bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissecting the Development of Clinical Reasoning in the Gross Anatomy Laboratory
Authors: Rae Guenevere , Karpinski Aryn C. , McGoey Robin
Published: November 2023

This project examined the relationship between the act of identifying pathologic lesions in the gross anatomy laboratory and the development of clinical diagnostic reasoning. During the first year gross anatomy course at Louisiana State University Health Science Center (LSUHSC), Pathology faculty and residents visited the laboratory four times (9.5 hours total) throughout the course and interacted with the first year medical students. During these interactive sessions, the Pathologists discussed grossly observable signs of pathology in the cadavers and discussed their potential role in the cadaver's cause of death. In situations where gross identification of the pathology was questionable, the pathology faculty and residents took tissue samples from the cadavers and histo‐pathologically confirmed the diagnoses. An assessment was designed for the measurement of the student's ability to assimilate the data in making an accurate diagnosis of the cadaver's cause of death. The assessment merged elements from two commonly used clinical reasoning assessments: a Script Concordance Test and a Triple Jump Test. Students were given the assignment to record data on the cadaver they were dissecting as if they were performing an autopsy. After the conclusion of the dissection procedures, the students (n=202) were asked to rate the degree to which they felt that each finding contributed to their “first patient's” cause of death. The ratings were compared to ratings given by a panel of clinical experts who reviewed the student's autopsy reports. The assessment is designed to detect clinical reasoning skills gained by first year medical students while dissecting within the gross anatomy laboratory environment.

Support or Funding Information

American Association of Anatomists Educational Scholarship
New Approaches and Insights into Cortical Microcircuits
Authors: Adesnik Hillel , Mardinly Alan , Pegard Nicolas , Oldenburg Ian , Waller Laura
Published: November 2023

The brain encodes and processes information through precise spatiotemporal patterns of neural activity. Despite recent technological advances, no approach offers sufficient resolution to decipher the neural code in order to reveal its logic and possible syntax. Doing so could offer unparalleled access to understanding brain function and disease. To address this technical gap, we developed and optimized a new approach to bi‐directionally edit neural populations codes in the neocortex during behavior using spatiotemporally precise multiphoton optogenetics. We overcame major barriers in temporal fidelity and spatial precision in order to add or remove action potentials from naturally active populations with single cell resolution and millisecond precision. By using ultrafast opsins, amplified infrared lasers, and optimized holographic stimulation, we present a method to arbitrarily write in or silence precise trains of action potentials in large populations of neurons and in three dimensions in the neocortex of behaving animals.

Support or Funding Information

Arnold and Mabel Beckman Foundation, New York Stem Cell Foundation Robertson Investigator, NIH New Innovator, McKnight Endowment for Neuroscience
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.
On the Way to Virtual
Authors: Ogilvie Robert W
Published: November 2023

In my experience as a career teacher of medical histology I have seen the medium for illustrating histological structure in lectures and labs evolve from intricate colored chalk drawings to dynamic Internet accessible virtual slides. My first exposure to histology was in a course taught by two professors at Ohio State University in 1958 that used intricate colored drawings on the classroom chalk board to illustrate the lectures. Since that time, during my 45+ year career in several medical schools directing medical histology courses, I have personally experienced the digital microscopy movement that began in the 1970's and today has a central place in the teaching of histology. In this presentation, the incremental application of new technologies during the digital microscopy movement to facilitate students learning histology will be highlighted concluding with an example of the application of virtual microscopy in teaching histology in an online histology course that originated at the University of South Carolina in the spring of 2011. This upper level course for college juniors, seniors and graduate students offered by the Department of Biological Sciences includes narrated video lectures and the utilization of two virtual lab resources. The narrated lectures that include interactive questions at the end for self‐assessment are available to students via Blackboard, the university's course management system. The lecture slides with the narrative text are provided to the students as PDF documents for study offline. All quizzes and exams are administered online. The virtual lab Internet accessible resources are WebMic that delivers nested static images with two opportunities for self‐assessment, and Virtual Slides that are dynamic and are posed to the students as unknowns during examinations. This course has completed 14 semesters with over 1500 students having completed the course. The majority of students enrolled in the course are planning to apply to medical or dental schools but there are students from most all of the disciplines and colleges on campus enrolling in the course each semester. A total of forty two students spread over four summer semesters, 2012 – 2015, took the course while enrolled in a one year certificate program designed for failed applicants to medical or dental school to improve their chance of being admitted. The online histology course is the first in the curriculum of this special program at the Medical University of South Carolina. The presentation will conclude with thoughts about how taking a histology course in college may impact the student's experience in the modern medical and dental school curricula.
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 Information

