bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and Anti‐Cancer Activity of Cysteine‐Deleted Tachyplesin Analogs
Authors: Eitel Anna , Hendrickson Nathan , Heyl‐Clegg Deborah , Evans Hedeel , Guthrie Jeffrey
Published: November 2023

Research investigating anti‐cancer agents is an exciting and important part of medicinal chemistry due to its prevalence in the population. Tachyplesin, an antimicrobial peptide isolated from the horseshoe crab, has been shown to kill cancer cells. The positive charges of the arginine and lysine residues allow for specificity for bacterial and cancer cell membranes, as they are attracted to negatively charged components that these cells express on the surface. The hydrophobic part then penetrates into the lipid membrane, disrupting membrane structure by creating holes and leading to cell death. Our lab has studied a linear version of this peptide, CDT, as an antibiotic, but it had not been tested for effects against cancer cells. In this study, the anti‐cancer effects of CDT and its analogs, where sequence and stereochemical configuration were modified, were studied using A549 lung cancer cells. An MTT assay was performed to calculate percent cell viability with relation to increasing concentrations of peptide. Results indicate that the D‐analogs of CDT have stronger anti‐cancer effects than CDT itself.
Withaferin A suppresses and inhibits hepatocellular carcinoma‐cell proliferation and tumor burden via activation of ERK1/2‐ELK1‐RSK‐DR5 axis
Authors: Kuppusamy Panjamurthy , Nagalingam Arumugam , Drachenberg Cinthia , Sharma Dipali , Saxena Neeraj K
Published: November 2023

Hepatocellular carcinoma (HCC) is the fifth most common highly lethal cancer and the third most common cause of cancer‐related mortality in the world. Available therapies are often limited by high toxicity, lower efficacy, therapeutic resistance and therapy‐related morbidity. Liver resection, ethanol injection, radiofrequency ablation, and chemoembolization have been used successfully for the local management of HCC; however, no single cytotoxic‐chemotherapeutic‐agent has been proven effective for systemic treatment. Therefore, more effective therapeutic strategies are needed to combat liver cancer related mortality. Withaferin A (WA) is a bioactive compound derived from Withania somnifera has been safely used for centuries in Indian‐Ayurvedic‐medicine against various ailments. For the first time, we demonstrated that WA displays substantial inhibitory activity against liver cancer cells in vitro and tumor burden in vivo in HCC‐cell‐xenograft‐nude mice and DEN induced HCC in C57BL/6.

Methods

Therapeutic efficacy of WA was assessed by determining cell viability of HCC cell lines, HepG2 and Huh7, using XTT reduction while apoptosis was quantified by TUNEL assay. Effect of WA on cell migration (spheroid assay), invasion (matrigel invasion), clonogenicity, anchorage independent growth (colony formation on soft agar) were determined. Additionally, in a quantitative real‐time, ECIS (Electric cell‐substrate impedance sensing)‐based assay was performed to evaluate efficacy of WA on migration and invasion of HCC cell lines. The mechanism for the inhibitory role of WA on HCC was determined by immunoblot assay for cleaved caspase 3, Bax, cleaved PARP, phosphorylated ERK, Elk1, RSK1 and DR5 in HCC cells, tumors derived from xenografts in nude mice and tumorous‐livers from DEN treated mice.

Results

WA treatment significantly inhibited HCC cell‐proliferation, migration, invasion, clonogenicity and anchorage‐independent growth. WA treatment significantly induced apoptosis to suppress cell and tumor growth as indicated by increased cleaved caspase 3 activity, cleaved PARP and Bax. Mechanistically, WA induced expression of death receptor 5 (DR5) that triggers apoptosis when overexpressed. Our study revealed that WA inhibits phosphorylation of Stat3 which in turn up‐regulates phosphorylation of Elk1. Inhibition of ERK or Elk1 phosphorylation inhibits WA‐induced DR5 induction, indicating that ERK and Elk1 cooperatively regulate DR5 expression.

Conclusions

Collectively, our studies show the efficacy of WA as a novel agent for HCC inhibition and provide mechanistic insight into the important role of ERK‐Elk1 axis in WA‐mediated HCC inhibition.
The cytoprotective effect of chlorogenic acid against oxidative stress‐induced cell damage via the ERK and PI3K/Akt‐mediated Nrf2/HO‐1 signaling pathways
Authors: Jang Miran , Kim Gun‐Hee
Published: November 2023

This study investigated the effect of chlorogenic acid of Cirsium japonicum on the expression and activity of heme oxygenase‐1 (HO‐1), an enzyme with potent antioxidant properties, as well as the molecular mechanisms involved. Chlorogenic acid markedly upregulated HO‐1 protein expression in PC12 cells. Chlorogenic acid also increased the protein level of transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2), which regulates HO‐1 expression by binding to the antioxidant response element (ARE) within the HO‐1 gene promoter, in a dose‐and time‐dependent manner. Exposure to H2O2 reduced the expression of HO‐1 in PC12 cells, the addition of chlorogenic acid restored HO‐1 expression protein levels. Moreover, chlorogenic acid decreased the expression of Kelch‐like ECH‐associated protein 1(Keap 1). Chlorogenic acid activated extracellular‐regulated kinase (ERK) and protein kinase B (PKB, Akt) in keratinocytes, while the ERK and Akt inhibitors attenuated chlorogenic acid‐enhanced Nrf2 and HO‐1 expression. The results suggested that chlorogenic acidactivates ERK‐and Akt‐Nrf2 signaling cascades in PC12cells, leading to the upregulation of HO‐1 and cytoprotection against oxidative stress. The phenolics in Cirsium japonicum leaf, particularly, chlorogenic acid can be utilized as effective and safe functional food substances, i.e., natural antioxidants, and may reduce the risk of neurodegenerative disorders.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2016R1A2B4014740)
Undergraduate Research Projects at the Community Colleges
Authors: Hess Brittany A , Fortner Alexia A , Chowdhury Syed A
Published: November 2023

