bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating Structural Drivers of Antigen Specificity
Authors: Singh Nishant K , Smith Sheena , Harris Dan , Kranz David , Baker Brian M
Published: November 2023

Cellular immunity and new immunotherapeutic approaches rely on interactions between T‐cell receptors (TCRs) and the peptide‐major histocompatibility complex (pMHC) to initiate successful immune responses. Achieving peptide specificity by the TCR while simultaneously engaging the pMHC is critical for proper recognition and T‐cell activation. Although early studies emphasized the role of CDR3α and CDR3β loops in determining antigen specificity, more recent work has shown that antigen specificity arises from a cooperative interplay between germline and hypervariable loops and the pMHC surface. To better understand the determinants of TCR specificity, we have been studying engineered variants of the human TCR A6, which recognizes the viral peptide Tax presented by the class I MHC HLA‐A2, but has also been re‐engineered to recognize the tumor associated antigen MART‐1 with high specificity. An additional variant shows a surprising loss of specificity, recognizing a large range of structurally and chemically distinct peptide/HLA‐A2 complexes with high affinity. Here, we describe the structural and biochemical features that correlate with this loss in specificity and discuss the implications for how TCRs achieve specificity and how it can be manipulated.
Muscle Growth by Activin Type II Receptor Blocking Ameliorates Weakness in GNE Myopathy Mice
Authors: Noguchi Satoru , Yonekawa Takahiro , Malicdan May Christine V , Miyakawa Miho , Lach‐Trifilieff Estelle , Nonaka Ikuya , Nishino Ichizo
Published: November 2023

Background

GNE myopathy is an autosomal recessive muscle disease clinically characterized by skeletal muscle atrophy and weakness. We have established that a key factor in the mechanism of disease is sialic acid deficiency, a target for recent and ongoing clinical trials. In GNE myopathy patients with existing severe muscle atrophy and degeneration, however, exploring additional therapeutic options may prove beneficial.

Objective

In this study, we demonstrate the efficacy of myostatin pathway blockade, which promotes muscle growth, on GNE myopathy model mice.

Methods

CDD866, a murinized anti‐activin type II receptor antibody, were administered to GNE myopathy model mice once weekly with subcutaneous injections for 8 weeks. Physiological examination and pathological observation on the skeletal muscles of treated mice were performed.

Results

CDD866, not only promoted body weight gain and increase in muscle size, but also led to the recovery of specific tetanic force, which is a functional indicator of muscle contractile devices. In addition, we demonstrate that increased S‐nitrosylation of contractile and metabolic proteins leads to muscle weakness in GNE myopathy mice. More importantly, CDD866 treatment in mice reversed S‐nitrosylation of the muscle proteins along with the recovery of muscle function.

Discussion

This results suggest that protein synthesis via myostatin pathway blockade has a beneficial effect by providing newly‐synthesized fresh muscle proteins to chronic myopathic muscles in addition to muscle size increment. Our results also suggest that the myostatin pathway blockade may be a promising therapy on GNE myopathy in addition to sialic acid supplementation.

Support or Funding Information

Intramural Research Grant (28‐6) for Neurological and Psychiatric Disorders of NCNP
Evaluating the activity of bacterial enzyme DXP synthase a potential target for newer antibiotics
Authors: Kessler Alicia M , Anand Sridhar , Callahan Kevin P
Published: November 2023

An emerging medical crisis involving the development of antibiotic resistant bacteria in the human body is compromising treatment of infections, leaving patients in critical condition. Infections that are becoming unresponsive to current treatment are creating a need for new antibiotics. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritol phosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would prevent or heavily reduce the rate of DXP production, which can then prevent or delay the continuation of the MEP pathway and thus is an ideal candidate for antibiotic development. In order to test the effectiveness of potential inhibitors of DXP synthase it is first necessary to establish a system to monitor the activity of DXP. We have purified Histidine tagged versions of DXP synthase and 1‐Deoxy‐D‐Xylulose 5‐Phosphate Reductoisomerase (IspC). The activity of DXP synthase cannot be directly measured using spectroscopic techniques, however it can be coupled to the activity of IspC, which in the process of converting DXP into MEP oxidizes NADPH. The establishment of this in vitro system will enable us to test the efficacy of novel inhibitors of DXP that are currently being developed.

Support or Funding Information

Supported by internal grants from St. John Fisher College School of the Arts and Wegmans school of Pharmacy
Exploring inhibitors of the periplasmic chaperone SurA using fluorescence anisotropy
Authors: Zheng Erica J , Bell Eric W , Ryno Lisa M
Published: November 2023

A requirement for cell homeostasis is the correct functioning of chaperones, which inhibit the aggregation of other proteins in the cell. The chaperone SurA, present in gram‐negative bacteria, prevents the aggregation of outer membrane porins as they traverse the aqueous periplasm. Disruption of SurA activity renders the bacterial cell more sensitive to agents that would normally be kept out by outer membrane porins. Here we describe an in vitro screen to test potential small molecule inhibitors of SurA that could be used to decrease the virulence of bacterial cells. Using fluorescence anisotropy, we conduct competitive binding assays using fluorescently‐tagged peptides that bind SurA with high affinity and small molecules discovered using in silico screening.