Supported by NINDS, various foundations and with collaborators identified during the lecture.
Everything That Matters in Biomedicine Builds on Basic Science
Authors: Collins Francis S.
Published: November 2023

In this presentation, the Director of the National Institutes of Health (NIH) will highlight the agency's past, present, and future support of basic science. Foundational research conducted by seven decades of grantees has been central to NIH's ability to fulfill its mission: to seek fundamental knowledge about the nature and behavior of living systems and to apply that knowledge to enhance health, lengthen life, and reduce illness and disability. History shows that major biomedical advances regularly spring from unexpected basic discoveries made through investigator‐initiated research. Furthermore, no one can foresee what threads of foundational knowledge will be woven together to produce new medical breakthroughs, or pave the way for innovative technologies that enable the biomedical research enterprise to tackle questions once beyond its reach. NIH‐funded basic science has set the stage for remarkable advances in the 21st century. To realize the full potential of these opportunities, NIH will require a steady, sustained stream of resources, and will need to be able to continue and strengthen its efforts to encourage, train, and support the next generation of basic scientists.
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 Information

This 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 Information

NIH grant GM 48413
Structural and Functional Analysis of Histone Chaperones
Authors: Gu Yajie , Bergeron Serge , Krzizike Daniel , Luger Karolin
Published: November 2023

Nucleosomes are large macromolecular complexes that are assembled and disassembled in a stepwise manner. Nucleosome assembly and disassembly processes are essential during DNA replication, transcription, and repair, and are orchestrated by multiple protein complexes. Histone chaperones are multifunctional proteins that bind and shuttle histones into the nucleus, assemble and disassemble nucleosomes in an ATP‐independent manner, and regulate transcription through largely unknown mechanisms. Several structurally unrelated types of histone chaperones, each with multiple family members, are known. Some histone chaperones operate equally well with all types of histones, whereas others are specific to types of histones, or even histone variants and function exclusively in replication‐independent pathways. Some histone chaperones act synergistically with ATP‐dependent chromatin remodeling factors. We will discuss various aspects of histone chaperone function during nucleosome assembly and disassembly, and highlight different mode(s) of histone chaperone – histone interactions with possible roles in histone folding.
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 Information

NIH 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 Information

This 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 Information

Foundation 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 Information

Research 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 Information

NIGMS, 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 Information

National Institutes of Health
Unconventional Protein Secretion and Protein Quality Control
Authors: Ye Yihong , Lee Jin‐Gu
Published: November 2023

To safeguard proteomic integrity, cells rely on the ubiquitin proteasome system to degrade aberrant polypeptides and autophagy to eliminate protein aggregates. However, it is unclear whether cells can remove defective proteins that escape degradation due to protein quality control (PQC) failure or proteasome dysfunction lest toxic protein aggregates be formed. Here we report a pathway termed Misfolding‐Associated Protein Secretion (MAPS), which utilizes the endoplasmic reticulum (ER)‐associated deubiquitinase USP19 to export aberrant cytosolic proteins that are inefficiently degraded by the proteasome. USP19 possesses both deubiquitinating and chaperone activity, allowing recruitment of misfolded proteins to the ER for deubiquitination, encapsulation into ER‐associated vesicles, and subsequent secretion. USP19 deficient cells are defective in growth after exposing to a proteasome inhibitor, suggesting a critical role in adaptation to proteasome dysfunction. Together, our findings delineate a novel PQC pathway that, unlike degradation‐based mechanism, promotes protein homeostasis by exporting misfolded proteins through an unconventional route.