About 45% of the nation's undergraduates experience their first post high school science education at a 2 year technical college. However, unfortunately almost none of these colleges have adequate undergraduate research opportunities for these students. Science education, by definition is, “the learning of scientific phenomena through investigation or research”. Research enhances our mental capacities and engages students in critical thinking. In a community college or a technical college setting, it is extremely difficult and also unlikely for students to spend a significant amount of time conducting research. Along with this, most instructors do not get enough time to write grants or participate in research when they have a heavy teaching load and often times serve on various committees. However, we have now created new opportunities for our undergraduate science students who show their interst in research. These newly added projects are concise, need little mentoring, and require minimal or no costs. With the help of our instructors and using current lab facilities, we have made dozens of projects readily available for our students. Any student can pick a project from those offered and get involved in simple yet extremely relevant research on campus. For instance, we have ‘testing attention and memory related brain functions’ which uses less expensive and much more portable equipment known as ‘PsychoToolKits’. We have also included, ‘Biopac’ to perform challenging experiments related to heart function, brain waves and muscle activities, or in order to examine the ‘behavior of microbes’ in multiple altered environments. Students have shown great interest in these projects and seem to enjoy investing time to understand and work through these labs thoroughly. We recommend that community as well as many other 2‐year colleges get involved in creating undergraduate research projects and we would love to see them offer these opportunities to students who are eager to study how the methods of planning, conducting, and gathering relevant information to research from numerous sources and then being able to analyze and present the data to their peers. Students will greatly benefit from being involved in these newly incorporated projects.

Support or Funding Information

Department of Science, Saint Louis Community College‐Wildwood
The Role of Glucose‐6‐Phosphate Dehydrogenase of Inflammasome Activation
Authors: Chiu Daniel Tsun‐Yee , Yen Wei‐Chen , Wu Yi‐Hsuan
Published: November 2023

Glucose‐6‐phosphate dehydrogenase (G6PD) is a rate‐limiting enzyme of pentose phosphate pathway to maintain cellular redox homeostasis via the production of NADPH. Through NADPH oxidase (NOX), NADPH modulates the generation of reactive oxygen species (ROS) against pathogen infection. G6PD deficient cells have low sensitivity to induce inflammation pathway for elimination of pathogen infection. Inflammasome activiation is a kind of inflammatory response and plays a regulatory role in innate immunity by producing pro‐inflammatory cytokines including interleukine‐1β and 18 (IL‐1β and IL‐18). However, the role of G6PD in the activation of inflammasome is remains elusive. Since well‐known G6PD genetic variation in Taiwan is 1376 (G>T; 42.3 ~ 52.3%) and 1388 (G>A; 10.5 ~ 34%), we collected peripheral blood from patients with either of these two point mutations. To investigate whether G6PD modulates inflammasome activation, peripheral blood mononuclear cells (PBMCs) were isolated and differentiated into macrophage‐liked cells by phorbol 12‐myristate 13‐acetate (PMA) treatment. The signal 1 and 2 pathways of inflammasome were activated by lipopolysaccharides (LPS) and adenosine triphosphate (ATP) stimulation, respectively. During inflammasome activation, a decreased secretion of IL‐1β was observed in PMA‐primed G6PD deficient PBMCs as compared with their counterparts. We further used human monocytic cells (THP‐1) as model to delineate whether the expression of molecules in inflammasome signal 1 and 2 pathway could be modulated by G6PD. Indeed, inflammasome activation‐induced IL‐1β secretion and NF‐κB phosphorylation of signal 1 pathway were downregulated upon G6PD knockdown in THP‐1 cells. Nevertheless, the effect of G6PD on signal 2 pathway is still unclear. Taken together, these results suggest that G6PD deficiency down regulates inflammasome activation and the underlying mechanism is currently under investigation.
NLRP3 Inflammasome Is Critical for Lipopolysaccharide‐Induced Depressive‐Like Behaviors
Authors: Lee Eunju , Jeon Seon‐A , Hwang Inhwa , Yu Je‐Wook
Published: November 2023

Cerebral inflammation is considered to contribute to the pathogenesis of depressive disorders, but the molecular target to intervene inflammation‐mediated depressive symptoms is still poorly elucidated. Recent studies intriguingly proposed the implication of NLRP3 inflammasome signaling in the depressive‐like disorders, although the molecular basis by which NLRP3 inflammasome activation contributes to depression is unclear. In this study, we examined lipopolysaccharide (LPS)‐induced depressive‐like behaviors in wild‐type and Nlrp3‐deficient mice. The levels of proinflammatory cytokines in the brain homogenate were not significantly different between wild‐type and Nlrp3‐deficient mice after LPS administration. However, significant reduction of active caspase‐1 was observed in the hippocampus of Nlrp3‐deficient mice brain compared to wild‐type mice. Of interest, LPS administration to mice caused a cerebral induction of Ido mRNA in a NLRP3‐dependent manner. Supporting this data, NLRP3 inflammasome‐activating stimulations, but not interleukin‐1β treatment, triggered a significant induction of IDO in mixed glial cells. Collectively, our findings suggest that IDO is a potential downstream mediator of the NLRP3 inflammasome in the progression of depressive‐like behaviors.

