bluuebunny/crossref_metadata_2025 huggingface dataset:
Lessons Learned from PKA: from Motifs to the Dynamic Assembly of Isoform‐specific Macromolecular Switches
Authors: Taylor Susan S.
Published: November 2023

Twenty‐five years after the first protein kinase structure, we are still learning about the fundamental properties that define this large enzyme family, and cAMP‐dependent protein kinase (PKA) continues to serve as a prototype for the superfamily. Each protein kinase has evolved to be a highly regulated and dynamic molecular switch. For most kinases the assembly of the active kinase is dynamically and transiently regulated by the alignment of a hydrophobic regulatory spine. In the case of PKA, however, the active catalytic (C) subunit is then packaged as an inactive R2C2 holoenzyme with dimeric regulatory (R) subunits that are receptors for cAMP. PKA thus combines two of the major regulatory mechanisms in biology, protein phosphorylation and cAMP signaling. Specificity in PKA signaling is achieved in large part by four functionally non‐redundant R‐subunits (RIa, RIb, RIIa, and RIIb), and targeting of these holoenzymes to specific sites in the cell creates yet another layer of specificity. The functional non‐redundancy of the R‐subunits is reflected in the structural diversity of the holoenzymes. Many diseases result from dysfunctional PKA signaling, and these diseases also reflect the diversity and unique functions of the R‐subunit isoforms. While we have learned much from the individual subunits, it was not until we solved the structure of the full length R2C2 holoenzyme that we could appreciate allostery and also why the four holoenzymes were functionally so different. Although each R‐subunit has the same domain organization that includes an N‐terminal dimerization/docking (D/D) domain and two tandem cAMP binding (CNB) domains, the quaternary organization of each holoenzyme is distinct due in large part to the intrinsically disordered linker that joins the D/D and CNB domains. Embedded in the linker is an inhibitor site that docks into the active site of the C‐subunit in the holoenzyme rendering it inactive, and these linkers in large part drive the conformational state of each holoenzyme. Our major challenge now is to understand how the flexible linkers drive the assembly and regulation of the holoenzyme. Capturing this dynamic space is a major challenge for PKA signaling and indeed a fundamental challenge for all protein kinases. Recognizing this critical roadblock, we are now using cryo Electron Microscopy (cryoEM) in parallel with crystallography and SAXS/SANS to explore the domain dynamics that contribute to PKA signaling.

Support or Funding Information

Funded in part by NIH grants GM34921 and DK54441.
Interactions between the G Protein‐coupled Estrogen Receptor and the Stromal Interaction Molecule 1
Authors: Tran Kim , Terry Lara E , Gebert‐Oberle Briana , Giles Jennifer , VerMeer Mark
Published: November 2023

The G protein‐coupled estrogen receptor (GPER/GPR30) participates in various Ca2+‐dependent cellular functions, yet its direct effects and mechanisms on components of Ca2+ signaling machinery are incompletely understood. Store‐operated Ca2+ entry (SOCE) is an essential Ca2+ signaling mechanism that regulates numerous cellular activities. Here we show that GPER/GPR30 expression and activation limits SOCE. Heterologous expression and gene silencing studies demonstrated that GPER/GPR30's expression puts a constitutive break on SOCE. Co‐immunoprecipitation and confocal microscopy studies indicated that GPER/GPR30 interacts with the stromal interaction molecule 1 (STIM1) at different interaction sites in distinct subcellular compartments. Pharmacological studies indicated that GPER/GPR30 activation acutely further suppresses SOCE in addition to the effect of physical interaction between GPER/GPR30 and STIM1. Site‐directed mutagenesis studies indicated that GPER/GPR30 activation inhibits SOCE via alteration in the phosphorylation status of STIM1. Taken together, the data demonstrate physical and functional interactions as mechanisms whereby GPER/GPR30 expression and activation regulates SOCE.

Support or Funding Information

NIH HL112184 (to KT)
A G Protein‐Biased Ligand of the Kappa Opioid Receptor is Antinociceptive and Antipruritic But Does Not Cause Sedation or Dysphoria
Authors: Brust Tarsis F , Morgenweck Jenny , Kim Susy A , Rose Jamie H , Locke Jason L , Schmid Cullen L , Zhou Lei , Stahl Edward L , Cameron Michael D , Scarry Sarah M , Aubé Jeffrey , Jones Sara R , Martin Thomas J , Bohn Laura M
Published: November 2023

The kappa opioid receptor (KOR) has been pursued as a target to treat pain and pruritis (itch). In contrast to opioid narcotics that target primarily the mu opioid receptor, KOR agonists are devoid of addictive and life‐threatening side effects, such as respiratory depression. However, the clinical use of KOR agonists has been limited by the onset of dysphoria and sedation. The KOR is a G protein‐coupled receptor (GPCR) that mediates its physiological actions through the activation of multiple signaling effectors, such as G proteins and βarrestins. Notably, the molecular pharmacology of GPCRs can be specifically modulated by functionally selective (or biased) ligands to induce preferential activation of a subset of the receptor's responses. The use of such ligands has been proposed as a strategy to improve drug therapies targeting GPCRs. For the KOR, selective activation of G proteins over βarrestins may present a means to promote analgesia and inhibit itch in the absence of dysphoria and sedation. In the current study, we evaluate a recently developed G protein‐biased KOR agonist in preclinical models of pain, pruritis, sedation, dopamine regulation and dysphoria. We show that while triazole 1.1 is antinociceptive and antipruritic, the compound does not induce sedation in mice. Additional studies show that, in contrast to the reference KOR ligand U50,488H, triazole 1.1 does not cause reductions in dopamine release or behaviors indicative of dysphoria in rodent models. These results illustrate that biased ligands may be useful to separate physiological responses downstream of GPCRs. Furthermore, our findings suggest that G protein‐biased ligands at the KOR, which inherently have reduced abuse potential, may present a means to treat pain and intractable itch without the side effects of dysphoria and sedation.

