bluuebunny/crossref_metadata_2025 huggingface dataset:
Placental expression of NFAT5, SLC5A3, and aldose reductase in gestational diabetes mellitus
Authors: Mejia Camilo A. , Reynolds Paul R. , Arroyo Juan A.
Published: November 2023

Gestational diabetes mellitus (GDM) is associated with a number of problematic elements that prevent the placenta from transporting necessary nutrients and oxygen important for fetal development. Hypertonic stress occurs in uncontrolled GDM, where complications such as fetal abnormalities, spontaneous abortion or stillbirth can occur. Our objective was to investigate the osmolarity factors in gestational diabetes, where we see significant glucose shifts, which could contribute to large swings in osmolarity. We obtained placental biopsies and paraffin embedded placental tissues slides for GDM‐I (gestational diabetes mellitus treated with insulin) and GDM‐D (gestational diabetes mellitus treated with diet), and term controls from the Research center for Women's and Infants’ Health BioBank, Ontario, Canada. After performing and nuclear and cytosolic extraction on the placental tissue, we were able to determine the cytosolic and nuclear expression of the factors involved in the hyperosmolar response. We observed no significant differences in maternal age, gestational weeks and fetal weight between control and GDM pregnancies. There was an upregulated expression of nuclear NFAT5 in GDM‐I (1.55‐fold, p<0.005) and GDM‐D (1.55‐fold, p<0.001) placentas when compared to control placental tissue. In contrast no significant differences were observed for cytosolic NAFT5. There was an upregulation of SMIT protein in GDM‐I (1.45 fold p<0.05) and GDM‐D (1.50‐fold, p<0.05) when compared to control placental tissue. Furthermore immunohistochemical staining confirmed that these changes were observed in the placental trophoblast. The expression of aldose reductase (AR) was not significantly different across GDM and control placentas. Our findings confirm that a hyperosmolar rescue response is necessary for the management of GDM and that this protection could be driven by an increase of inositol production in the placental trophoblast cells. Treatment variation between diet and insulin tissues was not significant and we therefore conclude that the mechanisms involved in correcting this pathology are similar in both treatments during GDM.
Conjugated Linoleic Acids (CLA) and Insulin Resistance: Differential Effects of CLA Isomers on Endoplasmic Reticulum (ER) Stress
Authors: Dammanahalli Jagadeesha K , Subbaiah Papasani V
Published: November 2023

Conjugated linoleic acids (CLA), the naturally occurring fatty acids prevalent in dairy products, are being used by the general public for potential health benefits, including weight loss, and cancer prevention, but they have also been reported to cause insulin resistance (IR) in animal models. However, the mechanism of action of CLA is not well understood. The current study tested the hypothesis that the development of insulin resistance after consumption of specific CLA isomers is due to the induction of endoplasmic reticulum (ER) stress. We tested the effects of four different CLA isomers, trans‐10,cis‐12 (t10c12)‐, cis 9, trans 11 (c9t11), trans 9 trans 11 (t9t11) and cis 9 cis 11 (c9c11) in three different cell lines‐ HepG2 (hepatocyte), c2c12 (myocyte) and 3T3‐L1 (adipocyte). The cells were treated with 0–100 μM CLA for 24 h, and analyzed for reactive oxygen species (ROS) by flow cytometry, and for ER stress markers by Western blotting. We found that both t9t11, and t10c12‐CLAs induced ROS and the expression of GRP78, the central regulator of ER stress, at 50 μM concentration in HePG2 and 3T3 cells. However these effects were elicited only by the t9t11 isomer in c2c12 cells. GADD153 (CHOP), another marker of ER stress, was induced by t10c12 in HepG2 and 3T3 cells whereas, c9t11, t9t11 and c9c11‐CLAs induced it in 3T3 and c2c12 cells but not in HepG2 cells. Further studies demonstrated that the t10c12 isomer inhibited the insulin‐stimulated phosphorylation of insulin receptor and AKT in HepG2 cells, whereas the c9t11, c9c11 and t9t11 isomers increased the phosphorylation of both proteins. These findings suggest that t10 c12 CLA, the isomer known to cause insulin resistance in mice, may do so by inducing ER stress.

Support or Funding Information

Supported by VA Merit Review BX001090
Decreasing O‐GlcNAc Levels Reverses Diabetic Nephropathy‐Associated Phenotypes
Authors: Peterson Sherket , Delannoy Michael , Banerjee Partha , Hart Gerald
Published: November 2023