Support or Funding Information

This work was supported by Oberlin College.
Diadenosine Polyphosphatases of the NUDIX Hydrolase Superfamily in M. tuberculosis
Authors: DiCola Andrew Michael , Knowles Alexander , O'Handley Suzanne
Published: November 2023

M. tuberculosis contains 11 potential Nudix hydrolases, and we are characterizing these enzymes as potential novel antibiotic targets. The diadenosine polyphosphatases (ApnAases)/mRNA decapping enzymes are a family of enzymes within the Nudix hydrolase superfamily. In M. tuberculosis there is the primary Nudix ApnAase and the secondary Nudix ApnAase. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis have been found to be important in each pathogen's ability to invade its host cells. It is of interest to know whether these enzymes act in the same way in M. tuberculosis. If they are all found to be involved in invasiveness and thus in virulence, then these enzymes could be novel antibiotic targets. We have cloned and overexpressed each protein and have subcloned each into a HisTag vector to optimize purification. We have purified the secondary Nudix ApnAase from M. tuberculosis so that we can complete its characterization. Thus far we have determined its substrate specificity (Ap4A is the best substrate), ApnAase activity, optimal pH (7.8), and divalent metal ion requirements (1 mM Mn2+ yields optimal activity, however Mg2+, Co2+, and Zn2+ yield activity as well).

Support or Funding Information

This research has been supported by an NIH AREA grant and RIT COS and honors summer undergraduate research fellowships.
Design and Analysis of a Microbiome Mock Community: Understanding and Mitigating Methodological Biases
Authors: Mosby Suquoia , Kiflezghi Michael , Edwards David , Brooks Paul J , Rivera Maria
Published: November 2023

The gut microbiome has been shown to influence immunity, to play a role in obesity, colorectal cancer and diabetes among others. Although much research has been done, there is still a lack of understanding of what factors in the gut microbiome are influencing the health and/or disease state of the host. Among the challenges facing microbiome research are the difficulties in accurately identifying and quantitating the components of a microbiome. A typical protocol for analyzing and comparing microbiomes is to conduct a taxonomic and phylogenetic survey or census of the members of the microbiome community. The microbiome survey is typically performed by high‐throughput sequencing of PCR amplification of the 16S rRNA gene from the community metagenome, followed by applying bioinformatics methodology to assign a taxonomic identification to each one of the sequenced fragments. These processing steps can introduce bias that can distort the true or original composition of the microbiome. The ultimate goal of the experiments presented here is to understand the type and magnitude of the bias introduced by the typical protocols used for microbiome characterization. In order to uncover potential biases affecting microbiome studies we are designing and analyzing a mock community or a synthetic microbiome. The mock community is designed by combining in vitro known proportions of a set of well‐characterized bacterial species. Processing the mock community with the same protocols used by microbiome experiments will allow us to generate a quality control for these types of experiments. We designed a low complexity mock community composed of 10 species of bacteria isolated from different hosts. These 10 bacterial species will be mixed at different ratios and the different mixes will be subjected to a full microbiome analysis. This analysis includes DNA extraction, PCR amplification, NGS sequencing and bioinformatics analysis. The goal of the experimental design is to determine our ability to recover the original input ratios. The ratios obtained by our experimental protocols will ultimately be used to develop mathematical models to predict the “true” composition of the microbiomes. In the initial phase of the project we grew each organism individually as a pure culture followed by PCR amplification and sequencing to assess the purity of the cultures. We analyzed the sequences by comparing them to the NCBI nucleotide database using BLASTN. The BLASTN results indicated we had to distinct cultures sharing only 95% sequenced identity; the sequence of one of the cultures showed a 99% sequence identity to Escherichia coli and the other showed 99% sequence identity to Shimwellia blattae. The same methodology will be used to analyze another 8 pure cultures.

Support or Funding Information

Initiative for Maximizing Student Development R25GM090084
The Metatranscriptome of the Rhesus Macaque: Investigating Potential Causes of Idiopathic Chronic Diarrhea
Authors: Westreich Samuel T , Ardeshir Amir , Kable Mary E , Korf Ian , Lemay Danielle G
Published: November 2023

The study of the gut microbiome—the collection of microbes within the intestinal tract and the genes they express—is growing in popularity as associations are found between diet, gut microbiome activity, and host health and disease. However, current metagenomic and ribosomal profiling approaches are unable to capture changes in activity within microbiome populations. In this environment, shifts in diet may not result in microbiome population changes, but may instead influence the activity of the existing microbial community. Metatranscriptomics, the study of microbial population activity based on RNA‐seq data, offers detailed data on the gene expression of all microbes within the sampled community. We have created a new bioinformatics pipeline, SAMSA (Simple Analysis of Metatranscriptome Sequence Annotations), designed specifically for metatranscriptome dataset analysis, with options for either in‐house or external server‐based computational processing. Designed for use by researchers with relatively little bioinformatics experience, SAMSA offers a breakdown of metatranscriptome activity by organism or transcript function, and is fully open source.