Support or Funding Information

This work was supported by NIH grants P01GM113852 and P50DA006634 (to T.J.M.), R01DA014030 and U01AA014091 (to S.L.J.), and R01DA031297 (to L.M.B. and J.A.).
Mechanistic Classification of Toxicants Using Differential Impedance‐Based Real Time Cytotoxicity Profiles in Primary Human Hepatocytes
Authors: Liguori Michael J , Sharapova Tatiana , LeRoy Bruce , Blomme Eric AG
Published: November 2023

Impedance‐based real time cytolethality profiling has become an important lead optimization toxicology tool in pharmaceutical discovery. This technique offers the advantage of continuous, non‐destructive monitoring of changes in electrical impedance (reported as cellular index or CI) that correlates to alterations in cell health, morphology, and proliferation of adherent primary cells as well as cell lines. In this study, we used a panel of reference toxicants representing several distinct mechanisms of hepatotoxicity and evaluated their impact on CI profiles of cultured primary human hepatocytes (PHH). The mechanisms of action included: apoptosis (staurosporine; ceramide c2), cytoskeletal disruption (phalloidin; colchicine), mitochondrial inhibition (antimycin A; carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP)), plasma membrane perforation (Triton X‐100), and general cellular detachment (trypsin digestion). PHH donor to donor variability (n= 6 donors) was assessed using chlorpromazine, which acts through two cytotoxicity mechanisms: oxidative stress via quinoneimine formation and changes in mitochondrial membrane potential. Cryopreserved PHH were thawed and cultivated using conventional conditions in Williams E‐based media. Cells were treated with eight concentrations of each toxicant or vehicle (0.1 to 0.5% DMSO) in electrode coated 96‐well plates and monitored with an xCELLigence real‐time cell analyzer (Acea Biosciences) for up to 3 days. Treatment of PHH with surfactant (Triton X‐100) or trypsin resulted in an immediate decline of normalized cellular index (NCI). Mitochondrial function inhibitors caused a rapid decrease of NCI at doses as low as 1.6 and 3.1 μM. Staurosporine and ceramide treatment (apoptosis inducers) decreased NCI (IC50 =1 to 6 μM), but over an extended time (>10 h) compared to the mitochondrial toxicants. Interestingly, exposure to cytoskeletal disruptors led to a gradual increase in NCI over 3 days, plausibly due to subtle alterations in cell shape. There was a high degree of inter‐individual variability in response to chlorpromazine treatment suggesting that cryopreserved PHH should be characterized using a positive control before an impedance‐based compound evaluation. Altogether, these results are useful to understand differences in impedance profiles based on compound toxicity mechanisms and can be compared to profiles from related liver‐derived primary cells and cell lines.

Support or Funding Information

All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Blood Pressure in the SHR Following Renal Denervation or Dopamine β‐Hydroxylase Inhibition with Etamicastat
Authors: Pires Nuno , Igreja Bruno , Moura Eduardo , Wright Lyndon , Serrão Paula , Soares‐da‐Silva Patrício
Published: November 2023

Overactivity of the sympathetic nervous system plays an important role in the development and progression of arterial hypertension. Catheter‐based renal nerve ablation for treatment of drug‐resistant hypertension has been recently developed. An alternative strategy for modulation of sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β‐hydroxylase (DβH), the enzyme that catalyses the conversion of dopamine to NE in sympathetic nerves.

Renal denervation (RDN) was performed in spontaneously hypertensive rats (SHR) to evaluate its effect on NE levels and blood pressure over a 28‐day period. Chronic oral treatment with the peripherally selective DβH inhibitor etamicastat (30 mg/kg/day) was performed in another cohort of SHR.

RDN and chronic oral treatment with etamicastat did not affect renal function, as assessed by urinary protein, creatinine and urea levels. RDN significantly decreased mean arterial blood pressure (MAP, in mm Hg) 7 days post‐surgery (139±6, P<0.01) versus baseline values (158±6). A gradual return of MAP to high baseline levels was observed over time; 139±7 at day 14, 151±12, at day 21 and 155±12 at day 28 after RDN. Treatment with etamicastat decreased baseline MAP (150±5) in a sustained and significant (P<0.01) manner at all‐time points: 141±5 at day 7, 139±5 at day 14, 138±5 at day 21 and 143±5 at day 28. At day 28, NE levels (in pmol/mg tissue) in renal tissue were significantly decreased in both RDN (0.12±0.08, P=0.001) and etamicastat‐treated groups (0.48±0.04, P=0.01) as compared to vehicle group (0.88±0.13). In contrast, left ventricle NE levels were decreased after treatment with etamicastat (1.24±0.12, P=0.04) but not in the RDN group (2.53±0.24, P=0.9) when compared to controls (2.59±0.58).

It is concluded that RDN produces transitory decreases in MAP, whereas prolonged down regulation of sympathetic drive with the DβH inhibitor etamicastat results in sustained decreases in MAP.

