bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological effects of Lactobacillus johnsonii N6.2 in healthy adults: A double‐blind, randomized trial
Authors: Lorca Graciela L , Marcial Guillermo , Ford Amanda , Gezan Salvador , Perry Daniel , Haller Michael , Wasserfall Clive , Brusko Todd , Atkinson Mark , Gonzalez Claudio , Dahl Wendy
Published: November 2023

The intestinal environment may play a central role in the development of Type 1 Diabetes (T1D). However, the use of bacteria to alleviate the onset of this disease has been restricted to studies in rodent models. To date, the ability of a Lactobacillus probiotic to prevent T1D onset in humans has not been evaluated. L. johnsonii N6.2 mitigated the onset of T1D when administered to Biobreeding diabetes‐prone rats by altering the activity of intestinal indoleamine 2,3‐dioxygenase‐1 (IDO), correlating with a Th17 bias in the mesenteric lymph nodes of animals protected from T1D onset. Translating this work toward the prevention of T1D in humans required a pilot study in healthy individuals. We performed a human trial to evaluate the immunological response in healthy individuals (n=42; F=30, M=12; 23.2±5.5 y). The 13‐week study was divided into three phases: 1‐week pre‐baseline, 8‐week intervention (1 capsule/d of L. johnsonii N6.2 at 108 CFU or placebo), and 4‐week wash‐out. Blood samples were collected and analyzed throughout the study. Leukocyte subpopulations were assessed at baseline, treatment, and washout by immunophenotyping using flow cytometry and serum blood markers were quantified by ELISAs. Consumption of the L. johnsonii N6.2 did not affect the B cells, dendritic cells or regulatory T cells subsets. However, significant changes were observed in Monocytes, Natural Killer cells, T cells, T effector and follicular helper T cells in the L. johnsonii group. Specifically, the administration of L. johnsonii N6.2 increased the population of activated effector T cells, Th1 (CD45RO+, CD183+, CD196−) after 8 weeks of treatment, an effect that was maintained after the wash‐out period (12 weeks). Based on these results, the concentrations of IL2, IL6, TNFa, INFa, IFNg, sCD25 (IL2Ra), C reactive protein, insulin and IgA were quantified in blood samples. It was found that the concentrations of IgA increased significantly over the time in the L. johnsonii group while no changes were observed for the others. In this study, we identified several biomarkers as a result of L. johnsonii N6.2 consumption in healthy subjects. The results of this pilot study provide a solid foundation for an investigation into prevention of T1D onset by L. johnsonii N6.2 in an at‐risk human population.

Support or Funding Information

These studies were funded by grants from the JDRF (1‐INO‐2014‐176‐A‐V to GLL and a Career Development Award 2‐2012‐280 to TMB) and grants from the National Institutes of Health P01 AI42288 (to MAA and TMB.).
Administration of Fluoxetine or a Small Molecule TPH1 Inhibitor Affects Litter Size, Pup Mortality, and Milk Yield During Murine Pregnancy and Lactation
Authors: Weaver Samantha , Fricke Hannah , Hernandez Laura
Published: November 2023

Non‐neuronal serotonin regulates milk synthesis in an autocrine‐paracrine manner during lactation. Selective Serotonin Reuptake Inhibitors (SSRI), including Fluoxetine, are the most commonly prescribed antidepressants during pregnancy and lactation in the United States. SSRI increase local activity of serotonin in both neuronal and non‐neuronal tissues, including the mammary gland. The objective of this study was to examine the effects of SSRI administration during lactation on milk yield, pup weight, pup mortality, and litter size. As a negative control, we used a small‐molecule inhibitor of the rate‐limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 (Tph1). Beginning the day after pregnancy (d1 after cervical plug), C57Bl/6J dams were administered 20 mg/kg of either saline or fluoxetine daily via intraperitoneal injection through d21 of lactation. Additionally, on d1 of pregnancy, dams were either maintained on a control diet or loaded on a control diet fortified with a small‐molecule inhibitor of Tph1. There were three treatment groups: injected with saline and receiving control chow (CON; n=15), fluoxetine/control chow (FLX; n=11), saline/Tph1 inhibitor chow (TPH1; n=14). Litter size on d0 was recorded, as was pup mortality throughout lactation. Milk yield per pup was determined using the weigh‐suckle‐weigh method and pup weight gain was recorded. Of all dams enrolled in the study (19 CON, 34 FLX, 26 TPH1), a certain proportion were excluded because all pups were found dead. By treatment, 21.1% (4/19) of CON dams, 67.6% (23/34) of FLX dams, and 46.2% (12/26) of TPH1 dams were excluded due to a dead/killed litter. Of those dams that kept all pups, FLX dams had smaller litters (P=0.01; 6.5 ± 0.4 vs. 4.6 ± 0.5 vs. 6.4 ± 0.5 pups for CON, FLX, and TPH1) and tended to have more dead pups (P=0.06; 0.7 ± 0.2 vs. 0.9 ± 0.3 vs. 0.2 ± 0.1 dead pups for CON, FLX, and TPH1) than CON or TPH1 dams. FLX dams also produced less milk (P=0.03) than TPH1 or CON dams and subsequently had pups that gained less weight throughout lactation (P<0.0001; 4.9 ± 0.5 vs. 4.4 ± 0.5 vs. 4.7 ± 0.5 g/pup for CON, FLX, and TPH1). Collectively, this data suggests that FLX administration throughout pregnancy and lactation may have adverse effects on pup development both in utero and during lactation. While previous literature has established that SSRI are the safest antidepressants due to minimal side effects, our data suggests that FLX treatment affects the dam's gestational and lactational capacity and therefore pup growth during pregnancy and lactation.

