bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microscopic evaluation of synaptic connectivity and network formation in primary hippocampal neurons
Authors: Verstraelen Peter , Timmermans Jean‐Pierre , Detrez Jan R , Pintelon Isabel , Nuydens Rony , Meert Theo , De Vos Winnok 
Published: November 2023

During brain development, orchestrated activation of genetic programs, as well as spontaneous electrical activity guide the correct wiring of neuronal networks. Mature neuronal networks are characterized by the expression of synaptic markers, synchronized electrical activity and the presence of dendritic spines, tiny protrusions from the dendritic shaft that compartmentalize single synapses to ensure optimal regulation of synaptic strength. While neurodevelopmental disorders such as schizophrenia are thought to result from defective neuronal network formation, neurodegenerative disorders like Alzheimer's disease are characterized by progressive synapse loss and associated mild cognitive impairment long before actual neurodegeneration occurs.We established an in vitro model based on primary hippocampal neurons that recapitulates features of mature neuronal networks. We optimized a set of microscopy workflows to quantitatively characterize both morphological (neurite outgrowth, synapse density, dendritic spine density) and functional (spontaneous electrical activity) aspects of the established neuronal networks (Cornelissen et al. 2013; Pani et al. 2014; Verstraelen et al. 2014). Neurite outgrowth and synapse density were quantified after immunostaining for β‐III‐tubulin and synaptophysin using an in‐house developed FIJI‐based image analysis script. Targeted photoconversion of mEos4b‐LifeAct in single neurons facilitated image analysis of dendritic spine density. Calcium fluctuations, serving as a proxy for electrical activity, were detected in neurons expressing the GCaMP6f reporter. A dedicated script was developed to obtain parameters describing both single neuron bursting patterns and correlated network events.Using calcium imaging, we pinpointed a critical period in which stochastic activity of individual neurons turned into robust, synchronized network activity, indicative of the formation of functional synapses. This synchronization coincided with an increase in neurite outgrowth and synapse density, while dendritic spine density increased mainly after synchronization of the activity, suggesting that the latter enables fine‐tuning of synaptic connections. We further showed that synchronized network activity is mediated by the NMDA receptor, and that interference with microtubule stability alters the bursting pattern.In brief, we have established a robust platform for pharmacological and/or genetic interrogation of synaptic connectivity in in vitro neuronal networks. Our approach is easily amenable to upscaling, which makes it an attractive model for high content screening in the context of neurodevelopmental or neurodegenerative disorders.
The Repairable Brain: An Anatomists View
Authors: Sladek John R 
Published: November 2023

Many neurological disorders result from progressive degeneration of specific anatomical pathways in the central nervous system (CNS). These include, but are not restricted to diseases such as Parkinson's (PD), amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and many others. Also, CNS trauma can result in paralysis, and cognitive decline, motor disability and neuroendocrine imbalance are common. The identification of neurotransmitters in the 1960's with the advent of monoamine histochemistry followed by immunohistochemical staining for neuropeptides a decade later made it possible to correlate loss of transmitters such as dopamine (DA) with specific diseases such as PD. Beginning in the 1980's we and other laboratories began to test whether cell replacement was possible in the CNS and discovered that endocrine, motor, visual, and cognitive functions could be improved in rodent models of neurological disorders. We then extended our PD studies to the non‐human primate and demonstrated that the functional integrity of the nigrostriatal DA pathway from the substantia nigra (SN) of the mesencephalon to the neostriatum could be restored by the implantation of embryonic DA neurons into DA‐deficient regions. Optimal results were obtained by knowledge of the anatomy and migratory pattern of the newly generated DA neurons during precise stages of ontogeny. Grafted neurons survived implantation, extended DA rich neurites into the host neuropil and released DA as measured biochemically in microdissected punches in the region of grafts. Animals improved for over a year and were studied morphologically to assess survival characteristics. These proof of principle studies led to clinical trials that showed efficacy and the value of a translational model closely related to humans.We next demonstrated the positive effect of concurrent gene therapy via the delivery of growth factors as well as the potential to reconstruct the nigrostriatal pathway with the use of helper co‐grafts of embryonic target tissue. Axons extended from DA rich neuronal grafts in the region of the substantia nigra to the neostriatum by placement of growth factor‐laden “stepping stones.” More recently, we studied another mechanism of potential brain repair, i.e. neurogenesis. In DA‐depleted monkeys we discovered a population of DA neurons that increases significantly in number and invades the caudate nucleus, most likely from the subventricular zone, a known neurogenic niche. Because vertebrates like the salamander can regenerate DA neurons in response to insult this wave of putative neurogenesis may represent a conserved biological mechanism in the primate brain. Research next will focus on mechanisms to stimulate and maintain these new DA neurons for neural repair.Support or Funding InformationSupported by NINDS, various foundations and with collaborators identified during the lecture.
DNA Excision Repair Map of the Human Genome at Single Nucleotide Resolution
Authors: Sancar Aziz 
Published: November 2023

