bluuebunny/crossref_metadata_2025 huggingface dataset:
The ISW1, CHD1 and RSC Remodelers Combine to Organize Chromatin in Yeast
Authors: Ocampo Josefina , Chereji Razvan V , Eriksson Peter R , Clark David J
Published: November 2023

In yeast, most genes have a nucleosome‐depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes that is phased relative to the transcription start site (TSS). We addressed the roles of four remodeling machines (ISW1, ISW2, CHD1 and RSC) in specifying this chromatin organization. We sequenced nucleosomes from isogenic strains with the essential RSC8gene linked to a GAL promoter and isw1Δ, isw2Δ or chd1Δ mutations in all combinations. Globally, RSC depletion results in a shift of the array into the NDR, isw1Δ shows reduced spacing and both isw1Δ and chd1Δ show weakened phasing, whereas isw2Δ has no effect. Generally, chromatin organization in the multiple mutants is the sum of the effects observed in the single mutants, indicating that these remodelers are not redundant but have distinct functions. Our data suggest that RSC determines the location of the first (+1) nucleosome relative to the TSS, which is then used as a reference nucleosome by the spacing enzymes, ISW1 and CHD1, to build nucleosomal arrays on genes. Our current focus is on the interactions between RSC and the nucleosome spacing enzymes, and their roles in transcription.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH (NICHD).
Roles for MED7 and MED21 in Regulating Assembly of the Mediator‐RNA Polymerase II Holoenzyme
Authors: Sato Shiego , Tomomori‐Sato Chieri , Saraf Anita , Florens Laurence , Washburn Michael , Tsai Kuang‐Lei , Asturias Francisco J. , Conaway Ronald C. , Conaway Joan W.
Published: November 2023

A key step in regulation of RNA polymerase II (Pol II) transcription is assembly of the Mediator‐Pol II holoenzyme. Here we dissect the mechanism of assembly of the holoenzyme. Our findings identify a subset of Mediator “head” module subunits that constitute a Pol II binding pocket. In addition, we present evidence that Mediator “middle” module subunits MED7 and MED21 play integral roles in promoting Mediator‐Pol II binding. Through structure‐functions studies of the MED7‐MED21 heterodimer, we identify an evolutionarily conserved “hinge” region critical for this process. A working model for how MED7 and MED21 function together to promote assembly of the Mediator‐Pol II holoenzyme will be presented.
Roles of the Histone Chaperone FACT in Drosophila Gene Regulation and Chromatin Architecture
Authors: Tettey Theophilus , Story Benjamin , Glaser Robert L , Blanchette Marco , Conaway Joan W , Conaway Ronald C
Published: November 2023

The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA polymerase II passage through chromatin. In yeast, mutation of FACT subunits leads to decreased nucleosome occupancy at the promoters and transcribed regions of genes. In addition, genetic experiments suggest that yeast FACT plays a critical role in restricting initiation of transcription to normal promoters, ensuring that only appropriate mRNAs are synthesized. However, FACT's roles in chromatin biology and transcriptional regulation in higher eukaryotes are not well understood. Using Drosophila S2 cells as a model, we observe that depletion of the FACT complex results in aberrant transcriptional regulation. Surprisingly, we observe in genome‐wide experiments that FACT depletion has little effect on total nucleosome occupancy or position, but dramatically alters the distribution of various histone modifications, including histone 3 lysine 4 trimethylation (H3K4me3), histone 2A variant (H2Av) and histone 3 lysine 36 trimethylation (H3K36me3), in the promoters and transcribed regions of genes. Our results thus far provide valuable insight into roles of FACT in transcriptional regulation and chromatin dynamics in higher eukaryotes.

Support or Funding Information

Stowers Institute for Medical Research
Structure of an MCM double‐hexamer
Authors: Meagher Martin
Published: November 2023

The MCM helicase is an integral component of the DNA replication machinery. This ring‐shaped enzyme binds double‐stranded DNA (dsDNA) as an inactive double‐hexamer at replication origins. Once activated, the two single hexamers proceed to unwind duplex DNA by encircling single‐stranded DNA (ssDNA) during this movement. Previous studies of the N‐terminal domain of archaeal Pyrococcus furiosis MCM (PfMCM) showed ssDNA bound perpendicular to the central channel of the ring; defined as a conserved MCM ssDNA‐binding motif (MSSB). Using this same model we seek to determine the mode in which dsDNA is bound within the central channel of the ring prior to activation. Initial findings show PfMCM in its double hexamer form and it is anticipated that further examination with various dsDNA substrates will reveal by what means this helicase interacts with dsDNA upon loading. These studies aim to define fundamental aspects of the DNA replication processes.
The Complex Role of Asn270 in Dianion Activation of Glycerol 3‐Phosphate Dehydrogenase‐Catalyzed Hydride Transfer
Authors: Reyes Archie C. , Amyes Tina L. , Richard John P.
Published: November 2023

