Polymerizing nanodroplets at solid-liquid interfaces is a facile solution-based approach to the functionalization of large surface areas with polymeric lens-shaped nanostructures. In this work, we have applied a one-pot approach to obtain polymeric nanolenses with controlled sizes and densities. We take advantage of the formation mechanism by the direct adsorption of nanodroplets from a surfactant-free microemulsion onto an immersed hydrophobic substrate. The interfacial nanodroplets were photopolymerized to produce polymeric nanolenses on the substrate surface. The surfactant-free microemulsion of the monomer nanodroplets was obtained through the spontaneous emulsification (i.e., ouzo effect) in the tertiary system of ethanol, water, and precusor monomer. The size of nanolenses on the surface was adjusted by the nanodroplet size, following a linear relationship with the ratio of the components in the microemulsion. This simple approach is applicable to produce nanolenses over the entire surface area or on any specific area at will by depositing a drop of the microemulsion. Possessing high optical transparency, the resulting substrates may have potential application as functional biomedical supporting materials or effective light-harvesting coatings.
Treatment with the tyrosine kinase inhibitor imatinib represents the standard of care for newly diagnosed patients with chronic myeloid leukemia. In recent years, several second generation inhibitors - such as dasatinib and nilotinib - have become available that promise to overcome some of the mutations associated with acquired resistance in these patients. Despite eliciting similar clinical responses, the molecular effects of these agents on different subpopulations of leukemic cells remain incompletely understood. Furthermore, the consequences of using high-dose imatinib therapy have not been investigated in detail. Here we utilized clinical data from patients treated with dasatinib, nilotinib, or high-dose imatinib, together with a statistical data analysis and mathematical modeling approach, to investigate the molecular treatment response of leukemic cells to these agents. We found that these drugs elicit very similar responses if administered front-line. However, the patient population displays significantly different kinetics when treated second line, both in terms of differences between front-line and second line treatment for the same drug, and among agents when used second-line. We then utilized a mathematical framework describing the behavior of four differentiation levels of leukemic cells during therapy to predict the treatment response kinetics for the different patient cohorts. The dynamics of BCR-ABL1 clearance observed in our study suggest that the use of standard or high-dose imatinib or a second generation TKI such as nilotinib or dasatinib elicit similar responses when administered as frontline therapy for patients with CML in chronic phase.
Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults.
Patients in complete cytogenetic response (CCyR) with detectable BCR-ABL1 after ?2 years on imatinib were randomized to nilotinib (400 mg twice daily, n = 104) or continued imatinib (n = 103) in the Evaluating Nilotinib Efficacy and Safety in clinical Trials-Complete Molecular Response (ENESTcmr) trial. By 1 and 2 years, confirmed undetectable BCR-ABL1 was achieved by 12.5% vs 5.8% (P = .108) and 22.1% vs 8.7% of patients in the nilotinib and imatinib arms, respectively (P = .0087). Among patients without molecular response 4.5 (BCR-ABL1(IS) ?0.0032%; MR(4.5)) and those without major molecular response at study start, MR(4.5) by 2 years was achieved by 42.9% vs 20.8% and 29.2% vs 3.6% of patients in the nilotinib and imatinib arms, respectively. No patient in the nilotinib arm lost CCyR, vs 3 in the imatinib arm. Adverse events were more common in the nilotinib arm, as expected with the introduction of a new drug vs remaining on a well-tolerated drug. The safety profile of nilotinib was consistent with other reported studies. In summary, switching to nilotinib enabled more patients with chronic myeloid leukemia in chronic phase (CML-CP) to sustain lower levels of disease burden vs remaining on imatinib. This trial was registered at www.clinicaltrials.gov as #NCT00760877.
In chronic myeloid leukemia (CML) patients, a breakpoint cluster region-Abelson (BCR-ABL1) value >10% at 3 months of therapy is statistically associated with poorer outcome, yet many of these patients still achieve satisfactory outcomes. We investigated 528 first-line imatinib-treated patients to determine whether patients with the poorest outcome can be better discriminated at 3 months. All outcomes were significantly superior for the 410 patients with BCR-ABL1 ?10% at 3 months (P < .001). However, the poorest outcomes among the 95 evaluable patients with BCR-ABL1 >10% at 3 months were identified by the rate of BCR-ABL1 decline from baseline, assessed by estimating the number of days over which BCR-ABL1 halved. Patients with BCR-ABL1 halving time <76 days (n = 74) had significantly superior outcomes compared with patients whose BCR-ABL1 values did not halve by 76 days (n = 21; 4-year overall survival, 95% vs 58%, P = .0002; progression-free survival, 92% vs 63%, P = .008; failure-free survival, 59% vs 6%, P < .0001; and major molecular response, 54% vs 5%, P = .008). By multivariate analysis, the halving time was an independent predictor of outcome in this poor risk group. Our study highlighted that the rate of BCR-ABL1 decline may be a critical prognostic discriminator of the patients with very poor outcome among those >10% at 3 months. The International Randomized IFN vs STI571 (IRIS) trial was registered at http://www.clinicaltrials.gov as #NCT00006343. The Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial was registered at http://www.clinicaltrials.gov as #NCT00124748. The Therapeutic Intensification in DE-novo Leukaemia (TIDEL) I trial was registered at http://www.ANZCTR.org.au as #ACTRN12607000614493. The TIDEL II trial was registered at http://www.ANZCTR.org.au as #ACTRN12607000325404.
