bluuebunny/crossref_metadata_2025 huggingface dataset:
ABSENCE OF MAST CELLS IN AN EXPERIMENTAL MODEL OF PULMONARY AND CARDIAC FIBROSIS, THE CUX‐1 MICE
Authors: Reddy Navya A. , Raza Farhan , Said Ahmed , Livingston Safia , Jacobsen David , Kearns Donovan , Baybutt Richard , Van Den Heuvel Gregory , Molteni Agostino , Hamidpour Soheila
Published: November 2023

Both pulmonary and cardiovascular (CV) fibrosis have a significant role in clinical medicine, however the pathophysiology of fibrosis in these organs is poorly understood (1). Previous rat models of pulmonary fibrosis have shown increased staining of renin and mast cells (MC) production in pulmonary tissue following injection with Triolein (T), a substance comprising 80% of human fat and which is experimentally used to induce fat embolism (FE) (2). MCs are known to produce renin in injured pulmonary tissue, leading to Angiotensin II mediated fibroblasts proliferation (3). The angiotensin II receptor blocker losartan is associated with reduced MCs in fibrotic lungs and a reduction of fibroblasts proliferation, also suggesting that MCs may have a significant correlation with the fibrosis (4).

We aimed to identify one of the mechanisms of CV fibrosis by determining if MC could be found within the lung and cardiac tissue of CUX 1 (CUT‐like homeobox) transgenic mice. Although the CUX‐1 gene is present in all metazoans, its constitutive expression such as in polycystic kidneys blocks cyclin dependent kinase inhibitors p21 and p27, leading to hepatic, pulmonary and cardiac fibrosis (5). Previous studies of CUX‐1 mice have shown severe inflammation of the pulmonary and coronary arteries with media thickening, luminal patency reduction, adventitial fibrosis and MCs presence (5).

In our study, 6 wild type and 6 CUX‐1 transgenic mice were sacrificed by isoflurane anesthesia and necropsied. Lungs and heart were collected, fixed in 10% formalin and stained with H & E (morphometric scoring), Trichrome (fibrosis), May‐Grünwald‐Giemsa and CP‐11‐C‐kit (MCs). In each organ slide, 10 photographs were taken at random at 400× magnification by 2 pathologists unaware of the slide identity who also reviewed the slides (5).

Mild lung and CV inflammation and fibrosis were observed in wild type mice while the CUX‐1 mice presented with very severe fibrosis, particularly in the bronchial basal membrane, the peribronchial pulmonary arteries and the coronary arteries. May‐Grünwald‐Giemsa staining showed very few MCs in the heart of wild type mice and none in the CUX‐1. MCD staining did not show MCs in either organs, not withstanding the severe damage. It remains to be seen whether the lack of MCs, as seen in this experimental model, has a pathogenetic role in the development of pulmonary and cardiac fibrosis. Further mechanisms of fibrotic damage should be explored to identify potential therapeutic options in patients with significant fibrotic conditions.

Support or Funding Information

Wheaton College Research Fund
Dimethyl Fumarate Ameliorates Pulmonary Hypertension in vivo and Prevents Fibrosis via βTRCP Mediated Degradation of β‐catenin and TAZ
Authors: Grzegorzewska Agnieszka , Han Rong , Seta Francesca , Stawski Lukasz , Feghali‐Bostwick Carol , Browning Jeffrey , Trojanowska Maria
Published: November 2023

Introduction

Pulmonary arterial hypertension (PAH) is a serious, progressive and fatal complication of systemic sclerosis (SSc). Oxidative stress and chronic inflammation have been shown to play important roles in pathogenesis of PAH. Dimethyl Fumarate (DMF) (Tecfidera®) is an anti‐oxidative and anti‐inflammatory agent approved by the FDA for treatment of multiple sclerosis. In this study we test DMF as a potential drug to treat PAH.

Methods

DMF was administered daily via intraperitoneal injections in a chronic hypoxia mouse model of PAH in preventive and therapeutic modes as well as preventively in a bleomycin lung fibrosis model. In vitro studies were performed on human primary arterial endothelial cells and primary human lung fibroblasts from SSc patients and healthy controls. Endothelial cells were exposed to hypoxic conditions or lipopolisaccharade (LPS) in combination with DMF or vehicle treatment. Pulmonary fibroblasts were treated with transforming growth factor beta (TGFβ) and DMF or vehicle.

Results

DMF prevented and reversed hemodynamic changes and right ventricular hypertrophy as well as reduced inflammation, oxidative stress and vascular muscularization in a mouse PAH model. DMF improved the function of cultured endothelial cells by normalizing hypoxia induced gene expression of pro‐inflammatory cytokines and growth factors via inhibition of NFκB, STAT3 and JNK signaling. DMF also suppressed TGFβ dependent pro‐fibrotic gene expression in cultured human lung fibroblasts. Studies of endothelial cells and fibroblasts indicated that Nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) ‐mediated anti‐oxidative pathway is dispensable for the anti‐inflammatory and anti‐fibrogenic properties of DMF. In fibroblasts, DMF had no significant effect on canonical TGFβ signaling, but instead led to ubiquitin ligase‐ βTRCP mediated degradation of the pro‐fibrogenic transcription factors Sp1, TAZ and β‐catenin. In vivo validation of anti‐fibrotic DMF properties in a bleomycin mouse model indicated that the drug prevented development of lung fibrosis in a manner correlated with a reduction in active β‐catenin levels by immunostaining.