Support or Funding Information

The research is funded by the intramural research program of NIDDK.
The E3 ligase Listerin/Ltn1 links ribosome‐associated protein quality control and neurodegeneration
Authors: Joazeiro Claudio
Published: November 2023

We had previously reported that a homozygous mutation in the lister gene leads to neurodegeneration in mice. lister encodes a RING domain E3 ligase, Listerin, which is conserved among eukaryotic organisms and is widely expressed in mouse tissues. To elucidate Listerin's function we took advantage of the genetic and biochemical tractability of S. cerevisiae and discovered that the yeast Listerin ortholog (Ltn1) is a key player in a novel pathway of protein quality control; more specifically, Ltn1 associates with the 60S ribosomal subunit, and acts as the critical E3 ligase mediating degradation of aberrant nascent polypeptides remaining stalled on ribosomes that are unable to either elongate or terminate translation (2, 3). Subsequent work from others revealed that Ltn1 functions together with other factors as part of the Ribosome‐Associated Quality Control (RQC) complex. I will present ongoing work in the lab towards dissecting mechanisms underlying Ltn1 and RQC function.

Support or Funding Information
Culture Independent Approaches for the Discovery of New Bacterial Metabolites
Authors: Brady Sean F.
Published: November 2023

One of the key revelations to arise from the large‐scale sequencing of bacterial genomic DNA is that traditional approaches used for the discovery of natural products only provide functional access to a small fraction of the natural product biosynthetic gene clusters present in nature. The sequencing of DNA extracted directly from soil samples indicates that as yet uncultured soil microbes outnumber their cultured counterparts by at least two to three orders of magnitude. Environmental bacteria no doubt produces secondary metabolites that could serve as molecular probes of biological processes and therapeutic agents. Uncultivated microorganisms are a very attractive source of potentially new natural products, but they are not amenable to the traditional approaches used to characterize natural products from microbes grown in pure culture. I will discuss culture independent approaches we are using to access novel natural products.
Nature Bank and Drug Leads for TB
Authors: Quinn Ron
Published: November 2023

Nature Bank is a unique storehouse of nature's chemical diversity. It consists of over 63,000 biota samples of plants, and marine invertebrates sourced from tropical and temperature Australia, China and Papua New Guinea. These samples have been processed using a 2‐step chromatographic procedure to produce over 200,000 fractions, optimised for high throughput screening. In addition, Nature Bank holds a collection of 3250 pure natural products that have been acquired over twenty years of natural product‐based research. Nature Bank is a core resource in the Eskitis Institute's drug discovery platform.

Natural product biosynthesis results in a significant number of compounds (~20 000) with molecular weight ≤ 250 Daltons. This set of small natural products contains around 66% of the pharmacophore space of those natural products with MW > 250 Daltons. Natural products with molecular weight ≤ 250 Da can be used in fragment‐based screening.

Multiple‐drug resistance tuberculosis is a serious health problem.

We have undertaken two orthogonal screening strategies. 1) HTS of the Nature Bank fraction library against Mycobacterium tuberculosis H37Rv. The HTS tested 202,983 natural product fractions, 452 fractions showed confirmed activity following MIC determination in an 11‐point dose response analysis (serial dilution starting at 1 μL of fraction in 40 μL M. tuberculosis broth). Activity was observed as low as 1 in 256 dilution (2 Hits) with 6 Hits at 1 in 128 dilution and 11 Hits at 1 in 64 dilution.

2) Fragment‐based screening using direct observation of ligand‐protein complexes in the ICR cell of a FT mass spectrometer. We screened potential TB drug targets and obtained Hits with 10 proteins. We constructed a Network Map to link the natural products with the proteins.

The resultant Network Map analysis has provided a novel approach to progress the fragment Hits.

