bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repurposing P‐glycoprotein Inhibitors as Modifiers of Sphingolipid Metabolism— Therapeutic Implications in Cancer
Authors: Cabot Myles C 
Published: November 2023

Chemotherapy efflux transporters such as P‐glycoprotein (P‐gp), a major player in multidrug resistance, remain attractive targets in laboratory and clinical studies in cancer therapy. Research in this area has largely been directed towards limiting the efflux capacity of these pumps with the goal of attaining lethal chemotherapy concentrations within tumor cells. One common avenue for reducing chemotherapy efflux is via the introduction of P‐gp inhibitors; however, whereas this goal remains promising, clinical use of a myriad of inhibitors has provided little progress over decades of trials. Herein we reveal that classical first, second, and third generation P‐gp inhibitors, in the single digit micromolar range, block sphingolipid (SL) metabolism at the juncture of ceramide glycosylation, a prominent ceramide detoxification pathway in cancer cells; this action effectively curbs the tumor‐suppressor properties of ceramide. Herein we sought to determine whether P‐gp inhibitors could be repurposed to circumvent the blunting effects elicited by ceramide glycosylation. Our work demonstrates that co‐administration of a ceramide therapeutic with a P‐gp inhibitor results in synergistic tumor cell killing. For example, in instances wherein short‐chain ceramides (C6‐ceramide) were supplied to the culture medium or when ceramide‐generating drugs like fenretinide were used, the inclusion of P‐gp inhibitors such as tamoxifen, cyclosporin A, biricodar, tariquidar, and the high‐affinity, specific P‐gp inhibitor zosuquidar, effectively depressed ceramide glycosylation in cancer cells by as much as 85%, resulting in magnification and perpetuation of ceramide's cancer‐suppressor activities. The C6‐ceramide‐P‐gp‐inhibitor‐containing regimens compared to the single agents, were more effective in inducing apoptosis in cellular models of human acute myeloid leukemia, including multidrug resistant HL‐60/VCR cells, colorectal, ovarian, and prostate cancer, and melanoma, with upstream mechanisms of action that enlisted mitochondrial dysfunction, ROS generation, inhibition of glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins. Although multidrug resistance modulators, when paired with chemotherapy drugs, have demonstrated little success clinically, the data herein suggest that repurposing these agents as modifiers of SL metabolism in ceramide‐centric drug regimens could provide novel therapeutic approaches, independent of drug resistance status, in treatment of cancer.Support or Funding InformationNational Institutes of Health (NCI) grant number 5 P01 CA171983
Phosphatidylinositol 3,5‐Bisphosphate acts as a Novel Regulator of Calcium Transport During Saccharomyces Cerevisiae vacuolar fusion
Authors: Miner Gregory E. , Fratti Rutilio A. 
Published: November 2023

Phosphoinositides (PIs) are a group of lipids that are differentially phosphorylated to provide specific protein‐lipid interactions that are necessary in maintaining eukaryotic homeostasis. In membrane traffic, different PIs can interact with nucleotide exchange factors, tethering molecules and SNARE proteins ultimately leading to membrane fusion and transfer of cargo material. Our work has shown that phosphatidylinositol 3‐phosphate (PI3P), PI4P and PI(4,5)P2 are essential for the fusion of yeast vacuoles/lysosomes. However, little is known about the role of PI(3,5)P2 in the fusion pathway. Others have shown that PI(3,5)P2 is necessary for fission to occur during osmotic shock that is coupled to the efflux of vacuolar Ca2+ into the cytoplasm. This is thought to occur through the interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Ca2+ efflux has also been linked to vacuole fusion and occurs upon the formation of SNARE complexes between partner vesicles. Our data show that increasing levels of PI(3,5)P2 (through the addition of exogenous short chain (diC8) lipid) led to inhibition of vacuolar fusion. Importantly, sub‐inhibitory concentrations of diC8‐PI(3,5)P2 completely blocked Ca2+ efflux without affecting fusion. This suggests that PI(3,5)P2 plays opposing roles in fission, fusion, and Ca2+ transport. It further suggests that the Ca2+ efflux channel used during fusion is different than Yvc1. Finally, these data indicate that Ca2+ efflux is not required for vacuolar fusion and may be a parallel response. To further elucidate the role of PI(3,5)P2 in fusion we examined the effect of inhibiting the PI3P 5‐kinase, Fab1 using the PIKfyve (human homolog of Fab1) inhibitor Apilimod. In accord with our other data, treatment of vacuoles with Apilimod led to enhanced Ca2+ efflux, illustrating that PI(3,5)P2 negatively regulates fusion‐linked Ca2+ efflux, however the mechanism through which this occurs remains unknown. In addition to effects on Ca2+ transport, PI(3,5)P2 has been shown to enhance vacuolar H+ pumping through its association with the V‐ATPase. This is further supported by our data showing that Apilimod inhibited H+ pumping. Taken together we now hypothesize that PI(3,5)P2 may affect the activity of the H+/Ca2+ exchanger, Vcx1, due to its effect on both H+ pumping and Ca2+ efflux.Support or Funding InformationNIGMS GM101132
Monitoring Organelle‐Specific Responses to Amphotericin B in Mammalian Cells and Candida albicans Biofilms
Authors: Tourville Claudia , Rigden Gabrielle , Lewis David , Hartsel Scott 
Published: November 2023

