bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic‐Modification of Adult Fibroblast Cells into OCT4+ and VEGFR‐2+ Angiogenic Cells
Authors: Wary Anita , Wary Neil , Baruah Jugajyoti , Wary Kishore
Published: November 2023

Rationale

The epigenome of fibroblast cells is plastic. Here, we address the hypothesis that fibroblast cells can be epigenetically modified into blood vessel forming cells.

Methods and Results

Human adult dermal fibroblast cells (hADFCs) were treated with optimal concentrations of chromatin‐modifying agents 5‐aza‐2′‐deoxycytidine (5‐Aza) and trichostatin A (TSA), thereafter subjected to activation of canonical Wnt signaling. We provide evidence that these epigenetically modified hADFCs increasingly expressed β‐catenin, pluripotency factor octamer‐binding transcription factor‐4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor‐2 (VEGFR‐2) and Lipid Phosphate Phosphatase‐3 (LPP3). Staining and microscopic analysis showed differential localization of β‐catenin and OCT4 proteins in epigenetically modified hADFCs. Accordingly, in a chromatin immunoprecipitation (ChIP) assay, OCT4 bound to the VEGFR‐2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a three‐dimensional (3D) Matrigel, a subset of modified cells formed branching point network‐like structures in VEGF‐dependent manner.

The epigenome of adult fibroblast cells is amenable to epigenetic modification, and has significant implications for the use of epigenetically modified cells to promote angiogenesis in vivo.

Support or Funding Information

Studies were supported by the RO1‐HL079356 and (GRNT25710129) and by the University of Illinois at Chicago Center for Clinical and Translation Science Award (UL1RR029879) to K.K.W. J.B was supported by AHA pre‐doctoral fellowship Mid‐West affiliate (15PRE22760004) and CCTS‐PECTs (1UL1TR002003). The UIC, RRC Core Imaging Facility, at the University of Illinois Chicago.
The homeodomain protein Cux1 regulates cell cycle progression in polycystic kidney disease through specific chromatin interactions
Authors: Livingston Safia , Carlton Carol , Sharma Madhulika , Baybutt Richard , Heuvel Greg Vanden
Published: November 2023

Cux1 is a homeodomain protein involved in cell cycle regulation and kidney development. Cux1 represses the cyclin kinase inhibitor p27 during early kidney development, promoting cell proliferation in the nephrogenic zone. Promoter reporter analysis of p27 revealed that Cux1 represses p27 in a concentration dependent manner, and immunoprecipitation showed that Cux1 interacts with the co‐repressor Grg4 and the histone deacetylases HDAC1 and HDAC3. Chromatin immunoprecipitation (ChIP) identified the interaction of Cux1, Grg4, HDAC1, and HDAC3 at two different sites in the p27 promoter. To determine whether there was an interaction between these two loci in the developing kidney, we performed chromatin conformation capture (3C) assay. Analysis of newborn kidney tissue with 3C and ChIP‐3C showed that the p27 promoter forms a loop intersecting at these two loci and that Cux1 bridges these two sites. To determine whether HDACs are required for Cux1 repression of p27 we analyzed p27 promoter activity in the presence of the HDAC inhibitor trichostatin A (TSA). TSA treatment completely relieved the repression of p27 by Cux1 and Grg4, demonstrating that Cux1 represses p27 in an HDAC dependent manner. To begin to test whether HDAC inhibitors could be used to target Cux1 repression of p27 for the treatment of PKD, we treated Pkd1 targeted pregnant mice with TSA or Valproic acid or vehicle beginning at embryonic day 10.5 until embryonic day 18.5. Newborn Pkd1 mutant mice that received vehicle exhibited extensive collecting duct cysts, while newborn Pkd1 mutant mice that received TSA showed a significant reduction in cysts. Moreover, p27 expression was upregulated in TSA treated Pkd1 mice. Taken together, these results suggest that HDACs are required for cyst growth, and raise the possibility that HDAC inhibitors may be an effective treatment for PKD.

Support or Funding Information

National Institutes of Health (NIDDK)
Ontogeny of Monocarboxylate Transporter 1 and CD147 Expression in Rat Liver
Authors: Ng Michael , Cao Jieyun , Felmlee Melanie A
Published: November 2023

Purpose

Monocarboxylate transporter 1 (MCT1) is integral in cellular energy metabolism and drug disposition. The ancillary protein CD147 (basigin) has been shown to complex with MCT1 for translocation of the transporter to the cell membrane. The maturation of MCT1 and CD147 expression is not fully understood. Differential hepatic expression levels and functional activities during development can lead to altered drug disposition of MCT1 substrates. The objective of the present work is to quantify the hepatic maturation of MCT1 and CD147.

Methods

Livers were collected from male and female Sprague Dawley rats from birth through sexual maturity (postnatal day (PND) 42) (N = 4 per sex at each age). Sex was assigned in animals less than PND 21 based on ano‐genital distance and confirmed by PCR analysis of the SRY gene on the Y chromosome. Whole cell protein extract and total membrane preparations were evaluated for MCT1 and CD147 expression using western blot, with expression levels normalized to the house‐keeping protein Na+/K+ ATPase.