Support or Funding Information

Karos Pharmaceuticals

National Science Foundation Graduate Research Fellowship Grant No. DGE‐1256259
Supplemental Zinc Decreases Fractional Zinc Absorption and Increases Lean Tissue Mass among Vietnamese Children with Tuberculosis
Authors: Hall Andrew G , Hoang Nga Thu , Hoang Van Thanh , Woodhouse Leslie , Tu Ngu , King Janet C , Vo Phuc Hanh
Published: November 2023

Childhood tuberculosis (TB) is a major health concern in Vietnam. Dietary Zn absorption is greater in children recovering from TB, possibly due to increased demand of Zn for immune function. Zn supplementation, therefore, may improve the effectiveness of TB treatment. Our objective was to determine changes in Zn homeostasis in response to Zn supplements among children admitted for treatment of TB at the National Lung Hospital in Hanoi, Vietnam.

After baseline, children matched by sex were randomized to 10 mg Zn/d (n=10) or placebo (n=11) for the first 2 mo of TB treatment. At baseline and after 2 mo, fractional Zn absorption (FZA) and exchangeable Zn pool size (EZP) were determined using Zn stable isotopes during an 8 d controlled‐diet metabolic study. After each of 3 meals on d 2, the child received 0.33 mg 67Zn orally and, 1 h following dinner, 0.1 mg 70Zn intravenously. FZA was determined from urinary Zn tracer‐tracee ratios (TTRs) in samples collected 5–7 d post isotope administration. EZP was estimated from the y‐intercept of the log‐transformed linear regression of urinary 70Zn TTRs. Height, weight, body mass index (BMI), mid‐upper arm circumference (MUAC), triceps skinfold thickness (TSK), and subscapular skinfold thickness (SSK) were determined. Mid‐upper arm muscle area (MUAM) and mid‐upper arm fat area (MUAF) were derived from MUAC and TSK.

21 children 9 to 15 y, 11 male and 10 female, enrolled. 14 had pulmonary TB. Height‐ and BMI‐for‐age z‐scores were −1.2 (−1.9, −0.6) and −1.7 (−2.4, −1.0), respectively. After 2 mo, controlling for baseline values, FZA was significantly lower among Zn‐supplemented children vs. placebo: 25.8% (21.5, 30.1%) vs. 32% (27.7, 36.3%), p < 0.05. EZP tended to be higher with Zn compared with placebo: 83.3 mg (69.2, 97.5 mg) vs. 70.1 mg (55.2, 85 mg), p = 0.19 (NS). No differences in weight, height, or BMI were observed. MUAC was higher among Zn supplemented, 21.1 cm (20.2, 22 cm), vs. placebo, 19.5 cm (18.7, 20.3 cm), p < 0.05. MUAM was also higher among Zn supplemented, 19.6 cm2 (17.3, 22.2 cm2) vs. 15.7 cm2 (13.9, 17.6 cm2) placebo, p < 0.05.

In sum, 10 mg Zn/d led to a homeostatic response and improved lean tissue gain in Vietnamese children with TB, and thus should further be evaluated as part of their treatment. Given the sensitivity of MUAC and MUAM, these indicators should be included for monitoring the TB recovery process in future studies.

Support or Funding Information

International Atomic Energy Agency, Contract 15859
The SELENOF Selenoprotein Localizes to the Outer Plasma Membrane in Human Prostatic Tissue and its Levels are Reduced in Prostate Cancer
Authors: Ekoue Dede , Ansong Emmanuel , Macias Virgilia , Deaton Ryan , Kajdacsy‐Balla Andre , Liu Li , Latcher Craig , Picklo Matthew , Nonn Larisa , Gann Peter , Freeman Vincent , Diamond Alan
Published: November 2023

SELENOF (also referred to as Sep15) is a selenium containing protein shown to reside in the endoplasmic reticulum (ER). Previous studies have linked polymorphisms in SELENOF to prostate cancer mortality. To investigate the role of SELENOF inprostate cancer, human prostatic tissue obtained from patients following prostatectomy was examined by immunohistochemistry. Unlike what we observe inhuman cancer‐derived cell lines, SELENOF partitioned predominantly to the outercell membrane in histologically benign glands distal to the tumor. Similarly, outer membrane staining was observed when primary human epithelial cells were examined by confocal microscopy. The pattern of expression in tumors was dramatically different than that observed in benign tissue with the levels of the protein being significantly lower and generally not in the outer membrane. AfricanAmerican men are at an increased risk of prostate cancer. A functional polymorphism in SELENOF expected to result in lower SELENOF levels is approximately 5‐fold more prevalent in African Americans. Therefore, the levels of SELENOF were compared between tumor tissue obtained from African Americans(n=33) and Caucasians (n=295). SELENOF levels were lower in tumor cells from African Americans when the signals were evaluated from the entire cell (P<0.02). These results indicate that SELENOF may have a non‐ER function in normal prostate epithelia and lower levels in tumor tissue may be contributing to prostate cancer risk. Tissue SELENOF levels are likely to be responsive to an individual's selenium status and SELENOF genotype. Selenium levels in plasma obtained from 129 men who had undergone a prostatectomy and participated in a Chicago‐based study were genotyped for SELENOF and plasma selenium levels were evaluated. The genetic data obtained confirmed the higher frequency of the allele likely to result in lower SELENOF levels in AfricanAmericans and this allele was associated with both higher levels of the Prostate Specific Antigen (P<0.05) and the Gleason Score, a measure of prostate cancer aggressiveness and prognosis. The selenium status of AfricanAmerican men (115.24 ±3.04 ng/mL) in this cohort was also significantly lower (P<0.01) than that determined for Caucasians (139.18 ±2.41 ng/mL). Collectively, the data obtained from these two approaches indicates that the SELENOF genotype and the selenium status of individuals may be contributing to the disparate incidence and outcome of prostate cancer experienced by African American men.