Support or Funding Information

This work was supported by BIAL ‐ Portela & Ca, S.A.
Investigation of the Mechanism of Cytotoxic Natural Product PateamineA analog
Authors: Kommaraju Sai Shilpa , Low Woon‐Kai , Romo Daniel
Published: November 2023

Natural products have served as a source for many modern therapeutics. Over the past decade, high throughput screening of libraries of small molecule compounds have been popular in drug discovery. One such natural product is PateamineA (PatA). The natural product PatA isolated from the marine sponge Mycale hentscheli has been found to be a highly potent antiproliferative agent with potential as a novel anti‐neoplastic agent. PatA and the simplified analog desmethyl, desamino PateamineA (DMDAPatA) have cytotoxicity selective for rapidly proliferating cells by inhibition of cap‐dependent translation initiation through binding to eIF4A (eukaryotic initiation factor 4A) of eIF4F complex. eIF4F complex consists of eIF4A (RNA helicase), eIF4E (cap‐binding protein) and eIF4G (Scaffolding protein). The consequence and impact of other eIF4F complex components on DMDAPatA action are investigated in vitro and and in cultured mammalian cells. Preliminary results demonstrate a role for eIF4E and cap‐binding in DMDAPatA mechanism of action.
Improving Therapy for Refractory Mantle Cell Lymphoma Using Small Molecule Inhibitors Vorinostat and Palbociclib to Target Histone Deacetylase and Cyclin Dependent Kinase 4/6
Authors: Hatch Nathan , Chaturvedi Nagendra , Kling Mathew , Joshi Shantaram
Published: November 2023

Mantle Cell Lymphoma is a B‐Cell non‐Hodgkin lymphoma that accounts for 10% of all non‐Hodgkin lymphoma cases. It is extremely aggressive with a median survival time of approximately 5 years. There is a high rate of relapse due to therapy‐resistant tumor cells. There is currently no cure for refractory MCL. Here we demonstrate that using the histone deacetylase (HDAC) inhibitor, Vorinostat, in conjunction with cyclin dependent kinase (CDK) 4/6 inhibitor, Palbociclib, can be an effective method of treatment. This is shown using multiple in vitro experiments including: MTT assay, Annexin‐V staining, and colony formation assay. This combination of small molecule inhibitors has been found to decrease tumor cell proliferation by at least 25% as well as increase the rate of tumor cell apoptosis by at least 60% when compared to the use of these drugs on their own at equivalent concentrations. Future research will consist of in vivo studies using murine models.

Support or Funding Information

This publication was made possible by grants from the Lymphoma Research Foundation New York, NY, the State of Nebraska LB506 Funds, and the National Institute for General Medical Science (NIGMS) (8P20GM103427), a component of the National Institutes of Health (NIH) and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
Employing Surface‐Displayed Proteins on Bacillus megabacterium Spores to Detect Environmental Pollutants
Authors: Wynn Daniel Toner , Mittal Rhiya , Knecht Leslie , Deo Sapna , Daunert Sylvia
Published: November 2023

Arsenic poses a major threat to both human populations and the environment. It is carcinogenic and a potent toxin. Because of its hydrophilicity, dangerous levels of arsenic can be found in the drinking water of many communities, especially in areas where water is collected from wells. As such, developing methods to quickly and reliably assess a wide variety of matrices for the presence of arsenic is very important. The current gold standard for arsenic detection is inductively‐coupled plasma‐mass spectrometry (ICP‐MS). Due to the limitations of ICP‐MS, chiefly its expense and inconvenience, many other methods have been developed, including biosensors.

We have developed a novel biosensor that employs the morphology of bacterial endospores to create spores engineered to be analytical tools (SEATs). In these biosensors, the sensing and reporting elements are displayed on the surface of Bacillus megaterium spores. This was accomplished by creating a triple fusion protein that joins the spore coat protein CotB, the arsenite binding protein ArsR, and the fluorescent reporter EGFP N‐terminus to C‐terminus, respectively. SEATs have greater sensitivity than traditional cell based biosensors, with limits of detection of 1 × 10‐11 M. We have also demonstrated that SEATs are suitable for use in complex matrices using both spiked samples and samples collected from local areas with arsenic contamination.

Support or Funding Information

Funded by the National Science Foundation.
Determination of total oxytocin in human serum by traditional LCMS
Authors: Franke Adrian , Menden Ariane , Li Xingnan
Published: November 2023

Oxytocin (OT) is a nanopeptide with hormone, neurotransmitter/neuromodulator and other activities. Interest in measuring OT has peaked in recent years due to its newly reported association with autism, schizophrenia, depression, social affiliation and many other conditions. Non‐protein bound (‘free’) OT is present in the human circulation but its measurement using traditional ELISA has been unreliable and inconsistent among different manufacturers partly due to its low innate concentrations (low pg/mL).

Liquid chromatography mass spectrometry (LCMS) represents an attractive alternative to ELISA, however, accurate measurements in the desirable concentration range often require labor intensive sample preparation and ultra‐sensitive expensive LCMS technology–issues which can be bypassed by simultaneously analyzing bound and unbound (total) OT levels, which are typically in the ng/mL range. Braendtzag et al. (2016, Nature: Scientific reports) measured total OT after chemical reduction and alkylation (R/A) prior to on‐line C18 solid‐phase extraction (SPE) and nano LCMS. This specialized setup was simplified in our method by applying a weak cation‐exchange column (Strata‐X‐CW 33 μm; 30 mg/ 1 mL) Phenomenex) for simple off‐line SPE purification of the R/A product followed by LCMS (orbitrap model Q‐Exactive, Thermo Scientific) using a Kinetex pre and analytical C18 column (5 × 3.0 mm, 2.6 μm and 150 × 3.0 mm, 2.6 μm; Phenomenex) and electrospray ionization followed by targeted selected ion monitoring MS in positive mode. Validation of our method using pooled serum samples (premenop. women) resulted in an intra‐ and inter‐day CV of 2.6 – 3.2 % and 2.9 % (1.67 ng/mL), and 0.7 – 11.9 % and 8.3%, respectively, for this pool spiked with 1 ng/mL with an average accuracy of 104% (n=3). The LLOD (S/N=3) and LLOQ (S/N=10) using 200 μL serum was 0.01 ng/mL and 0.033 ng/mL, respectively. Total OT levels in serum from healthy non‐pregnant young females (n=3), one healthy male as well as human pooled commercial serum from both genders (n=2) or a pool of female samples (n=2), ranged from 0.23–8.4 ng/mL with a median level of 1.78 ng/mL.