Results

Significant age‐related differences were observed in liver total and membrane MCT1 and CD147 expression, with the highest levels observed just prior to weaning. In whole cell proteins of male and female rats, MCT1 levels increased to peak at PND 14, before decreasing through PND42. Whole protein CD147 followed the MCT1 trend, with highest expression at PND 14 and 18. MCT1 membrane expression was significantly increased at PND 10 to 18 in males, and PND 14 and 18 in females. CD147 membrane expression for both sexes followed the same pattern as membrane‐bound MCT1. There was a significant difference in CD147 membrane expression between sexes, with males having increased CD147 levels at every age group. There is strong positive correlation between membrane‐bound MCT1 and CD147 expressions for both sexes.

Conclusions

Maturation of hepatic MCT1 expression occurs with a distinct pattern from typical drug transporters. Expression levels are highest just prior to weaning which may lead to increased hepatic drug uptake during this period. Liver membrane CD147 correlates significantly with membrane MCT1 indicating cellular trafficking is an important mediator of cell surface expression of the functionally active transporter during mammalian development. Further studies will evaluate the underlying regulatory mechanisms contributing to the age‐related changes in MCT1 expression, as well as drug disposition to observe developmental changes in transporter activity. As MCT1 is widely expressed, maturation will be assessed in additional tissues involved in drug disposition.
Docosahexaenoic Acid is More Effective than Eicosapentaenoic Acid in Increasing the Omega‐3 Index Measured in Red Blood Cell Membranes
Authors: Allaire Janie , Harris William , Vors Cécile , Tchernof André , Couture Patrick , Lamarche Benoît
Published: November 2023

Background

Whether eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have distinct effects on cardiometabolic risk remains unclear as most studies to date have used a mixture of the two fatty acids in various forms and proportions. The Omega‐3 Index (O3I) reflects the sum of EPA and DHA as percent of fatty acids in red blood cell (RBC) membranes and has been inversely associated with the risk of coronary heart diseases and coronary mortality in epidemiological studies. The objective of this study was to assess if supplementation with high dose EPA differentially modify the O3I compared with high dose DHA in men and women at risk of cardiovascular diseases.

Methods

Using a randomized double‐blind controlled crossover design, 48 men and 106 women with abdominal obesity and subclinical inflammation were randomized to a sequence of three treatment phases: 1–2.7g/d of EPA, 2–2.7 g/d of DHA, 3‐ and 0g/d of EPA+DHA (corn oil). All supplements were provided as 3×1g capsules for a total of 3g/d. Treatment phases had a duration of ten weeks each and were separated by nine‐week washouts. RBC membrane fatty acid composition was measured at baseline and the end of each phase. Differences in RBC membrane fatty acid composition and in the O3I between treatments were assessed using mixed models for repeated measures. Potential confounders of the response to treatments such as weight, sex, age and O3I baseline values were considered.

Results

The increase in the O3I after DHA (+87% vs. control, p<0.0001) was significantly greater than after EPA (+52% vs. control, p<0.0001; p<0.0001 between DHA and EPA). There was a significant sex*treatment interaction (p=0.046) in the O3I response to EPA and DHA, with similar O3I changes among men and women in response to EPA (0% difference between sexes), but slightly greater O3I increase after DHA among men (+9% compared with women). EPA supplementation increased docosapentaenoic acid (DPA) proportions in RBCs (+84% vs. control, P<0.0001) while DHA decreased DPA in RBCs (−28% vs. control, p<0.0001; p<0.001 between DHA and EPA).

Conclusions

High‐dose DHA may be more effective than high dose EPA in increasing the O3I, perhaps even more so among men than among women. Because DPA levels are not accounted for in the O3I, in vivo elongation of EPA to DPA may partly explain the difference between EPA and DHA in modulating the O3I. Long‐term intervention studies are needed to determine how this difference relates to cardiovascular risk in men and women.

Support or Funding Information

This study was supported by a grant from the Canadian Institutes for Health Research (CIHR, MOP‐123494). Douglas Laboratories provided the EPA, DHA and control capsules used in this study. Janie Allaire is a recipient of a PhD Scholarships from the CIHR and Fonds de recherche du Québec ‐ Santé.
Greater Stimulation of Postexercise Muscle Protein Synthesis after Consumption of Whole Eggs versus Egg Whites in Healthy Young Men
Authors: van Vliet Stephan , Shy Evan L. , Beals Joseph W. , Ulanov Alexander V. , Li Zhong , Paluska Scott A. , Moore Daniel R.
Published: November 2023

The majority of protein consumed in the diet is obtained from whole food sources that contain a mixture of macro‐ and micronutrients. Whereas the majority of research investigated the effect of isolated protein sources on protein metabolism, our aim was to assess the muscle anabolic potential of consuming a complete protein within its naturally occurring, nutrient dense food matrix during recovery from resistance exercise in young men. In crossover trials, 10 healthy resistance‐trained men (21±1 y; 88±3 kg; bodyfat: 16±1%) received primed continuous L‐[ring‐2H5]phenylalanine and L‐[1‐13C]leucine infusions. Repeated blood and muscle biopsies were collected before and after a single bout of resistance exercise (4 sets of 10 repetitions at 80% of 10‐RM for both leg press and leg extensions). Immediately after resistance exercise, participants ingested intrinsically L‐[5,5,5‐2H3]leucine labeled whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat) cooked in scrambled form. Exogenous leucine appearance rates increased (P<0.001) after protein ingestion with peak values of 513±57 nmol leucine·kg−1·min−1 occurring at 75 min after egg white ingestion compared with 496±38 nmol leucine·kg−1·min−1 at 120 min after whole egg ingestion. The total amount of protein derived leucine that became available in circulation over the 300 min postprandial period was similar (P=0.53) between whole egg (75±2.3%) and egg white ingestion (77±1.7%). Whole egg ingestion supported greater post‐exercise myofibrillar protein synthesis rates when compared to egg white ingestion (0.034%/h and 0.024%/h respectively; P=0.02). We conclude that whole egg ingestion stimulates the muscle protein synthetic response more effectively than the consumption of isonitrogenous amounts of egg whites during recovery from resistance exercise in healthy young men. This effect is not attributed to divergent postprandial protein‐derived amino acid availability, suggesting that some intrinsic factor within whole eggs and/or the divergent macronutrient profile may have modified the anabolic potential of this complete protein.