The use of indwelling medical devices such as catheters or dentures in combination with immunosuppressive therapies increase the body's susceptibility to fungal infections as a pathway to life threatening systemic infections. In particular, Candida albicans, an opportunistic pathogen existing in the normal microbial flora can produce serious systemic infections. This species is unique in its ability to exist in two phases: a yeast form and hyphal biofilm. Once it advances into its biofilm formation, Candida albicans is resistant towards drug treatment, making it immensely difficult to treat in clinical settings. Confocal laser microscopy using organelle‐specific fluorescent probes provides an internal view of Candida albicans along the stages of development and drug response. We have developed and deployed a series of our own naphthalimide‐based fluorescent probes targeted to mitochondria, lysosomes, and the Golgi apparatus to monitor organelle vitality in real time. The brightness, chemical and photo‐stability of our organelle probes allows us to image live cells in their appropriate culture media to observe cellular responses to drug treatments. The resistance to photobleaching makes them useful for longer periods of laser exposure as well as 3D imaging. We have investigated the various liposomal as well as micellar formulations of Amphotericin B including Fungizone, Abelcet, and Ambisome. All formulations of Amphotericin B display some level of toxicity, but lipid formulations such as Abelcet and Ambisome have shown a great reduction in human toxicity without significantly sacrificing their antifungal activity. Using our organelle probes in vitro, we are able to monitor the changes in specific organelles in both model human cells and Candida albicans yeast and hyphal cells to pinpoint the intracellular responses to Amphotericin B in both the target and host. Thus far we have found that Amphotericin B, especially Fungizone, leads to depolarization of mitochondria in some human cell lines while having little acute effect on mitochondria in Candida albicans biofilms. Comparable Amphotericin B doses in the forms of Abelcet and Ambisome, on the other hand, seem to allow for the maintenance of mitochondrial membrane potential in human cells as seen by persistent polarization following multiple hours of incubation. We are currently investigating the time‐dependent relationship between organelle vitality and the differing Amphotericin B formulations with all our probes and cell lines to provide insight as to how Amphotericin B affects mammalian, yeast and biofilm cells differently. Gaining this intracellular perspective of cell health will open doors for the optimization of antifungal drugs through pinpointing their effective mechanisms against fungi while minimizing cellular casualties resulting from organelle decline.Support or Funding InformationOffice of Research and Sponsored Programs‐UW‐Eau Claire
A Potential Role for Mitochondrial Ca2+ Uptake During B Cell Activation
Authors: Torres‐Quintanilla Alejandro , González‐Castillo Elena , García‐Rivas Gerardo 
Published: November 2023

