BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. ...Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.
Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring ...skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.
Bucket brigade is a linear order-picking process with one loading station and one unloading station. Here we model and quantify picker blocking in bucket brigade order picking systems (OPSs). A ...bucket brigade improves throughput and reduces variability in OPSs. However, each order picking trip fills different orders and creates workload variation per order. We show that bucket brigade order picking experiences picker blocking when there is a workload imbalance per pick face. We derive a closed-form solution to quantify the level of blocking for two extreme walk speed cases. Additional simulation comparisons validate the picker blocking model which includes backward walk and hand-off delays. We identify the relationship between picker blocking in bucket brigade OPSs and picker blocking in a circular-aisle abstraction of OPSs in which backward walk and hand-off delays as well as forward walk speed are considered. Our analytical model and simulations show that aggregating orders into batches smoothes the workload variation by pooling the randomness of picks in each order and that slowest-to-fastest picker sequencing modulates picker blocking between two pickers, i.e., the interaction between neighboring pickers.
The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified ...(SIRT1–SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2–3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.
Dendritic cells (DCs) sense microbes via multiple innate receptors. Signals from different innate receptors are coordinated and integrated by DCs to generate specific innate and adaptive immune ...responses against pathogens. Previously, we have shown that two pathogen recognition receptors, TLR2 and dectin-1, which recognize the same microbial stimulus (zymosan) on DCs, induce mutually antagonistic regulatory or inflammatory responses, respectively. How diametric signals from these two receptors are coordinated in DCs to regulate or incite immunity is not known. In this study, we show that TLR2 signaling via AKT activates the β-catenin/T cell factor 4 pathway in DCs and programs them to drive T regulatory cell differentiation. Activation of β-catenin/T cell factor 4 was critical to induce regulatory molecules IL-10 (Il-10) and vitamin A metabolizing enzyme retinaldehyde dehydrogenase 2 (Aldh1a2) and to suppress proinflammatory cytokines. Deletion of β-catenin in DCs programmed them to drive Th17/Th1 cell differentiation in response to zymosan. Consistent with these findings, activation of the β-catenin pathway in DCs suppressed chronic inflammation and protected mice from Th17/Th1-mediated autoimmune neuroinflammation. Thus, activation of β-catenin in DCs via the TLR2 receptor is a novel mechanism in DCs that regulates autoimmune inflammation.
Addressing global biodiversity loss requires an expanded focus on multiple dimensions of biodiversity. While most studies have focused on the consequences of plant interspecific diversity, our ...mechanistic understanding of how genetic diversity within plant species affects plant productivity remains limited. Here, we use a tree species × genetic diversity experiment to disentangle the effects of species diversity and genetic diversity on tree productivity, and how they are related to tree functional diversity and trophic feedbacks. We found that tree species diversity increased tree productivity via increased tree functional diversity, reduced soil fungal diversity, and marginally reduced herbivory. The effects of tree genetic diversity on productivity via functional diversity and soil fungal diversity were negative in monocultures but positive in the mixture of the four tree species tested. Given the complexity of interactions between species and genetic diversity, tree functional diversity and trophic feedbacks on productivity, we suggest that both tree species and genetic diversity should be considered in afforestation.
Relaxin‐3 has been proposed to modulate emotional–behavioural functions such as arousal and behavioural activation, appetite regulation, stress responses, anxiety, memory, sleep and circadian rhythm. ...The nucleus incertus (NI), in the midline tegmentum close to the fourth ventricle, projects widely throughout the brain and is the primary site of relaxin‐3 neurons. Over recent years, a number of preclinical studies have explored the function of the NI and relaxin‐3 signalling, including reports of mRNA or peptide expression changes in the NI in response to behavioural or pharmacological manipulations, effects of lesions or electrical or pharmacological manipulations of the NI, effects of central microinfusions of relaxin‐3 or related agonist or antagonist ligands on physiology and behaviour, and the impact of relaxin‐3 gene deletion or knockdown. Although these individual studies reveal facets of the likely functional relevance of the NI and relaxin‐3 systems for human physiology and behaviour, the differences observed in responses between species (e.g. rat vs. mouse), the clearly identified heterogeneity of NI neurons and procedural differences between laboratories are some of the factors that have prevented a precise understanding of their function. This review aims to draw attention to the current preclinical evidence available that suggests the relevance of the NI/relaxin‐3 system to the pathology and/or symptoms of certain neuropsychiatric disorders and to provide cognizant directions for future research to effectively and efficiently uncover its therapeutic potential.
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This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc
► We propose an augmented large neighborhood method for team orienteering problem. ► A shift improvement and a replacement improvement algorithms are developed. ► The method identifies the best known ...solution for 386 of 387 benchmark problems. ► It outperforms 12 existing approaches.
In the Team Orienteering Problem (TOP), a team of vehicles attempts to collect rewards at a given number of stops within a specified time frame. Once a vehicle visits a stop and collects its reward, no other vehicles can collect the reward again. Typically, a team cannot visit all stops and therefore has to identify the “best” set of stops to visit in order to maximize total rewards. We propose a large neighborhood search method with three improvement algorithms: a local search improvement, a shift and insertion improvement, and replacement improvement. Our proposed approach can find the best known solutions for 386 of the 387 benchmark instances, for the one instance which our solution is not the current best it is only varies by one from the best. Our approach outperforms all the previous approaches in terms of solution quality and computation time.
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Cancer immunotherapy has yet to reach its full potential due in part to limited response rates and side effects inherent to systemic delivery of immune-modulating drugs. Local ...administration of immunotherapy using drug-eluting embolic (DEE) microspheres as drug delivery vehicles for direct infusion into tumor-feeding arteries might increase and prolong tumor drug concentrations and reduce systemic drug exposure, potentially improving the risk-to-benefit ratio of these agents. The purpose of this study was to evaluate the ability of four immune modulators affecting two different immune pathways to potentiate replication of immune cells from a woodchuck model of hepatocellular carcinoma. DSR 6434, a Toll-like receptor agonist, and BMS-202, a PD-L1 checkpoint inhibitor, induced immune cell replication and were successfully loaded into radiopaque DEE microspheres in high concentrations. Release of DSR 6434 from the DEE microspheres was rapid (t99% = 0.4 h) upon submersion in a physiologic saline solution while BMS-202 demonstrated a more sustained release profile (t99% = 17.9 h). These findings demonstrate the feasibility of controlled delivery of immune-modulating drugs via a local DEE microsphere delivery paradigm.
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