A central alarm system that gates multi-sensory innate threat cues to the amygdala.

Perception of threats is essential for survival. Previous findings suggest that parallel pathways independently relay innate threat signals from different sensory modalities to multiple brain areas, such as the midbrain and hypothalamus, for immediate avoidance. Yet little is known about whether and how multi-sensory innate threat cues are integrated and conveyed from each sensory modality to the amygdala, a critical brain area for threat perception and learning. Here, we report that neurons expressing calcitonin gene-related peptide (CGRP) in the parvocellular subparafascicular nucleus in the thalamus and external lateral parabrachial nucleus in the brainstem respond to multi-sensory threat cues from various sensory modalities and relay negative valence to the lateral and central amygdala, respectively. Both CGRP populations and their amygdala projections are required for multi-sensory threat perception and aversive memory formation. The identification of unified innate threat pathways may provide insights into developing therapeutic candidates for innate fear-related disorders.

Divergent brainstem opioidergic pathways that coordinate breathing with pain and emotions.

Breathing can be heavily influenced by pain or internal emotional states, but the neural circuitry underlying this tight coordination is unknown. Here we report that Oprm1 (µ-opioid receptor)-expressing neurons in the lateral parabrachial nucleus (PBL) are crucial for coordinating breathing with affective pain in mice. Individual PBLOprm1 neuronal activity synchronizes with breathing rhythm and responds to noxious stimuli. Manipulating PBLOprm1 activity directly changes breathing rate, affective pain perception, and anxiety. Furthermore, PBLOprm1 neurons constitute two distinct subpopulations in a "core-shell" configuration that divergently projects to the forebrain and hindbrain. Through non-overlapping projections to the central amygdala and pre-Bötzinger complex, these two subpopulations differentially regulate breathing, affective pain, and negative emotions. Moreover, these subsets form recurrent excitatory networks through reciprocal glutamatergic projections. Together, our data define the divergent parabrachial opioidergic circuits as a common neural substrate that coordinates breathing with various sensations and behaviors such as pain and emotional processing.

Neural basis of opioid-induced respiratory depression and its rescue.

Opioid-induced respiratory depression (OIRD) causes death following an opioid overdose, yet the neurobiological mechanisms of this process are not well understood. Here, we show that neurons within the lateral parabrachial nucleus that express the µ-opioid receptor (PBL Oprm1 neurons) are involved in OIRD pathogenesis. PBL Oprm1 neuronal activity is tightly correlated with respiratory rate, and this correlation is abolished following morphine injection. Chemogenetic inactivation of PBL Oprm1 neurons mimics OIRD in mice, whereas their chemogenetic activation following morphine injection rescues respiratory rhythms to baseline levels. We identified several excitatory G protein-coupled receptors expressed by PBL Oprm1 neurons and show that agonists for these receptors restore breathing rates in mice experiencing OIRD. Thus, PBL Oprm1 neurons are critical for OIRD pathogenesis, providing a promising therapeutic target for treating OIRD in patients.

A Visual Analytics Approach for Ecosystem Dynamics based on Empirical Dynamic Modeling.

An important approach for scientific inquiry across many disciplines involves using observational time series data to understand the relationships between key variables to gain mechanistic insights into the underlying rules that govern the given system. In real systems, such as those found in ecology, the relationships between time series variables are generally not static; instead, these relationships are dynamical and change in a nonlinear or state-dependent manner. To further understand such systems, we investigate integrating methods that appropriately characterize these dynamics (i.e., methods that measure interactions as they change with time-varying system states) with visualization techniques that can help analyze the behavior of the system. Here, we focus on empirical dynamic modeling (EDM) as a state-of-the-art method that specifically identifies causal variables and measures changing state-dependent relationships between time series variables. Instead of using approaches centered on parametric equations, EDM is an equation-free approach that studies systems based on their dynamic attractors. We propose a visual analytics system to support the identification and mechanistic interpretation of system states using an EDM-constructed dynamic graph. This work, as detailed in four analysis tasks and demonstrated with a GUI, provides a novel synthesis of EDM and visualization techniques such as brush-link visualization and visual summarization to interpret dynamic graphs representing ecosystem dynamics. We applied our proposed system to ecological simulation data and real data from a marine mesocosm study as two key use cases. Our case studies show that our visual analytics tools support the identification and interpretation of the system state by the user, and enable us to discover both confirmatory and new findings in ecosystem dynamics. Overall, we demonstrated that our system can facilitate an understanding of how systems function beyond the intuitive analysis of high-dimensional information based on specific domain knowledge.

