bluuebunny/crossref_metadata_2025 huggingface dataset:
Mode of Ubiquitination Determines Complete or Partial Degradation of GFP Substrates by the 26S Proteasome
Authors: Chirico Giavanna Gabriella , Kraut Daniel Adam
Published: November 2023

The 26S proteasome is responsible for the bulk of intracellular protein degradation in eukaryotes. Most proteins are targeted to the proteasome by the addition of one or more polyubiquitin tags. K11‐and K48‐linked chains typically signal for degradation in the cell, although K63‐linked chains can also target proteins to the proteasome in vitro. After binding to the proteasome via the ubiquitin tag and engaging proteasomal ATPases via an unstructured region, substrates are deubiquitinated, processively unfolded, translocated into the core of the proteasome, and degraded into small peptides. The factors that allow the proteasome to successfully unfold a folded domain (or prevent it from doing so) are still poorly understood. Green Fluorescent Protein (GFP) is commonly used, both in vivo and in vitro, as a model substrate to follow protein degradation. However, there are also reports that GFP can be difficult to unfold and degrade, perhaps because of its stable β‐barrel structure. In particular, Saeki et. al. reported that GFP resists degradation when tagged with K63‐linked polyubiquitin chains, while other proteins were degraded efficiently. To investigate whether chain linkage or mode of ubiquitination affected the proteasome's ability to unfold and degrade GFP, we constructed a degron that could be ubiquitinated with either the E3 ligase Rsp5 (reported to give K63‐linked chains) or the E3 ligase complex Cul3/Rbx1/Keap1 (reported to give primarily K48‐linked chains) and attached it to a GFP domain. Although full‐length substrates ubiquitinated with either E3 were degraded by the proteasome, degradation of the Rsp5‐ubiquitinated substrate led to the production of a large protein fragment containing the GFP domain while degradation of the Keap1‐ubiquitinated substrate proceeded without fragment formation. Our results suggest that the ability of the proteasome to unfold its substrates is tuned by the specific polyubiquitin modifications used to target it to the proteasome, and give additional insight into why K63‐linked chains are not typically used for proteasomal degradation in vivo.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. 1515229
Conformational Switching in Proteasomal ATPases Allows for Proper Substrate Processing
Authors: Snoberger Aaron M. , Smith David M.
Published: November 2023

Virtually every cellular process relies on properly regulated protein degradation, and thus, improper regulation of this degradation can lead to a wide range of human diseases (e.g. Neurodegeneration, Cancers, Cardiomyopathies). The ubiquitin‐proteasome system is responsible for the majority of targeted degradation within eukaryotes, so it is critical to understand how this system functions to degrade proteins. However, due in part to the size and complexity of the proteasome, we still do not understand how it functions at a molecular level. The 26S proteasome from eukaryotes is a 2.5 megadalton macromolecular complex composed of 2 subcomplexes: a hollow 20S core particle with protease sites sequestered on its interior, and a 19S regulatory particle that caps one or both ends of the 20S. Proteins destined for degradation (e.g. polyubiquitinated proteins) bind and are recognized by the 19S regulatory particle, and then in an ATP‐dependent manner are unfolded and injected into the 20S core particle, where proteins are then degraded. The 19S ATPase is a heterohexamer, and although each of the 6 subunits are highly similar to one another, recent cryo‐EM structures reveal that that these subunits form an asymmetric “lockwasher” conformation. However the structural elements that give rise to this asymmetric conformation are unknown, and may come from the hetero‐oligomeric subunit arrangement of the ATPase ring. Furthermore, cryo‐EM structures reveal different conformations of the 19S ATPases in the ADP (i.e. ATPh) or ATPgS‐bound states. In the present study, we employ an exhaustive crosslinking approach to show that Proteasome Activating Nucleotidase (PAN), a homolog of the 19S ATPases, adopts similar conformations as the 19S ATPases in the ATP‐ and ADP‐bound states, even though PAN is composed of 6 identical subunits (i.e. a homohexamer). Thus, the asymmetrical state of the proteasomal ATPases appears to be an inherent property of the ATPase subunit and its allosteries that arise from oligomerization rather than the heteroligomeric nature of the 19S ATPases. Most interestingly, in the conformationally restricted variants that we designed we observed functional defects when the subunits are prevented from switching out of the different nucleotide‐bound conformations, suggesting that the asymmetrical subunit switching observed in the cryo‐EM structures is part of the proteasomal ATPase's normal mechanochemical cycle that allows for proper processing of substrates.

