bluuebunny/crossref_metadata_2025 huggingface dataset:
LONG NON‐CODING RNA HOTAIR ACTIVATION BY TGF‐BETA1 INDUCES EPITHELIAL MESENCHYMAL TRANSITION (EMT) IN T24 BLADDER CARCINOMA CELLS
Authors: de Oliveira Brito Rodrigo Barbosa , Malta Camila Soares , Matos Yves Silva Teles , Souza Diego Mota , Matheus Luiz Henrique Gomes , Dalboni Maria Aparecida , Dellê Humberto
Published: November 2023

HOTAIR is a long non‐coding RNA (lncRNA) which is implicated in cell programming for initiation of epithelial mesenchymal transition (EMT) in several types of cancer. In bladder carcinoma T24 cells, the TGF‐beta1 promotes invasion and metastasis through induction of EMT, however, there are evidences to believe that HOTAIR is a possible link between TGF‐beta1 and EMT in bladder cancer.

The aim of this study was to analyze the expression of IncRNA HOTAIR in human bladder carcinoma T24 cells incubated with TGF‐beta1, in order to correlate it with markers for EMT.

Bladder carcinoma cells (T24) were incubated with TGF‐beta1 (5 ng/ml) during 48h and then total RNA was extract to analyze the expression of Hotair, N‐cadherin (nCAD, a mesenchymal cell marker), E‐cadherin (eCAD, an epithelial cell marker), and Twist (an EMT transcription factor) by RT‐qPCR. The results are presented in mean±SEM, considering relative expression (RE).

T24 cells stimulated with TGF‐beta1 presented an increased Hotair expression (RE 2.80 ± 0.14 vs. 1.00 ± 0.17 in control, p<0.001). TGF‐beta1 upregulated the expression of nCAD gene (RE 2.22 ± 0.55 vs 1.00 ± 0.32 in control, p <0.0001), while decreased eCAD expression (RE 0.23 ± 0.15 vs 1.00 ± 0.07 in control, p <0.05). The Twist expression was significantly increased by treatment with TGF‐beta1 (RE 3.05 ± 0.54 vs 1.00 ± 0.32 in control, p <0.001).

We conclude that TGF‐beta1 upregulates Hotair expression in T24 cells, correlating with the EMT. It is possible that HOTAIR is involved in cell invasion and metastasis in bladder cancer.

Support or Funding Information

FAPESP (2012/04423‐0)
The Sensitivity and Specificity of Thyroglobulin as an Index of Iodine Status in Mildly Iodine Deficient Adults
Authors: Skeaff Sheila Anne , Ma Zheng Feei
Published: November 2023

Iodine deficiency is still one of the most common micronutrient deficiencies in the world. Iodine status is typically assessed by the determination of urinary iodine concentration (UIC) from a single spot urine sample obtained from a group of subjects. The median UIC of the group is then compared against population cut‐offs set by WHO and the Iodine Global Network. Typically only one spot urine sample is collected from each subject, but the large inter‐and intra‐individual variation means that the UIC in a single urine sample cannot be used as an index of iodine status in an individual. The collection of multiple spot urine samples from one subject has been shown to improve the precision of UIC, but this is associated with a increase in respondent burden. Serum thyroglobulin (Tg), a blood protein specific to the thyroid gland, holds promise as a biomarker of individual iodine status; higher levels of Tg are associated with lower UIC. The aim of this study was to assess the sensitivity and specificity of Tg as a index of iodine status.

The study was conducted in Dunedin, New Zealand in a group of adults (n=165) aged 18–40 years. From each participant, five spot urine samples were obtained over a one week period. The average UIC of the five samples was used as the “gold standard” to categorize each participant as iodine deficient (UIC<100 μg/L) or sufficient (UIC ≥100 μg/L). A blood sample was collected for the determination of Tg. Three Tg cut‐offs were used including >11 μg/L, >13 μg/L, and >15 μg/L; a Tg concentration >13 μg/L has been recommended for use in children. The sensitivity, specificity, positive predictive value, and negative predictive value of each cut‐off of Tg was subsequently determined.

As a group, the median UIC was 74 μg/L (range: 16–350 ) and median Tg was 16 μg/L (range: 0.2–179 ). The sensitivity was 73%, 65%, and 55% for a Tg >11 μg/L, >13 μg/L, and >15 μg/L, respectively. The specificity was 72%, 59%, and 53% for a Tg >11 μg/L, >13 μg/L, and >15 μg/L, respectively. The positive predictive value was 87%, 80%, and 74% for a Tg >11 μg/L, >13 μg/L, and >15 μg/L, respectively. The negative predictive value was 52%, 40%, and 60% for a Tg >11 μg/L, >13 μg/L, and >15 μg/L, respectively.

The results of this study indicate that Tg had relatively poor sensitivity and specificity, and a high negative predictive value. This suggests that Tg is not a good index of iodine status in individual adults, however, its usefulness for groups of adults has yet to be elucidated.

Support or Funding Information

Laurenson Award from the Otago Medical Research Foundation.
Vitamin B12 Status in Pregnant Women and their Infants in Southern India
Authors: Finkelstein Julia L. , Dwarkanath Pratibha , Mehta Saurabh , Thomas Tinku , Kurpad Anura V.
Published: November 2023

Objective

To determine the prevalence of vitamin B12 deficiency in a prospective cohort study of pregnant women, and its associations with infant vitamin B12 status and adverse birth outcomes in Southern India.