Conclusions

In conclusion, DMF has anti‐oxidative, anti‐inflammatory and anti‐fibrotic potential both in vitro and in vivo. Given the safety profile of Tecfidera® and its pleiotropic mechanism of action, it may provide an effective treatment for SSc‐PAH.

Support or Funding Information

CORT grant NIAMS P50 AR060780‐01
R‐Spondin2 is upregulated in idiopathic pulmonary fibrosis and affects fibroblasts behavior
Authors: Munguia Adrian , Becerril Carina , Mendoza Criselda , Balderas Yalbi , Ramirez Remedios , Melendez Jorge , Pardo Annie , Selman Moises
Published: November 2023

Backgroud

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disorder of unknown etiology characterized by fibroblast activation, excessive extracellular matrix accumulation and the subsequent destruction of the lung parenchyma. The R‐spondins family (RSPO) represents a recently described group of proteins with essential activities in development. RSPO2 is expressed primarily in the lungs and mutations of this protein cause severe defects in the respiratory tract. Three proteins of the G protein coupled family (LGR4, LGR5 and LGR6) have been identified as possible receptors. RSPO2 enhances the canonical Wnt signaling (a pathway involved in IPF) resulting in activation of b‐catenin.

Aim

To determine the role of RSPO2 in IPF.

Methods

The expression of RSPO2 and LGR6 was explored by qPCR in IPF lungs and fibroblasts. Cell source was analyzed by immunohistochemistry. The effect of RSPO2 on lung fibroblasts proliferation was evaluated with WST1 and on gene expression by RNAseq. The effect of RSPO2 on fibroblasts and epithelial cell apoptosis was examined by flow cytometry.

Results

RSPO2 and LGR6 were significantly increased (>100‐fold) in IPF compared with normal lungs. Both molecules were localized primarily in fibroblasts and epithelial cells. RSPO2 gene expression was also increased in fibroblasts derived from IPF lungs (2−DCT 2.59−5±3.6−6 vs 1.39−5±1.35−6). In vitro, RSPO2 modified the expression of 3116 genes in IPF and 795 in normal fibroblasts. Several pathways were revealed including apoptosis, cell cycle, Wnt pathway, and extracellular matrix metabolism. Treatment of fibroblasts with RSPO2 reduced cell proliferation and induced apoptosis in both normal and IPF fibroblasts. No effect was observed in epithelial cell apoptosis.

Conclusions

Our findings demonstrate that RSPO2 and its receptor LGR6 are upregulated in IPF lungs, primarily in fibroblasts and epithelial cells, although the exact role of this axis in the pathogenesis of this disease remains uncertain.

Support or Funding Information

Posgrado en Ciencias Biologicas, UNAM
Advancement in the Diagnosis and Treatment of Granulomatous/Lymphocytic Interstitial Lung Disease
Authors: Hoffmann Brian R. , Vallejos Andrew , Greene Andrew S. , Routes John M.
Published: November 2023

Common variable immunodeficiency (CVID) comprises the most clinically important primary immunodeficiency due to its prevalence, serious complications and long‐term costs of therapy. A form of interstitial lung disease known as granulomatous/lymphocytic interstitial lung disease (GLILD) occurs in 10–20% of patients with CVID. The causes of GLILD are unknown and there is no established standard of care for the treatment of GLILD. We determined that patients with active GLILD have an abnormally high percentage of activated T cells (CD3+) in the peripheral blood with the HLA‐DR+ protein biomarker (DR+ T cells). Through DNA sequencing we demonstrated that CD3+/DR+ T cells are enriched within the lung, suggesting that activated CD3+/DR+ T cells in the peripheral blood are homing to lung and contribute to active disease. We hypothesized that: 1) Specific cellular pathways are dysregulated in CD3+/DR+ T cells and contribute to the pathogenic inflammatory state in the lung and that 2) specific cytokine receptors will be upregulated in DR+ T cells compared to DR‐ T cells and contribute to homing to the lung. To test this, we characterized the transcriptome and proteome of CD3+/DR+ T cells and CD3+/DR‐ T cells isolated from peripheral blood of CVID‐GLILD patients. Numerous potential biomarkers and/or therapeutic targets were found to be up‐ or down‐regulated in CD3+/DR+ T cells compared to CD3+/DR‐ T cells. Target genes/proteins identified were involved in the regulation of important immune/inflammation responses, cell adherence, and cell migration processes important for the observed CD3+/DR+ T cell pathology in the lungs of CVID‐GLILD patients. Identification of the mechanisms behind this T cell pathogenesis has the potential to fundamentally change the management of this disorder and contribute to novel therapies for CVID‐GLILD patients.

Support or Funding Information

This project was supported by the Clinical & Translational Science Institute (CTSI) of Southeast Wisconsin through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Additional support was provided by the Medical College of Wisconsin Biotechnology and Bioengineering Center.
DNA Repair During DNA Replication
Authors: Cortez David
Published: November 2023

Successful DNA replication is essential to maintain genome integrity and prevent disease. In human cells, over 6 billion base pairs of DNA need to be replicated accurately and packaged into chromatin each cell division cycle. Furthermore, these processes must occur in the context of defects in the DNA template such as DNA damage, which present challenges to both genetic and epigenetic inheritance. The replisome is a highly regulated, dynamic machine that must work with speed and precision to duplicate the chromosomes successfully. Failures during DNA replication cause mutations, epigenetic changes, and other chromosomal aberrations that ultimately cause disease such as cancer. We recently developed a methodology called iPOND to isolate proteins on nascent DNA. Combining iPOND with quantitative mass spectrometry permits a detailed examination of the replisome and DNA damage response machineries. I will present how we have used this method to examine the functions of ATR in maintaining replisome stability and promoting replication fork restart. Also, I will demonstrate how monitoring protein abundance in multiple experimental conditions with iPOND is sufficient to identify protein complexes and new replisome and DNA damage response proteins. Finally, I will present data on how one of these new DNA damage response proteins functions to maintain genome integrity during DNA replication.