We have applied SAMSA to examination of Idiopathic Chronic Diarrhea (ICD), a long‐term inflammatory bowel condition in rhesus macaques that leads to significant mortality in captive populations. Previous studies on ICD have failed to identify a causative pathogen, and the disease is believed to be the result of a wider shift in the microbiome across multiple taxa. Through metatranscriptomic examination of both organism and functional activity shifts, we have identified underlying shifts in the gut environment of ICD‐afflicted animals, and observed changes in the activity of affected microbial communities. These results will offer new approaches for not simply providing palliative treatment for these affected animals, but permanently eliminating the ICD condition from captive rhesus macaque communities. These results demonstrate the value of a metatranscriptome‐based approach for studying microbiome environments.

Support or Funding Information

Funding provided by Signature Research in Genomics award.
Time Course Metabolic Profiling of Senescing Flight Muscles in The Hawk Moth, Manduca sexta
Authors: Wone Beate , Kinchen Jason M. , Kaup Elana R. , Wone Bernard W. M.
Published: November 2023

Biological aging profoundly impairs skeletal muscle function, performance, and metabolism. Because aging muscle in vertebrates has parallels with aging muscle in insects, we conducted a high‐resolution time course metabolic profiling of middle‐age to advanced age dorsolateral flight muscle in the hawk moth, Manduca sexta, to characterize the metabolic changes associated with aging muscles. These muscle samples were analyzed using non‐biased, global metabolomics profiling technology based on UHPLC/MS/MS2 and GC/MS platforms. A total of 535 metabolites were characterized across diel time (daytime/nighttime) and from middle‐age to advanced age in senescing muscle. We identified a number of differences, including changes in biochemicals related to energetics, extracellular matrix turnover, and polyamine metabolism. Metabolite over representation analyses indicated a greater than 2‐fold enrichment in biotin, fatty acid, purine, polyamine, branched chain amino acid, TCA cycle, glycolipid, and monoacylglycerol synthesis or function. Increased abundances of glycolytic metabolites suggest shifts toward activated glycolysis with age, whereas decreased abundances in acylcarnitines potentially reflect declining beta‐oxidative use. We have also seen a shift towards decreased polyamine metabolism with age, which might have downstream effects such as an age‐related decline in lipid metabolism because polyamines appear to regulate lipid metabolism through autophagy. Significant metabolite changes were detected between the metabolic profiles of males and females, revealing both similarities and differences across diel time and ages. Notably, 43 metabolites showed diel oscillations in their abundance levels in females and this pattern attenuated in advanced age. Males showed much dampened diel oscillations regardless of age, but overall abundances of these metabolites decreased with age for both sexes. Interestingly, biotin levels were elevated in senescing muscle of males. Collectively, our results obtained uncovered a cascade of biochemical regulation events coordinating senescence in muscle.

Support or Funding Information

This material is based upon work supported by the University of South Dakota start up funds.
Inhibition of Glycogen Metabolism as a Potential Strategy for Anticancer Therapy
Authors: Dukhande Vikas V , Barot Shrikant , Husein Suemaya , Palaguachi Christian
Published: November 2023

Cancer cells reprogram their energy metabolism to suit their growth and proliferation needs. Cancer cells upregulate glycolysis even in the presence of oxygen, a phenomenon termed Warburg effect. Glycolysis thus emerged as a promising target to kill cancer cells. Glycogen, a storage cache of glucose in the cell, could fuel the metabolic reprogramming in cancer cells. However, little is known about glycogen metabolism and its contributions to carcinogenesis, cancer growth, and metastasis. We aim to inhibit glycogen metabolism to arrest the growth of cancer cells and want to study molecular mechanisms of glycogen inhibition‐induced cancer cell death. We studied cell viability, apoptosis markers, autophagy markers, and cellular metabolism after glycogen metabolic inhibition using pharmacological agents in hepatoma and glioblastoma cells. Our results indicate that inhibition of glycogen metabolism induces cancer cell death and enhances the effects of anticancer drugs. Our ongoing studies will investigate the roles of apoptotic and autophagic processes in cancer cell death by glycogen metabolic inhibition. Our study is highly relevant for the understanding of the metabolic reprogramming in cancer cells and can elucidate potential targets for anticancer drugs.

Support or Funding Information

Supported by startup funds from the Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University to Vikas Dukhande.
Targeting Mitochondrial Molecular Chaperones as a Potential Therapeutic Target in Leukemia Kelly G. Bryant and Dario C. Altieri Prostate Cancer Discovery and Development Program and Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia PA 19104
Authors: Bryant Kelly G. , Altieri Dario C.
Published: November 2023