Our method for total OT in serum represents an attractive alternative to traditional ELISAs and labor‐intensive LCMS techniques as it is accurate, precise, simple, and requires MS instrumentation typically found in an analytical chemistry laboratory. It requires only 200 μL serum and can be easily applied to investigate OT as a biomarker, treatment agent and target in psychological disease plus provides an inexpensive tool to investigate OT's connections to other functions.

Support or Funding Information

P30 CA71789
Dissecting the Dual Activity of Cellular Retinoic Acid Binding Protein 2 (CRABP2)
Authors: Vreeland Amanda , Driscoll Donna , Noy Noa
Published: November 2023

Cellular retinoic acid‐binding protein 2 (CRABP2) displays two distinct functions. The classical function of this protein is to directly deliver retinoic acid (RA) to retinoic acid receptors (RARs), thereby enhancing the transcriptional activation of RARs and upregulating expression of target genes, including some involved in suppression of tumor growth. We have recently elucidated a second function of CRABP2, which is exerted in the absence of RA. We found that apo‐CRABP2 cooperates with the RNA‐binding and stabilizing protein HuR to upregulate and stabilize mRNAs. CRABP2 directly interacts with HuR and enhances its affinity for target mRNAs, including some involved in regulating oncogenic properties. Notably, while apo‐CRABP2 is cytosolic and binds to HuR, the complex dissociates upon binding of RA. Holo‐CRABP2 then translocates to the nucleus where it delivers RA to RAR. CRABP2 then returns to the cytosol and re‐assocaites with HuR, showing that CRABP2 is able to perform both of its functions in parallel. The goal of this work is to determine the structural charcteristics of CRABP2 that mediate it's interaction with HuR.

The RA‐responsiveness of the interaction between CRABP2 and HuR suggests that the residues on CRABP2 that stabilize these interactions can “sense” ligand‐binding, i.e. that they undergo conformational changes upon ligation of CRABP2. Analysis of the reported three dimensional X‐ray crystal structures of holo‐ and apo‐CRABP2 revealed two surface residues, Arg60 and Lys107, whose side chains are placed at different conformations in the absence and presence of the ligand. We have found that Arg60 is necessary for the RA‐responsiveness of the CRABP2‐HuR complex and mutation of this residue prevents CRABP2 from cooperating with RAR. Additionally, we have found that Lys107 is necessary for CRABP2 to cooperate with HuR, but mutation of this residue does not interfere with the ability of CRABP2 to cooperate with RAR. As CRABP2 displays tumor suppressive activities, current work is examining which of CRABP2's functions is responsible for the tumor supressive activity of the protein.

Support or Funding Information

This work was supported by NIH grant R01‐CA166955. A.V. was partially supported by NIH grant R25‐CA148052.
Characterizing the transcriptional regulation of importin α1 expression in non‐small cell lung cancer
Authors: Yu Chia‐Jung , Cheng Ya‐Yun , Feng Hsiang‐Pu , Li Po‐Yu , Wang Chih‐Liang
Published: November 2023

KPNA2 (importin α1), an importin α family member is overexpressed and associated with cancer invasiveness in several types of human cancer. However, the upstream signaling as well as the transcription factor responsible for the regulation of KPNA2 expression remains unclear. Previously, we identified KPNA2 as a potential biomarker for non‐small cell lung cancer (NSCLC) and that mTOR signaling positively regulated Dp1/E2F1‐mediated transcription of KPNA2. We also found that KPNA2 might be involved in response to DNA damage stimulation, DNA metabolic processes, DNA repair and cell cycle. We herein show that interferon regulatory factor 1 (IRF1), a transcription factor with antitumor activity would suppress the expression of KPNA2 in NSCLC. The binding site of IRF1 in Kpna2 promoter was examined by chromatin immunoprecipitation assay. The ATM serine/threonine kinase inhibitor treatment dramatically induced the expression of IRF1, whereas significantly down‐regulated the expression of KPNA2 in NSCLC cells. Importantly, IRF1 knockdown restored the reducing level of KPNA2 in ATM inhibitor‐treated cells, supporting the suppressive role of IRF1 in regulation of KPNA2 expression. We also found that inhibiting either mTOR or ATM pathway significantly reduced the E2F1 expression but induced the IRF1 expression in vivo. Our result collectively show for the first time that IRF1 suppressed the KPNA2 expression and that two pathways, mTOR and ATM signaling might be involved in this IRF1‐mediated transcriptional regulation of KPNA2 expression in NSCLC.