Support or Funding Information

This work was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, (2015M3A9B6073856)
Pseudomonas aeruginosa Attenuates Inflammasome Activation via Quorum Sensing‐Dependent Mechanism
Authors: Yang Jungmin , Lee Kang‐Mu , Park Sangjun , Yoon Sang‐Sun , Yu Je‐Wook
Published: November 2023

Quorum sensing (QS) is a bacterial communication mechanism that promotes coordinated adaptation by triggering expression of a wide range of genes. QS is thought to strongly contribute to the virulence of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, but the molecular effect of QS on host inflammasome defense is completely unknown. Here, we present evidence that P. aeruginosa dampens host inflammasome signaling by QS‐related products in mouse bone marrow‐derived macrophages. We demonstrate that QS‐regulated virulence factor pyocyanin and QS autoinducer 3‐oxo‐C12‐homoserine lactone directly suppress NLRC4‐ and NLRP3‐mediated inflammasome assembly and activation. In addition, bacteria‐free secretant from QS‐defective ΔlasR/rhlR mutant, but not from wild type bacteria, induces robust activation of the NLRC4 inflammasome. Extracellular P. aeruginosa‐released flagellin might be responsible for this inflammasome activation by ΔlasR/rhlR secretant, but QS‐dependent secretion of bacterial proteases degrade extracellular flagellin to evade NLRC4 inflammasome activation. P. aeruginosa–secreted proteases also impair inflammasome components and pro‐inflammatory cytokines in the extracellular space. Taken together, our data indicate that QS system of P. aeruginosa facilitates bacteria to evade host inflammasome‐dependent sensing machinery.

Support or Funding Information

This work was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, (2015M3A9B6073856)
Generating a novel cell line with a codon optimized LINE1 element at a single locus
Authors: Russell Brittany , Morales Maria , DeFreece Cecily
Published: November 2023

Long interspersed element‐1 (LINE1) is the only currently active autonomous retroelement within the human genome. LINE1 replicates itself through a process known as retrotransposition that requires the endonuclease and reverse transcriptase activities of the LINE1 protein ORF2. Further studies characterizing LINE1 endonuclease function demonstrated that LINE1 endonuclease induces formation of double‐stranded breaks (DSBs). Only a small fraction of LINE1‐induced DSBs results in a retrotransposition event, suggesting that the damage from the LINE1 endonuclease activity may be greater than previously considered. While LINE1 mutagenic insertion events have been associated with diseases such as breast and colon cancer as well as muscular dystrophy, the full effect of LINE1 endonuclease upon stability of the genome is undetermined. By inserting a codon‐optimized LINE1 element into a cell line at a unique genomic locus using the FRT system, we will investigate and track the effect of the endonuclease on the genome. Codon optimization of LINE1 will increase expression of the element and will aid in detection of novel retrotransposition events in the presences and absence of drugs. Here we are presenting the cloning of the FRT vector containing the codon‐optimized LINE1 element, the first step of project.

Support or Funding Information

The work presented is supported in part by monies from P20GM103424, TL4GM118968 and 5RL5GM118966‐03 as well as the Louisiana Cancer Research Consortium.
Direct and Specific Interaction of MIF and Bacterial Endotoxin May Play an Important Role in the Pathogenesis of Sepsis
Authors: Cho Thomas
Published: November 2023

Inflammatory response is triggered in the body as a result of infections by bacteria and other pathogens. Especially, a bacterial outer membrane component, lipopolysaccharide (LPS), is a major virulence factor. LPS activates the secretion of various inflammatory cytokines. Among the secreted cytokines, macrophage migration inhibitory factor (MIF) is a cytokine‐like protein that amplifies inflammation by activating the secretion of other inflammatory cytokines. MIF levels are elevated in severe sepsis and septic shock. MIF−/− mice demonstrated improved survival from sepsis. This implies that MIF plays an important role in sepsis, but the underlying molecular mechanism is unknown.

Our recent data demonstrated evidence that MIF dramatically improved CXCR4‐mediated cell responses only when it makes a complex with LPS. Based on this observation, we hypothesized that LPS released during bacterial infection forms a complex with MIF and the complex plays a deteriorating role in the pathogenesis of sepsis. Removal of bacterial endotoxin from recombinant MIF is challenging and it is controversial whether MIF alone or MIF complexed with a binding partner mediates cell responses leading to various inflammation diseases. We noticed that a conventional endotoxin‐removal column failed to separate endotoxin from MIF. This led us to investigate whether MIF and LPS directly bind each other. MIF has one tryptophan (W109) exposed to the solvent. We demonstrated that the intrinsic fluorescence of W109 was quenched when titrated with increasing concentrations of LPS. We confirmed the MIF‐LPS interaction by using isothermal titration calorimetry and determined the affinity (KD) of 2.9 micromolar. We also proved that MIF and LPS formed a complex during protein purification and in an animal model of sepsis. Finally, we identified a LPS binding site located at the trimeric interface of MIF by using NMR.

To elucidate potential biological consequences of the MIF‐LPS interaction, we tested whether this interaction alters the effect of each molecule on endothelial barrier function, which is damaged during conditions such as sepsis. Intriguingly, LPS together with MIF revealed less endothelial permeability than LPS alone. MIF alone did not significantly change endothelial barrier permeability. Our findings on the MIF‐LPS interaction could serve as a novel paradigm to better understand physiological functions of this important and enigmatic immune regulator.