Support or Funding Information

Vision 20/20 Grant, Division of Nutritional Sciences, University of Illinois, Urbana‐Champaign
Impact of Home Fortification of Complementary Foods Program on Child Anemia and Stunting in Bihar, India
Authors: Young Melissa Fox , Mehta Rukshan , Gosdin Lucas , Kekre Priya , Verma Pankaj , Larson Leila , Girard Amy Webb , Ramakrishnan Usha , Chaudhuri Indrajit , Srikantiah Sridhar , Martorell Reynaldo
Published: November 2023

Home fortification of complementary foods with multiple micronutrient powders (MMP) is recommended to reduce child anemia in resource‐poor settings. However, there is a lack of program effectiveness data in Bihar, India to guide policies. We conducted a cluster randomized effectiveness trial in West Champaran, Bihar in over 4000 children aged 6–18 months for a duration of one year, within the context of an ongoing program (CARE, India: Integrated Family Health Initiative) delivered by community front line workers. 70 health sub‐centers were randomized to intervention (MMPs and Infant and Young Child Feeding (IYCF) counseling) or control (only IYCF counseling). The MMPs included a daily RDA of iron, zinc, iodine, folic acid, and vitamins A, C and B12, for children. The study aimed to determine impact on measures of nutritional status (anemia, stunting, underweight and wasting) and child feeding practices and inform potential statewide scale‐up. A secondary objective was to examine potentially adverse outcomes (diarrhea, hospitalization and fever). We used a difference in difference approach using cross‐sectional baseline and endline surveys to evaluate impact and adjusted for caste, socioeconomic status, age, gender, maternal education, young mother status, and religion. The duration of the intervention was one year and at baseline, 72% of children were anemic and 33% were stunted. Although 86% of caretakers indicated that they would like to continue using the powders, only 38% of children had consumed MMPs in the last month and 24% were currently consuming MMP at endline. Anemia declined significantly by 7.8% (6.7% in adjusted models; p<0.05) with a greater decline among children 12–18 months (9.3%, p<0.05). There were no overall effects on the prevalence of stunting, underweight or wasting but there was a significant decline of 8.4% in stunting for children aged 12–18 months (7.2% in adjusted models; p<0.05). There were no significant differences in IYCF (age of initiation of complementary feeding, feeding frequency, dietary diversity or minimum acceptable diet). There were no differences in severe morbidity (bloody diarrhea, severe diarrhea, persistent diarrhea, hospitalizations or fever) and there was a significant 4% decrease in the prevalence of any diarrhea self‐reported in past 2 weeks in the intervention communities. In conclusion, home fortification of complementary foods is a promising intervention associated with modest improvements in anemia and possibly stunting within the existing government run program context in Bihar, India.

Support or Funding Information

Funding support provided by a BMGF grant through a subcontract with CARE‐India
The Role of Metastasis‐Associated Protein 1 (MTA1) in Breast Cancer Exosome‐Mediated Intercellular Communication
Authors: Hannafon Bethany N. , Gaskill Katy , Calloway Cameron , Ding Wei‐Qun
Published: November 2023

Background

Exosomes are small vesicles that mediate cell‐to‐cell communication and are thought to contribute to tumor progression and metastasis by modifying the tumor microenvironment at both local and distant sites. We recently identified Metastasis‐Associated Protein 1 (MTA1) in exosomes secreted from breast cancer cells, ductal fluids, and breast cancer patient plasma samples. MTA1 is a nuclear co‐regulatory molecule that is highly expressed in many cancers and correlates with tumor metastasis and progression. However, the precise role of MTA1 in breast cancer progression and metastasis is unclear.

Objective

Our objective was to determine the role of MTA1 in exosome‐mediated signaling in the tumor microenvironment of breast cancer.

Methods

An antibody array was used to identify MTA1 in breast cancer exosomes. Exosome‐MTA1 expression was confirmed by western blot. tdTomato‐tagged MTA1 expression and transfer were monitored by fluorescent microscopy and flow cytometry. Luciferase reporter constructs were used to monitor gene expression regulation by exosome‐MTA1 transfer. The CRISPR/Cas9 technique was used to knockout MTA1 in breast cancer cells. Endothelial cell tube‐formation assays were performed to ascertain the role of exosome‐MTA1 in angiogenesis.