Support or Funding Information

Ministry of Science and Technology, Taiwan, R.O.C. (105‐2320‐B‐182‐035‐MY3) and Chang Gung Medical Research Fund (BMRP894).
Characterization of a patient fibroblast cell line with Infantile Neuronal Ceroid Lipofuscinosis disorder
Authors: Balouch Bailey , Chu‐LaGraff Quynh
Published: November 2023

Infantile Neuronal Ceroid Lipofuscinosis (INCL) belongs to a family of pediatric neurodegenerative disorders collectively known as Batten Disease. INCL is caused by mutations in the palmitoyl protein thioesterase 1 (PPT1) gene, leading to retinal and CNS deterioration due to accumulation of autofluorescent storage materials in the lysosome. Although advances have been made in modeling the disease in various organisms, the elucidation of defined mechanistic pathways responsible for disease pathology and effective treatment remains a challenge. The goal of the present study is to characterize the cellular behavior of a human patient fibroblast cell line deficient in the PPT1 enzyme. PPT1 deficient cells were derived from a donor with Tyr247His and Met1Ile compound heterozygous mutations. The donor cells are characterized and compared to the two PPT1 expressing fibroblast cell lines, HFF and MRC‐5, using assays of cell morphology, cell viability, and autofluorescent accumulations. Future work aims to establish an Induced Pluripotent Stem Cell (iPSC) line using the Yamanaka technique, in order to continue studying the pathology of the disease. Specifically, neural progenitor cells deriving from these iPSC cells will be characterized and compared to wild type iPSCs to assess cell morphology, cell viability, autofluorescent accumulations, and neuronal function and pathology.

Support or Funding Information

Union College Student Research Grant
Modulation of Inflammasome Signaling by Advanced Glycation End Products (AGEs)
Authors: Son SeungHwan , Hwang Inhwa , Yu Je‐Wook
Published: November 2023

Deregulated immune response in obese condition leads to not only chronic inflammation but impaired protection against microbial infection such as influenza virus. The underlying mechanism by which obesity is related to the impaired host immune defense against pathogen invasion is poorly described. Here, we found that advanced glycation end products (AGEs), which are elevated in obese condition, specifically inhibit the activation of NLRP3 inflammasome in bone marrow‐derived macrophages (BMDMs). Pretreatment of AGEs attenuated diverse NLRP3 stimuli‐triggered caspase‐1 activation and interleukin‐1 beta (IL‐1β) secretion, but failed to affect AIM2‐ and NLRC4‐mediated inflammasome activation. AGEs also decreased ASC oligomerization and mitochondrial reactive oxygen species (ROS) production in response to NLRP3 inflammasome stimulator. However, treatment of receptor for AGEs (RAGE) antagonist FPS‐ZM1, did not alter the NLRP3 inflammasome‐suppressing effect of AGEs. Furthermore, AGEs abolished inflammasome activation triggered by poly I:C transfection or influenza virus infection. Taken together, our results suggest that AGEs potentially contributes to impaired innate immune response in obese condition by suppressing NLRP3 inflammasome signaling.

Support or Funding Information

This work was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean Government (2015M3A9B6073856)
Detection and Quantification of Probiotic Strains in Clinical Fecal Samples of Healthy Adults by Real‐time PCR
Authors: Nagulesapillai Varuni , Belvis Jocelyn , Tompkins Thomas , Girard Stephanie‐Anne
Published: November 2023

INTRODUCTION

A randomized, double‐blind, placebo‐controlled, crossover clinical study (NCT01879098) on healthy, middle‐aged adults investigated the effects of three probiotic strains, Bifidobacterium animalis subsp. lactis B94, Lactobacillus plantarum HA119, and Bacillus subtilis R0179 on bile acid metabolism, satiety, and inflammation. In order to substantiate positive outcomes, probiotic strains must persist during transit through the gastrointestinal tract of participants. The objective of this sub‐study was to detect and quantify HA119 and R0179 at the strain level in fecal samples using previously designed primers for real‐time PCR (qPCR).

METHODOLOGY

Participants of the clinical study were randomized to receive one capsule per day of a probiotic strain or placebo for six weeks. Following a four week washout period, participants were crossed to the other treatment (probiotic to placebo or placebo to probiotic). Fecal samples were collected at the baseline and final time points of each intervention for a total of four stool samples per participant. DNA isolated from fecal samples of participants in the L. plantarum HA119 (n = 136) and B. subtilis R0179 (n = 124) groups were analyzed by a SYBR‐green based qPCR method for HA119 and R0179, respectively. Strain‐specific primer targets included a gene encoding a phage protein for HA119, and a hypothetical protein for R0179. All fecal samples from participants in the B. lactis B94 group were not analyzed for B94 due to the lack of strain‐specific primers.

RESULTS

The HA119 strain was detected in 97% of the participants in the HA119 arm. The mean HA119 level in the feces from the final time point of probiotic intervention was 7.34 ± 0.86 log bacteria/g of feces. R0179 was detected in 97% of the participants in the R0179 group at mean 6.52 ± 0.38 log bacteria/g of feces in the final time point of probiotic intervention. The absence of the probiotic strains in the feces following the four week washout period suggests that the duration of the washout period was adequate between the intervention periods.

CONCLUSIONS

Overall, these results demonstrate the use of strain‐specific primers for the detection and quantification of probiotic strains HA119 and R0179 in complex fecal matrices containing numerous gut microbes. The ability to show persistence of specific probiotic strains in feces could help monitor participant compliance in clinical trials. This qPCR study also suggests that a four week washout period between intervention periods is sufficient for crossover clinical studies with probiotic intervention.