Support or Funding Information

NIH‐R01GM107129, NIH‐F31GM115171
Resveratrol Modulates Cytokine Expression in LPS‐induced Human Monocytes: Role of Proteasome Subunits
Authors: Silswal Neerupma , Reddy Nidhi , Qureshi Nilofer
Published: November 2023

Ubiquitin proteasome system (UPS) maintains the basic cellular processes of the eukaryotic system by catalyzing the degradation of proteins. The proteolytic active sites of proteasomes reside on three important constitutively expressed subunits: X (β5), Y (β1) and Z (β2) which exhibit chymotrypsin‐like (CT), post‐acidic (PA) and trypsin‐like (T) activities respectively. The constitutive subunits of proteasomes (P) can be induced to those containing immunosubunits (immunoproteasomes, IP) LMP7 (β5i), LMP2 (β1i) and LMP10 (β2i) after priming with LPS or cytokines. We have reported earlier that trans‐resveratrol decreases LPS‐induced inflammatory cytokine levels and proteasome enzyme activities by inhibiting NF‐κB activation in mouse macrophages. Recently, there has been an increased interest to develop natural, less toxic and potent proteasome inhibitors to reduce inflammation. Our objective here was to compare the effect of resveratrol and a known LMP7 inhibitor (ONX‐0914, obtained from UBPBio) on proteasome's activities in human monocytes/macrophages. Using fluorescence‐based assays, resveratrol (0–100 μM) inhibited all three protease activities of purified human blood IP and P, although predominantly IP as compared to P (n=2). In contrast, ONX‐0914 was more selective and potent for CT activity of IP and P (n=2). Similarly, using cell‐based luminescence assay (THP1, monocytes), resveratrol inhibited all three protease activities. Moreover, resveratrol (Ki 80 μM) and ONX‐0914 (Ki 0.25 μM), both inhibited expression of LPS‐induced inflammatory cytokines TNFα and ICAM1, as well as immuno‐proteasome subunits LMP7 and LMP2 in THP1 and CD14+ blood monocytes (p < 0.05, n=3). We observed that 90% cells (THP1) survived even at 80 μM dose of resveratrol, while < 50% survival was observed with 10 μM dose of ONX‐0914 after trypan‐blue staining as well as MTT‐assay. Collectively, our data suggest that resveratrol is an inhibitor of all three; CT (predominantly), T and PA protease activities and is less toxic to human monocytes/macrophages than ONX‐0914. In addition, resveratrol decreases the LPS‐induced inflammatory cytokine expression by decreasing the expression and activities of IP. Therefore, resveratrol, as a proteasome inhibitor can be used to curb inflammation in diseased states.

Support or Funding Information

This work was supported, in whole, by the National Institutes of Health Grants 1 R01 GM102631‐01 to Dr. Nilofer Qureshi.
Finding the Elusive Pathway to Promotion as an Educator
Authors: Lambert H. Wayne , Zdilla Matthew J. , Klinkhachorn Penprapa S.
Published: November 2023

Most faculty members are required to meet specific benchmarks in their yearly academic assignment in the areas of research/scholarship, teaching, and service. Teaching faculty are charged with providing the majority of the student instruction and support within an academic department, and these educators often struggle to find the time and a venue to produce the required scholarship to achieve promotion to the next academic rank. Therefore, this career development talk will highlight important steps an educator can take to achieve career success both personally and professionally.

Teaching faculty can follow varied pathways while progressing through the Promotion and Tenure Process. Most often, designing impactful scholarly projects around the teacher's personal passion is the best place to start. Activities, such as publishing in scholarly peer‐reviewed journals, developing effective teaching and assessments tools to be published in peer‐reviewed educational repositories like MedEdPortal, becoming involved with textbook projects, taking on administrative positions pertinent to the success of your department or school, and/or becoming a leader in national and international professional organizations, are a great place to carve out your own niche to derive satisfaction and enjoyment from your career.

General themes of this talk will include: 1) identifying mentors that can provides tools to ensure your career success; 2) attending professional meetings to share your scholarly endeavors; 3) building collaborative relationships with fellow faculty members; and 4) recognizing your career goals may change as you adapt to internal and external pressures that exist both within and outside your institution. The goal of this talk is to help teaching faculty advance their academic careers by unlocking their potential and seizing opportunities that will arise in teaching, scholarship, research, and service, while avoiding obstacles (or pitfalls) that are often present.
Downregulation of the G6PDH Pathway Can Promote Oxidative Stress‐Mediated Reversal of Drug Resistance in B‐Cell Lymphoma cell Lines
Authors: Mousavi‐fard Seyed Hossein
Published: November 2023

The Nrf2/G6PDH pathway plays a critical role in antioxidant defenses and cell signaling. A low oxidative stress state due to upregulation of Nrf2/G6PDH Pathway in cancer cells is one mechanism underlying acquisition of resistance to CHOP‐based chemotherapy in DLBCL.

Normally under oxidative stress, Nrf2 activates expression of the glucose‐6‐phosphate dehydrogenase (G6PDH) gene, which leads to increase production of reducing equivalents (NADPH) through the pentose phosphate shunt. NADPH is then utilized to increase production of reduced glutathione (GSH) to balance the redox state. The present study explored the role of the Nrf2 downstream pathway, G6PDH, in CHOP resistance in DLBCL cells.

The G6PDH inhibitor, DHEA, increased oxidative stress as evident by increases in ROS in CHOP‐resistant DBLCL cells. DHEA not only repressed G6PDH activity, but also G6PDH protein expression after 16 hrs. In addition, DHEA treatment decreased levels of NADPH and glutathione (GSH). Moreover, DHEA markedly increased sensitivity of chemoresistant cells to CHOP.

In conclusion, repression of the G6PDH pathway leads to decreases in both NADPH and GSH, and increases in ROS (oxidative state), thereby sensitizing chemoresistant cells to chemotherapeutics.