Diabetic nephropathy (DN) is a leading cause of morbidity and mortality in diabetic patients. DN is characterized by structural changes of the glomeruli in the kidney, such as mesangial cell expansion, podocyte foot process effacement, and apoptosis, with accompanying glomerular hyperfiltration. Hyperglycemia has been shown to initiate these glomeruli structural changes by stimulating flux through the hexosamine biosynthetic pathway (HBP). The major endpoint of the HBP is the formation of uridine diphosphate β‐D‐N‐acetylglucosamine (UDP‐GlcNAc), the donor for protein O‐GlcNAcylation, and complex extracellular glycosylation. O‐GlcNAcylation is a dynamic nutrient sensitive post‐translational modification that is characterized by the addition of single β‐D‐N‐acetylglucosamine (O‐GlcNAc) to the serine and/or threonine residues of almost every functional class of protein. O‐GlcNAc serves as a nutrient/stress sensor regulating signaling, transcription, cytoskeletal dynamics, and cell division. Altered O‐GlcNAc signaling is directly involved in the pathogenesis of diabetes and may have a role in diabetic complications. However, the molecular mechanisms of how hyperglycemia leads to glomerular dysfunction are largely unknown. Here we report that a dynamic sugar‐protein modification, O‐GlcNAcylation, which serves as a nutrient sensor, links hyperglycemia to defects in glomerular structure and function. By targeting the enzymes that either add or remove O‐GlcNAc (OGT and OGA, respectively), we show that O‐GlcNAcylation regulates mesangial ECM production. Increasing O‐GlcNAc distorts gene expression, protein expression, and subcellular distribution of key slit diaphragm specific proteins, whereas decreasing O‐GlcNAcylation, even in hyperglycemic conditions, restore podocytes to normal. Electron microscopy of the slit diaphragm reveals that decreasing O‐GlcNAc rescues the DN‐associated phenotype. This study provides evidence that O‐GlcNAc plays a role in glomerular structure and function and may contribute to the molecular etiology of DN‐associated phenotypes.

Support or Funding Information

This work is supported by NIH grants F32DK100199 to S. Peterson and R01DK61671, P01HL107153, and N01‐HV‐00240 to G. Hart.
Molecular Mechanism of Insulin Resistance in Offspring of Obese Fathers
Authors: Liu Xinhao , Ozbolt Joshua , Fuchs Ryan , Zhang Yizhu , Consitt Leslie , Slyvka Yuriy , Nowak Felicia
Published: November 2023

Parental diet and increased BMI are correlated with abnormal metabolic profile of offspring, often as a result of inherited differences in epigenetic marks. Much work has been done to investigate the effect of maternal metabolic abnormalities, such as increased BMI, or protein restriction with pregnancy, on offspring. However fewer studies have been done to investigate the effect of paternal metabolic status on offspring metabolism and disease susceptibility. Hypothesis of our study is insulin resistance developed in offspring is affected by paternal obesity via epigenetic route. Our present goals are 1) to establish the correlation between the paternal obesity induced by high fat diet (HFD) and insulin resistance in offspring; and 2) to investigate the synthesis and trafficking of GLUT4, as this insulin responsive glucose transporter greatly affects the glucose uptake by muscle and adipose cells. To achieve our objectives, male C57B/6 mice were placed on either LFD (Gr1) or HFD (Gr2) for 12 weeks, and then mated with females who had been on LFD. Offspring were maintained on regular chow. Glucose Tolerance Test GTT insulin level was obtained and HOMA was calculated based on blood insulin and glucose level. Gastrocnemius muscles were obtained from male and female offspring at 6 weeks, 6 months and 1 year. Total cell homogenates and subcellular plasma membrane and GSV fraction were analyzed for GLUT‐4 by Western blotting. Results showed there are significantly increased GTT insulin level and HOMA, a measure of inulin sensitivity, in 5–6 weeks in male offspring in Gr2 when compared with Gr1. Expression of total Glut‐4, 46kd band, shows no difference between offspring in Gr1 and Gr2. We have developed a subcellular fractionation protocol to measure the plasma membrane and Glucose Storage Vesicles (GSV) fraction to investigate Glut‐4 trafficking, which can affect GLUT‐4 function independent of a change in total GLUT‐4 protein. Future studies will include PCR quantification of mRNA for key genes involved in GLUT‐4 synthesis or trafficking and analysis of group differences in epigenetic marks. Identification of the underlying epigenetic mechanism of the change in insulin sensitivity will allow us to develop the diagnostic, therapeutic, and preventative strategies against obesity and diabetes in the offspring of obese parents.
Identification of Novel Antimicrobial Peptides to be used in Functional Foods
Authors: Doering Sean , Thomas Michael , Grant Jennifer E , Lee Eun Joo
Published: November 2023

Phosvitin, an egg yolk protein similar to casein in milk, is the most phosphorylated protein found in nature. The resulting anionic charge allows phosvitin to bind important bivalent ions such as zinc, iron, magnesium, and calcium. Because of its metal binding properties, phosvitin shows intriguing potential as a source of peptides that could be used in functional foods. Antibacterial properties of phosvitin have come to light as well. In this abstract we report on the antibacterial properties associated with phosvitin, and also discuss work to identify potential peptides that could be used in functional foods as either calcium delivery vehicles or antibacterial agents.

When bound to a bivalent cation such as zinc, phosvitin has previously been shown to elicit antimicrobial activity against bacterial species isolated from human hands (unpublished results). Since Serratia marsescens is a hand bacteria associated with human pathology, an isolate of this bacteria was utilized in antibacterial tests of phosvitin activity. Both Kirby Bauer and serial dilution antimicrobial tests were utilized to determine any potential antimicrobial activity. In Kirby Bauer antimicrobial tests, paper discs were soaked in 1mM ZnCl2 solutions with phosvitin in varying concentrations. These discs were then placed on LB plates seeded with S. marsescens and grown at 25 degrees Celsius to determine if rings of inhibition formed around the treated discs. In serial dilution tests, phosvitin was mixed in various concentrations in a 1mM ZnCl2 LB broth solution seeded with S. marsescens. These solutions were then incubated at 25 degrees Celsius to determine if bacterial growth occurred. Minimum inhibitory concentration (MIC) was determined to be around 5 mg/ml phosvitin in 1mM ZnCl2 LB broth solution. These experiments will be repeated using calcium to determine if antimicrobial effects extend across different phosvitin‐bound bivalent cations.

