth weight, gestational age, length of stay (as an indicator of severity of illness), gender, exposure to prenatal and postnatal steroids, presence of chorioamnionitis, sepsis, prolonged rupture of membranes (PROM), respiratory distress syndrome (RDS), bronchoFor the exposed infants the mean, minimum and maximum glucose levels, glucose intake and insulin infusion rate were calculated for the first 5 days after the onset of the hyperglycemia episode or until the end of the hyperglycemia episode if the episode lasted longer than 5 days. Additionally, duration of hyperglycemia, frequency of hypoglycemic periods, duration of insulin infusion, mean serum sodium concentration, maximum daily percentage of inhaled oxygen and maximum infusion rate of inotropic drugs (dobutamine and dopamine), were recorded.Outcome data collected at the corrected age of 2 years (± 3 months) included weight (kg), length (cm), head circumference (cm), and neurobehavioral development. Neurological outcome was classified into 3 groups: normal, mildly abnormal  and severely abnormal  according to the neurological examination by Hempel. BehavioData are reported as mean values ± standard deviation, numerical values or categories. Statistical analyses were performed with SPSS Version 16.0 . Numerical growth parameters were analyzed with an unpaired T-test. The categorical data for behavioural and neurological outcome were analyzed using a chi-squared test.Subgroup analyses were performed for subgroups in gestational age (24-28 and 29-32 weeks) and birth weight .Potential confounders in this study were gestational age, gender, birth weight, length of stay, exposure to prenatal and postnatal steroids, presence of severe RDS, sepsis, PROM, chorioamnionitis, NEC, BPD, PVL and IVH. Confounding was prevented by matching and using multivariable regression analysis for analyzing mortality and morbidity, as well as growth, neurological and behavioural development. Infants with missing baseline characteristics were excluded from regression analyses.Between January 2002 and December 2006 a total of 859 very preterm infants with a gestational age less than 33 weeks were admitted for neonatal intensive care. Mean gestational age was 29.4 ± 2.0 weeks and birth weight 1,323 ± 410 gram. Fifty-six percent was male. Prenatal steroids were administered to 63% of the mothers. Chorioamnionitis and PROM were present in 13 and 27% respectively. Twenty-nine percent of the infants developed sepsis, 6% IVH grade 3 or 4, 3% cystic PVL, 2% NEC grade 2 or 3, 19% RDS grade 3 or 4 and 14% BPD at 36 weeks postmenstrual age. Postnatal steroids were administered to 10% of the infants. Hyperglycemia occurred in 66 (8%) infants of whom 53 had a birth weight of ≤1,000 gram, the incidence in this subgroup was 25%. The mean age at onset of the hyperglycemia episode was 3.2 ± 3.7 days. Twenty-seven out of 66 infants with hyperglycemia (41%) died during admission versus 62/793 (8%) infants without hyperglycemia. Six of the remaining 39 infants with hyperglycemia were excluded from this follow-up study because of major congenital anomalies (1) and loss to follow-up (5). Emigration (1), placement in an unknown foster home (1) and no appearance at follow-up appointments (3) were the reasons for the loss to follow-up. These infants were excluded from the analysis together with their unexposed matches. For 3 exposed infants only 1 unexposed match was available. The complete follow-up population consisted of 96 infants; a flowchart can be seen in figure An overview of the baseline characteristics of included patients is given in table Table Analyses of the data on the hyperglycemia episodes table  showed sA multivariable regression analysis on all very preterm infants admitted between January 2002 and December 2006 (n = 798 infants with complete baseline characteristics), confirmed a significant increase in mortality in the exposed cohort (p = 0.001) Table . SubgrouIn the subgroup of infants with a birth weight >1,000 gram  hyperglycemia was associated with an increased incidence of IVH (p = 0.025), severe RDS (p = 0.012) and sepsis (p = 0.002). Sepsis was also more prominent in a subgroup of infants with a gestational age of 29-32 weeks . In the regression analyses for morbidity and mortality statistical correction was done for gestational age, gender, birth weight, exposure to prenatal and postnatal steroids, presence of severe RDS, sepsis, PROM, chorioamnionitis, NEC, BPD, PVL and IVH.The findings in this study indicate that hyperglycemia has no effect on long term growth. However, in addition to the already known short term effects, hyperglycemia has a distinct negative influence on neurological and behavioural outcome at 2 years of age. The observation by other investigators that hyperglycemia is a risk factor for early mortality in very preterm infants was confirmed. We found a firm association between neonatal mortality and the mean and maximum glucose levels on days 3 and 4 of the hyperglycemia episode. This association suggests that increased mortality is probably due to poorly controlled hyperglycemia. No association between mortality and duration of hyperglycemia was found. In subgroup-analyses we found relations between hyperglycemia and morbidity. Sepsis, severe RDS and IVH were more common in infants with hyperglycemia and a birth weight of >1,000 gram .Hays et al. demonstrated that blood glucose concentration had significant effects on both early death and the occurrence of severe IVH. To our This retrospective study has several limitations. An observer-bias cannot be excluded because several physicians did the follow-up consults at the age of 2 year. To reduce the observer-bias as much as possible strict cut off points were made and the follow-up information was interpreted by only one person. In the future it would be interesting to do a prospective study, in which Bayley scores will be collected from all participants. Furthermore, it was quite difficult to match unexposed controls for date of admission within 1 year to reduce the influence of changing policies. The composition of the unexposed control group was randomly chosen and purely based on the in advance defined matching criteria. Nonetheless, unintentional selection cannot be excluded and the matched controls may not 100% represent the complete population of very preterm