Methods

Pregnant women (n=419) participating in a perinatal cohort study in India were enrolled before 12 weeks gestation and monitored to assess vitamin B12 and folate status during pregnancy. Plasma vitamin B12 concentrations, methylmalonic acid, and homocysteine concentrations and erythrocyte folate concentrations were measured in maternal samples at enrollment and in infant cord blood at delivery. Binomial and linear regression models were used to evaluate the associations between maternal and infant vitamin B12 status.

Results

The median vitamin B12 concentrations in maternal and infant samples were 26.9 (IQR: 89.6, 172.2) and 173.7 (IQR: 108.6, 264.1) pmol/L. 64% of mothers were vitamin B12 deficient (<150 pmol/L) at their first trimester visit, and 41% of infants were vitamin B12 deficient at birth. Maternal vitamin B12 concentrations at baseline were associated with significantly higher infant vitamin B12 concentrations (p<0.0001) and lower risk of infant vitamin B12 deficiency (RR: 0.58, 95% CI: 0.49–0.69, p<0.0001). Maternal vitamin B12 deficiency was associated with significantly lower infant vitamin B12 concentrations (p<0.0001) and two‐fold higher risk of infant vitamin B12 deficiency (RR: 1.99, 95% CI: 1.44–2.75, p<0.0001) at birth. Maternal vitamin B12 deficiency was also associated with significantly higher risk of infant small‐for‐gestational age (RR: 1.48, 95% CI: 1.04–2.13, p=0.03).

Conclusions

Inadequate maternal vitamin B12 status during pregnancy predicted increased risk of infant vitamin B12 deficiency. Future interventions are needed to improve vitamin B12 status in women of reproductive age to reduce the risk of vitamin B12 deficiency in mothers and their infants.

Support or Funding Information

Division of Nutritional Sciences, Cornell University
Targeting the Enterohepatic Bile Acid Signaling to Modulate Hepatic Autophagic Activity in Non‐alcoholic steatohepatitis
Authors: Wang Yifeng , Ding Yifeng , Li Jibiao , Chavan Hemantkumar , Matye David , Ni Hongmin , Krishnamurthy Partha , Ding Wen‐xing , Li Tiangang
Published: November 2023

Non‐alcoholic steatohepatitis is associated with hepatic free cholesterol accumulation and defective autophagy activity, which sensitize hepatocytes to injury and inflammation. Bile acid synthesis is the only quantitatively significant mechanism for hepatic cholesterol catabolism. Stimulating bile acid synthesis prevented hypercholesterolemia and hepatic steatosis in mice. Autophagy delivers intracellularly stored cholesterol to the lysosome where cholesterol ester is hydrolyzed for subsequent cellular distribution, efflux and bile acid synthesis. The objective of this study is to investigate the molecular link between cholesterol and bile acid metabolism and autophagic activity in hepatocytes and in mouse models of fatty liver disease. Confocal microscopy, immunoblot and cathespin B activity assay showed that excessive intracellular free cholesterol accumulation caused by free cholesterol loading and inhibition of cholesterol esterification in the endoplasmic reticulum (ER) with an ACAT inhibitor blocked autophagosome/lysosome fusion and impaired lysosome function in cholesterol‐laden HepG2 cells. In contrast, cholesterol ester accumulation induced by purified human low‐density lipoprotein treatment increased lysosome function without inhibiting autophagy activity. In addition, bile acid treatment blocked autophagosome/lysosome fusion without affecting key autophagy gene expression in HepG2 cells. Interestingly, over‐expression of CYP7A1 by adenoviral gene delivery, which induced ER cholesterol catabolism and caused relative ER cholesterol depletion, strongly induced autophagy flux in both human hepatocytes and HepG2 cells. Based on this novel regulatory relationship between ER cholesterol sensing and autophagy activity, confocal microscopy, electron microscopy and immunoblot further revealed that cholestyramine administration strongly induced hepatic autophagy in lean mice, and restored hepatic autophagy activity and improved hepatic steatosis in obese mice. The cholestyramine‐mediated hepatic autophagy induction can be attributed to CYP7A1‐mediated ER cholesterol catabolism and decreased hepatic bile acid signaling. In conclusion, these results suggest that hepatic autophagy is regulated by an intracellular cholesterol sensing mechanism, and is an integral part of the complex regulatory network that maintains cholesterol homeostasis in hepatocytes. This study supports a new concept of targeting the enterohepatic bile acid‐CYP7A1‐cholesterol‐autophagy axis to selectively modulate hepatic autophagy activity in fatty liver disease.

