bluuebunny/crossref_metadata_2025 huggingface dataset:
EphA2 organization and dynamics in cancer cells
Authors: Bowman Deanna Meredith , Shi Xiaojun , Hapiak Vera , Zheng Ji , Muller‐Greven Jeannine , Lingerak Ryan , Buck Matthias , Wang Bing‐Cheng , Smith Adam
Published: November 2023

Eph receptors are a subfamily of protein tyrosine kinases (RTKs) that possess a sterile alpha motif (SAM) domain. These receptors are important for cell‐cell communication, proliferation, cell growth, and migratory responses. They are also over‐expressed in cancer cells and tumor vessels. Eph receptors are thought to be involved in tumor progression and metastatic spread. The behavior is dependent on expression density of the receptor and the presence of ephrin ligand in opposing cells. Oligomerization of the EphA2 receptor plays a key role in its function. Dimerization and oligomerization are part of the activation of RTKs and the response to ligand stimulation. Restricting the cluster size of active EphA2 affects the signaling output, and performance, ligand independent dimers are one possible source of aberrant signaling. In this study a fluorescence fluctuation method known as fluorescence correlation spectroscopy (FCS) is used to quantify oligomerization in live cells. Measurements were taken in COS7 cells as well as a prostate cancer model cell line (DU‐145). Both cell lines express endogenous EphA2 (without GFP), which could complicate the interpretation of the FCS measurements. We therefore developed an epithelial cell line from an EphA2/A1, knockout mouse, in which there is no endogenous receptor. By conducting FCS measurements across multiple cell lines we conclude that deletion of the SAM domain leads to an increase in oligomerization similar to the level observed in ligand‐activated, wild‐type receptors. Further investigation of the structural determinants of oligomerization will lead to a better understanding of the role that EphA2 plays in metastasis and tumorigenesis.
Suppressor of IKK epsilon (SIKE) acts as a bridge between innate immune signalling pathway and cytoskeletal rearrangements
Authors: Wittenberg Karli A , Slykas Frank A , Sonnenschein Halie A , Bell Jessica K.
Published: November 2023

Innate immunity coordinates the first line of defense against invading pathogens. This involves the recruitment and subsequent migration of nearby immune cells to the site of infection, allowing the pathogen to be consumed via phagocytosis to sequester the infection. These processes require the rapid disassembly, rearrangement, and repolymerization of the network of structural proteins known as the cytoskeleton. Migration and phagocytosis are one of the functional consequences of innate immune receptor triggered signaling pathways. Signals coalesce at catalytic hubs, such as kinases, which serve to amplify and regulate the signal. In the anti‐viral response, TANK binding kinase 1 (TBK1) functions as a catalytic hub. Suppressor of IKK epsilon (SIKE) acts as a high affinity, alternate TBK1 substrate, although SIKE's downstream function is not yet defined. The primary goal of this work was to assess the effect of SIKE on cytoskeletal rearrangements in migration and phagocytosis. For these studies, a CRISPR knockout cell line was derived, SIKE‐CR, from chronic myelogenous leukemia (CML) cell line KBM‐7 named HAP1. Wound healing assays of HAP1 parental cells and SIKE‐CR illustrated that the absence of SIKE significantly decreased cell migration. In the mouse macrophage RAW 264.7 cell line, SIKE expression was knocked down via lentiviral delivery of shRNA targeting SIKE. Phagocytosis was assessed in WT and SIKE knockdown cells via uptake of latex beads labeled with IgG‐FITC. Reduced SIKE expression increased phagocytic uptake. To determine SIKE interactions with cytoskeletal proteins, co‐immunoprecipitations (cIP), in vitro precipitation (IVP) reactions, and enzyme‐linked immunosorbent assays (ELISA) were completed and demonstrated SIKE:tubulin and SIKE:α‐actinin complexes. Together, these studies establish an interaction between SIKE and cytoskeletal proteins and suggest that SIKE functions in cytoskeletal rearrangement associated with migration and phagocytosis.

Support or Funding Information

Work supported by NIH grant R21AI107447 and USD SURE.
Educational Outcomes of Implementing Prosections as Educational Tools in the Medical Gross Anatomy Laboratory
Authors: Williams Sutton Ross , Thompson Kenneth , Sinning Allan
Published: November 2023

This research study aimed to identify the educational outcomes associated with the decision to utilize prosections (pre‐dissected specimens) rather than active dissection as the primary educational modality through which first year medical students were exposed to anatomical structures in a medical gross anatomy laboratory course. Participants in this study included 580 first year medical students enrolled in the medical gross anatomy course at The University of Mississippi Medical Center from 2013–2016. The educational outcome of interest was student performance on laboratory practical examination questions. Student performance on these questions was compared between and within groups based upon the educational modality through which they were exposed to the structure being assessed. As such, students were assigned into one of the following three cohorts for each question analyzed: dissector, non‐dissector, or prosector. Statistical analysis indicates a small but statistically significant difference between how the dissector and non‐dissector cohorts performed on all practical examination questions analyzed. The dissector cohort (M=82.8%) performed 1.5% (95% C.I‐ 1.1% to 2.1%) better on questions than the non‐dissector cohort (M=81.3%) X2(1, N= 85,300) = 36.11, p<0.001. Analysis also indicates that for both the hand and foot regions, those cohorts that were exposed to these regions through participation in the dissection process performed significantly worse on practical exam questions pertaining to these regions than those cohorts who were exposed to the hand and foot through interaction with prosected specimens. The average score on practical questions pertaining to the foot was 19.3% (95% C.I‐ 15.2% to 23.5%) lower for the dissector cohort (M=68.6%) than for the prosector cohort (M=87.9%) X2(1, N= 2,010) = 105.28, p<0.001. The average score on practical questions pertaining to the hand was 8.3% (95% C.I‐5.1% to 11.6%) lower for the dissector cohort (M=71.4%) than for the prosector cohort (M=79.7%) X2(1, N= 2,730) = 25.70, p<0.001. These results demonstrate that there is not a significant advantage for those medical students who learn human anatomy through the dissection process rather than through interaction with pre‐dissected specimens. Results also demonstrate that there is a significant advantage to using prosections rather than student dissection as the primary educational modality for learning the anatomy of the hand and foot regions.
Breast Tumor Cell‐derived FGF‐5 Induces MEKK1‐dependent Chemokine Expression in Mammary Fibroblasts; Implications for Tumor Microenvironment
Authors: Cuevas Bruce Daniel
Published: November 2023