We report long-term results in 40 patients with Philadlephia chromosome-positive (Ph+) acute leukemia who received an imatinib monotherapy window to evaluate in vivo effects on BCR-ABL signaling prior to induction chemotherapy. The first 25 patients (cohort 1) received the LALA-94 protocol without further imatinib (newly diagnosed Ph+ acute lymphoblastic leukemia [ALL]) or induction chemotherapy followed by single-agent imatinib. Subsequent patients (cohort 2) continued imatinib concurrently with either LALA-94 (newly diagnosed Ph + ALL) or other intensive chemotherapy regimens. Cohort 2 had a complete response (CR) rate of 93% and 5-year survival of 69%. For newly diagnosed Ph+ ALL, survival was superior in cohort 2 compared with cohort 1. Toxicity was similar to that expected for chemotherapy alone. Among 10 evaluable patients, rapid loss of phospho-CRKL occurred during the imatinib window in seven patients (all achieved CR) and incomplete inhibition in three patients (none with CR). In summary, a pharmacodynamic window design permitted biomarker assessment of BCR-ABL targeting without compromising clinical outcomes.
Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ? 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.
The use of alginate based microcapsules to deliver drugs and cells with a minimal host interaction is increasingly being proposed. A proficient method to track the position of the microcapsules during such therapies, particularly if they are amenable to commonly used instrumentation, would greatly help the development of such treatments. Here we propose to label the microcapsules with gold nanoparticles to provide a bright contrast on small animal x-ray micro-CT systems enabling single microcapsule detection. The microcapsules preparation is based on a simple protocol using inexpensive compounds. This, combined with the widespread availability of micro-CT apparatus, renders our method more accessible compared with other methods. Our labeled microcapsules showed good mechanical stability and low cytotoxicity in-vitro. Our post-mortem rodent model data strongly suggest that the high signal intensity generated by the labeled microcapsules permits the use of a reduced radiation dose yielding a method fully compatible with longitudinal in-vivo studies.
ANK3 gene variants have consistently been associated with bipolar spectrum disorder and schizophrenia spectrum disorder. However, the relevance of its encoded protein, ankyrin-3, in these disorders remains elusive. Here, we show that ANK3 gene expression in blood is significantly increased in bipolar disorder and schizophrenia compared with healthy controls. Additionally, we identified potential cis-acting expression quantitative trait loci located close to the transcription start site of one of the isoforms of the gene. These findings suggest that ANK3 mRNA is an interesting marker for further investigation of the underlying mechanisms in psychotic disorders.
Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5' untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression.
Treatment-free remission (TFR) has recently emerged as a goal of treatment in chronic myeloid leukaemia (CML). Molecular remission is sustained in around 30% of imatinib-treated patients who stop treatment after ?2 years with undetectable minimal residual disease (UMRD) by conventional real-time reverse transcription polymerase chain reaction. An additional 20-30% of patients will lose UMRD, but remain in stable major molecular remission off treatment. Most patients with molecular recurrence have a significant increase in BCR-ABL1 within the first 6 months off treatment, but there are also rare late relapses. As re-treatment with imatinib restores control, a trial of TFR is safe so long as careful molecular monitoring is provided to enable prompt re-treatment. The minimum eligibility criteria for a trial of TFR are not yet defined, but the available data support a MRD level of around a molecular response of 4.5 log for at least 2 years. Factors associated with a higher probability of TFR include low risk Sokal score, prior interferon treatment, longer total duration of imatinib treatment and higher numbers of natural killer cells at the time of imatinib discontinuation. Preliminary data suggest that the rate of TFR in patients treated with more potent tyrosine kinase inhibitors will probably be higher. The biology that underlies TFR is an area of active investigation.
Atomistic simulation of chemical systems is currently limited by the elementary description of electrostatics that atomic point-charges offer. Unfortunately, a model of one point-charge for each atom fails to capture the anisotropic nature of electronic features such as lone pairs or ?-systems. Higher order electrostatic terms, such as those offered by a multipole moment expansion, naturally recover these important electronic features. The question remains as to why such a description has not yet been widely adopted by popular molecular mechanics force fields. There are two widely-held misconceptions about the more rigorous formalism of multipolar electrostatics: (1) Accuracy: the implementation of multipole moments, compared to point-charges, offers little to no advantage in terms of an accurate representation of a system's energetics, structure and dynamics. (2) Efficiency: atomistic simulation using multipole moments is computationally prohibitive compared to simulation using point-charges. Whilst the second of these may have found some basis when computational power was a limiting factor, the first has no theoretical grounding. In the current work, we disprove the two statements above and systematically demonstrate that multipole moments are not discredited by either. We hope that this perspective will help in catalysing the transition to more realistic electrostatic modelling, to be adopted by popular molecular simulation software.
It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (?10 nucleotides in length) that alone can accurately predict AGO-mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity.
A reactive azobenzene based super organogelator was found to rapidly and reversibly transform a range of hydrophobic solvents from gels to solutions upon changes in temperature, light and shear force. More specifically they formed gels at concentrations as low as 0.08 wt%. Upon heating, exposure to UV light, or application of shear, the ?-? bonding was disrupted which resulted in a rapid drop of both modulus and viscosity. This was confirmed by (1)H NMR, SAXS, and rheological measurements. Although many examples of organogelators are known in the literature, this is the first time that a reactive group, a benzoyl chloride, has been incorporated in a supramolecular organogel structure. Moreover, this group is available for subsequent synthetic modifications. The presence of benzoyl chloride groups showed a remarkable effect on the formation and properties of the gels. Compared with other approaches, this strategy is advantageous in terms of structural design since it not only produces a multi-responsive soft material but also allows facile modifications which may expand the applications of organogels to other fields.