Support or Funding Information

NIH NIAMS
Spectroscopic Investigation of Novel New Delhi Metallo‐β‐Lactamase‐1 Inhibitors
Authors: Bergstrom Alex , Cheng Zishou , Miller Callie , Chen Allie , Thomas Pei , Bonomo Robert , Fast Walter , Cohen Seth M , Page Richard C , Tierney David L , Crowder Michael W
Published: November 2023

The prevalence of antimicrobial‐resistant pathogens is an ever‐growing threat to world health, and the pipeline supplying new classes of antibiotics has begun to run dry. The β‐lactams are a class of antibiotics that revolutionized healthcare and have been a mainstay in medicine for decades. However, their efficacy towards pathogenic bacteria is threatened by the transmission and evolution of β‐lactamases, enzymes capable of inactivating this important class of antibiotics. The metallo‐β‐lactamases (MBLs) are a class of β‐lactamases of which there exist no clinically known inhibitors. Yet, they are capable of hydrolyzing almost all β‐lactams. These enzymes rely on either one or two Zn(II) ions within their active site which are necessary for catalysis. New Delhi MBL‐1 (NDM‐1) is of particular concern due to its global spread and rapid transmission between pathogens. Recently, 2,6‐dipicolinic acid (DPA), as well as several derivatives, were identified as potent inhibitors of the MBLs. Here we report the spectroscopic techniques used to elucidate the mode of inhibition, which aided in the discovery process.

This investigation aimed to fit the mode of inhibition of NDM‐1 into one of two models: through metal scavenging, or through competitive inhibition. EDTA, a well‐known metal sequestering agent, was selected as a standard for interpreting spectroscopic data of an inhibitor fitting the first model. Alternatively, L‐captopril, a known competitive inhibitor of the MBLs, was selected for the second model. Using 1H NMR, CW‐EPR, and equilibrium dialysis coupled ICP‐AES on either the di‐Zn(II) or di‐Co(II) metalloforms of NDM‐1, it was discovered that DPA best fit a model of non‐selective metal scavenging. However, the data suggest that several of its derivatives form a stable ternary complex between NDM‐1:active site ions:inhibitor in a concentration dependent manner. At lower concentrations, closer to that which would be found in a cell, there is little evidence of metal scavenging effects. However, at high concentrations they better fit a metal‐sequestering model, though with a preference for targeting the Zn2 site before the Zn1 site. These spectroscopic techniques allowed for the minimization of non‐selective metal scavenging effects during the drug discovery process and may be useful for guiding future investigations into other Zn(II) metalloproteins.

Support or Funding Information

This work was supported in part by the National Institutes of Health (GM111926) and by the Robert A. Welch Foundation (F‐1572 to WF).
Spinal Cord Injury and the Gut Microbiome: Mechanisms and Therapies for Bowel Dysfunction
Authors: Jeffrey Elisabeth , O'Connor Greg , Deo Sapna , Dietrich Dalton , Daunert Sylvia
Published: November 2023

Both experimental and clinical studies have shown that Spinal Cord Injury (SCI) has detrimental effects on remote organs and systems, yet there have been limited studies focusing on these more widespread effects. Despite attention to the pathophysiology of SCI with a goal of protection and repair, there is very little known about these gastrointestinal side‐effects or their effect on the gut microbiome. We sought to characterize long term effects of SCI on the gut by identifying changes in bacterial communication, microbiome composition, and inflammation. Bacterial quorum sensing molecules (QSMs) regulate various communication processes including growth, biofilm formation, and virulence factor production, thus providing information on the state of gut microbiota. Despite a repeatedly observed link between the intestinal microbiome and health, there is very limited data characterizing the gut microbiome in adult SCI patients with bowel dysfunction. We have employed a SCI rat model to investigate changes in the intestinal microbiome and gastrointestinal tract which may potentially contribute to or cause the GI dysfunction experienced by patients with SCI. Specifically, we determined the changes in the intestinal microbiota post‐SCI, most notably there was a significant increase in the relative abundance of Bifidobacteriaceae and Clostridiaceae post‐SCI, and investigated the potential underlying mechanisms via levels of pro‐inflammatory cytokines (IL1‐β, IL‐12, MIP‐2, and TNF‐α) and QSMs. This study continues to provide new information regarding the injury‐dependent changes on the gut microbiome and revealing microbiome‐host interactions that may contribute to the long term effects of SCI on the GI tract function. Identifying mechanisms behind these changes will aid in the development of new therapeutic strategies such as dietary modulation to improve quality of life for people living with SCI.

Support or Funding Information

University of Miami Miller School of Medicine, Miami Project to Cure Paralysis, Department of Biochemistry and Molecular Biology
SP‐A2 contributes to miRNA‐mediated sex differences in response to ozone‐induced oxidative stress: pro‐inflammatory, anti‐apoptotic, and anti‐oxidant pathways are involved
Authors: Thorenoor Nithyananda , Noutsios George T , Zhang Xuesheng , Phelps David S. , Umstead Todd M. , Durrani Faryal , Floros Joanna
Published: November 2023

