bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimization of Reconstituted High Density Lipoprotein (rHDL) nanoparticles (NPs) for Short‐Interfering RNA (siRNA) Delivery
Authors: Mooberry Linda , Sabnis Nirupama , Lacko Andras
Published: November 2023

Objective

The objective of these studies was to characterize siRNA transporting rHDL NPs and to evaluate their suitability for translation toward clinical applications. Previous studies have shown that rHDL NPs are capable of promoting the delivery and retention of short‐interfering RNA (siRNA) by cancer cells and tumors (Shahzad et al., 2011).

Methods

Cholate dialysis and a facilitated mixing process through a microfluidics instrument (Precision Nanosystems NanoAssemblr) were used to prepare the siRNA containing rHDL NPs. Diameters of the NPs were estimated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Stability of the rHDL/siRNA formulations were examined before and after lyophilization. Western blotting was used to assess the expression of the transcription factor STAT‐3 in the SKOV‐3 ovarian cancer cell line following siRNA treatment.

Results

A series of neutralizers were evaluated to suppress the negative charges of siRNA. Oligolysine (MW=500–2000) was found to be most effective with an incorporation efficiency of 88.8 ± 10.1% by cholate dialysis and 88.6 ± 2.0% by microfluidics. The size of the complexes estimated by DLS was found to be 59.6 ± 6.7 nm for nanoparticles prepared by cholate dialysis and 39.6 ± 6.3 nm for NPs prepared by microfluidics. However, TEM images revealed uniform, spherical NPs with a diameter of 23.9 nm ± 2.9. Examination of diameter after lyophilization with a cryoprotective agent showed only a 6.8 ± 2.5 % increase in the size of the nanoparticle formulation with 90.5 ± 3.2% retention of the nucleic acid. The effective knockdown of the expression of the STAT‐3 target was achieved in an ovarian cancer (SKOV‐3) cell line.

Conclusions

This study is the first to report the preparation of NPs with a protein component, using the NanoAssemblr instrument. This rHDL/siRNA formulation has a small diameter, good efficiency at incorporating siRNA, and is biologically active. The siRNA containing rHDL NPs were found to be stable after lyophilization, with little change in diameter and siRNA content.

Support or Funding Information

The authors' research is being supported by Cancer Prevention and Research Institute of Texas, Wheels for Wellness, Fort Worth TX and the Rutledge Foundation.
Using CRISPR technology to edit the F508 deletion of the Cystic Fibrosis Transmembrane Regulator gene
Authors: Heavenor Kelsey , Roecklein‐Canfield Jennifer
Published: November 2023

The genetic disease Cystic Fibrosis is the result of a mutation in the Cystic Fibrosis Transmembrane (CFTR) protein. The CFTR protein is an important chloride ion channel in the cell membrane. The mutant protein results in a terminal disease characterized by breathing problems, digestive issues, and recurring lung infections. The ΔF508 mutation, results in the deletion of a phenylalanine at position 508 in the protein and the failure of the protein to fold into its functional conformation. This mis‐folded protein is targeted for degradation by the endoplasmic reticulum quality control system. The mutant CFTR protein is never transported to the apical membrane to carry out its function leading to severe consequences in the lungs and digestive system. Therapies currently available for Cystic Fibrosis are only able to alleviate symptoms to an extent; however, new strides in genetic technology have allowed researchers to now approach treating the disease on the genetic front, an imperative step to understanding and effectively treating Cystic Fibrosis. The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins) System is a new system derived from the prokaryotic immune system that can be used to precisely edit DNA. The CRISPR system can be designed to knockout or insert new genes into the genome, making it an ideal tool for manipulating the CFTR gene to learn more about the gene and the role of mutations in the progression of disease. We attempted to apply CRISPR technology in a gene knockout experiment and homology directed repair (HDR) of the mutant CFTR gene, using two MDCK cell lines, one expressing wt CFTR and the other expressing ΔF508 mutant. Confirmation of cellular expression of the proteins was confirmed by visualization of the Green Fluorescent Protein (GFP) fused to the N‐terminus. We used Cl‐ ion output to estimate how much of the protein is reaching the cell membrane as a measure of protein function. Regain of function in ΔF508 mutant will determine if the gene editing was a success. The data will determine which CRISPR design is most effective at repairing the gene, for example determining what is the ideal length for homologous arms in HDR repair that yields best results. Perfecting the art of CRISPR design in the laboratory is essential to the eventual creation of a functional CRISPR design for use in humans. Many diseases cannot be fully controlled through drugs; the editing of the genome offers an extraordinary pathway for eliminating disease.