The relationship between the delivery of dexamethasone and the composition of silicone hydrogel materials was investigated. Two hydrophilic monomers (2-hydroxyethyl methacrylate or N,N-dimethylacrylamide), a siloxy methacrylate-based monomer (1-(Bis(trimethylsiloxy)methylsilyl)propoxy-3-metacryloxy-2-propanol, a polysiloxane (monomethacryloxypropyl-terminated polydimethylsiloxane) and a polymerizable silicone surfactant (Silmer ACR A008-UP) were used to synthesize silicone hydrogels of variable composition. The materials properties, such as surface wettability and equilibrium water content, were highly dependent on polymer composition. All dexamethasone-loaded hydrogels showed uptake that was driven primarily by sorption to the polymer phase. Furthermore, a positive correlation between loading mass and equilibrium water content was established. The duration of drug release from the hydrogels ranged from one to greater than two weeks depending on the monomer composition and relative contribution of hydrophilic and hydrophobic monomers. Higuchi model rate constants for the release showed strong correlation with the equilibrium water content, signifying that the release is likely controlled by aqueous phase diffusion.
In a randomized, phase III trial of nilotinib versus imatinib in patients with newly diagnosed Philadelphia chromosome positive chronic myeloid leukemia in chronic phase, more patients had suboptimal response or treatment failure on front-line imatinib than on nilotinib. Patients with suboptimal response/treatment failure on imatinib 400 mg once or twice daily or nilotinib 300 mg twice daily could enter an extension study to receive nilotinib 400 mg twice daily. After a 19-month median follow up, the safety profile of nilotinib 400 mg twice daily in patients switching from imatinib (n=35) was consistent with previous reports, and few new adverse events occurred in patients escalating from nilotinib 300 mg twice daily (n=19). Of patients previously treated with imatinib or nilotinib 300 mg twice daily, respectively, 15 of 26 (58%) and 2 of 6 (33%) without complete cytogenetic response at extension study entry, and 11 of 34 (32%) and 7 of 18 (39%) without major molecular response at extension study entry, achieved these responses at any time on nilotinib 400 mg twice daily. Estimated 18-month rates of freedom from progression and overall survival after entering the extension study were lower for patients switched from imatinib (85% and 87%, respectively) versus nilotinib 300 mg twice daily (95% and 94%, respectively). Nilotinib dose escalation was generally well tolerated and improved responses in about one-third of patients with suboptimal response/treatment failure. Switch to nilotinib improved responses in some patients with suboptimal response/treatment failure on imatinib, but many did not achieve complete cytogenetic response (clinicaltrials.gov identifiers: 00718263, 00471497 - extension).
The TOPS trial evaluated high- (800 mg/day; n = 319) versus standard-dose (400 mg/day; n = 157) imatinib in patients newly diagnosed with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase. Patients had a minimum follow-up of 42 months or discontinued early. Major molecular response (MMR) rates were similar between arms at (51.6 vs 50.2 % for 400 and 800 mg/day, respectively; P = 0.77) and by (75.8 vs 79.0 %; P = 0.4807) 42 months. There were no differences in event-free survival (EFS), progression-free survival(PFS), or overall survival (OS) between arms. The estimated rates of PFS on treatment and OS at 42 months were significantly higher in patients with MMR at 6, 12, and 18 months compared with those without MMR.Adverse events were more frequent with high-dose imatinib. Patients with B1 treatment interruption (vs [1) and those able to maintain imatinib C600 mg/day (vs\600 mg/day) in the first year of treatment had faster and higher response rates, but no improvement in EFS or PFS. Adherence to prescribed dose without interruption may be more important than initiation of therapy with higher doses of imatinib. Achievement of MMR correlated with longterm clinical outcomes.
Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA-binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.
Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ?1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ?34% of the ?170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.
Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.
Genetic, dietary and immune factors contribute to the pathogenesis of atherosclerosis in humans and mice. Complement activation is an integral part of the innate immune defence but also shapes cellular responses and influences directly triglyceride synthesis. Deficiency of Factor B of the alternative pathway (AP) of complement is beneficial in LDLR(-/-) mice fed a high fat diet. The serum glycoprotein properdin is a key positive regulator of the AP but has not been studied in experimental atherosclerosis. Atherosclerosis was assessed after feeding low fat (LFD) or high fat (HFD) Western type diets to newly generated LDLR(-/-) Properdin(KO) (LDLR(-/-)P(KO)) and LDLR-/-PWT mice. Lipids, lymphocytes and monocytes were similar among genotypes, genders and diets. Complement C3, but not C3adesarg, levels were enhanced in LDLR(-/-)P(KO) mice regardless of diet type or gender. Non-esterified fatty acids (NEFA) were decreased in male LDLR(-/-)P(KO) fed a HFD compared with controls. All mice showed significant atherosclerotic burden in aortae and at aortic roots but male LDLR(-/-) mice fed a LFD were affected to the greatest extent by the absence of properdin. The protective effect of properdin expression was overwhelmed in both genders of LDLR(-/-)mice when fed a HFD. We conclude that properdin plays an unexpectedly beneficial role in the development and progression of early atherosclerotic lesions.
Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long-term, which may be partly due to cytokine-mediated resistance. We evaluated the expression of the IL-3 receptor ? subunit (CD123), an established marker of acute myeloid leukemia (AML) stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared to normal donors CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only healthy donor allogeneic natural killer (NK) cells were able to mount an effective CSL362-mediated ADCC response, but also CML patients autologous NK cells. In addition, CSL362 also neutralized IL 3-mediated rescue of TKI-induced cell death. Notably, combination of TKI and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal haematopoietic stem and progenitor cells. Thus, our data supports the further evaluation of CSL362 therapy in CML.