Results

MTA1 protein expression was detected in exosomes derived from breast cancer cells, ductal fluids, and breast cancer patient plasma samples. MTA1‐tdTomato expression and exosome‐mediated transfer of MTA1 was observed by fluorescent microscopy and flow cytometry. Importantly, we found that exosome‐MTA1 regulates hypoxia signaling via exosome‐transfer between breast cancer and endothelial cells. Furthermore, exosome‐MTA1 modified the tube formation by endothelial cells.

Conclusions

This is the first report showing that breast cancer exosomes contain MTA1 and that exosome‐MTA1 alters signaling events in the tumor microenvironment. These observations suggest that exosome‐mediated transfer of MTA1 contributes to breast cancer progression and exosome‐MTA1 may serve as a biomarker and therapeutic target for breast cancer.

Support or Funding Information

Grant Support: Oklahoma Center for the Advancement of Science and Technology (HR14‐147) and the Oklahoma Shared Clinical and Translational Resources (NIGMS #U54GM104938).
Revisiting the Evolution of Low Trabecular Bone Density in Modern Humans
Authors: Chirchir Habiba , Dean Hannah , Carlson Kristian , Ruff Christopher
Published: November 2023

Evidence suggests that recent modern humans have low trabecular bone density (i.e., bone volume fraction) compared with other primates and fossil hominins. This has been linked to the increased sedentism observed at the beginning of the Holocene with the advent of agriculture. Additionally, among recent humans, sedentary agricultural populations have been shown to have lower density skeletons compared with foragers. However, these findings relied primarily on either comparisons between sedentary populations and fossil hominins, or between limited samples of agricultural and foraging populations. Thus, the extent to which trabecular bone density in recent human populations with varying subsistence strategies (i.e., farmers, foragers, and populations transitioning from foraging to farming) differs from that of different fossil hominins, and, in turn, how hominins differ from extant anthropoids is unclear. Therefore, we test the hypothesis that populations with subsistence strategies demanding high physical activity will exhibit greater trabecular density than sedentary populations, and will be more similar to fossil Homo, and australopiths will be more similar to anthropoids due to the greater mechanical loading on their skeletons. We measure trabecular density using pQCT and microCT in lower and upper limb joints in recent modern humans (five populations), anthropoids (Chlorocebus aethiops, Papio anubis, Pongo pygmaeus, Pan troglodytes), and fossil hominins (Australopithecus africanus, Australopithecus sediba, Paranthropus robustus, Homo neanderthalensis, and early Homo sapiens).

The results show that in the lower limb joints, there is a significant and uniform decline in trabecular density among all recent humans in the Holocene, while fossil hominins and extant anthropoids show similar, higher trabecular density values. In contrast, in the upper limb, there is no significant shift in trabecular density among humans; active recent human populations display trabecular density that is similar to the fossil Homo. In addition, some anthropoids are not significantly greater in density than the active recent human populations. These results suggest that trabecular density of the lower limb only reflects a reduction in mobility as humans became more sedentary throughout the Holocene. The lack of a significant decline in trabecular density in the upper limb in recent humans may reflect the active use of the arms and hands that is needed for food production and processing even among sedentary populations. While the results support the recent evolution of low trabecular density in modern humans resulting from reductions in activity levels in the Holocene, it is important to compare multiple skeletal elements across a diverse set of recent modern humans with varying subsistence strategies when drawing conclusions about changes in bone density in the human skeleton.
Association of Snacking Frequency with Patterning of Daily Energy Intake and Weight Among US Toddlers and Preschoolers
Authors: Fisher Jennifer Orlet , Davey Adam , Kachurak Alexandria , Bailey Regan K
Published: November 2023

Snacking is nearly universal among young children but associations with weight status are not well studied especially among young children for whom snacks are believed to be important for growth. The objective of this research was to evaluate the cross‐sectional association of snacking frequency with weight status and daily energy intake among 4,271 US toddlers and preschoolers aged 12–71 months participating in the 2007–2014 National Health and Nutrition Examination Survey. Snacking and nutrient intakes were estimated using the mean of 2, 24‐hr dietary recalls. Overweight among toddlers was defined as weight‐for‐length ≥ 97.7th percentile and overweight/obesity among preschoolers was defined as BMI z‐score ≥ 85th percentile. Models used American Academy of Pediatrics recommendations of 2–3 snacks/d as the reference group; data were adjusted for survey design and sample weights, demographics and energy reporting accuracy. In the total sample, 8.7% of children 12–23 mo and 23.8% of children were overweight or obese. Children consumed, on average, 28% (419.3 ± 6.1 kcal) of daily energy from snacks eaten at 2.91 ± 0.03 occasions/d. Children consuming 4+snacks/d (n=610, 13.5%) had greater odds of being OW/OB (OR=1.50, 95CI=2.09–0.02, p<0.05), consumed slightly greater daily energy (Mean (SE)= 1495 (14.0) vs.1462 (4.9), p<0.05), and consumed a greater proportion of daily energy from snacks (42% (0.7) vs. 27% (0.5), p<0.0001) than children consuming 2–3 snacks/d (n=2189, 55.1%). Risk of OW/OB and energy intake among children consuming 0–1 snacks/d did not differ from children consuming 2–3 snacks/d in the full sample. Among preschoolers, however, those children consuming 0–1 snacks/d had a lower risk of OW/OB than children consuming 2–3 snacks/d (OR=0.75, 95CI=0.57–0.98, p<0.05). These findings provide first evidence of associations between snacking frequency and weight status among young US children < 5 years. These findings indicate that US toddlers and preschoolers who snack more frequently than recommended guidelines may consume greater daily energy and be at elevated risk of OW/OB than those who do not. That preschoolers who consumed fewer than 2–3 snacks/d had the lowest risk of OW/OB points to potential age‐related changes in the role of snacking in healthy weight. Longitudinal and experimental studies are needed to evaluate causal influences of snacking on appetite and growth in early childhood.