Work is also underway to identify potential calcium‐binding peptides derived from phosvitin and identify potential antibacterial activity that may be associated with them. Our peptide of interest is residues 2–13 of phosvitin with a phosphorylation post‐translational modification on residue 12 resulting in the peptide of AEFGTEPDAKpTS. The binding capacity and antimicrobial activity of this peptide will be compared to phosvitin as a potential bioavailable replacement.

Support or Funding Information

This work was supported by the University of Wisconsin Stout Student Research Grant, as well as a Student Jobs Program Award, to S.D.
Progress Towards Addressing Gram Negative Bacterial Resistance
Authors: Koleva Bilyana N. , Takrouri Khuloud , Cooper Harold D. , Spaulding Andrew , Zucchi Paola , Cleary Dillon , Tear Wesley , Hirsch Elizabeth B. , Aggen James B. , Beuning Penny J.
Published: November 2023

Multidrug antibiotic resistance poses an increasingly urgent threat to global health and the world economies. Among antibiotic resistant species, gram negative bacteria present the utmost urgency as they have become resistant to nearly all available treatment options. Combating antibacterial resistance should include continuous introduction of new antibiotic classes; however over the last 40 years there has been a void in antibiotic drug discovery. Extending the spectrum of activities of current classes of antibiotics is another approach. The oxazolidinones are a class of antibiotics that work by interfering with protein translation and are mainly active against gram‐positive bacteria. Linezolid, marketed under the trade name Zyvox®, was the first FDA‐approved oxazolidinone for the treatment of pathogenic gram‐positive bacteria. One focus of this work is to optimize the properties of oxazolidinones in order to penetrate the lipopolysaccharide layer of gram‐negative bacteria while maintaining their target potency. The rational lead optimization campaign based on the linezolid scaffold involves keeping a low molecular weight (MW <400 Da), and high polarity (clog D <1) to allow passage through the porins and to be able to escape the bacterial efflux pumps. A focused library of oxazolidinone analogs was synthesized and evaluated for activity in a panel of wild‐type and outer membrane‐ and or efflux pump‐compromised Escherichia coli and Staphylococcus aureus. Several C‐ring modification analogs offered moderate activity against gram negative bacteria and seem to improve membrane permeability and evasion of the efflux pumps. A subset of these compounds was tested in the context of an in vitro transcription and translation assay and most were found to be comparable to linezolid in terms of efficacy.
Genesis of Antibiotic Resistance XII: Trifling Role of SMARXT DISPOSAL TM to Mitigate the Evolution of Antibiotic Resistance
Authors: Kannan Subburaj , Ambriz Paloma , Flores Oscar , Fernandez Priscilla , Carrales Nallely , Mata Eduardo , Moreno Analicia , Herrera Estefania , Hernandez Jazania
Published: November 2023

SMARXT DISPOSAL has been identified as public‐private partnership between the U.S. Fish and Wildlife Service, the American Pharmacists Association, and the Pharmaceutical Research and Manufacturers of America and North Plains Groundwater Conservation District in Texas) (NPGCD‐Texas). Methodical disposal of unused prescription drugs in NPGCD‐Texas monitored by multiple agencies has been suggested to mitigate the contamination of ground water by the Pharmaceuticals and Personal Care Products (PPCPs) including the antibiotics and antimicrobials. Systematic analysis of the data on file for the pharmaceuticals including the antibiotics and antimicrobials in the water quality data analysis of NPGCD‐Texas has been completed. The analysis of the water quality data show that neither the pharmaceuticals nor the antibiotics/antimicrobials were performed in the Ogallala aquifer. The Ogallala aquifer contains groundwater, with little to no recharge and over 8000 active wells within this district. As identified by the State of Texas, it is a finite and dwindling resource of water in Texas Panhandle. United States Geological Survey (USGS), and Texas Water Development Board(TWDB) presented an antiquated water quality analysis in NPGCD which failed to provide any relevant data on PPCPs in particular antibiotics concentration in the soil and water. Sedimentation analysis on residual metabolite intermediates has not yet been performed. TCEQ's data bases is devoid of either antibiotics or metabolite intermediate or its derivative in NPGCD. Coincidental, lack of water quality analysis for PPCP including antibiotics/antimicrobials is a viable threat to the public health. Data analysis for PPCP, on data communicated by the NPGCD‐Texas will be presented at EB 2016.