infants admitted to the nursery during the study period. An inevitable complicating factor in this study was the concomitant occurrence of hyperglycemia and insulin therapy, through which the individual influence of hyperglycemia or insulin therapy cannot be demonstrated. Although this study does not have the strength of a prospective follow-up study, the results will give some support and direction for the prognosis of the development of very preterm infants with hyperglycemia.Treatment with insulin is not unequivocally associated with better outcome. The first study in adults by van den Berghe showed very promising results. HoweverSeveral follow-up studies in infants with diabetes mellitus type 1 may explain our findings concerning neurobehavioural outcome. Infants with longer episodes of hyperglycemia seem to have an impaired cognitive development, though the specific impaired elements of cognitive development vary between the studies. Compared with healthy siblings, infants with hyperglycemia had a lower verbal intelligence. A folloThe results of this retrospective follow-up study suggest that hyperglycemia and insulin therapy are not only associated with increased mortality and short term morbidity, but also exert long term effects on development. Given the definition of hyperglycaemia, including the use of insulin for >12 hours, one can not assume that the association is due to poorly controlled hyperglycemia in this design, but may be the effect of insulin.BPD: Bronchopulmonary dysplasia; IVH: Intraventricular hemorrhage; NEC: Necrotizing enterocolitis; NICU: Neonatal intensive care unit; PVL: Periventricular leukomalacia; PROM: Prolonged rupture of membranes; RDS: Respiratory distress syndromeThe authors declare that they have no competing interests.NMvdL participated in the design of the study, collected the data, performed the statistical analysis and drafted the manuscript. VEHJS-W collected the data and helped to correct the manuscript. PHTvZ collected the data and drafted the manuscript. FJW conceived of the study, participated in its design and coordination and helped to draft and correct the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/10/52/prepub


Compound microsatellites are a special variation of microsatellites in which two or more individual microsatellites are found directly adjacent to each other. Until now, such composite microsatellites have not been investigated in a comprehensive manner.in silico survey of microsatellite clustering in genomes of Homo sapiens, Maccaca mulatta, Mus musculus, Rattus norvegicus, Ornithorhynchus anatinus, Gallus gallus, Danio rerio and Drosophila melanogaster revealed an unexpected high abundance of compound microsatellites. About 4 – 25% of all microsatellites could be categorized as compound microsatellites. Compound microsatellites are approximately 15 times more frequent than expected under the assumption of a random distribution of microsatellites. Interestingly, microsatellites do not only tend to cluster but the adjacent repeat types of compound microsatellites have very similar motifs: in most cases (>90%) these motifs differ only by a single mutation (base substitution or indel). We propose that the majority of the compound microsatellites originates by duplication of imperfections in a microsatellite tract. This process occurs mostly at the end of a microsatellite, leading to a new repeat type and a potential microsatellite repeat track.Our Our findings suggest a more dynamic picture of microsatellite evolution than previously believed. Imperfections within microsatellites might not only cause the "death" of microsatellites they might also result in their "birth". Microsatellites or simple sequence repeats (SSR) are DNA stretches consisting of a tandemly repeated short DNA motif ≤ 6 bp). Due to the special mutation mechanism of microsatellites termed "DNA replication slippage", these sequences often exhibit length hypervariability with respect to the number of motifs being repeated [reviews: -3]. Owin bp. Due According to Chambers et al.  the folln and [AG]m can be found in four different arrangements. The [AG]m microsatellite might be located 5' or 3' to the [AC]n microsatellite and either the poly-TC or the poly-AG tract of the [AG]m microsatellite might be found on the same DNA strand as the poly-AC tract of the [AC]n microsatellite. For these reasons, four different motif standardizations were introduced by Kofler et al. Rs Table . The numRs Table . We founRs Table . The comable n1 mn2 [m1]n3To test whether the occurrence of compound microsatellites can be attributed to mere chance, we determined whether microsatellites tend to aggregate with respect to an assumed random distribution of microsatellites in the genome.dmax), which can be part of a more complex compound microsatellite. For example a tri-SSR compound microsatellite could be viewed as two overlapping SSR-couples. SSR-couples containing two microsatellites with an identical motif were not considered n than with any other microsatellite motif. For simplicity, we confined this analysis again to SSR-couples. We define SSR-couples having the form [m1]n [m2]n as SSR-couples of motif m1–m2, e.g.: the SSR-couple [AT]12 [AC]9 has the motif AT-AC he form mn [m2]n an microsatellite consists of a poly-AC and a poly-TG tract on the complementary strand. The SSR-couple [AC]8 [AG]9 can be found in two conformations, the poly-AC tract of the [AC]8 microsatellite may either be found on the same or on the complementary DNA-strand as the poly-AG tract of the [AG]9 microsatellite. We call the former plus-conformation and the latter minus-conformation In the whole genome of all eight species the most abundant SSR-couple motifs are AT-AC, AC-AG and AAAG-AAGG Table . Differen microsatellite usually are only about 40 fold overrepresented. A few SSR-couples have an overrepresentation of ≈ 1, which suggests that they have emerged by chance. Most SSR-couples, however, are mainly found in only one of the two possible conformations  SSR-couples in D. rerio and O. anatinus have such complementary motifs . Interestingly, both conformations can be found in all examined species with relativly equal frequencies . Bachtrog et al. 67. Assuming that compound microsatellites predominately originate through the process described by Jakupciak and Wells n