Support or Funding Information

NIH R21 HD085137‐01A1
A Child‐Administered Food Security Scale is Associated with Household Socio‐Economic Status, Household Food Security and Diet Diversity
Authors: Jamaluddine Zeina , Choufani Jowel , Sahyoun Nadine R , Ghattas Hala
Published: November 2023

Recent studies have shown that children are able to accurately describe their own experiences of food insecurity, however few studies have quantified this experience, and none have done this in the Middle Eastern context. Informed by a previous qualitative analysis of child food insecurity in Lebanon, we developed a set of items to quantify child food insecurity experience, aiming to develop and validate an Arabic child food security scale (CFSS).

Fourteen items were developed and administered as part of a study on food security and nutrition in schools, to 6–16 year old children attending four UNRWA schools for Palestinian refugee (n=1381). We used statistical methods based on Item Response Theory (Rasch model) to assess internal validity of the proposed CFSS. Further validation of the scale was conducted by examining associations through linear and logistic regression with socio‐demographic, economic, household food security and diet diversity variables. STATA 13 and R 3.1.3 were used for data analysis.

Three items were dropped from the scale due to high infit statistics and high residual correlation with other items. The resulting 11‐item scale had acceptable reliability (0.77), sensitivity (0.86) and specificity (0.96). CFSS was correlated with household food security score (p<0.001). When categorized into food secure (score 0–2) and food insecure (score 3+), there was 63.6% alignment between child food insecurity and household food insecurity. Food insecure children came from households with lower income (p=0.02) and expenditures per capita (p<0.001), higher household crowding (p=0.004), had less educated mothers (p<0.001), lower household diet diversity (p=0.001) and lower household consumption of meat (p<0.001) and fruit (p<0.001).

Child food security in Palestinian refugee schoolchildren in Lebanon was accurately measured through an 11‐item questionnaire and was associated with household socio‐economic status, household food security and diet diversity. This tool is appropriate to assess child food insecurity experience directly with children, and can be used in school‐based interventions, where obtaining data from parents may be cumbersome.

Support or Funding Information

This research was funded by The Nestlé Foundation for the Study of Problems of Nutrition in the World
Zip14‐Mediated Zinc Transport Contributes to Regulation of Glucose Homeostasis in Intestine, Pancreas and Liver
Authors: Aydemir Tolunay Beker , Kim Min‐Hyun , Cousins Robert J.
Published: November 2023

Zinc influences signaling pathways through controlled targeted zinc transport. ZIP14 is a SLC39 family member zinc transporter which traffics zinc into the cytoplasm. We have previously shown that ZIP14 was the most up‐regulated transporter in models of liver inflammation. Using a Zip14 knockout (KO) mouse model, we further demonstrated that ZIP14 was essential for controlling signaling events for glucose homeostasis and inflammation.

Metabolic endotoxemia is one of the major factors contributing to obesity and diabetes, two of the most prevalent metabolic disorders with incidence that is increasing rapidly worldwide. Zip14 KO mice present with signatures of metabolic endotoxemia including elevated serum endotoxin levels due to impaired intestinal barrier function leading to low grade chronic inflammation; increased cytokine expression by metabolic tissues; increased production/secretion of leptin with insulin insensitivity in adipose tissue; and enlarged pancreatic islets with greater serum insulin levels through activation of the MYD88 pathway. In Zip14 KO mice, elevated serum endotoxin levels; insulin insensitivity in adipose tissue; and greater biosynthesis and secretion of insulin from pancreas were returned to control level when mice were treated with antibiotics.

Hyperglycemia in type 2 diabetes and obesity is caused by insulin resistance. Insulin resistance results in inhibition of glucose uptake by liver and other peripheral tissues, principally adipose and muscle and with concurrently higher hepatic glucose production. We demonstrate that during glucose uptake the cell surface abundance of zinc transporter ZIP14 and mediated zinc transport increases. Zinc is distributed to multiple sites in hepatocytes through sequential translocation of ZIP14 from plasma membrane to early and late endosomes. Endosomes from Zip14 KO mice contained zinc but were zinc deficient as activities of the zinc‐dependent insulin degrading proteases, insulin degrading enzyme and cathepsin D, were impaired, hence insulin receptor (IR) activity increased. Transient increases in cytosolic zinc levels are concurrent with glucose uptake and suppression of glycogen synthesis. In contrast, Zip14 KO mice exhibited greater hepatic glycogen synthesis and impaired gluconeogenesis and glycolysis related to low cytosolic zinc levels.

In conclusion, ZIP14‐mediated zinc transport contributes to regulation of intestinal barrier function, biosynthesis and secretion of insulin by pancreas and endosomal IR activity and glucose homeostasis in hepatocytes. Therefore, modulation of ZIP14 transport activity presents a new target for management of diabetes and other glucose‐related disorders.

