bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amoxicillin decreases intestinal microbial diversity and increases stress‐associated behaviors in zebrafish
Authors: Deprey Kirsten L. , Uno Jennifer K 
Published: November 2023

Anxiety disorders affect approximately 18% of the population in the U.S. Research indicates that antibiotic exposure may contribute to the onset of anxiety. The gut‐brain axis is a bidirectional communication pathway between the gut and the brain. Evidence supports the role of the microbiota in this relationship. This study employs zebrafish to observe the impact of amoxicillin‐induced changes to the microbiota on stress‐related behaviors. We hypothesize that a decrease in intestinal microbial diversity will increase anxious behavior. Adult zebrafish were administered amoxicillin or vehicle and challenged via death‐induced odors and a net‐stress. Top‐to‐bottom and side‐to‐side movement over time was recorded. Intestinal bacterial‐DNA was extracted and bacterial phyla was examined and quantified by qPCR. Results confirmed amoxicillin significantly decreased the quantity and diversity of intestinal bacterial communities, indicated by a 20% change in universal gut bacteria in the treated fish (n=14, p value <0.05), and a slight increase in Lactobacillus. Antibiotic treatment also correlated to behavioral changes in the fish, illustrated by a significant decrease in top‐to‐bottom and side‐to‐side movement in fish (35%, 54% decrease in top‐to‐bottom and side‐to‐side, respectively, n=12 p value <0.01, p value <0.001). Behavioral studies after reestablishment of the gut microbiota also demonstrated a significant decrease in top‐to‐bottom swimming (n = 12, p value <0.01). Collectively, these results signify that decreased intestinal microbial diversity may elicit anxiety in zebrafish. These data warrant further investigation into the mechanism behind which microbiota‐induced behavioral changes are associated with antibiotics. This study was supported by the APS and Elon University.Support or Funding InformationThis study was supported by the APS and Elon University.
MicroRNAs Target Decidual Angiogenic Factors During Early Pregnancy in BPH/5, a Spontaneous Mouse Model of Preeclampsia
Authors: Xia June , Baxter Samuel , Grenier Jen , Davisson Robin L , Sones Jenny L 
Published: November 2023

Preeclampsia (PE) is a hypertensive disease that affects up to 10% of pregnant women worldwide. It presents as late gestational hypertension/proteinuria and only resolves upon delivery of the fetus and placenta. Despite being a leading cause of maternal and fetal morbidity/mortality, the underlying mechanism(s) is unknown. Previous research indicates that abnormal placental development plays a key role in PE. Proper placental formation is dependent on successful implantation and decidualization, the essential process of uterine angiogenesis prior to trophoblast invasion and placentation. Using the BPH/5 spontaneous genetic mouse model of PE, we have previously described profound periimplantation defects in early pregnancy with the principle dysregulated factor being cyclooxygenase 2 (Cox2). Preliminary data indicates that uterine Cox2 misexpression plays a causal role in PE. As Cox2 is heavily regulated by micro(mi)RNAs during the periimplantation period in mice, we hypothesized that miRNAs play a key role in the periimplantation defects seen in the preeclamptic BPH/5 mouse. To test this hypothesis, miRNA sequencing was performed on BPH/5 and C57 control implantation sites collected at e7.5 of pregnancy, the day of peak decidualization. Twenty‐one differentially expressed miRNAs were identified (≥1.5‐fold difference, p < 0.05) with the top 9 being downregulated in BPH/5 implantation sites. Bioinformatic analyses identified potential targets related to angiogenesis. Interestingly, one of these was miR‐146a (−2.2‐fold vs C57), which targets vascular endothelial growth factor (VEGF)‐A. Vegf mRNA levels in e7.5 BPH/5 implantation sites are comparable to C57 as measured by qRT‐PCR, however VEGF164 protein is significantly upregulated in BPH/5 e7.5 implantation sites (1.013 ± 0.25 vs C57: 0.1580 ± 0.1, n =7, p<0.05). Further studies are warranted to confirm if miR‐146a is playing a causal role in the VEGF164 expression pattern. PE is characterized by an imbalance in placental angiogenic factors; however, regulation of these factors during decidualization in the context of PE is unknown. This will provide better understanding of the relationship of miRNAs and angiogenic factors in the decidua before placenta formation and the onset of the maternal syndrome in BPH/5 mice. These findings may provide insights into the origins of the angiogenic imbalance observed in PE pregnancies.Support or Funding InformationStudies described herein were supported in part by a seed grant from the Cornell Center for Reproductive Genomics, using funds obtained as part of the NICHD National Centers for Translational Research in Reproduction and Infertility (NCTRI), award number P50HD076210 (PI: P.E. Cohen).
The Essence of Quiescence: Understanding the roles of histone modification H4K20me3 and the histone modifying enzyme Suv4‐20h2 in cellular quiescence
Authors: Corvalan Adriana Z , Evertts Adam G , Lowry William E , Coller Hilary A 
Published: November 2023