Abbreviations used: Nrf2, Nuclear factor erythroid–related factor 2; G6PDH, glucose‐6‐phosphate dehydrogenase; CHOP, (C)yclophosphamide, (H)ydroxydaunorubicin, (O)ncovin, (P)rednisone; DLBCL, Diffuse large B‐cell lymphoma; NADPH, Nicotinamide adenine dinucleotide phosphate; GSH, Glutathione; DHEA, didehydroepiandrosterone; ROS, reactive oxygen species;

Support or Funding Information

Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843‐1114, USA
Identification of small molecule inhibitors of the LINE1 endonuclease
Authors: Meyers Jade , DeFreece Cecily
Published: November 2023

Long interspersed element‐1 (L1) is the only active autonomous retroelement in the human genome. Studies characterizing L1 function show that the L1 endonuclease activity is associated with the formation of DNA double strand breaks (DSBs). While L1 mutagenic insertion events have been associated with diseases such as breast and colon cancer as well as muscular dystrophy, the full effect of L1 endonuclease upon stability of the genome is undetermined. Data suggests that the damage from L1 endonuclease activity may be greater than previously considered seeing as though only a small fraction of L1‐induced DSBs results in a retrotransposition event. We have utilized an in vitro fluorescence based assay to identify novel small molecule inhibitors of the L1 endonuclease. A short term cellular viability assays was also used to determine the toxicity of the drugs to cells. A cellular retrotransposition assay was performed to determine if the drugs prevent LINE1 retrotransposition. We have identified three small molecules that inhibit L1 endonuclease activity in our fluorescence‐based assay. The drugs also prevent L1 retrotransposition but do not appear toxic under the conditions examined. This study shows that novel small molecule inhibitors have been identified that can be further modified to create potent and selective inhibitors of the L1 endonuclease.

Support or Funding Information

This work was funded by an Institutional Development Award (IDeA) from the NIGMS under grant number P20GM103424 and from the Louisiana Cancer Research Consortium, the NIH‐RCMI grant #8G12MD007595‐05.
Novel Cell‐Penetrating Peptides Overcome Endosomal Escape and Deliver Protein Cargos into the Cell
Authors: NGWA Verra , Salerno John , Nowak Scott , Chrestensen Carol , McMurry Jonathan
Published: November 2023

Over the last decade a number of peptides that are rapidly internalized by mammalian cells have been discovered or designed. Cell‐penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargoes to the cell interior. We have developed a novel CPP‐adaptor protein technology that allows any user‐defined cargo delivery and release into the cytoplasm. Our hypothesis is that a CPP‐adaptor with a moiety allowing high‐affinity but reversible non‐covalent cargo binding would lead to more efficient penetration and release than current CPP delivery strategies. Delivery of proteins to the interiors of cells has many applications. In addition to detecting and mapping the location of the components of living cells with fluorescent tags in real time, the availability of our system will enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers.

CPP‐adaptors and cargo proteins (horse radish peroxidase, myoglobin and Beta‐galactosidase) were expressed in and purified from E. coli BL21 (DE3)pLysS. Optical biosensing experiments demonstrated that affinity and kinetics between the novel CPP and cargo proteins did not significantly differ from wild‐type interactions; all had subnanomolar affinities. Cargo proteins were labelled with DyLight 550. CPP‐cargo complexes or cargo alone were incubated with subconfluent baby hamster kidney (BHK) cells. After washing, cells were imaged by fluorescence confocal microscopy. All user define cargos exhibited penetration and release to the cytoplasm whereas cargo‐only controls exhibited no measurable penetration (though some adherence to the outside of the cells was observed.) Time courses and dose‐dependency studies characterizing penetration and release kinetics will be presented as well as initial efforts to deliver cargo that will alter cell‐signaling pathways will be described.

The present results demonstrate the feasibility of delivery of a wide variety of cargo proteins and open up an array of potential research, diagnostic and therapeutic applications.

Support or Funding Information

NIH grant R25GM111565
Regulation of the Unfolded Protein Response by Fic‐mediated Adenylylation/AMPylation
Authors: Mattoo Seema , Sanyal Anwesha
Published: November 2023

Maintenance of ER (endoplasmic reticulum) homeostasis is a critical aspect of determining cell fate, and requires a properly functioning unfolded protein response (UPR) to combat such stress. We have discovered a hitherto unknown role for adenylylation/AMPylation, a post‐translational modification that involves the covalent attachment of an adenosine monophosphate (AMP) to the target protein, in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this reaction. A single Fic protein, called HYPE (Huntingtin yeast interacting protein), exists in humans. We reported that the ER molecular chaperone, BiP, is a target of HYPE‐mediated adenylylation. BiP serves as a sentinel for activating UPR and maintaining ER homeostasis by monitoring protein folding in cells. Our data indicate that HYPE localizes to the ER lumen where it interacts with BiP and adenylylates it, to alter BiP's ATPase activity in vitro. Further, HYPE expression increases upon induction of UPR stress. Accordingly, we demonstrated that cells knocked down for HYPE are unable to mount an appropriate UPR and are more susceptible to stress induced cell death. As such, HYPE represents a new player in UPR signaling. Here, we assess the cellular consequences of BiP adenylylation during UPR progression and describe the structural and kinetic parameters that govern the HYPE‐BiP interaction.

