bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Nucleotide Exchange Factor Sil1 Modulates Redox Signaling through the Molecular Chaperone BiP
Authors: Siegenthaler Kevin , Pareja Kristeen , Wang Jie , Sevier Carolyn 
Published: November 2023

The endoplasmic reticulum (ER) is responsible for the secretion of approximately one third of the eukaryotic proteome. The oxidizing environment within the ER lumen allows for the formation of disulfide bonds present in many secreted proteins. However, oxidative protein folding generates hydrogen peroxide, which can over oxidize the ER and disrupt protein folding. Since protein flux through the ER can vary widely in a cell depending on developmental phase and environmental factors, the oxidative status of the organelle can fluctuate. It is critical that cells maintain the fidelity of protein folding despite the fluctuating redox environment. One way cells do this is by regulating the activity of the lumenal Hsp70 chaperone, BiP. When hydrogen peroxide accumulates in the ER, a conserved cysteine residue in BiP is oxidized to cysteine sulfenic acid. This decouples the normally ATPase driven chaperones nucleotide hydrolysis cycle from its peptide binding, resulting in constitutively higher peptide binding by the chaperone. BiP therefore tightly binds translocating proteins and maintains them in a folding‐competent state until oxidative stress levels subside.While BiP oxidation is protective during periods of oxidative stress, constitutive modification decreases cell fitness, raising the question of how BiP is reduced post stress. Unexpectedly, we have found that Sil1, one of BiP's nucleotide exchange factors, is able to reduce BiP and restore progression through the chaperones ATPase cycle. Sil1's reductase activity is tractable to a catalytic cysteine pair near the proteins N‐terminus and is distinct from its activity as a NEF. We are currently studying the role Sil1's reducing activity plays in the cells oxidative stress response and how this connects to human health. Interestingly, mutations in the Sil1 gene are associated with the cerebellar ataxia Marinesco Sjögren syndrome (MSS), suggesting loss of Sil1's reducing activity may cause ER dysfunction that manifests itself in a multi‐system disorder.Support or Funding InformationR01 GM105958 T32 GM007273
Synthesis of a Novel Endoperoxide and Evaluation of the Drug's Ability to Induce Apoptosis in Colon Cancer Cells
Authors: Devlin Jason R , Gornick Emily S , Vavrek Tyler R , Rubush David M , Sarathy Jayashree 
Published: November 2023

Artemisinin is currently the frontline treatment for malaria and it has recently demonstrated potential as an anticancer drug. The key pharmacophore in artemisinin is an endoperoxide which generates ROS in the presence of reducing agents. It has been shown to work by decreasing cell proliferations, inducing apoptosis and cell death, and might involve ROS generation in intracellular compartments. In this study, in an effort to probe this reactivity and develop more potent endoperoxides, we developed a simple and economical synthesis of novel endoperoxide containing small molecules. Our unique methodology combines imines and peroxyquinols to form novel 1,2,4‐dioxazinanes in a one‐pot cascade reaction. 1,2,4‐dioxazinanes are a largely unexplored class of molecules with potential anticancer activity. One therapy to control cancer with minimal side effects would be to use non‐toxic natural products or their derivatives that could cause cell death by inducing apoptosis. Therefore, the efficacy of one of the synthetic derivatives, namely DMR‐1, to induce apoptosis in cancer cells was studied using a human colon carcinoma cell line, T84. Cells were grown to confluency in 12 well plates in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle medium supplemented with 2.5 mM L‐‐glutamine and fetal bovine serum, 5%. Cells treated with increasing doses (5 – 100μM) of DMR‐1 in serum‐free media for 18 hrs were labeled with Annexin V and propidium iodide to assess apoptosis and cell death using an Accuri Flow cytometer. We report that DMR‐1 increased apoptosis at concentration as low as 5μM; however, there was no dose‐dependent increase in apoptosis [AnnexinV positive cells, in %; Control: 9±1; 5 μM: 20±0.4; 10 μM: 15±2; 50 μM: 12±1; 100 μM: 11±1]. Other artemisinin derivatives have been shown to be more effective in rapidly growing cell lines and at higher concentrations (>200 μM). T84 cells are slow growing cells and also have a tendency to secrete mucin, thereby requiring higher doses. Therefore, our future studies will include using higher doses of DMR‐1 and also studying the effect of DMR‐1 (and other derivatives synthesized in our laboratory) on rapidly growing cancer cells such as A549 lung cancer cells. If DMR‐1 is found to be effective in inducing apoptosis in these cells, the mechanism of action will be determined.Support or Funding InformationThis research is supported by NSF ‐ MRI: DBI‐1427937 and Institutional Funds, Ben. U. to JS and to DMR
Mapping calmodulin‐induced oxidase domain interactions of neuronal nitric oxide synthase using H/D exchange mass spectrometry
Authors: Barrett Morgan F , Carley Jeffrey R , Underbakke Eric S 
Published: November 2023