The ability to transition between a proliferative state and quiescence, a reversible exit from the cell cycle, is essential for proper development. During the formation of tumors by cancer cells, the ability to exit and re‐enter the cell cycle in response to environmental cues is lost. This shift from the proliferative to quiescent state is associated with changes in gene expression and chromatin landscape, with quiescent cells exhibiting increased chromatin compaction. Using a high throughput mass spectrometry‐based approach to monitor histone modifications in proliferating and quiescent cells, we discovered that histone H4K20 is differentially methylated in proliferating versus quiescent fibroblasts, with an 8‐fold increase of H4K20me3 in quiescent cells. Levels of H4K20me3 are regulated by Suv4‐20h2 and loss of this enzyme results in decreased chromatin compaction, an increase in the fraction of cells in S phase, and a defect in entry to quiescence. Using ChIP‐Seq, we found that H4K20me3 modifications are present in intergenic regions and promoters of genes that are important for proliferation and pluripotency in a cultured cell line. By investigating the hair follicle stem cells isolated directly from mouse skin, we found that H4K20me3 levels are higher in quiescent hair follicle stem cells isolated from skin during the resting phase of the hair follicle cycle than when the same cell lineage is proliferating in the anagen phase of the hair follicle cycle. Further, H4K20me3 levels are higher in the stem cells of the hair follicle compared to other cell types in the skin such as the basal epidermal cells and dermal fibroblasts. These results support a model in which Suv4‐20h2‐mediated H4K20me3 deposition plays a role in entry and exit of the cell cycle and chromatin architecture.Support or Funding InformationRuth L. Kirschstein National Research Service Award GM007185.
LncRNA H19 inhibits gut epithelial regeneration via Wnt co‐receptor LRP6 by interacting with RNA‐binding protein HuR
Authors: Chen Gang , Rao Jaladanki N. , Zou Tongtong , Xiao Lan , Chung Hee Kyoung , Gorospe Myriam , Wang Jian‐Ying 
Published: November 2023

Long noncoding RNAs (lncRNAs) are involved in a variety of physiological processes and disease states by modulating gene expression through a wide range of distinct mechanisms. H19 is a conserved lncRNA in mammalian tissues and acts as a molecular sponge or primary template for given microRNAs to regulate proliferation and differentiation. This study tested the hypothesis that H19 regulates intestinal epithelial cell (IEC) proliferation by altering Wnt‐signaling pathway.MethodsStudies were conducted in human colonic epithelial cells (Caco‐2 line) and H19 function was examined by its ectopic overexpression.ResultsH19 overexpression by transient transfection decreased the level of Wnt co‐receptor LRP6 protein but it did not alter cellular abundances of Lrp6 mRNA, suggesting the repression of LRP6 translation by H19. Since H19 exon1 encodes miR‐675, we investigated the possibility that H19 derives its functionality from miR‐675 and demonstrated that ectopic overexpression of either miR‐675‐3p or miR‐675‐5p failed to inhibit LRP6 expression. We further found that H19 directly bound to the RNA‐binding protein HuR (activator of LRP6 expression) and inhibited HuR‐induced stimulation of LRP6 translation. Elevation of H19 also inhibited IEC proliferation.ConclusionsThese data indicate that H19 inactivates Wnt‐signaling by inhibiting LRP6 translation through competitive inhibition of HuR binding to Lrp6 mRNA, thus down‐regulating intestinal epithelial renewal.Support or Funding InformationNIH (JYW) and Department of Veterans Affairs (JYW, JNR)
Role of lncRNAs in Regulating Endothelial Function
Authors: Huang Tse‐Shun , Wang Kuei‐Chun , Li Julie Yi‐Shuan , Chang Ting‐Yu , Subramaniam Shankar , Shyy John , Wang Hsei‐Wei , Chien Shu 
Published: November 2023