We explored the impact of early molecular response (EMR; BCR-ABL ? 10% on the international scale [BCR-ABL(IS)] at 3 or 6 months) on outcomes in patients with newly diagnosed chronic myeloid leukemia in chronic phase treated with nilotinib or imatinib, based on 4 years of follow-up in ENESTnd (NCT00471497). Patients (N = 846) received nilotinib 300 mg twice daily, nilotinib 400 mg twice daily, or imatinib 400 mg once daily. At 3 months, more patients had EMR failure (ie, BCR-ABL(IS) > 10%) on imatinib (33%) than on nilotinib (9%-11%); similarly at 6 months, 16% of patients in the imatinib arm vs 3% and 7% in the nilotinib arms had EMR failure. In all arms, EMR failure was associated with lower rates of molecular response, an increased risk of progression, and lower overall survival compared with EMR achievement. We also analyzed patient and treatment characteristics associated with EMR and found distinct patterns in the nilotinib arms vs the imatinib arm. High Sokal risk score was associated with a high rate of EMR failure on imatinib, but not on nilotinib. In contrast, reduced dose intensity and dose interruptions were strongly associated with EMR failure in nilotinib-treated, but not imatinib-treated, patients. This study is registered at clinicaltrials.gov, identifier: NCT00471497.
With the approval in many countries of nilotinib and dasatinib for frontline therapy in chronic myeloid leukemia, clinicians now have to make a difficult choice. Because none of the 3 available tyrosine kinase inhibitors (TKIs) have shown a clear survival advantage, they all represent reasonable choices. However, in individual patients, the case may be stronger for a particular TKI. In the younger patient, in whom the prospect of eventually achieving treatment-free remission is likely to be of great importance, dasatinib or nilotinib may be preferred, although their advantage over imatinib in this setting remains to be proven. In patients with a higher risk of transformation (which is currently based on prognostic scoring), the more potent TKIs may be preferred because they appear to be more effective at reducing the risk of transformation to BC. However, imatinib still represents an excellent choice for many chronic myeloid leukemia patients. All of these considerations need to be made in the context of the patients comorbidities, which may lead to one or more TKIs being ruled out of contention. Whatever first choice of TKI is made, treatment failure or intolerance must be recognized early because a prompt switch to another TKI likely provides the best chance of achieving optimal response.
Despite vast improvements in the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML) in chronic phase (CP), advanced stages of CML, accelerated phase or blast crisis, remain notoriously difficult to treat. Treatments that are highly effective against CML-CP produce disappointing results against advanced disease. Therefore, a primary goal of therapy should be to maintain patients in CP for as long as possible, by (1) striving for deep, early molecular response to treatment; (2) using tyrosine kinase inhibitors that lower risk of disease progression; and (3) more closely observing patients who demonstrate cytogenetic risk factors at diagnosis or during treatment.
Identifying genes in the genomic context is central to a cells ability to interpret the genome. Yet, in general, the signals used to define eukaryotic genes are poorly described. Here, we derived simple classifiers that identify where transcription will initiate and terminate using nucleic acid sequence features detectable by the yeast cell, which we integrate into a Unified Model (UM) that models transcription as a whole. The cis-elements that denote where transcription initiates function primarily through nucleosome depletion, and, using a synthetic promoter system, we show that most of these elements are sufficient to initiate transcription in vivo. Hrp1 binding sites are the major characteristic of terminators; these binding sites are often clustered in terminator regions and can terminate transcription bidirectionally. The UM predicts global transcript structure by modeling transcription of the genome using a hidden Markov model whose emissions are the outputs of the initiation and termination classifiers. We validated the novel predictions of the UM with available RNA-seq data and tested it further by directly comparing the transcript structure predicted by the model to the transcription generated by the cell for synthetic DNA segments of random design. We show that the UM identifies transcription start sites more accurately than the initiation classifier alone, indicating that the relative arrangement of promoter and terminator elements influences their function. Our model presents a concrete description of how the cell defines transcript units, explains the existence of nongenic transcripts, and provides insight into genome evolution.
We describe novel lyotropic liquid-crystalline (LLC) materials based on photoresponsive amphiphiles that exhibit rapid photoswitchable rheological properties of unprecedented magnitude between solidlike and liquidlike states. This was achieved through the synthesis of a novel azobenzene-containing surfactant (azo-surfactant) that actuates the transition between different LLC forms depending on illumination conditions. Initially, the azo-surfactant/water mixtures formed highly ordered and viscous LLC phases at 20-55 wt % water content. Spectroscopic, microscopic, and rheological analysis confirmed that UV irradiation induced the trans to cis isomerization of the azo-surfactant, leading to the disruption of the ordered LLC phases and a dramatic, rapid decrease in the viscosity and modulus resulting in a 3 orders of magnitude change from a solid (20?000 Pa) to a liquid (50 Pa) at rate of 13?500 Pa/s. Subsequent exposure to visible light reverses the transition, returning the viscosity essentially to its initial state. Such large, rapid, and reversible changes in rheological properties within this LLC system may open a door to new applications for photorheological fluids.
Developmental transcription factors are key players in animal multicellularity, being members of the T-box family that are among the most important. Until recently, T-box transcription factors were thought to be exclusively present in metazoans. Here, we report the presence of T-box genes in several nonmetazoan lineages, including ichthyosporeans, filastereans, and fungi. Our data confirm that Brachyury is the most ancient member of the T-box family and establish that the T-box family diversified at the onset of Metazoa. Moreover, we demonstrate functional conservation of a homolog of Brachyury of the protist Capsaspora owczarzaki in Xenopus laevis. By comparing the molecular phenotype of C. owczarzaki Brachyury with that of homologs of early branching metazoans, we define a clear difference between unicellular holozoan and metazoan Brachyury homologs, suggesting that the specificity of Brachyury emerged at the origin of Metazoa. Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. This finding suggests that functional specificity between different T-box classes is likely achieved by interaction with alternative cofactors, as opposed to differences in binding specificity.