Breast tumors contain both transformed epithelial cells and non‐transformed stroma cells, and accumulating experimental evidence strongly suggests that this heterogeneous tumor microenvironment influences tumor growth and metastasis. Previously, we demonstrated that MAPK/ERK kinase kinase 1 (MEKK1) regulates gene expression in fibroblasts and breast tumor cells, and that transgene‐induced breast tumor metastasis is markedly inhibited in MEKK1‐deficient mice. In this work, we utilized expression array analysis of heterotypic cell cultures to determine the role of MEKK1 in stroma gene expression induced by breast tumor cells. We discovered that the breast tumor cell line MDA‐MB 231 constitutively expresses fibroblast growth factor 5 (FGF‐5), and that exposure to either tumor cell‐conditioned media or recombinant FGF‐5 induces chemokine expression in mammary fibroblasts. We demonstrate that FGF‐5‐induced expression of C‐C Motif Chemokine Ligand 5 (CCL5/RANTES) is markedly reduced in MEKK1‐deficient mammary fibroblasts, and that CCL5 expression requires MEKK1 kinase activity but is independent of MEKK1 ubiquitin ligase activity. Furthermore, we report that FGF‐5 initiates MEKK1 kinase activation and signaling in mammary fibroblasts. Finally, we show that breast tumor cell chemotaxis induced by fibroblasts is markedly inhibited by exposure to the CCL5 receptor antagonist Maraviroc, and that MEKK1‐deficient mammary fibroblasts are significantly less effective at promoting tumor cell chemotaxis than cells that express MEKK1. Overall, our findings suggest that MEKK1 is a key regulator of the stroma cell response to tumor cell‐derived FGF‐5. We conclude that MEKK1 is part of the signaling mechanism regulating a tumor/stroma paracrine loop that influences function of both transformed cells and stroma cells within breast tumors. Future studies in our lab will investigate the utility of the FGF‐5/CCL5 signaling axis as a set of targets that can be therapeutically inhibited to reduce tumor growth and progression.

Support or Funding Information

American Cancer Society, Illinois Div.
Mir‐16 and Mir‐34a Suppress Growth of a Variety of Human Cancer Cells
Authors: Jones Eyone , Mazirka Pavel , McNurlan Margaret A , Brink Peter , Caso Giuseppe
Published: November 2023

MicroRNAs (miRs) can have a pivotal role in the regulation of tumor growth and progression and could represent important therapeutic agents. MiR‐16 and miR34a levels are reduced in many human cancers and have potential as tumor suppressors.

The aim of this study was to test the direct effect of miR‐16 and miR34a on the growth of different types of human cancer cells. Human prostate (PC3) and breast cancer (MBA‐MD‐231) and melanoma (UACC62) cells were transfected with miR‐16 or miR34a. Cell proliferation was assessed over a 120 h period using an MTT based assay. MiR‐induced apoptosis was also evaluated in transfected PC3 cells by flow cytometry using Annexin V and PI staining.

Increasing intracellular levels of miR‐16 had a profound suppressive effect on the growth of all tumor types. After 120 h cell number was decreased by 86% in PC3, by 56% in MDA‐MD‐231, and by 80% in UACC62 cells transfected with miR‐16 compared to controls (P<0.001). MiR‐34a significantly depressed growth in prostate and breast cancer cells. 120 h after transfection with mir‐34a cell number was 56% in PC3 and 66% lower in MBA‐MD‐231 cells than controls (P<0.001). No significant effect of miR‐34a was detected on melanoma cell growth. Both miRs increased apoptosis in PC3 prostate cancer cells, with miR‐16 showing a larger effect. The number of apoptotic cells increased from 6.4 % (control) to 20.5 % and 16.2% after treatment respectively with miR‐16 and miR34a.

In summary, miR‐16 showed a profound suppressive effect on growth of human prostate and breast cancer, and melanoma cells. MiR34 depressed the growth of breast and prostate cancer cells but did not show a significant effect on UACC62 melanoma cells. The negative effect on growth of miR‐16 and miR‐34a on PC3 prostate cancer cells results from both suppression of cell proliferation and enhancement of apoptosis.