Long non‐coding RNAs (lncRNAs) are shown to carry out functions in wide varieties of pathological and physiological processes such as progress of cancer and maintenance of homeostasis in circulation system. The roles of lncRNAs in atherosclerosis (vessel wall thickening) remain unknown. We propose that lncRNAs regulate endothelial functions to control vascular homeostasis. The chemical (such as therapeutic drug Statins) and mechanical (such as blood flow pattern) stimuli modulate lncRNAs to regulate endothelial status.With the state‐of‐the‐art RNA sequencing technique, we identified several lncRNAs that are enriched in the endothelial cells under various hemodynamic flow patterns. Among these flow‐regulated lncRNAs, we demonstrated that LINC00341 was up‐regulated by atheroprotective flow, which led to the reduction of vascular adhesion molecule‐1 (VCAM1) expression and the consequential monocyte‐endothelial interactions. Overexpression of LINC00341 repressed the VCAM1 expression, as well as monocyte adhesion, caused by TNFα and atheroprone oscillatory shear stress; knocking down LINC00341 abrogated the atheroprotective effect of Statin (i.e. the repression of VCAM1 and monocyte adhesion). Thus, we conclude that LINC00341 plays an important role in vascular homeostasis. Several modes of action for lncRNAs have been purposed, including alteration of protein complexes targeting specificity by direct incorporation. To investigate the molecular mechanisms by which LINC00341 represses VCAM1 expression, we found that LINC00341 is associated with the immunoprecipitates of Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 that transfers a methyl group to K27 amino acid of histone H3. We further used the systems biology approach that combines computational and molecular biology analyses to investigate the integrated system of lncRNAs and their regulated gene networks to elucidate the mechanisms by which lncRNAs regulate vascular functions. The knowledge gained in this study will help us to understand the roles of lncRNAs in atherosclerosis and benefit the development of therapeutic strategies.Support or Funding InformationThis study is supported by the Ministry of Science and Technology, Taiwan (103‐2917‐I‐564‐030) and the National Institutes of Health, US (HL106579)
Cranial Neural Crest Migration Sculpts Endothelial Patterning in Avians with Anti‐Angiogenic Factors
Authors: McKinney Mary Cathleen , Kulesa Paul 
Published: November 2023

The embryonic neural crest and endothelial cells arise in separate locations but migrate to form intimately‐linked distinct structures in the vertebrate head. The lack of in vivo observations of these cell populations simultaneously in the same embryo has led to several unanswered mechanistic questions. Here, we take advantage of fluorescent labeling of cranial neural crest cells within Tie1 transgenic quail embryos Tg(tie1:H2B‐eYFP) to examine the dynamics of cranial neural crest and endothelial cell migration. In vivo time‐lapse analysis reveals that migrating neural crest and endothelial cells 1) move in opposing directions along a common dorsolateral migratory pathway, 2) display physical interactions that result in changes mainly in endothelial cell trajectories, 3) sort onto discrete pathways after interacting. Deletion of premigratory cranial neural crest cells results in changes to the endothelial cell movements and the vascular pattern. To examine the underlying molecular signals, we find that neural crest cells express anti‐angiogenic factors confirmed by fluorescence in‐situ hybridization and protein analysis. We specifically test the role of Angiopoetin‐2 and show that gain‐ or loss‐of‐function in neural crest cells leads to alterations in the migration patterns of the neural crest and endothelial cells. Together, our data support a model hypothesis that migrating neural crest cells express anti‐angiogenic factors in order to prevent occlusion of their migration by premature vessel formationSupport or Funding InformationStowers Institute for Medical Research
Regulation of liver energy balance by nuclear receptors
Authors: Moore David D 
Published: November 2023

