bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of renal proximal tubule function by CREB‐regulated transcriptional coactivators and salt‐inducible kinase
Authors: Taub Mary , KIm Dongwook , Cutuli Facundo 
Published: November 2023

The kidney plays a central role in blood pressure regulation, via its ability to control sodium reabsorption via locally produced natriuretic, and anti‐natriuretic factors. Previously, natriuretic and anti‐natriuretic factors, including dopamine and norepinephrine, have been observed to alter sodium reabsorption by changing the phosphorylation state of Na,K‐ATPase in the renal proximal tubule (RPT). Protein Kinase A and calcium‐mediated signaling pathways are involved. These same signaling pathways control transcription of the Na,K‐ATPase β subunit gene atp1b1 in RPT cells. In this report, evidence is presented that (1) recently discovered cAMP‐regulated transcriptional coactivators (CRTCs), and salt‐inducible kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in RPT cells and (2) renal effectors, including norepinephrine, dopamine, prostaglandins, and sodium, play a role. Evidence for the role of CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant‐negative CRTC, and a CREB mutant with an altered CRTC binding site. In a number of experimental systems, SIK1 phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK1 in primary RPT cells, atp1b1 transcription increased in the presence of a dominant‐negative SIK1. Regulation by dopamine, norepinephrine, and monensin was also disrupted by a dominant‐negative SIK1. These latter observations can be explained if these renal effectors phosphorylate and inactivate SIK1. Our results support the hypothesis that Na,K‐ATPase in the RPT is regulated at the transcriptional level via SIK1 and CRTCs, in addition to the previously reported control of the phosphorylation of Na,K‐ATPase.Support or Funding InformationNHLBI
Analysis of Differentially‐Expressed Genes in a Gamma‐Secretase Conditional Knockout Mouse
Authors: Biefeld Chelsea , Harvey Amanda , Zhu Julia , Yu Gang , Dries Daniel 
Published: November 2023

gamma‐secretase is an intramembrane protease involved in Alzheimer's disease and neurodevelopment. Here, a conditional knockout mouse was made in which a component of the gamma‐secretase complex (Nicastrin) was deleted from oligodendrocytes. These mice displayed schizophrenia‐ and compulsive‐like phenotypes. In order to identify genes and pathways that elicit these phenotypes, RNA‐Seq was used to perform transcriptome analysis. A bioinformatics pipeline was created to process this data and search for differentially expressed genes within the conditional knockout mice. Gene ontology programs were used to evaluate the genes and identify biological processes and functions that may play a role in the behavioral aspects of these mice.Support or Funding InformationWe would like to thank the Howard Hughes Medical Institute, the National Institutes of Health (Grant R01 AG029547), and the International Healthspan Institute for providing funding.
Determining the effect environmental conditions have on the activity of the 3′–5′ exoribonuclease Rrp6
Authors: McAdoo Brooke 
Published: November 2023

Eukaryotic cells contain quality‐control systems that monitor RNA biogenesis. These systems feature ribonucleases that prevent the accumulation of nonfunctional RNAs as well as regulate normal mRNAs. Rrp6 is a 3′–5′ exoribonuclease that plays a critical role in the 3′‐end formation and degradation pathways in the nucleus. In Sacchromyces cerevisiae Rrp6 physically and functionally interacts with the exosome, a highly conserved RNA‐processing complex. Rrp6 possesses activities that are both dependent and independent on its association with the exosome. It has been well documented that the deletion of RRP6 (rrp6‐Δ) leads to increased levels of both coding as well as non‐coding RNAs. However few experiments have addressed how changes in nutrient source, temperature, and under hypoxic conditions affect the activity of Rrp6, specifically its ability to regulate coding and non‐coding RNAs. To address this question we evaluated the level of several Rrp6 regulated RNAs in wild‐type and rrp6‐Δ cells grown in media containing either galactose or glucose, grown at 30 degrees and 37 degrees and in the presence or absence of oxygen. We calculated the growth rate of the yeast under these conditions and used quantitative real‐time polymerase chain reaction (qRT‐PCR) and ΔΔ Ct analysis to determine changes in RNA expression. The goal of these experiments is to better understand the activity of Rrp6p, which is critical regulator of RNA levels in all eukaryotes.Support or Funding InformationSupported by internal funding by St. John Fisher College.
Resolving specificity over a large footprint distinguishes androgen and glucocorticoid receptor DNA binding
Authors: Zhang Liyang , Martini Gabriella D. , Rube H. Tomas , Bussemaker Harmen J. , Pufall Miles A. 
Published: November 2023