Support or Funding Information

Supported by the Stony Brook Foundation TRO‐Catacosinos Cancer Translational Researcher Award, and by the Dept. of Surgery, Stony Brook University Medical Center (Small Grant Program)
Subpopulations of Neurotensin Neurons in the Lateral Hypothalamic Area Respond to Distinct Cues and Contribute to Energy Balance by Discrete Mechanisms and Projections
Authors: Brown Juliette Anne , Wright Anna , Bugescu Raluca , Woodworth Hillary L , Kurt Gizem , Leinninger Gina M
Published: November 2023

In the lateral hypothalamic area (LHA), some neurons, express the neuropeptide neurotensin (Nts) and regulate feeding, drinking and physical activity. Many LHA Nts neurons also contain the inhibitory neurotransmitter GABA, but some modulate glutamate (glut) signaling. This suggests that there are different subtypes of LHA Nts neurons. LHA Nts neuronal terminals have been seen within both the Ventra Tegmental Area (VTA) and Substantia Nigra Compacta (SNc) implying that subpopulations of LHA Nts neurons have distinct midbrain projection targets and mechanisms of action. Furthermore, some LHA Nts neurons co‐express the long form of the leptin receptor (LepRb) and are activated by the anorectic hormone leptin (NtsLepRb neurons) while others are activated by dehydration (NtsDehy neurons). I examined the hypothesis that NtsLepRb and NtsDehy neurons are distinguishable from each other based on their expression of GABA or glut and whether they project preferentially to the VTA or SNc. Until now, the lack of reagents to simultaneously identify Nts, GABA and glutamate prevented this analysis. To overcome this, we designed a dual genetic recombinase approach in order to simultaneously label Nts and GABA or glutamate‐containing neurons. We first generated mice that express FlpO recombinase specifically in Nts neurons (NtsFlpO mice) which we crossed with mice that express Cre recombinase in GABA or glutamate neurons. The resulting offspring expressed FlpO recombinase in Nts neurons and Cre in GABA/Glutamate Neurons; NtsFlpO/vGatCre or NtsFlpO/vGlutCre mice respectively. I injected vectors for FlpO‐inducible green fluorescent protein (GFP) and Cre‐inducible red fluorescent protein (RFP) in the LHA these mice with causes GFP expression only within LHA Nts neurons and RFP expression only in GABA/Glut neurons enabling the simultaneous detection of Nts and GABA and/or glutamate containing neurons via fluorescent microscopy. To examine the midbrain projections of each subtype of LHA Nts neurons, Fluorogold (FG) retrograde tract tracer was injected into the VTA or SNc of mice that express GFP only in Nts neurons (NtsGFP mice) and followed with leptin, or dehydration treatment. Brains were analyzed via immunostaining to label NtsLepRb, or NtsDehy neurons and FG. Our data shows that many LHA Nts neurons contain GABA and some express glutamate. Additionally, both LHA NtsLepRb neurons project to the VTA to the SNc but LHA NtsDehy neurons do not project to either midbrain region. The neurotransmitter content of these cue‐distinct subpopulations will be determined using Leptin and Dehydration treatment too.

Support or Funding Information

This work is supported by NIH awards to JAB (1F31 DK107081‐01A1, 5 T32 ES007255‐27) and GML (RC105025)
Elucidating Peptide Pheromone Signaling in Pathogen Conversations to Understand Advantages of Social Cooperation
Authors: Federle Michael J
Published: November 2023

Cell‐to‐cell communication (quorum sensing) of Gram‐positive bacteria commonly employs peptide pheromones that transmit information between bacterial cells to facilitate coordinated gene expression across a population. In bacterial pathogens, quorum‐sensing pathways are found to contribute to virulence, biofilm development, antibiotic resistance and species evolution. In Firmicutes, pheromone receptors that are located within the cytosol encompasses a large protein family, designated RNPP (Rap/Npr/PlcR/PrgX), that contains a common structural domain responsible for binding pheromones. Though related by structure, primary amino acid sequences of RNPP proteins and their corresponding peptide ligands are disparate. Likewise, the interactions between receptors and ligands, and the mechanisms of activation resulting from these interactions are empirically unique individually for the few prototypical systems studied and thus, overall, the family remains underexplored. It is our goal to elucidate the sensory‐transduction mechanisms of these molecular switches to facilitate methodologies that can be used to modulate communication pathways to treat or prevent disease. Using genetic, biochemical, structural and chemical‐biology approaches, we have begun to elucidate signaling pathways among streptococcal species, have characterized receptors and their interactions with ligands, and have identified modulatory compounds that interfere with normal signaling. For the Rgg2/3 signaling pathway in Streptococcus pyogenes, pheromones induce phenotypes that are consistent with an ability for bacteria to colonize the host asymptomatically, and we hypothesize that social coordination of this behavior reinforces conformity to avoid host innate‐immunity stimulation.

Support or Funding Information

NIH R01AI091779 and R01AI425452, and the Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases
Genotypically distinct Helicobacter pylori causes Gastric and Peptic Ulcer diseases in Kashmir Indian patients
Authors: Rehman Shabina , Bharti Alok C , Das Bhudev C , Talukder Jamil
Published: November 2023

Background

Although 50% of the world population is infected, only a small fraction of people infected with H. pylori suffers from active gastrointestinal (GI) diseases such as gastric ulcer, peptic ulcer and gastric cancer. Genotypically varying virulent H. pylori such as cytotoxin associated gene (Cag A) and vaculating assoctiated cytotoxin (VacA) have been shown associated with gastric and duodenal ulcer patients living in different countries, respectively. Significant Kashmir population has been identified to have GI diseases. However, it is not known whether Kashmir patients with gastric and peptic ulcer diseases are infected with different or same virulent H. pylori.

Aim

This study was initiated to identify whether patients with gastric and peptic ulcers, and gastric cancer are infected with same or different H. pylori genotypes.

Methods

263 patients scheduled for upper endoscopy and 50 healthy volunteers were recruited for this study. Endoscopy examination divided the patients into non‐ulcer Dyspepsia (NUD), Peptic ulcer Disease (PUD) and Gastric cancer (GC). Biopsy specimens were taken from stomach (antrum and fundus) and duodenum from each patient. H. Pylori infection was confirmed by Rapid urease test (RUT), glm ureC and culture. H. pylori genomic DNA was isolated from H. pylori culture and biopsy specimen using phenol chloroform method. CagA and VacA genotypes were confirmed by polymerase chain reaction (PCR). H. pylori antigen was detected by immunohistochemistry.

