bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationNIH 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 InformationNIH 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 InformationNational 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 InformationThe Intramural Research Program of the NIAID, NIH and the Bill and Melinda Gates Foundation.
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 InformationThe 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 InformationNSF under the CCI Center for Enabling New Technologies through Catalysis (CENTC) CHE‐0650456 and CHE‐120518Agency 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 InformationThis 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 InformationNIH 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 InformationFunding for this research was from GM34921 to sst and GM100310 to gv
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

BackgroundHuman 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 DesignCSE1L 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.ResultsCSE1L 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.ConclusionCSE1L 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 InformationGrant 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).