Support or Funding Information

NIDDK and NIAAA
High MUC2 Biosynthesis Induce ROS Production which Increases Goblet Cell Susceptibility to ER stress and Apoptosis
Authors: Tawiah Adelaide , Moreau France , Chadee Kris
Published: November 2023

MUC2 is a large glycoprotein, produced by goblet cells, which forms the protective mucus blanket overlying the intestinal epithelium. Apart from its role as a physical barrier, other functions of MUC2 have not been explored. During pathological conditions such as inflammatory bowel disease (IBD), MUC2 biosynthesis and secretion are accelerated. There is however little known on how MUC2 production is regulated and whether goblet cells undergo increased endoplasmic reticulum (ER) stress and apoptosis following high MUC2 biosynthesis and secretion. We studied the role of MUC2 in goblet cell ER stress and apoptosis using a high MUC2‐producing goblet cell line (HT29‐H) and a clone of HT29‐H (HT29‐L) in which MUC2 has been silenced using lentivirus shRNA technology. Cells were treated with ER stress‐inducing agents, and markers for ER stress (GRP78, ATF4, sXBP1 and CHOP) and apoptosis (caspase 3 cleavage, DNA fragmentation and PARP cleavage) were quantified by western blotting, confocal microscopy and transmission electron microscopy. Compared to HT29‐L, HT29‐H cells showed significant increase in ER stress and apoptosis in response to ER stress and apoptosis‐inducing agents. HT29‐H cells also exhibited higher mitochondrial activity and reactive oxygen species (ROS) production than HT29‐L although the cells had similar mitochondrial mass. Inhibition of ROS abrogated basal ER stress levels, tunicamycin and thapsigargin‐induced ER stress, and apoptotic events including cytoplasmic calcium influx, loss of mitochondrial membrane potential, and caspases 3 and 4 activation. This indicates that high MUC2 production specifically increases ROS production, which drives ER stress and apoptosis. These findings were corroborated in isolated colonic epithelial cells and colonic tissues from Muc2+/+ and Muc2−/− mice that showed significant increase in ER stress in Muc2+/+ mice as compared to Muc2−/− mice. Finally, goblet cells in Muc2+/+ mice exhibited severe ER stress and apoptosis during progressive development of DSS‐induced colitis leading to disruption of the mucus barrier. We conclude that high ROS output, as a consequence of high MUC2 production, induces ER stress and apoptosis in goblet cells that could subsequently impair mucus barrier function in disease pathogenesis.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
Hydroxylase inhibition reduces transforming growth factor‐β1 induced fibrosis associated with colitis
Authors: Manresa Mario , Tambuwala Murtaza , Radhakrishnan Praveen , Harnoss Jonathan , Cavadas Miguel , Keogh Ciara , Cheong Alex , Cummins Eoin , Barrett Kim , Schneider Martin , Taylor Cormac
Published: November 2023

Intestinal fibrosis is a severe complication of inflammatory bowel disease (IBD) that often leads to surgical interventions, highlighting an urgent need for alternative therapies. The inhibition of key oxygen‐sensing hydroxylases that regulate the response to hypoxia ameliorates inflammation in mouse models of colitis. Here we investigated the impact of hydroxylase inhibition on intestinal fibrosis. Intestinal fibrosis was induced in mice secondary to colitis through exposure to dextran sodium sulfate (DSS) in drinking water. Cultures of human colonic (CCD‐18Co) and mouse embryonic (MEF) fibroblasts were used to investigate the actions of hydroxylase inhibitors on the response to transforming growth factor‐β1 (TGF‐β1). Mice allowed to recover for 8 or 14 days after exposure to DSS developed fibrosis with profound alterations in the pattern of collagen deposition and infiltration of α‐smooth muscle actin (α‐SMA)‐positive fibroblasts. Treatment with the pan‐hydroxylase inhibitor, dimethyloxalylglycine (DMOG) during the recovery period reduced signs of fibrosis. In contrast, exposure of prolyl hydroxylase‐2+/− (PHD‐2+/−) mice to DSS resulted in severe intestinal fibrosis suggesting that the effects of DMOG are independent of the PHD‐2‐HIF axis. In vitro, hydroxylase inhibitors reduced TGF‐β1 mediated production of key fibrosis markers such as α‐SMA, Connective Tissue Growth Factor (CTGF) and collagen‐1‐α, in both CCD‐18Co and MEF, strongly suggesting an effect on TGF‐β1 signalling. Furthermore, these antifibrotic effects were present in PHD‐2+/− fibroblasts even though these cells showed enhanced responsiveness to TGF‐β1. In order to investigate the molecular mechanisms of the anti‐TGF effects of hydroxylase inhibitors, we analyzed their impact on the activation of TGF‐β1 signalling pathways. While no major effects were detected on the ability of TGF‐β1 to activate the Smad pathway, hydroxylase inhibition strongly inactivated TGF‐ERK signalling. Furthermore, areas of the intestinal mucosa of mice exposed to DSS where α‐SMA positive fibroblasts were concentrated, exhibited abundant positive phosphoERK nuclear staining. This observation highlighted the importance of the activation of the ERK pathway in fibrotic colons. Taken together, our studies showed that hydroxylase inhibition ameliorates intestinal fibrosis, likely via effects on TGF‐ERK signalling. We propose hydroxylase inhibition as a potential new therapeutic approach for the treatment of intestinal fibrosis

Support or Funding Information

This work was funded by Science Foundation Ireland
Absence of Mast Cells in the Kidneys of Rats Subjected to Fat Embolism Despite their Presence in the Lungs and Heart
Authors: Patel Chirag , Fletcher Amanda N , Poisner Alan M , Siddiqi Ahsan , Arif Dauod , Hamidpour Soheila , Ponnapureddy Rakesh , Molteni Agostino
Published: November 2023

In a rat model of fat embolism (FE) induced by i.v. injection of triolein (T), we have shown that the lungs, hearts and kidneys demonstrate severe histopathological damage characterized by organ inflammation and arterial constriction. 1,2,3 Such damage is present up to 10 weeks after injection.1 The renin angiotensin system (RAS) plays a role in the pathogenesis of the damage since captopril (an ACE inhibitor) and losartan (Los), an angiotensin‐II type 1 receptor blocker, prevented the damage.4 After T injection, an increase in renin stain was observed in the kidney juxta‐glomerular apparatus (JXTG)5 and in the lungs.6 The renin overexpression may be related to an increased number of mast cells (MCs),7 and indeed we found an increased number of these cells 10 weeks post T injection in the lungs of the rats.8 MCs in lower numbers were also observed in hearts of the same rats, but LOS did not reduce their number.9 On the basis of these observations, we decided to evaluate the potential presence of MCs in kidneys after T injection.