Results

Endoscopic examination grouped the 213 patients into NUD (65), PUD (50) and GC (98). glm ureC analyses established 69%, 80% and 65% of NUD, PUD and GC patients were H. pylori positive, respectively. PCR analysis revealed that: 1) 78%, 80% and 75% of the NUD, PUD and GC patients were infected with CagA positive strains of H. Pylori, respectively; and 2) 76%, 85% and 80% of the NUD, PUD and GC patients were infected with VacA positive strains of H. pylori, respectively. Of the 50 normal healthy volunteers 15 (30%) were also identified H. pylori positive.

Conclusion

The most virulent genotype VacA is significantly over‐expressed in all the disease groups indicating a strong association of this genotype with H. pylori ‐ associated disease pathogenesis.

Age distribution of different gastro‐duodenal disease groups in JK and Delhi region.

Region
NUD
PUD
GC
Normal
p value

JK
38.8±8.7 (n=65)
44.1±12.6 (n=50)
52.9±11.95 (n=98)
46.94±9.42 (n=50)
0.07a<0.001b<0.001b

Delhi
38.1±9.6 (n=55)
43.7±11.5 (n=62)
48.3±12.9 (n=50)
36.1±11.7 (n=50)
0.05a<0.00 lb<0.22c

NUD‐ non ulcer dyspepsia; PUD‐peptic ulcer disease; GC‐gastric cancer; JK – Jammu and Kashmir.

p value for NUD vs. PUD,

NUD vs. GC; and

PUP vs. GC
Oral Supplementation of Soybean And Hop Restored Bone Mass And Bone Turnover in Ovariectomized Rats
Authors: Lee Hansol , Noh Dasom , Lim Yeni , Kwon Oran
Published: November 2023

A decline in the level of circulating estrogen induces reduction in bone mass and microarchitectural deterioration of bone tissue, placing postmenopausal women at a greater risk of osteoporosis. However, hormone replacement therapy (HRT) often provides the risk of oncogenicity and the adverse outcomes on cardiovascular disease. Thus, alternatively dietary intervention have been received a growing attention for their use with no accompanying adverse effects. The most significant body of evidence comes from the isoflavone flavonoids. Soybeans (Glycine max L.) and hops (Humulus lupulus L.) are the important sources of isoflavones, but provide different chemical profiles each other. Soybeans provides genistein and daizein, while hops provides prenylflavanone, isoxanthohumol, and xanthohumol. The aim of this study was to test whether a mixture of soybeans and hops (GH) alleviate bone changes found in ovariectomized rats. Eight‐week female Sprague‐Dawley rats were randomly divided into five groups (n = 10/group): sham‐operated control (CON), ovariectomized (OVX) control (O), OVX + 30 mg/kg BW of GH (OL), OVX + 100 mg/kg BW of GH (OM), and OVX + 300 mg/kg BW of GH (OH). After oral administration of each diet for 8 weeks, bone microarchitecture, bone turnover markers (osteocalcin, alkaline phosphatase, CTX, and NTX in plasma and urine), and osteoclastic and resorptive gene markers in the bone were measured. The GH supplementation mitigated the OVX‐induced increase in bone turnover rate and induced favorable architectural modifications of trabecular bone. Furthermore, GH suppressed osteoclastic gene expression as determined by RANKL/OPG ratio. The data suggest that the GH supplementation may be a promising alternative to HRT for the management of postmenopausal osteoporosis.

Support or Funding Information

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01169502)” Rural Development Administration and the Bio‐Synergy Research Project (NRF‐2012M3A9C4048761) of the Ministry of Science, ICT and Future Planning through the National Research Foundation.
Persimmon Tannin Regulates Gene Expressions Important for Lipogenesis And Cholesterol Efflux in HepG2 Cells
Authors: Oh Eun Kyoung , Ahn Youngsook , Kwon Oran
Published: November 2023

Persimmon (Diospyros Kaki) is a deciduous fruit and largely cultivated in the world, containing an array of bioactive compounds such as phenolic acids, tannin, flavonoids and dietary fiber. The health benefit of persimmon tannin has recently been reported in relation to the hypocholesterolemic, hypolipidemic and antioxidant properties in animals. However, the regulatory mechanisms have yet not to be elucidated. Here, we tested the hypothesis that persimmon tannin exerts inhibitory effects against the accumulation of both triglycerides and cholesterol in fatty‐acid challenged HepG2 cells. Tannin‐enriched persimmon extract (PT) was prepared by pressurized water extraction (121°C, 90 min), filtration, and concentration (20 mmHg, 125 rpm, 30°C). Tannin concentration of PT was enriched by 20 times compared with that in the persimmon powder. HepG2 cells were treated with fatty acid in the presence of PT at 0, 0.25, 0.5, and 1 mg/mL. Inhibitory effect of PT on lipid accumulation was confirmed by Oil‐Red‐O staining. Gene expressions related to triglyceride synthesis (fatty acid synthase, FAS; and sterol regulatory element binding protein‐2, SREBP‐2), cholesterol synthesis and excretion (LDL receptor, LDLR; proprotein convertase subtilisin kexin 9, PCSK9; 3‐Hydroxy‐3‐methylglutaryl CoA reductase, HMGCR; and ATP‐cassette binding proteins G5/8, ABCG5/8), and cholesterol efflux via bile acid synthesis and excretion (cholesterol 7‐alpha‐hydroxylase, CYP7A1; and bile salt export pump, BSEP) were measured by real‐time quantitative PCR. The results revealed significant decreases either in lipogenesis (FAS and SREBP‐2) or in cholesterol synthesis (HMGCR and PCSK9) and a significantly decrease in cholesterol excretion transporters gene (ABCG5, ABCG8) expression, whereas a significant increase in cholesterol uptake and efflux (LDLR, CYP7A1 and BSEP). These results suggest that PT may have a potential to modulate both lipid and cholesterol metabolism. The physiological relevance test of this in vitro observation is ongoing in rats treated with a high fat and cholesterol diet.