An efficient MRI T2-weighted contrast agent incorporating a potential liver targeting functionality was synthesized via the combination of superparamagnetic iron oxide (SPIO) nanoparticles with multiwalled carbon nanotubes (MWCNTs). Poly(diallyldimethylammonium chloride) (PDDA) was coated on the surface of acid treated MWCNTs via electrostatic interactions and SPIO nanoparticles modified with a potential targeting agent, lactose-glycine adduct (Lac-Gly), were subsequently immobilized on the surface of the PDDA-MWCNTs. A narrow magnetic hysteresis loop indicated that the product displayed superparamagnetism at room temperature which was further confirmed by ZFC (zero field cooling)/FC (field cooling) curves measured by SQUID. The multifunctional MWCNT-based magnetic nanocomposites showed low cytotoxicity in vitro to HEK293 and Huh7 cell lines. Enhanced T2 relaxivities were observed for the hybrid material (186 mM(-1) s(-1)) in comparison with the pure magnetic nanoparticles (92 mM(-1) s(-1)) due to the capacity of the MWCNTs to "carry" more nanoparticles as clusters. More importantly, after administration of the composite material to an in vivo liver cancer model in mice, a significant increase in tumor to liver contrast ratio (277%) was observed in T2 weighted magnetic resonance images.
The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.
The complement system is a potent component of the innate immune response, promoting inflammation and orchestrating defense against pathogens. However, dysregulation of complement is critical to several autoimmune and inflammatory syndromes. Elevated expression of the proinflammatory cytokine IL-1? is often linked to such diseases. In this study, we reveal the mechanistic link between complement and IL-1? secretion using murine dendritic cells. IL-1? secretion occurs following intracellular caspase-1 activation by inflammasomes. We show that complement elicits secretion of both IL-1? and IL-18 in vitro and in vivo via the NLRP3 inflammasome. This effect depends on the inflammasome components NLRP3 and ASC, as well as caspase-1 activity. Interestingly, sublethal complement membrane attack complex formation, but not the anaphylatoxins C3a and C5a, activated the NLRP3 inflammasome in vivo. These findings provide insight into the molecular processes underlying complement-mediated inflammation and highlight the possibility of targeting IL-1? to control complement-induced disease and pathological inflammation.
Advances in chronic myeloid leukemia treatment, particularly regarding tyrosine kinase inhibitors, mandate regular updating of concepts and management. A European LeukemiaNet expert panel reviewed prior and new studies to update recommendations made in 2009. We recommend as initial treatment imatinib, nilotinib, or dasatinib. Response is assessed with standardized real quantitative polymerase chain reaction and/or cytogenetics at 3, 6, and 12 months. BCR-ABL1 transcript levels ?10% at 3 months, <1% at 6 months, and ?0.1% from 12 months onward define optimal response, whereas >10% at 6 months and >1% from 12 months onward define failure, mandating a change in treatment. Similarly, partial cytogenetic response (PCyR) at 3 months and complete cytogenetic response (CCyR) from 6 months onward define optimal response, whereas no CyR (Philadelphia chromosome-positive [Ph+] >95%) at 3 months, less than PCyR at 6 months, and less than CCyR from 12 months onward define failure. Between optimal and failure, there is an intermediate warning zone requiring more frequent monitoring. Similar definitions are provided for response to second-line therapy. Specific recommendations are made for patients in the accelerated and blastic phases, and for allogeneic stem cell transplantation. Optimal responders should continue therapy indefinitely, with careful surveillance, or they can be enrolled in controlled studies of treatment discontinuation once a deeper molecular response is achieved.
As intermolecular interactions such as the hydrogen bond are electrostatic in origin, rigorous treatment of this term within force field methodologies should be mandatory. We present a method able of accurately reproducing such interactions for seven van der Waals complexes. It uses atomic multipole moments up to hexadecupole moment mapped to the positions of the nuclear coordinates by the machine learning method kriging. Models were built at three levels of theory: HF/6-31G(**), B3LYP/aug-cc-pVDZ and M06-2X/aug-cc-pVDZ. The quality of the kriging models was measured by their ability to predict the electrostatic interaction energy between atoms in external test examples for which the true energies are known. At all levels of theory, >90% of test cases for small van der Waals complexes were predicted within 1kJmol(-1), decreasing to 60-70% of test cases for larger base pair complexes. Models built on moments obtained at B3LYP and M06-2X level generally outperformed those at HF level. For all systems the individual interactions were predicted with a mean unsigned error of less than 1kJmol(-1).
Cholesterol is implicated in the development of late-onset Alzheimers disease (AD). We sought to determine the associations between beta amyloid (A?) plaque deposition in vivo using Pittsburgh compound B (PiB) and several indices of cholesterol homeostasis (i.e., total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein E (ApoE), clusterin, oxysterol metabolites of cholesterol, and previously reported genes associated with late-onset AD) in 175 nondemented elderly subjects. High A? deposition was associated significantly with a lower Mini-Mental State Examination score (<27 points, p = 0.04), high systolic blood pressure (p = 0.04), carrying the apolipoprotein E epsilon 4 allele (p < 0.01), and lower plasma ApoE levels (p = 0.02), and variation in the ABCA7 (p = 0.02) and EPHA1 genes (p = 0.02). Cholesterol measures were not related to A? deposition in this cohort of nondemented elderly adults. However, plasma and genetic factors relating to cholesterol transport were associated with A? deposition in the brain. A better understanding of cholesterol transport mechanisms may lead to the design of potential targets for the prevention of A? deposition in the brain.