Support or Funding Information

NIDDK Grant RO1 DK94244
Hepatic Fibrosis is Independent of the Effects of Endotoxin (Lipopolysaccharide) on Hepatic Stellate Cells
Authors: Gandhi Chandrashekhar R
Published: November 2023

Background

Binding of Gram‐negative bacterial endotoxin lipopolysaccharide (LPS) to CD14 is critical for its cellular effects via TLR4. Activation of hepatic stellate cells (HSCs) from its quiescent phenotype to α‐smooth muscle actin (α‐sma)‐expressing fibrogenic and proliferative myofibroblast‐like phenotype is the major mechanism of liver fibrosis of various etiologies. LPS/TLR4 interactions in HSCs were reported to play an essential role in hepatic fibrosis in experimental murine models. However, LPS was found to induce synthesis of nitric oxide and inflammatory cytokines in HSCs without the aid of CD14 or TLR4, suggesting that CD14/TLR4‐independent pathways may play an important role in hepatic pathophysiology. To address this hypothesis, we investigated effects of LPS on HSCs from CD14‐knock‐out (KO) mice and carbon tetrachloride (CCl4)‐induced fibrosis in them.

Methods

HSCs derived from wild type (WT) or CD14‐KO mice were stimulated with LPS (100 ng/ml) following their culture‐induced activation. Parameters of fibrogenic activity (expression collagen, α‐sma and fibrogenic cytokine TGFβ1and its receptor) and inflammatory cytokines/chemokines were measured. In vivo, mice were subjected to CCl4 treatment for 4 weeks, and then administered PBS or LPS (5 mg/kg; ip). Liver injury, inflammation and fibrosis were determined.

Results

LPS‐stimulated TNFα expression was greater, IL6 expression similar and IL10 expression lower in CD14‐KO HSCs compared to WT HSCs. Expression of the chemokines Ccl2, Cxcl1 and Cxcl9 also increased similarly in LPS‐stimulated CD14‐KO and WT HSCs. LPS down‐regulated Acta2 (encodes α‐sma), TGFbr1 and collagen 1 expression in both WT and CD14‐KO HSCs, and the inhibitory effects of LPS on α‐sma and collagen 1 in HSCs were not reversed by exogenous TGFβ1. In vivo, CCl4 treatment induced similar liver injury (histology and serum ALT) and fibrosis (Sirius red staining and hydroxyproline content) in WT and CD14‐KO mice. However, administration of LPS following CCl4 treatment increased liver injury and inflammation in WT mice but not in CD14‐KO mice. Essentially similar results were obtained when TLR4‐KO mice were treated with CCl4 and HSCs from TLR4‐KO mice were challenged with LPS.

Conclusion

These data suggest that the direct effects of LPS on HSCs are not essential for hepatic fibrosis. In contrast, LPS may play an important role in limiting fibrosis by down‐modulating the critically responsible components in HSCs.

Support or Funding Information

This work was supported by a VA Merit Review Award 1IO1BX001174.
QUANTIFICATION OF SUBSTANCE P IN DORSAL ROOT GANGLION NEURONS IN YOUNG AND ADULT RATS, AFTER NOCICEPTIVE STIMULATION DURING THE NEONATAL PERIOD
Authors: Simoes Ana Leda Bertoncini , Silva Greice Anne Rodrigues , Carmo Elisabeth Cassia , Giorgetto Carolina , Fazan Valeria Paula Sassoli
Published: November 2023

Substance P (SP) is widely used as a marker for peripheral neurons with unmyelinated fibers. Understanding the plasticity in peripheral pain pathways, even before the stimulus reach the central nociceptive areas is crucial, particularly when noxious stimuli are applied to neonates, when the nervous system is still under remodeling process. We quantified SP immunostained neurons in the dorsal root ganglion (DRG) of male and female Wistar rats 15 and 180 days after nociceptive stimulation [1] during the neonatal period. Right and left L5 DRG were removed, frozen and 8 μm thick criossections were incubated in rabbit polyclonal anti‐substance P antibody (1:5.000; 24hr). Immunostaining protocol was used as described [2]. Labeled neurons were counted and compared between ages, genders and groups. Differences were considered significant if p<0.05. Gender differences were present in both ages, being the number of SP positive neurons in the DRG larger in males on both sides, in animals 15 days old. After 6 months, males showed a larger number of SP positive neurons only on the right side that received the nociceptive stimulus, compared to females. An increased number of SP positive neurons in the DRG of the stimulated side in females was present, immediately after nociceptive stimulation but not after 6 months. In conclusion, neonatal noxious stimulation caused a permanent increase in the SP positive neurons in males that was not observed in females, suggesting that differences in pain sensitivity between genders could be related to morphological alterations of the nervous system.

Support or Funding Information

FAPESP, CNPq, CAPES and FAEPA
Studying interaction of the immune and vascular system using intravital multiphoton microscopy
Authors: Zinselmeyer Bernd H , Saunders Brian T , Huang Li‐Hao , Runci Daniele , Williams Jesse W , Randolph Gwendalyn J
Published: November 2023

With the beginning of the new millennium intravital multiphoton imaging of the immune system has become a necessary tool for understanding cellular behavior in steady state and models of disease, in vivo. The first studies visualized dynamics of antigen presenting cells and T‐cells. Followed by development of fluorescent reporter mice that allowed tracking of neutrophils, monocytes and macrophages, however the spatiotemporal resolution achieved wasn't sufficient enough to study physiological parameters like blood or lymph flow.