Support or Funding Information

Simmons SURE Grant
Molecular Genotyping of Transposable Element Insertions in a Population of Uniform Mu Events
Authors: Martinez Gerardo Antonio Arroyo , Springer Nathan
Published: November 2023

Uniform Mu is a maize strain that was developed as a model for genetic studies. Mutator transposable elements (TE) have been activated within an inbred W22 background and the movement of these transposons results in novel mutations. Researchers can identify Uniform Mu events that contain transposon insertions within a gene of interest. TE insertion events within the organism's genome often result in loss‐of‐function mutant alleles. This study aimed to genotype the presence of a TE insertion in several genes predicted to be involved in the regulation of gene expression or chromatin structure using Uniform Mu crops. Molecular genotyping was performed by using Polymerase Chain Reaction (PCR) and gel electrophoresis. We determined that 43.89% of the population contained a homozygous wild‐type genotype. On the other hand, 7.24% of the individual scored had a homozygous mutant genotype, while 11.08% of the rows assessed presented heterozygous alleles. In future experiments, we expect to analyze these genes further and identify their possible role in causing abnormalities in the chromosomes. As an integral part of people's diet around the world, these results will lead to a better understanding of the agriculture of maize crops.

Support or Funding Information

I would like to thank the support of Springer Lab at the University of Minnesota for all the help and guidance this summer. Also, I would like to acknowledge the RISE program in the University of Puerto Rico at Ponce for their funding. NIH‐NIGMS #2R25GM096955
Characterization of a Plasmid Isolated from Enterococcus faecalis Found in the Fecal Material of a Blue Whale
Authors: McLaughlin Richard , Kopanic Robert
Published: November 2023

Enterococcus faecalis is widely distributed in nature being found in soil, water, plants, and foods. It is also found in the gastrointestinal tract of humans and animals including Blue Whales. Despite its well‐reported existence in animal species, very few plasmids isolated from E. faecalis have been described in the literature. Therefore, the purpose of this study was to more completely describe the genomics of E. faecalis. The complete 52325 bp sequence of the plasmid pGTC2 was determined using Next‐Generation sequencing technology. Harbored by a strain of E. faecalis present in the fecal material of a Blue Whale, this plasmid does not contain any antibiotic or heavy metal resistance genes. Instead multiple mobile element proteins, relaxase and transposon sequences are found throughout. Since these genes are present it may indicate that horizontal gene transfer may occur frequently in pGTC2.

Support or Funding Information

This project was supported by funding provided by Gateway Technical College and by the Gateway Foundation.
Unique Transposon Genome Rearrangements Identified in Elizabethkingia anophelis Outbreak Strains in the Midwest US
Authors: Flores Rita , Torres Nathanial , Matts Jessica , Gustafson John , Canaan Patricia , Hoyt Peter
Published: November 2023

The genus Elizabethkingia is comprised of aerobic, non‐fermenting, non‐motile and non‐spore‐forming gram‐negative rods that are widely distributed in nature. Elizabethkingia is rarely reported to cause infections in humans with only a few isolated incidents reported each year typically confined to healthcare facilities. However, a recent outbreak of an Elizabethkingia strain (sp. anophelis) occurred in Wisconsin with additional single infections in Michigan and Illinois, resulted in an unusually high morbidity (20 deaths/65 cases). Patients diagnosed with this strain of Elizabethkingia anopheles lived in unassociated areas and were present in different health facilities across the state. As of November 9, 2016, the source of this most recent outbreak is yet to be determined. Elizabethkingia is known to have multiple genes coding for antibiotic resistance, but these do not seem to have contributed to the unusual outbreak. Patients affected are exclusively immune‐suppressed with most infections occurring in the blood, leading to sepsis. One hypothesis for this change in phenotype could be due to mobile genetic elements such as transposons, integrases, plasmids and phages all of which function to facilitate DNA rearrangements. We have identified a homologous double transposon region conserved in only a few strains of Elizabethkingia suggesting a recent conjugative event. This conserved homologous region (~62,871bp) is bordered by mobile genetic elements, and found exclusively within E. anopheles NUHP‐1, PW2809, CSID_3015183678, CSID_3000521207, CSID_3015183681, CSID_3015183684, Genomospecies 2, Genomospecies 3 and E. miricola ATCC. The shortest version of this conserved region is in E. anopheles PW2809 with a slightly larger conserved region present in both Genomospecies 2 and 3. However, the largest version of this double transposon was found in E. anophelis NUHP‐1, miricola ATCC and the four anophelis CDC outbreak strains. The increased homology of this transposon in a select number of strains seems to suggest a relatively recent evolutionary event. However, the location of this highly conserved transposon (termed “Tnpx2”) changes specifically within the CDC outbreak strains the result of which causes a disruption in the gene sequence used for coding of an A/G‐specific adenine glycosylase. This potential disruption of glycolytic function along with the identification of toxin‐antitoxin complexes unique to the CDC outbreak strains may have caused a phenotypical change within the bacteria that increased its survival. We propose that the genome rearrangement, found only in the CDC outbreak strains isolated from Wisconsin, could have influenced epidemiological changes and the subsequent severity of the most recent outbreak of Elizabethkingia anophelis.
Liver and Serum miRNAs in Toxic Liver Fibrosis
Authors: Permenter Matthew Grant , Ippolito Danielle L , Trabosh Valerie A
Published: November 2023

Toxic chemical exposure is a risk factor for toxic liver injury in the military, chemical plant workers, and the general public. Early indicators of disease would facilitate medical and strategic decisions involving treatment, protective equipment, and/or relocation. Here, we map liver miRNA to their predicted mRNA targets in a rodent model of hepatotoxicity. We report a panel of serum miRNA that may serve as early, blood‐based indicators of toxic liver injury or fibrosis.