Most patients with chronic myeloid leukemia (CML) treated with imatinib will relapse if treatment is withdrawn. We conducted a prospective clinical trial of imatinib withdrawal in 40 chronic-phase CML patients who had sustained undetectable minimal residual disease (UMRD) by conventional quantitative polymerase chain reaction (PCR) on imatinib for at least 2 years. Patients stopped imatinib and were monitored frequently for molecular relapse. At 24 months, the actuarial estimate of stable treatment-free remission was 47.1%. Most relapses occurred within 4 months of stopping imatinib, and no relapses beyond 27 months were seen. In the 21 patients treated with interferon before imatinib, a shorter duration of interferon treatment before imatinib was significantly associated with relapse risk, as was slower achievement of UMRD after switching to imatinib. Highly sensitive patient-specific BCR-ABL DNA PCR showed persistence of the original CML clone in all patients with stable UMRD, even several years after imatinib withdrawal. No patients with molecular relapse after discontinuation have progressed or developed BCR-ABL mutations (median follow-up, 42 months). All patients who relapsed remained sensitive to imatinib re-treatment. These results confirm the safety and efficacy of a trial of imatinib withdrawal in stable UMRD with frequent, sensitive molecular monitoring and early rescue of molecular relapse.
Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.
Visceral fat is a significant cardiovascular risk factor. Because visceral fat has not been measured systematically in patients with adrenal incidentalomas, we have tested the hypothesis that visceral fat volume may be associated with cutoffs for serum cortisol levels post dexamethasone.
The membrane attack complex of complement (MAC), apart from its classical role of lysing cells, can also trigger a range of non-lethal effects on cells, acting as a drive to inflammation. In the present study, we chose to investigate these non-lethal effects on inflammasome activation. We found that, following sublytic MAC attack, there is increased cytosolic Ca(2+) concentration, at least partly through Ca(2+) release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This increase in intracellular Ca(2+) concentration leads to Ca(2+) accumulation in the mitochondrial matrix via the mitochondrial calcium uniporter (MCU), and loss of mitochondrial transmembrane potential, triggering NLRP3 inflammasome activation and IL-1? release. NLRP3 co-localises with the mitochondria, probably sensing the increase in calcium and the resultant mitochondrial dysfunction, leading to caspase activation and apoptosis. This is the first study that links non-lethal effects of sublytic MAC attack with inflammasome activation and provides a mechanism by which sublytic MAC can drive inflammation and apoptosis.
To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.
To determine if regional gray matter volume (GMV) differences in middle-aged adults with and without type-1 diabetes (T1D) are localized in areas most vulnerable to aging, e.g. fronto-subcortical networks; and if these differences are explained by cardiovascular risk factors and diabetes complications.
Gene duplication results in two identical paralogs that diverge through mutation, leading to loss or gain of interactions with other biomolecules. Here, we comprehensively characterize such network rewiring for C. elegans transcription factors (TFs) within and across four newly delineated molecular networks. Remarkably, we find that even highly similar TFs often have different interaction degrees and partners. In addition, we find that most TF families have a member that is highly connected in multiple networks. Further, different TF families have opposing correlations between network connectivity and phylogenetic age, suggesting that they are subject to different evolutionary pressures. Finally, TFs that have similar partners in one network generally do not in another, indicating a lack of pressure to retain cross-network similarity. Our multiparameter analyses provide unique insights into the evolutionary dynamics that shaped TF networks.
Recent studies have demonstrated that some patients with chronic myeloid leukemia (CML) can maintain remission after discontinuation of imatinib. A prerequisite is stable, undetectable BCR-ABL1. It is not known how many patients achieve this response or the factors associated with its achievement. We examined 423 de novo imatinib-treated patients to determine the cumulative incidence of achieving the discontinuation criteria as defined in the CML8 study (?2 years of undetectable BCR-ABL1 [Stable MR(4.5)]), and predictive factors. After 8 years of imatinib, the cumulative incidence of Stable MR(4.5) was 36.5%. Therefore, 9% to 15% of first-line imatinib-treated patients would maintain remission after discontinuation. The BCR-ABL1 level at 3 months and factors at diagnosis were examined for association with Stable MR(4.5): Sokal risk, age, sex, and assigned imatinib dose. The only independent predictors were female sex (54.4% vs 27.2%; P = .018) and the 3-month BCR-ABL1 (P < .001). The highest cumulative incidence of Stable MR(4.5) after 8 years was 78.2% for patients with BCR-ABL1 ? 0.10%(IS) at 3 months (n = 38). Time to major molecular response (MMR) influenced the time to reach Stable MR(4.5) (P < .001), suggesting slower dynamics of response with a delayed MMR. The findings justify the focus on rapid reduction of BCR-ABL1 as a strategy to maximize potential suitability for imatinib discontinuation studies. The Iris trial was registered at http://www.clinicaltrials.gov as NCT00006343. The Tops trial was registered at http://www.clinicaltrials.gov as NCT00124748. The TIDEL I trial was registered at www.ANZCTR.org.au as ACTRN12607000614493. The TIDEL II trial was registered at www.ANZCTR.org.au as ACTRN12607000325404.