Support or Funding Information

This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development Rural Development Administration (PJ01169502) and the Bio‐Synergy Research Project (NRF‐2012M3A9C4048761) of the Ministry of Science, ICT and Future Planning through the National Research Foundation.
A Novel Assay for Cardiolipin Quantification in Isolated Mitochondria and Cell Lysates
Authors: Bahl Vasundhra , Saddar Sonika
Published: November 2023

Cardiolipin is a phospholipid present in the inner mitochondrial membrane. It plays an essential role in regulating the enzymes involved in mitochondrial respiration and processes such as biogenesis, fission, fusion, protein transport and apoptosis. Diabetes, heart failure and aging are linked to changes in levels of cardiolipin. Exposure to toxicants like cigarette smoke and organophosphates causes an alteration in cardiolipin levels and composition, and adversely affect health. Cardiolipin abnormalities are associated with various diseases such as Barth's syndrome, a genetic disorder affecting the gene responsible for cardiolipin synthesis that can lead to cardiomyopathy, underdeveloped muscles and growth delay. Since cardiolipin is crucial to mitochondrial functions there is increasing interest in assays for determining levels of cardiolipin in biological samples.

Until now, cardiolipin has been detected using mass spectrometry which requires very sophisticated instrumentation and expertise in the technique. BioVision's Cardiolipin Quantification Assay is a simple, straight‐forward and versatile plate‐based fluorometric assay that uses a proprietary probe. Our assay is highly specific for cardiolipin and no cross‐reactivity is observed with other lipids such as phosphatidylcholine, phosphatidylserine and sphingomyelin. The kit can detect as low as 0.2 nmol of cardiolipin in isolated mitochondria as well as cell lysates.
HIF‐1a‐Mediated Induction of Insig‐2 Regulates Cholesterol Metabolism by Accelerating Degradation of HMGCR
Authors: Hwang Seonghwan , Jo Youngah , Wolff Nicholas , Brugarolas James , DeBose‐Boyd Russell
Published: November 2023

HMG CoA reductase (HMGCR) catalyzes a rate‐limiting reaction in cholesterol synthesis. In cultured cells, sterols exert tight feedback control on HMGCR by inhibiting its gene transcription and by accelerating its proteasomal degradation. Both of these processes require endoplasmic reticulum membrane proteins called Insig‐1 and Insig‐2. Although the transcriptional component of the HMGCR regulatory system has been scrutinized in livers of mice through various transgenic and knockout animals, regulated degradation of HMGCR in animal livers has not been studied in detail. HMGCR protein accumulates disproportionately to its mRNA in livers of mice genetically deficient in both Insigs, suggesting that Insig‐mediated degradation significantly contributes to feedback regulation of the enzyme. As cholesterol synthesis is an oxygen‐intensive process, the current study is aimed at elucidating whether hypoxia modulates Insig‐dependent degradation of HMGCR in liver of mice.

To determine if hypoxia affects Insig‐dependent degradation of HMGCR in vivo, a liver‐specific transgenic mouse model was developed which expresses a T7‐tagged membrane domain of HMGCR, which is necessary and sufficient for degradation of HMGCR. As the transcription of the transgene is driven by a constitutive apoE promoter, the feedback regulation of the transgene product is exclusively dependent on proteasomal degradation. In this mouse model, we demonstrated that mice exposed to hypoxia stabilized hypoxia‐inducible factor (HIF)‐1a, increased Insig‐2 mRNA and protein, and accelerated degradation of HMGCR. Similar results were observed in mice administered with dimethyloxalylglycine (DMOG), a pharmacological inhibitor of prolyl hydroxylase that mimics hypoxia. DMOG failed to reduce the levels of HMGCR protein in mice that express the mutant HMGCR which is resistant to Insig‐induced ubiquitination, indicating that degradation of the enzyme significantly contributes to DMOG‐induced inhibition of HMGCR.

In cultured human cell lines, DMOG treatment stabilized HIF‐1a, up‐regulated Insig‐2, and accelerated degradation of HMGCR in a dose‐dependent manner. RNAi‐mediated knockdown of HIF‐1a, but not of HIF‐2a, impaired DMOG‐induced expression of Insig‐2 and degradation of HMGCR. Moreover, DMOG‐induced degradation of HMGCR was blocked in cells lacking Insig‐2, whereas DMOG continued to augment HMGCR degradation in Insig‐1 deficient cells. Using chromatin immunoprecipitation and luciferase promoter assays, we show that Insig‐2 is a target gene of HIF‐1 in human and mouse cells. This is consistent with the presence of a putative HRE in the promoters of the human and mouse Insig‐2 gene. Overall, these findings suggest that Insig‐2 is a direct transcriptional target of HIF‐1 and that hypoxia‐induced degradation of HMGCR is mediated through HIF‐1a stabilization and subsequent up‐regulation of Insig‐2.