Rats were orally administered vehicle control or the hepatotoxicant 4, 4′‐methylenedianiline (MDA; 22, 60 or 162 mg/kg) for 1 or 5 consecutive days. Serum and liver tissue were collected at necropsy, 24 hours after the last exposure. Liver sections were stained with hematoxylin and eosin for histology. Liver tissue and serum miRNA expression was determined by next‐generation sequencing for selected doses of MDA. mRNA gene expression in the liver was measured by microarray. Known and novel miRNAs were mapped to the rat genome using miRDeep2 and differential expression was determined by DESeq (FDR<0.05). mRNA changing by at least 1.5 fold were used for the target analysis using Ingenuity Pathway Analysis (IPA) software. Pathway enrichment analysis was also conducted in IPA.

MDA poisoning caused moderate liver fibrosis and bile duct hyperplasia after 5 consecutive doses, as shown by histological analysis. One day after the final dosing, we identified 0, 7, and 38 differentially expressed liver miRNA at 22, 60, and 162 mg/kg, respectively. After 5 doses, 0, 126, and 127 liver miRNA were differentially expressed at 22, 60, and 162 mg/kg, respectively. Sixteen miRNAs were modulated in the serum of the rats exposed to 162 mg/kg of MDA. We also identified novel, previously undescribed differentially expressed miRNAs which may play a role in liver injury progression. We predicted mRNA targets of each differentially expressed miRNA. Pathway enrichment analysis of the predicted targets identified pathways involved in the liver fibrosis adverse outcome pathway, including inflammation, collagen accumulation, and stellate cell activation. Many of the modulated mRNAs targeted by these miRNAs have been previously associated with liver fibrosis.

In conclusion, we identified liver miRNAs that may target and regulate gene expression contributing to liver fibrosis. The miRNAs in the serum may serve as prognostic indicators of disease progression.

Support or Funding Information

Disclaimer: Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.
High Resolution Melting Analysis for the Study of Single Nucleotide Polymorphisms
Authors: Beuschel Grant , Diez Christina
Published: November 2023

Pharmacogenetics is the study of how inherited genetic variations correspond to an individual's specific reaction to drugs and their metabolism. An ideal pharmacogenetic test would provide information about an individual's genotype which would then inform the the doctor and patient about drug efficacy, provide specific dosage recommendations, and warn of the risk of adverse side effects.

The purpose of this project is to determine how to use high resolution melting analysis to characterize the different alleles caused by single nucleotide polymorphisms (SNPs). The SNPs being tested are in the VKORC1 and CYP2D6 genes which code for enzymes involved in the metabolism of warfarin and codeine, respectively. In addition, to refine our DNA extraction and amplification method and to provide support for an existing introductory biology laboratory exercise, we are also working on the SNP for PTC (Phenylthiocarbamide) tasting as well. The PTC SNP is currently being genotyped by students using restriction enzyme digestion. However, results of the restriction enzyme digest have conflicted with direct observations of PTC tasting ability. Therefore, we are interested in developing a reliable genotyping method using high resolution melting analysis. We were able to extract DNA from cheek cells and amplify the SNP alleles. We are presently developing a protocol for high resolution melting analysis of SNPs using the Roche LightCycler 480. The high resolution melting analysis should be sensitive enough to distinguish between the different SNPs. We will verify our SNPs results with DNA sequence analysis. Once we have developed a reliable assay, we will explore the possibility of creating an introductory biology laboratory module to expose students to pharmacogenetics.

The overall goal is to optimize a protocol to make such testing more effective and frequent. This way when a patient is in need of medication a simple analysis of their genes can be made to determine the correct type and dosage of drug that will be most beneficial to them. Perfecting a pharmacogenetic testing using high resolution melt curve analysis would extinguish the current and less effective method of trial and error.

Support or Funding Information

Lawrence Technological University, Department of Natural Sciences
Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy But Age‐dependent Exhaustion of Satellite Cells
Authors: Yue Feng , Bi Pengpeng , Wang Chao , Li Jie , Liu Xiaoqi , Kuang Shihuan
Published: November 2023

Skeletal muscle stem cells (satellite cells, SCs) are normally maintained in a quiescent (G0) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting GAlert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long‐term benefits to SCs is unknown. Here we report that embryonic myogenic progenitors lacking Pten exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten‐null SCs are predominantly in the GAlert state even in the absence of injury. The GAlert SCs are deficient in self‐renewal and subjected to accelerated depletion during regeneration and aging, and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G0‐entry of SCs, and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.