Support or Funding Information

This work was supported by grants from the National Institutes of Health [Grant # RO1CA127943, R21CA182103] and a Research Supplement to Promote Diversity in Health‐Related Research to AMD.
Serum 25‐Hydroxyvitamin D Concentration and Leukocyte Telomere Length in US Adults: A Nationwide Population‐Based Study
Authors: Bao Wei , Oleson Jacob J. , Snetselaar Linda G.
Published: November 2023

Background

Vitamin D status has been associated with a variety of health outcomes, beyond bone health. The role of vitamin D in aging and longevity remains controversial. We examined the association between serum 25‐hydroxyvitamin D (25[OH]D) concentration and telomere length, an indicator of cellular aging and biological longevity, in US adults.

Methods

We used data from a nationally representative subsample of 4215 adults in the 2001‐2002 National Health and Nutrition Examination Survey (NHANES). This was the only NHANES cycle when both serum 25(OH)D and leukocyte telomere length were measured. Serum 25(OH)D concentration was measured using a radioimmunoassay method. The leukocyte telomere length relative to standard reference DNA (T/S ratio) was measured in purified DNA samples using the quantitative polymerase chain reaction method. We used linear regression to estimate the relationship between serum 25(OH)D levels and telomere length. Both serum 25(OH)D levels and telomere length were log‐transformed prior to modeling to satisfy regression assumptions. The model was adjusted for covariates including age, gender, race/ethnicity, education, family income to poverty ratio, smoking status, alcohol intake, physical activity, total energy intake, and body mass index. Survey weights, strata, and primary sampling units were used in all analyses to account for the complex sampling design.

Results

After adjustment for covariates, although there was no main effect (β [SE] 0.017 [0.014], P=0.24), the relationship between serum 25(OH)D levels and leukocyte telomere length differed by age. Specifically, there was a significant association between serum 25(OH)D levels and leukocyte telomere length in adults of 45–64 years old (0.086 [0.022], P=0.001), but not in adults of 20–44 years old (−0.024 [0.021], P=0.26) or adults ≥ 65 years old (−0.004 [0.027], P=0.87). We observed no effect modification on the association by gender or race/ethnicity.

Conclusions

In this large nationwide population‐based study, we observed a significant association between serum 25(OH)D levels and leukocyte telomere length in middle‐aged adults, but not in younger or elder adults.
Desmoglein 2 Regulates Pancreatic Cancer Cell Migration through Modulation of cAMP Levels
Authors: Zeiler Julian , Sachs Lena , Spindler Volker
Published: November 2023

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor prone to early metastasis, but the mechanisms underlying the movement of pancreatic cancer cells are poorly understood. All epithelial tissues rely on desmosomes to acquire strong cell‐cell adhesion and to modulate cellular behavior. Here, we studied the role of the desmosomal cadherin‐type adhesion molecule Desmoglein 2 (Dsg2) for migration of pancreatic cancer cells. Silencing of dsg2 as well as CRISPR/Cas9‐mediated dsg2 knockout resulted in faster migration and a pronounced mesenchymal phenotype of PDAC cell lines in a MAPK‐dependent manner. In line with this observation, RNA‐Seq‐based transcriptome analysis revealed upregulation of mesenchymal markers in shdsg2 AsPC‐1 cells compared to non‐target controls. Increased migration as well as the enhanced mesenchymal phenotype were abolished by pharmacologic elevation of cAMP levels using the adenylyl cyclase activator forskolin and the phosphodiesterase IV inhibitor rolipram (F/R). F/R also reduced the activation of ERK in shdsg2 cells, providing a mechanism how cAMP elevation reduces AsPC‐1 cell migration. Surprisingly, shdsg2 cells displayed increased cAMP levels compared to non‐target controls. This indicates that cAMP elevation in response to loss of Dsg2 represents a salvage pathway in an attempt to reduce cell migration and to keep cells in an epithelial and adhesive state. Elevation of cAMP levels thus represents a promising approach to reduce the invasive behavior of pancreatic cancer cells.
Adducin regulates migration and invasion of normal lung epithelial cells and lung cancer cells
Authors: Lechuga Susana , Amin Parth H , Ivanov Andrei I
Published: November 2023