We have developed methods for genome‐wide analysis of damage formation and repair in humans. These methods, which are named Damage‐seq and Excision Repair‐seq (XR‐seq), were used to map Ultraviolet (UV) light‐induced DNA damage and repair by nucleotide excision repair at single nucleotide resolution. The high resolution damage formation and repair maps have enabled us to quantitatively describe the effects of chromatin states, including transcription, histone marks, and sequence elements, on DNA repair with potential applications to carcinogenesis and chemotherapy.Support or Funding InformationThis work was supported by National Institutes of Health grant R01GM32833.
Mechanism and Regulation of the SWI/SNF Family of ATP‐dependent Chromatin Remodelers
Authors: Bartholomew Blaine , Sen Payel , Kundu Soumyadipta , Hailu Solomon , Persinger Jim , Hada Arjan , Lu Yue , Luo Jie , Ranish Jeff 
Published: November 2023

In yeast there are two SWI/SNF complexes and in mammals there is a family of an estimated ~100 complexes, each with different combinations of related subunits. Efforts in sequencing many cancer genomes has revealed that mammalian SWI/SNF complexes is one of the most frequently mutated epigenetic factors found in a broad range of cancers. In order to understand more about the subunit organization of SWI/SNF and because yeast and mammalian SWI/SNF complexes share many conserved subunits and domains; we have mapped the inter‐subunit and intra‐subunit interactions of the yeast SWI/SNF complex with lysine specific homo bi‐functional crosslinkers and mass spectrometry. The A‐T motif was shown to extensively interact with the ATPase domain, and is well conserved throughout all eukaryotes and targeted in colorectal and endometrial cancers. Deletion of the A‐T hook in yeast SWI/SNF reduced the DNA‐stimulated ATPase and consequently the nucleosome mobilizing activities of the complex without affecting the subunit composition or nucleosome binding affinity. Loss of the A‐T hook effects only a small subset of the genes regulated by SWI/SNF as shown by RNA‐seq analysis and suggests that mutation of the A‐T hook creates an aberrant form of SWI/SNF that impacts a subset of its target genes. Further analysis using MNase‐seq has found that in the absence of the A‐T hook domain the occupancy of the +1 nucleosome, immediately downstream of the transcription start site, is decreased and implicates a role of SWI/SNF in helping to assemble or maintain nucleosomes at this position. The A‐T hook of murine Brg1 was deleted in embryonic stem cells using CRISPR‐Cas9 and the role of the A‐T hook in mammalian SWI/SNF analyzed by RNA‐seq and ChIP‐seq as will be discussed in more detail. In addition the Snf5 subunit of yeast SWI/SNF was found to interact with the ATPase domain of the catalytic subunit. When the Snf5 subunit was deleted several other subunits were also lost from the complex and formed an aberrant version of the SWI/SNF complex due to changes in subunit composition. We have carefully mapped the changes in nucleosomal interactions and nucleosome mobilizing activities that occur with the loss of the SNF5 sub‐module. The Snf5 subunit is required for the stable interactions of the catalytic subunit with nucleosomal DNA in the absence of ATP. The mammalian ortholog of SNF5 or SMARCB1 is a tumor suppressor gene and the loss of this subunit is a driver mutation in pediatric rhabdoid sarcomas. Our data suggest how the loss of SNF5 might alter the activity of SWI/SNF, which in turn could lead to oncogenesis.Support or Funding InformationNIH grant GM 48413
Mapping Dynamic Protein Interaction Landscapes in Saccharomyces cerevisiae Using a Novel Whole Network Enrichment Approach
Authors: Yates John , Stein Benjamin D. , Calzolari Diego , Lavallee‐Adam Mathieu 
Published: November 2023