Our understanding of the role played by mitochondria during immune cell activation has passed from just an energy‐producing organelle to an active modulator of cell signaling. Ca+2 handling and redox homeostasis are intracellular signals modulated by mitochondria. For example, mitochondrial Reactive Oxygen Species (mROS) have recently been described as signaling molecules that fine‐tune activation pathways by allowing full nuclear translocation of NFAT in T cells. Moreover, mitochondria have received special attention as buffers of cytosolic Ca+2 dynamics during activation, through MCU‐dependent Ca+2 uptake. Here, we suggest that mitochondria participate during B cell activation through Ca+2 overload. Primary murine B cells were purified and subjected to BCR‐dependent activation using anti‐IgM antibody and BCR‐independent activation using thapsigargin. Thapsigargin induces store operated Ca+2 entry by emptying endoplasmic reticulum Ca+2 storage. After 7hrs of stimulation, flow cytometry was used to determine activation through CD69 surface expression and mROS levels using a mitochondrial superoxide fluorescent probe. Mitochondrial Ca+2 content and uptake was determined in permeabilized lymphocytes using Fluo‐4 and Ca+2 Green 5N after activation. We found that after BCR‐independent stimulation, CD69 surface expression was equivalent to activation via BCR. Importantly, pretreatment with a mitochondrial antioxidant reduced CD69 surface expression after stimulation with both anti‐IgM or thapsigargin. This result suggested that mitochondrial Ca+2 uptake occurs during activation to trigger mROS production. Lastly, we found that activated cells show higher contents of mitochondrial Ca+2. Together, these results suggest that B cell activation induces an important uptake of Ca+2 by mitochondria, which might participate in the activation cascade by increasing mROS production.Support or Funding InformationATQ is supported by CONACyT MSc scholarship
Differential Effects of 1,25D3‐MARRS Expression on Mammary Gland Growth and Development Depend on the Vitamin D3 Dose
Authors: Wilkin Allison , Trinh Thao , Edson Michael , Meckling Kelly 
Published: November 2023

1,25 dihydroxyvitamin D3 (1,25D3) is one of the most potent biologically active forms of vitamin D3. Primary activities include regulation of blood calcium and phosphate homeostasis and a plethora of other effects including cell growth inhibition, induction of differentiation, and anti‐cancer effects, depending on the target tissue. Most of the activities of 1,25D3 continue to be ascribed to the classic vitamin D receptor despite the identification of another membrane receptor for 1,25D3. The membrane‐associated rapid response steroid‐binding (MARRS) receptor is receiving more attention as a novel vitamin D receptor working through rapid, non‐genomic pathways. The purpose of this study was to explore the role of MARRS in the mammary gland using a conditional knockout mouse model and a vitamin D3 dietary intervention. 1,25D3‐MARRS expression was reduced in epithelial cells of mammary glands (MG) using the Cre/loxp system. Dams were weaned onto semi‐purified diets with 10,000 IU/kg, 1,000 IU/kg, or 0 IU/kg of 1,25D3 for 4 weeks before being set‐up to breed. 4th MGs were collected from 6‐week old female MMTV‐Cre mice (n=73). MGs were whole‐mounted and stained with carmine alum. Tissue growth was assessed by counting the number of terminal end buds (TEB) of alveolar branches, measuring the length of the longest ductal extension, and measuring total MG area covered ducts. Whole body weight was similar between all groups. There was a significant interaction between genotype and diet. Knockout mice on the 1,000 IU/kg diet had significantly fewer TEBs (p=0.016) and reduced ductal growth and extension (p=0.037, p=0.023 respectively) compared to controls on the same diet. Mice on the 0 IU/kg diet had similar TEB counts across all genotypes. Knockout mice on the 10,000IU/kg diet had significantly more TEBs than the other mice of the same diet (p=0.042), and more extensive growth and coverage of alveolar branches than knockouts on the 1,000 IU/kg diet. These results suggest that there is an effect of 1,25D3‐MARRS that is dependent on vitamin D3 dose. Because the dose‐response is not linear in this animal model, it suggests caution when making recommendations for vitamin D3 nutrition in animals, and possibly humans as well.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada
Vitamin A Sugar Fortification in Zambia: Opportunities and Challenges
Authors: Greene Matthew , Kabaghe Gladys , Musonda Mofu , Palmer Amanda C 
Published: November 2023