Support or Funding Information

Professional development funds provided to S.Kannan at SWTJC for 2014–2015
Genesis of Antibiotic Resistance XIII: Critical Appraisal of incineration of Prescription Drug en masse from National Drug Take Back day (NDTBD) by United States Drug Enforcement Administration – Department of Justice (US‐DEA‐DOJ)
Authors: Kannan Subburaj , Munoz Janet , Luna Jennifer , Rosales Ramiro , Rodriguez Jennifer , Ruiz Vanessa , Velez Micaela , Rodriguez Lizzeth , Chavez Dariela , Subburaj Prabavathy
Published: November 2023

An effort has been to demonstrate that the incineration of Prescription Drug en masse impart potential environmental hazard. According to http://www.natracare.com/en‐GB/ the current data shows that even in “state‐of‐the‐art” incinerators, burning hazardous waste will lead to the release of three types of dangerous pollutants into our environment: heavy metals; unburned toxic chemicals; and new pollutants ‐ entirely new chemicals formed during the incineration process (Verbatim). According to the US Environmental Protection Authority: “Fugitive emissions and accidental spills may release as much or even more toxic material into the environment than direct emissions from incomplete waste incineration.” According to Greenpeace, studies have identified up to 43 different semi‐volatile, organic chemicals in incinerator ash, and at least 16 organic chemicals in scrubber water from hazardous waste incinerators. Ash is commonly buried in landfill, while effluent is often treated before being discharged into rivers or lakes. Fragments of partially burned waste chemicals recombine within incinerator furnaces, smokestacks, and/or pollution control devices. One study identified 250 volatile organic compounds, many of which are known to be highly toxic or carcinogenic. Among the possible compounds are dioxins and furans, often referred to as just dioxins suppression of the immune system, diabetes, organ toxicity and a wide range of effects on hormones. It is estimated that for every three tonnes of waste that is incinerated, one tonne of ash is generated. Projection analysis on the amount of fly ash generated due to the incineration of drugs from National Drug Take Back day (NDTBD) by United States Drug Enforcement Administration – Department of Justice (US‐DEA‐DOJ) mount 3 fold increase in the environmental contamination. However, taking into account of anaerobic digestion of the drugs from NDTBD – US‐DEA‐DOJ could potentially decrease the methane emissions by 5 – 7 folds compare to the industrial chemicals disposal standard. Taken together, the projection analysis data indicate that there is a statistically significant increase in the environmental contamination by incineration the drugs from NDTBD – US‐DEA‐DOJ. Data analysis for the environmental impact assessment of chemical pollutant will be presented at EB 2016.

Support or Funding Information

Professional Development Funds provided to S. Kannan at SWTJC for 2014–2015
Genesis of Antibiotic Resistance XVI: “Role of Lax Regulations on Pharmaceuticals and personal care products (PPCPs) in AR Pandemic” – Antibiotics, and Estradiol are detected in four surface water sites of Edwards Aquifer Authority (EAA), San Antonio, Texas
Authors: Kannan Subburaj , Carrizales Diana , Carrizales Tania , Hernandez Martin , Moody Martin
Published: November 2023

EAA, Hydrologic Data Report, Report No:14‐02, December 2014 for 2013, for the calendar year 2013 was reviewed regarding the detection of PPCPs. In view of the public sentiments, information pertaining to detection of PPCPs, reported on page 58–59 of the report is presented verbatim: Water samples collected from four surface water sites and four springs were analyzed for PPCPs in 2013. PPCPs compounds were detected in all four surface sites, and none of the spring samples tested positive for PPCP compounds. These detections are in the nanogram per liter (ng/L) range. Detections are summarized by sample site below. Surface Water PPCPs: Blanco River at Wimberley: Trimethoprim—(antibiotic) detected at 5.45 J ng/L ; Cibolo Creek (near the Nature Center in Kendall County): 17a‐Estradiol—(synthetic estrogen hormone) detected at 1.57 J ng/L ; Trimethoprim—(antibiotic) detected at 10.6 ng/L; Triclocarban—(ingredient in antibacterial soaps) detected at 3.95 J ng/L; Sulfamethoxazole— (antibiotic) detected at 391 ng/L ; Triclosan—(ingredient in antibacterial soaps) detected at 9.41 J ng/L ; Frio River at Concan: Diltiazem—(blood pressure medication) detected at 0.997 J ng/L; San Geronimo Creek: 17a‐Estradiol—(synthetic estrogen hormone) detected at 1.27 J ng/L ; Taken together, presence of PPCP in EAA, pose a insurmountable and also a vital contributing factor for development of AR in the consumers. The prospect and scope of lax regulations (in Texas! / United States!) on PPCPs level in water quality analysis and its role in AR pandemic shall be discussed in EB2016. Acknowledgement: http://www.edwardsaquifer.org/scientific‐research‐and‐reports/hydrologic‐data‐reports & Technical Staff of Water Quality – EAA, San Antonio, Texas)

Support or Funding Information

Professional development funds provided by SWTJC to S.Kannan for 2014–2015
Inclusion of Chicken Broth AAC1 as a Dietary Supplement Modulates the Gut Microbiome: Results of Next‐Generation DNA Sequencing
Authors: Hawkins Jordan , Norton Rhy , Durham Paul
Published: November 2023

Objective

The focus of this study was to characterize shifts in the rodent gut microbiota that occur as a result from the oral administration of the commercial chicken broth formulation AAC1.

Background

There is emerging evidence of the importance of maintaining a healthy gut microbiome. Changes in the ratio of the phylum Bacteroidetes to the phylum Firmicutes are associated with various metabolic disorders, obesity, and aging. Diet plays a key role in helping to promote the growth of bacteria that function to modulate inflammation in the gut and other tissues including the nervous system. Although many cultures report the benefit of chicken broth, the effect on the gut microbiome is not well understood.