Support or Funding Information

This work was supported by a grant from the US National Institutes of Health (R01AR060652 to S.K.) and Purdue incentive grant from Purdue University Office of Vice President for Research (OVPR) to S.K.
Modulation of MEK/ERK and mTOR pathways by Na+‐K+‐ATPase and AMPK inhibitors in ovarian cancer cells
Authors: Clark Matthew P , Madigan Audrey , Gallant Benjamin Paul , Zhang Wei , Lu Hongguang , Wan Yinsheng
Published: November 2023

In addition to ion transport activities, Na+‐K+‐ATPase acts as a cellular signal transduction molecule. Its inhibitors, such as Ouabain, induce the EGFR/ERK pathway as well as the PI3K/AKT pathway. AMPK is involved in cellular metabolism and inhibiting the mTOR pathway. Activation of AMPK leads to cell growth inhibition and apoptosis in cancer cells. Reports have demonstrated that activation of the AMPK stimulates Na+‐K+‐ATPase. In this study, we investigated the effects of both the Na+‐K+‐ATPase inhibitor Ouabain and the AMPK inhibitor Compound C on ovarian cancer cells. MTT assay results showed that both Ouabain and Compound C alone decrease cell viability. The combination of both yields a synergistic effect. Western blot analysis results indicated that Ouabain induces AKT/mTOR and ERK activation in a time and dose dependent manner. Interestingly, while Compound C inhibits Ouabain‐induced mTOR activation as measured by S6 phosphorylation in a dose dependent manner, Compound C enhances Ouabain‐induced ERK activation. Compound C alone induces ERK activation. Further studies showed that the MEK/ERK inhibitor U0126, but not the mTOR inhibitor Rapamycin, inhibits Ouabain‐induced S6 phosphorylation. ER redox assay data showed that Compound C treatment alters the redox level. Mito‐Tracker test data showed that both Ouabain and compound C increase mitochondria activities. Taken together, our data indicates that both Na+‐K+‐ATPase and AMPK are important for MEK/ERK and mTOR pathways in ovarian cancer cells. Modulation by inhibitors of both may reveal molecular targets for cancer treatments.

Support or Funding Information

RI‐INBRE
Activation of the E3 ligase Cbl by Neutrophil Cathepsin G Impairs CXC chemokine receptor 4 Signaling in Cardiomyocyte Degeneration.
Authors: Shukla Sanket , Sikder Kunal , Sarkar Amrita , Liu Weijing , Rafiq Khadija
Published: November 2023

Objective

Inflammatory cells and their proteases contribute to tissue repair at the site of inflammation. Although beneficial in the early stage, excessive inflammation leads to cell death and tissue damage. Increasing evidence demonstrates the role of inflammatory cells and their proteases in cardiac remodeling during the progression to heart failure; however the mechanisms by which inflammation contribute to myocyte death and subsequent alteration in the geometry and mechanical properties of the heart are still largely unknown. Therefore, we hypothesized that neutrophil‐derived protease Cat.G regulates CXC chemokine receptor (CXCR) 4 protein stability and turnover in myocytes through activation of Casitas b‐lineage lymphoma (c‐Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity.

Methods and Results

We have previously shown that neutrophil serine protease Cat.G promotes cardiomyocyte death through c‐Cbl activation and interaction with tyrosine receptors that result in their ubiquitination and degradation. We next assessed whether Cbl activation was involved in CXCR4 signaling downregulation and myocyte apoptosis secondary to exposure of Cat.G. Acute stimulation with Cat.G (5 min to 30 min) induced CXCR4 phosphorylation along with its interaction with Cbl. However, chronic stimulation with Cat.G (1–4hrs) increased both CXCR4 ubiquitination and degradation. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced CXCR4 degradation induced by Cat.G. In contrast, adenoviral expression of wild type Cbl enhanced CXCR4 ubiquitination and degradation in response to Cat.G. Restoration of CXCR4 signaling, by adenoviral expression of CXCR4, attenuated CXCR4 downstream signaling downregulation, myofibril degeneration and myocyte apoptosis induced by Cat.G.

Conclusion

These data support a model in which neutrophil derived serine proteases promote c‐Cbl interaction with CXCR4 protein, resulting in enhanced c‐Cbl‐mediated CXCR4 protein degradation, myofibril degradation and subsequent down‐regulation of myocyte survival signaling.
CARMA2sh and Its Psoriasis‐Linked Variants Regulate Inflammatory Pathways in Human Keratinocytes
Authors: Scudiero Ivan , Mazzone Pellegrino , Telesio Gianluca , Pizzulo Maddalena , Vito Pasquale
Published: November 2023

OBJECTIVE

The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain‐containing adapter molecule BCL10, and MALT1 represent a central hub in regulating activation of the pleiotropic transcription factors NF‐κB. Recently, dominant germline mutations in CARMA2sh have been shown to cause with high penetrancy psoriasis and pityriasis rubra pilaris, two related skin inflammatory disorders. Aim of our work was to dissect the molecular mechanisms by which CARMA2sh and its variants associated with psoriasis control NF‐κB activity in the skin.

METHODS

Molecular partners of CARMA2sh were searched by a yeast two‐hybrid screening and tested by co‐immunoprecipitation in HEK293T cells. Immunoblot analysis, in vitro kinase and phosphatase assays were used to monitor the phosphorylation status of CARMA2sh. Deletion mutants of CARMA2sh served to map the phosporylation region and its functional significance. Murine strains expressing psoriasis‐linked mutants of CARMA2sh in their keratinocytes were generated via homologous recombination in the Rosa26 locus. NF‐κB activation signaling and the expression of inflammatory genes were investigated by biochemical and genetic assays in human and murine keratinocytes. A BCL10 inhibitory peptide was delivered by liposome in NHEK cells exposed to heat‐killed bacteria or fungi and NF‐κB target genes expression levels were assessed by Real Time RT‐PCR.