Large‐scale food fortification is a major strategy to improve dietary vitamin A (VA) intakes and control VA deficiency. Zambia introduced mandatory sugar fortification with VA, at a target of 10 mg/kg, in 1998; however, there are conflicting reports as to the program's impact on VA status. Our objective was to assess the current state of the program from the policy level to the household. We conducted key informant interviews with stakeholders regarding the program's history, implementation, impact, and perceived challenges. In one district, we mapped formal and informal sugar vendors and recorded coverage of available brands and packaging options. In a random sample, we conducted a Knowledge, Attitudes, and Practices survey of both vendors and consumers. We obtained 50 g samples of sugar from vendors for quantitative VA analysis using an iCheck portable fluorimeter and, in an ongoing surveillance cohort in this community, collected ~1 g samples for qualitative analysis. Key informant interviews revealed a strong commitment to sugar fortification in Zambia, but also several concerns: potential for excess VA intake, poisoning or infertility, cost of fortificant, and limited resources available for monitoring and evaluation of program impact. Market assessments revealed a variety of sources for fortified sugar. The distribution of VA in sugar in market samples was highly skewed, with a median of 2.4 mg/kg (25th percentile, 75th percentile: 1.5, 5.6) and a range from 0.6 mg/kg to 18.0 mg/kg. VA levels in sugar varied by source. Overall, the commitment of stakeholders to VA fortification of sugar is promising for the program's ability to adjust course, as needed, and for its long‐term sustainability. While limited to one area of the country, our data suggest wide variability in VA content, with median levels falling below the 10 mg/kg target. Given observed differences in VA content, it is possible that this is related to varied storage and packaging—a challenge that could be addressed by targeted messages to vendors. Additional research is needed to better understand patterns of sugar consumption at the household level, both to inform the contribution of fortified sugar to overall dietary VA intakes, but also as a baseline in a country that is experiencing a nutrition transition.Support or Funding InformationFunded by Sight & Life, with support from CBM Internationa
Determinants of Dietary Supplement Use and Compliance by US Adults
Authors: Bogale Alemtsehay , MacKay Douglas , Mitmesser Susan H 
Published: November 2023

IntroductionEvidence suggests that dietary supplement (DS) use, as a preventive measure to nutrient shortfalls, is among one of the components of a healthy life style.ObjectiveTo investigate consumer related factors that may affect DS use and compliance, and to recommend consumer centered tools and strategies to improve dietary supplement compliance.MethodData from The Council of Responsible Nutrition (CRN) Consumer Survey, published NHANES data and literature review was used to determine the prevalence, gender and age distribution, reasons for DS use and non‐use, and compliance and noncompliance by US adults. These data sources were utilized to compile a comprehensive list of reasons for DS use, reliable information sources of DS, and comparisons of nutrient profiles of DS users versus non‐users.ResultsOverall about 68% Americans use DS. In contrary to earlier findings that suggested increase, DS use remained stable at 68 % for five consecutive years from 2011–2015. Seventy five percent of DS users take multivitamin/mineral supplements followed by Vitamin D (37%), vitamin C (34%), calcium (29%), and vitamin B/B complex (24%). DS use tended to be higher in females (71%) but, compliance was higher in males (69%) than females (60%). Older adults age 55+ had both higher user prevalence (71%) and compliance (75%). Higher DS compliance was observed in the Northeast (68%) and South (69%) than Midwest (59%) and West (58%).The main reasons of DS use were: overall health (43%), energy (30%), to fill nutrient gap (28%), immune health (23%), bone health (22%), heart health (21%) healthy aging (20%), skin, hair & nails (19%) digestive health (17%) and joint health (17%). Physicians (55%), pharmacists (33%) and nutritionists (30%) were listed as a trusted source of information about DS. The primary reason indicated for noncompliance and non‐use was forgetfulness and not feeling the need. Among DS users, 10% for calcium and 25% for vitamin D did not meet the recommended daily intake, while 38% and 96% non‐users did not meet the daily requirement respectively. Other nutrients that fall short for both DS users and non‐users include potassium, magnesium, zinc, and, vitamins A, C, E and K.ConclusionAlmost two thirds of Americans use DS for overall health, energy, and to fill nutrient gaps. DS users are more likely to meet the daily recommended nutrient amount than non‐users, however nutrient gaps continue to exist amongst DS users. Non‐compliance appears to be a potential barrier to meeting nutrient recommendations. Improved systems of DS delivery such as simplified regiment with improved sensory attributes and personalized products that target individual nutrient shortfalls may improve DS compliance and contribute to achieving recommended nutrient intakes. Imparting scientific research findings through trusted sources such as health care providers and nutritionists should be considered among the primary strategies in nutrition intervention programs to improve compliance.
Interrelationship between Total and Undercarboxilated Osteocalcin Levels, and Body Mass Index in Women
Authors: Zago Liliana , Infantino Carlos Alfredo González , Zeni Susana , Río María Esther , Felipoff Ana Lía , Weisstaub Adriana , Dupraz Hernán , Presner Natalia 
Published: November 2023