The glucocorticoid (GR) and androgen receptors (AR) share an identical DNA binding interface, bind a similar core motif, yet occupy largely distinct genomic loci to regulate different gene programs. Their interplay is particularly important in the context of some castration resistant prostate cancers (CRPC), where GR can functionally substitute AR to drive the growth of tumor in the absence of androgen signaling. Some differences in the genomic localization of AR and GR can be explained by selective association with binding partners, however it is not clear how they discriminate the remaining sites. Examination of the crystal structures suggested that specificity might extend over a larger footprint (≥20bp) which had not been accessible to experimental and computational techniques previously. We therefore developed a refined version of SELEX‐seq coupled to an iterative algorithm, SelexGLM, to model the free energy of binding over a ~30bp binding site. The position specific affinity matrices (PSAMs) for these two factors reveled difference both within and outside the 15bp core motif, with AR being less tolerant to changes and thus more specific. Part of this specificity was derived from readout not just of specific base pairs, but also the shape of DNA. AR also exhibited preference for A‐tracts flanking the core sequence that narrow minor groove width. In contrast, GR specificity was largely restricted to the core sequence, with preference for a wider minor groove within that motif. The enhanced specificity of AR appears to be intrinsic to the protein, and thermodynamically driven. Isothermal titration calorimetry revealed that AR DNA‐binding is more enthalpically driven than GR binding, which is more entropically driven. This demonstrates, on a comprehensive scale, that, much like direct readout, DNA shape driven binding is enthalpically driven, and that entropically driven binding is associated with decreased specificity. Thus, this work provides a biophysical rationale for how GR can functionally substitute for AR in some castration resistant prostate cancers: in the absence of AR, overexpression of the less selective GR is able to bind AR sites. Further, this work demonstrates that combining high‐throughput and classical biophysical approaches provides the deep understanding of specificity that is necessary to dissect the regulation of fine‐tuned systems.Support or Funding InformationAcknowledgments: This research was supported by the National Institutes of Health (NIH) K99/R00 CA149088, the Roy J. Carver Charitable Trust 01–224, and the National Science Foundation CAREER grant 1552862 (MAP), and grant R01HG003008 (HJB).
Angiotensin II‐mediated Repression of Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression Involving CREB, HSF4a, and HDAC1/2 in Mesangial Cells
Authors: Arise Kiran K , Kumar Prerna , Pandya Krishna , Pandey Kailash N. 
Published: November 2023

Atrial natriuretic peptide (ANP) is an endogenous cardiac hormone that binds to guanylyl‐cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and regulates sodium excretion, water balance, and steroidogenesis; all directed toward the reduction of blood pressure and blood volume. The present study was aimed at understanding the mechanisms involved in angiotensin II (Ang II)‐mediated repression of Npr1 (coding for GC‐A/NPRA) gene transcription. The mouse mesangial cells (MMCs) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with Ang II for 24 h. The luciferase assay of the Npr1 promoter deletion constructs showed that the region approximately –1182/–916 bp upstream of the transcription start site contains the cis‐elements involved in ANG II‐mediated transcriptional repression of Npr1. Real‐time quantitative PCR and Western blot analyses revealed that Npr1 mRNA expression and NPRA protein levels were reduced by 60 – 65% in Ang II‐treated MMCs. Treatment with Ang II also significantly decreased ANP‐stimulated intracellular accumulation of cGMP as compared with ANP‐stimulated cells. Gel mobility shift assays confirmed that Ang II enhanced the binding of transcription factors cAMP‐response element‐binding protein (CREB) and heat shock factor 4a (HSF4a) to the Npr1 promoter. Increasing concentrations of Ang II also decreased the acetylation levels of histone H3 at lysine 9 and 14 and histone H4 at lysine 8. In contrast, there was significant increase in protein expression of prohypertrophic Class 1 histone deacetylase (HDAC) 1 and 2. Treatment of MMCs with Ang II increased the activity of HDAC in a dose‐dependent manner. The present findings provide a novel regulatory mechanism of Ang II‐mediated repression of Npr1 transcription and expression, which plays an important role in the control of hypertension and cardiovascular homeostasis.Support or Funding InformationThis work was supported by NIH grants (HL057531 and HL062147).
CRISPR‐mediated removal of antibiotic resistance genes in Enterococcus faecalis populations
Authors: Rodrigues Marinelle , Hullahalli Karthik , Palmer Kelli 
Published: November 2023