In patients with chronic myeloid leukemia, BCR-ABL mutations contribute to resistance to tyrosine kinase inhibitor therapy. We examined the occurrence of treatment-emergent mutations and their impact on response in patients from the ENESTnd phase 3 trial. At the 3-year data cutoff, mutations were detected in approximately twice as many patients (21) on imatinib 400 mg once daily as on nilotinib (11 patients each on nilotinib 300 mg twice daily and nilotinib 400 mg twice daily). The majority of mutations occurred in patients with intermediate or high Sokal scores. Most mutations (14 [66.7%]) emerging during imatinib treatment were imatinib-resistant and nilotinib-sensitive. Incidence of the T315I mutation was low (found in 3, 2, and 3 patients on nilotinib 300 mg twice daily, nilotinib 400 mg twice daily, and imatinib, respectively) and mostly occurred in patients with high Sokal scores. Of the patients with emergent mutations, 1 of 11, 2 of 11, and 7 of 21 patients on nilotinib 300 mg twice daily, nilotinib 400 mg twice daily, and imatinib, respectively, progressed to accelerated phase/blast crisis (AP/BC) on treatment. Overall, nilotinib led to fewer treatment-emergent BCR-ABL mutations than imatinib and reduced rates of progression to AP/BC in patients with these mutations. (Clinicaltrials.gov NCT00471497).
Current guidelines for managing Philadelphia-positive chronic myeloid leukemia include monitoring the expression of the BCR-ABL1 (breakpoint cluster region/c-abl oncogene 1, non-receptor tyrosine kinase) fusion gene by quantitative reverse-transcription PCR (RT-qPCR). Our goal was to establish and validate reference panels to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate global standardization on the international scale (IS).
The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110? by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.
The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the ?c family due to their receptors sharing the signalling subunit ?c, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the ?c cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the ?c receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.
BACKGROUND: We evaluated BCR-ABL1 kinetics in patients treated with nilotinib and analyzed whether a dynamic model of changes in BCR-ABL1 levels over time could be used to predict long-term responses. METHODS: Patients from the nilotinib registration trial (CAMN107A2101; registered at www.clinicaltrials.gov as NCT00109707) who had imatinib-resistant or -intolerant Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase (CP) or accelerated phase with BCR-ABL1 greater than 10% (on the international scale [IS]) at baseline and, in the first 6 months, had at least three BCR-ABL1 transcript measurements and an average daily dose of at least 720 mg were included in this analysis (N equals 123). RESULTS: More than half of patients (65/123; 53 percent) had a slow monophasic response and the remainder (58/123; 47 percent) had a biphasic response, in which patients had a rapid initial decrease in BCR-ABL1 transcripts followed by a more gradual response. The biphasic response type strongly correlated with improved event-free survival (EFS). Data in the first 6 months of follow-up were sufficient to predict EFS at 24 months. CONCLUSIONS: Unlike newly diagnosed patients with Ph+ CML-CP--in whom the majority had a biphasic response--approximately half of patients with imatinib-resistant or -intolerant CML had a slower, monophasic response. Second-line patients who did have a biphasic response had an EFS outlook similar to that of newly diagnosed patients treated with imatinib. Our model was comparable to using BCR-ABL1 (IS) less than or equal to 10 percent at 6 months as a threshold for predicting EFS.
Genomic analyses often involve scanning for potential transcription factor (TF) binding sites using models of the sequence specificity of DNA binding proteins. Many approaches have been developed to model and learn a proteins DNA-binding specificity, but these methods have not been systematically compared. Here we applied 26 such approaches to in vitro protein binding microarray data for 66 mouse TFs belonging to various families. For nine TFs, we also scored the resulting motif models on in vivo data, and found that the best in vitro-derived motifs performed similarly to motifs derived from the in vivo data. Our results indicate that simple models based on mononucleotide position weight matrices trained by the best methods perform similarly to more complex models for most TFs examined, but fall short in specific cases (<10% of the TFs examined here). In addition, the best-performing motifs typically have relatively low information content, consistent with widespread degeneracy in eukaryotic TF sequence preferences.
Research on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH.
Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.
Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103(+) DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.
RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.
HnRNP (heterogeneous nuclear ribonucleoprotein) proteins are a large family of RNA-binding proteins that regulate numerous aspects of RNA processing. Interestingly, several paralogous pairs of hnRNPs exist that exhibit similar RNA-binding specificity to one another, yet have non-redundant functional targets in vivo. In this study we systematically investigate the possibility that the paralogs hnRNP L and hnRNP LL have distinct RNA binding determinants that may underlie their lack of functional redundancy. Using a combination of RNAcompete and native gel analysis we find that while both hnRNP L and hnRNP LL preferentially bind sequences that contain repeated CA dinucleotides, these proteins differ in their requirement for the spacing of the CAs. Specifically, hnRNP LL has a more stringent requirement for a two nucleotide space between CA repeats than does hnRNP L, resulting in hnRNP L binding more promiscuously than does hnRNP LL. Importantly, this differential requirement for the spacing of CA dinucleotides explains the previously observed differences in the sensitivity of hnRNP L and LL to mutations within the CD45 gene. We suggest that overlapping but divergent RNA-binding preferences, as we show here for hnRNP L and hnRNP LL, may be commonplace among other hnRNP paralogs.
The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. DOI:http://dx.doi.org/10.7554/eLife.00675.001.
Specific imatinib-resistant BCR-ABL1 mutations (Y253H, E255K/V, T315I, F317L, and F359V/C) predict failure of second-line nilotinib or dasatinib therapy in patients with chronic myeloid leukemia; however, such therapy also fails in approximately 40% of patients in the chronic phase of this disease who do not have these resistant mutations. We investigated whether sensitive mutation analysis could identify other poor-risk subgroups. Analysis was performed by direct sequencing and sensitive mass spectrometry on 220 imatinib-resistant patients before they began nilotinib or dasatinib therapy. Patients with resistant mutations by either method (n = 45) were excluded because inferior response was known. Of the remaining 175 patients, 19% had multiple mutations by mass spectrometry versus 9% by sequencing. Compared with 0 or 1 mutation, the presence of multiple mutations was associated with lower rates of complete cytogenetic response (50% vs 21%, P = .003) and major molecular response (31% vs 6%, P = .005) and a higher rate of new resistant mutations (25% vs 56%, P = .0009). Sensitive mutation analysis identified a poor-risk subgroup (15.5% of all patients) with multiple mutations not identified by standard screening.