Support or Funding Information

NIH R01CA102729, R01GM116616, and the Breast Cancer Research Foundation
Structural Basis for Divergent Reaction Outcomes in Iron(II)/2OG‐dependent Oxygenases
Authors: Boal Amie
Published: November 2023

Iron(II)/2‐oxo‐glutarate (Fe/2OG)‐dependent oxygenases are a large and diverse family of enzymes best known for their ability to install –OH groups into a wide variety of substrates. The mechanism by which these transformations occur is well understood and involves hydrogen atom abstraction from a specific site on the substrate via a potent FeIV=O intermediate followed by attack upon the resulting FeIII‐OH complex to yield a hydroxylated product. Fe/2OG enzymes are also capable of other reactivities including stereoinversion, double bond formation, cyclization, and alternative group transfer reactions such as halogen installation. It remains unknown how these other outcomes are favored over the more typical hydroxyl group transfer reactions, in part due to lack of comprehensive structural information. X‐ray crystal structures of enzyme‐substrate complexes for many of these non‐canonical systems suggest new unifying themes in promotion of other reactivities including use of distinct substrate/cosubstrate binding sites and optimization of protein structural features to take advantage of properties of the substrate that promote new reaction pathways.

Support or Funding Information

NIH grant GM100011 and the Searle Scholars Program
Ubiquitin And ISG15 Conjugation At The Ribosome
Authors: Huibregtse Jon , Canadeo Larissa , Swaim Caleb , O'Connor Hazel
Published: November 2023

Nascent polypeptides are subject to a variety of modifications while they are ribosome‐associated, including ubiquitylation and ISGylation. Ubiquitylation of nascent chains occurs on both stalled ribosomes, via the RQC/Ltn1‐dependent pathway, as well as on actively translating ribosomes in a process we refer to as co‐translational ubiquitination (CTU). CTU appears to be a conserved process in that it occurs in both yeast and human cells, and CTU is increased in both yeast and human cells in response to treatments that result in misfolding of nascent chains, such as incorporation of amino acid analogs. mTOR inhibitors (e.g., rapamycin, torin) lead to a decrease in CTU, consistent with a model where decreased translational load and/or slowing of translation may allow for increased translation fidelity and cotranslational folding efficiency. CTU products contain primarily K48‐linked polyubiquitin chains, consistent with a proteasomal targeting function. We will report on progress in identification of targets and ligases involved in CTU. Conjugation of the interferon‐induced ubiquitin‐like protein ISG15 is mediated by the Herc5 HECT domain ligase. The similarities and differences in CTU and CTI (cotranslational ISGylation) with respect to factors that influence the efficiency of each will be discussed, as well as the determinants of Herc5 that mediate ribosome association. In addition to its intracellular conjugation function, extracellular ISG15 cooperates with IL12 in the inducing the secretion of IFN‐gamma from cells within the PBMC population, particularly Natural Killer (NK) cells. We will report on progress in characterizing this unique signaling pathway.

Support or Funding Information

National Institutes of Heatlh, NIAID (AI096090) and NIGMS (GM103619)
Exploiting the biology of trehalose to develop novel imaging probes for tuberculosis
Authors: Barry Clifton
Published: November 2023

Mycobacterium tuberculosis uses trehalose esters to form the outer leaflet of its cell envelope. These esters are formed by the three secreted proteins of the Antigen 85 complex that catalyze transesterification of long chain mycolic acids to form both trehalose mono‐ and di‐mycolates. We have studied the substrate specificity of these enzymes for various trehalose analogs and discovered them to be surprisingly promiscuous. Taking advantage of this promiscuity we have developed fluorescent analogs that are important tools for cell biology as well as positron emitting analogs allowing whole body imaging of TB‐infected animal species including rabbits, marmosets and cynomolgous macaques. 18‐F‐2‐deoxytrehalose labeling of infected animals revealed that sites of active bacterial replication in the lungs are discrete from sites of active inflammation as assessed by serial scanning with 18‐F‐2‐deoxyglucose. Sites of intense inflammation are non‐overlapping with sites of active bacterial metabolism suggesting TB infection is a summation of many discrete localized focal lesions.