Twenty‐two Sprague Dawley rats (~300 gm BW) received either T (0.2 mL i.v.) or saline. Six weeks later, half of the rats were given i.p. injection of Los and the remainder received saline. Four weeks later (10 weeks post T injection), rats were anesthetized with isoflurane, sacrificed, and necropsied. Tissues were fixed in 10% formalin and stained with H&E (morphometric scoring), Trichrome (fibrosis) and CD 11‐c kit (MCs). On each slide, 10 photographs were taken at random at 400× and MCs evaluated by two pathologists unaware of the slide identity.

After T injection, we observed severe arterial damage with increased medial thickness and reduced lumen patency. Glomeruli showed increased cellularity, thickening of basal membrane, and crescent formation. Scattered chronic inflammatory cells and erythrocytes were seen in the tubuli. The damage was moderately reduced by Los treatment. MCs were not seen in any of the animals.

In the kidneys, despite the high presence of renin in the JXTG and severe vasculitis after T injection, we could not identify MCs at 10 weeks even with a very specific staining method. This is in contrast with the presence of MCs that we have found in the lungs and heart. The absence of MCs in the kidneys at 10 weeks may reflect different pathogenic mechanisms or a time course different from that of the lungs or heart.

Support or Funding Information

M. K. Geldmacher Foundation, St. Louis, MO; This work was supported by a CTSA grant from NCRR and NCATS awarded to the University of Kansas Medical Center for Frontiers: The Heartland Institute for Clinical and Translational Research # TL1TR000120. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH, NCRR, or NCATS.
Increased Indoleamine 2,3‐dioxygenase expression in the renal fibrogenesis induced by unilateral ureteral obstruction
Authors: Matheus Luiz Henrique Gomes , Simão Gislene Mendes Simão , Amaral Taíssa Altieri do , de Oliveira Brito Rodrigo Barbosa , Malta Camila Soares , Matos Yves Silva Teles , Souza Diego Mota , Dellê Humberto
Published: November 2023

Chronic kidney disease (CKD) is a progressive and high prevalent disease worldwide. Its prevalence doubled on USA on the last decade and as such, deserves attention and a worry to better know its mechanisms.

Renal fibrosis is a lead event happening in CKD, and is characterized by an extensive inflammatory process with vast macrophage migration and extracelular matrix accumulation, those leading to damages on the kidney architecture and renal function, and thus worsening the patient condition.

In this frame, Indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐catabolizing enzyme with immunomodulatory properties, seems to be deeply associated with mechanisms leading to a bad prognostic to fibrotic and inflammatory conditions. Because IDO may be modulated by Transforming growth factor‐β (TGF‐ β), a key cytokine in the development of renal fibrosis, we hypothesized the relationship between IDO and renal fibrogenesis.

In the present study, our objective was to analyze IDO activity and expression in a model of renal fibrogenesis induced by unilateral ureteral obstruction (UUO), and correlate it with markers of epitelial‐mesenchymal transition.

Wistar rats were submited to 7 days of UUO. The unobstructed kidneys of the UUO (CL) and the kidneys from SHAM operated rats were used as control. Increased interstitial collagen deposition was remarkable in UUO kidneys. Immunohistochemistry analysis revealed a significant increase in the number of macrophages in UUO kidneys, accompanied by reduction of tubular e‐cadherim expression. Markers of mesenchymal cells (alphaSMA and vimentin) were increased in UUO kidneys, in particular in the renal interstitium and in tubules. These findings characterize the epitelial‐mesenchymal transition process and were accompained by increased TGB‐beta 1 expression (qRT‐PCR). Finally, IDO was markedly expressed in cortical and medular tubules of the UUO kidneys. In addition, IDO activity was accessed from renal tissue, being significantly higher in UUO kidneys. These data demonstrated that IDO is increased during renal fibrogenesis and associated with an increased number of macrophages as well as an upregulation of mesenchymal cell markers, sugesting an association between IDO and EMT.

Support or Funding Information

FAPESP (2012/04423‐0)
Clomiphene Citrate and Male Infertility: Investigation of Treatment on Spermatogenesis and Testicular Histomorphology in a Rat Model
Authors: Gibson‐Corley Katherine , Kogan Paul , Wald Moshe
Published: November 2023