Nitric oxide (NO) is a short‐lived, freely diffusible gas that serves as an omnidirectional neurotransmitter. As such, NO signaling must be controlled through regulation of localization and activation. Neuronal nitric oxide synthase (nNOS) generates NO at a heme‐containing active site upon Ca2+‐induced activation mediated by calmodulin (CaM). We sought to apply hydrogen/deuterium exchange mass spectrometry (HDX‐MS) to investigate how CaM influences catalysis at the heme domain. HDX‐MS can reveal conformational changes and binding sites by monitoring perturbations in amide proton exchange rate changes. To assess how CaM impacts the electron transport and NO production at the heme domain, we compared inactive nNOS with Ca2+‐CaM‐activated nNOS.HDX‐MS analysis showed several localized perturbations due to Ca2+/CaM‐binding. Here, we highlight two major effects. First, the CaM‐binding linker exhibited profound decreases in exchange rate, indicating burial and helix formation. Activated nNOS also exhibited exchange rate changes in the oxidase domain suggesting a delivery mechanism for reducing equivalents into the active site. Ultimately, HDX‐MS revealed several important nNOS conformational changes and inter‐domain dynamics responsible for Ca2+/CaM‐gated activation of NO production.
Aspirin: A Regulator of Tumor Angiogenesis in Breast Cancer
Authors: Maity Gargi , Chakraborty Jinia , Banerjee Snigdha , Banerjee Sushanta K 
Published: November 2023

Background and ObjectiveTumor neovascularization / angiogenesisis a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs to supply adequate nutrition to cancer cells for metastatic growth. Therefore, controlling tumor angiogenesis is an attractive target for cancer therapy. Structural abnormalities of vasculature (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA)has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis.MethodsWe investigated the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA‐treated(2.5mM) conditioned media (231‐CMASA) or vehicle‐treated conditioned media (231‐CMVT) of MDA‐MB‐231 cells on different endothelial cell physiology. These include endothelial cells' migration towards serum using modified Boyden chamber assay, in vitro capillary‐like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes‐endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on invivo tumor angiogenesis using in vivo Angiogenesis Assay.Results and ConclusionsWe found that 231‐CMASA significantly blocks in vitro migration, the formation of in vitro capillary‐like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231‐CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF‐A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis.Support or Funding InformationThis work was supported by VA Merit Review Grants (S. Banerjee and S.K. Banerjee).
Genetic variants of tumor necrosis factor‐α (rs1800629) gene enhances susceptibility to malaria infection in West Africa
Authors: Snyder Tara , Noble Jenelle , Funwei Roland , Falade Catherine , Ojurongbe Olusola , Thomas Bolaji N 
Published: November 2023

Malaria is still a disease that causes untold human death and suffering in many parts of the world. Despite the fact that the parasite genome has been sequenced, much remain to be deciphered, especially on factors that exacerbates or limit susceptibility to infection. We hypothesize that host genetics factors, with particular focus on genetic polymorphisms that affects gene transcription and variability in immune response, are major contributors to host susceptibility, resistance or disease severity. Therefore, we elucidated the genomic diversity and haplotype frequency of tumor nectrosis factor‐a (snp rs1800629) genes among and between malaria‐infected and control groups in West Africa, and extrapolated their role, if any, on parasitemia. To do this, genomic DNA samples obtained from 107 malaria‐infected (microscopy and PCR‐confirmed) patients and 380 uninfected controls from Nigeria, were subjected to a PCR‐RFLP analysis. We report that there is no significant difference in the genotypic (p=0.15; 55.2% versus 50.0% for malaria‐infected and controls respectively) or allelic frequency (p=0.21; 3.8% versus 8.4% for malaria‐infected and controls respectively) of the mutant variant of the vitamin D receptor (rs731236) gene. However, we found a statistically significant difference in the genotypic (p=2.20E‐16) and allelic frequencies (p=2.20E‐16) of the tumor nectrosis factor‐a (snp rs1800629) gene between malaria‐infected and control groups. The highest frequency of the mutant variant (77.6%) was found in the malaria‐infected group with the opposite reported among controls. In addition, haplotype frequencies were different between groups, with the most common haplotypes combining one form of the tumor nectrosis factor‐a mutant variant with a combination of the VDR mutant or wild type variant. Our observation clearly shows that tumor necrosis factor, as a proinflammatory cytokine, necessary to intitiate innate immune response is a major factor in susceptibility to malaria in West Africa. The preponderance of the mutant gene implies these patients cannot mount an effective immune response leaving parasitemia to increase uncontrolled until the initiation of adaptive immune response, thereby worsening disease. This, more than any other, may be the major factor delineating who is susceptible to disease or not, or the degree of infectivity and parasite load post‐infection. Future studies elucidating this genetic data in clearly delineated mild, uncomplicated versus severe disease groups viz a viz serum assays are ongoing.Support or Funding InformationFunding provided by College of Health Sciences and Technology Faculty Development and Laboratory Support Fund, Rochester Institute of Technology
Effect of the Mitochondrial DNA 4977‐bp “Common Deletion” on Metabolism and Cell Growth in Human Lymphoblast Cell Lines
Authors: Keck Christopher , Gulfo Michelle , O'Donnell Robert , Hoops Harold , Pogozelski Wendy 
Published: November 2023