Methods

Young adult male Sprague‐Dawley rats received 0.5% solid (w/v) AAC1 dissolved in their drinking water for three weeks. Basal fecal samples were collected prior to the inclusion of chicken broth into the diet. An additional control group was included that only received tap water. Fecal samples were collected from each animal at day 0 (basal) and day 21 (end of feeding). Also on day 21, animals were euthanized and caecal samples were collected. Total DNA was extracted and amplified from the V3‐V4 region of the 16S rRNA gene with sample‐specific barcode sequences prior to 454‐pyrosequencing. Returned sequences were assigned taxonomic classification using the SILVA rRNA database. Microbial community analysis was performed using phylogenetic packages in R. Only returned sequences greater than 96% identical to a known, published sequence were included in this analysis.

Results

A phylogenetic analysis of this raw data produced a total of 106 operational taxonomic units. Importantly, AAC1 did not alter the Bacteroidetes: Firmicutes ratio in fecal samples after 21 days while promoting the growth of Proteobacteria. Inclusion of AAC1 in the diet resulted in an increase in several operational taxonomic units including Faecalibacterium, Parasutterella, Morganella, and Oscillospira. In the cecum samples, AAC1 selectively increased expression of Lactobacillus as well as Odoribacter, Gastranaerophilales, Faecalibacterium, Allabaculum, Coprococcus, and Oscillibacter while decreasing the levels of Intestimonas.

Conclusions

Results from our study provide evidence that dietary inclusion of AAC1 chicken broth causes a shift in composition of the rodent gut microbiota to increase the percentage of bacteria reported to aid in overall digestive health, decrease inflammation, mitigate the disruptive effects of antibiotics, and reduce the number of sick leave days in adults. Thus, our findings support the notion that AAC1 might be useful as a therapeutic prebiotic.

Support or Funding Information

International Dehydrated Food
Protein Dynamics Defines Allostery in the Conserved EPK Core
Authors: Ahuja Lalima G , Kornev Alexandr P , McClendon Christopher , Veglia Gianluigi , Taylor Susan
Published: November 2023

The Eukaryotic Protein Kinases (EPKs) share a conserved hydrophobic core that conceptualizes our understanding of their regulation and catalysis. This EPK core was first described by 12 subdomains, which were later mapped to conserved elements of secondary structure in the EPK core when the first structure of PKA kinase became available. Much biochemistry, thermodynamics and biophysics has been subsequently done on many EPKs since then to explore similarities and differences in their allosteric control. The discovery of the Hydrophobic Spines in recent years has provided a novel tool for understanding both entropy driven allostery and catalysis. Both entropy driven allostery and catalysis are essentially dependent on internal protein dynamics that has been described by NMR as slow motions in the millisecond time frame. Although time and cost intensive, these NMR studies are essential and provide information for residue side chain and main chain dynamics. However, a number of sites in the EPK core evade labeling, and it becomes difficult to study them. To understand these protein dynamics further we have hence ventured into the in‐silico system of Molecular Dynamics simulations that are increasingly providing convincing information on par with NMR details. Our Graph theory methods applied to these simulations have allowed us to define dynamic clusters of residues whose correlated motions define the allosteric network of the EPK core in the PKA catalytic subunit. We call these clusters as ‘Communities’ and the cluster network is defined by a ‘Community Map’. Community maps of PKA at different steps of the catalytic cycle have allowed for dissection of internal protein dynamics that must work alongside catalysis. These community maps also allow for understanding allosteric mutations such as Y204A in PKA that are far from the active site but affect EPK function. Many such sites in EPKs, which remain unexplained, are found to be mutated in cancer genes. These Community maps hence allow for a novel, yet unexplored dynamics‐based criterion for understanding EPK modulation in molecular medicine.

Support or Funding Information

NIH grant GM19301 and the Howard Hughes Medical Institute
PKA and Calcineurin Scaffolding on AKAP79: A Small Angle Xray/Neutron Scattering Profile
Authors: Hall James , Copps Jeffrey , Zhang Ping , Heck Albert , Heller William , Blumenthal Don , Taylor Susan
Published: November 2023

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIa, RIb, RIIa, and RIIb). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIb holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIb2C2:CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Furthermore, we have identified a functional interaction between RIIb and CN using peptide arrays. It involves the flexible linker regions of RIIb including both of LxVP and RRxSV motifs. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that provide tight spatiotemporal control.

Support or Funding Information

Support for this work was provided through NIH GM034921
An Optogenetic Toolkit for Spatial and Temporal Control of the cAMP Dependent Protein Kinase
Authors: OBanion Colin Padraic , Hughes Robert , Priestman Melanie , Lawrence David
Published: November 2023

Cellular signaling is highly compartmentalized in both time and space as exemplified by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. PKA and associated signaling proteins are sequestered to specific subcellular compartments by A‐kinase anchoring proteins to generate distinct signaling microenvironments. These signaling nodes provide spatial specificity to PKA so that this otherwise ubiquitous signaling pathway is only activated in the right location and at the right time. Although many tools are available to manipulate cellular signaling on a global scale, it is difficult to control intracellular signaling with any degree of spatiotemporal resolution. Here, we present a set of optogenetic tools to control PKA signaling at the plasma membrane, cytoskeleton, and outer mitochondrial membrane. We used molecular engineering approaches in conjunction with biochemical and cell biology assays such as western blotting and fluorescent microscopy to show that activation of our optogenetic toolset in cells results in compartment specific PKA phosphorylation events upon stimulation with light, and that activity is localized to discrete locations within the cell using a PKA reporter system generated by our group.