Molecular chaperones of the Heat Shock Protein‐90 (HSP90) family play an important role in proper protein folding, protein complex formation, as well as minimizing aggregate formation. The discovery of HSP90 and its ortholog, tumor necrosis factor receptor‐associated protein‐1 (TRAP‐1) abundantly localized to the mitochondria of tumor, but not the majority of normal cells has broadened the field to include a role in cancer progression, and importantly, a therapeutic candidate in many solid tumors. We sought to determine whether targeting the mitochondrial HSP90s with small molecule inhibitors would be effective in leukemia. Fractionation studies of several different types of leukemia cell lines show both TRAP‐1 and HSP90 proteins in the mitochondria, thus providing a target for the mitochondrial targeted HSP90 inhibitor Gamitrinib (GA‐mitochondrial matrix inhibitors). Accordingly, a variety of different leukemia cell lines were treated with Gamitrinib and for each line tested, we found growth inhibition in a dose and cell‐dependent manner. Treatment also induced cell death, PARP cleavage, and caspase activation at lower doses than is required for solid tumors. Treatment with low doses of Gamitrinib did not affect protein levels of the mitochondrial electron transport chains I through V. Additionally there were not significant differences in the levels of lactate produced by these cells, and only a slight decrease in glucose consumption. Metabolic studies also show that treatment leads to a decrease in ATP levels in the leukemia cell lines, as well as a decrease in the oxygen consumption rates of these cells. Utilizing several primary Acute Myeloid Leukemia Patient derived samples, Gamitrinib treatment resulted in a dose‐dependent decrease in viability and increase in apoptosis as determined by FACS analysis for Annexin V/Propidium Iodide, as well as protein markers of cell death. Current ongoing experiments are focused on determining the efficacy of this inhibitor in vivo by utilizing whole body luciferase imaging in MV‐411 GFP/Luciferase xenografts. Taken together, Gamitrinib results in robust cell death in all of the leukemia cells tested, and these studies provide ample evidence that Gamitrinib could be a strong clinical candidate as an alternative HSP90 inhibitor for the treatment of different types of leukemia.

Support or Funding Information

Wistar Institute T32 Training Grant
Human Hepatocellular Carcinoma Cells Adapt to ASCT2 and LAT1 Amino Acid Transporter Silencing by shRNA and CRISPR‐Cas9
Authors: Bothwell Paige , Bode Barrie
Published: November 2023

Amino acid transporters ASCT2 and LAT1 are coordinately enhanced in human cancers where, among other roles, they are thought to drive mammalian target‐of‐rapamycin (mTOR) growth signaling. To assess the value of ASCT2 and LAT1 as therapeutic targets in hepatocellular carcinoma (HCC), nine unique shRNA sequences in lentiviral vectors were used to produce stable transporter suppression in mesenchymal and epithelial HCC cell lines (SK Hep and Hep 3B, respectively). In addition, direct editing of the ASCT2 and LAT1 genes was performed using six CRISPR‐Cas9 vectors in SK Hep and HUH7 HCC cell lines. Glutamine and leucine transport rates were reduced proportionally to diminished transporter protein expression in each cell line, and a greater degree of knockout was achieved by the CRISPR‐Cas9 gene editing system versus the stable shRNA expression system (75–85% vs. 20–80% respectively). Contrary to expectations, diminished glutamine and leucine influx failed to affect cellular proliferation. Analysis of mTOR complex 1 (mTORC1) signaling output assessed by 4E‐BP1 showed no compromise to basal or amino acid‐stimulated mTORC1 signaling. Quantitative RT‐PCR indicated no significant upregulation of other transporters or glutamine synthetase (GLUL) to supplement intracellular availability of glutamine. Prolonged uptake of radiolabeled glutamine and leucine revealed that diminished transport rates did not affect the total intracellular accumulation of either glutamine or leucine. Further in vivo analysis will be essential to understanding upregulation of ASCT2 and LAT1 across cancers, and these findings suggest that, in an in vitro context, HCC cell lines are able to adapt to decreased rates of amino acid transport.

Support or Funding Information

NIH Grant# R15CA108519 (BPB)
Role of iNKT cells in obese adipose tissue
Authors: Park Jeu , Huh Jin Young , Ji Yul , Kim Jae Bum
Published: November 2023

Immune cells actively regulate inflammatory responses and affect whole‐body energy metabolism. Various immune cells are present in adipose tissue and they affect adipose tissue homeostasis. Obesity‐induced inflammation is considered as one of the key factors to promote insulin resistance. Immune cells also contribute to control adipose tissue remodeling, adipocyte differentiation, and lipolysis. In particular, adipose iNKT cells have various roles in protecting against obesity‐induced inflammation, weight gain, and hypertrophic adipocyte. Upon high fat diet iNKT cell deficient mice such as Ja18−/− and Cd1d1−/− show increased pro‐inflammatory responses, more body weight gain with larger adipocytes. To date, most studies in adipose iNKT cells have focused on increased pro‐inflammatory responses. In this study, we have investigated the direct crosstalk between iNKT cells and adipocytes to maintain adipose tissue homeostasis.

Support or Funding Information

This work was supported by grants from the National Creative Research Initiative Program (2011‐0018312) funded by the Ministry of Education, Science and Technology. J.P. and Y.J. were supported by BK21 program.
Improvement of diabetic cardiomyopathy by Eryngium carlinae
Authors: García‐Cerrillo Diana , Noriega‐Cisneros Ruth , Salgado‐Garciglia Rafael , Manzo‐Avalos Salvador , Clemente‐Guerrero Mónica , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo
Published: November 2023

Diabetes mellitus (DM) is a metabolic disease that has increased significantly in recent decades. DM and its complications are powerful risk factors for cardiovascular diseases. Diabetic heart damage is closely related to mitochondrial dysfunction and an increase in the generation of reactive oxygen species. The external supply of antioxidants can counteract the effect of such species on the body, and prevent the onset of many underlying diseases.