Human host defense molecules, surfactant protein‐A1 (SP‐A1) and SP‐A2 differentially affect alveolar macrophage (AM) function and their proteome. We hypothesized, that SP‐A genes differentially regulate the AM miRNome. SP‐A knockout (KO), humanized transgenic mice expressing SP‐A1 and SP‐A2 and SP‐A2 gonadectomized mice were exposed to O3 or filtered air (FA) and AM isolated. Of the miRNAs (n=372) studied, 11 miRNAs were differentially expressed under O3‐induced oxidative stress (OxS) in SP‐A2 males. Their gene targets included IL‐6, STAT3, BCL2, NF‐kB, and FOXO1. a) We observed increase in STAT3 mRNA (p < 0.05) in SP‐A2 after 4hr of O3 exposure (4hrO3). To account for half‐life differences, we measured mRNA after 18hrO3. BCL2 increased (p < 0.05) in SP‐A2 AM, whereas, the other genes significantly decreased compared to FA. TUNEL assay revealed significant decrease and increase in apoptotic cells under OxS in SP‐A2, and KO, respectively, indicating a role for SP‐A2 in apoptosis. b) The IKKβ mRNA level after 18hrO3 decreased significantly in SP‐A2, but not in KO; a number of SP‐A2‐downregulated miRNAs correlated with the upregulation of target genes, TNF‐α, IL‐1β, and IL‐6, indicating a role in pro‐inflammatory gene expression. c) Although FOXO1 did not change, SOD2 and CAT decreased and increased significantly in SP‐A2 and KO, respectively, after 18hrO3, indicating that SP‐A2 contributes to ROS homeostasis by decreasing the expression of anti‐oxidant enzymes. d) Sex hormones contribute to differential miRNA expression, as demonstrated by gonadectomy. Thus, SP‐A2 affects miRNA‐mediated sex‐specific differences in response to OxS by involving inflammatory, anti‐apoptotic, and anti‐oxidant pathways.

Support or Funding Information

CHILD fund, Dept of Pediatrics, Penn State University, College of Medicine
Tales from the Crypt: Malic Enzyme 1 (ME1) Links Metabolism to Intestinal Cancer
Authors: Fernandes Lorenzo , Al‐Dwairi Ahmed , Marji Meera , Brown Dustin , Simmen Rosalia C.M , Simmen Frank A.
Published: November 2023

Colorectal cancer (CRC) is the third leading cause of cancer‐associated deaths in the USA. CRC has significant metabolic underpinnings but the connections between metabolic dysregulation and CRC remain elusive. Our laboratory studies the metabolic pathways involving Malic Enzyme 1 (ME1), the cytosolic enzyme that provides NADPH for fatty acid and cholesterol biosynthesis. Up‐regulation of ME1 has been implicated in oncogenesis, however, its role in CRC remains unknown. Here, we established a new CRC mouse model by crossing mice overexpressing ME1 in the intestinal mucosa (ME1‐Tg) with Apcmin/+ mice that bear a mutation in the Adenomatous Polyposis Coli gene (Apc). Both male and female Apcmin/+/ME1‐Tg offspring exhibited an increase (P≤0.05) in the numbers of adenomas in their duodenum, jejunum and ileum compared to Apcmin/+nontransgenic mice. Immunohistochemical analysis revealed increases in the sizes and numbers (P≤0.05) of β‐catenin‐stained adenomas in ilea of Apcmin/+/ME1‐Tg compared to Apcmin/+ mice. BrdU staining showed no significant differences in proliferation between Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. However, TUNEL staining demonstrated a tendency for a decrease (P=0.08) in apoptotic cell death in the Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. Immunostaining for ME1 revealed a greater (P≤0.05) percentage of positive staining in cells of the villi and adenomas of ilea from Apcmin/+/ME1‐Tg vs. Apcmin/+ mice, while crypt cells exhibited a tendency (P=0.06) to have higher percentage of positive ME1 staining. Fatty Acid Synthase (FASN), a cognate lipogenic pathway partner of ME1, was abundantly expressed in ilea of both Apcmin/+/ME1‐Tg and Apcmin/+ mice, but without significant differences between the groups. To determine the mechanism(s) responsible for higher tumor burden, we performed targeted gene expression analysis of intestines from the two groups. Transcription factor Sp5 mRNA was significantly up‐regulated (by 2.5 fold; P≤0.05) in the jejunums of Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. Immunostaining for Sp5 corroborated our gene expression results revealing a greater percentage of Sp5 staining (P≤0.05) in the cells of the villi of ilea from Apcmin/+/ME1‐Tg compared to Apcmin/+ mice. Our findings are consistent with previous reports of increased Sp5 expression in CRC. To determine a direct role of ME1 in CRC proliferation, we performed in‐vitro assays on HCT116 CRC cell lines using siRNA Knockdown. A significant reduction in HCT116 cell proliferation (P≤0.05) was observed with siRNA‐mediated ME1 knockdown relative to scrambled siRNA treatment. Our collective data establish a link between ME1 up‐regulation and higher tumor burden in the duodenum, jejunum and ileum in a mouse model and suggest the Wnt target and stem cell modulator Sp5 as a potential mediator in these events.

Support or Funding Information

Grant Support: NIH R01CA136493, NIH UL1TR000039
Reliability and Validity of Accelerometry Methods Used to Assess Facet Joint Crepitus and Cavitation
Authors: Cramer Gregory D , Budavich Matthew , Bora Preetam , Koo Terry , Madigan Dana , Braden Amanda , Ross Kim
Published: November 2023

Objective

Novel accelerometry methods have been developed to assess and localize vibrations from zygapophyseal (Z) joint audible sounds (crepitus and cavitations) during normal lumbar motion and side‐posture spinal manipulation. This study assessed the reliability and validity of the methods.

Methods

A lumbar spine was embedded in silicone in the prone position. Before pouring the silicone, tunnels were created from the posterior aspect of the Z joints to the surface. Once the spine phantom was completed, a specialized mechanical device was lowered into each tunnel and was used to strike the Z joints with a force of approximately 7 N to simulate joint crepitus, and 67 N to simulate cavitation (2 experiments). Ten accelerometers applied in a previously developed pattern were used to localize the specific origin of the joint vibrations. For each experiment (crepitus and cavitation), each joint was struck on three different passes (n=30) while recordings from the accelerometers were made. The order of joint strikes was randomized twice for use in two observation sessions. Two observers blinded to the joint strikes, results of one another, and to their previous results, analyzed the oscilloscope recordings of the accelerometers on two separate occasions, separated by a minimum of 2 hours, to identify the joint from which the vibrations originated. Intra‐ and inter‐observer reliability and validity (actual joint struck vs. observer determination from recordings) were calculated.