We have improved our two‐photon imaging approach by decreasing photo toxicity and generated a combination of fluorophores which are low in cytotoxicity, able to label morphological structures and cell populations of interest. This combination of dyes is based on high excitation efficiency capable to be excited with low laser power and bright emission properties. Together with our customized dual femtosecond pulsed laser system (InSight DS+ together with an Mai Tai DeepSee) featuring an ultra‐fast resonance scanner and sensitive hybrid detectors, we can image up to five fluorophores in intravital settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo examining changes in flow dynamics and cellular interaction (Fig 1).

For decades blood vasculature has been studied extensively, but our understanding of lymphatic functions remains poor. Lymphatics interact systemically and locally with the blood vasculature (Fig 2) and using these technique we have recently shown that dendritic cells are an important regulator in lymphatic function (Ivanov et. al. J Clin Invest. 2016 Apr 1;126(4):1581–91.). Many immune‐vasculature interactions can only be proven in the living animal, showing the interactions and impact of immune cells on vascular flow dynamics and vice versa. Our most recent intravital imaging studies allow the visualization of arterial resident macrophage dynamics within atherosclerotic plaque. Additionally, we are utilizing our intravital vascular imaging approaches to address the challenge of determining the role of distinct subsets of myeloid cells in glomerular disease and in murine tumor models (Fig 3). Furthermore, we are hoping these studies help to identify functional interactions of the vasculature and different immune cell populations as therapeutically targets for inflammatory diseases and for the enhancement of cancer immune therapy.

Support or Funding Information

BHZ was awarded a Scientist Development Grant (16SDG30480008) from the American Heart Association (AHA) 07/01/16‐06/30/19
Efficacy of Undergraduate Medical Education in Anatomical Sciences for Surgical Residency Preparation
Authors: Klaus Rachel , Lee Lisa
Published: November 2023

Undergraduate medical education has transformed over the past decades into a more integrated, clinically oriented, and team‐based curricula, guided by the core competencies. Contact hours for foundational basic sciences have seen a steady and drastic decrease. The implications of these changes on preparedness of interns for graduate medical education have been of concern to residency program directors for some time. The purpose of this study was to assess preparedness of interns starting a surgical residency program, in terms of basic sciences knowledge and other core competencies. Two 18‐item surveys were created in Qualtrics; one for faculty and one for residents. The survey assessed perceived importance of basic sciences to the discipline, areas of deficiency, and changes in preparedness and quality of interns over the past 10 years. The survey was distributed to all surgical residency programs in the U.S. Survey results were compared between the two cohorts and comments were thematically organized and analyzed for emerging patterns. Faculty and residents from over 25% of U.S. surgical residency programs completed the survey. For all items assessing intern preparedness level at the onset of the program, residents consistently rated themselves as more prepared than faculty did. Majority of the faculty reported a decline in preparedness of incoming residents over the last decade; listing deficiencies in basic skills, basic sciences knowledge, clinical experience, and independence. Residents and faculty both identified interns' deficiencies in integrating foundational knowledge with clinical practice and in effective interaction with, and treatment of patients. More residents than faculty identified basic sciences as the most valuable subject a surgical intern should acquire in medical school. More faculty than residents identified competencies such as interpersonal skills, self‐directed learning, critical thinking, and exposure to clinical practice as the most valuable skill or subject to acquire during medical school. In terms of basic sciences content, both cohorts rated anatomy, physiology, and microbiology as the most important subjects for success in surgical residency. Biochemistry, embryology and histology were rated higher by faculty than residents. The results of this study report a perceived decline in preparedness of surgical residency interns in the past decade. Both faculty and residents report the importance of basic sciences education in medical schools but faculty placed more emphasis on other core competencies. These findings suggest further evaluation of the changing medical curricula is necessary to ensure that students acquire knowledge and skill sets necessary to be successful in graduate medical education.
Remarkable functional convergence: Type I and II toxin‐antitoxins induce multidrug tolerance by (p)ppGpp‐dependent mechanisms
Authors: Gerdes Ken
Published: November 2023

Using single‐cell technology, we showed previously that, in Escherichia coli, the ubiquitous bacterial stress alarmone (p)ppGpp (Magic Spot) is a central regulator of both spontaneous and environmentally induced persistence1. The (p)ppGpp level varied stochastically in a population of exponentially growing cells and the high (p)ppGpp level in the rare cells induced persistence. Persister cell formation depended on 10 type II toxin – antitoxin (TA) modules encoding RNases that reversibly inhibit translation by cleavage of mRNA or rRNA2.

More recently, another research group showed that a type I TA module (hokB/sokB) of E. coli can induce persistence by a mechanism that also depends on (p)ppGpp and, and surprisingly, the highly conserved GTPase Obg3. Type I TAs encode small proteins that depolarize the cell membrane and confer membrane damage and rapid cell killing when overexpressed. By contrast, moderate expression of Hok toxins depletes the ATP pool and induce drug tolerance4. Expression of these highly toxic proteins is repressed by cis‐acting antisense RNAs5. A complex folding pathway allows the mRNA to escaping irreversible inactivation by the antisense and expression of the toxin in the absence of transcription6,7. However, it is not yet known how (p)ppGpp and Obg induce HokB expression and how the cell recover from Hok‐induced ATP‐depletion and stasis.