RESULTS

1) We identified a serine/threonine kinase, termed CIK (CARMA2sh Inhibitory Kinase), which binds to and phosphorylates CARMA2sh. Phosphorylation of CARMA2sh is required to promote lysosomal degradation of BCL10 and to turn off CARMA2sh‐mediated NF‐κB signaling. 2) We demonstrate that CARMA2sh mutants associated with psoriasis escape CIK inhibition. 3) We show that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway connecting Pathogen Associated Molecular Patterns recognition to NF‐κB activation. 4) A peptide blocking CARD‐mediated BCL10 interactions reduces the capacity of psoriasis‐linked CARMA2sh mutants to activate NF‐κB.

CONCLUSIONS

Our work elucidates a basic signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.

Support or Funding Information

Qatar National Research Fund NPRP 7‐466‐3‐119
Disruption of the cereblon gene prevents bleomycin‐induced pulmonary fibrosis in mice
Authors: Lee Kyoung‐Hee , Koo Yoon‐Jung , Lee Chang‐Hoon , Yoo Chul‐Gyu
Published: November 2023

Pulmonary fibrosis is a progressive and lethal lung disease characterized by proliferation and differentiation of lung fibroblast and deposition of extracellular matrixes. Currently there are no proven effective therapeutic and preventive strategies. In a bleomycin (BLM)‐induced lung fibrosis model, cereblon (CRBN) mRNA and protein decreased. However, the role of CRBN in lung fibrosis is not known. In this study, we elucidated the role of CRBN protein on bleomycin‐induced pulmonary fibrosis in mice and TGFβ‐induced differentiation of lung fibroblast. Bleomycin was applied via intratracheal instillation (5units/kg) in wild type (WT) and Crbn‐deficient mice. Crbn‐deficient mice treated with BLM showed lower level of lung fibrosis on histologic analysis and decreased expressions of collagen, collagen type 1 and fibronectin mRNA in lung tissue compared with WT mice. To assess the role of CRBN in lung fibroblast, cells were transiently transfected with CRBN overexpressing vector and then stimulated with TGFβ. CRBN overexpression increased cell survival and enhanced TGFβ‐induced activation of SMAD2/3. Subsequently, the expression level of α‐smooth muscle actin, vascular endothelial growth factor, and collagens increased. Recently, CRBN was known to interact with adenosine monophosphate‐activated protein kinase (AMPK) and negatively regulate AMPK activity. AMPK activation was reported to suppress the inflammation and fibrotic process of BLM‐induced pulmonary fibrosis. We showed that CRBN co‐localized with AMPK in lung fibroblast by immunofluorescence staining. CRBN overexpression inactivated AMPK. When cells were treated with AMPK activator, metformin, CRBN‐induced activation of SMAD3 was significantly suppressed, which suggest that increased TGFβ‐induced activation of SMAD by CRBN overexpression was associated with inactivation of AMPK. Taken together, these data suggest that CRBN is one of pro‐fibrotic regulators and may be a potential target for lung fibrosis.
Chronic Low‐grade Inflammation in Obesity and Diabetes associated Colorectal Cancer Development.
Authors: Kumar Narendra , Mishra Jayshree , Kumar Premranjan
Published: November 2023

Epidemiological studied identified that colorectal cancer (CRC) is a worldwide health problem, being the third most commonly identified cancer in males and the second in females. Increasing CRC incidence trends are associated with life‐style and calorically excessive high‐fat diet and obesity. Recent, studied have evidenced an important correlation between obesity and diabetes‐associated inflammations in colorectal cancer development. The regulatory pathway by which metabolic disorders induce inflammation in obesity leading to development and progression of CRC is remain largely unknown. Here, we demonstrate the intestinal specific chronic low grade inflammation is associated with obesity and diabetes, which impact CRC development. Our data shows that Jak3 facilitates prevention of CLGI through multiple mechanism that involves down‐regulation of the receptors of innate immunity, the toll like receptors (TLR), promotion of regulatory T‐cell expansion and suppression of Th17 effector functions in both in human and mouse model of obesity. Jak3 expression also suppresses TLR‐mediated activation of NF‐kB through activating PI‐3K‐AKT axis.