microsatellite a compound microsatellite having the form [CA]n [AT]n [CA]n would result. This example illustrates that, first the resulting compound microsatellite would be a tri-SSR compound microsatellite and that second the two microsatellites flanking the central microsatellite would share the same motif. Only about 13% of the compound microsatellites contain three or more microsatellites , Pan troglodytes , Maccaca mulatta , Mus musculus , Rattus norvegicus , Ornithorhynchus anatinus , Gallus gallus, Danio rerio  and Drosophila melanogaster  were downloaded from the Ensembl ftp-server .The genomic pseudomolecules of . The sequences obtained with BioMart, were pretreated to remove empty sequences and to ensure that each sequence has an unique identifier (fasta ID). This is an important prerequisite for the identification of compound microsatellites with SciRoKo. Table Since sequence information of the Y-chromosome is not available for all examined species, only the autosomes and the X-chromosomes were used unless stated in the text. Non-chromosomal DNA was not considered. The 5' untranslated region (5'-UTR), coding sequence (CDS) and 3' untranslated region (3'-UTR) were obtained with Ensembl BioMart dmax (maximum distance between adjacent microsatellites as to account as compounded) of 10 bp was used. All microsatellite motifs and SSR-couple motifs were standardized as described by Kofler et al. [The microsatellite search was done with the software SciRoKo 3.3 . The folr et al.  [see alsSince the content of the letter 'N' varies between 4 – 20% in the pseudochormosomes of the eight taxa, only the letters 'A','T','C' and 'G' were considered to calculate the sequence length dependent variables (e.g SSR density or compound microsatellite density). The sequence length dependent variables were not adjusted for the cds, 5'-UTR and 3'-UTR.The microsatellite search results generated with the software SciRoKo were processed with a number of console applications. All console applications were written in C# or Perl. All programs can be obtained from the corresponding author upon request.Che.exp: equation 2) was estimated, by calculating the total number of expected SSR-couples (Cexp: equation 1) and subtracting, for each microsatellite motif, the expected number of SSR homocouples (equations 1 & 2), i.e pairs of microsatellites sharing the same motif. The overrepresentation (Or: equation 3) is calculated by dividing the observed number of microsatellite heterocouples by the expected one:Calculation of the expected number of SSR heterocouples, i.e. pairs of microsatellites not sharing the same motif, are based on a random distribution of microsatellites within DNA sequence space. The expected number of SSR heterocouples  and the frequency of the second motif (m2). To test whether the observed number of SSR heterocouples significantly deviates from the expected one, a both sided Poisson Distribution was used and cumulative probabilities were calculated for P(x ≥ Che.obs).All parameters are as described above, except for the frequency of the first motif  test was done with SPSS 15.0. The influence of recombination was tested separately by using the two groups 'Y' and 'not-Y' instead of the category chromosome. The data used for CatReg test can be found in Additional file H. sapiens as published by Kong et al. [To identify the parameters governing compound microsatellite density we determined the SSR and the compound microsatellite density along the chromosomes of g et al. . We usedRK, CS and TL designed the study. RK wrote the software and conducted the bioinformatic analysis. EL conducted the statistic analysis. RK, CS, EL and TL wrote the manuscript.Standardization of microsatellite motifs and compound microsatellite motifs. Describes in detail, the methods used in this publication, for the standardization of microsatellites and compound microsatellites.Click here for fileAdditional tables. Contains the additional tables S1 – S11.Click here for fileCatReg raw data. Contains the raw data used for the CatReg-test.Click here for fileRecombination vs compound microsatellite density. Contains the raw data for calculating the correlation between recombination and compound microsatellite density. Additonally contains the source code of the perl script used for calculating this raw data.Click here for fileSignificant overrepresentation of SSR-couples in all eight species. Contains the expected number of SSR-couples, the observed number of SSR-couples and the significance of the overrepresentation. The analysis has been conducted for each sliding window (size 5 Mbp) in all eight species.Click here for file


Based on the observation of an increased number of paralogous genes in teleost fishes compared with other vertebrates and on the conserved synteny between duplicated copies, it has been shown that a whole genome duplication (WGD) occurred during the evolution of Actinopterygian fish. Comparative phylogenetic dating of this duplication event suggests that it occurred early on, specifically in teleosts. It has been proposed that this event might have facilitated the evolutionary radiation and the phenotypic diversification of the teleost fish, notably by allowing the sub- or neo-functionalization of many duplicated genes.In this paper, we studied in a wide range of Actinopterygians the duplication and fate of the androgen receptor , a nuclear receptor known to play a key role in sex-determination in vertebrates. The pattern of AR gene duplication is consistent with an early WGD event: it has been duplicated into two genes AR-A and AR-B after the split of the Acipenseriformes from the lineage leading to teleost fish but before the divergence of Osteoglossiformes. Genomic and syntenic analyses in addition to lack of PCR amplification show that one of the duplicated copies, AR-B, was lost in several basal Clupeocephala such as Cypriniformes (including the model species zebrafish), Siluriformes, Characiformes and Salmoniformes. Interestingly, we also found that, in basal teleost fish (Osteoglossiformes and Anguilliformes), the two copies remain very similar, whereas, specifically in Percomorphs, one of the copies, AR-B, has accumulated substitutions in both the ligand binding domain (LBD) and the DNA binding domain (DBD).The comparison of the mutations present in these divergent AR-B