The functional activity of the organic cation transporter 1 (OCT-1) protein (OCT-1 activity) is an excellent predictor of molecular response and progression-free survival in patients with newly diagnosed chronic phase chronic myeloid leukemia treated with imatinib as front-line therapy.
This study was untaken to investigate the association of micro brain infarcts (MBIs) with antemortem global cognitive function (CF), and whether brain weight (BW) and Alzheimer lesions (neurofibrillary tangles [NFTs] or neuritic plaques [NPs]) mediate the association.
The yeast Saccharomyces cerevisiae is a prevalent system for the analysis of transcriptional networks. As a result, multiple DNA-binding sequence specificities (motifs) have been derived for most yeast transcription factors (TFs). However, motifs from different studies are often inconsistent with each other, making subsequent analyses complicated and confusing. Here, we have created YeTFaSCo (The Yeast Transcription Factor Specificity Compendium, http://yetfasco.ccbr.utoronto.ca/), an extensive collection of S. cerevisiae TF specificities. YeTFaSCo differs from related databases by being more comprehensive (including 1709 motifs for 256 proteins or protein complexes), and by evaluating the motifs using multiple objective quality metrics. The metrics include correlation between motif matches and ChIP-chip data, gene expression patterns, and GO terms, as well as motif agreement between different studies. YeTFaSCo also features an index of expert-curated motifs, each associated with a confidence assessment. In addition, the database website features tools for motif analysis, including a sequence scanning function and precomputed genome-browser tracks of motif occurrences across the entire yeast genome. Users can also search the database for motifs that are similar to a query motif.
Cellular-based therapies for insulin-dependent diabetes are potential means of achieving and maintaining normal blood glucose levels (BGL) without the need for insulin administration. Islets isolated from donor pancreases have been the most common tissue used to date, but supply is a limiting factor. The use of human embryonic stem cells (hESC) as a therapy became a possibility with the report that these cells could be differentiated to pancreatic progenitors (PP) over 12 days in vitro. Conversion of PP to glucose-responsive insulin-secreting cells can be achieved by transplanting the progenitors in vivo where cell maturation occurs. To date this step has not been shown under in vitro conditions.
Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.
BCR-ABL1 mutation analysis is recommended to facilitate selection of appropriate therapy for patients with chronic myeloid leukemia after treatment with imatinib has failed, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. However, mutations could be present below the detection limit of conventional direct sequencing. We developed a sensitive, multiplexed mass spectrometry assay (detection limit, 0.05% to 0.5%) to determine the impact of low-level mutations after imatinib treatment has failed.
Imatinib induces a durable response in most patients with Philadelphia chromosome-positive chronic myeloid leukemia, but it is currently unclear whether imatinib reduces the leukemic stem cell (LSC) burden, which may be an important step toward enabling safe discontinuation of therapy. In this article, we use mathematical models of BCR-ABL levels to make inferences on the dynamics of LSCs.
Nilotinib has shown greater efficacy than imatinib in patients with newly diagnosed Philadelphia chromosome-positive chronic myeloid leukaemia (CML) in chronic phase after a minimum follow-up of 12 months. We present data from the Evaluating Nilotinib Efficacy and Safety in clinical Trials-newly diagnosed patients (ENESTnd) study after a minimum follow-up of 24 months.
We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.
There are three currently identified secondary resistance mechanisms observed in patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs). These are BCR-ABL kinase domain (KD) mutations, increased BCR-ABL expression, and overexpression of drug-efflux proteins (ABCB1 and ABCG2). To investigate the interplay between these three modes of resistance, three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib to 2 ?M, or dasatinib to 200 nM. Eight imatinib- and two dasatinib-resistant cell lines were established. Two imatinib-resistant K562 lines both had increased BCR-ABL expression as the apparent mode of resistance. However, when a dasatinib-resistant K562 culture was generated we observed gradually increasing BCR-ABL expression which peaked prior to identification of the T315I mutation. BCR-ABL overexpression followed by mutation development was observed in a further 4/10 cell lines, each with different KD mutations. In contrast, three imatinib-resistant K562 Dox lines exhibited only a further increase in ABCB1 expression. All TKI-resistant cell lines generated had increased IC(50) (dose of drug required to reduce phosphorylation of the adaptor protein p-Crkl by 50%) to imatinib, dasatinib, and nilotinib, regardless of which TKI was used to induce resistance. This suggests that currently available TKIs share the same susceptibilities to drug resistance.
Next-generation sequencing (NGS) techniques have already shown their potential in the identification of mutations underlying rare inherited disorders. We report here the application of linkage analysis in combination with targeted DNA capture and NGS to a Norwegian family affected by an undiagnosed mental retardation disorder with an autosomal recessive inheritance pattern. Linkage analysis identified two loci on chromosomes 9 and 17 which were subject to target enrichment by hybridization to a custom microarray. NGS achieved 20-fold or greater sequence coverage of 83% of all protein-coding exons in the target regions. This led to the identification of compound heterozygous mutations in NAGLU, compatible with the diagnosis of Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo Syndrome type B). This diagnosis was confirmed by demonstrating elevated levels of heparan sulphate in urine and low activity of ?-N-acetyl-glucosaminidase in cultured fibroblasts. Our findings describe a mild form of MPS IIIB and illustrate the diagnostic potential of targeted NGS in Mendelian disease with unknown aetiology.
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