Epithelial cell migration represents a critical mechanism underlying normal tissue morphogenesis and disease‐related processes such as epithelial restitution and tumor metastasis. In the lungs, epithelial cell migration is essential to repair the airways damaged by environmental and inflammatory insults. Furthermore, this process is critical for metastasis of non‐small cell lung cancer (NSCLC) cells. Migration of epithelial cells depends on orchestrated dynamics of the plasma membrane and the underlying actin cytoskeleton, which implicates the membrane/cytoskeleton interface in cell motility. The plasma membrane is linked to the actin cytoskeleton by a spectrin‐adducin polymeric lattice. The aim of this study was to investigate the roles of adducin in migration and invasion of normal lung epithelial cells and NSCLC cells. We used 16HBE14o immortalized lung epithelial cells and two NSCLC cell lines, H1573 and H1299, which express two different isoforms of adducin, ADD1 and ADD3. CRISPR/Cas9 mediated knockout of either ADD1, or ADD3 significantly increased Boyden chamber migration of individual16HBE14o and NCI‐H1573 cells without affecting their collective motility in a wound closure assay. Overexpression of ADD1 in H1299 cells significantly decreased their wound healing, Boyden chamber migration, and Matrigel invasion. The described effects of adducin knockout and overexpression on epithelial cell migration were not due to changes in cell proliferation. ADD1‐overexpressing H1299 cells demonstrated markedly accelerated cell adhesion and spreading on the collagen matrix. These effects were accompanied by the increased size of focal adhesions at the spreading cell edges, higher Src phosphorylation, and the assembly of long filopodia‐like protrusions enriched in F‐actin and non‐muscle myosin IIB. The protrusions had a slow rate of contraction and detachment from the matrix. Our findings reveal a novel function of adducin as a negative regulator of migration and invasion of normal lung epithelial cells and NSCLC cells by mechanisms, involving remodeling of the actin cytoskeleton, and modulation of cell adhesion to the extracellular matrix.

Support or Funding Information

Suported by NIH R01 grant DK108278 to AII.
Tryptophan Metabolite Activation of the Aryl Hydrocarbon Receptor in Intestinal Epithelia Promotes Mucosal Healing
Authors: Lanis Jordi M , Alexeev Erica , Colgan Sean
Published: November 2023

Inflammatory bowel disease (IBD) is a chronic inflammation of the intestine affecting more 1 million people in the United States. The pathogenesis of IBD is not completely understood, but complex risk factors such as genetics, environmental exposures, diet, and changes to the microbiota all play a role in the establishment of this disease. Regardless of the triggering factors, it is clear that the response of intestinal epithelia to the various inflammatory signals is crucial to restore homeostasis. Significant shifts in tissue metabolism are detected during inflammation and include a notable increase in tryptophan (Trp) metabolites. In vivo work has demonstrated that inflammation stimulates the induction of epithelial indoleamine 2,3‐dioxygenase (IDO1), the enzyme that catalyzes the conversion of Trp to kynurenine (Kyn). Consequently, elevated levels of intracellular kynurenine trigger activation of the Aryl Hydrocarbon Receptor (AHR). Numerous pathways are mediated by the transcription factor AHR, but preliminary data suggests that AHR activation during colonic inflammation is important for IL10 receptor (IL10R1) expression and wound healing. Based on these findings, we hypothesized that epithelial AHR signaling is protective in colitis. To that end, in vivo studies have revealed that Kyn mediates protective actions via the AHR to ameliorate colitis in an IL10R1 dependent manner. In contrast, colitis in intestinal epithelia conditional AHR knockout mice is not improved with exogenous Kyn, and the isolated colonic epithelial cells are deficient in IL10R1. Together, these results provide evidence on the importance of AHR signaling in intestinal epithelia and implicate AHR in the regulation of IL10R1 expression in the colon.

Support or Funding Information

This work was supported by NIH grants DK099452, DK50189, DK095491, VA Merit BX002182 and by the Crohn's and Colitis Foundation of America.
Neutrophils Induce Pro‐angiogenic T cells with a Regulatory Phenotype in Pregnancy
Authors: Nadkarni Suchita , Smith Joanne , Sferruzzi‐Perri Amanda , Kishore Madhav , Mauro Claudio , Williams David , Marelli‐Berg Federica , Perretti Mauro
Published: November 2023

Whilst fundamental in controlling innate immune responses, the role of neutrophils in regulating adaptive immunity is just being appreciated. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4+ T‐cells displaying a GARP+CD127loFOXP3+ phenotype following antigen activation. Neutrophil‐induced (ni)T‐cells produce IL‐10, IL‐17 and VEGF, and promote vessel growth in vitro. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal‐maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT‐cell numbers in draining lymph nodes. Using CD45 congenic cells, we show that induction of niT‐cells and their regulatory function occurs via transfer of apoptotic neutrophil‐derived proteins including FOXO1 to T‐cells. Unlike in healthy pregnancies, neutrophils from blood and placental samples of pre‐eclamptic women fail to induce niT‐cells as a direct consequence of their inability to transfer FOXO1 to T‐cells. Finally, neutrophil‐selective FOXO1 knockdown leads to defective placentation and compromised embryo development similar to that resulting from neutrophil depletion. These data define a novel, non‐redundant function of neutrophil‐T‐cell interactions in the regulation of vascularisation at the maternal‐fetal interface.

Support or Funding Information

SN and MP were supported by the Wellcome Trust (Programme 086867/Z/08/Z). FMB is supported by the British Heart Foundation (CH/15/2/32064).
Proteostasis function and disfunction: the delicate art of maintaining a healthy proteome
Authors: Frydman Judith
Published: November 2023

Correct protein folding and quality control are essential for normal cellular function. The accumulation of misfolded proteins is emerging as central to a wide range of disease states, including many neurodegenerative disorders such as Huntington's and Prion Disease. A complex network of molecular chaperones facilitate protein folding and assembly and monitor all aspects of protein homeostasis. Chaperones assist the folding of newly translated and stress‐denatured proteins, as well as affects protein quality control.