Natural sources of antioxidants may be safer to use with regard to synthetic antioxidants, due to lower toxicity and side effects. Eryngium carlinae (EC) is a plant used in traditional Mexican medicine, whose hexanic extract has been shown to possess in vitro antioxidant capacity. The serum and heart mitochondria of male Wistar rats were analyzed. Previously experimental diabetes was induced with streptozotocin (STZ) and they were treated with hexanic extract of EC for seven weeks (30 mg/kg body weight). Cholesterol, triacylglycerides and glucose and lipoperoxidation levels were determined in serum. In addition, in the left ventricle heart mitochondria, oxidative and nitrosative stress levels were determined through the quantification of parameters such as thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, and tissue histological analysis was performed. The results of the biochemical parameters of glucose and triacylglycerides show a significant decrease of the diabetic group treated with extract with respect to the diabetic group without treatment. Similarly, there was a significant decrease in lipoperoxidation levels in the diabetic group treated with extract compared to the untreated diabetic group.

Histological observation shows signs of hypertrophy of the left ventricle of diabetic rats which appears to be diminished by treatment with EC extract. In conclusion, EC hexanic extract possesses antioxidant activity in vivo in the groups in addition to possessing the ability to lower glucose and triglyceride levels in diabetic rats, which may prevent or delay the development of diabetic cardiomyopathy.

Support or Funding Information

Acknowledgments: Special thank for the grants of CIC‐UMSNH: 2.10 to RSG; 2.16 to ASM, and 2.37 to SMA.
Generating a Transgenic Mouse Line Containing the Plasma Membrane Phosphatidylinositol 4,5‐Bisphosphate Depletion System
Authors: Gulyás Gergő , Szalai Bence , Geiszt Miklós , Balla Tamás , Hunyady László , Várnai Péter
Published: November 2023

Phosphoinositides are key regulators in many signaling cascades and several cellular processes. These lipids are located in cellular membranes containing different forms of these lipids. The plasma membrane (PM) is enriched in two phosphorylated variants, phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate (PIP2).

To discover the role of these lipids, several applications have been developed. One of them is the rapamycin inducible heterodimerization system, based on the rapid and irreversible interaction of the FRB and FKBP12 proteins. In our system FRB was targeted to the PM using the localization sequence of the Fyn tyrosine kinase, whereas the FKBP was fused to an enzymatically active cytoplasmic 5‐phosphatase. After rapamycin treatment, the enzyme recruited to the PM, where rapid depletion of PIP2 could be achieved. Our goal was to generate a genetically modified mouse line containing this lipid depletion system, which can be highly capable to investigate the physiological role of PIP2 in primary prepared cells or in intact tissues and organs.

Our plasmid used for the transgenic application contained both protein coding sequences of the depletion system in the same vector. To reach the stochiometric expression of the two distinct fragments, a viral T2A sequence was inserted between the protein coding sequences. For further detection of the transgene the PM targeted protein was fluorescently tagged. The transgenic construct was driven by a CMV promoter to reach a general expression in various animal tissues. The transgenic modification was carried out by applying the Sleeping Beauty transposase in FVB/Ant mouse strain. Based on the genotyping data of the founder generation, two distinct lines with low and high copy number were selected for the further investigations. Interestingly, transgene showed detectable expressions only in pancreatic acinar cells and in keratinocytes.

To investigate the functional effect of the PIP2 depletion we carried out cytoplasmic calcium ion measurements. Using freshly prepared pancreatic acinar cells of the F2 generation rapamycin treatment did not resulted a reduced cytoplasmic calcium signal upon stimulation the cells with carbachol, indicating the low expression level of the PIP2 depletion system. A mathematical model was created, which predicts of the increase of protein expression, so we decided to select the animals with the highest copy numbers in each generation and use them as parents of the further generations. The efficiency of lipid depletion is monitored by measuring the translocation of PLCdelta1‐PH‐domain based PIP2 sensors, as well as by measuring other PIP2‐dependent cellular responses.

Support or Funding Information

National Research, Development and Innovation Fund (NKFI K105006)
Orthogonal lipid sensors determine differential signaling roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2
Authors: Wang Zhi‐Gang , Liu Shu‐Lin , Cho Wonhwa
Published: November 2023

Activation of Class I phosphoinositide 3‐kinase (PI3K) leads to formation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Although many pleckstrin homology (PH) domains bind both PtdIns(3,4,5)P3 and PtdIns(3,4)P2, spatiotemporal dynamics of these lipids and their differential signaling roles have not been fully defined. To address these questions, we developed orthogonal ratiometric sensors for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 that allowed the simultaneous, spatiotemporally resolved in situ quantification of these lipids in mammalian cells upon growth factor stimulation. Quantitative correlation of spatiotemporal dynamics of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with the differential plasma membrane recruitment and activation of Akt isoforms shows how differentially PtdIns(3,4,5)P3 and PtdIns(3,4)P2 activate Akt isoforms. Further structural and biophysical studies provided the mechanistic basis for these roles and led to the development of Akt isoform‐selective inhibitors.
Biological Inspiration of Salt Exclusion Membranes in Mangroves toward Fouling‐Resistant Reverse Osmosis Membranes
Authors: Wood Adam Richard , Justus Kyle , Parigoris Eric , Russell Alan , LeDuc Philip
Published: November 2023