ObjectiveTo determine the effects of elevated levels of the common deletion ΔmtDNA4977 on glucose metabolism, oxidative phosphorylation, and cell growthIntroductionHuman mitochondria (mt) contain circular DNA molecules of 16549 base pairs. Because replication of mtDNA is asynchronous, and because the sequence contains repeats, mispairing can occur during replication, resulting in large deletions. One particular deletion of 4977‐bp is associated with several sporadic and degenerative diseases and is therefore called the “Common Deletion” (ΔmtDNA4977),. The deleted sequence codes for small regions of Complexes I, IV and V in the mitochondrial respiration chain. While resulting in truncated respiratory complexes and reduced cellular lifetimes, mtDNA bearing this deletion are nonetheless tolerated; cells can be functional due to the redundancy of mtDNA copies and the fact that normal and abnormal mtDNAs co‐exist in a heterplasmic state. However, if the level of mtDNA bearing this common deletion (ΔmtDNA4977), rises above a threshold, energy production is compromised enough to result in physiological symptoms. What has not been well‐understood are the details of how metabolism and cell growth are affected.MethodsWe are using lymphoblasts derived from a family with mitochondrial disease. Using real‐time PCR, we previously showed that cells derived from the son of the family (the 848 cell line) had 7% of the mtDNA bearing the common deletion. The boy's sister (849 cell line) had undetectable levels of the deletion and thus her cell line serves as a normal control. We compared the growth and metabolism of these cell lines in media of varying glucose content, measured the rates of glycolysis with a lactate assay, measured oxidative phosphorylation via oxygen consumption, and studied the shapes of the cells via microscopy. We also investigated the ability of succinic acid to rescue metabolically deficient cells.ResultsWhen compared with normal cells, initial results showed that the cells with elevated ΔmtDNA4977 1) grew more slowly; 2) grew poorly in media with low glucose; 3) showed higher rates of glycolysis; 4) formed spheroids more readily; and 5) were more often polarized in shape. Normal control cells grew well in all media types tested; however, when grown in high glucose, normal control cells were polarized more often. Succinic acid supplementation did not substantially affect growth of either cell type. Initial results suggest that the cell line with the increased levels of the common deletion ΔmtDNA4977 has an increased dependence on glucose metabolism and experiences activation of mechanisms that result in projections and polarization.Support or Funding InformationFunding is acknowledged from the Geneseo Foundation and Student Foundation
Hydrodynamic Delivery of Isocitrate Dehydrogenase II for the Treatment of Acute Kidney Injury
Authors: Kolb Alexander , Atkinson Simon , Bacallao Robert 
Published: November 2023

Mitochondrial Respiration is a critical process for normal kidney function. Kidneys that undergo an Acute Kidney Injury (AKI), specifically those injuries that result from ischemia, lead to changes in mitochondrial function and respiration. These changes in respiration are due to changes in TCA, Electron Transport Chain complexes, and regulation of ROS levels. To study mitochondrial changes in the kidney as a result of an AKI a proteomic screen was conducted on kidneys two weeks after ischemic injury. The screen indicated that in response to an ischemic event there was an upregulation of proteins that may play a pivotal role in kidney recovery and subsequent protection from future ischemic events. One such protein, Isocitrate Dehydrogenase 2 (IDH2) plays an important role in the TCA and ROS clearance. Treatment of AKI is limited to supportive care, which was reported to cost patients approximately 10 billion dollars in 2012. The limitations and cost of treatment lead to the idea of gene therapy as a possible means of treatment. However, delivering genes directly to the kidney and getting sufficient levels of expression has proven difficult. Recently a novel technique, hydrodynamic delivery, has shown promising results as a possible route of delivery for our genes. This technique uses pressure as a means to deliver genes, dyes, and vectors into the kidney. Our data indicates that hydrodynamic delivery of genes, such as IDH2, block creatinine increase, increase mitochondrial membrane potential, respiration, NAD(P)H and ATP levels compared to control or saline only models.Support or Funding InformationNIH/NIDDKVA merit
Diabetic Nephropathy Accelerated by Imbalance of Renal Renin‐Angiotensin System Components in db/db Mice with High Fat Diet
Authors: Wang Guan‐Hua , Cho Ching‐Chang , Lin Chung‐I , Wu Hsi‐Tien , Lin Chih‐Sheng 
Published: November 2023