with those known in human to be implicated in complete, partial or mild androgen insensitivity syndrome suggests that the existence of two distinct AR duplicates may be correlated to specific functional differences that may be connected to the well-known plasticity of sex determination in fish. This suggests that three specific events have shaped the present diversity of ARs in Actinopterygians: (i) early WGD, (ii) parallel loss of one duplicate in several lineages and (iii) putative neofunctionalization of the same duplicate in percomorphs, which occurred a long time after the WGD. Takifugu rubripes and Tetraodon nigroviridis genomes [Tetraodon nigroviridis were assigned to syntenic groups of duplicated genes, demonstrating the genomic scale of the duplication. It was further shown that each pair of duplicated genes was homologous to one non-duplicated human chromosomal region [Actinopterygian fishes have provided the first clear demonstration of an ancient whole genome duplication (WGD) in vertebrate evolution . This ev genomes ,7. The d genomes ,9. In adl region .et al. [Direct dating of fish gene duplications based on molecular clock was hampered by saturation of synonymous changes at the time scales considered, as well as by differences in evolutionary rates between mammals and fishes ,10-13. Let al.  have scret al. ,16.et al. [i.e. they share the ancestral function of their non duplicated ancestor) and some others undergo neofunctionalization (i.e. they change their function when compared to their ancestor). In most cases the sub- or neofunctionalization events are classically considered to have occurred relatively soon after the duplication event. We recently suggested that a biased subset of genes was retained as duplicates after the genome duplication and that gene retention was biased with regard to biological processes [One basic question regarding gene duplication is the fate of duplicated genes. According to the Duplication – Degeneration – Complementation (DDC) model proposed by Force et al. , duplicarocesses . Most norocesses ,18. In arocesses -21). Of rocesses -24.i.e. regular androgens (DHT) versus 11-oxygenated androgens (11KT) [Oncorhynchus mykiss [ Gambusia affinis [Astatotilapia burtoni  : a hypers (11KT) . In Actis mykiss , the mos affinis , Astatot1KT : a hjaponica ,32 and iculeatus . Interesndulatus  and the athratus . These tathratus . In addiathratus .The case of AR in fish is particularly interesting to study as sex determination mechanisms are known to be particularly plastic in Actinopterygians ,38. For Interestingly, in human, mutations of the AR gene represent the molecular basis of androgen insensitivity syndrome (AIS) . AIS is In this paper, we reconstructed the evolutionary history of AR in Actinopterygians. We observed a complex history shaped by three successive events well separated in time: (i) an ancestral duplication event specific to teleost fishes corresponding to the WGD; (ii) a parallel loss of one duplicated copy (AR-B) in basal Clupeocephala and (iii) a major sequence divergence indicative of a change in functional constraints in the AR-B duplicate of Percomorphs. This evolutionary history together with the striking mutation patterns is indicative of a putative neofunctionalization event that took place late during AR-B evolution It is tempting to link this neofunctionalization event to the plasticity of sex determination in Percomorphs.in silico search against various databases, we were able to characterize 26 different new AR cDNA fragments from 20 different fish species. Along with these AR cDNA fragments, we also identified other steroid receptors (NR3C group) in A. baerii and E. stoutii (Genbank accession numbers ABF50787 and ABF50785). This is probably due to a combination of the high conservation of the C and E domains used to design primers among all steroid nuclear receptors and the low stringency of touchdown PCR procedure that we used. In this study, five new sequences of AR were also identified using database searches and 21 new sequences of AR were isolated by RT-PCR with degenerated primers, the majority of them being teleosteans (20), one being chondrichthyan, one being Dipnoi and one being chondrostean (Table Using a combination of RT-PCR with degenerate primers designed in the conserved C and E domains and Oryzias latipes was found to match with the 5' end region of the divergent AR in Haplochromis burtoni (AF121257). The corresponding clone was further sequenced and was confirmed to be a medaka AR. No more than one gene was identified in zebrafish by searching both EST databases and the whole genome sequence (see below). Additional file Of note, one expressed sequence tag in urtoni AF21257. ThUsing the 30 full length sequences identified in our screens, we reconstructed the phylogeny of the Actinopterygian ARs using Neighbor-Joining, Maximum Parsimony, Maximum Likelihood and Bayesian methods Figure . The shoAcipenser baerii and this sequence is, as expected, clearly located at the base of the Actinopterygian sequences, suggesting that it corresponds to a non-duplicated version of the AR gene  that would allow us to confirm more accurately that the WGD occurred specifically in teleosts. Nevertheless, our data are fully consistent with this likely scenario.In all cases, only one AR sequence was identified in each tetrapod. Based on partial sequence analysis, the AR from the west African lungfish branched at the root of this tetrapod cluster (not shown). We obtained only one AR sequence in the sturgeon, e Figure . In contar genes ,53, we f[et al., . In contThat the two sequences AR-A and AR-B are indeed the product of the ancestral WGD specific of teleosts is further supported by the chromosomal location of the two tetraodon AR sequences. Indeed, these two genes are located on chromosomes T1 and T7 in the tetraodon genome Figure . From a Heterotis, Anguilla) as well as in many Percomorphs but is missing in many basal Clupeocephala lineages.When analyzing the tree presented in Figure etc...) are available. First, we carried out RT-PCR experiments using different batches of primers and several RNA extracts of zebrafish embryos at various developmental stages, as well as adult organs. In all cases, we detected only one AR sequence whereas our primer batches were able to detect divergent NR3C steroid receptors