Support or Funding Information

Health‐SBIR (GM109528)
The Effect of Proline Rich Tyrosine Kinase 2 Activity on the Na+‐H+ Exchanger Isoform 1 Regulation of Cell Proliferation and Migration
Authors: Bagnell Kyle P , Provost Joseph J , Wallert Mark A.
Published: November 2023

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain where various kinase and proteins bind, altering transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in driving cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602/S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon activation, while other studies have shown PyK2 to enhance NHE1 activity. In PSN cells stimulated with LPA, it has been shown that inhibition of PyK2 increases the LPA induced increase in pHi. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Our results suggest that serum stimulation enhances cell proliferation and PF43I396 inhibition of PyK2 in serum stimulated cells decreases proliferation rate. To further evaluate these responses we will evaluate PSN and PyK2 mutant cells for proliferation and migration stimulated by serum and LPA.
Melatonin modifies peripheral blood cell oscillators in humans
Authors: Kostovski Emil , Frigato Elena , Dahm Anders , Skretting Grethe , Mowinkel Marie‐Christine , Sandset Per Morten , Iversen Per Ole , Bertolucci Cristiano
Published: November 2023

Objective

The blood concentrations of melatonin are highly associated with daylight patterns, with peak concentrations late at night. In healthy adults, the average daytime and peak night‐time values range from 10 to 200 pg/ml, however in tetraplegic persons the melatonin levels is blunted, ranging from 2 to15 pg/ml. Mounting evidence suggests that biological clocks, exist outside the central circadian pacemaker. We here explored how melatonin may influence oscillators in peripheral blood mononuclear cells (PBMCs) in tetraplegic and control persons.

Study design and setting

A double‐blind, randomized, placebo‐controlled cross‐over study, was conducted at Sunnaas Hospital, Nesoddtangen, Norway.

Methods

Six subjects with long‐standing complete tetraplegia received 2 mg of melatonin or placebo 4 days before sampling. We also included six able‐bodied men sleeping or being kept awake during the night. Plasma samples were collected from 4 to 10 times during 24‐hours. Expression of the period circadian clock‐1 (PER1) and −2 (PER2) genes, aryl hydrocarbon receptor nuclear translocator‐like protein 1 gene (BMAL1) and the nuclear receptor subfamily 1, group D, member 1 (REV‐ERB) gene was quantified in PBMCs using quantitative RT‐PCR (qRT‐PCR). The data were analysed using linear mixed models.

Results

Melatonin substitution increased night time melatonin plasma levels from 10 to 500 times in tetraplegic persons. The 24‐h profiles of all clock genes tended to differ (P < 0.09) or differed (P < 0.05) between tetraplegic patients receiving melatonin and tetraplegic patients receiving placebo, shifting and normalizing the mean maximum from a delayed peak (0400 h) in the tetraplegic patients receiving placebo towards the time of melatonin substitution (2200 h) in the tetraplegic persons receiving melatonin. There were no differences in the 24 h profile‐ or level‐expressions in able‐bodied staying awake compared to those sleeping or in between able‐bodied (all groups) compared to tetraplegic persons (all groups).

Conclusions

We here show differences in circadian variation of several clock genes related to melatonin concentration fluctuations in tetraplegic persons, suggesting that melatonin play a role in regulation of peripheral oscillators in humans.

Support or Funding Information

Sponsorship: Financial support was provided from the Throne Holst Foundation.
The Phosphorylation of the Na+‐H+ Exchanger Isoform I (NHE1) and Its Role in the Regulation of Cell Proliferation and Migration
Authors: Smith Tayler M , Provost Joseph J , Wallert Mark A.
Published: November 2023

The hallmarks of cancer are a collection of phenotypic changes that define cancer development and progression. Two of these hallmarks, sustaining proliferative growth and the activation of invasion and metastasis are regulated in part through the phosphorylation of the Na+‐H+ Exchanger Isoform 1 (NHE1) in response to changes in the tumor microenvironment. NHE1 is a transmembrane protein that exchanges one extracellular Na+ for one intracellular H+. The rate of transport activity can be altered through the phosphorylation of the cytoplasmic regulatory domain of the protein. Here we evaluate the role of NHE1 bring phosphorylated by five distinct protein kinases in seven defined locations. These kinases and the locations they phosphorylate are: 1) Pyk2 at both S602 and S605, 2) AKT at S648, 3) ROCK at T653, 4) RSK at S703, and 5) ERK at both S770 and S771. To evaluate the role of these phosphorylation sites in the regulation of cell proliferation and migration, we have created a series of Chinese hamster lung fibroblast cell lines expressing human NHE1. The PSN cells line expresses normal human NHE1 while the PSN‐7A cell line expresses human NHE1 with all seven of phosphorylation sites mutated to an alanine, thus removing that ability for NHE1 to be phosphorylated at these locations. Additionally, we have created PSN‐T653A which have the Rock phosphorylation site mutated to an alanine as well as a PSN‐7A‐T653 cell line which has the T653 site added back into the PSN‐7A mutants. We will present data on the production of PSN‐7A and PSN‐7A‐T653 cell lines and evaluate the impact of these mutations on cell proliferation and migrating in an effort to evaluate the role of Rock phosphorylation of NHE1 in these cellular functions.
Structure of the two‐component response regulator RcsB‐DNA complex
Authors: Filippova Ekaterina V. , Zemaitaitis Bozena , Wolfe Alan J. , Anderson Wayne F.
Published: November 2023