Support or Funding Information

This work is supported by NINDS 1R21NS093617‐01
SIRT3 prevents bleomycin induced lung fibrosis in mice by blocking mitochondrial DNA damage and ROS synthesis
Authors: Bindu Samik
Published: November 2023

Background and objective

Tissue fibrosis is one of the most critical mechanisms in the pathogenesis of wide range of diseases. Fibrosis is also hallmark of organ dysfunction associated with aging. The process of fibrosis begins with transformation of fibroblast into myofibroblast, a cell which deposits extracellular matrix proteins and contributes to scar formation. Recently a group of NAD‐dependent deacetylases, called sirtuins, have been identified which are capable of regulating variety of cellular processes including extending lifespan of the organism. One of them is SIRT3, which is considered guardian of mitochondria. SIRT3 activation has been shown to ameliorate many aging‐associated diseases including development of metabolic syndrome, heart failure, hearing loss and cancer. SIRT3 activation has been also linked to longevity of the man. This study was undertaken to examine the role of SIRT3 in protecting the lungs from development of fibrosis.

Methods and Results

One of the most important contributors of tissue fibrosis is activation of transforming growth factor‐β (TGFβ1) signaling. To determine the role of SIRT3 in the induction of fibrosis, we treated human fetal lung fibroblasts (IMR‐90) with TGFβ1 (10ng/ml) for 48 hours. The results showed that there was a significant increase in the expression of fibrotic markers, including smooth muscle alpha‐actin (α‐SMA), type I collagen and fibronectin in TGFβ1 treated cells. TGFβ1 treatment also caused depletion of endogenous SIRT3 levels, which correlated with increased ROS production, DNA damage and subsequent reduction in levels of OGG1, a major DNA glycosylase which hydrolyzes oxidized‐guanine (8‐Oxo‐dG) and protects the DNA from oxidative damage. We also found increased expression of inflammosome markers such as caspase‐1, and IL‐1β in TGFβ1 treated IMR‐90 cells. These changes were blocked when cells were infected with adenovirus expressing wild‐type SIRT3, but not the mutant, thus suggesting the ability of SIRT3 to block transformation of lung fibroblasts to myofibroblasts. To determine the anti‐fibrotic effect of SIRT3 in vivo, we induced lung fibrosis in mice by intra‐tracheal instillation of bleomycin for three weeks. As compared to wild‐type controls, SIRT3‐KO mice showed exacerbated fibrosis after bleomycin treatment, as measured by increased expression of fibrotic marker collagen‐1 and inflammasome marker caspase 1 and subsequent decreased levels of OGG1. However, the transgenic mice having whole body SIRT3 over expression were protected from bleomycin‐induced lung fibrosis.

Conclusion

These data demonstrate that loss of mitochondrial SIRT3 promotes transformation of lung fibroblasts to myofibroblasts and development of lung fibrosis. Therefore, SIRT3 activation may be considered a medical therapy for the management of lung fibrosis.

Support or Funding Information

This study was supported by the NIH‐RO1 grants HL117041 and HL111455.
Characterizing the functions of WHIMP as a new WASP‐family actin nucleation factor
Authors: Kabrawala Shail , Zimmer Margaret , Campellone Kenneth
Published: November 2023

Actin nucleation factors from the Wiskott‐Aldrich Syndrome Protein (WASP) family cooperate with the Arp2/3 complex to assemble branched filament networks during cell migration, vesicle trafficking, and many other vital cellular functions. To initiate actin assembly, conserved WH2‐Connector‐Acidic (WCA) domains within the eight known mammalian WASP‐family proteins bind both to actin monomers and the Arp2/3 complex. In the current study, based on sequence similarity to WCA domains, we have identified a new protein in the family, WHIMP (WAVE Homology In Membrane Protrusions). Immunoblotting of mouse tissue extracts indicated that WHIMP was expressed in multiple organs, including heart and kidney. Additionally, RT‐PCR and immunoblotting demonstrated that WHIMP was expressed in several different mouse cell lines. Separation of NIH3T3 cells into their nuclear, membrane, and cytosolic fractions revealed that endogenous WHIMP was found predominantly in the cytosol. A further examination of WHIMP localization by fluorescence microscopy confirmed that GFP‐tagged WHIMP was dispersed throughout the cytoplasm. However, after treatment of cells with epidermal growth factor, WHIMP became enriched at membrane ruffles. Similar recruitment of WHIMP near the plasma membrane was observed when cells adhered to a substrate. Overexpression of WHIMP also caused a small, yet statistically significant increase in cytoplasmic actin filaments. Additional biochemical analyses using purified proteins in pyrene‐actin assembly assays suggested that recombinant WHIMP was a weaker nucleation‐promoting factor than other WASP‐family proteins. Taken together, these results provide insight into the expression, localization, and actin assembly properties of WHIMP as a new member of the WASP family.
Investigations of Functional Interactions Between Env7 and TOR Complex 1 in Saccharomyces cerevisae
Authors: Shaoulian Jonathan , Gharakhanian Editte
Published: November 2023