such as GR, MR or PR. PCR experiments based on DNA amplification of short fragments contained in only one exon also failed. Finally, we intensively screened the release Zv7 (13 July 2007) of the zebrafish genome using various fragments of the AR gene as baits without any significant hit. Of note, no sequence reminiscent of a pseudogene was detected.As this observation could be due to an experimental bias linked to a failure to amplify a divergent gene by PCR, we then first checked that this observation was not due to an artifactual lack of detection of the AR-B gene. To this end, we focused our analysis on zebrafish for which a large number of data . The Tetraodon AR-B gene map to chromosome T1 and the region encompassing the gene corresponds mainly to the zebrafish chromosome Z14. The mapping of the region containing the Tetraodon AR-B sequence on the zebrafish genome shows that this region has been scrambled during evolution. Many gene orders are not conserved and large fragments are missing or were exchanged (data not shown). The same consideration is reached when the medaka genome is considered , Characiformes (1 species) and Siluriformes (1 species) that altogether form the well-supported clade Otophysi , we alsoar-alpha and ar-beta known in rainbow trout [Strikingly, the exact same situation was observed in another independent lineage of Clupeomorph: the Salmoniformes. In the 2 species analyzed one more time, we found only AR-A and not AR-B. Of note, in Salmoniformes two AR-A sequences are observed (corresponding to the two subtypes named ow trout ). These ow trout ). It is It is clear that loss of duplicated genes is a very common fate after a genome duplication event but the present analysis nicely illustrates a late case of neofunctionalization. Our data suggest that basal teleosts and percomorphs kept two functional copies of AR whereas "intermediate" lineages such as Otophysi and Salmoniformes lost it secondarily (see below). According to the topology of teleost fish phylogeny presented in Figure It is difficult to speculate with the data available why the Otophysi and the salmonids apparently do not need a second AR-B gene. Given the major function of AR in sex determination and sex organ differentiation, it is tempting to link these events with these processes but given that these data on Otophysi and salmonids are limited to some specific models such as zebrafish, salmon and trout, it is up to now difficult to find an obvious connection. It is striking that zebrafish and salmonids are extremely different regarding sex determination and sex differentiation. In addition, as discussed above, the exact phylogenetic range of this loss of AR-B is still unclear.i.e., the seabass D. labrax, the sand goby, P. minutus, the nile tilapia, O. niloticus and A. burtoni, a scorpaeniforme with M. scorpius, the shorthorn sculpin,; a beloniforme with the medaka, O. latipes, a cyprinodontiforme with the mosquitofish, G. affinis, and 2 tetraodontiformes with the tetraodon, T. nigroviridis and the fugu, T. rubripes. Indeed, when we considered the sequence alignment  clearly contained AR-A and AR-B paralogs. For AR-A, this is not difficult to establish given that this gene is present in a wide phylogenetic range of species. For AR-B, the assignment is less obvious since, as discussed above, this gene has been lost in basal Clupeocephala. The fact that the AR-Bs from Anguilla and Heterotis are indeed orthologs of the percomorph AR-B is indicated by several features: (i) these sequences exhibit a few key sequence signatures that represent synapomorphies of AR-B sequences  secondary loss of AR-B in basal Clupeocephala  explaining the restricted phylogenetic occurrence of AR-B when compared to AR-A; (iii) a late specific divergence of AR-B. The long branch connecting percomorph AR-B to the basal AR-B sequences is indicative of the accumulation of numerous mutations and we thus proposed that it corresponds to a functional shift that has affected the AR-B protein.The most likely scenario accounting for the data available concerning Actinopterygian AR evolution is therefore a three step model Figure : (i) ancversus the other were compared to answer this question: are they significantly more different than they would be if branches were picked at random among the two subtrees? Expected numbers of substitutions were estimated for all branches of the tree and all sites of the alignment [e.g. sturgeon), duplicated AR with a non-divergent AR-B (e.g. eel) and duplicated AR with a divergent AR-B (e.g. medaka) this neofunctionalization cannot yet be formaly proved and should be regarded as only putative.We therefore wanted to test whether the two groups of paralogous genes AR-A and percomorph AR-B were under different selective pressures. We reasoned that if selective pressures differed between the two groups, there should be sites undergoing substitutions in the AR-A subtree and constrained in the percomorph AR-B subtree, and symmetrically sites constrained in the AR-A subtree undergoing substitutions in the AR-B subtree. Patterns of evolutionary rates in one subtree lignment . A non-slignment . A signiThis pattern of a late spectacular divergence of a duplicated gene in a precise taxonomic group is an interesting case in which the duplication and the functional shifts are clearly two recognizable events that were decoupled in time. The AR-B gene will thus be a very interesting model to study the precise functional and biological impact of these two events since we have sequences of non-duplicated fish AR (sturgeon), duplicated AR-A (in eel and medaka for example), duplicated and non divergent AR-B (eel) and duplicated and divergent AR-B (medaka). In addition, we have other interesting cases for comparison such as a unique zebrafish AR-A gene with secondary loss of AR-B. The fact that AR is a gene encoding a nuclear hormone receptor with a known ligand, a clear biological role and for which several functional tests are available renders this gene particularly suitable for a precise integrated study of the consequences and respective roles of duplication and evolutionary sequence divergence. For example, it may be very interesting to study if, as proposed recently at a broader scale for nuclear receptors, sequence divergence is correlated to expression divergence .versus AR-A. We first