Results

Crepitus, Observer 1: validity Test (Run) 1 = 0.93; validity Test 2 = 0.96; intra‐observer reliability (Test 1 vs. Test 2) = 0.96; Crepitus, Observer 2: validity Test 1 = 0.89; validity Test 2 = 0.96; intra‐observer reliability = 0.93; Crepitus, interobserver reliability (Observer 1 vs. Observer 2) = 0.94. Cavitation, Observer 1: validity Test 1 = 1.00; validity Test 2 = 1.00; intra‐observer reliability (Test 1 vs. Test 2) = 1.00; Cavitation, Observer 2: validity Test 1 = 1.00; validity Test 2 = 1.00; intra‐observer reliability (Test 1 vs. Test 2) = 1.00; Cavitation, interobserver reliability (Observer 1 vs. Observer 2) = 1.00.

Conclusions

The methods as tested in a highly controlled environment were in almost perfect agreement for both simulated crepitus and simulated cavitation. The methods should be further developed and refined for use in human subjects.

Support or Funding Information

NIH/NCCIH Grant # 3R01AT000123‐06S2
P311, an Intrinsically Unstructured Protein, in Adipogenesis
Authors: Badri Kameswara Rao , Muppuru Kesavulu Muni , Samuel Raymond E
Published: November 2023

Obesity is becoming a global pandemic with about one third of obese adults reported in United States. This debilitating condition decreases the quality of life, which can ultimately influence the lifestyle leading to various disorders including insulin resistance, diabetes mellitus, dyslipidemia and cardiovascular diseases. There are very limited drugs and invasive procedures available to treat obesity. Hence, there is every necessity to explore and understand novel molecular mechanisms of adipogenesis to control this global pandemic. Recent developments identified white adipose tissue as endocrine organ and brown adipose tissue in thermogenesis recognizing potential complex regulation of adipose tissue in health and disease. P311 is a small 8kDa protein that is abundant in the brain, smooth muscle, skeletal muscle and lungs. We recently showed the role of P311 in the regulation of blood pressure. Here we studied the role of P311 in adipogenesis and elucidated the structure of P311. For the current research, we used 3T3‐L1 in‐vitro cell culture model to study the role of P311 in adipogenesis. We treated 3T3‐L1 cells with adipogenic cocktail (1μM dexamethasone, 10 μg/mL insulin and 0.5mM 3‐isobutyl‐1‐ methylxanthine), let them differentiate into adipocytes for different days (0 to 13 days) and determined the levels of P311 and key adipogenic marker, PPARγ2. We observed significant increase in the expression of P311 as fibroblasts differentiated into adipocytes. In addition, P311 expression is positively correlated with the expression of key adipogenic transcriptional regulator, PPARγ2. In dual luciferase promoter reporter assays, P311 activated the PPARγ2 promoter mediated luciferase expression indicating the importance of P311 in PPARγ2 transcription. Our studies identify P311 as an intrinsically disordered protein and a key molecule that mediates adipogenesis.

Support or Funding Information

U54MD008621 (HU‐160026) to Hampton Univeristy
Yeast Pah1 Phosphatidate Phosphatase Regulates the Expression of Phosphatidylserine Synthase for Membrane Phospholipid Synthesis
Authors: Han Gil‐Soo , Carman George M.
Published: November 2023

Pah1 phosphatidate phosphatase (PAP), which catalyzes a committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of PA required for de novo phospholipid synthesis. In this study, we examined the Pah1 PAP‐mediated regulation of phosphatidylserine (PS) synthase, which catalyzes a committed step for the synthesis of major membrane phospholipids. PS synthase activity was highly increased by the lack of Pah1 PAP with a growth‐dependent upregulation from the exponential to the stationary phase. Immunoblot analysis showed that the elevation of PS synthase activity results from an increase in the level of the enzyme encoded by CHO1. By promoter truncation analysis and site‐directed mutagenesis, the induced expression of CHO1 in the pah1Δ mutant was shown to occur through the inositol‐sensitive upstream activation sequence (UASINO, −163 ~ −154), a cis‐acting element for the PA‐controlled Ino2‐Ino4/Opi1 regulatory circuit. The abrogation of CHO1 induction by the UASINO mutation suppressed the pah1Δ phenotypes related to the increase of PA levels (e.g., increased phospholipid synthesis, nuclear/ER membrane expansion, and reduced lipid droplet formation) with its effect stronger in the exponential phase than in the stationary phase. These results demonstrate that Pah1 PAP regulates the expression of PS synthase through its control on the PA level.

Support or Funding Information

Supported by NIH grant GM028140
Evidence for Muscle Pump Enhancement of Capillary Filtration as a Redundant Mechanism for Bone Fluid Flow
Authors: Winet Howard
Published: November 2023

The current popular paradigm for generating bone fluid flow (BFF) sufficient to stimulate osteocytes combines gravity, poroelastic bone bending and glycocalyx receptors to amplify fluid shear stress to values sufficient for stimulation of bone growth[1]. Given the well‐established influence of muscle pumps on circulation we tested the hypothesis that they can modulate trans‐luminal pressures that drive capillary filtration in cortical bone, thereby serving as a redundant mechanism for BFF.