We are working to understand the mechanism for the strong phosphite dianion (HPi) activation of proton transfer reactions catalyzed by triosephosphate isomerase (TIM), decarboxylation reactions catalyzed by orotidine monophosphate decarboxylase (OMPDC), and hydride transfer reactions catalyzed by glycerol 3‐phosphate dehydrogenase (GPDH). The phosphodianion of substrate dihydroxyacetone phosphate (DHAP) for GPDH provides 11 kcal/mol stabilization of the transition state for hydride transfer. Eight kcal/mol of this stabilization is observed as HPi activation of GPDH for catalysis of the reduction of glycolaldehyde (GA). The X‐ray crystal structure of the nonproductive complex between GPDH, DHAP and NAD+ shows interactions of Arg269 and Asn270 with the substrate phosphodianion. The R269A mutation of GPDH results in a large 9.1 kcal/mol destabilization of the transition state for enzyme‐catalyzed reduction of DHAP from elimination of the enzyme‐substrate ion‐pair; and 6.7 kcal/mol of this stabilization is recovered when the reaction is carried out in the presence of 1.0 M guanidinium cation.1 Similar effects were reported for K12G and R235A mutations of TIM2 and OMPDC,3 respectively, consistent with a similar architecture of the dianion activation sites for these enzymes. However, the following effects of N270A and R269A/N270A mutations require an unexpected greater mechanistic complexity for dianion activation of GPDH, compared with OMPDC and TIM. 1.) The 5.6 kcal/mol effect from N270A mutation on transition state stability is more than twice as large as that predicted for the deleted phosphodianion‐amide interaction. 2.) The effects of consecutive R269A and N270A mutations depend strongly on the order of these mutations. 3.) The N270A mutation results in a surprising 40‐fold increase in the second‐order rate constant for GPDH‐catalyzed reduction of GA, and causes HPi to change from a strong activator to a weak inhibitor of this GPDH‐catalyzed reaction. These large and complex changes in the activation barriers for hydride transfer show that the amide side chain of Asn270 functions as a “linchpin” in maintaining the structural integrity of the dianion activation site for GPDH.
Analysis of the Iron‐Sulfur Cluster in Human Cytoplasmic Aconitase by Electron Paramagnetic Resonance Spectroscopy
Authors: Smith Onanong , Byrd Kenneth F. , Smith Virginia F.
Published: November 2023

Human recombinant cytoplasmic aconitase, also known as human iron regulatory protein 1 (hIRP‐1), was studied in vitro by electron paramagnetic resonance (EPR) spectroscopy to determine the effects of urea on iron‐sulfur cluster assembly. The bifunctional protein functions as either an enzyme that catalyzes the isomerization of citrate in the cytoplasm or as an iron‐responsive element‐binding protein in human iron metabolism and regulation. Protein behavior is regulated by the assembly and disassembly of the [4Fe‐4S]2+ cluster, which is retained and active at high cellular iron concentrations. At low iron levels, the cluster is disassembled and the protein loses its enzymatic function. Although the [4Fe‐4S]2+ is diamagnetic, alternate forms of the cluster are paramagnetic and may be observed by EPR as the protein is denatured. EPR spectra obtained at high and low fields were used to characterize the iron‐sulfur cluster. Titration with urea reveals the existence of a paramagnetic species that appears at about 2.0 M urea and disappears above 2.5 M urea, consistent with the gradual disassembly of the Fe‐S cluster. The changes in the Fe‐S cluster reported by EPR correspond closely to protein conformational changes detected by intrinsic tryptophan fluorescence and active‐site structure monitored by enzyme activity. Experiments are underway to determine the effects of substrate and inhibitor on stability of the cluster in the presence of urea.
Impact of Microbicide Formulation on Glycosylation and Innate Immunity in Cervicovaginal Fluid
Authors: Koppolu Sujeethraj , Wang Linlin , Dezzutti Charlene S , Isaacs Charles E , Moncla Bernard , Hillier Sharon L , Rohan Lisa Cencia , Mahal Lara K
Published: November 2023

Glycosylation is a key component in the establishment of healthy symbiotic bacterial colonization patterns and is involved in the maintenance of mucosal barriers to infection, regulating both adaptive and innate immunity. Changes in glycosylation state have powerful effects on the stabilization and activation of antibodies and can alter the microbicidal effects of immune molecules. Thus glycome is both a marker and modulator of innate immunity. We recently showed a strong correlation between glycosylation status in the cervicovaginal (CVL) fluid and microbiome status, using lectin microarray analysis. Here, we report on the impact of thin film versus hydrogel placebos, for vaginal delivery of drugs, on the glycosylation status and innate antiviral immunity of CVL fluids. CVL samples from 52 women were studied for glycosylation status using lectin microarray analysis and their impact on the infectivity of herpes simplex virus 1 & 2 (collectively HSV) and HIV‐1 using Plaque and TZM‐bl assays respectively. In our studies, we identified lower levels of high‐mannose, as indicated by the high‐mannose binding lectins griffithsin (GRFT) and scytovirin (SVN) that correlated with lower anti‐HIV‐1 (SVN, p = 0.02; n= 90; t‐test) and anti‐HSV activities (GRFT, p=0.001; n= 139; t‐test). We also found that the drug delivery mode has a significant impact on the glycan composition of the CVL, which in turn may affect innate immunity. Gel use had a greater impact on the glycome with a significant loss of high‐mannose signature (GRFT, p = 0.008; SVN, p = 0.01; n= 53; paired t‐test). GRFT immobilized on agarose beads were used to isolate GRFT‐binding glycoproteins in the CVL and a proteomic analysis on those samples identified a number of protease inhibitors that were previously associated with reduced susceptibility to HIV infection. Collectively, our data suggests that high‐mannose in the CVL is a marker for anti‐viral activity and that thin films do not impact the innate anti‐viral immunity in the CVL.