detected how the various mutations observed in Actinopterygian ARs are located in function of the complex evolutionary history described above. We thus categorized the mutations in four classes  muts Figure ; (ii) mus Figure ; (iii) ps Figure ; and fins Figure . From ths Figure  specificWe thus studied in more detail the 38 mutations found in the divergent AR-B sequences in order to see if some of them could have obvious functional consequences. Of note, and not surprisingly, none of these mutations affect the positions known to directly interact with the ligand or implicated in coactivator binding as determined in the 3D structure of the AR LBD complexed with various ligands -63. Few Then, we scrutinized the positions in the DBD and LBD divergent in AR-B and we checked whether these mutations are affecting amino acids found mutated in human pathologic conditions Figure . We wereWe thus compared the mutation pattern observed in the divergent percomorphs AR-B gene (that is 38 mutations found in the LBD as well as 22 mutations in the DBD) which are linked to a neofunctionalization event to the mutation pattern occurring in the pathological conditions Figure . When thin vitro and in vivo analysis of the role of AR-A and AR-B in suitable fish models. How this functionality is precisely affected remains therefore an open question. The teleost duality in terms of active androgens involved in reproduction [in vivo binding studies carried out in two perciform species, i.e. the Atlantic croaker, Micropogonias undulates and the kelp bass, Paralabrax clathratus, that demonstrated the existence of two different nuclear androgen receptors that may mediate the physiological actions of different androgens [A. burtoni in which it has been shown that AR-A and AR-B have distinct expression patterns in the brain [The above analyses suggest that these sequence differences between AR-A and AR-B will affect the functionality of these receptors and are linked to a putative neofunctionalization event. This remains of course to be directly addressed through oduction  is of grndrogens -36. The he brain , with a he brain . Taking he brain . One mayPerca fluviatilis, Lepomis gibbosus, Pomatoschistus minutus, Myoxocephalus scorpius, Protopterus annectens, Labeo rohita and Eptatretus stoutii), bred in captivity in experimental or aquaculture facilities  or from local aquarium fish retailers (Gymnocorymbus ternetzi and Acipenser baerii). For Ginglymostoma cirratum, we used the Epigonal Nurse Shark cDNA Library provided by Dr. M.F. Flajnik at the University of Miami School of Medicine [® reagent  following the manufacturer's instructions and mRNA was further purified from 500 μg of total RNA using the OligoTex mRNA kit (Qiagen).Common and scientific names of all fish species used in this study are given in Table Medicine . All ani® , in a total volume of 25 μl.For cDNA synthesis, 2 μg of mRNA were denatured in the presence of oligo (dT) (0.5 μg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 hour using M-MLV reverse transcriptase  as described by the manufacturer. Namely, 2 μg of mRNA were reverse transcribed with 200 units of M-MLV reverse transcriptase in the presence of 1.25 μl of each dNTP at 10 mM, 5 μl of M-MLV 5× reaction buffer and 25 units of RNasinH. burtoni AR-B  and steroid LBD (E domain) regions. The alignment of the divergent et al. [Taq Polymerase (Sigma), 200 μM of dNTPs, 30ρmol of each degenerated primer and 2.5 μl of Taq buffer 10× (Sigma). The total volume of the reaction was 25 μl and the cycling Touchdown PCR conditions were: 94°C for 1 min, 20 cycles of regularly decreasing annealing temperature from 50°C to 40°C for 30 sec and 72°C for 30 sec, and 30 cycles at the annealing temperature of 40°C, ending at 72°C for 30 sec. PCR products were analyzed on agarose gel (1%) and amplified DNA fragments of the anticipated length (average between 370 and 450 bp) were subsequently subcloned into pCR 2.1 plasmid. Bacteria  were transformed by electroporation, spread on LB-ampicillin agar plates and incubated overnight at 37°C. From 10 to 20 randomly selected recombinant colonies were then screened either using PCR with primers amplifying the inserts (T7 and M13 reverse primers) or by hybridization of nitrocellulose membrane lifts with a rainbow trout AR radiolabelled (dCTP32) probe. Positive clones were sequenced using a dideoxy cycle-sequencing method with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and reaction sequences were read on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). A secondary nested PCR was carried out for RNA samples from G. ternezi, D. rerio, P. annectens, and A. baerii species , we retrieved AR sequences belonging to the Ensembl family ENSF000000000291.Homologous DNA and protein fish ARs were searched on available public databases  using the various BLAST programs available through the network servers at the National Center of Biotechnology Information http://mrbayes.csit.fsu.edu/index.php.Multiple alignments of the deduced amino acid sequences were generated with Muscle using the default parameters . Phylogeversus the AR-B one in percomorphs were compared. Expected numbers of substitutions per site and per branch were estimated with the CoMap program [To test whether the two groups of paralogous genes AR-A and AR-B were under different selective pressures, patterns of substitutions estimated in the AR-A subtree  program  based on program . This pr program  in the Rhttp://abacus.gene.ucl.ac.uk/software/paml.html[In order to determine any change in the selective pressure along the branch leading to the percomorphs AR-Bs beside of this previous test, we also used PAML version 4 paml.html.http://androgendb.mcgill.ca/[http://pymol.sourceforge.net/).Location of all the substitutions found in the LBD of the human AR was retrieved from Bruce Gottlieb's database at his web site cgill.ca/. Amino aVD performed most of the experimental work , FB performed the bioinformatic analysis , BB studied the selective pressures acting on the ARs, IA and BH contributed to the cloning/sequencing steps, VVG and FB compared the pattern of mutations present in fish AR with those found in human patients, VL and YG designed the study and wrote the paper.ARs sequences alignment. Alignment of the DBD and LBD sequences of ARs compared to that of human  with PhyML with 