Developing fouling‐resistant membranes for reverse osmosis (RO) seawater desalination would provide many advantages for reducing energy consumption. As a potential avenue to inspire such membranes we are studying mangrove trees. Mangrove trees inspire a membrane‐based approach due to their natural desalination of untreated seawater in a process similar to RO. We have focused on biological mimetic systems and are identifying the locations in mangroves where salt exclusion occurs when intact stem‐and‐root complexes are used as a semipermeable membrane in an RO system. We have developed an RO system capable of sealing and testing intact stem‐and‐root complexes and individual root tissue (i.e. – root epidermis) at pressures up to 50 atm and 20 atm, respectively. Using our custom fabricated systems we have extruded xylem sap from mangroves and confirmed that they are naturally desalinating a saline growth solution. We are continuing to investigate our RO systems to enable us to identify the salt exclusion regions in mangroves and investigate potential fouling resistance in these tissues. This work will have impact in a diversity of fields including biologically inspired approaches to membrane processes, plant biology, and material science.
Glucose Transporter (HXT) Homeostasis in Saccharomyces cerevisiae
Authors: Swain Evelyn
Published: November 2023

The proposed research explores the regulation of glucose metabolism in Saccharomyces cerevisiae. Specifically, the focus is on how the loss of endocytic function results in aberrant glucose homeostasis through the deregulation of glucose transporter localization/function. By combining methods of yeast genetics, molecular biology, and cell biology, novel features regulating transporter function and localization will be studied to uncover mechanisms for maintaining glucose homeostasis. Data show that the BAR domain protein Rvs161 is required for proper transporter localization and function.1 Recent reviews describes BAR domain containing proteins as curvature inducing/stabilizing proteins essential for affecting a wide variety of membrane remodeling processes, including endocytosis.2 Endocytosis and degradation of transporters is critically important in maintaining glucose homeostasis in yeast as well as mammals. Saccharomyces cerevisiae is a well‐established model organism for the study of endocytosis.3 Additionally, yeast share similar components of glucose signaling and glucose transporter pathways with mammalian counterparts making yeast a useful model system for studies of glucose transporter biology.4, 5 Yeast strains lacking the RVS161 gene fail to grow under conditions of glucose limitation and harbor endocytosis defects. Our data indicate that overexpression of specific glucose transporters can suppress this defect. Additionally, our data suggests that glucose transporter interactions are required for proper transporter function and that Rvs161p is required for proper transporter localization and function.

Support or Funding Information

NA
YHM2 from S. cerevisiae and Y. lipolytica; Comparison of their Role in Oxidative Stress
Authors: Hartnett James , Pierson Paige N. , Nicaud Jean‐Marc , Trotter Pamela J.
Published: November 2023

Oxidative stress causes cell death, which progresses diseases such as Alzheimer's disease and Parkinson's disease. This occurs in the presence of elevated levels of reactive oxygen species (ROS), which are typically regulated by antioxidants. A known antioxidant precursor is NADPH, which in the Saccharomyces cerevisiae is maintained, in part, by the citrate and oxoglutarate mitochondrial transport protein scYhm2p. In the oleaginous yeast Yarrowia lipolytica there is a homologous protein ylYhm2p, which is believed to have a similar function. In this study, ylYHM2 was disrupted through the process of homologous recombination and the growth effects were observed in the presence of hydrogen peroxide, a known ROS. The mutant had difficultly growing in the presence of hydrogen peroxide, confirming the loss of antioxidant function and suggesting scYhm2p and ylYhm2p have similar roles. The ylYHM2 gene was then amplified by PCR, inserted into a multicopy vector under the control of the GAL1 promoter of S. cerevisiae, and transformed into E. coli. Transformation plasmids then underwent a restriction digest as well as sequencing to confirm that the ylYHM2 insert was present in the plasmid vector as well as in the correct orientation. The vector containing ylYHM2 was then introduced into S. cerevisiae lacking scYHM2 to observe if antioxidant functionality was restored. Despite the homology in gene sequence and similar growth defects of the Y. lipolytica disruption mutants, ylYHM2 did not restore antioxidant function to S. cerevisiae lacking scYHM2, suggesting that the expression level was too low or that scYhm2p and ylYhm2p are not interchangeable. Further experiments are required to understand ylYHM2 function, including a quantitative analysis of NADPH production and the insertion of ylYHM2 into a Yarrowia lipolytica disruption mutant to see if function is restored.
Regiospecificity of Galactan Polymerization by Divergent GlfT2 Orthologs
Authors: Sadecki Patric W. , Justen Alexander M. , Ho Jordan S. , Kiessling Laura L.
Published: November 2023

Despite their ubiquity of polysaccharides, we lack detailed mechanistic understanding toward how carbohydrate polymers are assembled. Unlike the predictable linkage patterns used in other major molecules, polysaccharides can draw upon a large bevy of diverse glycosidic linkages to form complex polymers. To better understand polysaccharide assembly, our research group has turned toward using the mycobacterial galactan as a model. Composed of galactofuranose (Galf) residues, this essential polymer serves as a covalent connector between the mycolic acids and peptidoglycan of mycolated bacteria. The processive glycosyltransferase GlfT2 uses the activated form of galactofuranose, UDP‐Galf, to continuously add residues onto the galactan until reaching a mature length.