Support or Funding Information

This work was funded by the National Institutes of Health, Grant # U191AI082639.
LEPTIN LEVELS ATTENUATED BY COCONUT WATER IN HIGH‐FAT AND HIGH‐FRUCTOSE DIET FED ANIMAL MODELS
Authors: Imaga Ngozi A , Obafemi Adepoju , Onadeko Oluwadamilola , Okafor Uzoma A
Published: November 2023

Coconut water is the liquid endosperm of coconut fruitrich in important nutrients such as vitamins, phytohormones, amino acids andphytochemicals. Tender coconut water, the fluid present in young green coconuts and mature coconut water, the clear liquid present in the older brown coconut have been shown to significantly reduce hyperlipidemia in cholesterol fed rats. Leptin is a 16 KDa peptide hormone that is the product of ob gene in adipose tissues. Leptin level in circulation is directly related to adipose tissue mass. The effect of tender and mature coconut water on the leptin level of high‐fat and high‐fructose diet fed rats was investigated in this study. 55 rats were separated into 11 experimental groups of which 4 groups each had high‐fat and high‐fructose feeding respectively with and without coconut water treatment. The rats were fed for 8 weeks followed by 4 weeks of treatment with tender coconut water, mature coconut water and atorvastatin as reference standard. Leptin level was significantly reduced(p<0.01) by tender and mature coconut water in a manner that was comparable to atorvastatin in both high‐fat and high‐fructose diet fed rats. Leptin lowering in the high‐fat fed rats was similar in both mature and tender coconut water, but the lowering effect was more in the mature coconut water (MCW) than in the tender coconut water (TCW) in the high‐fructose fed rats. Tender and mature coconut water thus, have a lowering effect on leptin level in circulation. This lowering effect might be due to the reduction of adipose tissue mass by the metabolic action of tender and mature coconut water or the interplay of their phytochemical and proximate constituents in a synergistic fashion or the contribution of all the factors.

Support or Funding Information

na
Finding Inhibitors for The Type II Phosphatidylinositol 4‐kinase Alpha Enzyme
Authors: SENGUPTA NIVEDITA , JOVIC MARKO , BARNAEVA ELENA , Baumlova Adriana , Southall Noel , Hu Xin , Marugan Juan , Ferrer Marc , Boura Evzen , Balla Tamas
Published: November 2023

Type II phosphatidylinositol 4‐kinases (PI4K2A and 2B) are two of four lipid kinases that synthesize phosphatidylinositol 4‐phosphate (PI4P) in vertebrates. PI4K2A and PI4K2B are highly homologous only differing in their NH2 termini. PI4K2A is tightly membrane bound via palmitoylation and is the most active PI4K associated with various cellular membranes. It has been shown to play important roles in trafficking of several cargoes from the plasma membrane to the sorting endosomes and beyond. The enzyme is localized to endosomes and the trans‐Golgi network (TGN) where it was shown to recruit various clathrin adaptor proteins. Given its central function in vesicular trafficking, and possibly controlling viral replication and assembly, development of PI4K2A‐specific inhibitors would greatly facilitate research in understanding the role of the protein in controlling all of these processes. Few compounds have been described to inhibit these enzymes and they lack selectivity and access to intracellular compartments. The aim of this study was to identify potent and specific small molecule inhibitors for this enzyme.

A high throughput inhibitor screen was performed with ~350,000 compounds from the molecular library repository using a bacterially expressed human PI42A enzyme. Based on primary screen results and counter‐screens with PI4KB (a type III PI4K that belongs to a different class of proteins) 14 inhibitors were short listed for further studies. The kinase activity of the human PI4K2A is strongly dependent on a cysteine‐rich sequence that is palmitoylated in the vertebrate enzyme providing stronger membrane association. Hence, all 14 inhibitors were re‐tested using expressed enzymes isolated from COS‐7 cells or using assays that tested the activity of the enzyme in situ. We found a strong correlation between the potency of inhibitors between the enzymes isolated from COS‐7 cell or expressed in bacteria. In situ kinase assays were then performed in the presence of type III PI4K inhibitors in permeabilized cells showing two inhibitors that reduced PI4P and PIP2 phosphorylation, thus confirming the effect of these inhibitors on the intracellular PI42A activity. Lastly, confocal microscopy revealed that these two inhibitors reduced the Golgi and endosomal pool of PI4P. Further studies are in progress to improve the selectivity and potency of these lead compounds.
Selective inhibitors epigenetically modify and eradicate tumor‐initiating stem‐like cells through downregulating microRNA 22‐mediated TET induction and apoptosis
Authors: Machida Keigo , Chen Chia‐Lin NONE , Kumar Dinesh Babu Uthaya , Swaminathan Suresh , French Samuel W , Panyam Jayanth
Published: November 2023

A critical barrier to improved cancer therapy is the recurrence of drug‐resistant tumors expanded from tumor‐initiating stem‐like cells (TICs). Discovery of a drug that specifically targets the TIC population is critical for effective treatment. We first performed drug screening on TICs for the identification of cell‐type specific drugs and found that all‐trans retinoic acid (ATRA) specifically inhibited cell viability. Additionally, transduction of human TICs with a lentivirus Nanog‐GFP reporter was used to perform high‐throughput screening for Nanog‐inhibitory drugs. HDAC inhibitor (SAHA), among several candidates, suppressed Nanog expression. Three high‐throughput screenings identified the best combination of repurposed FDA‐approved drugs (ATRA and SAHA). Moreover, combination of RA with SAHA synergistically reduced Nanog expression and inhibited the self‐renewal abilities of TICs resulting in apoptosis in vitro and in vivo. Genome‐wide transcriptome analysis by using of RNA‐seq showed that combined treatment reduced microRNA‐22, which induced phosphatase and tensin homolog (PTEN) and ten‐eleven translocation (TET). PTEN‐mediated FOXO activation promotes BIM‐mediated apoptosis. TET induction demethylates p53‐binding sites within the Nanog promoter proximal region. The drug combination epigenetically altered DNA methylation of Nanog promoter leading to inactivation of Nanog in TICs. Taken together, ATRA and SAHA may serve as a novel strategy for HCC treatment.