Support or Funding Information

This work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Core Pilot Grant, 2) a Showalter Research Trust Award, 3) an American Cancer Society (ACS) Institutional Grant, and 4) the National Institute Of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.
Effects of simulated microgravity on the microbial physiology of Ralstonia pickettii isolates from the International Space Station
Authors: Fergione Sarah J
Published: November 2023

Bacterial growth in the potable water system aboard the International Space Station is currently controlled using iodine. While iodine has been shown to be an effective biocide, it can accumulate in the thyroid of some individuals, impart a bad taste to drinking water, and is incompatible with the biocidal silver system used in the Russian segment. In comparison, silver is effective as an antimicrobial at very low concentrations, poses health risks only at very high intake levels, and is readily excreted from the body. In addition to controlling bacteria‐related risks to crew and spacecraft health, biocide is also necessary to prevent interplanetary transport of microorganisms. In this study, we investigated the effects of various stress assays on the bacterial isolate Ralstonia pickettii in a simulated microgravity environment. Isolates were subjected to acid, thermal, and silver biocide stress assays at three different concentrations. A rotating wall vessel culture apparatus was used to provide a low‐shear culture environment, simulating certain aspects of spaceflight. This environment is known as low‐shear modeled microgravity (LSMMG). Isolates used in this study were originally identified by 16S ribosomal gene sequencing of return water samples from long‐duration expeditions 29 and 35 to the International Space Station. Isolates were grown in sterile water to simulate the potable water environment aboard the ISS. The thermal stress and silver biocide assays showed significant effectiveness in bacterial control, while the acid stress assay showed minimal effectiveness. All three concentrations of silver biocide produced a zero percent survival rate in both control and LSMMG environments. An increased growth rate in isolates grown in the LSMMG environment was shown. No differences between the control and LSMMG environments were evident in the response of R.pickettii to stress assays. A ten percent difference in survival rate was seen in the acid stress assay results, with LSMMG culture having a lower survival rate. These results suggest silver as a promising candidate to replace iodine as we move towards long duration spaceflight.
Obesity Risks Associated with Low‐ and High Birth Weight and They Varied by Population Obesity Prevalence: A systematic Review and Meta‐analysis
Authors: Song Taixia , Wang Youfa , Huang Wenjie , Lu Lu , Zhao Yong , Sharma Manoj , Zhang Yong
Published: November 2023

Objectives

This systematic review examined obesity susceptibility in populations with different obesity prevalence, assessed obesity risk associated with low‐ and high birth weight( ie, tested “fetal origins” of obesity). Nutrition affects birth weight and obesity, while during prenatal stage it may modify metabolisms in that fetus can adapt to maternal nutritional status. Therefore, individual with different birth weight might have different susceptibility to obesity and its susceptibility might change in different nutrition conditions.

METHODS

Pubmed and Embase databases were searched for relevant studies. Relative risk (RR) of overweight/obesity (OW/OB) compared with normal birth weight was analyzed by description and meta‐analysis. During meta‐regression, the OW/OB prevalence of samples was introduced to analyzing of association of birth weight and risk of OW/OB.

RESULTS

Forty‐one studies met inclusion criteria. The pooled RR of OW/OB for low birth weight (LBW) was 0.80 (95%CI, 0.75–0.84, I2=73.0%), and 1.52 (95%CI, 1.45–1.60, I2=87.2%) for high birth weight (HBW). Meta‐regression showed that RR of OW/OB for LBW was positively associated with OW/OB prevalence (β=0.496, SE=0.244, p=0.046), and, for HBW, it was negatively associated with OW/OB prevalence (β=−0.722, SE=0.268, p=0.009).

CONCLUSIONS

LBW was associated with lower risk of subsequent OW/OB; the relation was stronger in countries with high obesity prevalence. HBW incases risk of subsequent OW/OB, and the relations was weaker in countries with high obesity prevalence. The findings support “fetal origins” hypothesis in obesity development.

Support or Funding Information

National Natural Science Foundation of China (No. 30901189) and NIH U54 center grant, HD070725
Effect of Daily Iron Supplementation in Healthy Exclusively Breastfed Infants: A Systematic Review with Meta‐Analysis
Authors: Cai Chenxi , Friel James
Published: November 2023

Background

Maintaining adequate iron is critical to infants’ physical and neurological development. The literature on iron requirements of exclusively breastfed infants contains conflicting data and contrary views. We summarized the evidence for benefits and risks of daily oral iron supplementation with regard to hematologic, growth, cognitive parameters and adverse effects in exclusively breastfed infants.

Methods

Structured electronic searches were conducted until August 26, 2015. We searched PubMed, Cochrane Library databases and Google Scholar for randomized controlled trials (RCTs) involving daily iron supplementation in healthy term, exclusively breastfed infants. Random‐ and fixed‐effects models were used to calculate the pooled estimates. Heterogeneity was assessed by Q‐test.