Support or Funding Information

The Intramural Research Program of the NIAID, NIH and the Bill and Melinda Gates Foundation.
A Potential Mediator of Diabetes‐associated Cardiovascular Disease: the Enzyme FMO3
Authors: Biddinger Sudha
Published: November 2023

Despite the well‐documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. To identify the mechanisms by which insulin resistance promotes cardiovascular disease, we performed metabolomic and transcriptional profiling in Liver Insulin Receptor Knockout (LIRKO) mice. The hepatocytes of these mice are entirely resistant to insulin due to a liver‐specific deletion of the insulin receptor. Interestingly, on an atherogenic diet, 100% of these mice, but none of their controls, develop atherosclerosis. By performing transcriptional and metabolomic profiling on the livers of these mice, we found the enzyme flavin‐containing monooxygenase 3 (Fmo3) and its product, trimethylamine N‐oxide (TMAO), to be highly induced. TMAO has recently been suggested to promote atherosclerosis in mice and humans. We found FMO3 to be suppressed by insulin in vitro, increased in obese/insulin resistant male mice and increased in obese/insulin‐resistant humans. Knockdown of Fmo3 in insulin‐resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycemia, hyperlipidemia and atherosclerosis. Taken together, these data suggest that insulin resistance may drive atherosclerosis via FMO3.
Science Policy: Making a Big Difference in the Future of Discovery
Authors: Wilson David R.
Published: November 2023

At this very moment science is under fire. Law makers are slashing budgets and funding opportunities are becoming more and more competitive. So who fights for the scientists? Who helps inform policy makers about the importance of the scientific endeavor? They are people commonly referred to as science policy professionals. Science policy is a loosely defined profession with potentially tremendous impacts. The impact of science policy can occur at the local, state and federal/national level. In this session I will cover the importance of connecting all three levels and how your scientific training has uniquely prepared you for this career away from the bench.
Proteins and Carbs – The Balanced Diet of a Complex and Unusual Enzyme Family
Authors: Boraston Alisdair , Noach Ilit , Ficko‐Blean Elizabeth , Stuart Christopher , Brochu Denis , Gilbert Michel
Published: November 2023

Many host‐adapted bacteria have the capability to colonize mucosal layers. To do so, these bacteria have often developed the capacity to breakdown the mucin component of the protective layers that cover epithelial cells. As mucins are glycoproteins that are up to 80% carbohydrate by mass the most common feature of bacterial mucin degrading systems is an arsenal of enzymes that breakdown carbohydrates. However, how such bacteria may target the protein backbone of mucins remains a poorly investigated question. We have identified mucin‐degrading proteins in Clostridium perfringens, Bacteroides thetaiotaomicron, and Pseudomonas aeruginosa, which are all host‐adapted a bacteria notable their ability to colonize the mucus rich environments of human and animal tissues. These proteins are zinc‐metalloproteases and thus cleave the peptide backbone. Remarkably, our structural and functional analyses of these enzymes reveal that O‐linked glycans accommodated in the zinc‐metalloprotease active site is a determinant of specificity, and this is dependent on the particular protease. This work provides the first detailed molecular insight into a large family of glycoproteases found in host‐adapted microbes and highlights the potential complexity of substrate specificity in glycoprotein specific proteases.
Genome Regulation by Long Noncoding RNAs
Authors: Chang Howard Y.
Published: November 2023

The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. I will discuss genome‐scale strategies to discover and characterize lncRNAs. Genome‐wide mapping of RNA secondary structures, termed the structurome, provides important clues to potential functions of regulatory RNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, long noncoding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in developmental patterning and cancer.
The Primary Target Of Let‐7 MicroRNA
Authors: Pasquinelli Amy
Published: November 2023

The discovery that regulatory RNAs control almost every biological pathway has revolutionized our understanding of gene expression. At the forefront, miRNAs have proven to be an essential class of RNA molecules with mis‐regulation of miRNA expression underlying a variety of human diseases, including cancer. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65‐nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22‐nucleotide forms. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in mRNA transcripts, leading to translational repression and destabilization of the target mRNAs. Because of the imperfect nature of miRNA‐target duplexes, identifying biologically relevant target sites is an outstanding challenge. To address this problem, we have used cross‐linking immunopurification with high‐throughput sequencing (CLIP‐seq) to capture miRNA target sequences in vivo. Focusing on the last larval stage in C. elegans development, we found that more than 3,000 mRNA transcripts have sequences bound by ALG‐1 (Argonaute Like Gene 1). Unexpectedly, the non‐coding let‐7 primary transcripts (pri‐let‐7), which are processed into the mature let‐7 miRNA, were among the RNAs targeted by Argonaute. We found that association of ALG‐1 with pri‐let‐7 promotes processing and this interaction is mediated by mature let‐7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive‐feedback loop. Argonaute also binds let‐7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non‐coding RNAs in addition to protein‐coding messenger RNAs across species. Our studies reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation.

Support or Funding Information

The Pasquinelli lab is supported by NIH grant GM071654
Expanding The Boundaries Of Biocatalysis
Authors: Zhao Huimin
Published: November 2023

Biological catalysis and chemical catalysis are generally considered as two separate research areas, each with its unique challenges and opportunities.[1] However, the boundary between these two areas has been becoming increasingly blurred due to rapid advances in synthetic biology and development of new water‐compatible chemical catalysts. In this talk, I will discuss our recent efforts in exploring the synergy between biological catalysis and chemical catalysis and highlight three examples. The first example involves the design and development of a tandem catalytic process consisting of an olefin metathesis catalyst and a cytochrome P450 enzyme in a biphasic system, which converts a mixture of alkenes to a single epoxide.[2] The metathesis catalyst leads to an equilibrating mixture of alkenes in the organic phase, and the P450 enzyme converts one member of the mixture of alkenes to the corresponding epoxide in the aqueous phase. The second example involves the design and development of a tandem catalytic process consisting of a thermophilic glucose isomerase enzyme for glucose isomerization to fructose and a solid acid catalyst for fructose dehydration to 5‐hydroxymethylfurfural.[3] The third example involves the metabolic engineering of a recombinant E. coli strain which converts D‐galacturonic acid released from sugar beet residues by enzymatic hydrolysis to mucic acid. The resulting mucic acid is chemically converted to adipic acid through oxorhenium‐complex‐catalyzed deoxydehydration reaction and subsequent Pt/C‐catalyzed transfer hydrogenation reaction.[4] These examples have clearly demonstrated the great potential of combining biological catalysts and chemical catalysts for synthesis of chemicals, fuels, and drugs.