Due to its virulence, one of the most extensively studied mycobacteria is Mycobacterium tuberculosis (M. tb). M. tb GlfT2 makes alternating β‐(1→5) and β‐(1→6) linkages in the galactan biosynthesis. Previous work from our research group sought to evaluate whether M. tb GlfT2 strictly adheres to this linkage pattern. When given UDP‐Galf with a floro‐ substituted 5 or 6 position, the enzyme is unable to elongate the polymer past two units.1 This suggests that the alternating β‐(1→5), β‐(1→6) linkage pattern is maintained with high fidelity. Curiously, linkage patterns within galactan isolated from other mycolated bacterium, such as Nocardia and Rhodococcus species differ from those in Mycobacterium. Data suggests that Nocardia brasiliensis (N. bras) only synthesizes β‐(1→5) linkages and Rhodococcus equi (R. equi) synthesizes β‐(1→3), β‐(1→5), and β‐(1→6) linkages.2 Since GlfT2 is responsible for much of galactan biosynthesis, we propose that GlfT2 from these organisms contain differences from M. tb GlfT2, allowing them to synthesize galactan with different glycosidic linkages.

Our research group is keenly interested in gaining mechanistic insight toward what variation in the active site of GlfT2 leads to control over glycosidic linkage formation. We are specifically interested in understanding differences and similarities to the mechanism used by M. tb GlfT2. Since our research group has demonstrated two ‘DXD’ motifs are critical to GlfT2 catalysis, we seek to examine the active site of Nocardia brasiliensis and Rhodococcus equi GlfT2 by mutating these motifs. We hope that these studies will better illuminate which features of glycosyltransferases guide glycosidic linkage formation.
Three‐dimensional structure of Xyloglucan Xylosyltransferase I and proposed mechanism of catalysis
Authors: Culbertson Alan Thomas , Tietze Alesia , Smith Adrienne , Zabotina Olga
Published: November 2023

Xyloglucan is the most abundant hemicellulose component of the primary cell wall of dicotyledonous plants. It is synthesized by multiprotein complex localized in Golgi. Three xyloglucan xylosyltansferases (XXT1, XXT2, XXT5) are essential for xylosylation of the xyloglucan backbone. Experiments on these enzymes have mainly focused on characterization of their function in vivo using reverse genetics and characterization of their products in vitro. Currently, there is no information about their atomic structure, the modes of substrate binding, catalytic mechanisms, or interacting surfaces. Here, we report a 3‐D structure of XXT1 using high‐resolution X‐ray crystallography and a homology model of XXT2. Site‐directed mutagenesis was used to investigate the catalytic mechanism and critical residues involved in substrate binding. We confirmed that the DxD motif is critical for the coordination of the manganese ion that coordinates UDP‐Xylose. Additional residues were identified as being important for UDP‐Xylose binding. We have also predicted possible points of protein‐protein interactions based on the surface characteristics of these proteins and their assessment using Isothermal Titration Calorimetry is in progress.

Support or Funding Information

NSF‐MCB grant #1121163
Low‐income Women in Iowa Lack Awareness of the Health Benefits of Beans
Authors: Winham Donna M. , Palmer Shelly M. , Baier Jenny L. , Roe Taylor A
Published: November 2023

Beans are noted for their healthful nutrient profile, diversity in flavors, and role in sustainable agriculture. Pinto, black, white, kidney, and others, can lower LDL cholesterol, aid in glycemic control for type 2 diabetes, and reduce some cancers. Despite these benefits, few Americans meet the recommended intake of 1.5–2 cups of beans per week. Limited resource populations in the US have higher chronic disease rates – often related to poor dietary intakes. Empowering low‐income women with knowledge about the health benefits of beans could have far‐reaching results. They are often responsible for decisions made about the family diet, and influence children's food preferences. We collected baseline survey data on attitudes towards knowledge of the health benefits of beans from 178 low‐income women. This convenience sample came from agencies that provide health, nutrition, or community services to low income people in three central Iowa counties (Polk, Marshall, Story). Most questions were asked as Likert scales with options for: strongly disagree, disagree, do not know, agree, and strongly agree.

The average age of respondents was 36.4 ± 12.5 years, and 55% self‐identified as Hispanic. Cholesterol reduction by bean consumption is one of the most widely known health benefits, yet 59% “did not know”, with 34% reporting “agree/strongly agree.” A similar lack of knowledge was observed with blood glucose control (62% do not know; 27% agree/strongly agree). Cancer risk reduction is a lesser known association with bean consumption. It was not surprising that 70% did not know of this potential benefit from eating dry beans. A few significant differences were observed by Hispanic ethnicity categories. More Hispanic women agreed that beans improved your nutrition (p =0.025), and helped you feel full (p =0.018) than their non‐Hispanic peers.