The Rcs phosphorelay is a complex two‐component system in enteric pathogens that achieves transmission of molecular signals through a phosphorylation cascade between two hybrid sensor kinases RcsC‐RcsD and the response regulator RcsB. The latter can form either homodimers or heterodimers with other transcriptional factors. These heterodimers can be phosphorylation‐dependent or –independent. This complexity allows RcsB to regulate the transcription of a diverse array of genes essential for pathogen survival and host invasion. Despite increasing appreciation of the importance of RcsB in pathogenicity, the molecular mechanisms by which RcsB forms homo/heterodimers to facilitate DNA binding in response to different environmental stresses are not well understood. Here, we present the first X‐ray crystal structure of the response regulator RcsB from Escherichia coli in complex with DNA determined at a resolution of 3.4 Å. The structure reveals that non‐phosphorylated RcsB forms an unusual asymmetric dimer in the DNA‐bound state. This asymmetry likely provides conformational flexibility that allows RcsB to interact with a large number of genes and to form heterodimers with other transcriptional regulators. Combining our structural data with electrophoretic mobility shift assays, surface plasmon resonance, and size exclusion chromatography with multi‐angle light scattering analysis provides important details about the molecular mechanism by which the universal two‐component response regulator RcsB recognizes DNA.

Support or Funding Information

CSGID is funded by the National Institute of Allergy and Infectious Diseases of NIH under Contracts No. HHSN272200700058C and HHSN272201200026C
Novel Genetic Variants in the Oxytocin Receptor Predict Oxytocin Non‐Responsiveness in Pregnant Women
Authors: Lee Grace Y. , Raghuraman Nandini , Reinl Erin L. , England Sarah K.
Published: November 2023

Background

Oxytocin is used on 43% of all women in the US to induce or augment labor. The maximum oxytocin dose varies between women from ≤2mU/min to ≥20mU/min. Those requiring ≥20mU/min of oxytocin are defined as “non‐responsive.” The biological cause underlying these individual differences in oxytocin response is unknown, however, maternal genetics is suspected to be a factor. Our laboratory has identified three gene variants (H173R, R150L, and R151C) in the oxytocin receptor (OXTR) gene in women who are non‐responsive to oxytocin.

Hypothesis

We hypothesize that variants H173R, R150L, and R151C of the OXTR gene impair ligand binding and receptor signaling and underlie oxytocin non‐responsiveness seen in certain pregnant women.

Methods

OXTR variants were created via mutagenesis and transfected into HEK293T cells. The oxytocin binding and signaling abilities of the wild‐type OXTR and OXTR variants were then assessed with an ELISA assay using IP1 accumulation as the readout for OXTR signaling.

Results

H173R, R150L, and R151C are located in the binding and signaling domains of the OXTR and were predicted by the Sorting Intolerant From Tolerant (SIFT) algorithm to be damaging to protein function. Wild‐type OXTR were functional as measured by ELISA and Ca2+ imaging. Increasing concentrations of oxytocin led to greater stimulation of the OXTR and greater IP1 accumulation.

Conclusion

Our studies will provide evidence as to whether oxytocin responsiveness can be predicted. This can help improve clinical methods and treatment of women during labor and delivery.

Support or Funding Information

Supported by the American Physiological Society (to G.Y.L) NIH 1R21 HD076677 (to S.K.E.) and F31 HD079148 (to E.L.R).

Transfected with:
Oxytocin concentration
IP1 concentration (nM)

Wild‐type OXTR
0nM
17.57

30nM
27.42

100nM
29.35

1uM
35.07

Un‐transfected
0nM
8.42

1uM
7.97
Wnt and Ca2+ signalling changes in LRRK2 Parkinson's disease models
Authors: Harvey Kirsten , Wetzel Andrea , Hughes Michael , McKay Tristan , Waddington Simon , Rahim Ahad
Published: November 2023

Objective

Examination of the influence of leucine‐rich repeat kinase 2 (LRRK2) on Wnt and Ca2+ signalling changes in Parkinson's disease.

Background

Mutations in LRRK2 are a major cause of Parkinson's disease. Changes in LRRK2 activity were shown to cause alterations in several cell signalling pathways. Wnt and Ca2+ signalling changes are some of the most reproducible reported observations to date. LRRK2 was shown to modulate Ca2+‐dependent immune responses, autophagy and mitochondrial function. In addition, LRRK2 modulated canonical Wnt signalling was shown to be affected in opposite ways by protective and pathogenic genetic Parkinson's disease variants, suggesting a clear correlation between Wnt signalling activity and neurodegenerative disease. This suggests the prospect of targeting Wnt signalling pathways to slow or halt the progression of Parkinson's disease.

Methods

Use of LRRK2 knockout and G2019S knock‐in models to conduct functional Wnt signalling assays. We introduced lentiviral biosensors into mouse brains to record Wnt and Ca2+ cell signalling changes in the brains of live animals from one week to six months of age using an advanced imaging technology (IVIS).

Results

Here, we present the analysis of Wnt and Ca2+ signalling activity using the exciting new technology of tissue‐targeted delivery of lentiviruses containing a transcription factor activated luciferase reporter to newborn mice. This allows quantification of transcription factor activity over the lifetime of an animal. We compare wild type mice with LRRK2 knockout mice and with mice harbouring the most prevalent familial LRRK2 G2019S mutation. Our data collectively support the idea of LRRK2 as a modulator of Wnt and Ca2+ signalling.