Results

Four RCTs with 511 infants were included in the meta‐analysis. Iron interventions had no significant effect on iron deficiency and iron deficiency anemia (OR=0.56; 95% CI, 0.23–1.33), serum ferritin level, or hemoglobin level. Iron interventions did result in a significant increase in Bayley psychomotor developmental indexes in later life (MD= 7.00, 95% CI, 0.99 to 13.01) and mean corpuscular volume (MD=2.17 fL; 95% CI, 0.99–43.5 fL). Iron supplementation was associated with slower growth in the exclusive breastfeeding period, but its long‐term effect is unclear.

Conclusions

Limited available evidence suggests that daily iron supplementation has beneficial effects on the hematologic parameter and later cognitive development, but delays the physical growth in healthy term, exclusively breastfed infants. There was no evidence to suggest iron supplementation could cause adverse effects.
Mouse Carnitine Palmitoyltransferase 2 (CPT2) is required to sustain cardiac function
Authors: Ellis Jessica M , Hasek Like Y , Yurovich Elizaveta J , Harris Kate L , Goergen Craig J
Published: November 2023

Carnitine palmitoyltransferase 2 (CPT2) is an enzyme required for the acyl‐carnitine shuttle that controls mitochondrial long‐chain fatty acid oxidation. The role of CPT2 in fatty acid metabolism is well established. However, patients with early‐onset CPT2 deficiency suffer cardiac complications and it has remained unknown how the loss of CPT2 in the heart alters cardiac function due to the lack of a physiological model. Here we present a novel mouse model deficient in cardiac CPT2 (Cpt2M−/−) which causes severe cardiac hypertrophy and eventual cardiac failure. Cpt2M−/− hearts weigh up to 4‐fold that of control hearts within 8–12 weeks of age and the mice fail to survive beyond 15 weeks of age. The Cpt2M−/− mice maintain a normal body weight and adiposity during their short lifespan. The loss of cardiac CPT2 reduced long‐chain fatty acid oxidation in heart homogenates by 93%. The loss of cardiac CPT2 increases the abundance of mitochondrial proteins, protein acetylation, activation of the mTOR pathway, and reduces insulin‐stimulated induction of the insulin‐signaling pathway. Together, these data suggest that CPT2‐mediated flux of fatty acid oxidation is required to retain insulin sensitivity and to sustain cardiac function.
Metabolic Profiling Reveals Differences in Plasma Concentrations of Arabinose and Xylose after Consumption of Fiber‐rich Pasta and Wheat Bread with a Differential Rate of Systemic Appearance of Starch‐derived Glucose in Healthy Men
Authors: pantophlet andre , eelderink coby , vonk roel , wopereis suzan , dijk‐stroeve annelies , bijlsma sabina , van stee leo , bobeldijk ivana , priebe marion
Published: November 2023

Consumption of starchy products with slowly digestible starch is implicated with a lower risk of metabolic diseases. Previously, it has been shown that pasta consumption resulted in a lower rate of appearance of exogenous glucose (RaE) and lower glucose clearance rate quantified with a dual isotope technique, which was in accordance with a lower insulin and GIP response after pasta intake. To get more insight in the acute metabolic consequences of the intake of products with differential rate of appearance of starch‐derived glucose, plasma metabolic profiles in 9 healthy men were analyzed after consumption of wheat bread and pasta, both enriched with wheat‐bran.

Differences in postprandial responses of 134 plasma metabolites were analyzed using a GC‐MS ‐based metabolomic approach. Six samples were analyzed per subject; one baseline sample and 30, 60, 90, 120 and 180 min after consumption. A two‐way ANOVA was used to analyze the effect of time, treatment, and their interaction. Additionally, pathway analysis was performed.

The observed differences occurred primarily in pathways related to protein and energy metabolism. Profiles of various amino acids had similar postprandial patterns and showed lower and attenuated responses after pasta, which could be explained by a slower absorption (parallel to the lower RaE) as well as the lower insulin response after pasta. Metabolites of glucose metabolism showed similar profiles as RaE, whereas metabolites of lipid metabolism were suppressed for a longer time after pasta intake. Remarkable higher plasma concentrations of arabinose (A) and xylose (X) after consumption of pasta were found. A and X are derived from arabinoxylans (AX), which are important components of wheat bran. The higher bioavailability of A and X coincided with a lower rate of appearance of glucose and amino acids. This is possibly due to higher viscosity of AX in the small intestine after pasta consumption, which is hindering absorption. The lower rate of glucose appearance is thus apparently not caused by slow starch digestion, but by slow absorption of starch‐derived glucose. As both food products contained the same amount of AX, different methods of processing may explain the difference in viscosity. It is speculated that the higher bioavailability of A and X after pasta is due to higher degradation of AX by small intestinal microbiota, facilitated by higher viscosity of AX after pasta. These results suggest that also wheat bran, depending of processing, may increase viscosity of the meal bolus in the small intestine and interfere with macronutrient absorption, thereby influencing postprandial glucose and insulin response.

Support or Funding Information

The project was supported by UMCG, TNO, TIFN and ZON‐MW
Respiratory effects of weight loss or gain over 1 year in obese children
Authors: Bhammar Dharini M , Stickford Jonathon L , Kim Roy J , Marines‐Price Rubria , Bassett J. Todd , Roman Maria C , Kruetzer Andreas , Babb Tony G
Published: November 2023

Introduction

The purpose of this case series was to investigate the respiratory effects of weight loss or gain in obese children over 1 yr.