Support or Funding Information

This project was supported by NIH research grants R01AA018857, P50AA011999 (Southern California Research Center for ALPD and Cirrhosis, pilot project, program, animal core, morphology core), Lee‐Summer‐Project funding, P30DK048522 (USC Research Center for Liver Diseases, pilot project program), Non‐Parenchymal Liver Cell Core (R24AA012885) and UO‐021898. This research was also supported by a Research Scholar Grant (RSG‐12‐177‐01‐MPC); pilot funding from American Cancer Society (IRG‐58‐007‐48); The Cell and Tissue Imaging Core ‐ USC Research Center for Liver Diseases (P30 DK048522).
A study of putative genetically determined defects of de novo purine synthesis using CRISPR‐Cas9 genome‐edited HeLa cells
Authors: Skopova Vaclava , Baresova Veronika , Souckova Olga , Krijt Matyas , Kmoch Stanislav , Zikanova Marie
Published: November 2023

Objectives

The diagnosis of patients with neurological impairment can be set down only in 2% of studied cases. The reason might be the inability to capture new, to date unknown, diseases. One of the metabolic pathways whose malfunctions could lead to neurological impairment is de novo purine synthesis (DNPS). Currently, two genetically determined defects of DNPS enzymes, ADSL deficiency and AICA‐ribosiduria, are known. Therefore, the existence of other defects manifested by neurological symptoms and by accumulating DNPS intermediates in body fluids, is highly presumable.

Methods

We knocked out the expression of DNPS enzyme‐coding genes in HeLa cells using GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit. We tested the cells for presence of mutations by DNA sequencing, for the presence of knocked‐out proteins by Western blot, and we analyzed DNPS substrates accumulation in cell lysates and growth media by LC‐MS/MS. We enzymatically prepared the substrates of DNPS enzymes for use as standards. We also analyzed the ability of the knockout cells to form purinosome by immunofluorescence.

Results

We prepared CRISPR‐Cas9 genome edited HeLa cells deficient for particular steps of DNPS to model possible genetically determined defects of the DNPS enzymes. We developed an analytical method for screening of these defects based on LC‐MS/MS analysis of the accumulated enzyme substrates. The precise detection of the metabolites was accomplished using the prepared DNPS intermediates. In all prepared model cells except of one we detected an accumulation of the substrate(s) for knocked‐out enzyme. The purinosome formation was disrupted in cell lines where the DNPS enzymes GART, ADSL and ATIC were knocked‐out. In cell lines with knock‐out of the other proteins of DNPS was the purinosome formation reduced.

Conclusion

We conclude that, similar to ADSL deficiency and AICA‐ribosiduria, the deficiency of other DNPS enzymes will manifest with accumulation of substrates for these enzymes in the body fluids of patients. Our data allow the detection of PAICS and PFAS deficiency in a population of patients with severe neurological impairment that have not yet been diagnosed.

Support or Funding Information

This work was supported by grants AZV 15‐28979A from the Ministry of Health, CR, PRVOUK‐P24/LF1/3 programs of Charles University in Prague, CR, and NPU from the Ministry of Education, Youth and Sports, CR.
Role of Wnt Signaling in Colon Cancer Cell Metabolism
Authors: Tanio Stephanie Sprowl , Pate Kira , Waterman Marian L.
Published: November 2023

Over eighty percent of colon cancers derive from mutations that overactivate the Wnt pathway and cause cell transformation. These mutations stabilize β‐catenin protein, leading to its accumulation and nuclear translocation. Nuclear localization enables interaction with LEF/TCF transcription factors and the transcriptional overactivation of Wnt target genes, or gene programs. Identifying these gene programs and defining how they drive both the cancer cell phenotype and the tumor response to drug treatment is critical for developing therapies that block tumor progression. We and others have discovered that metabolism is one of the gene programs regulated by Wnt. Constitutively active Wnt signaling drives cells toward a glycolytic phenotype even in the presence of oxygen, a phenomenon known as Warburg metabolism. Using Gene Expression and ChIP‐seq analysis of colon cancer cells expressing dominant negative LEF‐1 and TCF‐1 isoforms (dnLEF/TCFs) we identified direct Wnt target genes that regulate this metabolic phenotype. Gene ontology analysis identified a subset of metabolism‐linked genes, including PDK1 (Pyruvate Dehydrogenase Kinase 1) and SLC16A1/ MCT1 (monocarboxylic acid transporter 1). PDK1 is a mitochondrial kinase that suppresses respiration and increases rates of glycolysis. MCT1 is a membrane‐bound transporter critical for maintaining the homeostasis of glycolytic cancer cells through import and/or efflux of lactate. PDK1 and MCT1 RNA and protein levels are significantly and specifically down regulated by dnLEF/TCFs in multiple colon cancer cell lines (SW480, SW620, HCT116 and DLD1). ChIP‐seq analysis with dnTCF1 identified regulatory regions with Wnt Response Elements (WREs) in distal and promoter proximal locations for each locus. Each peak of occupancy could confer Wnt regulation in luciferase reporter assays. c‐Myc, a Wnt target gene and known regulator of SLC16A1 transcription, regulates the locus additively but not synergistically with LEF/TCFs. Work by others has shown MCT1 to be necessary for the import of 3‐bromopyruvate, a potential cancer therapeutic that inhibits glycolysis through a mechanism not well understood. Our data suggests that colon cancer cells are sensitive to 3‐bromopyruvate in a Wnt dependent manner, and that co‐treatment of cells with Wnt inhibitors (e.g. XAV939, a tankyrase inhibitor) promotes resistance and recovery from 3‐bromopyruvate treatment. Overall this data suggests that Wnt signaling is important for maintaining a Warbug phenotype through PDK1 and MCT1, and that MCT1 may serve as an important indicator for drug sensitivity in colon cancer therapy.