Support or Funding Information

NSF under the CCI Center for Enabling New Technologies through Catalysis (CENTC) CHE‐0650456 and CHE‐120518

Agency for Science, Technology and Research, Singapore
Monitoring Cell Death in Real‐Time/Time‐Lapse Studies
Authors: Edward Roy
Published: November 2023

Personalised medicine, chronic exposure drug toxicity and environmental exposure amongst others have created the demand for in vitro assays which are more physiologically relevant. One aspect of this may be to run assays for many days and monitor them in real‐time or time‐lapse mode. Therein, it can be useful to assess the general cell survival or perhaps a specific cell death pathway in the context of the treatment or insult under observation.

Depending on the sample under analysis, to date, this can be done by either marking the cells that remain functionally competent or measure release of ATP at a bulk level. It would be preferable if, conversely, only dead/damaged/apoptotic cells were marked in a binary manner, with a convenient and spectrally separated emission signature and with specificity for a predictable intracellular target such as gDNA, and cell‐by‐cell.

To explore this need a novel far‐red DNA binding viability probe, DRAQ7, has been developed. It has been shown to have undetectable toxicity in long‐term/real‐time cell based assays as validated in recent publications (Akagi et al., 2013, Marciniak et al., 2013, Wang et al., 2015, Liang et al., 2015, and internal data) including ultra‐sensitive bioassays for DNA intercalators, ex‐plant tissue culture and nano‐particle toxicity, time‐lapse apoptosis assays and importantly in the presence of toxicants/anti‐cancer compounds.

Thus, cells can be exposed to it at any stage of an assay to permit real‐time monitoring of loss of membrane integrity (in apoptosis, death). Being DNA specific it allows monitoring cell‐by‐cell while its spectral properties mean it can be incorporated into multi‐colour flow cytometry experiments, or with Hoechst 33342 or CyTRAK Orange (for simple cell health assays) or with mitochondrial membrane potential probes such as TMRM.

DRAQ7 is truly cell impermeant yet retains the DNA targeting and far‐red fluorescence of the parent DRAQ chromophore. Accordingly, it likewise can be used on HCS imaging platforms, fluorescence microscopes, cytometers and sorters. Its spectral properties (long wavelength) are particularly suited to penetrative imaging of multi‐cellular structures and thick ex‐plant tissue sections.

The presentation will focus on the deployment and application of DRAQ7 to demanding real‐time and 3D micro‐tissue assays including patient‐derived samples that enable strategies for personalised medicine, underpinned by core performance data that define its unique properties.

Support or Funding Information

This submission is funded by my employer, Biostatus Limited.
Reversing The Paradigm: Protein Kinase C As A Tumor Suppressor
Authors: Newton Alexandra
Published: November 2023

Protein kinase C (PKC) isozymes transduce the myriad of signals resulting from receptor mediated hydrolysis of phospholipids, playing critical roles in diverse cellular functions. Its activity must be precisely tuned for cellular homeostasis and too much, or not enough, activity results in pathophysiologies. The discovery in the 1980s that PKCs are receptors for the potent tumor promoting phorbol esters led to the dogma that activation of PKC by phorbol esters promotes tumorigenesis. However, clinical trials with PKC inhibitors have not have not only failed but, in some cases, resulted in worsened outcome. Analysis of approximately 10% of the >500 mutations identified in human cancers reveals that most mutations are loss‐of‐function and none are activating. Loss‐of‐function mutations occur in all PKC subgroups and occur by disrupting diverse mechanisms: they impede second‐messenger binding, prevent phosphorylation, or inhibit catalysis. Thus, PKC functions as a tumor suppressor and its loss of confers a survival advantage. In contrast, we show that gain‐of‐function mutations accompany degenerative diseases such as Alzheimer's disease. Our results reveal that therapeutic strategies should focus on restoring PKC activity in cancer and inhibiting its activity in neurodegenerative diseases.

Support or Funding Information

NIH GM43154
Dysfunctional PKA Drives Diverse Disease Phenotypes
Authors: Taylor Susan S. , del Rio Jason , Bruystens Jessica , Veglia Gianluigi , Stefan Eduard , Ilouz Ronit , Zhang Ping
Published: November 2023