The liver is crucial for maintaining whole body energy balance. For example, the liver responds to fasting by secreting glucose and to feeding by storing it as glycogen. In response to overnutrition, the liver stores excess fat, which is correlated with insulin resistance. The nuclear receptors FXR and PPARα are activated in the fed or fasted liver, respectively, and regulate many metabolic processes. Among such processes, autophagy is a catabolic pathway that recycles nutrients upon starvation and maintains cellular energy homeostasis. We have found that both FXR and PPARα regulate hepatic autophagy. Pharmacologic activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, while pharmacologic activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy.Neonatal cholestasis is a potentially life‐threatening condition. We have identified four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the bile acid receptor FXR. Clinical features of NR1H4‐related cholestasis include neonatal onset with rapid progression to end‐stage liver disease, vitamin‐K independent coagulopathy, low‐to‐normal serum gamma‐glutamyl transferase activity, elevated serum alpha‐fetoprotein, and undetectable liver bile salt export pump (ABCB11) expression. The coagulopathy is due to FXR transcriptional regulation of multiple components of the coagulation pathway. Genome wide binding studies indicate that FXR may also regulate a number of other liver secreted proteins. The process of secretion is very energy intensive, and it is well known that fasting suppresses secretion via post‐transcriptional mechanisms. We suggest that nuclear receptors also provide nutrient sensitive transcriptional input to liver secretion, with FXR licensing this process while PPARα suppresses it. Overall, we conclude that nuclear receptors act via complementary, interlocking mechanisms to maintain proper energy balance in the liver.
An active microrheological approach to determine material properties of early avian embryos
Authors: Kosa Edina , Akos Zsuzsa , Filla Michael , Isai Dona G , Ghazvini Saba , Dhar Prajna , Czirok Andras 
Published: November 2023

To understand how cells facilitate large‐scale tissue rearrangements during morphogenesis, we utilize high resolution time‐lapse microscopy of early embryonic tissues (cells and ECM) of avian embryos. To determine the material properties of embryonic tissues, we microinject magnetic microparticles that can be rotated by an external magnetic field. The ECM microenvironment is visualized by fluorescently labeled antibodies, microinjected into the extracellular space. We calibrate our microrheology method in hydrogels. Our data demonstrate that tissues harden gradually both (i) in time (at the same anatomical location) as tissues mature, and (ii) at more anterior locations within the same embryo. (iii) The apparent elastic modulus strongly depends on the size of the probe: large aggregates of magnetic crystals co‐rotate with the surrounding ECM. In contrast, individual, 5–10 um long ferromagnetic crystals often appear to rotate freely. The posterior embryo is, in particular, a very loose, fluid‐like assembly of cells and ECM.Support or Funding InformationR01‐GM102801
Co‐Incident Upregulation of Shh and Hoxa 13 by Fgf in The Developing Limb Bud
Authors: Real Daniel , Watson Billy A , Feenstra Jennifer M , Pira Charmaine U , Oberg Kerby C 
Published: November 2023

Sonic hedgehog (Shh) is secreted from the zone of polarizing activity (ZPA) within the distal‐posterior limb bud mesoderm and regulates pattern and proliferation along the anterior‐posterior limb axis. Fibroblast growth factor 8 (Fgf8) is expressed in the distal limb bud from a rope‐like thickening called the apical ectodermal ridge (AER) and regulates proximal‐distal outgrowth and patterning. Once established, Shh and Fgf8 maintain each other's expression via a reciprocal loop, tightly regulating progressive limb development. Intermediates between Fgf and Shh are still being characterized. Gene arrays following application of Fgf2 to the posterior margin of developing chick wings showed up‐regulation of not only Shh, but also Hoxa13. Hoxa13 expression is a marker of the developing autopod or handplate. In the absence of Hoxa13, there is increased anterior apoptosis and disruption of the normal anterior‐posterior patterning. We hypothesized that the upregulation of Hoxa13 by Fgf in the posterior margin of the limb bud is important for the up‐regulation of Shh.To test this hypothesis Fgf2 or PBS soaked beads were implanted in the posterior limb margin of stage 23 chick wing buds. Embryos were harvested 24 hours after bead implantation for Hoxa13 whole mount in‐situ hybridization (WMISH).The results showed that Fgf induced ectopic Hoxa13 expression 24 hours after bead implantation. This confirmed the gene array data that showed a 13 fold increase in Hoxa13 following Fgf2 application. Limb‐specific Shh expression is regulated by the ZPA regulatory sequence (ZRS) which contains Hox binding sites important for full regulatory activity. Future studies are needed to establish whether Hoxa13 binding to the ZRS can facilitate Shh up‐regulation.
Lmx1b Binds to a Gdf5‐Associated Enhancer Element Active During Limb Dorsalization
Authors: Haro Endika , Pernu Lauren , Pira Charmaine U , Oberg Kerby C 
Published: November 2023