Nevertheless, these results together reveal Magic Spot as the central regulator and toxin ‐ antitoxins as the central effectors of persister cell formation in E. coli.

Support or Funding Information

This work was supported by the Danish National Research Foundation (grant identifier DNRF120), a Novo Nordisk Foundation Laureate Research Grant and the ERC Advanced Investigator Grant PERSIST (294517).
Daily Mango (Mangifera Indica L.) Consumption for 42 Days Differentially Modulates Metabolism and Inflammation in Lean and Obese Individuals
Authors: Fang Chuo , Kim Hyemee , Barnes Ryan , Talcott Stephen T , Mertens‐Talcott Susanne U
Published: November 2023

Excess weight and body fat increase the risk of developing multiple medical conditions, including Type 2 Diabetes, cardiovascular diseases, and cancer, which pose major threats to public health. Diets rich in fruits and vegetables have been recommended for preventing metabolic disorder and cardiovascular disease. Mangoes are reported to have high concentrations of polyphenols that exert potent anti‐inflammatory activities relevant to the treatment of chronic diseases and cancer, as demonstrated in preclinical studies. However, the number of human clinical trials examining the metabolic effects of mango polyphenols is limited. This study was carried out to investigate the effects of daily mango consumption for 42 days in lean and obese individuals. In this study, 21 healthy lean (BMI 18–25kg/m2) and obese (BMI>30kg/m2) subjects aged 18–55, were continuously provided with 400g of mango pulp for 42 days. Inflammatory cytokines, metabolic hormones, and lipid profiles were examined in plasma at a baseline and after 42 days of treatment. A three‐day food record was filled out for dietary assessment prior to each clinical visit. Results revealed that 42 days of mango consumption decreased systolic blood pressure (mean ± SD: 119.83±13.16 vs. 115.42±12.33; p<0.05) in lean subjects, but had no significant effect in obese subjects. The level of hemoglobin A1c was improved significantly in obese but not lean subjects. Reduced expression of PAI‐1, associated with reduced risk of atherosclerosis and thrombosis, was observed in both groups (In lean group, mean ± SD: 30.93±18.12 vs. 23.69±17.58; p<0.1; In obese group, mean ± SD: 31.34±8.09 vs. 24.93±12.20; p<0.05). There was a non‐significant trend towards lowered levels of C‐reactive protein, and elevated levels of IL‐10 in both groups. No significant changes were observed in lipid profiles for either group. The consumption of mango did not improve the plasma levels of adipokines. In conclusion, daily mango consumption lowers blood pressure in lean individuals, and benefits obese individuals by maintaining long‐term glucose homeostasis. Galloyl‐derivatives from mango may possess therapeutic potential in the prevention and treatment of obesity and metabolic disorders, which remain to be confirmed in a larger‐size human clinical trial.

Support or Funding Information

Partial funding for this research was provided by: National Mango Board
Dairy Products and Iodine‐Containing Supplements Use are Positively Associated with Iodine Status in Childbearing Age Women in the United States
Authors: Arsenault Michelle Ann , Lee Kyung Won , Song Won O.
Published: November 2023

Iodine is a critical nutrient in the synthesis of thyroid hormone in the body. Although recommended dietary intake of iodine has been established, food composition data of iodine are not available and thus iodine status in population is determined by urinary iodine concentration (UIC). By using food group‐based approach, we reported that dairy products are positive contributors to adequate iodine intake in general adult population in the US. Inadequate iodine status during pregnancy is serious due to fetal neurodevelopment that begins the first trimester of pregnancy. However, little is known on dietary determinants of iodine status in childbearing age women of different race/ethnic groups whose intake of dairy products vary widely. We aimed to describe iodine status and its determinants in childbearing age women of different race/ethnic groups and their associations with dairy product intake. Data from the National Health and Nutrition Examination Survey 2003–2012 were utilized. A total of 2,722 non‐pregnant and non‐lactating women of childbearing age (15–44 years) with information on UIC, single 24‐hour dietary recall, iodine‐containing dietary supplements, salt use, and sociodemographic characteristics were included. Iodine status was assessed by two different WHO criteria (median UIC and the prevalence of UIC <50 μg/L) from single spot urine sample. Multivariable linear regression and logistic regression analyses were conducted after adjusting for covariates. Iodine status of childbearing age women had borderline iodine adequacy (median UIC: 126.6 [117.6–135.6] μg/L). Childbearing age women in the highest tertile of dairy product consumption had lower odds of UIC <50 μg/L (adjusted odds ratio=0.52; 95% confidence interval: 0.30–0.91), compared with those in the lowest tertile. An increase in dairy product intake by 100 g/day led to an increase in UIC by 5% (P <0.01). Taking iodine‐containing supplements and using salt in food preparation were positively associated with UIC (all, P <0.05). However, those older, non‐Hispanic blacks, and highly educated tend to have a lower UIC than their counterparts (all, P <0.05). We found the major driving forces for improving UIC among US childbearing age women were dairy products and dietary supplements with iodine. Childbearing age women should be advised to increase dairy food consumption and iodine‐containing supplement use for achieving adequate iodine intake during subsequent pregnancies. Further studies are needed to identifying dietary and behavioral determinants of iodine status among pregnant and lactating women.

Support or Funding Information

This study was conducted by the generous financial support of the College of Agriculture and Natural Resources Undergraduate Research Program at Michigan State University.