Cellular physiology is dynamic in nature, especially in response to perturbation. Despite significant advances in genetics, biochemistry and cell biology, signal transduction networks and protein effectors that mediate specific cellular responses have mostly been annotated under static (i.e. basal) cellular conditions, both at the genetic and physical interaction level. These approaches provide little information as to the dynamics of a given pathway or its constituents under varying patho‐physiological contexts. Proteins that compose these cellular networks are largely organized into protein complexes that work in concert to enable desired cellular responses. Therefore, defining the architecture and response of these complexes to perturbation is vital to our mechanistic understanding of fundamental cellular biology, and may provide critical insight into disease pathology.We have developed and applied methods to annotate the architecture and dynamics of protein complex networks by utilizing a novel whole‐network affinity pull‐down method coupled to novel computational analysis tools. This method involves generation of TAP‐tagged node proteins, which are expressed, pooled and affinity‐purified in parallel. This approach allows for efficient system‐wide identification and quantification of protein complex members and novel interactors in response to perturbation in a highly efficient manner. This proteomic platform has been enabled through development and application of biochemical and proteomic methodologies to study network response to known perturbations in established protein networks. Specifically, as a proof‐of‐principle, the developed platform has been employed to study changes in nutrient sensing protein complexes in response to rapamycin treatment in Saccharomyces Cerevisiae.This concept has been extended to the DNA damage response as well as energy homeostasis networks in yeast, and the latter correlated to a homologous mammalian network for investigation of metabolic dysregulation in disease. By measuring the dynamics of protein networks, we have identified novel associations within and between components that have otherwise been missed in traditional protein‐by‐protein Affinity Purification‐Mass Spectrometry (AP‐MS) methodologies, and this is the first proteomic platform to enable dynamic interaction measurements at a network‐wide scale.Support or Funding InformationNIH P41 GM103533, R01 MH67880
Defining interaction and structural dynamics of protein complexes using integrated mass spectrometry approaches
Authors: Huang Lan 
Published: November 2023

Protein complexes are dynamic and functional entities that are of critical importance for various biological processes in living cells. Protein‐protein interactions are vital for modulating the composition, assembly, structure, stability and function of macromolecular protein complexes. Disruption of normal protein‐protein interactions has been linked to various human disease and recent studies have described protein interaction interfaces as a new class of attractive targets for drug development. Therefore, comprehensive characterization of interaction dynamics of protein complexes in living systems not only improves our understanding of cellular processes but also provides potential targets for future therapeutics. However, such tasks remain challenging due to limitations in existing methodologies. In recent years, we have developed several novel mass spectrometry‐based proteomic strategies by integrating new sample preparation techniques, affinity purification methods, quantitative mass spectrometry with bioinformatics tools to obtain a comprehensive proteomic profile of protein complexes from living cells. In addition, we have developed new cross‐linking strategies to identify protein interaction interfaces for structural elucidation of large protein complexes. To evaluate these strategies, we have characterized the proteome dynamics of proteasome complexes that are responsible for the degradation of ubiquitinated substrates. The results have provided new mechanistic insights on the regulation of proteasomal degradation at different physiological states. The methodology presented here can be applied for the study of other protein complexes.Support or Funding InformationThis work is supported by NIH RO1GM074830 to L.H, and R01GM106003 to L.H. and S. R..
Targeting Tumors Driven by VHL or IDH Mutations
Authors: Kaelin William 
Published: November 2023