Our research investigates the mechanisms and pathways by which chaperones carry out these diverse functions. Systems approaches identified a chaperone network linked to the protein synthesis apparatus assists protein biogenesis. The emergence of this translation‐linked chaperone network likely underlies the elaborate co‐translational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells. A stress‐inducible chaperone network protects cells from environmental stress and assists quality control. These chaperones also communicate with the ubiquitin‐proteasome pathway to clear misfolded proteins from the cell. Protein quality control in the eukaryotic cytosol relies on the sequestration of misfolded cytosolic proteins in specific quality control compartments. Our studies of chaperone function provide a framework to understand the link between protein misfolding and human disease.
Structure‐based discovery of new chemotypes conferring new biology
Authors: Shoichet Brian
Published: November 2023

Receptor binding sites are evolved to recognize a few endogenous ligands, but the degeneracy of molecular recognition ensures that multiple other ligands and chemotypes can bind to the same site. Sometimes these non‐biological molecules will confer activities that the native ligands do not.

Structure‐based docking is a fruitful way to find such novel ligands, by screening large libraries of compounds for new chemotypes that physically complement a binding site, but do not resemble the endogenous ligands. If these searches are coupled to the right biological assays, they can find new molecules conferring unprecedented biology. This will be illustrated with a screen for novel agonists of the m‐opioid receptor. New opportunities arising from a great increase in the number of available, interesting molecules, now well‐over 108 of which are readily available and dockable, will be considered.

Support or Funding Information

Supported by GM71896 and GM106990
Organelles without membranes: intrinsically‐disordered proteins bring order to the cytoplasm
Authors: Seydoux Geraldine , Smith Jarrett , Calidas Deepika , Schmidt Helen , Rasoloson Dominique
Published: November 2023

RNA granules are non‐membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We have identified two intrinsically disordered proteins MEG‐3 and MEG‐4 that function redundantly to scaffold the assembly of P granules in the posterior cytoplasm. We will describe how MEG‐3 and MEG‐4's ability to phase separate with RNA, bind to other RNA‐binding proteins, and be phosphorylated by DYRK MBK‐2 contribute to localized P granule assembly.

Support or Funding Information

We gratefully acknowledge support from JHMI, HHMI and NIH
The role of intracellular lipolysis in thermogenesis and metabolic disease
Authors: Zechner Rudolf , Schweiger Martina , Breinbauer Rolf , Zimmermann Robert
Published: November 2023

During times of food deprivation, lipolysis of triglyceride (TG) stores in white adipose tissue provides unesterified fatty acids (FAs) for the rest of the body. The majority of lipolytic, circulating FAs is absorbed by the liver, reesterified to TG, and deposited as cytoplasmic lipid droplets or re‐secrated as TG‐rich VLDL. We tested how inhibition of the primary TG‐hydrolase adipose triglyceride lipase (ATGL) by a specific, small molecule inhibitor (Atglistatin) affects parameters of metabolic disease in high fat diet (HFD)‐treated mice. Results show that a partial inhibition of lipolysis has preventive and therapeutic effects on HFD‐induced obesity, insulin resistance and fatty liver disease. Potential negative effects on cardiac function or thermogenesis were not observed. Thus, inhibition of lipolysis may represent a potential target to treat metabolic disease.
Novel Mechanisms of Regulation of Bioactive Sphingolipids in Cancer Biology
Authors: Obeid Lina M. , Senkal Can , Pulkoski‐Gross Michael
Published: November 2023

Our laboratory focuses on studying the role and regulation of bioactive sphingolipids in cancer. Specifically, we are interested in metabolic pathways of ceramide ‐‐ the growth suppressor, proapoptotic, prosenescense lipid, and sphingosine‐1‐phosphate (S1P) ‐‐ the antiapoptotic, proangiogenic, promitotic lipid. Our recent studies have led us to uncover a novel metabolic pathway involving de novo ceramide acylation to lead to acylceramide generation by a protein complex of ceramide synthase (CerS), acyl‐CoA synthetase long‐chain family member 5 (ACSL5), and diglyceride acyltransferase 2 (DGAT2). This metabolic pathway leads to sequestration of acylceramide in lipid droplets thus making it unavailable for ceramide action in cell death pathways. The implications of this pathway to fatty liver and cancer will be discussed.

In other recent studies, we have also uncovered a novel mechanism of regulation for the enzyme sphingosine kinase 1 by phosphatidic acid (PA) acid binding, activation and translocation to the cell membrane leading to several biologic consequences including ezrin phosphorylation and cell invasion. Moreover, this regulation of sphingosine kinase 1 appears to have implications to its role in endocytosis and other membrane structural changes.

Taken all together these studies underscore the important role of bioactive sphingolipids and their metabolizing enzymes in cell regulation and cancer biology.

Support or Funding Information

5I01BX000156‐0 (Obeid ‐ PI) 1.2 cal. mos. 10/1/2010 – 9/30/2017

Veteran's Administration Merit Award $258,300 “Bioactive Sphingolipid enzymes as targets in inflammation”

The long‐term goal of this project is to define the role of ceramidases and sphingosine kinase in inflammation and to target these enzymes for novel anti‐inflammatory therapy.

2P01CA097132‐11A1 (Hannun – PI, Obeid ‐ Project 3 Leader) 7/01/2002‐8/31/2019

Agency: NIH/NCI 1.2 cal. mos. $133,257

Sphingolipids in Cancer Biology and Therapy

The aims of this proposal are to study the role of sphingosine kinase in p53 null and mutant cancer development.