Support or Funding Information

NIH R01 CA108697 (PI: Waterman), President's Dissertation Year Fellowship, UC Irvine 2015–2016
Allyl‐isothiocynate (AITC) ameliorates LPS induced NF‐κB mediated neuroinflammation
Authors: Subedi Lalita , Lee Taek Hwan , Chae Jae Kyoung , Venkatesan Ramu , Kim Sun Yeou
Published: November 2023

Toxins‐induced microglial activation can initiate the inflammatory cascade in most of the neurodegenerative as well as neuroinflammatory conditions that leads to the neuronal cell death. Chronic activation of microglia is dangerous and the strategy to control the microglial over activation might be protective in neurodegenerative diseases. Natural compounds especially nutraceuticals are good examples for the treatment of such condition without cellular toxicity. In this study, we investigated the promising antineuroinflammatory activity as well as neuroprotective efficacy of allyl‐isothiocynate (AITC) from W. japonica against LPS activated BV‐2 cells and its toxicity to neuroblastoma cells called N2a cells. We evaluated the neurotrophic effect of AITC in neuronal astrocytes as well. AITC inhibited the LPS activated BV2 induced inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), nitric oxide (NO), prostaglandin E2 (PGE2), Tumor necrosis factor‐a (TNF‐a), interleukin‐6 (IL‐6), MAPK signaling, and NF‐κB mediated transcription. Additionally, AITC reduced the activated microglia induced neuronal toxicity via downregulating the neuronal apoptosis together with enhanced neurite outgrowth in neuronal cells. Furthermore AITC also increased the neurotrophins production through neuronal astrocytes. In conclusion, AITC might be a potential neuroprotective agent.

Support or Funding Information

This research was supported by High Value‐added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (114006041HD020)
Metabolome Dynamics Platform Reveals Endocrine‐metabolic Crosstalk in PCOS and Its Reprogramming of Lipid Metabolic Pathways with 3‐Iodothyronamine
Authors: Assadi‐Porter Fariba M , Selen Ebru , Bolandnazar Zeinab , Sabatin Martina , Rogowski Michael , Chiellini Grazia
Published: November 2023

Objectives

Polycystic ovary syndrome (PCOS) is a complex metabolic and endocrine disorder, and a leading cause of infertility in women. The PCOS metabolic phenotype in women and mice is associated with defective lipid and glucose metabolism that is linked to dysregulation of steroidogenesis through unknown mechanism(s). Here, we investigated tissue specific treatment effects of 3‐Iodothyronamine (T1AM), a natural analog of thyroid hormone, action on metabolic pathways and associated transcriptional and gene signaling in vivo in order to understand the crosstalk between endocrine and lipid metabolism as drug targets in PCOS pathophysiology.

Methods

We analyzed plasma and tissue extracts using multidisciplinary omics and biochemical approaches. We used 1H‐NMR spectroscopy using metabolome dynamics platform (MDP) to analyze metabolomics profiles and complemented with monitoring transcription levels of major targeted metabolic regulatory genes and proteins.

Results

Administering with the low sub‐chronic application of T1AM (10mg/Kg/Day) in vivo induces a profound anti‐lipogenic effect by lowering gene expression of key regulators of lipid metabolism, Ptpb1 and Plin2, and significant increased levels of metabolites (glucogenic amino acids, carnitine and acetate) in a tissue specific manner, while enhancing protection against oxidative stress in liver and muscle. In contrast, T1AM has an opposing effect on the regulation of estrogenic metabolic and gene pathways in the ovary by upregulating Star, Cyp11a1 and Cyp17a1s. Biochemical measurements provide further evidence of a significant reduction of plasma total cholesterol and triglycerides after T1AM treatment.

Conclusions

Our MDP analysis indicate a novel mechanism for T1AM in regulating lipid metabolism, which untangles endocrine‐metabolic pathways, thus elucidating the intricacies within the pathophysiology of PCOS at the tissue specific level in contrast to overall systemic effect. This study opens new avenues to design new approaches for essential therapeutic targets in order to improve quality of life in complex diseases such as PCOS with endocrine‐metabolic dysfunction.

Support or Funding Information

This research was supported by RC4 EY021357 and a Wisconsin Institute of Discovery Grant (WID‐135A039) to F.A‐P. This study made use of the National Magnetic Resonance Facility at Madison for data collection, which is supported by NIH grant P41RR002301 (NIGMS).
A structural imbalance of the brain and the whole body spinal column associated with the temporomandibular disorders
Authors: Lee Somin , Jung Jeeyoun , Jerng Ui Min , Kwon O sang , Choi Kwangho
Published: November 2023

Background

The most commonly reported that the temporomandibular disorders (TMD) refer to the causes symtoms are numerous include systemic, iatrogenic disease, occlusal trauma and mental health disorders. These TMD symptoms are related to muscle sensitivity through palpation, restricted mouth opening, asymmetric mandibular movements, joint sounds, pain and otologic signs and symptoms. However, it is not fully reports about correlation of TMD and the systemic spinal column.

Methods

A survey was performed using the electronic databases (PubMed, SCOPUS, Oasis, Korean TK, RISS, KISS) between 2006 and 2016. The following search keywords were selected using TMD pain, TMD, TMJ, TMJ disorders, systemic spinal column, occlusal splint, TMD physiotherapy, TMJ rheumatoid.