Loss of the VHL tumor suppressor protein (pVHL) is the signature event in clear cell renal cancer, which is the most common form of kidney cancer. pVHL is the substrate recognition subunit of an ubiquitin ligase that targets the alpha subunits of the HIF transcription factor for proteasomal degradation. Deregulation of HIF, especially HIF2, drives pVHL‐defective tumor growth in preclinical models. Although HIF2α would classically be deemed “undruggable”, Rick Bruick and Kevin Gardner at UTSW identified a potentially druggable pocket in HIF2α that formed the basis for a drug discovery effort at Peloton Therapeutics. I will present preclinical data with a tool compound, PT2399, that inhibits HIF2 and suppresses orthotopic and metastatic VHL‐/‐ renal carcinomas in mice. Our recent work with lenalidomide suggests an additional strategy for undruggable transcription factors, namely, small molecule mediated target destabilization.Some cancers, including a subset of acute leukemias and gliomas, are driven by IDH1 or IDH2 mutations. These mutations led to the accumulation of mM amounts of R‐2 hydroxyglutarate (R‐2HG). R‐2HG can interfere with (usually inhibit) the activities of various 2‐oxoglutarate (2‐OG)‐dependent enzymes, including the JmjC histone demethylases, the TET DNA hydroxylases, and the EglN prolyl hydroxylases. We showed that the effects of R‐2HG in leukemia are reversible and drugs that inhibit R‐2HG production appear promising in early clinical trials in leukemia patients. It is less clear whether the effects of R‐2HG will be reversible in glioma. Nonetheless, we have identified vulnerabilities created by high R‐2HG levels that might be exploited therapeutically.
Post Transcriptional Regulation of Lipogenesis
Authors: Horton Jay D 
Published: November 2023

Obesity and insulin resistance are strongly associated with the development of nonalcoholic fatty liver disease (NAFLD). The excess triglyceride that accumulates in hepatocytes is derived from multiple sources, one of which is de novo lipogenesis. Lipogenesis in liver is regulated by SREBP‐1c, a transcription factor that activates genes involved in fatty acid synthesis. The first committed enzyme in fatty acid synthesis, acetyl‐CoA carboxylase (ACC), is also regulated by phosphorylation/dephosphorylation, and protein polymerization. Previously, we showed that MIG12, a 22 kDa cytosolic protein, binds to ACC and lowers the threshold for citrate‐induced ACC activation. In vitro, recombinant MIG12 induced recombinant ACC polymerization and increased ACC activity by >50‐fold. In vivo, adenoviral overexpression of MIG12 induced ACC polymerization, increased fatty acid synthesis, resulting in triglyceride accumulation and fatty liver. A second protein, Spot14 (S14) shares significant homology with MIG12, is also highly expressed in liver and is regulated by SREBP‐1c. The mechanism for S14's ability to modulate lipogenesis has not been completely elucidated. In vitro biochemical studies and in vivo studies show that S14 can form a heterodimer with MIG12. Recombinant MIG12;S14 heterodimeric proteins also had no effect on ACC polymerization or activity and reduced overall flux through the fatty acid synthesis pathway, suggesting that S14 has an additional role in regulating fatty acid synthesis. Here, we further explore the interrelated molecular and physiological functions of lipogenic regulators in the regulation of lipogenesis.Support or Funding InformationFoundation LeDucq Network Award NIH‐HL020948
Prospects for Noncoding RNA Discovery in Bacteria
Authors: Breaker Ronald R. 
Published: November 2023