9R01GM097741‐13A1(Obeid ‐ PI) 1.2 cal. mos. 8/1/2015‐6/31/2019

Agency: NIGMS $250,000

A novel ceramide metabolic pathway in cell regulation

The aim of this proposal is to study our newly discovered pathway of ceramide metabolism involving o‐acyl ceramide generation and its association with lipid droplets and regulation of cell death pathways.
Signaling through hydrogen sulfide
Authors: Banerjee Ruma
Published: November 2023

Despite the excitement about the varied physiological effects mediated by H2S and the consequent profusion of literature on H2S biology, there are large gaps in our understanding of how cells maintain very low steady‐state levels of H2S and amplify the signal as needed (1). Three enzymes in the sulfur network are important for its biogenesis. Two catalyze well‐described non‐H2S producing reactions in the transsulfuration pathway and also synthesize cysteine persulfide from cystine (2), raising questions about how the decision between these competing reactions is made in the cell. The pathway for H2S oxidation resides in the mitochondrion where the enzymes successively oxidize sulfide to sulfate. While sulfate is innocuous, a number of the intermediates in the sulfide oxidation pathway are reactive and their role in sulfide‐based signaling remains to be assessed (3). We have recently discovered a noncanonical sulfide oxidation pathway (4, 5) and the challenging heme‐dependent oxidation chemistry will be discussed. We will also discuss how H2S signals via protein persulfidation in the context of cancer.

Support or Funding Information

This work was supported in part by the NIH (HL58984 and GM112455
Proliferation of Transplanted Hepatocytes Drives Efficient Repopulation in Juvenile Host Rat Livers
Authors: Stock Peggy , Hempel Madlen , Hsu Mei‐Ju , Christ Bruno
Published: November 2023

To improve the engraftment of donor hepatocytes is a central goal to enhance the therapeutic efficacy of hepatocyte transplantation as an alternative to whole liver transplantation. We reported previously that efficient liver repopulation by transplanted hepatocytes was hampered in livers of old as compared to juvenile animals 6 weeks after transplantation. To investigate, whether this was due to a better initial entry into the host parenchyma of juvenile as compared to senescent donor cells, or to differences in proliferation, we determined repopulation rates in juvenile and senescent host livers 1 week after transplantation of juvenile and senescent hepatocytes by flow cytometry and quantification of DPPIV enzyme activity in liver sections. Proliferation was assessed in the liver parenchyma by the expression of Ki67 six weeks after cell transplantation.

1 week after hepatocyte transplantation comparable numbers of scattered cell clusters appeared both in juvenile and senescent host livers, irrespective of the age of the donor hepatocytes. However, quantification by flow cytometry of isolated primary hepatocytes from these livers revealed significantly higher numbers of juvenile as compared to senescent transplanted hepatocytes in juvenile hosts and as compared to either cell type in senescent host livers. This indicates that no differences in the parenchymal entry were obvious but rather higher proliferation of juvenile hepatocytes for higher numbers of transplanted cells in the juvenile host livers. Indeed, 6 weeks after hepatocyte transplantation significantly higher proliferation rates of transplanted hepatocytes were observed in juvenile host livers (4.2%) as compared to senescent hosts (zero), respectively.

Hence, irrespective of the age of donor hepatocytes the parenchymal entry is similar but the efficient repopulation by juvenile hepatocytes seemed to rely on higher proliferation rates during liver regeneration.
Rat Liver Repopulation by Transplanted Late Gestation Fetal Hepatocytes
Authors: Sanders Jennifer , Boylan Joan , Francois‐Vaughan Heather , Thomas Paul M , Sikora Jacek , Abshiru Nebiyu , Gruppuso Philip , Kelleher Neil
Published: November 2023

Hepatocyte transplantation has the potential to be an effective therapy for liver failure. However, the application of this therapy has been hampered by identification of an appropriate cell population and limited understanding of the factors accounting for the ability of cells to repopulate injured liver. Late gestation (E19) fetal hepatocyte proliferation in the rat is mitogen‐independent in vivo and in vitro. Fetal hepatocytes also differ from adult hepatocytes with regard to ribosome biogenesis, translation control, gene expression and cell cycle regulation. Our current studies are based on the hypothesis that histone posttranslational modifications (PTMs) acting through effects on chromatin structure account for the fetal hepatocyte signaling phenotype and that this phenotype will result in a selective growth advantage for fetal cells in the injured adult liver microenvironment. Late gestation fetal hepatocytes were isolated using antibodies against hepatic and ductal cell surface markers. The engraftment and repopulating ability of these cells was assessed using the mitomycin C dipeptidyl peptidase IV (DPPIV) transplant model. Liver harvested at 4 and 42 weeks posttransplantation contained well defined donor hepatocyte and endothelial colonies. Morphologic and phenotypic analysis using antibodies against well characterized markers revealed the formation of ductal colonies by 10 months. The average size of the donor colonies increased so that by 10 months up to 30% of the liver was repopulated by donor‐derived cells. Characterization of the donor cells pretransplant revealed that a large subpopulation of the hepatocytes also expressed ductal markers. Transcriptomic analysis revealed that these cells represent a distinct subpopulation of less well differentiated hepatocytes. Mass spectrometry‐based profiling of 77 modifications across 26 lysines on Histones H1, H2A, H3 & H4 revealed 23 significant changes including differential methylation of H3K27, H3K36 and H4K20 as well as acetylation of H3K4 comparing fetal to adult hepatocytes. In contrast, only 5 sites were significant comparing the two subpopulations of fetal hepatocytes. In conclusion, late gestation fetal hepatocytes are capable of extensive long‐term liver repopulation. These cells possess a histone PTM profile that is distinct from that of adult hepatocytes and may contribute to the maintenance of a fetal hepatocyte phenotype upon transplantation.