Methods

Two obese children filled out the physical activity questionnaire for children (PAQ‐C) and underwent pulmonary function testing, dual‐energy x‐ray absorptiometry, maximal exercise testing, and constant load cycling at 40W before and after either 1 yr of: 1) exercise training and weight loss, or 2) free‐living weight gain. The children were not provided with a supervised intervention for weight loss or gain.

Results

Subject 1 was a girl, age 11.8yr, who engaged in a program of diet and regular exercise. The child's PAQ‐C score increased from 1.71 to 3.40. Height increased from 147.4 to 152.1cm, weight increased from 57.6 to 58.1kg, and BMI decreased from the 96th percentile (z‐score = 1.84) to the 93rd percentile (z‐score = 1.52). The percent body fat decreased from 44.1 to 36.3%, fat mass decreased from 25.4 to 21.1kg, and lean body mass increased from 32.2 to 37.0kg. FEV1/FVC ratio increased from 85 to 89%, TLC increased from 96 to 102% of predicted, and FRC (% TLC) increased from 34 to 40%. VO2peak (% predicted based on ideal body weight) increased from 106 to 117%. During constant load exercise at 40W, ratings of perceived exertion (RPE) reduced from 12 to 8, and ratings of perceived breathlessness (RPB) did not change (2 to 2).

Subject 2 was a boy, age 10.8yr, who continued to gain weight. PAQ‐C score reduced from 2.12 to 1.94. Height increased from 150.2 to 157.6cm and weight increased from 76.3 to 91.9kg. BMI stayed over the 99th percentile, and the BMI z‐score increased from 2.53 to 2.60. The percent body fat increased from 48.3 to 50.6%, and fat mass and lean body mass increased from 36.9 to 46.5kg and 39.4 to 45.4kg, respectively. FEV1/FVC ratio decreased from 90 to 81%, TLC decreased from 110 to 102% of predicted, and FRC (% TLC) decreased from 32 to 22%. VO2peak (% predicted based on ideal body weight) decreased from 103 to 101%. During constant load exercise at 40W, RPE reduced from 11 to 10 and RPB reduced from 4 to 2.

Discussion

An exercise training and weight loss program in children could substantially alter body composition and improve respiratory function and aerobic fitness, while a continued weight gain trajectory worsens body composition and could negatively impact respiratory function and aerobic fitness. Furthermore, body composition determination may be an important tool to help clinicians track progress of children undergoing lifestyle interventions during maturation in addition to measurements of BMI percentile or z‐score.

Support or Funding Information

Supported by unrestricted grant funds from Dr. Pepper Snapple, King Foundation, and Texas Health Presbyterian Hospital Dallas.
Characterization of the Pro‐inflammatory Cytokine IL‐1β on Butyrate Oxidation in Colorectal Cancer Cells
Authors: Johnstone Megan , Han Anna , Bennett Natalie , Standifer Cynthia , Smith Alexis , Donohoe Dallas
Published: November 2023

Intake of a high‐fiber diet has been proposed to protect against colorectal cancer development. Butyrate, a short‐chain fatty acid that is derived from the bacterial fermentation of ingested fiber, inhibits cellular proliferation and induces apoptosis in colorectal cancer cells at physiologically relevant concentrations. Butyrate serves as the primary energy source for the non‐cancerous colonocyte. In contrast, colorectal cancer cells shift cellular metabolism toward glycolysis and increased glucose uptake and utilization (the Warburg effect). Here, we sought to determine how butyrate oxidation is impacted by the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) in colorectal cancer cells. In this study, we used a colon cancer cell line (HCT116 cells) in a substrate‐limited media (1X KHB with 2.5 mM glucose) and butyrate oxidation was measured with or without IL‐1β [1ng/mL] treatment. Colorectal cancer cells pre‐treated for 3 hrs with IL‐1β show diminished oxidation of butyrate. On the other hand, we also find that acute treatment (less than 3 hrs) did not alter butyrate oxidation, suggesting that an IL‐1β treatment time of at least 3 hrs is required to see the effect. Treatment with interleukin 1 receptor antagonist (IL‐1RA at [50 ng/mL]) promotes a partial recovery of butyrate oxidation. Furthermore, IL‐1β suppression of butyrate oxidation was not observed in non‐colonic cell lines that were tested, including prostate (22rv1) and melanoma (A375) cells. IL‐1β altered mitochondrial function and proton leak. These data begin to link the inflammatory response in IL‐1β with altering cellular metabolism and decreasing the oxidation of butyrate in the cancerous colonocyte.
Calcium‐binding Peptides Isolated from Casein Hydrolysates Enhances Intestinal Calcium Uptake
Authors: Miao Jianyin , Liu Guo , Luo Zhen , Xia Zumeng , Liu Fei , Chen Yunjiao , Dixon William , Xiao Hang , Cao Yong
Published: November 2023