Clomiphene citrate (CC) is used off‐label as a treatment for selected cases of male infertility, acting as a competitive selective‐estrogen receptor modulator. Although CC is used clinically, there have only been a small number of randomized controlled human studies that have yielded inconclusive findings. Here we have used a prospective animal model for studying CC treatment to determine if this drug increases spermatogenesis for treatment of male infertility. We treated sexually mature Sprague‐Dawley rats with CC via oral gavage for either 1 or 2 rat spermatogenic cycles (13 days for 1 cycle or 26 days for 2 cycles) at a dose of 0.6 mg/kg. This dose was chosen as it is a similar dose to what is clinically used in humans. Testes were weighed and sperm counts were measured from rats treated with CC and control animals. Testes were fixed in modified Davidson's solution and stained with HE for histopathological and morphometric analysis. No differences in sperm count and no histomorphologic differences were noted in the testes treated with CC as compared to control. Seminiferous tubule minimum diameter and tubular epithelial height were measured and the number of retained spermatozoa was also scored with no significant differences compared to control. Although previous studies have shown that rats treated with much higher doses of CC (>20 mg/kg) have decreased spermatogenesis, we can conclude that CC treatment at a therapeutic dose of 0.6 mg/kg in rats does not appear to have an effect on spermatogenesis. That said; CC may play a more indirect role in male infertility which may include changes in accessory reproductive organs or alterations in hormonal levels.

Support or Funding Information

Internal funding; Department of Urology, University of Iowa
The Effect of the Baby Friendly Hospital Initiative on Exclusive Breastfeeding Rates in Hospitals
Authors: Patterson Julie Ann , Olson Beth H , Keuler Nicholas S
Published: November 2023

Background

Exclusive breastfeeding is associated with an increased level of protection from maternal and infant morbidity and mortality. The Baby Friendly Hospital Initiative (BFHI) was launched in 1991 to positively influence breastfeeding outcomes by recognizing hospitals with optimal practices in mother/infant care. There is limited research examining the influence of the BFHI on exclusive breastfeeding rates, and how these rates may be influenced by other factors.

Study Objective

The aim of this study is to examine the relationship between population demographics, including poverty and race, and exclusive breastfeeding outcomes in hospitals with and without the BFHI designation.

Methods

Data was obtained from the Joint Commission, a leading agency in hospital accreditation, and included 283 BFHI hospitals and 1071 hospitals without the BFHI designation. Demographic variables were applied to the data set and included total population; proportion of white, African American, Hispanic, Asian; educational status and household income, as computed for the zip code surrounding each hospital. Relationships were explored using linear regression. We additionally built a multiple linear regression model in an attempt to determine how well we can predict exclusive breastfeeding rates.

Results

The proportion of whites, number of residents with a bachelor's degree, and income were all positively related to exclusive breastfeeding rates within the hospital (p<0.0001). By contrast, the proportion of African Americans, proportion of Hispanics, and number in the population at less than 200% of the poverty line were negatively related to exclusive breastfeeding rates within the hospital (p<0.0001). Hospitals with BFHI designation had exclusive breastfeeding rates that were on average 10–15% higher than hospitals without BFHI designation, regardless of demographics. Using a multiple linear regression model we were able to explain 22% of the variability in exclusive breastfeeding rates.

Conclusions

BFHI designation is associated with significantly higher exclusive breastfeeding rates independent of demographic variables. However, we recognize that our model was limited in its ability to explain the variability in the exclusive breastfeeding rates. We suggest areas for improvement of the model, including additional possible explanatory variables, which could be the subject of future research.

Support or Funding Information

No funding was received for this research.
Physical Mechanisms of Cell Organization on Micron Length Scales
Authors: Rosen Michael K.
Published: November 2023

Cells are organized on length scales from Angstroms to microns. However, the mechanisms by which Angstrom‐scale molecular properties are translated to micron‐scale macroscopic properties are not well understood. We have shown that interactions between multivalent proteins and multivalent ligands can cause liquid‐liquid demixing phase transitions, resulting in formation of micron‐sized liquid droplets in aqueous solution and micron‐sized puncta on membranes. These transitions appear to occur concomitantly with sol‐gel transitions to form large, dynamic polymers within the droplets/puncta. I will discuss how such transitions may control the spatial organization and biochemical activity of actin regulatory signaling pathways, and contribute to formation of PML nuclear bodies in the mammalian nucleus. Our data suggest a general mechanism by which cells may achieve micron‐scale organization based on interactions between multivalent macromolecules.
Organic Cation Transporter Novel Type‐1 (OCTN‐1) And Pulmonary Responses To Secondhand Tobacco Smoke (SHS)
Authors: Milner Dallin C. , Lewis Joshua B , Winden Duane R , Gassman Jason , Monson Troy , Broberg Dallin , Arroyo Juan A , Reynolds Paul R
Published: November 2023

Oxidative stress is involved in the pathogenesis of lung disorders such as asthma, COPD, acute lung injury, pulmonary fibrosis and lung cancer. The antioxidant amino acid ergothioneine and its highly specific transporter, OCTN‐1 (organic cation transporter novel type‐1) play important roles in antioxidant cellular defence. The goal of this project was to understand the role of OCTN‐1 in protecting the lung from oxidative stress caused by secondhand tobacco smoke (SHS). The current research evaluated the in vivo effects of SHS exposure in OCTN‐1 null and control mice compared to identical animals exposed to room air only for three months. Mice were subjected to daily secondhand tobacco smoke (SHS) via a nose only inhalation system (Sireq industries) from PN20‐110. Q‐PCR and immunoblotting confirmed no OCTN‐1 expression in nulls regardless of exposure, but a significant decrease in expression following SHS exposure. Bronchoalveolar lavage fluid (BALF) procured from OCTN‐1 null and control animals revealed SHS‐induced increases in total cell counts and protein compared to controls. Inflammatory mediators including TNF‐alpha and IL‐1beta were also significantly increased in SHS‐exposed OCTN‐1 nulls compared to smoke exposed controls. As a general theme, inflammatory status and remodeling were increased in OCTN‐1 nulls exposed to SHS compared to smoked controls. Such differences included histological simplification of the distal lung, decreased abundance of non‐ciliated cells in the proximal lung, elevated PMNs, and augmented inflammatory cytokines including TNF‐alpha and IL‐1beta. Of particular interest, increased thickness and volume density of alveolar septa and inflammatory cell infiltration were observed in OCTN‐1 lungs exposed to SHS compared to controls. These data reveal intriguing insight into potential mechanisms related to oxidative stress that may be key factors in SHS‐mediated inflammation.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Molecular Mechanism Of DNA Interstrand Crosslink Repair
Authors: Smogorzewska Agata , Kottemann Molly , Conti Brooke , Thongthip Supawat , Rickman Kimberly , Lach Frank
Published: November 2023