Support or Funding Information

The work was supported by Bill & Melinda Gates Foundation Grand Challenges Explorations Grants OPP1008376 and OPP1035218 and the Australian Research Council (ARC) Linkage Project (LP120100485). We thank the Australian Research Council (ARC) for support toward NMR and MS equipment (Grant LE0668477 and LE0237908).
Arginine Methylation Modulates RNA Biology Of African Trypanosomes
Authors: Read Laurie K.
Published: November 2023

Kinetoplastid parasites, including Trypanosoma brucei, T. cruzi, and Leishmania spp. share unique biological features. Prominent among these is the lack of transcriptional control. Instead, kinetoplastids regulate gene expression during development and in response to internal and external cues almost entirely by posttranscriptional processes, including mRNA stability and translation. Thus, RNA binding proteins (RBPs) are the key gene regulatory factors in these organisms. Arginine methylation is a posttranslational modification that disproportionately targets RBPs in humans, yeast, and, T. brucei, the causative agent of Human African Trypanosomiasis. To begin to define the impacts of arginine methylation on T. brucei gene expression, we focused on DRBD18, an abundant and essential protein that contains three methylarginine residues between its two RNA binding domains. DRBD18 has opposing effects on the stabilities of two different cohorts of mRNA, such that DRBD18 depletion leads to significant increases and decreases in the abundances of hundreds of mRNAs, including those encoding several kinases, phosphatases, and RBPs. By analysis of DRBD18 knockdown parasites complemented with wild type, hypomethylated (RK), or methylmimic (RF) DRBD18 we demonstrated that methylation promotes the stabilization of those mRNAs that are typically stabilized by DRBD18, whereas hypomethylated DRBD18 is crippled for this function. In direct contrast, destabilization of a different subset of mRNAs relies on hypomethylated DRBD18, and methylmimic DRBD18 cannot perform this function. DRBD18 interacts with the protein arginine methyltransferase, TbPRMT1, and TbPRMT1 knockdown parasites display phenotypes analogous to those of cells complemented with hypomethylated DRBD18, confirming regulation of DRBD18 by methylation. Our data indicate that arginine methylation impacts the association of both mRNAs and effector proteins with DRBD18. For example, RIP/qRT‐PCR revealed differential association of DRBD18(RF) and DRBD18(RK) with target mRNAs. Through tandem affinity purification and mass spectrometry, we isolated several RBPs that interact with hypomethylated or methylmimic DRBD18 in a mutually exclusive manner. Because DRBD18 target mRNAs have unusually long 3′ UTRs, this suggests that methylation regulates association of DRBD18 with other RBPs, which together lead to combinatorial effects on target mRNA fate. In addition, methylmimic and hypomethylated DRBD18 differentially interact with proteins involved in numerous aspects of RNA biology, including RNA transport, deadenylation, and translation. Together, our results demonstrate that arginine methylation acts a molecular switch towards a major trypanosome RBP, DRBD18, highlighting the power of this posttranslational modification to dramatically remodel the transcriptome.

Support or Funding Information

This work was supported by NIH RO1 AI060260
Effects of Acute n‐3 Fatty Acid Administration on Cardiac Lipid Metabolism and Ca2+ Homeostasis during Ischemia‐Reperfusion
Authors: Zirpoli Hylde , Quadri Nosirudeen , O'Shea Karen M , Ananthakrishan Radha , Abdillahi Mariane , Rosario Rosa , Deckelbaum Richard J , Ramasamy Ravichandran
Published: November 2023

Omega‐3 fatty acids (n‐3 FA) are bioactive nutrients exerting cardioprotective effects. In our previous study, we showed that acute n‐3 FA emulsion administration after myocardial ischemia/reperfusion (I/R) injury provides cardioprotection by preserving cardiac function and decreasing infarct size. To address molecular mechanisms responsible for their cardioprotective effects we focused on two main pathways: lipid metabolism pathways and Ca2+ signaling, both playing a crucial role during early reperfusion in the metabolic and functional recovery of post‐ischemic myocardium.

C57BL/6 murine hearts were perfused using Langendorff technique and the administration of n‐3 triglyceride emulsion (300mgTG/100ml) was performed during the reperfusion. We then studied lipid metabolism and Ca2+ signaling.