Optical bone chambers were implanted in tibias of 21 female NZW rabbits divided into control and stimulated cohorts, and allowed to fill with bone slivers. Weekly intravital microscope observations were video‐recorded and images processed as usual[2]. During the last observation, 2.54cm TENs electrodes were attached directly to the exposed and released gastrocnemius/soleus muscle of the implanted leg. It was stimulated with a 0–4V biphasic waveform at 4Hz. The one hour stimulation was stopped at intervals after injection of fluorescent dyes to record capillary filtration of the dyes. Dye concentrations as a function of fluorescence intensity were calibrated by the method of Wu et al.[3]. Bending strain on the tibia during stimulation was measured and compared to that of the controls to monitor gravity effects. Measurements were evaluated with Students t‐test.

Capillary filtration in stimulated rabbits in the absence of bone bending was significantly greater than in controls. There was no significant gravity‐generated bone strain in either group.

We conclude that tibial muscle pump‐generated pressure waves in cortical bone significantly enhance capillary filtration independently of poroelastic effects of bone bending by muscle or gravity. Since fluid flow through bone interstitium or in canaliculi was not directly measured here, no assessment could be made of shear stress on osteocytes or osteoblasts.

Published poroelastic models are optimization—for engineers—or adaptationist—for natural selection biologists—in nature. As such they do not allow for redundant mechanisms that can assist or substitute if the original is disabled. As Gould and Lewontin have shown[4], adaptation‐limited models ignore three of the four mechanisms of Neo‐Darwinian evolution paradigms. Evolution includes redundancy and pleiotropy. One consequence of this oversimplification is the absence of a BFF mechanism to explain bone healing in a nailed or casted long bone.

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Supported by UCLA Dept. Bioengineering
Regulatory Mechanisms of Sec1/Munc18 Proteins in Intracellular Vesicle Fusion
Authors: Yu Haijia , Liu Yinghui , Shen Jingshi
Published: November 2023

Intracellular vesicle fusion is mediated by a conserved core machinery composed of SNAREs (soluble N‐ethylmaleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins. The core domains of the vesicle‐rooted SNARE (v‐SNARE) and the target membrane associated SNAREs (t‐SNAREs) zipper into a four helix coiled‐coil bundle between two apposed bilayers. The SNARE bundle assembles toward the membranes and provides the energy to bring the two membranes into close apposition to fuse. SM proteins are soluble factors that promote membrane fusion through binding to their cognate trans‐SNARE complexes. It is well known that SM proteins interact with multiple domains on their cognate SNAREs and contribute to the compartmental specificity of intracellular vesicle fusion. However, the molecular mechanism of how SM proteins regulate the vesicle fusion has not been fully elucidated. In a reconstituted fusion system, we observed that the SNAREs themselves were inefficient in driving membrane fusion. The fusion rate was strongly increased by either the cognate SM protein or a Vc peptide corresponding to the C‐terminal half of the v‐SNARE. Further analysis showed that SM proteins promote membrane fusion in a similar manner as the Vc peptide, through restructuring the C‐terminal part of the t‐SNAREs.

Support or Funding Information

National Institutes of Health grants GM102217
Peroxynitrite Produced in Isolated Guinea Pig Cardiac Mitochondria: Role of Mitochondrial NOS
Authors: Gerdes Harrison John , Heisner James S , Yang MeiYing , Camara Amadou KS , Stowe David F.
Published: November 2023

The presence of an endogenous nitric oxide synthase (NOS) isoform in cardiac mitochondria remains controversial. NOS catalyzes production of the gaseous signaling molecule nitric oxide (NO•) from arginine. During cardiac ischemia/reperfusion (IR) injury, NO• can react with superoxide (O2•−), derived from a free electron and O2, to form the strong oxidant peroxynitrite (ONOO−), which initiates deleterious post‐translational modifications of cellular proteins and can cause tissue damage. We hypothesized that isolated mitochondria contain endogenous NOS. Our aim was to assess production of ONOO− in isolated cardiac mitochondria under conditions of oxidative/nitrosative stress, and to determine if ONOO− production is dependent on the presence of endogenous mitochondrial NOS. To investigate first if isolated guinea pig cardiac mitochondria contain NOS, Western blot analyses were performed using whole‐heart tissue and percoll‐purified cardiac mitochondria samples with anti‐eNOS and anti‐nNOS antibodies. The bands showed that the reacted eNOS antibody (140 kDa), but not the nNOS, antibody, was detected in both the whole‐heart and isolated mitochondria samples, where band density was much higher. Additionally, mitochondria were suspended in respiratory buffer (pH 7.15), and treated with succinate, a complex II substrate, and CaCl2 followed by menadione, a redox cycler that generates large amounts of O2•−. In physiological buffer, added L‐tyrosine is oxidized by ONOO− to form the fluorescent dimer dityrosine (diTyr). We found that exposing isolated mitochondria energized with succinate to CaCl2 and menadione resulted in an increase in the diTyr fluorescence signal, indicating that the simulated IR injury conditions enhanced mitochondrial ONOO− production. To verify that mitochondrial ONOO− production was dependent on NOS, mitochondria were incubated with the NOS inhibitors L‐NAME/L‐NNA, PTIO, a potent global NO• scavenger, and TEMPOL, a superoxide dismutase (SOD) mimetic. L‐NAME, L‐NNA and PTIO significantly decreased succinate, Ca2+ and menadione‐mediated ONOO− production. TEMPOL also decreased ONOO− levels by converting O2•− to H2O2, as assessed by the HRP/amplex red assay. Some reports furnish evidence that NOS migrates from the cytosol to locate in mitochondrial outer membrane. These results suggest that mitochondrial NOS is responsible for the production of ONOO− during simulated oxidative/ nitrosative stress.