The focus of our research is to understand the mechanism of DNA repair during DNA replication. I will discuss how DNA interstrand crosslinks (ICLs) are repaired as well as describe our early studies to understand the response of replication fork machinery to stalling at difficult to replicate regions.

Most of our research concentrates on ICL repair with the overall goal to gain insights into the cellular and organismal consequences of deficiencies in this type of repair. I will discuss the role of FAN1 in assuring proper DNA repair. FAN1 is a nuclease important for ICL repair, whose deficiency results in kidney disease. To help elucidate the cellular and tissue maintenance functions of FAN1, we generated Fan1 knockout mice. Fan1−/− mouse embryonic fibroblasts (MEFs) display Mitomycin C (MMC)‐induced proliferation defect. The crosslink sensitivity of Fan1−/− cells is not as significant as the level observed in Fanca, Fancd2 or Slx4 (Fancp)‐deficient cell lines. Wild type mouse FAN1 cDNA but not the variants with inactivating mutation in the SAP or nuclease domains complement ICL sensitivity in Fan1−/− MEFs. As we previously described for a human protein, the mutation in the UBZ domain, which is essential for the interaction with FANCD2, is completely dispensable for the crosslink‐repair function of FAN1. At high level of crosslink insult, deficiency in Fan1 can additively enhance the degree of MMC‐induced chromosomal abnormality in Fancd2−/− MEFs. Karyomegalic nuclei are detected in the kidney and liver of Fan1 mice from the age of 6 months with the level of renal karyomegaly intensifying with age. Fan1−/− mice, when treated with MMC, display hypocellular bone marrow and are hypersensitive to MMC treatment.

In the second part of my talk, I will discuss proteins at the active replication fork that are important for proper response to replication stress and describe a regulatory network that is called upon when DNA damage is encountered during replication.

Support or Funding Information

Supported by NIH RO1grant HL120922, grant # 8 UL1 TR000043 from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH) Clinical and Translational Science Award (CTSA) program, and by Rita Allen Foundation Scholars grant.
Chromatin Replication And Epigenome Maintenance
Authors: Groth Anja
Published: November 2023

In dividing cells, faithful duplication of the genome must be accompanied by reproduction of the chromatin landscape on newly synthesized DNA. Our research focuses on how human cells replicate chromatin and ensure transmission of genetic and epigenetic information during cell division.

Recently, we have developed an explorative proteomic approach, Nascent Chromatin Capture (NCC), to profile chromatin replication in human cells (Alabert et al., 2014). NCC relies on biotin‐dUTP labeling of replicating DNA and cross‐linking, followed by affinity‐purification of tagged chromatin and quantitative proteomics. By comparing nascent newly synthesized chromatin with mature post‐replicative chromatin, we have provided an extensive resource to understand DNA replication and restoration of chromatin organization (Alabert et al., 2014). In addition, we have used NCC technology combined with pulsed SILAC labeling to comprehensively address transmission of histone marks across the cell cycle. We find that histone PTMs are propagated by two distinct mechanisms in human cells (Alabert et al., 2015), which hasimplications for how cell proliferation is linked to cell fate decision. To understand how histones are handled at active and damaged replication forks, we are doing histone chaperone structure‐function analysis (Huang, Stromme et al., 2015). I will discuss the roles of MCM2, ASF1 and TONSL‐MMS22L in handling histones at the fork and our latest progress in understanding how functional chromatin domains are maintained in dividing cells.
Enzymes Involved In Secondary Metabolism And RNA Modification
Authors: Bandarian Vahe
Published: November 2023

Deazapurine‐containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogs found throughout biology, that include various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as antibiotic, antiviral, and antineoplastic agents, the biosynthetic route toward deazapurine production had remained largely elusive for more than 40 years. This presentation will provide an overview of the pathway and discuss recent advances in understanding the structures and mechanisms of enzymes that catalyze key transformations.
Twists and turns in Ubiquitin Conjugation Cascades
Authors: Schulman Brenda
Published: November 2023

