13273 research outputs found
Sort by
Accurate measurement of microsatellite length by disrupting its tandem repeat structure
Full text linkTandem repeats of simple sequence motifs, also known as microsatellites, are abundant in the genome. Because their repeat structure makes replication error-prone, variant microsatellite lengths are often generated during germline and other somatic expansions. As such, microsatellite length variations can serve as markers for cancer. However, accurate error-free measurement of microsatellite lengths is difficult with current methods precisely because of this high error rate during amplification. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure of initial templates so that their sequence lengths replicate faithfully. In this work, we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring agreement from two independent first copies of an initial template, we reach error rates below one in a million. We apply this method to a thousand microsatellite loci from the human genome, revealing microsatellite length distributions not observable without mutagenesis
A neural theory for counting memories
Full text linkKeeping track of the number of times different stimuli have been experienced is a critical computation for behavior. Here, we propose a theoretical two-layer neural circuit that stores counts of stimulus occurrence frequencies. This circuit implements a data structure, called a count sketch, that is commonly used in computer science to maintain item frequencies in streaming data. Our first model implements a count sketch using Hebbian synapses and outputs stimulus-specific frequencies. Our second model uses anti-Hebbian plasticity and only tracks frequencies within four count categories ("1-2-3-many"), which trades-off the number of categories that need to be distinguished with the potential ethological value of those categories. We show how both models can robustly track stimulus occurrence frequencies, thus expanding the traditional novelty-familiarity memory axis from binary to discrete with more than two possible values. Finally, we show that an implementation of the "1-2-3-many" count sketch exists in the insect mushroom body
A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation
No full textTargeted protein degradation (TPD) has risen as a promising therapeutic modality. Leveraging the catalytic nature of the ubiquitin-proteasome enzymatic machinery, TPD exhibits higher potency to eliminate disease-causing target proteins such as oncogenic transcription factors that may otherwise be difficult to abrogate by conventional inhibitors. However, there are challenges that remain. Currently, nearly all degraders engage CUL4CRBN or CUL2VHL as the E3 ligase for target ubiquitination. While their immediate efficacies are evident, the narrowed E3 ligase options make TPD vulnerable to potential drug resistance. In addition, E3 ligases show differential tissue expression and have intrinsic limitations in accessing varying types of disease-relevant targets. As the success of TPD is closely associated with the ability of E3 ligases to efficiently polyubiquitinate the target of interest, the long-term outlook of TPD drug development will depend on whether E3 ligases such as CUL4CRBN and CUL2VHL are accessible to the targets of interest. To overcome these potential caveats, a broad collection of actionable E3 ligases is required. Here, we designed a macrocyclic degrader engaging CUL3KLHL20 for targeting BET proteins and validated CUL3KLHL20 as an E3 ligase system suitable for TPD. This work thus contributes to the expansion of usable E3 ligases for potential drug development
Multisensory integration of social signals by a pathway from the basal amygdala to the auditory cortex in maternal mice
Full text linkSocial encounters are inherently multimodal events, yet how and where social cues of distinct modalities merge and interact in the brain is poorly understood. For example, when their pups wander away from the nest, mother mice use a combination of vocal and olfactory signals emitted by the pups to locate and retrieve them. Previous work revealed the emergence of multisensory interactions in the auditory cortex (AC) of both dams and virgins who co-habitate with pups (‘surrogates’). Here we identify a neural pathway that integrates information about odors with responses to sound. We found that a scattered population of glutamatergic neurons in the basal amygdala (BA) projects to the AC and responds to odors, including the smell of pups. These neurons also exhibit increased activity when the surrogate female is searching for pups. Finally, we show that selective optogenetic activation of BA-AC neurons modulates responses to pup calls, and that this modulation switches from predominantly suppressive to predominantly excitatory after maternal experience. This supports an underappreciated role for the amygdala in directly shaping sensory representations in an experience-dependent manner. We propose that the BA-AC pathway integrates olfaction and audition to facilitate maternal care, and speculate that it may carry valence information to the AC
Understanding the neuronal substrates of sensorimotor transformations via a novel closed-loop olfactory task (Smellocator) for mice
No full textThe order code in the olfactory bulb: can odorants be represented by the order of glomerular activation?