Conclusions

Increasing our understanding of the role of LRRK2 in Wnt and Ca2+ signalling pathways during neuronal development and aging might contribute to the generation of novel PD treatments targeting early events in the pathogenesis of PD. Decrease in Wnt signalling activity has previously been linked to neurodegeneration and provides therefore a plausible mechanism and a good therapeutic target in the Parkinson's disease process.

Support or Funding Information

Medical Research Council, Wellcome Trust
Small size of membrane‐tethered ligands regulates kinase‐phosphatase segregation in gamma/delta TCR triggering.
Authors: Choudhuri Kaushik
Published: November 2023

Ligand recognition by the T cell antigen receptor (TCR) is a central event in initiating T cell‐mediated immunity. While ab T cells recognize small peptides in association with major histocompatibility complex molecules (pMHC), gd T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co‐receptors CD4/8. Some of these unconventional T cells recognize invariant stress‐inducible membrane‐tethered ligands, and their rapid activation and recruitment to sites of inflammation is thought to serve as a ‘first line of defense’ against incipient infection. In light of the substantial structural and stoichiometric similarities between ab and gd TCRs, we sought to determine if gd TCR ligand‐recognition shared basic physicochemical constraints to triggering that have been identified for conventional ab TCRs. We chose as a model system the stress‐inducible surface protein T22 that is recognized by the G8 gd TCR, since this receptor‐ligand pair has been characterized biophysically, and the T22/G8 structure has been solved at atomic resolution. Since T22 is similar in size to pMHC molecules, we tested whether ‘size‐based’ kinase‐phosphatase segregation, following engagement of G8 TCR by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation, and IL‐2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell‐cell contacts. We are testing whether truncation of the ectodomain of the large membrane phosphatases CD45 and CD148, which are normally sterically segregated from engaged TCR, abrogates TCR phosphorylation by membrane‐associated Src‐kinases. Finally, we are adapting these strategies, to investigate the principles of triggering in gd T cells which recognize small‐molecule chemical haptens.

Support or Funding Information

AI093884 (NIH/NIAID)
Multi‐generational silencing dynamics control cell aging
Authors: Hao Nan , Li Yang , Jin Meng , O'Laughlin Ricky , Tsimring Lev , Pillus Lorraine , Hasty Jeff
Published: November 2023

Cell aging is a universal biological phenomenon, but mechanisms that regulate aging remain unclear. Using novel microfluidic and imaging technologies, we tracked the replicative aging of single yeast cells and found a dramatic loss of heterochromatin silencing leading to cell aging and death. The dynamics of silencing loss during aging can be dissected into an early phase with sporadic silencing loss, followed by sustained silencing loss preceding cell death. Although the length of the later phase is relatively constant, the length of the early phase is highly variable among cells and largely determines lifespan. The intermittent silencing dynamics during the early phase depends on a conserved histone deacetylase Sir2 and is important for longevity, whereas either sustained silencing or loss of silencing shortens lifespan. These findings reveal that the temporal dynamics of key molecular processes can directly influence cell aging.
TAK1/TRAF6 signalling in regulation of skeletal muscle mass
Authors: Kumar Ashok , Hindi Sajedah M
Published: November 2023

Loss of skeletal muscle mass is a devastating consequence of a large number of chronic disease states and several other conditions such as starvation, denervation, immobilization, and aging. However, the signaling and molecular mechanisms that regulate skeletal muscle growth and atrophy remain less understood. It is now evident that a complex array of signaling pathways regulates skeletal muscle mass primarily through modulating the rate of protein synthesis and degradation. Transforming growth factor β (TGF‐β) activated kinase 1 (TAK1), a member of the MEK kinase family, is an important signaling protein which activates multiple signaling pathways in response to cytokines, growth factors, and microbial products. In many signaling cascades, TRAF6 ubiquitylates and activates TAK1. We have previously reported that TRAF6 mediates skeletal muscle atrophy in response to denervation, starvation, and cancer growth. We have now investigated the role of TAK1 in the regulation of adult skeletal muscle mass. Our results demonstrate that inducible inactivation of TAK1 leads to severe muscle wasting and development of kyphosis in adult mice. Moreover, denervation‐induced muscle atrophy is exacerbated in skeletal muscle‐specific TAK1 knockout mice. TAK1 is required for the activation of specific intracellular signaling pathways which promote skeletal muscle growth and prevent atrophy. Inactivation of TAK1 inhibits protein synthesis, induces protein degradation, and causes oxidative stress in skeletal muscle of adult mice. We have also found that deletion of TAK1 leads to accumulation of dysfunctional mitochondria in skeletal muscle potentially due to the inhibition of mitophagy. Intriguingly, phosphorylation of TAK1 is dramatically induced in skeletal muscle of mice undergoing load‐induced hypertrophy. Importantly, targeted inactivation of TAK1 blunts skeletal muscle hypertrophy in response to functional overload. Overall, our experiments suggest that TAK1 is a key regulator of skeletal muscle mass.

Support or Funding Information

National Institute of Health grants AR059810, AR068313, and AG029623