Post‐translational modification by ubiquitin and ubiquitin‐like proteins (collectively referred to as UBLs) is a predominant eukaryotic regulatory mechanism. The vast reach of this form of regulation extends to virtually all eukaryotic processes. UBL modifications play critical roles in controlling the cell cycle, transcription, DNA repair, stress responses, signaling, immunity, plant growth, embryogenesis, circadian rhythms, and a plethora of other pathways. UBLs dynamically modulate target protein properties including half‐life, subcellular localization, enzymatic activity, conformation, and intermolecular interactions. Moreover, the enzymatic process of UBL ligation to and removal from proteins is itself dynamic, involving cascades of E1, E2, and E3 enzymes antagonized by deubiquitinating enzymes. The UBL C‐terminus is first activated, then transferred between multiple enzyme active sites, and ultimately ligated via an E3 enzyme to a target. With roughly 300 members in humans, the largest E3 family consists of the multisubunit Cullin‐RING ligases (CRLs), which regulate a staggering number of biochemical pathways and biological processes. We have found that Cullin‐RING E3 activities are modulated through protein‐protein interactions influencing enzyme conformation. In my presentation, I will discuss dynamic mechanisms underlying regulation of and by this large family of E3 ligases
Imaging Mass Spectrometry ‐ The Pyrazinamide Enigma
Authors: Prideaux Brendan , Sarathy Jansy , Via Laura , Gopal Pooja , Barry Clifton , Dick Thomas , Dartois Veronique
Published: November 2023

Pyrazinamide is a 60‐year old anti‐tuberculosis (TB) drug, the mechanism of which has been the focus of intense research and yet largely remains a mystery. Today, PZA would fall under the category of fragment rather than drug, and would likely be missed because it is not active under standard in vitro conditions despite its remarkable sterilizing activity against human TB. At the time of its introduction, PZA reduced therapy duration from 9 to 6 months, and has been part of the first‐line and most second‐line drug regimens since then. However, resistance to PZA continues to emerge, threatening one of the most valuable drugs in our current arsenal.

Given the striking disconnect between PZA's activity in vitro and in vivo, we have developed pharmacological tools to study the pharmacokinetics, tissue distribution, metabolism, and activity of PZA at the site of action, in patients and in animal models. In pulmonary TB, the pathogen is both intracellular and extracellular, and resides in multiple types of lung lesions that essentially comprise layers of immune cells often surrounding necrotic foci. Decades of clinical trials have taught us that the presence of cavities – large necrotic lesions that are connected with an airway – correlates with poor treatment outcome. We also know that the spectrum of lesions seen in human TB is inhabited by multiple bacterial populations with differential susceptibilities to any given drug. We thus asked “which bacterial subpopulations are reached and killed by PZA?”

Using MALDI mass spectrometry imaging and classical mass spectrometry, we have discovered that PZA and its active metabolite have the unique ability to distribute rapidly and homogeneously in all lesion types and lesion compartments, including cavities. Lesion‐centric efficacy studies in animal models have been conducted to measure the killing activity of PZA and its active metabolite POA against specific subpopulations, either in vitro or ex vivo. Characterization of resistant mutants isolated in vitro revealed complex metabolic networks affected by PZA's active metabolite. We will discuss what can be learned from these collective results to develop assays and screening strategies that have the potential to capture the “next‐gen PZA” and overcome ramping resistance.
To Stay (At The Bench) Or Go? Determinants Of Career Decision‐Making In Recent Ph.D. Graduates
Authors: Gibbs Kenneth D , Griffin Kimberly
Published: November 2023

Recent biomedical workforce policy efforts have centered on the challenges of enhancing career preparation for trainees and increasing workforce diversity. While these issues have been addressed separately, little work considers them concurrently. This study addresses two questions: (a) what is the process of career interest formation for recent Ph.D. graduates, and (b) how does this process differ across lines of gender and race‐ethnicity? We report results from a mixed‐methods study, including a survey of 1500 American biomedical Ph.D. graduates (n=276 from underrepresented minority (URM) backgrounds), and interviews with 70 respondents pursuing diverse career pathways. Survey data showed that relative to Ph.D. entry, on average, scientists from all backgrounds reported (i) increased knowledge about career options (ii) significantly decreased interest in faculty careers at research universities, and (iii) significantly increased interest in non‐research careers. There were differences in the overall levels of career pathway interest (at Ph.D. entry, completion, and postdoc), and the magnitude of change in interest in these careers across social identity. Multiple logistic regression showed that when controlling for career pathway interest at Ph.D. entry, first‐author publication rate, faculty support, research self‐efficacy, and graduate training experiences, women and URM men remained 36–54% less likely than White/Asian men to express high interest in faculty careers at research‐intensive universities, and URM women were 93% more likely than all other groups to report high interest in non‐research careers. Qualitative data revealed how vicarious learning experiences during graduate school, systemic dynamics within the biomedical enterprise, and unique personal motivations for pursuing scientific research play determinative roles in the career pathways Ph.D. graduates pursued. Findings point to the need for enhanced career development programs earlier in the training process, and interventions that are sensitive to the distinctive patterns of interest development across social identity groups.