Obesity and osteoporosis are two of the main common chronic diseases in women. Both are interconnected and contribute to glucose homeostasis. The objective of this study was to evaluate the effect of BMI on serum levels of total (OC) and undercarboxilated osteocalcin (ucOC), in 100 non diabetic overweight or obese adult women. Women were divided according BMI (range 25.0 to 52.8 kg/m2) into quartiles, and according to their menopausal status: premenopausal (PreM: ≤45 y; n=33), perimenopausal (PeriM: 46–54 y; n=18), postmenopausal (PostM: 55–65 y; n=34) and late menopausal (LM: >65 y; n=15). Total OC, ucOC and leptin were determined by ELISA kits. There were not significant differences between PreM and PostM from the same BMI quartile both in OCn and ucOCn. No significant changes were found in total OC mean values among BMI quartiles in either PreM or PostM (From the 1st to 4th quartiles: PreM: 37.5±3.0; 33.9±9.9; 27.2±12.2 and 25.1±17.4 ng/ml; PostM: 35.9±13.2; 43.6±21.5; 28.5±12.8; 28.3±16.3). In both menopausal status, the mean OCn levels presented a tendency to have lower values in the 3rd and 4th quartiles as compared to the 1st and 2nd. Conversely, there was a significant increase in ucOC mean levels as BMI increase, both in PreM and in PostM groups (from 1st to 4th quartiles: PreM: 1.53±1.08; 1.93±1.25; 3.25±1.72 and 4.27±0.86 ng/ml; ANOVA P=0.0003, Tuckey post‐test: P<0.01 between 1st and 4th and between 2nd and 4th quartiles; PostM: 1.09±1.04; 1.88±1.62; 2.74±1.51; 4.21±1.83; ANOVA P=0.0009, Tuckey post‐test: P<0.001 between 1st and 4th and P<0.05 between 2nd and 4th). Leptin levels also increased with BMI in both PreM and PostM.ConclusionThe present results confirm that BMI presents a significant influence on OC decarboxylation through leptin levels. Moreover, as ucOC is involved in glycemic control, the present results suggest that glucose homeostasis is affected by the increase in BMI.Support or Funding InformationSupported by Universidad de Buenos Aires, UBACyT 20720130100006BA
Effect of Histone Deacetylase Inhibitors on Vitamin D Dependent Gene Expression in Human Colorectal and Breast Cancers
Authors: Hossain Sharmin 
Published: November 2023

There are little evidence on the effect of histone deacetylation by a specific histone deacetylase inhibitor (HDACI), sulforaphane, on vitamin D dependent genes in human colorectal and breast cancers. Sulforaphane is a bioactive compound present in cruciferous vegetables like broccoli, Brussel sprouts etc. In the present study, we examined the influence of sulforaphane on expressions of vitamin D dependent genes, VDR and TRPV6, via inhibition of histone deacetylation with TSA as a positive control. We also examined DKK1, one of the inhibitors of Wnt signaling pathway, to see any potential effect of Vitamin D, sulforaphane (SFN) and/or the combination treatment on its regulation. The effects of SFN and TSA with and without the presence of vitamin D were tested on Caco‐2 and TMX2–28 (breast cancer) cells. Although not statistically significant, we found vitamin D to increase the expressions of VDR (2.3 fold) and TRPV6 (2.8 fold) compared to control. The expression of TRPV6 was increased two‐fold by the combination of vitamin D+TSA and three‐fold by TSA alone. DKK1 expression was increased upon treatment with vitamin D+TSA (fold increase 2.8) and TSA alone (fold increase 3.4). DKK1 expression was also increased by SFN (2.7 fold) suggesting a stand‐alone effect. Expression of DKK1 was also increased in TMX2–28 cells upon treatment with sulforaphane (1.5 fold) and the combination of vitamin D + sulforaphane (1.4 fold). Vitamin D (alone or in combination) with sulforaphane can be an effective nutrigenomic intervention for colorectal and/or breast cancer through the inhibition of histone deacetylation‐ a crucial epigenetic modification process. This research, therefore, has the potential to create a prospective model for epigenetic control in cancer.Support or Funding InformationHatch Grant# MAS00992