Results

Results of the number of reports, TMD correlation between systemic spinal column. These data were TMD with cervical spine disorders has been reported by several researches. Furthermore, TMD relevant reported that functional cerebrospinal therapy (FCST) (Lee young Jun clinics who developed) is markedly improvement of systemic imbalances in the spine.

Conclusion

In the conclusion, improvement of the temporomandibular joint disorder can be utilized for fundamental treatment that can improve the imbalance of the whole body spinal column.

Support or Funding Information

This work was supported by a Korea Institute of Oriental Medicine (C16100).
Markers of Muscle Protein Synthesis and Breakdown in Slow‐twitch Skeletal Muscle of Rodents Aged 3 to 24 months
Authors: Haun Cody T. , Mumford Petey W. , Mobley Brooks C. , Kephart Wesley C. , Holland Maleah , Osburn Shelby C. , Martin Jeffrey S. , Young Kaelin C. , Kavasis Andreas N. , Lowery Ryan P. , Wilson Jacob M. , Roberts Michael D.
Published: November 2023

PURPOSE

Muscle protein synthesis (MPS) and muscle protein degradation (MPD) are well known modulators for muscle mass maintenance. Given that slow‐twitch skeletal muscle atrophies with aging, we sought to examine the effects of aging on MPS and MPD markers in the soleus muscle of rodents ranging from 3 to 24 months.

MEHTODS

Male Fischer 344 rats (300–600g) were successfully aged to 3, 6, 12, 18 and 24 months, were injected with puromycin (0.02 mg/g body mass), euthanized, soleus muscle was extracted and wet skeletal muscle weights were obtained. Muscle tissue was then processed for biochemical processing. MPS was assessed via the SUNsET method, and MPD was assessed using whole lane densitometry via ubiquitin immunoblotting.

RESULTS

Relative soleus masses revealed significant between‐group differences throughout the lifespan (p=0.004); specifically, masses were 20% greater in 3 versus 18 month old rats (p=0.010) and 20% greater in 3 versus 24 month old rats (p=0.013). Interestingly, MPS levels were not different between any of the age groups. Poly‐ubiquinated protein levels were trending (p=0.051), but not different between any of the age groups.

CONCLUSION

It appears that soleus muscle atrophy occurs with aging. However, this does not appear to be reflected via decrements in MPS and increases in protein poly‐ubiquination. Mechanisms related to slow‐twitch skeletal muscle atrophy remain to be determined and should be further investigated.

Support or Funding Information

ACKNOWLEDGEMENTS: This study was supported in part by a contract from The University of Tampa (to M.D.R. from J.M.W.) as well as laboratory start‐up funds from M.D.R.
“Translation Speed is Genetically Encoded and Influences Protein Folding”
Authors: Spencer Paige , Siller Efrain , Anderson John , Barral Jose
Published: November 2023

The degeneracy of the genetic code allows most amino acids to be encoded by multiple codons. The distribution of these so‐called synonymous codons among protein coding sequences is not random and multiple theories have arisen to explain the biological significance of such non‐uniform codon selection. Many ideas revolve around the notion that certain codons allow for faster or more efficient translation, whereas the presence of others result in slower translation rates. The presence of these different types of codons along a message is postulated in turn to confer variable rates of emergence of the nascent polypeptide from the ribosome, which may influence its capacity to fold towards the native state, among other properties. Previous studies have reported conflicting results with regards to whether certain kinds of codons correlate or not with particular structural or folding properties of the encoded protein. We believe this has arisen, in part, because different criteria have been traditionally used for predicting whether a codon will be translated quickly or slowly in a given organism, including its frequency of occurrence among highly expressed genes and the concentration of tRNA species capable of decoding it, which do not always correlate. We have developed a metric to predict organism‐specific polypeptide elongation rates of any mRNA based on whether each codon is decoded by tRNAs capable of Watson‐Crick, non‐Watson‐Crick or both types of interactions. We demonstrate by pulse‐chase analyses in living E. coli cells that sequence engineering based on these concepts predictably modulates translation rates due to changes in polypeptide elongation and show that such alterations significantly impact the folding of proteins of eukaryotic origin. We also demonstrate that sequence harmonization based on expression‐host tRNA content designed to mimic ribosome movement of the original organism can significantly increase the folding of the encoded polypeptide. This body of work could provide insight into how synonymous nucleotide substitutions result in altered protein function and disease.

Support or Funding Information

The Welch Foundation and The Sealy Center for Structural Biology and Molecular Biophysics
Profiling stage‐related tissue proteomes to discover potential diagnostic and prognostic biomarkers for lung adenocarcinoma
Authors: Hsu Chiung‐Hung , Wu Chih‐Ching , Wang Chih‐Liang , Hsueh Chuen , Wu Yi‐Cheng , Hsu Chia‐Wei , Liu Chin‐Ching , Lai Ping‐Chang , Yu Jau‐Song , Chang Yu‐Sun , Yu Chia‐Jung
Published: November 2023

Lung cancer is the leading cause of cancer‐related death worldwide. Both diagnostic and prognostic biomarkers are urgently needed to increase patient survival. In this study, we identified/quantified 1763 proteins from paired adenocarcinoma (ADC) tissues with different extents of lymph node (LN) involvement using an iTRAQ‐based quantitative proteomic analysis. Based on a bioinformatics analysis and literature search, we selected six candidates (ERO1Lα, PABPC4, RPS25, TARS, NARS, and RCC1) from 151 proteins with a 1.5‐fold increase in ADC tumors without LN metastasis compared with adjacent normal tissue. These six proteins underwent further verification using immunohistochemical staining and western blotting. The protein levels of these six candidates were overexpressed in tumor tissues compared with the adjacent normal tissue. The area under the receiver operating characteristic curve was 0.864 for the combination of the two potential biomarkers (ERO1Lα and NARS) in discriminating tumor tissues without LN metastasis from adjacent normal tissue. Importantly, ERO1Lα and NARS levels were positively associated with LN metastasis, and ERO1Lα overexpression was correlated with poor patient survival. Moreover, we found that the knockdown of either ERO1Lα or NARS reduced the viability and migration ability of ADC cells. Our results collectively provide a potential biomarker dataset for ADC diagnosis/prognosis and reveal novel roles of ERO1Lα and NARS in ADC progression.