Support or Funding Information

Lallemand Health Solutions
Generation of Leigh Syndrome Cell Models
Authors: Alvarado Joshua
Published: November 2023

Leigh Syndrome (LS) is a neurological disease that stems from mutations in the subunits of mitochondrial respiratory chain subunits. These mutations decrease electron transport chain (ETC) efficiency, leading to decreased cellular ATP levels and increased reactive oxygen species (ROS) levels. There is currently no therapeutic approach to combat this degenerative disorder, and patients typically die by 8–10 years of age. There are also very few models with which to study this disease. We have generated LS cell models using both a chemical and genetic approach. Chemically, we inhibit ETC activity with low levels of rotenone, an ETC poison, in order to suppress ETC‐dependent ATP generation to levels seen in LS patients. Genetically, we use siRNA to knock down the ETC subunits that are mutationally inactivated in LS patients, also disrupting ETC function. Through these approaches, we have constructed LS cell models that can now be used to investigate potential therapeutics.

Support or Funding Information

CSUPERB New Investigative Grant
The optical spectroscopic properties of glycation induced changes in hemoglobins
Authors: Sriramoju Vidyasagar , Dudley Raymond , Secor Jeffrey A , Ashrafi Solyman , Alfano Robert R
Published: November 2023

The glycation induced changes in human hemoglobin is studied using absorption, fluorescence, Raman and time resolved fluorescence spectroscopic methods. The non‐enzymatic glycosylation of hemoglobin as a result of sustained elevated blood sugar levels changes the chemistry of its native chromophore and fluorophore moieties as a result it's tertiary structure gets altered significantly. These post translational molecular modifications would induce changes in optical properties like absorption, fluorescence emissions, fluorescence lifetimes and Raman finger prints. In this study the optical properties of normal hemoglobin are compared with glycated hemoglobin under non‐denaturing conditions in order to evaluate their potential as diagnostic and/or investigative biomarkers for chronic hyperglycemic conditions such as Diabetes mellitus Type 1 and 2.

Support or Funding Information

NxGen Partners, Ross Tower, 500 N. Akard Street, Suite 1820 Dallas TX 75201
Identification of Metabolites of Maresin 1 in Human Neutrophils
Authors: Gijón Miguel A , Almstrand Ann‐Charlotte , Johnson Chris A , Murphy Robert C , Zarini Simona
Published: November 2023

The resolution of inflammation, once considered to be rather passive, is now understood to be a complex, regulated process. One of the components in this process is the action of pro‐resolving lipid mediators, including lipoxins, resolvins, protectins and maresins. Maresin 1 (MaR1), derived from the oxidation of docosahexaenoic acid (22:6, n‐3), exhibits biological activities relevant to the resolution of inflammation, such as inhibiting accumulation of neutrophils and enhancing efferocytosis by macrophages.

Measurements of lipid mediators derived from arachidonic acid (20:4, n‐6) or docosahexaenoic acid have been quite successful in tissue exudates or cell culture‐based systems. However, efforts to measure these lipids in blood as a correlate to their production in vivo during an inflammatory episode have not been as straightforward and are somewhat controversial. In fact, much of our understanding of the role of prostaglandins and leukotrienes produced in vivo has come from measurement of urinary metabolites rather than plasma measurements.

This work is aimed at identifying metabolites of MaR1 and their accumulation in urine, which will then allow us to develop sensitive and specific mass spectrometry‐based assays for their quantitation. As a first approach, we have investigated the metabolism of MaR1 by human neutrophils, which are key cells recruited to inflammatory sites where MaR1 is known to be produced in the first place. Using a combination of liquid chromatography, gas chromatography, UV spectrometry and mass spectrometry, we have identified five novel metabolites of maresin 1, which are produced in a time‐ and dose‐dependent manner by human neutrophils We present here the chromatographic and spectral properties of these metabolites, as well as their proposed chemical structures. Four of these metabolites are oxidized forms of MaR1, and contain one or two hydroxyl groups. Preliminary evidence suggests that at least one of these metabolites may also be present in the urine of mice injected with MaR1.

These results open the possibility of investigating the role of MaR1 in experimental animal models and in human disease by analyzing its urinary metabolites. This will provide insight into the progression and possible treatment of several human diseases that include inflammation as a component in their progression.

Support or Funding Information

This work is supported in part by a grant from the Cayman Biomedical Research Institute.
Skin of the Catfish (Arius Bilineatus, Val.) Contains Lipid Compounds that Regulate NET Formation and NET‐Mediated Inflammation
Authors: Pace‐Asciak Cecil , Al‐Hassan Jassim M. , Afzal Mohammad , Paul Bincy , Oommen Sosamma , Khan Meraj , Liu Yuan Fang , Palaniyar Nades
Published: November 2023

Neutrophil Extracellular Traps (NETs) are networks of DNA fibers that are coated with cytotoxic components, released by neutrophils. NETs are beneficial for killing microbial pathogens, extracellularly, or by ensnaring toxins and cytokines. In different disease context, NETs can either be pro‐inflammatory or anti‐inflammatory. Therefore, identifying compounds that could regulate NET formation is important. Two major pathways of NETosis have been identified: NADPH oxidase‐dependent and calcium‐mediated NADPH oxidase‐independent NETosis. We have recently shown that NETosis is stimulated by a natural lipid eicosanoid, hepoxilin A3, which has pro‐inflammatory properties. Depending on the concentration, hepoxilin A3 induces either one or both types of NETosis. Preparations from the skin of the catfish contain lipids and proteins which have several important biological properties, such as anti‐thrombotic, anti‐inflammatory and wound healing activities. Our goal is to isolate and characterize products present in the lipid fractions of the preparations and study their biological activities. The lipids were separated from the proteins and fractionated by column chromatography. The neutral lipid fraction was separated into several sub‐fractions by thin layer chromatography (TLC). These sub‐fractions were tested in a battery of bioassays including the NETosis assays. We discovered that various isolated TLC fractions regulated NETosis, and importantly suppressed NETosis induced by the calcium ionophore A23187 to various degrees. This discovery suggests a novel mechanism for the anti‐inflammatory actions of the preparations.