Support or Funding Information

NIH Grants HL20948, GM112409
A Novel Yeast‐Based Screening System Identifies Signal Motifs that Regulate Membrane Protein Trafficking
Authors: Bernstein Joshua , Okamoto Yukari , Shikano Sojin
Published: November 2023

The cell surface density of membrane proteins dictates their corresponding cellular activity. Many diseases, such as cystic fibrosis, arise from defects in cell surface trafficking of membrane proteins. Intracellular trafficking of membrane proteins is often controlled by the distinct signal motifs on the cargo proteins. A systematic approach to identify trafficking signals would facilitate our understanding of membrane protein trafficking. Here we report development of a new yeast‐based screening system that enables identification of the sequence motifs that down‐regulate surface expression of membrane proteins. We show that B31, a mutant S. cerevisiae strain lacking potassium (K+) efflux transporters, is unable to grow in high K+ media when expressing the mammalian K+ uptake channel Kir2.1 due to overloaded K+. However, B31 expressing the Kir2.1 channel defective in post‐Golgi trafficking or those fused with intracellular targeting motifs were rescued in high K+ media. Thus a growth rescue of B31 cells in high K+ media represents the reduction in the surface expression of the reporter Kir2.1 protein. Using this system, we have developed a method of screening a random C‐terminal peptide library fused to Kir2.1 channel for signals which reduce the levels of Kir2.1 on the cell surface. We have conducted a pilot screening, and identified sequences that down‐regulated cell surface expression of Kir2.1. We demonstrate that the identified sequences are functionally transplantable into different protein contexts such as CD4 and CD8. Furthermore, we have characterized the molecular mechanism of transport caused by these sequences and show that the screening system can identify signal sequences that operate through multiple distinct trafficking pathways such as ER retrieval and endocytosis. Thus our system offers a unique tool to discover potentially novel sequence motifs that regulate cell surface expression of membrane proteins

Support or Funding Information

NIH R01GM099974
Type I Interferons Block Extracellular Vesicle‐Mediated Cargo Transfer
Authors: Hantak Michael P , Qing Enya , Gallagher Tom
Published: November 2023

Extracellular vesicles (EVs) transfer transmembrane and cytosolic macromolecules between cells. EVs must fuse with target cell membranes to deliver cargo, either at the plasma membrane or after endocytosis, in a manner analogous to enveloped virus entry. The factors mediating and regulating EV‐cell fusion are unknown. We hypothesized that EV‐cell and virus‐cell fusions are similarly regulated. To address this hypothesis, we developed a novel assay to monitor EV membrane fusions. In the assay, EVs and target cells are loaded with complementary split luciferase fragments, such that EV‐cell fusion is required for fragment complementation and luciferase formation, which can be easily measured. Using this assay, we discovered that the antiviral type I interferon response, through its induction of interferon – induced transmembrane protein 3 (IFITM3), potently inhibited EV‐cell fusions. IFITM3 was incorporated into EVs, which prevented the EVs from fusing with themselves, and also with target cells. These findings demonstrated that IFITM3 regulates EV‐mediated inter‐cellular protein transport. Notably, the endosomal sorting complexes required for transport (ESCRT) were required to incorporate IFITM3 into EVs, and were also required more generally for EV‐mediated inter‐cellular protein transport. These findings demonstrated that ESCRT – generated EVs are the agents of inter‐cellular protein transport. These results identify regulators of EV‐mediated cargo transfer and reveal machinery necessary to produce cargo transfer‐competent EVs. The findings may translate into more effective methodologies for controlled delivery of hydrophilic macromolecules into cells.
Drug Delivery in the Eye
Authors: Hosten Justin , Penumutchu Swathi , Lavik Erin
Published: November 2023

On average, 1.4 million Americans suffer a head related injury every year. Of these injuries many can result in eye injury, in particular retinitis pigmentosa(RP). Recent innovation has lead to the creation of biodegradable microspheres and nanoparticles. One such use of these spheres/particles is to be a carrier for various drugs and be used for delivery to the human body, where oral drugs fail. For optic nerves, the blood brain barrier, which protects the brain from various chemicals that could have major negative effects, blocks many oral drugs from reaching the area where they are located. This poses a problem from optic related injuries. The drug All‐Trans Retinoic Acid (ATRA), is a compound that has the ability to repair damaged optic nerves. This drug can be loaded and directed to the optic nerves through poly (lactic‐co‐glycolic acid) (PLGA) microspheres. PLGA is a biodegradable polymer that allow for control and targeted release for various drugs. We hypothesize that loading ATRA with the addition of a co solvent, trifluoroethanol (TFE), methanol, and acetone, will increased the amount of ATRA within the microspheres, due to the hydrophilic properties of ATRA. This increased loading of the drug will allow for sustained release over long person of time, improve patient compliance, and patient treatment.

Support or Funding Information

Funded by NIH and Glaucoma Foundation
Localization of the Platelet‐Activating Factor Receptor on the Intact Human Periodontal Ligament
Authors: Cerutis D. Roselyn , Nichols Michael G. , Khan Shakeel A. , Miyamoto Takanari
Published: November 2023

Our laboratory has reported the presence of the lysophosphatidic acid receptor subtypes (LPARs) LPA1 and LPA3 on the intact human periodontal ligament (huPDL) using an in situ technique we developed (Cerutis et al. 2016). PAF (platelet‐activating factor) is another lipid mediator that also plays key homeostatic and inflammatory roles. Like LPA, it acts at nM concentrations and is found in significantly higher concentrations in saliva and gingival crevicular fluid from patients with chronic periodontal disease (PD), as we have reported for LPA (Bathena et al. 2011). It is now established that members of the LPA, PAF and prostaglandin (EP) GPCRs can localize to the nucleus in mammals (Gobeil et al., 2003).The LPAR and PAFR GPCRs are expressed in nuclear membranes in other systems, where they share signal transduction pathways to activate inflammatory gene transcription like COX‐2 and inducible nitric oxide synthase. Therefore, we hypothesized that the PAFR would also be expressed throughout healthy and PD PDL, where they would likely interact with LPARs to regulate the inflammatory response to periodontal pathogens.