Multidrug resistant infections caused by Enterococcus faecalis are serious threats in hospitals where immunocompromised individuals are at elevated risk of infection. A major cause for the rise of drug resistance in E. faecalis is the horizontal transfer of antibiotic resistance genes on mobile genetic elements including integrative and conjugative elements and the species‐specific pheromone‐responsive plasmids. The transfer of these potentially beneficial mobile elements among the population can be impeded by genome defense systems such as the prokaryotic adaptive immune system CRISPR‐Cas. However, multidrug‐resistant strains of E. faecalis circumvent this by compromising their genome defense systems, leaving them vulnerable to invasion by mobile DNA. In this study, the cas genes of a native E. faecalis CRISPR‐Cas system were introduced into a MDR strain, leading to the reactivation of host genome defenses. When self‐targeting CRISPR spacers were employed on conjugative elements in heterogenous populations of E. faecalis, we found that certain antibiotic resistance genes could be selectively removed from the population under pressure to maintain the conjugative element. Taken together, this study demonstrates that CRISPR‐mediated alteration of E. faecalis population structure may be a viable option for depleting undesirable traits from E. faecalis human gut populations.Support or Funding InformationThis project is supported by R01AI116610 to KP.
Conserved DNA motifs in the type II‐A CRISPR leader region
Authors: Rajan Rakhi , Van Orden Mason , Klein Peter , Kesavan Babu , Najar Fares 
Published: November 2023

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‐associated (Cas) proteins consist of RNA‐protein complexes that provide bacteria and archaea sequence‐specific immunity against bacteriophages and conjugative plasmids. The bacteria or archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA site‐specifically into the leader‐repeat (repetitive units of CRISPR RNA gene) junction, in a process called adaptation. The hypothesis of the present work is that sequence/structural motifs present in the leader‐repeat junction recruits type‐specific Cas proteins for successful adaptation. To test this, we analyzed 167 type IIA CRISPR loci belonging to different bacterial genera. Our dataset consisted of 150 nucleotides of the CRISPR leader, the first repeat, and the protein sequences of Cas1, Cas2, Cas9, and Csn2 proteins. After performing multiple sequence alignments using MUSCLE, phylogenetic trees were generated in UGENE. Our results indicate two highly conserved DNA motifs at the 3′ leader end; 5′‐ATTTGAG‐3′ (indicated as group 1) and 5′‐CTRCGAG‐3′ (indicated as group 2). A third group with a very short 5′‐CG‐3′ DNA conservation at the 3′ leader end is observed mostly in Lactobacilli. Interestingly, the 3′‐leader end of group 1 is associated with the CRISPR‐1 locus of Streptococcus thermophilus DGCC7710, while that of group 2 is associated with the CRISPR‐3 locus of S. thermophilus DGCC7710. Since the CRISPR loci 1 and 3 of S. thermophilus DGCC7710 have been established as two distinct type IIA locus, our results demonstrate the association of these two loci with distinct 3′‐leader end sequences. A phylogenetic analysis of the repeat and Cas proteins revealed segregation reflecting the 3′ leader end conservation and pointed to the coevolution of the leader region and the CRISPR components. Previous studies have shown that the 3′‐leader end sequences are important for adaptation in S. thermophilus (group 1) and S. pyogenes (group 2) type IIA systems. Our results expanded this and demonstrated that the 3′‐leader end sequence conservation is more universally conserved covering several (~50 in this study) bacterial genera. Studies are ongoing to experimentally prove the importance of the 3′ leader end sequences and its co‐relation to locus‐specific Cas proteins. Similar studies can be used to identify conserved leader sequences in other CRISPR types and sub‐types.Support or Funding InformationResearch was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under the grant number P20GM103640.
Structural Characterization of the Recognition of U6 snRNA by the Yeast U6 Biogenesis Protein Usb1
Authors: DeLaitsch Andrew T , Didychuk Allison L , Montemayor Eric J , Larson Matthew A , Lucarelli Stefani , Butcher Samuel E 
Published: November 2023