Lmx1b is a homeodomain transcription factor expressed in the dorsal limb mesoderm that mediates dorsal patterning. Mice lacking functional Lmx1b develop double ventral limbs, whereas ectopic over‐expression in chicken exhibits double dorsal limbs. Although, Lmx1b‐regulated genes have been identified in developing limb buds by gene arrays, no direct targets have been confirmed.To elucidate direct target genes we performed an Lmx1b‐specific chromatin immunoprecipitation followed by massive parallel sequencing (Lmx1b‐ChIP‐seq). From the recovered genomic intervals, potential regulatory sequences (PRS) were determined by “in silico” comparison to published enhancer‐associated (H3K27ac, P300, RNAPolII or Med12 ) ChIP‐seq data in stage‐matched limbs. Since dorsal‐ventral patterning is a conserved process across divergent vertebrates, PRS were screened for conservation. Finally, intervals matching these criteria were compared to genes differentially expressed in the presence of Lmx1b. Functional validation of selected PRS was performed using an enhancer‐reporter construct in a chick limb bioassay.Of the 1287 identified genomic intervals bound by Lmx1b, nearly half were intergenic, suggesting that Lmx1b acts at distant regulatory regions rather than at gene promoters. 1022 Lmx1‐bound intervals correlated with enhancer‐associated markers and conservation was evident in 316 intervals that were associated to 121 differentially expressed genes in the presence of Lmx1b. Within the selected PRSs we identified an active enhancer located 78 Kb from Gdf5 that drives reporter activity in the limb and joints. The activity of this Lmx1b‐bound enhancer overlaps Gdf5 expression suggesting a role for Lmx1b in its regulation. Further studies are needed to characterize this enhancer and the role of Lmx1b in regulating Gdf5 expression during limb dorsalization.
Differential changes in trophoblast lineage allocation in rats deficient in uterine natural killer cells
Authors: Renaud Stephen J , Scott Regan L , Chakraborty Damayanti , Rumi M.A. Karim , Soares Michael J 
Published: November 2023

Uterine natural killer (uNK) cells are the most prevalent uterine leukocyte population during early pregnancy. They are recruited to the uterus soon after embryo implantation, and “prime” the uterine spiral arteries in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification occurs by invasive trophoblast cells. Invasive trophoblast cells originate in a specialized compartment of the placenta ‐ called the junctional zone in rodents ‐ and progressively migrate into the uterus en route to the spiral arteries. This “two‐wave” transformation of the uterine vasculature by both NK cells and trophoblast cells occurs in humans and rats. The purpose of this study was to generate and characterize a genetic model of uNK cell deficiency in rats, and observe the resulting consequences on trophoblast development and reproductive outcomes. To accomplish this task, we utilized zinc finger nuclease (ZFN)‐mediated genome editing of the interleukin 15 (Il15) gene. The Il15 locus was targeted because it encodes a cytokine required for NK cell maturation and survival. ZFNs were microinjected into zygotes, and transferred to pseudopregnant dams. Following genotypic characterization of pups, we identified a founder animal containing a seven base‐pair deletion within exon 2 of the Il15 locus. The deletion resulted in a frame shift within the coding sequence of Il15, and caused a functional loss of IL15 protein. In comparison to wild‐type pregnant female rats, whose uteri contained massive quantities of perforin‐positive NK cells, uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable uNK cells. At midgestation, an expansion of the junctional zone of the placenta was observed, as well as depth and quantity of trophoblast cells invading within the spiral arteries. Accordingly, the internal luminal diameters of these vessels were substantially larger in rats lacking uNK cells. In summary, we demonstrate here significant differences in the size of the junctional zone and the number of invasive trophoblast cells in uNK cell‐deficient rats. These studies will enhance our understanding of the role of NK cells on uterine spiral artery remodeling, trophoblast invasion, and hemochorial placental development.Support or Funding InformationSupported by NIH HD020676