Methods

Polyclonal antibodies (Abs) against LPA3 and PAFR were covalently tagged with R‐Phycoerythrin and used to label both receptors on PDL from healthy and PD patients (IRB‐approved, consented donors), using our in situ technique.

Results

Confocal microscopy confirmed nuclear LPA3, and nuclear labeling was detected with PAFR Abs; similar but different patterns of distribution were seen for each GPCR. In conclusion, to the best of our knowledge, this is the first report of the presence of the PAFR on intact huPDL. In inflamed periodontal tissue, PAF may play a significant role, as it induces vasodilation and platelet aggregation; LPA is produced by activated platelets, so they are likely to interact at the molecular level. We reported that LPA regulates the expression of >60 PD‐associated inflammatory transcripts in human oral fibroblasts (Cerutis et al. 2015), supporting LPARs as having a regulatory role in chronic PD. This confirmed presence of PAFRs on the huPDL suggests that further investigation of the interplay of these two receptor families is needed to determine their role in both PDL homeostasis and in chronic PD.
Fix the Wrecks, RecA
Authors: Dubiel Katarzyna , Liang Adream , Sandholm Lily , Keck James L
Published: November 2023

Accurate and complete DNA replication is essential to all life. However, collisions and obstacles that lead to DNA breaks are common. There are many proteins that have the essential job of correcting these mistakes. Some do this by trimming the broken ends of the strands and reconnecting them. RecA instead assists a more accurate method of repair called homologous replication. In this process, RecA binds to the broken strand of DNA and locates the same sequence on an intact duplicate strand. The ssDNA bound RecA then catalyzes a synapsis reaction between the filament and homologous double stranded DNA (dsDNA). Other proteins add complementary bases to synthesize a second copy of the missing section of the original single stranded DNA (ssDNA). Finally, the strands are separated to form a complete DNA and restore the missing information (Cox, MM. (2007)).

Homologs of RecA are found in almost all organisms and are a critical part of genome maintenance and DNA repair. It is crucial for accurate homologous recombination which, when done incorrectly can lead to cancer and genome instability (Chen, Z., et al (2008)). When Rad51, the human homolog of RecA is dysregulated, it increases chances of cancer. Specifically, Rad51 is underexpressed in 100% of renal cell carcinomas (Liu, S., et al (2016)). Understanding the implications of cell repair regulation may allow for future cancer treatments.

The Madison West High School SMART (Students Modeling A Research Topic) Team is Modeling RecA bound to DNA to learn more about the process of homologous recombination. Specifically, how the RecA filament interacts with DNA. Current science dictates that three RecA molecules interact with each nucleotide triplet in a DNA strand (Chen, Z., et al (2008); PDB: 3CMX), which we will be investigating in more detail. DNA replication is necessary for life, and when breaks occur in the DNA, it needs to be repaired. RecA recognizes the DNA damage, and performs homologous recombination. Understanding this process may aid in the development of novel cancer therapies, allowing for more cost‐effective treatments.
Kinesin, The Workhorse of the Living Cell
Authors: Blythe Justin , Burns Morgan , Fuchs Kelsey , Uttley Zach
Published: November 2023

For a living cell to stay vital, it must transport molecules and organelles to their designated positions within the cell. Similar to motorized interstate transport systems, cells achieve their transport using motor proteins. One fascinating motor protein, kinesin, hauls its cargo on microtubule tracks at a speed of 2 μm/sec, a process fueled by ATP hydrolysis. The structure of kinesin consists of two heads (motors) each with an ATP binding pocket and a microtubule‐binding domain that spans the length of its motor subunit. The two motors are attached to the neck domain, which consists of a neck linker and a neck coil. As it begins its journey, kinesin docks one motor head at a time to a microtubule unit. ATP binds to the docked head (the leading head) and undergoes hydrolysis, causing a power stroke. Detailed studies showed that this sequence of events leads to a kinesin's hand‐over‐hand moving pattern. Other structural elements in kinesin include a coiled‐coil stalk with hinge regions that allow kinesin to fold on itself, an energy saving process adopted when the protein is idle, a tail domain and a light chain subunit that contain cargo binding sites. Mutated kinesin has been linked to multiple diseases including, Alzheimer's, neurofibromatosis, Huntington's disease and cancer. The Pittsburg High School SMART TEAM used 3D technology to design a model of kinesin's motor and neck domains to better understand their structure‐function link. This knowledge may shed light on how mutations in these regions can lead to diseases linked to kinesin's role in cellular transport.
Neuroglobin Gone Wild
Authors: Bresniker Catherine Lynn , Martinez Sandra Ann , Anderson Poetry , Bartolomei Barrett , Bingham Tanner , Bogdon Phoebe , Dulay Jaclyn , Mergens Josh , Shipley Ashley Willow
Published: November 2023