Although cAMP‐dependent protein kinase (PKA) was the second regulatory protein kinase to be discovered, it was the first to be crystallized and has served ever since as a prototype for the large and diverse protein kinase superfamily. PKA, ubiquitous in mammalian cells, is a critical signaling node and regulates a plethora of biological processes. Unlike most protein kinases, which are regulated by turnover of an activation loop phosphate, PKA is synthesized as a stable phosphoprotein and then assembled as an inhibited holoenzyme complex with two cAMP‐controlled and functionally non‐redundant regulatory (R) subunits (RIa, RIb, RIIa, RIIb). Although each inactive R2C‐2 holoenzyme consists of an R‐subunit dimer and two catalytic (C) subunits, the quaternary structures of each R2C2 holoenzyme are remarkably different indicating unique but overlapping pathways for allosteric regulation. There are many recently discovered autosomal dominant mutations in PKA signaling that are directly linked to disease phenotyes, and these can be classified into distinct categories. Cushings Disease and Carney Complex (CNC) Disease are caused by gain‐of‐function mutations in the catalytic and regulatory subunits, respectively. A number of single mutations in either subunit lead to a phenotype where PKA activity is not tightly regulated. In these patients the basal PKA activity levels are high even in the absence of cAMP. In contrast, hormone resistance and lowered PKA activity was observed in acrodysostosis (ACRDYS) patients, a disease that is associated with severe skeletal defects. Developmental delay and intellectual disability also accompany ACRDYS patients. ACRDYS1 patients harbor mutations in the RIa gene but these patients, in contrast to CNC patients, have a loss‐of‐function phenotype; the RIa holoenzyme is more difficult to activate. Perhaps the most striking PKA‐linked disease mutation so far is a fusion protein between DNAJB1 and the PKA Ca subunit, which was discovered to be a driver of a childhood liver cancer, fibrolamellar hepatocellular carcinoma (FLHCC). In other cases, inhibition of PKA can lead to an invasive phenotype suggesting that PKA can function as both a tumor driver and a tumor suppressor. Recently, a point mutation in the RIb dimerization/docking domain was found in patients diagnosed with a neurodegenerative disease. This mutation disrupts dimerization, prevents AKAP binding and thus abolishes targeting even though the residual activity of the complex is robustly regulated by cAMP. The variety of mutations that lead to dysfunctional PKA signaling is consistent with its central role in regulating so many biological events. The consequences of dysfunctional PKA signaling will be both diverse and cell‐specific.

Support or Funding Information

Funding for this research was from GM34921 to sst and GM100310 to gv
Optimization of a Series of Novel Chalcone Derivatives for Anti‐Cancer Activity
Authors: Whited Tawna L. , Stang Christopher R.T. , Adams Bryce R. , Khupse Rahul S. , Dudley Richard W. , Schneider Ryan A.
Published: November 2023

Chalcones are natural products that have been studied for their numerous pharmacological properties including anti‐cancer, antioxidant, and anti‐inflammatory effects. An alpha‐beta unsaturated ketone at the core of the chalcone scaffold provides the basis for the multitude of pharmacological effects. However, synthetic modifications to the periphery of the scaffold seemingly afford diverse activity. We synthesized a small library of chalcone derivatives to screen for anti‐cancer activity in vitro. Additionally, we sought to determine the required pharmacophore and optimize structural features in order to improve activity. We previously reported preliminary data with two of these compounds, RK6 and RK7. Here we report our findings for two additional derivatives (RK8 and RK9) and compare them to the previous data for optimization of anti‐cancer activity. These four compounds were screened for anti‐cancer activity in multiple, diverse, human cancer cell lines (A375 melanoma, CRL‐1620 glioblastoma, HT‐29 colon carcinoma, and MCF‐7 breast adenocarcinoma). GI50 values for each of the compounds in all four cancer cell lines were determined using the CyQUANT® NF assay. Treatments were administered with increasing concentrations (1–100 μM) of each compound separately for 48 hours. RK6 and RK7 demonstrated activity in each of the four lines tested with GI50 values ranging from 16–48 μM. RK8 and RK9 showed activity in some, but not all cell lines, with GI50 values ranging from 36 to greater than 100 μM. A Caspase‐Glo® 3/7 assay and western blotting for cleaved caspases‐3/7 and cleaved poly(ADP‐ribose) polymerase were done to determine potential mechanisms of cancer cell death. Collectively, results from our experiments suggest that these chalcones induce cell death in a non‐caspase‐dependent manner. Additional studies are ongoing to optimize the chalcone scaffold and to determine the mechanisms of anti‐cancer activity.
Targeting CSE1L in Colorectal Cancer
Authors: Pimiento Jose , Neill Kevin G , Henderson‐Jackson Evita , Eschrich Steven , Chen Dung‐Tsa , Husain Kazim , Shibata David , Coppola Domenico , Malafa Mokenge
Published: November 2023

Background

Human CSE1L gene plays a role in nuclear‐to‐cytoplasm transport and chromosome segregation during mitosis, cellular proliferation and apoptosis. CSE1L may play a role in the progression of human colorectal cancer.

Study Design

CSE1L gene expression was assessed by surveying three different datasets using Affymetrix U133+gene chips on normal human colonic mucosa (NR), adenomas (AD), and colorectal carcinoma (CRC). CSE1L protein expression in CRC, AD and NR from the same patients was measured by immunohistochemistry (IHC) using a tissue microarray (TMA). We evaluated CSE1L expression in CRC cell lines (HCT116, SW480 and HT29) and its biologic functions.

Results

CSE1L mRNA was significantly increased in all ADs and CRCs as compared to NR (p<0.001 and p=0.02 respectively). CSE1L expression by IHC was significantly increased in AD and CRC when compared to NR in a CRC TMA (p=0.01 and p<0.0001 respectively). CRC cell lines expressed CSE1L protein at baseline. CSE1L knockdown by siRNA induced inhibition of this protein, resulting in decreased cell proliferation, reduced colony formation in soft agar and induction of apoptosis.