Support or Funding Information

This work was supported by grants from the Ministry of Science and Technology, Taiwan, R.O.C. (101‐2320‐B‐182‐035‐MY3 and 104‐2320‐B‐182‐027), the Chang Gung Medical Research Fund (CMRPD3E0081‐2 and CMRPD1C0092) and the Ministry of Education, Taiwan, R.O.C. (EMRPD1E1401).
Site‐Specific Covalent Labeling of RNA with RNA‐TAG: A Platform for the Elucidation of RNA Function
Authors: Busby Kayla N , Alexander Seth C , Devaraj Neal K
Published: November 2023

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Here, we demonstrate the site‐specific covalent labeling of RNA transcripts using the RNA‐TAG (transglycosylation at guanosine) methodology. This technology takes advantage of a bacterial tRNA transglycosylase (TGT) capable of incorporating nucleobase derivatives bearing functional probes into an encodable RNA stem loop recognition motif. Using this methodology, we have successfully labeled mRNA transcripts with a wide variety of probes including biotin, BODIPY, and Cy7 in a single enzymatic step. Endogenously transcribed and processed RNAs containing the RNA‐TAG recognition hairpin have been successfully visualized in cell imaging studies. Furthermore, we have demonstrated that an RNA of interest can be selectively biotinylated and affinity purified from a complex mixture of RNAs and cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient platform for the elucidation of RNA function via cellular localization studies and affinity purification of RNA‐protein complexes.

Support or Funding Information

This work was supported by the Department of Defense Army Research Office Contract W911NF‐13‐1‐0383. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1144086 (fellowship to K.N.B.).
Nonstop Decay in C. elegans: Examination of a Possible Role for Endogenous siRNAs
Authors: Youngman Elaine M , Gu Weifeng , Mello Craig C
Published: November 2023

In C. elegans, small noncoding RNAs known as 22G RNAs are synthesized by RNA‐dependent RNA polymerases and target thousands of protein‐coding mRNAs, repetitive sequences, and cryptic transcripts throughout the genome. In some cases, 22G RNA synthesis appears to be triggered as a secondary event downstream of targeting by C. elegans piRNAs (known as 21U RNAs) in a pathway that defends against non‐self RNA species. (1, 2) However, the synthesis of many 22G RNAs is unaffected by loss of 21U RNAs, and the triggers for biosynthesis of these 21U‐independent 22Gs remain elusive. Using a comparative genomic approach in geographically distributed wild isolates of C. elegans, we have identified a locus (F43E2.6) at which loss of the stop codon is correlated with increased production of 22G RNAs. The F43 mRNA has a short 3′UTR with no further in frame stop codons, raising the intriguing possibility that 22G RNAs could be involved in a nonstop decay pathway in C. elegans. F43 is also targeted by an unusual Dicer product; however this Dicer product does not appear to act as a primary siRNA to trigger F43 22G RNAs. First, the Dicer product is expressed equally in strains with large differences (more than 10 fold) in 22G RNA levels. Second, this product is expressed only during embryogenesis, whereas F43 22G RNAs are expressed most strongly in adults. We have demonstrated that the 22G RNA expression phenotype at the F43 locus is genetically linked to the locus itself, and are currently assaying transgenic worms carrying F43 reporter constructs that bear or lack a stop codon to determine whether the loss of a stop codon is indeed causal for increased 22G RNA production. In addition, we are generating reporter constructs that will allow us to ask whether 22G RNAs play a functional role in the nonsense‐mediated and no‐go decay pathways.
Production and Characterization of Polyclonal Generic Phosphotyrosine‐specific Antibodies
Authors: Pelech Steven , Yue Lambert , Lai Shenshen , Winkler Dirk , Shi Jane , Zhang Hong
Published: November 2023