Support or Funding Information

The study was Supported by: A grant from the Kuwait Foundation For Advancement of Sciences (KFAS 2013‐120701 A‐C), Kuwait University (KU SL03/14) and the Hospital for Sick Children and University of Toronto.
Effect of 8‐Hour Time Restricted Feeding on Body Weight in Obese Subjects
Authors: Gabel Kelsey , Hoddy Kristin K , Haggerty Nicole , Varady Krista A
Published: November 2023

Background

It has been speculated that time restricted feeding (TRF) could offer a non‐pharmacological intervention for weight loss in obese patients. No studies to date have examined the effect of TRF on body weight and coronary heart disease (CHD) risk factors in obese adults.

Objective

Accordingly, this study assessed the effects of 12 weeks of 8‐hour time restricted feeding (10am–6pm) on body weight and CHD risk factors.

Methods

In this pilot study, obese subjects (n = 9) were instructed to eat only between the hours of 10am–6pm daily for 12 weeks.

Results

Body weight decreased (P = 0.003) by −4.9% ± 1.9 (4.9 ± 1.9 kg) from baseline. Heart rate decreased (P = 0.037) by 4.7 ± 5.6 bpm from baseline. Fat mass decreased from baseline (P = 0.003) by −3.7% ± 3.1. Blood pressure, plasma lipids, and fat free mass remained unchanged.

Conclusion

These findings suggest that consuming food within an 8‐hour window can facilitate weight loss. The effect of TRF on CHD risk warrants further investigation.

Support or Funding Information

Campus Research Board Grant at University of Illinois at Chicago
Genetic and Epigenetic Signature Identifies Individuals with Elevated Response to Vitamin B12 Supplementation
Authors: Johnson Jessie Nicodemus , Brown Mark
Published: November 2023

Vitamin B12 is essential for many aspects of human health. Deficiencies are largely caused by inadequate intake or reduced absorption, and are associated with increased incidence of neurological, cognitive and cardiovascular decline. Using a previously published dataset of B12/ folic acid supplementation in elderly individuals (GSE74548), we investigated the effect of a variant in the methylenetetrahydrofolate reductase gene (MTHFR; rs1801133) on serum B12 and folate levels at baseline and after 2 years of supplementation. As well as, the association between baseline genome‐wide DNA methylation (Infinium 450K DNA methylation chip) in peripheral blood mononuclear cells (PBMCs) and supplementation induced changes in circulating B vitamin levels. After supplementation, we observed that those with the rs1801133 CC genotype had elevated circulating B12 but not folic acid levels, relative to the rs1801133 TT genotype (P = 0.0073 and P = 0.47, respectively). Moreover, individuals with the CC genotype clustered into two distinct B12 response groups (P = 2.8 × 10−6), termed high and low responders. Additionally, we observed an enrichment of low p‐values among baseline DNA methylation patterns and change in circulating B12 levels after supplementation in CC individuals only (empirical P < 1 × 10−5). These sites are not near genetic variants or genes known to influence vitamin B12 uptake. Overall, we present evidence that epigenetic signatures at baseline may predict an individual's response to B12 supplementation and may highlight novel pathways of regulation. Elucidation and further understanding of pathways associated with elevated response to B12 supplementation, may aid in the treatment of chronically deficient individuals.

Support or Funding Information

USANA Health Sciences
Maternal rumen‐protected methionine supplementation during late‐pregnancy affects calf development and growth during early postnatal life
Authors: Alharthi Abdulrahman Salem M , Batistel Fernanda , Parys Claudia , Helmbrecht Ariane , Loor Juan
Published: November 2023

Pregnancy and early life are critical periods of plasticity during which the fetus and neonate may be influenced by environmental factors such as nutrition. Evidence indicates that the maternal nutrition during pregnancy can affect offspring development. However, it is not clear if this is an effect acquired during pregnancy (e.g., greater maternal‐fetus nutrient transport) or a colostrum effect (e.g., high‐quality colostrum). Thus, the objective of this study was to investigate if increasing Met (a methyl donor) supply during late‐pregnancy affects developmental parameters at birth and subsequent growth and if it is an utero‐placenta or colostrum‐dependent response. The study included 39 female calves born to cows fed a basal control [CON; 1.47 Mcal/kg dry matter (DM) and 15.3% crude protein (CP)] diet with no added methionine or CON plus ethyl cellulose rumen‐protected methionine (MET; Mepron®, Evonik Industries AG, Germany). The MET was fed during the last 28 days of pregnancy at a rate of 0.09% of diet DM. At birth, calves were randomly allocated considering dam treatment and colostrum as follows: 1) calves from CON cows and colostrum from CON cows (n=9); 2) calves from CON cows and colostrum from MET cows (n=9); 3) calves from MET cows and colostrum from MET cows (n=11); and 4) calves from MET cows and colostrum from CON cows (n=10). Cows were milked within 4 hours and colostrum amount and quality were recorded. Calves were fed 3.8 L of colostrum. All calves were housed and managed in the same fashion during the first 9 weeks of life. Body weight, hip and wither height, hip width and body length were measure at birth and weekly through weaning (42 days of age) and until 9 weeks of age. Compared with CON, calves from dams fed MET had greater hip height (P = 0.02; 81.0 vs 78.5 cm) at birth. However, body weight at birth (P = 0.67; 41.7 vs 41.1 kg), hip width (P = 0.15; 15.8 vs 16.4 cm), wither height (P = 0.11; 77.3 vs 75.8 cm), body length (P = 0.51; 110 vs 108.9 cm) as well as colostrum quality (P = 0.95; 55.4 vs 52.4 IgG) and quantity (P = 0.80; 5.9 vs 5.7 kg) were not affected by maternal treatment. Over the first nine weeks of life, there was no colostrum effect for any of the growth variables measured. However, compared with CON, calves from dams fed MET had greater body weight (P = 0.03; 60.8 vs 57.3 kg), hip height (P = 0.02; 87.7 vs 85.7 cm), wither height (P = 0.05; 83.5 vs 81.9 cm), and average daily gain (P = 0.01; 0.69 vs 0.60 kg/day). Hip width (P = 0.73; 19.7 vs 19.6 cm) and body length (P = 0.24; 122.8 vs 124.8 cm) were not affected by maternal MET. Results indicate that neonatal heifer calf growth during the pre‐weaning and early post‐weaning period were enhanced by increasing MET supply of the cow during late‐gestation.
Cortical Bone Development is Lower in Ad36 Seropositive Versus Seronegative Adolescents: A 5‐Year Prospective Study
Authors: Belcher Staci L , Kindler Joseph M , Laing Emma M , Tripp Ralph A , Lewis Richard D
Published: November 2023