The D614G mutation in the SARS-CoV2 Spike protein increases infectivity in an ACE2 receptor dependent manner.

The SARS-CoV2 coronavirus responsible for the current COVID19 pandemic has been reported to have a relatively low mutation rate. Nevertheless, a few prevalent variants have arisen that give the appearance of undergoing positive selection as they are becoming increasingly widespread over time. Most prominent among these is the D614G amino acid substitution in the SARS-CoV2 Spike protein, which mediates viral entry. The D614G substitution, however, is in linkage disequilibrium with the ORF1b P314L mutation where both mutations almost invariably co-occur, making functional inferences problematic. In addition, the possibility of repeated new introductions of the mutant strain does not allow one to distinguish between a founder effect and an intrinsic genetic property of the virus. Here, we synthesized and expressed the WT and D614G variant SARS-Cov2 Spike protein, and report that using a SARS-CoV2 Spike protein pseudotyped lentiviral vector we observe that the D614G variant Spike has >1/2 log10 increased infectivity in human cells expressing the human ACE2 protein as the viral receptor. The increased binding/fusion activity of the D614G Spike protein was corroborated in a cell fusion assay using Spike and ACE2 proteins expressed in different cells. These results are consistent with the possibility that the Spike D614G mutant increases the infectivity of SARS-CoV2.

Circularity in fisheries data weakens real world prediction.

The systematic substitution of direct observational data with synthesized data derived from models during the stock assessment process has emerged as a low-cost alternative to direct data collection efforts. What is not widely appreciated, however, is how the use of such synthesized data can overestimate predictive skill when forecasting recruitment is part of the assessment process. Using a global database of stock assessments, we show that Standard Fisheries Models (SFMs) can successfully predict synthesized data based on presumed stock-recruitment relationships, however, they are generally less skillful at predicting observational data that are either raw or minimally filtered (denoised without using explicit stock-recruitment models). Additionally, we find that an equation-free approach that does not presume a specific stock-recruitment relationship is better than SFMs at predicting synthesized data, and moreover it can also predict observational recruitment data very well. Thus, while synthesized datasets are cheaper in the short term, they carry costs that can limit their utility in predicting real world recruitment.

Time varying causal network reconstruction of a mouse cell cycle.

BACKGROUND: Biochemical networks are often described through static or time-averaged measurements of the component macromolecules. Temporal variation in these components plays an important role in both describing the dynamical nature of the network as well as providing insights into causal mechanisms. Few methods exist, specifically for systems with many variables, for analyzing time series data to identify distinct temporal regimes and the corresponding time-varying causal networks and mechanisms. RESULTS: In this study, we use well-constructed temporal transcriptional measurements in a mammalian cell during a cell cycle, to identify dynamical networks and mechanisms describing the cell cycle. The methods we have used and developed in part deal with Granger causality, Vector Autoregression, Estimation Stability with Cross Validation and a nonparametric change point detection algorithm that enable estimating temporally evolving directed networks that provide a comprehensive picture of the crosstalk among different molecular components. We applied our approach to RNA-seq time-course data spanning nearly two cell cycles from Mouse Embryonic Fibroblast (MEF) primary cells. The change-point detection algorithm is able to extract precise information on the duration and timing of cell cycle phases. Using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle. CONCLUSIONS: The temporal dependence of cellular components we provide in our model goes beyond what is known in the literature. Furthermore, our inference of dynamic interplay of multiple intracellular mechanisms and their temporal dependence on one another can be used to predict time-varying cellular responses, and provide insight on the design of precise experiments for modulating the regulation of the cell cycle.

BRCA1/BARD1-dependent ubiquitination of NF2 regulates Hippo-YAP1 signaling.

Coordination of growth and genomic stability is critical for normal cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player in maintenance of genomic stability, its role in growth signaling remains elusive. Here, we show that BRCA1 facilitates stabilization of YAP1 protein and turning "off" the Hippo pathway through ubiquitination of NF2. In BRCA1-deficient cells Hippo pathway is "turned On." Phosphorylation of YAP1 is crucial for this signaling process because a YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 kinase recognition sites not only resists degradation but also rescues YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, an ectopic expression of the active Mt-YAP5SA, but not inactive Mt-YAP6SA, promotes EGF-independent proliferation and tumorigenesis in BRCA1-/- mammary epithelial cells. These findings establish an important role of BRCA1 in regulating stability of YAP1 protein that correlates positively with cell proliferation.