1000 bootstrap replicates; maximum parcimony (MP) and neighbor joining (NJ) as implemented in Seaview.Click here for fileOrthology relationships between medaka, tetraodon and zebrafish chromosomes. As referenced in legend Figure Click here for fileStatistical distributions of the AR-A and AR-B substitutions in fish. Statistical representation of the distributions of the AR-A and AR-B substitutions in fish. The bell distribution is a random distribution of the substitutions, the diamond shows that the specificity of AR-A and AR-B versus the other AR is statistically clearly not random.Click here for fileHuman and fish substitutions along the DBD of the human AR. Representation of the DBD of the human AR modified from [ied from . The aa Click here for fileList of human substitutions in AR leading to AIS phenotypes and prostate cancer. List of substitutions in human AR leading to CAIS, PAIS, MAIS and prostate cancer phenotype, as referenced into Bruce Gottlieb's database (androgendb.mcgill.ca/AR23C.pdf) [23C.pdf) , and lisClick here for fileDetailed analysis of Table Click here for file


While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.50 (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC50  of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr ICin vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.Together, these data demonstrate that AR-42 has  Inhibitors of these enzymes were originally reported to relieve transcriptional repression and subsequent epigenetic silencing caused by histone deacetylation. It is now evident that the targets of these enzymes also include a broad array of proteins such as transcription factors, chaperones, signaling components, and cytoskeletal proteins. Thus, the effects of DAC inhibitors are diverse and incompletely understood, and likely vary by cell type and context. Adding to the complexity of reported DAC inhibitor activities is the different, but occasionally overlapping, effects on class I and II DACs. Class I DACs  are primarily found in the nucleus, although DAC3 is found in both the nucleus and cytoplasm. Class II DACs  are generally reported to shuttle in and out of the nucleus, depending on intracellular signals. DAC6 is a cytoplasmic enzyme that deacetylates tubulin Chronic Lymphocytic Leukemia (CLL) is immunophenotypically defined as a malignancy of CD5/CD19/CD23 positive, CD20 and Ig dim B cells that manifests with bone marrow failure, lymphadenopathy and infections as a consequence of disease-associated immune suppression. While recent advances in chemoimmunotherapy strategies have improved options for CLL patients, the median overall survival for fludarabine-refractory patients is just 13 months. Mantle cell lymphoma (MCL), an aggressive B cell malignancy, is characterized by the abnormal proliferation and accumulation of CD5/CD20/CD22 positive, CD23 negative B cells in various hematopoietic tissues, with or without peripheral blood involvement. While MCL comprises approximately 8% of Non-Hodgkin lymphoma cases, it is associated with a disproportionate number of deaths and a mean survival of only three years Multiple studies have demonstrated that DAC inhibitors including romidepsin, entinostat (MS-275) and valproic acid can alter histone acetylation status in CLL and lead to selective cytotoxicity in these cells N-acetyldinaline , romidepsin Clinical trials with class I DAC inhibitors in vitro and in vivo activity in multiple solid tumor models In vivo, AR-42 suppressed PC-3 tumor xenograft growth by 67%, whereas vorinostat at the same dose suppressed growth by 31% in vitro and in vivo using CLL and related B-cell malignancies.AR-42  is a novel hydroxamate-tethered phenylbutyrate derivative 50) of AR-42 at 48 hr was 0.61 µM  in Raji Burkitt's lymphoma cells, 0.22 µM  in 697 acute lymphoblastic leukemia cells, and 0.21 µM  in JeKo-1 MCL cells (n = 3 each). In simultaneous assays, the IC50 values of vorinostat were 3- to 6-fold higher, consistent with results in prostate cancer cell lines 50 of 0.76 µM , similar to what we observed with the class I DAC inhibitor entinostat (MS-275) in vitro work presented here, AR-42 was used at the LC50 of 0.90 µM that was initially calculated using a smaller number of CLL samples. Although this LC50 was found to be moderately lower when additional CLL samples were included, we continued to use the initial LC50 of 0.90 µM for consistency among experiments.In MTT assays . However, cytotoxicity with a 16 hr exposure was similar to that observed when samples were incubated continuously for 48 hr  , we found that AR-42 has similar cytotoxic effect regardless of whether human or bovine serum matrices are used. Importantly, we demonstrate that AR-42 efficacy in CLL cells is not compromised by co-culture with stromal cells, which have been widely shown to prevent spontaneous apoptosis and mediate drug resistance in CLL tumor cells et al. AR-42 is a novel class I and II DAC inhibitor that has shown pre-clinical activity in a variety of solid tumor in vivo activity in murine models of Burkitt's lymphoma, MCL, and CLL. With all three models, increased survival with AR-42 is observed compared to the vehicle control. Interestingly, in the Raji Burkitt's lymphoma model, the class I/II DAC inhibitor vorinostat administered at its maximum tolerated dose (MTD) lacked activity, whereas AR-42 showed statistically significant activity without discernable toxicity. It should be noted that our selection of doses of each agent were based on MTD in SCID mice as determined by weight loss greater than 20%. We acknowledge that direct comparison of AR-42 and vorinostat in vivo, even within the same model, is complicated by potentially differing pharmacologic properties such as oral absorption and half-life, as well as toxicities unrelated to weight loss. Thus it remains to be determined whether this difference in efficacy will be observed in leukemia patients. However, these data collectively support future clinical development of AR-42 in the treatment of lymphoid malignancies.Importantly, AR-42 demonstrates An important consideration with DAC inhibitors in the treatment of hematologic malignancies is the development of combination strategies with other targeted therapies. As has been reported with other DAC inhibitors, AR-42 significantly sensitizes CLL patient cells to TRAIL. This finding