Neuroglobin (Ngb) has an important role in maintaining homeostasis through processes such as the detoxification of nitrogen, the storage/transport of oxygen, and the protection of neurons at risk of unwanted apoptosis. One of the fascinating differences between neuroglobin and all the other globins is that it has a hexa‐coordinated heme group. In neuroglobin, all six coordination positions are bound, creating a slightly different mechanism for when oxygen has to bind. Protein concentration of Ngb is tissue dependent. In retinal cells, where there is a high demand for oxygen, Ngb concentrations can be up to 100 times greater than in neurons. Levels of expression determine the role Ngb is going to have, such as a regulator of metabolic functions in the hypothalamus or involvement in the signaling pathways in neuronal development. Oxidative stress may lead to disruption of the mitochondria causing the release of cytochrome c, the beginning of apoptosis. In addition to its role in oxygen delivery, Ngb is a neuroprotective protein released by astrocytes, which reside between the neurons, that detect changes in the environment. It is believed that Ngb may slow and possibly stop cell death. Ferrous Ngb performs a redox reaction with ferric cytochrome c while binding with amino acids glu 60,87 on Ngb and the cytochrome c amino acids lys 72,25. Ngb binds to the same site on cytochrome c where the cytochrome would bind with other apoptotic molecules to form an apoptosome that would activate executioner caspases. Research has been done to link intrinsic characteristics with conditions presented by the cell in stress, such as acidosis or hypoxia have led to identifying a probably role of Ngb as a neural protector. Cell death is associated with mitochondrial dysfunction and is common in neurological disorders. Degenerative brain disorders ‐ amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's disease, among others affect more than 45 million people worldwide. An understanding of these actions may provide a basis for new research and drugs to inhibit excessive neuronal cell death. The El Capitan High School CBM MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of neuroglobin.
Histidine Kinase CheA: You Spin Me ′Round
Authors: Chou Chris , Martyr Richard , Barila Diana , Chaney Michael , Drewes Jake , Hak Iain , Harroun Haleakala , Kamenides Theodore , Lippard Maya , Martyr Cara , Merrill Jack , Mugunga Tol , Neuhard Zoe , Real Sophia , Swope Danielle , Wilkinson Tyler , Falke Joseph J
Published: November 2023

Annually, 600 cases of necrotizing fasciitis, caused by wound seeking bacteria, are documented in the United States. One in four patients who contract this infection die. Researchers seek a method to block bacterial chemosensing, to prevent wound seeking and infection. Histidine kinase CheA (CheA) is central to chemosensing and chemotaxis. CheA is coupled to a transmembrane methyl‐accepting chemoreceptor, which detects attractants and repellants. CheA is a homodimer and each subunit contains five domains: substrate (P1), CheY binding (P2), dimerization (P3), kinase (P4), and regulation (P5). P1 contains the histidine residue that is the site of autophosphorylation. Attractant or repellent binding to the chemoreceptor inhibits or activates autophosphorylation of the histidine, respectively. Following autophosphorylation the phosphate is transferred, yielding phospho‐CheY. Low phospho‐CheY maintains the current swimming direction, while high phospho‐CheY causes the bacteria to stop and reorient. Hydrophobic dimerization interfaces in CheA create an energy barrier to dissociation of the subunits, ensuring a stable dimer for proper regulatory and kinase functions. The dimerization domain is linked to the kinase domain by a highly conserved flexible link. Due to their 3D orientation, the kinase and regulatory domains of the monomer do not interact, rather they interact with the complementary monomer. The kinase domain contains a deep cleft that is the ATP binding site. The Longmont High School SMART (Students Modeling a Research Topic) Team modeled histidine kinase CheA using 3D printing technology to investigate structure‐function relationships. By understanding the way that bacteria move, we can more effectively fight pathogenic bacteria.
Microvascular Corrosion Casting of the Ovary in Non‐pregnant and Pregnant Does
Authors: Hafez Shireen , Caceci Thomas
Published: November 2023

The ovary is one of the most dynamic examples of cyclic angiogenesis. Continuous changes in its structures are accompanied by equally significant modifications in its microvasculature. Morphological changes on the caprine ovarian surface in non‐pregnant and pregnant does at different stages of pregnancy were examined using scanning electron microscopy. No significant differences were observed on the surface of ovaries from non‐pregnant or pregnant does; nor between stages of pregnancy, and/or between the right and left ovaries. In all ovaries, the proper ovarian branch of the ovarian artery ran toward the hilus of the ovary, where it penetrated the ovary. Subdivisions of the proper ovarian branch of the ovarian artery coiled around the proper ovarian tributary of the ovarian vein from the level of their origin to within the substance of the ovary. The arterial and venous branches of large vessels penetrating the ovary showed distinct microvascular patterns characteristic of the follicles and corpora lutea. In the early preantral follicle, these appeared as a spherical, basket‐like network of capillaries. At later stages, follicles possessed a highly complex wreath of capillaries, which tend to be arranged into layers. Late well‐developed antral follicles acquired a well‐defined multilayered basketlike network, the inner wall of which was characterized by development of capillary sinusoids. Capillary sprouts and evidence of intussusceptive capillary growth due to enhanced angiogenesis were seen in inner wall of the late developed follicles. The outer wall was formed from larger size vessels. Ovulatory follicles were characterized by the presence of an avascular apical area at the location of the stigma. Leakage of casting medium was observed around the border of the stigma due to increased vascular permeability in this region. Early formation of the corpus luteum was characterized by inward growth of vessels from the apical part of the ovulating follicle, followed by extensive new formation of capillaries, which filled up the corpus luteum and closed the apical area. Corpora lutea could be recognized by their complex vasculature. The vascular bed of the corpus luteum consisted of a dense, conglomerated capillary plexus, and large vessels distributed on its outer surface. Large flat veins could be observed on the outer surface of the corpus luteum. Coiling of vessels might represent a local channel required for product transport needed to allow the transfer of substances produced in the ovary from the ovarian vein to the ovarian artery. In other words, physiological processes occurring in the ovary can be controlled by the ovary itself; this should be investigated further.