The spliceosome is an essential molecular machine responsible for the removal of non‐coding introns from precursor messenger RNA in eukaryotic cells. An essential component of the catalytically active spliceosome, the U6 snRNA, must undergo a biogenesis pathway prior to incorporation into the spliceosome. Maturation of U6 snRNA is mediated in part by the highly conserved U six biogenesis protein (Usb1), a 3′‐5′ exoribonuclease responsible for the post‐transcriptional removal of uridines from U6 RNA. In addition, the enzyme is responsible for leaving behind a specific 3′ end modification in the form of a terminal phosphate, which is required for high affinity binding of a component of the U6 snRNP, the Lsm2–8 protein complex. Mutations in the gene encoding Usb1 give rise to the autosomal recessive disease poikiloderma with neutropenia (PN) in humans. I expressed and purified Usb1 from S. cerevisiae, which crystallized and diffracted X‐ray to 1.8 Å. The structure is remarkably similar to the structure of human Usb1 despite less than 20% sequence identity. A sulfate ion occupies the active site in a manner that may mimic coordination to the terminal phosphate of the RNA. Understanding how Usb1 recognizes RNA will give us insight into the mechanism of Usb1 processing and its role in U6 snRNA biogenesis.To biochemically characterize Usb1, I incubated recombinant Usb1 with a fluorescent RNA and monitored exoribonuclease activity via denaturing polyacrylamide gel electrophoresis. The assays show that activity is inactivated upon mutation of active site residues. In addition, fluorescence polarization binding experiments were used to monitor binding of RNA with variants of Usb1. The results of these experiments will help determine conditions most suitable for structural determination by NMR, SAXS, or x‐ray crystallography methods. I will present our progress towards determining a co‐structure of Usb1 with RNA in its active site with the goal of characterizing how the enzyme recognizes and subsequently processes U6 RNA for incorporation into the spliceosome.
Exploring Dib1's Role in pre‐mRNA Splicing
Authors: Schreib Christian Cody , Hernandez Cody , Bowman Emily , Lucas Amber , Maeder Corina 
Published: November 2023

Pre‐messenger RNA splicing is a molecular process conserved throughout all eukaryotic organisms in which non‐protein coding regions of the pre‐messenger RNA are removed. This process is facilitated by the spliceosome, a large multi‐component molecular machine made of five small nuclear RNAs and ~100 proteins. One such protein is Dib1, a small protein in the U4/U6‐U5 triple snRNP that has been shown to be essential to cell viability and splicing. Previous work suggests that Dib1 leaves the splicing machinery once it assembles, and recent cryo‐EM structures indicate that Dib1 is housed in a central location in the U4/U6‐U5 triple snRNP.To better understand what role Dib1 plays in splicing, we aimed to identify essential regions of the protein by identifying temperature sensitive mutations. Conserved residues found in hydrophobic pockets, basic regions, and loop regions of Dib1 were mutated to alanines utilizing site‐directed mutagenesis. These Dib1 mutants were transformed into a S. cerevisiae dib1Δ strain. Growth phenotypes of these yeast were surveyed using a serial dilution growth assay. Two Dib1 mutations caused decreased cell viability at 37°C. One of these mutations is located in a hydrophobic pocket and the other in an external loop region. The temperature sensitive mutations were further characterized by assaying their effect on splicing efficiency through in vitro splicing assays. Both mutations were found to cause severe splicing defects, blocking splicing prior to the first step. We then assayed the assembly of the spliceosome in the presence of these temperature sensitive Dib1 mutants through native gel analysis. Because splicing is an intricate process that involves many complexes coming on and off the pre‐mRNA, knowing the specific complexes on the pre‐mRNA can tell us when in the process of spliceosome assembly the Dib1 mutations had an effect, and in turn tell us when in the spliceosome assembly Dib1 is essential. The results of these assays suggest that Dib1 plays a vital role in the binding of the U4/U6‐U5 triple snRNP to the pre‐mRNA.Support or Funding InformationArnold and Mabel Beckman Foundation, NIH NIGMS R15GM120720, Welch Foundation
Investigation of ck/MyosinVIIA interactors using affinity tagged C‐terminal binding domains
Authors: Halverson Thomas M , Sallee Jennifer L 
Published: November 2023