Fat accumulation is associated with insulin resistance that results from overactivation of angiotensin converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 receptor (AT1R) axis of renin‐angiotensin system (RAS). Ang II induces macrophage infiltration into adipose tissue and increases pro‐inflammatory cytokines (TNF‐alpha and IL‐6) to inhibit insulin receptor that causes insulin resistance and hyperglycemia.Several studies support antihypertensive efficacy of RAS blockade in the patients with type 2 diabetes and obesity. Future studies should address the role of kidney‐specific deficiency of RAS components to definitively determine the relevance of renal RAS to the disease progression related with diabetic or obesity‐related nephropathy. Due to BKS db/db mice is similar in human type 2 diabetes which can be spontaneously induced diabetic nephropathy. Therefore, male BKS db/db mice were fed a high‐fat diet (HFD; 60% of fat in diet) from 8 to 12 weeks of age and fed a normal diet (ND) from 8 to (12 or 20) weeks of age then the animals were sacrificed for the further assays in this study. The changes of serum and urine biochemistry, renal MAPK and STAT3 signaling pathways, RAS components, and pathology associated with diabetic nephropathy were evaluated in these mice.According to the results of body and kidney weight, serum (triglycerides, total cholesterol, glucose, creatinine, blood urea nitrogen, and albumin) and urine (creatinine, blood urea nitrogen, and albumin) biochemical assays, our data confirm the previous reports that db/db mice can be spontaneously induced hyperglycemia at 8 week of age and progressive nephropathy at age of approximate 20 weeks in the mice fed with ND. However, nephropathy and severe diabetic dyslipidemia was occurred early at age of 12 weeks in the db/db mice fed with HFD. Increasing expression levels of renal ACE and chymase would synergistically generate more Ang II and then abnormally flare‐up ACE/AT1R axis that leads local inflammation and promote the progression of nephropathy. Vascular remodeling via MAPK signaling factor, p‐MEK and p‐ERK1/2, an inflammatory signaling factor, p‐STAT3, in the kidney of HFD‐fed db/db mice were significantly higher than those in the ND‐fed db/db mice. The pathological changes also show that diffuse glomerulosclerosis and mesangial matrix fraction in the kidney tissue of HFD‐fed db/db mice were more serious than those in the db/db mice with ND.In conclusion, HFD treatment accelerate impaired kidney function and progressive diabetic nephropathy by increasing renal ACE and chymase activity of RAS in the db/db mice.
Prohibitin has sex dimorphic role in adipose and immune functions
Authors: Xu Yang Xin Zi , Ande Sudharsana , Mishra Suresh 
Published: November 2023

The promoter of a gene that is selectively expressed in just a few cell types provides unique opportunities to study the pleiotropic function of a protein in two different cell types including the cell compartment specific function. This is not possible with a ubiquitous or a highly specific gene promoter. The adipocyte protein‐2 (aP2) is one such gene. It is primarily expressed in adipocytes, but also selectively in monocytic macrophages and dendritic cells among various immune cell types. Thus, the aP2 gene promoter provides an opportunity to simultaneously manipulate adipose and immune functions in a transgenic animal model.Prohibitin (PHB) is a pleiotropic protein with cell compartment specific functions such as mitochondria and the plasma membrane. New evidence suggests that PHB has roles in adipocyte and immune cells. To discern the cell compartment specific functions of PHB in adipocyte and immune cell, we developed two novel transgenic mice by expressing wild type PHB and a phospho mutant PHB (Y114F or m‐PHB) from the aP2 gene promoter, separately. Both transgenic mice develop obesity in a sex‐neutral manner suggesting that m‐PHB retains mitochondria‐related adipogenic function. However, both transgenic mice develop obesity‐related metabolic dysregulation in a male sex‐specific manner. Interestingly, the male PHB mice spontaneously developed T2D and liver cancer, whereas the male m‐PHB mice developed lymph node tumors, or T1D in a context‐dependent manner. This would imply that plasma membrane‐related phosphorylation of PHB at Tyr‐114 has a role in immune cell functions.Collectively, our data suggests that PHB has sex dimorphic roles in adipose and immune functions, which are mediated in a cell compartment specific manner. Also, we provide evidence that sex differences in adipose and immune functions contribute to sex differences in diabetes and cancer, which warrant further investigations.Support or Funding InformationSM is supported by funds from Natural Sciences and Engineering Research Council of Canada (NSERC) and Research Manitoba.