Support or Funding Information

The studies were supported by NIH grants R01DK100301 and P41GM108569. Support was also provided by the Rhode Island Hospital Department of Pediatrics.
Cell‐specific Wnts regulate liver regeneration after partial hepatectomy
Authors: Preziosi Morgan , Yang Jing of , Okabe Hirohisa , Diegel Cassandra , Williams Bart , Monga Satdarshan
Published: November 2023

Our lab has previously shown that β‐catenin regulates liver regeneration in a partial hepatectomy (Phx) model, under the control of Wnt and the canonical signaling pathway. This is true at early timepoints up to 40–72 hours. Using cell‐specific Wntless (Wls) knockout mouse models, which lack Wls and prevent Wnt secretion, we have previously determined epithelial cells are not the source of these Wnts. However, macrophage‐specific Wls knockouts (MP‐KO) have a regeneration deficit at 40 hours that is not seen earlier, suggesting they are a contributing source of Wnts in a temporal manner. Considering removing Wls from all endothelial cells was embryonic lethal, we sought to elucidate contributions of Wnts using hepatic endothelial cell‐specific Wls knockouts, EC‐KO1 affecting venous endothelial cells, and EC‐KO2 affecting sinusoidal endothelial cells. Initially, EC‐KO1 showed minimal deficits in β‐catenin target genes and zonation markers GS, Cyp2e1, and Cyp1a2 by protein level. EC‐KO1 show reduction in Cyclin D1 24 hours after PHx which rebounded by 48 hours. Intriguingly, EC‐KO2 demonstrate a marked reduction in zonation markers at baseline, which has not yet been reported using protein levels. After PHx, EC‐KO2 display less proliferation than littermate controls, overall emphasizing the role of endothelial cell‐specific Wnts in liver regeneration. In vivo, MP and EC demonstrate high levels of Wnt2 and 9b at baseline, which significantly increase after Phx. There is an increase in portal pressure and shear stress after Phx, so we tested whether shear stress was sufficient to increase Wnt mRNA in vitro, and saw an increase in mRNA in three separate endothelial cell types including primary liver endothelial cells. Taken together, we hypothesize Wnt2 and Wnt9b secretion is initiated early after Phx in part via shear stress, to temporally regulate β‐catenin's contribution to liver regeneration.
Serotonin Receptor Regulation of Eosinophil Transendothelial Migration
Authors: Walker Matthew , Green Jeremy , Ferrie Ryan , Cook‐Mills Joan
Published: November 2023

The tryptophan amino acid metabolite, 5‐hydroxytryptophan (5HTP), is a metabolic precursor of serotonin. 5HTP reduces allergic inflammation in the lungs of mice and blocks eosinophil migration across endothelial monolayers in vitro. Blocking the metabolic conversion of 5HTP to serotonin decreases inhibition of eosinophil transendothelial migration, indicating that its metabolism is required for inhibition. Currently it is not known if serotonin receptors are involved in mediating this regulation by 5HTP. We determined which serotonin receptors are expressed by endothelial cells and eosinophils by western blot and qPCR. There was expression of the serotonin receptors 1A, 1B, 3A, and 6. The function of the receptors in eosinophil transendothelial migration was analyzed in vitro using serotonin receptor inhibitors. Selective receptor inhibitors for serotonin receptors 1A and 1B blocked 5HTP inhibition of migration. This suggests that these serotonin receptors are involved in 5HTP inhibition of transendothelial migration and thus may serve as a target for intervention during allergic inflammation. These results will help design clinical studies addressing whether 5HTP supplementation reduces allergic asthmatic responses.

Support or Funding Information

NIH R01HL111624
Force‐Induced α4 Integrin‐Dependent Monocyte Adhesion Strengthening and F‐Actin Remodeling Requires Actomyosin Contractility and Talin‐1
Authors: Ibrahim Hisham M , Hyduk Sharon J , Cybulsky Myron I
Published: November 2023

During inflammation, monocytes roll and arrest on inflamed endothelium, at which point they are subjected to increased hydrodynamic forces imparted by the flowing blood. We showed previously that stabilization of α4 integrin‐mediated monocyte adhesion and resistance to detachment involves rapid actin polymerization and accumulation of filamentous actin (F‐actin) at upstream adhesion sites and in the adjacent cortical actin cytoskeleton (J Cell Biol 2012; 197:115–129). Using parallel plate flow chamber assays with a VCAM‐1‐coated adhesion surface, U937 monocyte‐like cell line and confocal imaging we further investigated mechanisms of adhesion strengthening and actin cytoskeleton dynamics in cells undergoing α4 integrin‐mediated adhesion. Myosin ATPase, calcium/calmodulin, myosin light chain kinase and Rho kinase signaling were required for α4 integrin‐mediated adhesion strengthening in the presence of hydrodynamic force. Upon introduction of fluid flow (2 dynes/cm2), adherent U937 cells flattened and the cell contact area with the adhesion surface increased both parallel and perpendicular to the direction of fluid flow. In addition, F‐actin at the adhesion surface redistributed towards the periphery of the adhesion surface contact area. Increased cell adhesion surface contact area and F‐actin redistribution were highly reproducible responses observed in >90% of adherent cells. Unlike F‐actin, α4 integrins remained localized throughout the adhesion contact area, and formed clusters in response to flow. Inhibition of myosin ATPase prevented force‐induced lateral spreading of U937 cells and F‐actin redistribution at the adhesion surface. siRNA knockdown of talin‐1, an adaptor molecule that binds to the integrin β‐chain cytoplasmic tail and mediates outside‐in‐signaling as well as interaction with the actin cytoskeleton, also inhibited α4 integrin‐dependent adhesion stabilization, lateral spreading and redistribution of F‐actin. These data suggest that integrin‐dependent adhesion stabilization and cytoskeletal remodeling induced by hydrodynamic force require both actomyosin contraction and talin‐1. When U937 cells were adhered to poly‐L‐lysine, we observed that hydrodynamic force induced a similar increase in the cell adhesion surface contact area and F‐actin redistribution; however, in this setting neither myosin ATPase nor talin‐1 were required. This suggests that adherent cells can respond to hydrodynamic force in an integrin‐independent manner, but adhesion mediated by α4 integrins inhibits the force‐induced, integrin‐independent response.