Reversible protein‐tyrosine phosphorylation plays critical roles in cell regulation under normal and pathological conditions, which has made generic phosphotyrosine (pY) antibodies invaluable tools for biomedical research. A recent study that compared the specificities of three widely‐used monoclonal pY antibodies has raised concerns about strong sequence motif selectivity and low overall coverage rates. These issues can potentially introduce significant bias in pY site identification and quantification. In this study we describe a novel strategy of generating a pool of polyclonal antibodies from a large set of physiological pY‐site sequences. Over 400 peptides spanning 7 to 20 amino acids in length, each featuring 1 to 7 phosphorylation sites and representing over 1000 phosphosites, were used to immunize over 100 rabbits. The sera from these rabbits were subjected to ammonium sulphate fractionation, pooled, and then affinity‐purified on pY‐agarose columns to produce the PYK antibody preparations. The specificity of the purified PYK antibody was assessed with phosphopeptide microarrays, including the Jerini Peptide Technologies Phosphatase Peptide Microarray, which features 6,099 peptides representing diverse human pY‐sites. The rabbit polyclonal PYK phosphotyrosine antibody proved to perform better than the well known 4G10, PY20 and PY100 mouse monoclonal antibodies. PYK was extremely stable to repeated freeze thaw, and found to be 4‐fold or more sensitive for detection of phosphopeptides on arrays and for proteins following Western blotting of EGF‐treated A431 cell lysates than the monoclonal antibodies. Like the other monoclonal pY antibodies, PYK reactivities with phosphothreonine and phosphoserine were over 200‐ and 300‐fold less, respectively, when compared to phosphotyrosine on phosphopeptide microarrays. PYK also detected a larger number of diverse pY‐containing peptides than 4G10, PY20 and PY100 on phosphopeptide microarrays, and unlike these other antibodies, acidic amino acid residues surrounding the pY‐sites were not negative determinants for antibody recognition. Since acidic amino acids are important positive determinants for substrate recognition for most protein‐tyrosine kinases and flank most protein‐tyrosine phosphosites, our findings indicate that the PYK antibody may be more useful for enrichment and analysis of physiological pY‐sites than the commonly used mouse monoclonal antibodies for such purposes. Further purification of the PYK antibody over an IgG‐agarose column also allowed development of preparation of a reporter antibody that was suitable for use in sandwich antibody microarrays to assess changes in the tyrosine phosphorylation status of over 500 signaling proteins simultaneously with as little as 50 μg of crude lysate protein from cells and tissues.

Support or Funding Information

Supported by Kinexus Bioinformatics Corporation.
Successful Diagnosis and Treatment of a Case of Arterial Adventitial Cystic Disease (ACD)
Authors: Liu Ye , Wang Long , Zhang Feng
Published: November 2023

Purpose

Arterial adventitial cystic disease (ACD) is a rare type of non‐atherosclerotic peripheral vessel disease which results in intermittent claudication. There has been only a few cases reported in the past 50 years and its pathogenesis has been poorly understood. In ACD, it is the challenging goal of surgeons to reach a quick and correct diagnosis operation. A series of observations and treatment in one case of typical ACD were conducted to better understand the development and the diagnostic procedure of ACD.

Methods

A 55‐year‐old female patient visited the hospital with a complaint of claudicating on the right lower extremity that had begun three months earlier. Computed tomography angiography (CTA) and digital subtraction angiography (DSA) was performed for exact diagnosis. Tissue samples obtained from surgeries were observed histopathologically.

Results

The patient was admitted in hospital for suspecting occlusive artery on right lower extremity based on a physical examination and CTA and the treatment plan for arterial occlusion was stent placement. After carefully reviewed the result of CTA, the authors found the intima of popliteal artery was smooth and a mass with uniform density could be seen near the adventitia. Digital subtraction angiography (DSA) was further performed for exact diagnosis and the result indicated adventitial cystic disease as well. Thus, a cystic mass excision was performed. Stent‐graft bypass implantation was performed because the cyst was 8cm long which was not fit for partial excision repair. In the end, a cystic mass obtained from surgeries were observed histopathologically and final ACD diagnosed based on the macroscopic findings. The patient was recovered and the claudicating on the right lower extremity was resolved.

Conclusions

Arterial adventitial cystic disease is a rare type of non‐atherosclerotic peripheral vessel disease. It could be misdiagnosed as occlusive artery and stent placement would aggravate the condition. Therefore, the diagnosis is critical for ACD and the cystic mass excision must be performed.
Development and Delivery of Robust Biosensors for Measuring Signal Transduction in Living Cells
Authors: Tillo Shane , Tewson Paul , Martinka Scott , Hughes Thom , Quinn Anne Marie
Published: November 2023

We present a set of robust, easy to use, genetically encoded sensors for the detection of cyclic AMP (cAMP), diacyl‐glycerol (DAG), and PIP2 in a variety of living cell types. The sensors fluoresce in either the green or red part of the visible spectrum, and they can be combined with one another, or other colored sensors, for the simultaneous detection of multiple components of signal transduction pathways. The sensors are packaged in BacMam, a modified baculovirus, under the control of CMV, synapsin, EF1a, or CAG promoter systems for ease of use in a wide variety of cells.

We present examples of the simultaneous imaging of cAMP and DAG, cAMP and PIP2, and DAG and PIP2 kinetics. We use the cAMP sensors to detect, quantify, and compare Gi and Gs mediated signaling across CHO, HEK293, and NIH 3T3 cells. The cAMP sensor is remarkably sensitive, and it can be used to detect either Gs activation of adenyl cyclase or Gi inhibition of adenyl cyclase in the same cells. Notably, our technology allows for Gi detection without the need for forskolin or IBMX pre‐treatment. These sensors are not only easy to use on standard microscopes, but they excel on automated plate readers, producing Z’ values > 0.8.
Genomic analysis and comparison of the extremely halophilic archaea using next‐generation sequencing
Authors: Rhee Jin‐Kyu
Published: November 2023

Extremely halophilic microorganisms have potentially valuable physiological properties to grow at high salt concentrations; they produce enzymes that have potential biotechnological applications especially for food engineering. These three strains extremely halophilic archaea, Halorubrum halophilum B8T, Halapricum salinum CBA1105T and Halococcus sediminicola CBA1101T, had been collected from saline samples and their genomes were sequenced through a next‐generation sequencing platform. The draft genomes of strain B8T, CBA1105T and CBA1101T contain 3,677,984 bp, 3,451,492 bp and 3,764,367 bp, respectively, with G + C contents of more than 60% and include genomic information on various carbohydrate‐active enzymes. Based a detailed comparative analysis with the haloarchaeal genome data, these novel halophilic enzymes were investigated further to find practical opportunities for biotechnological applications in food engineering.