In eukaryotic cells, deficiencies in proteins involved in lysosomal trafficking and function result in serious human disorders like Tay‐Sach's Disease or Alzheimer's. Our lab has identified ENV7, a novel gene involved in the late‐endosome‐to vacuole interface in Saccharomyces cerevisae. ENV7, (a homolog of human STK16) encodes Env7, a vacuolar membrane kinase that negatively regulates vacuolar fusion. Tor1 is another vacuolar membrane kinase, and has been identified as a vacuolar fragmentation factor. Activated Tor Complex 1 (TORC1) stimulates vacuolar fragmentation, as well as cell growth and proliferation. Tor1 and TORC1 are also evolutionarily conserved, and in mammals, hyperactivity of TORC1 plays a major role in cancers. Furthermore, inhibition of TORC1 is associated with the initiation of autophagy, which itself involves a number of fusion events at the vacuole. Because of this, we hypothesized that there is a functional interface between the vacuolar membrane kinases Env7 and Tor1 in TORC1. Here, using confocal microscopy techniques, we report that while the absence of TOR1 is not sufficient to affect the localization of exogenously expressed GFP‐Env7, when TORC1 is inhibited by rapamycin in the same cells, there is a significant mislocalization of the GFP‐Env7 fusion protein to the plasma membrane. Cells of the tor1Δ strain treated by rapamycin also display an enlarged vacuole morphology. Additionally, this enlarged vacuole morphology in tor1Δ seems to be associated with slightly increased levels of autophagy. Therefore, the results thus far suggest a functional interaction between Env7 and TORC1. We are currently investigating the localization of native levels of Env7‐GFP when TORC1 is inhibited by rapamycin. We are also further exploring the TORC1 and Env7 interface by studying additional deletion mutants of TORC1 components.

Support or Funding Information

This project is supported by funding from the NIH (grant SC1 GM112560‐01 to Editte Gharakhanian)
ER‐associated SNAREs are involved in atlastin‐mediated ER membrane fusion
Authors: Lee Miriam , Moon Yeojin , Kim Su Yoon , Jun Youngsoo
Published: November 2023

The endoplasmic reticulum (ER) is a key player in the maintenance of cellular functions. Its functions are thought to be closely related with its unique structure, which forms a network of sheets and tubules that are maintained by homotypic ER membrane fusion. Although the exact mechanism and players of homotypic ER fusion is yet to be unveiled, the dynamin‐like GTPases of the atlastin family are thought to mediate homotypic ER membrane fusion. In this study, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p‐dependent ER fusion. Using this assay, we found that Sey1p‐mediated ER fusion requires ER SNAREs Sec22p and Sec20p. Consistently, ER fusion was significantly inhibited by Sec18p and Sec17p, which regulate SNARE‐mediated membrane fusion. The physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE, further suggested the involvement of SNAREs in Sey1p‐dependent ER fusion. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25‐fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE‐mediated membrane fusion is involved in atlastin‐initiated homotypic ER fusion.

Support or Funding Information

This research was supported by grants from the Silver Health Bio Research Center at GIST and the BK21 plus program of Korea.
NHE1 Phosphorylation and Steady State Intracellular pH
Authors: Hasle Dan , Wallert Mark , Provost Joseph John
Published: November 2023

The Sodium‐Hydrogen Exchanger 1 (NHE1) is a transmembrane protein important for several important cell processes including migration and intracellular pH (pHi) homeostasis. The activity of NHE1 is modulated upon phosphorylation by eleven kinases at 14 known and predicted sites. Four known kinases and their NHE1 phosphorylation sites include ERK 1/2, protein kinase B (AKT), RhoA kinase (Rock) and ribosomal s6 kinase (RSK). Various agonists show different degrees of NHE1 stimulation, however we do not have an understanding of how these sites coordinate or if there is a hierarchical phosphorylation regulation of NHE1. The potential phosphorylation‐dependent regulation of NHE1 on pHi is determined by measuring the impact of these four kinases. To accomplish this, cells will be treated with three different agonists; lysophosphatidic acid (LPA), platelet‐derived growth factor (PDGF), and phenylephrine (PE); each which involves signaling to all four kinases through diverse pathways. Dose response assays for the mentioned agonists in fibroblasts deficient in NHE1 expression (PS120 cells) and in fibroblasts stably expressing human NHE1 in PS120 cells (PSN cells). To quantify the magnitude of stimulation each agonist has on NHE1 basal activity, cells were treated with each agonist and steady state pHi was determined 15–20 min after addition of agonist using the pH‐dependent dual excitation, single emission dye, 2′,7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐Carboxyfluorescein, Acetoxymethyl Ester (BCECF) and fluorescent microscopy. Increases in pHi of 0.25 to 0.28 (n=12) were observed for the agonists. To selectively analyze the extent each kinase has upon NHE1 activation, cells were introduced with specific cell permeable kinase inhibitors (1uM MK‐2206, 10uM BI‐D1870, 10uM Y27632 and 0.5uM SCH772984) prior to the agonist. To investigate the effect of direct phosphorylation for each agonist, mutant PSN cells expressing human NHE1 with Ser/Thr to Ala mutations for each kinase site will be subjected to the same assays. Our project will provide better understanding of the relative contributions each signaling cascade has upon NHE1 basal function.
Purification and characterization of the Drosophila melanogaster (Dm) IKKβ/IKKγ complex
Authors: Rogers William Eric , Huxford Tom
Published: November 2023