Our findings support the need for increased education about the health benefits of bean consumption as part of a healthy lifestyle for limited‐resource women engaged in nutrition or health assistance programs. Promotion of beans for improving diet quality, cholesterol reduction, and normalization of blood glucose are important health education messages for all populations, but especially for those at high health risk. Increasing awareness of these health benefits might help retain beans in the diets of Hispanics as they experience acculturation, and increase intakes among all citizens as well for better health.

Support or Funding Information

Partial funding provided by: Iowa State University ‐ Human Sciences Extension and Outreach, College of Health Sciences, and Food Science and Human Nutrition – Science with Practice Seed Grant Program.
NUTRITION EDUCATION ON A GLOBAL SCALE: EXTENDING REACH AND INFLUENCE
Authors: Truby Helen , Adamski Melissa , Dart Janeane , Volders Evelyn , Blumfield Michelle , Murgia Chiara , Devey Adrian , Gibson Simone
Published: November 2023

Background/Aims

Widespread web access, use of social media, and blogs combined with celebrity endorsed diets provide a noisy background in which evidence based nutrition information has to compete.

Methods

A free open access course designed for those with no prior science knowledge called ‘Food as Medicine’ was developed in Melbourne Australia and delivered via the MOOC FutureLearn platform, (UK). The 3‐week on‐line course covered topics relating to food, diet and health, aimed to give participants skills to synthesise and critique information about food and provide evidence based nutritional information.

Results

In 2016, 83,000 participants from 158 countries enrolled: Australia/New Zealand (43%), UK (22%) and North America (9%). Learners were predominately female, older than average MOOC participants: 22% being 18–35 and 40% over 56 years. Over half had not participated in an on‐line course previously. Sixty percent were the target group (no previous academic or professional knowledge) with 29% reporting work or study in this field. Consistent with other MOOCs, over a third competed the whole course and spent 3–4 hours per week on‐line. Over 105,000 comments were made by 9100 learners. Eighty two percent of completers reported the course met or exceeded their expectations.

Conclusions

Free open access courses provide an opportunity to disseminate evidence based unbiased knowledge about food and health with extensive reach. More research is required as to their effectiveness as an education tool.

Support or Funding Information

Office of the Vice‐Provost Learning &Teaching and the School of Clinical Sciences, Monash University.
A Mindful Eating Intervention to Improve Self‐Management of Dietary Intake for Early Stage Chronic Kidney Disease Patients
Authors: Tahir Muna J , Timmerman Gayle M , Lewis Richard M , Samoson Deborah , Temple Holli , Forman Michele R
Published: November 2023

Objective

Over 10% of the US population suffers from Chronic Kidney Disease (CKD), a condition with major financial and emotional burdens. Dietary management has the potential to improve disease prognosis, particularly through self‐management programs which help patients have a major role in managing their disease. However, few self‐management interventions have been tested in this population and none have incorporated mindful eating as a modality to improve dietary adherence and enhance the eating experience. Therefore, we examined the effects of a Self‐Management of Dietary Intake using Mindful Eating (SM‐DIME) intervention on dietary adherence of participants with mild to moderate chronic kidney disease.

Methods

In a prospective, single group pretest‐posttest pilot study, participants (n=19) with Stage 1–3 CKD (aged 45–78 years) were recruited from primary care and nephrology offices. SM‐DIME, conducted at the University of Texas at Austin, consisted of six weekly, 2‐hour classes focusing on: a) practice of mindful eating, goal‐setting, problem‐solving, and food label reading; b) CKD dietary recommendations; and c) tailoring the intervention to individual dietary recommendations. Weight, body mass index (BMI), dietary intake from 3‐day 24‐hour recalls (kcal, fat, protein, carbohydrates, sodium, calcium, potassium, phosphorus) and fasting blood samples (eGFR, serum albumin, creatinine, BUN, lipid panel, carotenoid panel) were collected and analyzed at baseline and six weeks.

Results

The sample was ethnically diverse with most participants being male (73.7%) and having at least some college or baccalaureate degree (47.4%). The majority of participants had Hypertension (94.7%) and Type 2 Diabetes Mellitus (52.6%). Participants experienced significant reductions from pre‐to post‐intervention in mean weight (203.21 ± 42.98 vs 199.91 ± 40.36 lbs; P = 0.03) and BMI (32.02 ± 5.22 vs 31.57 ± 5.27 kg/m2; P = 0.04). No significant changes in dietary intake or blood measures were evident except a significant increase in cis‐β‐Carotene (0.020 ± 0.012 vs 0.026 ± 0.012 mcg/mL; P = 0.008), which was correlated with number of fruit and vegetable servings (r = 0.42; P = 0.076).

Conclusion

At the completion of the six‐week pilot intervention, participants improved in weight, BMI, and cis‐β‐Carotene levels. These improvements are promising and warrant further testing of the intervention with larger, randomized control trials.

Support or Funding Information

Center for Transdisciplinary Collaborative Research in Self‐Management. NIH, NINR, P30 NR01533511