No full textWhole Genome Sequencing Comparison of Acute Myeloid Leukemia at Presentation and Remission Predicts Patient Outcome
No full textUnique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions
No full textTranslesion DNA synthesis (TLS) polymerases evolved to tolerate DNA damage that bypasses DNA lesions, thus preventing genomic instability. Multiple TLS polymerases exist with different damage tolerance capabilities, since they have low fidelity their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein-protein interactions are used to regulate TLS polymerase activity. A major knowledge-gap in elucidating the roles of these polymerases is that it is difficult to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2 -benzyl- 2'-deoxyguanosine (EBndG) that is highly selective toward DNA polymerase kappa (Pol κ), a Y family TLS polymerase. Pol k can bypass bulky lesions in the minor groove generated by a metabolite benzo[a]pyrene diolepoxide (BPDE), an environmental carcinogen. Although Pol κ has been identified to have multiple cellular roles, the mechanisms regulating its different cellular activities are unknown. To interrogate the identity of proteins surrounding the Pol κ active sites, we performed an extensive study using modified iPOND (isolation of proteins on nascent DNA), called iPoKD (isolation of proteins on Pol kappa synthesized DNA). Human cell lines were treated with BPDE, subsequently 5-ethynyl-2'-deoxyuridine (EdU) or EBndG was added and proteins bound to the DNA containing EdU and EBndG were analyzed by mass spectrometry. In addition, we performed quantitative analysis of the Pol κ active sites interactome using iPoKD followed by iTRAQ (isobaric tags for relative and absolute quantitation). Our data identified DNA replication and repair proteins previously identified with EdU pull-downs; and interestingly enrichment of RNA binding, ribosome biogenesis, nucleolar proteins and transcriptional repressive complex(es) associated with EBndG pull-downs. Chromatin modifiers, histone chaperones and histone variants are identified suggesting changes in chromatin structure that facilitates Pol κ-mediated DNA lesion bypass, repair and restorative process. identification of unique proteins associated with EBndG-containing DNA, suggests novel roles for Pol κ's activity in the cell. Using super-resolution confocal microscopy, Pol κ activity is identified in the nucleolus after BPDE damage. EBndG is observed in the nucleolar DNA and Pol κ 's activity regulated by the canonical polycomb-complex recruited by the PARylation of PARP1. BPDE lead to transcriptional stress and repression that is gradually recovered. We are investigating whether Pol κ maintains ribosomal DNA integrity after BPDE damage and is required for TLS DNA synthesis or repair. Pol κ active site associated candidate proteins are being validated using CRISPR-Cas9 knockout or siRNA knockdown strategies. These data will provide first insight into Pol κ's core interactome, it's regulation, chromatin surrounding Pol κ active sites and its novel cellular roles
UBP12 and UBP13 deubiquitinases destabilize the CRY2 blue light receptor to regulate Arabidopsis growth
No full textLight is a crucial exogenous signal sensed by cryptochrome (CRY) blue light receptors to modulate growth and the circadian clock in plants and animals. However, how CRYs interpret light quantity to regulate growth in plants remains poorly understood. Furthermore, CRY2 protein levels and activity are tightly regulated in light to fine-tune hypocotyl growth; however, details of the mechanisms that explain precise control of CRY2 levels are not fully understood. We show that in Arabidopsis, UBP12 and UBP13 deubiquitinases physically interact with CRY2 in light. UBP12/13 negatively regulates CRY2 by promoting its ubiquitination and turnover to modulate hypocotyl growth. Growth and development were explicitly affected in blue light when UBP12/13 were disrupted or overexpressed, indicating their role alongside CRY2. UBP12/13 also interacted with and stabilized COP1, which is partially required for CRY2 turnover. Our combined genetic and molecular data support a mechanistic model in which UBP12/13 interact with CRY2 and COP1, leading to the stabilization of COP1. Stabilized COP1 then promotes the ubiquitination and degradation of CRY2 under blue light. Despite decades of studies on deubiquitinases, the knowledge of how their activity is regulated is limited. Our study provides insight into how exogenous signals and ligands, along with their receptors, regulate deubiquitinase activity by protein-protein interaction. Collectively, our results provide a framework of cryptochromes and deubiquitinases to detect and interpret light signals to control plant growth at the most appropriate time