We first investigated whether acute treatment with n‐3 FA emulsion could modulate lipase activity. However, either myocardial ATGL or HSL expression was not altered by n‐3 FA administration during I/R injury, demonstrating that the uptake of n‐3 FA did not involve lipase‐mediated pathways. AMP‐activated protein kinase (AMPK) and peroxisome proliferator‐activated receptor (PPAR) are critical regulators of lipid metabolism. We then examined whether the activities of these molecules were coordinately regulated by n‐3 FA. After ischemic injury, n‐3 FA induced phosphorylation of AMPK (p<0.05); in contrast, PPARα showed no changes in protein expression after n‐3 FA administration.

I/R injury also alters Ca2+ homeostasis through several mechanisms. Na+/Ca2+ exchanger (NCX) activity increases cytosolic Ca2+, causing cell damage. Also, in I/R conditions sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA2a) dysfunction impairs myocardial contractility and Ca2+ handling. Thus, we questioned whether NCX and SERCA2a might be potential targets for n‐3 FA. n‐3 FA administration after ischemic injury increased both mRNA and protein levels of SERCA2a by 60% (p<0.05). n‐3 FA administration significantly decreased protein level of NCX by 50% (p<0.05), with no effect on its transcript level.

Our results suggest that n‐3 FA partially affect lipid metabolism by activating AMPK and regulate intracellular Ca2+ homeostasis through their influence on SERCA2a and NCX modulations in I/R conditions.

These effects induced by n‐3 FA may contribute to develop new therapeutic approaches in myocardial infarction.
The Portion Control Strategies Trial: a Year‐long Randomized Controlled Trial on the Impact of Portion Control on Weight Change
Authors: Rolls Barbara J. , James Brittany L. , Sanchez Christine E. , Roe Liane S.
Published: November 2023

Controlling food and beverage portion sizes can help reduce energy intake, but there are few data on the longer‐term effects on body weight. We tested the efficacy of two portion‐control strategies on weight change over one year. In a randomized controlled trial, overweight and obese women were randomly assigned to one of three equally intensive interventions: two focused on portion control and one provided standard weight loss advice. The Portion Selection Group learned to select appropriate portions based on energy density and received tools such as food scales, and the Pre‐portioned Foods Group learned to structure meals around pre‐portioned foods and received vouchers for commercial frozen entrées. The Standard Advice Group learned to manage weight by following the US Dietary Guidelines to eat less and to make nutritious choices. Participants met individually with trained interventionists weekly in Month 1, biweekly in Months 2–5, and monthly in Months 6–12; all groups received the same advice on increasing physical activity and managing behavior change. The participants were 186 women with a mean (±SEM) age of 50±0.8 y and weight of 201±2.1 lb; 19% were overweight and 81% were obese. A total of 151 women (81%) had their weight measured at the end of the trial. Rates of attendance and withdrawal from the trial were similar across groups. Results were analyzed with a statistical model that estimated a weight loss trajectory for all participants regardless of the number of contacts. Participants in all groups lost weight during the first six months (13.0±0.9 lb) and maintained most of this loss during the second six months (11.4±1.2 lb). In the first three months, however, the Pre‐portioned Foods Group lost weight at a greater rate (P<0.01; 11.0±1.1 lb) than the Portion Selection Group (9.1±1.0 lb) and the Standard Advice Group (8.4±1.0 lb), and subsequently regained more weight. At the end of the year, weight loss did not differ significantly across groups; 45% had lost ≥ 5% of body weight and 20% had lost ≥ 10% of body weight. Physical activity (step counts) increased over time but did not differ across groups. Changes in blood pressure over the course of the trial did not differ across groups. In all groups, waist circumference and fasting glucose decreased, HDL cholesterol increased, and LDL cholesterol did not change over the trial. These results show that for all groups, individual instruction on diet and physical activity facilitated weight loss and maintenance of that loss. In addition, limiting exposure to large portions by using pre‐portioned foods enhanced initial weight loss, but this was not sustained in the longer term.

Support or Funding Information

National Institutes of Health grant DK059853