Support or Funding Information

This work was supported, in part, from a generous grant from the Burroughs Wellcome Fund. KG is supported by the NCI Cancer Prevention Fellowship Program.
Human Gut Bacteroidetes
Authors: Gilbert Harry
Published: November 2023

The human large bowel is colonized by a community of microbes, the microbiota, which has a significant impact on human health and nutrition through the production of short chain fatty acids (SCFAs), and by interaction with the host immune system. The major nutrients available to these organisms are dietary glycans, also known as complex carbohydrates. Thus, dietary and nutraceutical strategies, based on complex carbohydrates, can, potentially, be deployed to encourage the dominance of beneficial microbes in the microbiota, particularly those producing health promoting SCFAs such as propionate and butyrate, and bacteria that have an anti‐inflammatory impact through its interaction with the human immune system, ensuring that this microbial ecosystem has a positive influence on human health. This approach, however, is greatly restricted by a critical lack of understanding of the mechanisms by which complex glycans are metabolized by the microbiota. Significantly, the wealth of genomic and metagenomic microbiota sequence presents an exciting, but so far unfulfilled, opportunity to make decisive advances in our understanding of glycan metabolism in the human large bowel. This seminar will review our biochemical, genetic and microbiological strategies, in harness with metagenomic and genomic data, to understand the mechanisms of complex glycans utilization by the human microbiota1,2,3. The models established, will trigger the development of novel dietary strategies that are designed to maximize human health through manipulation of microbiota structure.
Interactions between Campylobacter jejuni and AB5 toxins in the gut
Authors: Szymanski Christine M. , Patry Robert T. , Stahl Martin , Sacher Jessica C , Vallance Bruce A.
Published: November 2023

Campylobacter jejuni is a ubiquitous commensal in chickens, but a major cause of human diarrheal disease worldwide. To avoid immune detection by its host, most C. jejuni isolates express human ganglioside‐mimicking lipooligosaccharide (LOS) structures and it has been shown that host recognition of these antigens as non‐self results in the development of Guillain‐Barré syndrome. In addition to immune evasion, expression of ganglioside mimics in C. jejuni has been associated with improved host cell invasion and resistance to human serum. Although only positive benefits for ganglioside mimic expression have been described for C. jejuni, the organism has the ability to quickly switch off the mimicry through a phase‐variable galactosyltransferase, CgtB, involved in LOS biosynthesis. Vibrio cholerae secretes the bacteriophage‐encoded cholera toxin (CT) which binds to GM1 ganglioside structures on human cells as well as to C. jejuni GM1 ganglioside‐mimics. Enterotoxigenic Escherichia coli secretes heat‐labile enterotoxin I (LT), which also binds to these structures. Since C. jejuni, V. cholerae and E. coli can co‐infect the same niche, we examined the effects of these AB5 toxins on C. jejuni biology in order to determine if this relationship could represent a new mechanism for interpathogen competition in the intestine. We demonstrate that CT and LT specifically bind only to C. jejuni strains expressing GM1 mimics. Interestingly, our experiments show that addition of CT or LT to C. jejuni strains expressing GM1‐mimicking LOS partially clears cell growth on agar. LOS from bacteria remaining inside these zones of clearance was isolated and compared to LOS from untreated bacteria. SDS‐PAGE and silver staining of the LOS showed the loss of a band from the exposed cells compared to untreated cells, and Western blot analysis revealed reduced binding of LOS to CT. Sequencing cgtB in these two groups demonstrated that this enzyme is “on” or “off” in the unexposed group, while cgtB in the CT‐ and LT‐treated cells showed a frameshift mutation, causing premature truncation of the galactosyltransferase. Examination of an engineered E. coli strain expressing GM1 mimics shows that CT is capable of binding to the mimicking cells without inducing growth clearance, pointing toward a Campylobacter‐specific mechanism of action. These results suggest that CT and LT have a negative influence on C. jejuni and could contribute to competition between these pathogens. Further studies describing the interactions between these zoonotic pathogens, the resident gut microbiome and poultry will be described.

Support or Funding Information

CMS is an Alberta Innovates Technology Futures iCORE Strategic Chair in Bacterial Glycomics.
The Future of Biobased Chemicals
Authors: Shanks Brent
Published: November 2023

The replacement of crude oil‐derived chemicals with biobased chemicals is both a technical and economic challenge. From a technical perspective the challenge is how to selectively remove oxygen from a highly oxygenated substrates. However, the economic challenge is even more significant in that the biobased chemicals must compete against a highly efficient petrochemical production system. The serial approach of targeting one biobased chemical product at a time, which is the most common technical approach, is both time intensive and expensive. To mitigate costs, a need exists to develop a technological framework in which a range of biobased chemicals can be produced from a common platform. One such generalized platform being developed by the NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), depends on the exploitation of the fatty acid/polyketide metabolic pathway leading to a diversity of intermediate chemicals that are subsequently converted to chemical products using chemical catalysts. An overview of the technical strategy being used by CBiRC to achieve a generalized chemical production platform will be discussed using several specific examples involving varying chain length carboxylic acids and triacetic acid lactone (TAL). The TAL system will be discussed in greater technical depth to demonstrate the power of biological and chemical catalyst researchers interacting.
Engineering Microbial Fatty Acid Biosynthesis As An Industrial Biotechnology Platform
Authors: Schirmer Andreas
Published: November 2023

REG Life Sciences acquired LS9 in January of 2014 and is advancing its microbial fatty acid technology platform. Microbial fatty acid metabolism has been demonstrated as a versatile platform for the production of diverse renewable chemicals and fuels. From novel raw materials for the oleochemical and food industries to drop‐in and advantaged products for the fuel, commodity, and high‐value chemical industries, new applications of this pathway continue to emerge. REG Life Sciences is focused on the systematic control of microbial fatty acid metabolism to synthesize new chemical families, control structure within each family, and optimize production efficiency to support scalable commercial processes. This talk shall briefly review the REG Life Sciences technology platform and highlight specific examples of new product pathways and the optimization of our most advanced product platforms.