Support or Funding Information

Funded by CIHR
Student‐Produced Videos Enhance Learning in a Freshman Cellular and Molecular Biology Course
Authors: Chen He Tian , Ellenbogen Yosef , Ng Maxwell , Brar Karanbir , Lee Jinny , Lee Catherine , Lee Brian , Nastos Stash
Published: November 2023

Increasing student engagement in the learning process is a good way to enhance the educational experience, but the mechanics of doing so are difficult to define. Putting students in the role of teachers can help to introduce a student‐centered perspective on learning, provided this is done in a way that is appropriate to their strengths. Undergraduate students who had completed a freshman course in cellular and molecular biology were encouraged to develop multimedia resources (particularly videos) to supplement the lectures given in this class. The purpose of the videos was to provide the students with an alternative way of looking at the course content, from a student's point of view. Attention was focused on one specific component of the course — an assessment tool designed to test a students' abilities to solve problems based on course content. These assessments, Rapid Problem‐Solving Exercises (RAPSEs) were given to students in two variants. In the hypotheses generation RAPSEs, students were provided limited information (usually experimental data) and asked to frame possible solutions within a limited time period (20 minutes). In the other variant, students were given limited data but provided with a specific hypothesis for which they were required to design suitable experiments. These RAPSEs are challenging evaluations but provide a meaningful learning experience, and we wanted to see if we could aid in the learning process through educational tutorial videos. Specifically, we sought to develop videos tailored to student needs that consisted of students working through sample RAPSEs to highlight the thought process required to successfully navigate the assessment. Additionally, we produced animations of cellular mechanisms to help students visualize our thought processes. These videos went through several iterations with instructor feedback. These videos were shown to the class and were also available for individual student study. A voluntary questionnaire was sent out to the students following the RAPSE assessment. Preliminary responses to the questionnaire (n=109) revealed that 95% responded with ‘agree’ or ‘strongly agree’ when asked if the videos provided a valuable learning resource. The implementation of multimedia in problem‐solving through student‐centered learning is an effective manner of filling gaps endemic in standard didactic teaching. Student‐generated multimedia resources provide a valuable opportunity, not only to enhance student learning, but also foster faculty‐student collaboration in the learning experience.
Redesigning a Comprehensive Integrated Pharmacology Course for Active Learning in a Competency‐Based Curriculum
Authors: Marsh Susan A. , Paine Mary F. , Gibson K. Michael
Published: November 2023

The Washington State University (WSU) Doctor of Pharmacy (PharmD) program has undergone extensive changes in recent years, including consolidating the program to the health sciences campus in Spokane, expanding class sizes, extending the program to a satellite campus and moving to a competency‐based curriculum. These changes necessitated that all didactic courses be transformed to an evidence‐based active learning delivery format. A four‐course integrated pharmacology series, a core requirement of our PharmD program, mandated complete redesign to accommodate not only the program's many changes, but also to account for curricular drift. After consulting with both students and other instructors, several challenges became apparent during the redesign process, including a demanding student schedule that was not conducive to copious pre‐class work, student resistance to move from a traditional lecture‐based delivery to an active learning approach and the need to reduce the cost of course materials. All four semester‐ long courses in the series integrate the following critical areas: pathophysiology, pharmacology, toxicology, therapeutics and medicinal chemistry. The courses differ based on organ systems covered. For example, the third course covers cardiovascular, pulmonary and renal systems. We recently completed the second semester of this redesigned course, thus serving as an example of our efforts to completely redesign an integrated pharmacology course for active learning in a competency‐based curriculum. The course was divided into 24 self‐contained modules such that each module replaced what had historically been delivered as a two‐hour live lecture. Within each module, students were required to complete one hour of pre‐class work that typically consisted of viewing several instructor‐prepared videos lasting at most 30 minutes in total length per module, briefly reviewing the module's learning objectives and study guide, and completing five open‐book multiple choice questions. In‐class sessions began with a short informal question and answer period to clarify concepts or problems from the pre‐class work, with the remainder of the first hour working through guided case studies, first in small groups and then as a class. The second hour was student‐driven and focused on instructor‐prepared review questions that specifically addressed each of the learning objectives; this optional hour greatly reduced the need for review sessions and office hours. Student and instructor feedback and participation in teaching and learning workshops will continue to be used as means to refine this active learning strategy to improve student engagement, retention of fundamental knowledge, and critical thinking. Future plans include analyzing the effectiveness of this strategy by comparing markers of academic success and retention to historical student cohorts in the pharmacology series.