Support or Funding Information

National Heart, Lung, and Blood Institute Training Grant T35 HL072483
Antidiabetic activity and gene expression profiling of db mice treated with Catharsius molossus (a type of dung beetle) glycosaminoglycan
Authors: Ahn Mi Young , Kim Ha Jeong , Kim Ban Ji , Hwang Jae Sam
Published: November 2023

Anti‐diabetic activity of Catharsius molossus (Ca, a type of dung beetle) glycosaminoglycan (G) was evaluated to reducing glucose, creatinine kinase, triglyceride and free fatty acid levels in Db mice. Diabetic mice were orally administered with 5 groups: Db heterozygous, (PBS, control), Db homozygous (PBS, control), CaG (5 mg/kg), Bombus ignitus (a type of bumble bee) queen (IQG5, 5 mg/kg) and metformin (10 mg/kg), for 1 month. CaG's sero‐biochemial levels were evaluated to determine their anti‐diabetic, and anti‐inflammatory in rats after 1 month CaG or IQG treatments in Db mice. Blood glucose level was decreased by treatment of CaG. CaG produced meaningful anti‐diabetic effect showing inhibition of creatinine kinase and alkaline phosphatase levels. As diabetic parameters, sero‐glucose level, total cholesterol and triglyceride were significantly decreased in CaG5 treated group compared to control's. Dung beetle glycosaminoglycan, compared to control, could be potential therapeutic agents for anti‐diabetic effect in diabetic mice. CaG5 treated rat group, compared to control, showed that 48 genes including cytochrome P450, family 8/2, subfamily b, polypeptide 1 (Cyp8b1), 5′‐nucleotidase, ecto (Nt5e) and myo‐inositol1‐phosphatesynthase A1 (Isyna1) were up‐regulated and 79 genes including S100 calcium binding protein A9 (S100a9), immunoglobulin kappa chain complex (Igk), interferon‐inducible GTPase 1‐like (LOC630751) and immunoglobulin heavy constantmu (Ighm) were down‐regulated. Data suggest mitochondrial thymidine kinase (mt‐TK), was up‐ regulated, and calgranulin A (S100a9) were down‐regulated by CaG5 treatment indicating to be potential therapeutic markers for anti‐diabetic agent.

Support or Funding Information

This work was supported by the Rural Development Administration Basic Research project, PJ011853.
Anti‐cancer effect and gene expression profiling of melanoma induced mice treated with dung beetle glycosaminoglycan
Authors: Ahn Mi Young , Kim Ha Jeong , Kim Ban Ji , Hwang Jae Sam
Published: November 2023

Dung beetle (Catharsius molossus) glycosaminoglycan (CaG) possessed anti‐cancer activities of elongation in survival time showing reduced melanoma tumor sizes in treated CaG melanoma induced mice. Thus microarray analyses were performed with a rat 28K cDNA clone set array to identify the gene‐expression profiles for C57BL/6N mice treated with the CaG, 5 mg/kg, over a month for investigation of anti‐cancer effect compared with queen of Bombus ignitus glycosaminoglycan (IQG, 5 mg/kg). CaG5 or IQG5 produced meaningful anti‐cancer effect showing inhibition of melanoma cell growth in XTT assay. In addition, treated theses glycosaminoglycan increased tissue inhibitor of metalloprotease 2 (TIMP‐2) activities in HMVEC cells pretreated by TNF‐alpha or melanoma cells. CaG5 treated rat group, compared to control, showed that 192 genes including collagen, type XII, alpha1 (Col12a1), and hemoglobin, beta adult minor chain (Hbb‐bt), amino levulinic acid synthase2, erythroid (Alas2) and heparanase (Hpse) were up‐regulated and 152 genes including nuclear RNA export factor (Nxf3), hyaluronan and proteoglycan link protein1 (Hapln1), G protein‐coupled receptor17 (Gpr17) and myelin protein zero (Mpz) were down‐regulated. Data suggest collagen, type XII, alpha1, was up‐regulated, and nuclear RNA export factor (Nxf3) were down‐regulated by CaG5 treatment indicating to be potential therapeutic markers for anti‐cancer agent

Support or Funding Information

This work was supported by the basic research of National Academy of Agricultural Science, Rural Development Administration, PJ011853.
Biological roles of oligosaccharides on recombinant eel LH and functions of eelLH receptors
Authors: Byambaragchaa Munkhzaya , Kang Myung‐Hwa , Kim Dae‐Jung , Min Kwan‐Sik
Published: November 2023

The goandotropins (GTHs) follicle‐stimulating hormone (GTH1) and luteinizing hormone (GTH11) are produced in the pituitary gland of all vertebrates, including fish species, and they are key regulators of gonadal development and reproduction. In this study, we produced the deglycosylated mutants of rec‐eelGTHll and analyzed their biological activity using eel LH receptor cell lines. In the hormone secretion, the glycosylation site as Asnα56 dosen't affect, while glycosylation sites at Asnα79 and Asnβ10 have a little effect at the hormone expression into the medium. In western blotting analysis, rec‐LH was detected bands of 32 kDa and the molecular weight was decreased about 2–3 kDa in the single glycosylation site mutants. Deglycosylation treatment by PNGase F was decreased to 25 kDa at the molecular weight of all mutants. Oligosaccharides at Asn56 on α subunit and at Asn10 on β subunit are crucial for eelLH biological activity by using cell lines expressing eel LH receptor.

Support or Funding Information

This work was supported by the project “Seeding production of eel, Anguilla japonica by artificial induction of sexual maturation” (R2017003).