The aim of this study was to isolate calcium‐binding peptides from casein hydrolysates and to determine their effects on intestinal uptake of calcium in Caco‐2 cell monolayers. Five peptide fractions (P1 to P5) with calcium‐binding capacity were isolated from casein hydrolysates using HPLC. All five fractions promoted calcium uptake by Caco‐2 cell monolayers with fraction P5 showing the highest potency in enhancing calcium uptake. Within 1 hour of treatment, fraction P5 increased the calcium uptake by more than 7‐fold compared to the control group. The chemical composition of P5 was elucidated using LC‐MS/MS analysis and the amino acid sequence was determined to be RELEELNVPGEIVESpLSpSpSpEESITR. This calcium‐binding peptide was derived from β‐casein and contained four characteristic phosphorylated seryl residues. Our results demonstrated that synthetic P5 also displayed capacity to increase calcium uptake in Caco‐2 cell monolayers. Overall, our results provide novel insight into the identity and potency of bioactive phosphopeptides from casein hydrolysates that may enhance intestinal calcium uptake.

Support or Funding Information

Theauthors would like to express their gratitude to the Infinitus(China) Company Ltd for the financial support.
Testing the Use of Urinary Biomarkers to Assess Added Sugar Intake in Obese Postmenopausal Women
Authors: Arnold Kristen , Bomser Joshua , Andridge Rebecca , Orchard Tonya
Published: November 2023

Rationale

Assessment of dietary intake is most frequently done by self‐reported measures such as 24‐hour recalls and food frequency questionnaires. Although these measures are useful, there is error associated with self‐report. This is found to be particularly true in overweight/obese individuals and for specific foods perceived as having negative health outcomes (e.g. added sugars) which are often under‐reported. Therefore objective measurements are needed to more accurately assess dietary intake of certain nutrients. Urinary sucrose and fructose excretion measured in urine samples collected over 24 hours has been validated as a biomarker of added sugar intake in controlled feeding studies. However, whether a single spot urine sample can be used to assess self‐reported added sugar intake is less clear, especially in overweight/obese individuals.

Objective

Test correlations between urinary biomarkers from a single spot urine sample and self‐reported dietary intake of added sugars in obese postmenopausal women.

Methods

Fasting urine samples were collected at the baseline visit from 8 postmenopausal women with a BMI >30, who were enrolled in a dietary intervention study aimed at lowering added sugars and increasing fiber and fatty fish. Urine samples were transferred to cryovials and frozen at −20 C until assayed in batches. Urinary sucrose and fructose excretion was measured using a modified enzymatic assay (Rosche BioPharm). Participants completed 24 hour recalls at the baseline visit with the guidance of a Registered Dietitian using the USDA 5‐step multiple‐pass method designed to enhance accuracy of dietary data collection. Self‐reported added sugar intake (g/d) was estimated using Nutritional Data System for Research (NDSR). Pearson correlations were used to examine associations of added sugar intake with urinary sucrose and fructose excretion.

Results

Preliminary baseline data from 24‐hour recalls revealed a mean added sugar intake of 40.1 g/d (SD 25.2), with a range of 0 – 97 g/d. Mean urinary sucrose excretion was 0.024 g/L (SD 0.004), range 0.000 – 0.108 g/L. Fructose in the urine was undetectable. A statistically significant correlation (r=0.74, p=0.04) was observed between baseline urinary sucrose excretion and added sugar intake from 24 hour recalls. There was no significant correlation (r= 0.15, p=0.81) between baseline urinary sucrose excretion and self‐reported sucrose intake, which includes sucrose naturally found in plant foods.

Conclusion

Preliminary cross‐sectional data from a small sample of postmenopausal obese females suggest that urinary sucrose excretion can be used as an objective biomarker of added sugar intake. This warrants further testing in a larger sample.

Support or Funding Information

Internal Ohio State University Funds
iTRAQ‐Based Quantitative Proteomic Analysis of the Anticancer Mechanism of a Novel Ent‐kaurane Diterpenoid from RubuscorchorifoliusL.f
Authors: Chen Xuexiang , Gao Zili , Ou Yan Wen , Liu Guo , Gao Xiangyang , Liu Xiaojuan , Chen Yunjiao , He Lliping , Xiao Hang , Cao Yong
Published: November 2023

Our previous study has identified (16R)‐16,17‐dihydroxy‐ent‐ kaur‐2‐one (a novel ent‐kaurane diterpenoid) from Rubuscorchorifolius L.f.as a potential anti‐cancer agent. Herein, we determined the mechanism of action of the diterpenoid in inhibiting the growth of human colon cancer cells. The results from flow cytometry analysis showed that the diterpenoid induced extensive apoptosis and cell cycle arrest in human colon cancer cells. iTRAQ‐Based Quantitative Proteomic analysis revealed that treatment with the diterpenoid led to upregulation of 67 proteins and down regulation of 53 proteins. These proteins play important roles in multiple cellular processes such as enzyme activity adjusting, electron transferring, anti‐oxidation and protein transcription. These proteins also are critical in path ways related to carcinogenesis such as oxidative phosphorylation pathway, cell cycle regulation, and p53 pathway. Treatment with the diterpenoid modulated the expression levels of APC, DCC, TGFβRII, Smad2m, Bax, p53 and 14‐3‐3σ, which may contribute to its inhibitory effects on colon cancer cells. Overall, our work produce dinitial mechanistic information on the action of (16R)‐16,17‐dihydroxy‐ent‐ kaur‐2‐one in inhibiting the growth of colon cancer cells, which provided important scientific basis for further investigation.