Conclusion

CSE1L protein is expressed early and across all stages of CRC development. RNA knockdown of CSE1L was associated with inhibition of tumorigenesis in CRC cell lines. CSE1L may represent a marker for early detection of CRC and a target for prevention and treatment of CRC.

Support or Funding Information

Grant information: This study was supported in part by the National Institutes of Health Grants 1R01 CA‐129227‐01A1, 5RO1 CA‐098473‐05, DAVOS 69‐15099‐99‐01 (to Mokenge Malafa).
The ghrelin/leptin imbalance during cholestasis is driven by peripheral S1P2R‐dependent bile acid signaling
Authors: DeMorrow Sharon , Zigman Jeffrey , McMillin Matthew
Published: November 2023

During the course of chronic cholestatic liver diseases cholangiocytes exhibit marked proliferative capacity followed by cholangiocyte loss. Dysregulation in the hormones controlling energy expenditure and food intake have been demonstrated as a consequence of cholestatic liver diseases. Specifically, in patients with primary biliary cirrhosis, circulating leptin levels are increased whereas, ghrelin, its counterpart, is decreased, although the cause of this imbalance is largely unknown. Increased serum bile acids are a key feature of cholestasis and are blamed for some of the extrahepatic consequences of these diseases. Bile acids are capable of signaling via a number of receptors including the sphingosine‐1 phosphate 2 receptor (S1P2R). The aims of this study were to assess the role of bile acid signaling in the imbalance between ghrelin and leptin. In a rat model of cholestasis (bile duct ligation; BDL) circulating levels of leptin was increased and ghrelin levels were suppressed. Ghrelin and leptin are weakly expressed in the liver and expression levels are unchanged during cholestasis, suggesting that ghrelin and leptin are originating from extrahepatic sources. Total ghrelin protein in the stomach was decreased after BDL surgery, as was the protein required for bioactivation of ghrelin (ghrelin O‐acyltransferase; GOAT). Furthermore, treatment with recombinant ghrelin decreased cholangiocyte proliferation (as assessed by cytokeratin‐19 expression) and markers of biliary fibrosis (a‐smooth muscle actin, collagen 1a1, and fibronectin 1) after BDL. Conversely, leptin expression in adipose tissue was increased after BDL and contributed to cholangiocyte proliferation and fibrosis. The imbalance between ghrelin and leptin after BDL could be attenuated in rats fed a diet enriched in the bile acid sequestrant cholestyramine. In vitro, treatment of mouse gastric ghrelin‐expressing cells with the conjugated bile acids taurocholic acid (TCA) and glycochenodeoxycholic acid (GCDA) inhibited ghrelin mRNA expression and could be prevented by pretreatment with the S1P2R antagonist JTE013. Similarly, the S1P2R antagonist prevented TCA‐induced upregulation of leptin in differentiated adipocytes. Taken together our data supports the observation that an imbalance between ghrelin and leptin exists during cholestasis that contributes to the resulting liver pathology. Furthermore our data suggests that this imbalance is as a consequence of S1P2R‐mediated bile acid signaling in the periphery.

Support or Funding Information

The following study was funded by an NIH R01 award (DK082435), and a VA Merit award (BX002638‐01) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (BLR&D) to Dr. DeMorrow
Regulation of mesenchymal phenotypes by microRNA‐21 during alcohol‐induced liver injury
Authors: Meng Fanyin , Francis Heather , McDaniel Kelly , Alpini Gianfranco
Published: November 2023

Background

MicroRNAs belong to the heterogeneous class of non‐coding RNAs (ncRNAs) that regulate gene expression in a sequence specific manner. The activation of miR‐21 and mesenchymal signaling is recognized as the key event of alcoholic liver injury and hepatic fibrosis. The current study was aimed to characterize the functional role of microRNA‐21 regulated mesenchymal phenotypes in mice liver with chronic and binge ethanol feeding (the NIAAA model) in vivo and activated human hepatic stellate cells (HSCs) in vitro.

Methods

miRNA expression in TGF‐β treated HSCs, as well as in 5 weeks chronic and binge ethanol feeding mouse liver specimen (with or without anti‐miR‐21 Vivo‐Morpholino treatment) and control HSCs and liver tissue, was assessed using a hybridization based microarray and by real‐time PCR, respectively. The mesenchymal phenotypes and the activation of HSCs were monitored by Western blot and real‐time PCR analysis through specific markers such as vimentin, S100A4, α‐SMA, laminin, fibronectin and MMPs.

Results

The total liver histopathology score increased in chronic ethanol feeding group relative to control mice, whereas Sirius red staining revealed increased liver fibrosis with hepatic stellate activation after 5 weeks ethanol feeding. We identified that ethanol feeding significantly up‐regulated miR‐21 by 5.3±0.8 fold, which was further verified by Taqman real‐time PCR assay. The expression of miR‐21 was also significantly increased during human hepatic stellate cell activation when cultured in uncoated plastic culture dishes for 5 weeks. Treatment of HSCs with TGF‐β (10 ng/ml) for 24 hours induced a significant increase of miR‐21 expression in both activated and control state. Transfection of anti‐miR‐21 inhibitor markedly attenuated the expression of S100A4, laminin and fibronectin mRNAs and additionally blunted the increased expression of α‐SMA, MMP‐2 and MMP‐9, which are key genes involved in the activation of HSCs. Furthermore, the expressions of TGF‐β, as well as mesenchymal and fibrosis markers, including vimentin, S100A4, α‐SMA, laminin, fibronectin, MMP‐2, MMP‐9 and LIN28, were significantly reduced after miR‐21 Morpholino/AS treatment in chronic ethanol feeding mouse liver specimens compared to controls.