Glioblastoma Model Using Human Cerebral Organoids.

We have developed a cancer model of gliomas in human cerebral organoids that allows direct observation of tumor initiation as well as continuous microscopic observations. We used CRISPR/Cas9 technology to target an HRasG12V-IRES-tdTomato construct by homologous recombination into the TP53 locus. Results show that transformed cells rapidly become invasive and destroy surrounding organoid structures, overwhelming the entire organoid. Tumor cells in the organoids can be orthotopically xenografted into immunodeficient NOD/SCID IL2RG-/- animals, exhibiting an invasive phenotype. Organoid-generated putative tumor cells show gene expression profiles consistent with mesenchymal subtype human glioblastoma. We further demonstrate that human-organoid-derived tumor cell lines or primary human-patient-derived glioblastoma cell lines can be transplanted into human cerebral organoids to establish invasive tumor-like structures. Our results show potential for the use of organoids as a platform to test human cancer phenotypes that recapitulate key aspects of malignancy.

Lentiviral vectors for retrograde delivery of recombinases and transactivators.

Lentiviral vectors pseudotyped with the rabies virus (RV) envelope glycoprotein efficiently infect via axon terminals to stably deliver transgenes to distant neurons projecting to an injection site, but the resulting expression levels are too low and variable for most neuroscientific applications. If used to deliver recombinases or transactivators, however, lentiviral vectors are excellent means of targeting projection neurons when used in reporter mice or in combination with a second virus to express "payload" transgenes at high levels. For retrograde infection of significant numbers of neurons, high virus titers are critical. Here we present reagents and a protocol for generating high-titer supernatants that can be concentrated 1000-fold for final titers in excess of 10(10) infectious units per milliliter. We demonstrate the usefulness of these vectors by selectively targeting corticothalamic and corticotectal neurons for high-level expression of a fluorophore in knock-in reporter mice.

Role of BRCA1 in brain development.

Breast cancer susceptibility gene 1 (BRCA1) is a breast and ovarian cancer tumor suppressor whose loss leads to DNA damage and defective centrosome functions. Despite its tumor suppression functions, BRCA1 is most highly expressed in the embryonic neuroepithelium when the neural progenitors are highly proliferative. To determine its functional significance, we deleted BRCA1 in the developing brain using a neural progenitor-specific driver. The phenotype is characterized by severe agenesis of multiple laminated cerebral structures affecting most notably the neocortex, hippocampus, cerebellum, and olfactory bulbs. Major phenotypes are caused by excess apoptosis, as these could be significantly suppressed by the concomitant deletion of p53. Certain phenotypes attributable to centrosomal and cell polarity functions could not be rescued by p53 deletion. A double KO with the DNA damage sensor kinase ATM was able to rescue BRCA1 loss to a greater extent than p53. Our results suggest distinct apoptotic and centrosomal functions of BRCA1 in neural progenitors, with important implications to understand the sensitivity of the embryonic brain to DNA damage, as well as the developmental regulation of brain size.

Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration.

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration.

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl.

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

BRCA1 tumour suppression occurs via heterochromatin-mediated silencing.

Mutations in the tumour suppressor gene BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of Brca1 in mice results in transcriptional de-repression of the tandemly repeated satellite DNA. Brca1 deficiency is accompanied by a reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions and ubiquitylates H2A in vivo. Ectopic expression of H2A fused to ubiquitin reverses the effects of BRCA1 loss, indicating that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA de-repression was also observed in mouse and human BRCA1-deficient breast cancers. Ectopic expression of satellite DNA can phenocopy BRCA1 loss in centrosome amplification, cell-cycle checkpoint defects, DNA damage and genomic instability. We propose that the role of BRCA1 in maintaining global heterochromatin integrity accounts for many of its tumour suppressor functions.

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration.

BACKGROUND: Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. RESULTS: Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences. CONCLUSION: Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Genic regions of a large salamander genome contain long introns and novel genes.

BACKGROUND: The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 x 10(9) bp) were isolated and sequenced to characterize the structure of genic regions. RESULTS: Annotation of genes within BACs showed that axolotl introns are on average 10x longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86%) of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5x larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! CONCLUSION: This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein.

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.