Inhibitor of KappaB Kinase (IKK) is a major regulation point in the activation of innate immunity. The kinase subunit of the complex has undergone extensive study including biophysical and structural characterization. The IKK complex that includes the regulatory subunit has been extremely difficult to study due to its inherent instability, and propensity to aggregate. Analysis of this type of complex in a Dm background has allowed for the purification of a stable single particle containing both the kinase and regulatory subunits. Here we present initial purification and characterization of the in vivo functional IKK complex from Drosophila melanogaster.

Support or Funding Information

W.E.R is a recipient of an Arne N. Wick pre‐doctoral research fellowship from the California Metabolic Research Foundation.
Overcoming challenges in the diagnosis of Schistosoma mansoni infections using recombinant protein technology
Authors: Queiroz Rafaella F G , Silva‐Moraes Vanessa , Almeida Nathalie B F , Harn Donald A , Coelho Paulo Marcos Z
Published: November 2023

Control constraints of Schistosomiasis include the lack of diagnostic methods with high sensitivity. We develop a prospective study in southeast Brazil to standardize new sensitive diagnostic methods for Schistosoma mansoni infection. Currently, we are investigating 6 endemic areas (>1000 individuals with chronic infection) and 84 travelers infected in a freshwater pool (with acute infection). Sera, urine, feces, saliva and spinal cord liquid samples were used for the standardization/validation of innovative methods, including acute, chronic, post‐treatment and myeloradiculopathy schistosomiasis patients. Comparisons are performed with eggs in feces, IgG titers, encephalomyelitis by NMR and clinical symptoms. With our new method using a selected recombinant protein called CCAr, we were able to detect the disease early as 10 days post‐infection and more than 95% of positive cases from chronic and low endemicity areas were obtained. Recombinant protein technology allowed a superior detection method when comparing it to the purified glycoprotein and/or its synthetic peptides. Now, monoclonal antibodies anti‐CCAr are been produced for improvement of the diagnosis by promoting direct diagnosis, together with implementation of large scale recombinant production. Data showed 100% of sensitivity of chronic patients and 98% of acute patients.

Support or Funding Information

Fapemig, CNPq, Capes, Fiocruz, Fiotec, PDTIS (Brazil). Fulbright, NIH, University of Georgia (USA).
Autophagy regulates the early secretory pathway through interaction between ULK1 and Sec23
Authors: ZHAO Shan , Gan Wenjia , Chan Hsaio Chang , YU Sidney
Published: November 2023

Autophagy is an autodigestive process that maintains the intracellular homeostasis under starvation. Although it has been intensely studied, how it affects the early secretory pathway has not been well characterized. In this study, we found that autophagy changes the morphology of ER exit sites and causes delay in the export of transport cargo proteins from ER exit sites. The serine/threonine kinase ULK1 is a kinase regulating the activation of autophagy. We found that the morphological and functional changes of ER exit sites are mediated by the phosphorylation of Sec23 by ULK1. This event weakens the interaction between Sec23 and Sec31 but strengthens the binding of Sec23 to Sar1. These results uncover connection between the autophagic pathway and the exocytic pathway such that ULK1 decreases traffic of protein vesicles at the stage of ER exit and such regulation helps save energy for the cells when they are under starvation condition.

Support or Funding Information

This project was supported by Research Grant Council (RGC 14118914)
Ate1‐mediated posttranslational arginylation is essential for stress response regulation and mutagenesis suppression
Authors: Kumar Akhilesh , Birnbaum Michael D , Morgan William , Zhang Fangliang
Published: November 2023

Arginylation is a posttranslational modification which leads to addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence and the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in these conditions. In our study by using multiple types of eukaryotic cells as test models, we found that Ate1/arginylation is essential for stress response, apoptosis and mutagenesis suppression. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of stressing factors including heat shock, oxidative and osmotic stress, heavy metal and radiation. We demonstrated that Ate1 and its arginylation activity are up‐regulated during stress and activate the intrinsic apoptotic pathway with dependence on the mitochondria‐related apoptosis‐inducing factor (Aif1), but not directly on the ubiquitin‐proteasome system or Caspase (Yca1). Moreover, over‐expression of Ate1 also leads to cell death which depends on its enzymatic activity. Furthermore, we found that Ate1/arginylation is required to suppress mutagenesis in the population during DNA damaging stress. Our data thereby explain the basis for the involvement of arginylation in physiological and diseased conditions by its regulation of cell death and/or mutagenesis. We also uncover a novel mechanism by which a posttranslational modification exerts a global effect on mutagenesis.

Support or Funding Information

NIH R01GM107333

DoD/CDMRP PC140622 and PC141013

ACS IRG‐98‐277‐13

Sylvester Comprehensive Cancer Center Developmental Grant