Actin‐based protrusions play a variety of roles in cells from motility via filopodia to increasing surface area in microvilli to providing the molecular basis of hearing in humans and other vertebrates with stereocilia. Loss of function mutations in the unconventional myosin motor protein Myosin VIIA result in shortened stereocilia which lack correct polarity of the protrusion. Theses mutations are seen in seventy‐five percent of patients afflicted with Usher Syndrome, the leading genetic cause of hereditary deaf‐blindness. Drosophila melanogaster contain a homologous protein to Myosin VIIA called crinkled (ck/MyoVIIA). Mutations in ck/MyoVIIA result in shortened, split, and malformed hairs and denticles, actin based protrusions similar to vertebrate microvilli and stereocilia. Our project aims to identify proteins that interact with ck/MyoVIIA to help determine the molecular mechanism of ck/MyoVIIA in actin‐based protrusion formation. In this study, we are investigating the binding of ck/MyoVIIA to Cad99C. Cad99C is the Drosophila homolog to Protocadherin 15, a protein necessary in the formation of stereocilia that is known to interact with MyoVIIA in humans. In addition, Cad99C has been shown to interact with ck/MyoVIIA in the formation of follicle epithelial cells in Drosophila oocytes. We want to investigate whether this interaction occurs in embryos due to the known effect of loss of ck/MyoVIIA on actin‐based denticle formation in embryos. To test this protein interaction we used a transgenic Drosophila expressing the C‐terminal tail binding domains (MyTH4, FERM, and SH3) of ck/MyoVIIA fused to an N‐terminal triple affinity tag (FLAG, His, STREPII tags). The triple tag ck‐tail and HA‐Cad99C were expressed using a Squash‐GAL4 driver and isolated using a Talon metal affinity pull‐down procedure for the HIS tag. Proteins were separated using SDS‐page gel electrophoresis and identified using anti‐FLAG antibody for the ck‐tail and anti‐HA antibody for Cad99C.Support or Funding InformationBeta Beta Beta Undergraduate Research Grant
Nanobody Mediated Neutralization of Listeria monocytogenes
Authors: Brooks Cory , Huh Ian , Toride Moeko , Shenai Akhelish 
Published: November 2023

Listeriosis represents a significant public health concern, as consumption of food contaminated with Listeria monocytogenes can result in fatal meningitis and termination of the fetus during pregnancy. A series of recent outbreaks originating from foods historically considered to be low risk for Listeria infection highlights the need for novel strategies to improve food safety. The first step in bacterial invasion of host cells is interaction of Listeria cell surface proteins called Internalins with host cell receptors triggering receptor‐mediated endocytosis. Internalin B (InlB) binds the receptor tyrosine kinase cMET, an interaction that has been demonstrated to be crucial for bacterial invasion of the placenta. Given the importance of this interaction for bacterial virulence it represents a target for therapeutic intervention. We have isolated several nanobodies, the single domain antibodies derived from the unique heavy‐chain antibodies of camels, which bind to InlB with high affinity. A combination of gentamicin protection assays and florescent microscopy demonstrate that InlB specific nanobodes can block the invasion of Listeria cell in vitro. Furthermore, a high resolution X‐ray structure of one of the nanobodies in complex with InlB reveals that the nanobody binds the cMet interaction site, acting as a competitive inhibitor preventing bacterial invasion. The results presented point to the potential of nanobodies as a novel class of therapeutics for the prevention of Listeriosis.Support or Funding InformationProjected is funded by a NIH SCORE (SC3) grant (SC3GM112532)