Conclusion

Our results show that miRNAs are critical regulators of mesenchymal transition, human hepatic stellate activation and transdifferentiation during alcoholic liver injury. The findings provide new insight into the function of specific miRNAs and the mechanisms of alcohol induced liver injury and fibrosis.
Paradoxical Role of Yes‐Associated Protein (YAP) in Liver Injury and Regeneration following Acetaminophen Overdose
Authors: Poudel Samikshya , Manley Michael W. , Bhushan Bharat , McGill Mitchell R. , Jaeschke Hartmut , Apte Udayan
Published: November 2023

Acetaminophen (APAP) overdose is the major cause of Acute Liver Failure (ALF) in the Western world. Treatment options for APAP‐induced ALF are limited. Studies have shown that stimulating liver regeneration could be a potential treatment for ALF after APAP overdose. However, the mechanisms of liver regeneration after APAP overdose are not completely understood, which is the focus of research in our laboratory. We investigated the role of Yes associated protein (YAP), the downstream co‐activator of Hippo Signaling Pathway, in liver injury and regeneration following APAP overdose. Previous studies have shown that YAP is involved in stimulation of hepatocyte proliferation. Treatment of two‐three months old C57BL/6 male mice with 300 mg/kg APAP (APAP 300) resulted in significant liver injury and regeneration over a time course of 0 to 96 hr. Increased activation of YAP was observed during initial time points (3, 6 and 12 hr) after APAP administration which coincided with both injury and regeneration. To determine whether YAP activation plays a role in injury or regeneration, we generated hepatocyte specific YAP knockout mice (hYAP‐KO) using YAP‐floxed mice treated with AAV8.TGB.Cre. Hepatocyte specific deletion of YAP did not change hepatic CYP2E1, the enzyme involved in APAP bioactivation, and hepatic glutathione (GSH) levels. Consistent with these data, no difference in APAP‐protein adducts was observed between control and hYAP‐KO mice after APAP treatment. Liver injury measured by serum ALT and histopathology showed extensive and similar liver injury up to 12 hr after APAP treatment in both control and hYAP‐KO mice. However, the progression of the liver injury beyond 12 hr after APAP administration was significantly lower in hYAP‐KO mice as compared with control mice. The decrease in progression of liver injury was accompanied with an earlier decrease in JNK activation and faster GSH recovery in hYAP‐KO mice. Additionally, the hYAP‐KO mice showed an early induction of proliferative markers demonstrating early liver regeneration as compared to the control mice. These data indicate that hepatocyte specific activation of YAP may be involved in progression of liver injury and may inhibit liver regeneration after APAP overdose. These findings indicate a novel paradoxical role of YAP in APAP overdose and highlight YAP as a potential therapeutic target for the treatment of APAP‐induced ALF.

Support or Funding Information

NIH (5P20RR021940‐07, T32ES007079‐32, R01DK098414) And the Liver Scholar Award by AASLD/American Liver Foundation
Role of post‐translational modifications in prion strain diversity
Authors: Aguilar‐Calvo Patricia , Bett Cyrus , Eraña Hasier , Castilla Joaquin , Nilsson Peter , Sigurdson Christina
Published: November 2023

Prion diseases are caused by the post‐translational conversion of the cellular prion protein (PrPC) into a pathogenic β‐sheet rich misfolded isoform (PrPSc) that accumulates mainly in the brain. Prions can misfold into a wide variety of PrPSc conformations, known as strains, that correlate to diverse biochemical properties and result in distinctive disease phenotypes, including differences in incubation times, clinical signs, histopathological lesion profiles, and PrPSc deposition patterns in the brain within the same host genotype. Post‐translational modifications in PrPC include two N‐linked glycans and a glycophosphatidylinositol (GPI) anchor that may increase the conformational spectrum of PrPSc. Previous studies have shown that passage of GPI‐anchored prion oligomers into mice expressing GPI‐anchorless PrPC switches the prion morphology from diffuse aggregates to dense, fibrillar plaques, yet it is unclear whether the original prion conformation is maintained within the anchorless PrPSc fibrils.

Here we aim 1) to determine the impact of the GPI‐anchorless state on the properties of the prion strain and 2) to identify the properties of the original GPI‐anchored prions that are maintained within the GPI‐anchorless fibril. We compared the biological, histological, and biochemical properties of four prion strains before and after three serial passages through mice expressing GPI‐anchorless PrPC. We found that the four GPI‐anchorless prion strains seemed to converge into a similar strain, as mice showed equivalent incubation periods and morphologically‐identical vasotropic fibrillar plaques in the brain. However, remarkable biochemical differences reminiscent of their respective original GPI‐anchored strains were also identified among anchorless prions. Interestingly, when passaged back into wild type mice expressing GPI‐anchored PrPC, some GPI‐anchorless prions recovered the properties from their original GPI‐anchored strain, indicating prion conformational fidelity. In contrast, other anchorless prions changed their properties markedly, consistent with the emergence of a new strain. Considering that GPI‐anchorless PrPC is normally present at low levels in the brain, these data suggest that alternate post‐translationally modified PrPC states may be a source of prion strain diversity within an individual.

Support or Funding Information

NIH grant R01NS069566