Modern cells likely descended from ancient RNA World organisms that employed a great diversity of noncoding RNAs, and therefore many types of catalytic and ligand‐sensing RNAs might still await discovery. Indeed, ongoing bioinformatics analyses are revealing the existence of many novel ribozyme and riboswitch candidates, and some of these still await further biochemical and biological validation studies. Recently we have discovered several classes of self‐cleaving ribozymes and ligand‐binding riboswitches that broaden the known functional and structural diversity of bacterial noncoding RNAs. In rare instances, unusually large and highly‐conserved noncoding RNAs are identified. We speculate that some of these large noncoding RNAs are ribozymes that have novel biochemical functions. In this presentation, I will summarize some of the most recent ribozyme and riboswitch discoveries, and highlight the prospects for future noncoding RNA discoveries in bacteria.Support or Funding InformationResearch in the Breaker Laboratory is supported by HHMI, the NIH, and by Yale University.
When Good Ribosomes Go Bad
Authors: Baserga Susan J. , McCann Kathleen L. , Teramoto Takamasa , Zhang Jun , Tanaka Hall Traci M. 
Published: November 2023

Ribosome biogenesis, beyond being a critical requirement for growth in all eukaryotic cells, has a broad impact on the etiology of numerous human disorders. Human genetic diseases caused by mutations in structural components of the ribosome or in factors required to make ribosomes in the cell nucleolus are called ribosomopathies. The congenital ribosomopathy, ANE syndrome, is caused by a single amino acid substitution (L351P) in the third RNA recognition motif (RRM) of the nucleolar protein, RBM28. While patients with ANE syndrome have reduced numbers of mature ribosomes, it is unclear how this single point mutation disrupts nucleolar function in ribosome synthesis and assembly. To investigate the molecular basis of ANE syndrome, we utilized yeast as a model system and determined how the orthologous mutation (L306P) in RRM3 of Nop4, the yeast ortholog of RBM28, contributes to defective ribosome biogenesis. We find that introduction of the ANE syndrome mutation into Nop4 confers both growth and pre‐rRNA processing defects in yeast. Recently, we showed that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome (Genes & Dev. 2015). Here we demonstrate that the presence of the L306P mutation abrogates several, but not all, of Nop4's protein‐protein interactions, thereby disrupting Nop4's hub protein function. Surprisingly, RRMs 3 and 4 of Nop4 are necessary and sufficient for growth, pre‐LSU rRNA processing and hub protein function. Circular dichroism and NMR demonstrate that introduction of the ANE syndrome mutation into RRM3 of human RBM28 disrupts domain folding, consistent with the nucleolar dysfunction of the mutated full‐length protein. We conclude that the ANE syndrome mutation generates defective protein folding which, in turn, abrogates protein‐protein interactions and causes defective pre‐LSU rRNA processing, thus revealing the underlying molecular basis of this human disease.Support or Funding InformationNIGMS, NIEHS, Intramural Research Program of the NIH
The Nanodisc Platform for Determining the Structure and Function of Membrane Proteins
Authors: Sligar Stephen G. 
Published: November 2023

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction, cell‐viral interaction, neuronal connections and key steps in many metabolic pathways. Integral membrane proteins account for >30% of the human proteome and are the targets of more than half of all currently marketed therapeutic agents. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists and present a challenge in avoiding denaturation or inactivation and in finding inhibitors, activators and specific antibodies. Often, since membrane proteins are inherently insoluble and prone to aggregation and oligomerization when removed from their native membrane environment, the active function of interest is obscured. The Nanodisc platform circumvents these challenges by providing a generally applicable self‐assembled system that renders all classes of membrane proteins, such as receptors, transporters, enzymes, and viral antigens, soluble in aqueous media. Because Nanodisc constructs provide a native‐like bilayer environment that maintain a target's functional activity, they are a versatile tool in the study of ion channels, biased signaling by GPCRs, integrins, cytochrome P450s, various toxins and viral entities. Importantly, the Nanodisc also provides a controlled surface for protein – membrane recognition such as is involved in RAS signaling and blood coagulation. Numerous recent advances in the structural biology of membrane proteins, particularly cryoelectron microscopy and NMR, have been enabled through the use of Nanodiscs. In addition to these opportunities, Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. In my presentation I will summarize recent successes using the Nanodisc platform.Support or Funding InformationNational Institutes of Health