is important, as TRAIL alone has little activity in CLL but also shows little or no toxicity toward non-tumor cells. Thus, the combination of AR-42 and TRAIL receptor agonists may provide improved clinical benefit without substantial side effects. In particular, antibodies targeting DR5 are quite attractive, as they have shown extended half-life.e.g. romidepsin et al. determined that the pattern of gene expression is largely similar between these two groups. In addition we observed no cytotoxic effect of DAC6 inhibition in CLL patient cells, suggesting that acetylation of tubulin and/or HSP90 is not required for DAC inhibitor-mediated cytotoxicity in these cells. However AR-42 may influence other pathways controlled by class II DACs, although these are not well defined. For example, class II DACs can function as transcriptional co-repressors, and it is possible that inhibition of these enzymes allows expression of genes with pro-apoptotic effects The importance of dual inhibition of DAC classes I and II is unclear. Most investigations of DAC inhibitors in B-cell malignancies have used class I-specific inhibitors  in a study population that included multiple types of leukemia/lymphoma in vitro as well as in vivo compared to vorinostat. These observations suggest an improved potency and therapeutic index of AR-42 that will be of key importance in the development of this agent, given the widely observed constitutional symptoms observed with this class of drugs. Also, AR-42 shares with vorinostat and panobinostat the favorable property of oral availability, allowing far greater feasibility and flexibility of administration. Pre-clinical pharmacology and toxicology also support clinical development (data not shown), and an investigational new drug application has been approved for a first-in-man study of AR-42. Based on these collective findings, a phase I clinical trial of AR-42 in patients with B-cell lymphoid malignancies including CLL is now underway.Previous work by members of our group Blood was obtained from CLL patients after obtaining written, informed consent according to an Ohio State University Institutional Review Board-approved protocol, in agreement with the principles of the Declaration of Helsinki. All animal research was reviewed and approved by The Ohio State University Institutional Animal Care and Use Committee.2.All patients previously received a diagnosis of CLL as defined by National Cancer Institute (NCI) criteria, had elevated leukocyte counts , and were without treatment for at least four weeks prior to blood collection. CD19-positive cells were isolated from peripheral blood using Rosette-Sep reagents  and isolated by density gradient centrifugation . 697 cells were obtained from DSMZ . Raji and HS-5 Romidepsin (depsipeptide) was obtained from the NCI. Tubacin was the kind gift of Drs. Ralph Mazitschek and Stuart Schreiber, The Broad Institute and Harvard University, Cambridge MA. Vorinostat and AR-42 were synthesized in the laboratory of Dr. Ching-Shih Chen, OSU College of Pharmacy. Recombinant human TRAIL/Apo2L, used at 100 ng/mL, was obtained from Cell Sciences, Inc. .50 and IC50 values were calculated using Prism software .MTT  assays were performed as described After exposure to AR-42, cells were resuspended in buffer containing annexin V-FITC and propidium iodide (PI) according to the supplier's instructions . Annexin binding and PI positivity were assessed by flow cytometry on a Coulter EPICS-XL. For caspase inhibition, 100 µM Z-VAD-fmk  was added to cultures 15 minutes prior to drug addition.Cell extracts were prepared as previously described The use of C.B-17 SCID mice  as a lymphoma model has been described 7 cells/ml in PBS at room temperature, and 2×106 cells were inoculated via tail vein. Treatment began 3 days after engraftment. AR-42 and vorinostat were dissolved in vehicle . In pilot studies, the maximum tolerated dose (MTD) of AR-42 and vorinostat in these mice was determined to be 75 mg/kg and 50 mg/kg, respectively, when administered daily by oral gavage. MTD was defined as the maximum dose resulting in weight loss of less than 20% over the course of treatment. After engraftment, mice were randomly placed into three groups that received the following treatments: (a) vehicle alone, (b) AR-42 at 75 mg/kg every other day, (c) vorinostat at 50 mg/kg daily. Mice received treatment by oral gavage (10 µl/g body weight) for the duration of the study. The mice were monitored daily and were sacrificed if hind-limb paralysis, respiratory distress, or weight loss greater than 20% was observed. Survival (absence of the above criteria) was used as an endpoint for this study.Cells were resuspended at 107 JeKo-1 cells results in a disseminated tumor after 3–4 weeks post injection and, without intervention, mice have a mean survival of 28 days Similar to the previous models, 6–8 week old female C.B-17 SCID mice were used. Mice were depleted of murine NK cells with intraperitoneal injections of 0.2 mg rat anti-mouse interleukin 2 (IL-2) receptor β monoclonal antibodies (TMβ1), one day before engraftment and then every week, as described Development and validation of the Eμ-Tcl1 transgenic mouse as a CLL model has been described P values of less than 0.05 were considered significant. All analyses were performed using SAS/STAT software, Version 9.2 .To test for differences between AR-42-treated cells in the presence or absence of Z-VAD-fmk, a linear mixed effects model was used to account for dependencies among samples from the same patient. Main effects and differences were estimated from this model. Linear mixed effect models were also used to test for significant interactions between AR-42 and TRAIL. For assessments of the effect of AR-42 pretreatment in CLL cells alone or co-cultured with HS5 cells and differences in tumor load in Eμ-TCL1 mice, outcomes were natural log-transformed to stabilize variabilities among conditions and mixed effects models were then applied to the data. From these models, relevant estimates with 95% confidence intervals were obtained. For survival assessments, Kaplan-Meier estimates of the survival function for control and AR-42-treated mice were generated. Median survival times with 95% confidence intervals were calculated, and the log-rank test was used to compare the overall survival between the two groups.