Adenovirus 36 (Ad36) is a common cold virus that infects between seven and 73% of children worldwide. Ad36 seropositivity is more prevalent in obese versus normal weight children and, in obese adolescents, is associated with compromised bone strength. Prospective data examining the relationship between Ad36 infection and secondary health outcomes, specifically relating to bone health, are lacking. The aim of this 5‐year prospective study was to examine the relationship between Ad36 seropositivity and musculoskeletal development in children during growth. Participants included black and white boys and girls, 9–13 years of age at the baseline time point. At baseline and follow‐up, fasting blood draws, dual‐energy X‐ray absorptiometry (total body), and peripheral quantitative computed tomography (tibia 66% site relative to the distal growth plate) were performed. Antibodies for Ad36 were detected from baseline fasting blood samples using an indirect ELISA immunoassay. To compare changes in body composition and bone outcomes between Ad36 seropositive (Ad36 (+)) and negative (Ad36(−)) participants, ANCOVA was performed controlling for baseline race, sex, and baseline sexual maturation rating (SMR) stage. Change in MCSA and change in height were also included as additional covariates. Thirty‐six (50.7%) children were Ad36(+) and 34 were Ad36(−). There were no significant baseline group differences in age, height, weight, SMR, sex, race, or any musculoskeletal endpoints. Overweight/obesity prevalence was 38.9% and 42.9% in the Ad36(+) and Ad36(−) groups, respectively (p=.734). Change in tibia MCSA (p=.016), periosteal circumference (p=.021), and total area (p=.035) were significantly lower in the Ad36(+) versus Ad36(−) children. After correcting for change in MCSA or change in height as a covariate, change in cortical bone endpoints did not differ between Ad36(−) and Ad36(+) groups. Skeletal muscle is a strong determinant of pediatric skeletal development. The more optimal cortical bone changes in the Ad36(−) versus Ad36(+) groups may have been attributed to corresponding differences in skeletal muscle accretion.
Effects of an Egg‐based, Carbohydrate‐restricted Diet on Body Composition, Fat Distribution, and Metabolic Health in Older Adults with Obesity: Preliminary results from a randomized controlled trial.
Authors: Goss Amy Miskimon , Gower Barbara A , Soleymani Taraneh , Stewart Mariah , Fontaine Kevin
Published: November 2023

Background

Age‐related loss of lean mass, accumulation of visceral adipose tissue (VAT), and insulin resistance place aging adults with obesity at particularly high risk of cardio‐metabolic disease. By reducing insulin demand, carbohydrate (CHO)‐restricted diet patterns that include foods such as whole eggs may promote loss of body fat, specifically from the visceral cavity, without compromising lean mass and improve metabolic health in aging adults with obesity.

Objective

The objective of this study was to determine if consumption of an egg‐based lower‐CHO/high‐fat diet (EBD) vs a standard CHO‐based/low‐fat diet (STD) results in loss of total and visceral fat, preservation of lean mass, and improvements insulin resistance and the lipid profile.

Design

In a randomized clinical trial, 26 men and women age 60–75yrs. with obesity (BMI 30–40kg/m2) consumed either an EBD (<25:25:>50% energy from CHO:protein:fat) or STD (55:25:20) for 8 weeks. Participants were not asked to restrict total energy intake. The EBD group was provided eggs throughout the study, whereas the STD group was provided breakfast bars to consume. Body composition by DXA, resting energy expenditure (REE) by indirect calorimetry, and insulin, glucose, HOMA‐IR and lipids by fasting blood draw were assessed at baseline and after the 8‐week intervention.

Results

Participants lost 11.0% and 2.3% total fat following the EBD and STD diet, resp. (p<0.01 for difference between diets). The EBD group experienced ~3‐fold greater loss in VAT compared to the STD group (−23.3% vs −7.1%, p<0.01 for difference between diets) and greater decrease in HOMA‐IR (p<0.01), whereas there were no significant between‐group differences in change of total lean mass or REE. Among those in the EBD group, there was a significant decrease in triglycerides and increase in HDL.

Conclusions

When compared to a standard high‐CHO, low fat diet, consumption of a diet lower in CHO supplemented with 3 whole eggs per day resulted in loss of fat mass, specifically from the visceral cavity while preserving lean mass and resting metabolic rate. This diet pattern also resulted in significant improvements in indices of metabolic health including insulin resistance, triglycerides and HDL cholesterol.

Support or Funding Information

Egg Nutrition Center