Support or Funding Information

The authors are grateful for the financial support from the University of International Cooperation in Science and Technology Innovation Platform Project of Guangdong (2013gjhz0003), the Joint Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20114404120022),
Unfermented and Fermented Cocoa Extracts as Inhibitors of Pancreatic α‐Amylase, α‐Glucosidase, and Pancreatic Lipase
Authors: Ryan Caroline , Ye Liyun , O'Keefe Sean , Neilson Andrew Peter
Published: November 2023

Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of flavan‐3‐ols, which may aid in prevention of obesity and type‐2 diabetes. Our objective was to determine the impact of fermentation and roasting on the ability of cocoa to inhibit digestive enzymes. Polyphenol profiles of unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) extracts were characterized by colorimetric and chromatographic methods. Inhibitory activities of the cocoa extracts against pancreatic α‐amylase, α‐glucosidase, and pancreatic lipase were measured in vitro. Overall, there was a significant decrease (p<0.05) in total polyphenols, flavan‐3‐ols, and anthocyanins between the two sets of unfermented and fermented cocoa samples. HPLC and thiolysis demonstrated a decrease in catechins between unfermented and fermented samples as well as an increase in average flavanol degree of polymerization from UB to FB. For α‐glucosidase, UL, FL, Acarbose (+ control), FB, and UB had IC50 values of 90.0, 109, 158, 200, and 218 μg/mL, respectively. This is a novel finding that suggests the potential utility of cocoa, particularly that of cocoa liquors, in blunting increases of blood glucose similar to the drug Acarbose. For α‐amylase and pancreatic lipase, Acarbose and Orlistat (+ controls) had the lowest IC50 values (54.3 and 181 μg/mL, respectively) compared to the cocoa samples. However, at 500 μg/mL concentrations, Acarbose, FB, and FL inhibited α‐amylase 71.0%, 53.6%, and 38.4% respectively, all of which were not significantly different (p<0.05). The same was true for inhibition of pancreatic lipase by Orlistat (62.9%), UB (47.6%), and FB (41.5%) at 200 μg/mL concentrations. Thus, at high concentrations, inhibition of fermented cocoa and Acarbose against α‐amylase and that of cocoa beans and Orlistat against lipase are similar. Although processing induced significant changes in the polyphenol profiles of these cocoas, each enzyme was affected differently by fermentation and roasting of the samples. For α‐glucosidase, roasting of cocoa improved its inhibitory activity. For α‐amylase, both fermentation and roasting decreased cocoa's inhibition abilities, whereas these processes improved its inhibition of pancreatic lipase. Inhibition of these enzymes delays the digestion of carbohydrates and lipids, potentially dallying or blunting increases in postprandial blood glucose levels, reducing fat accumulation, and increasing satiety. These results suggest that compositional changes in cocoa during processing, other than alterations to polyphenols, influence potential inhibition of digestive enzymes.
The Elements of Story Telling
Authors: Poznanski Ann
Published: November 2023

It has been said that stories are remembered 22 times more than facts alone. Last year at American Association of Anatomists meeting we heard an eloquent appeal for teaching science through story telling by Sean Carroll of the Howard Hughes Medical Institute. What are the elements of a good story? Drawing from diverse perspectives such as Andrew Stanton, the screenwriter of WALL‐E and Finding Nemo, Oliver Sachs, Rita Charon, Nicole Woods, Raphael Luna and others, this talk will review and give examples of useful elements that work together to make a good story. We will see that there is not one formula but many that are offered. A good story can be told in many different lengths as well. We will also find pearls that can be taken from this variety of voices to help us build our skills and cultivate the art of story telling in education.
Your 15 Minutes on the Podium have Finally Arrived…
Authors: Laitman Jeffrey Todd
Published: November 2023

While our “15 minutes of fame” may never actually occur, most of us will get our 15 minutes at the podium. Indeed, that is the usual time allotted for presentations at most national or international meetings, give or take a few for questions. These 15 minutes in the spotlight can be some of the most important of our careers whether we are putting forward our latest, exciting science (and hoping that there are those in the audience on our grant review panel), advancing the next‐best‐innovation in education (ditto the above), or, putting our self forward when looking for a job (with prospective employers out there.) How to approach ‐ and how not to approach ‐ the “golden 15” is something that must be carefully planned so as to maximize execution. Like the D‐day invasion of World War II, every aspect must be carefully scripted with a clear focus, story, and understandable ending; and like landing on a D‐Day beach, the terrain must be known beforehand to avoid pitfalls or derailments that take too much time to explain. Although 15 minutes may not seem long, if used properly, there is ample time that allows one to: bond with the audience, set the tone, define the topic and its importance, present your hypothesis and story, and, give the take‐home message. An important part of the 15‐minute talk is knowing your audience and making a bond with them. While you may have heard of the “Five‐Minute Mentor” a 15‐minute talk gives time to produce the “15‐Minute Family.” A group that has warmed to a speaker will open their ears more readily than one that is apathetic. Remember, a lecture is not a journey but an invitation to the journey, and if we do a good job in our 15 minutes, those in the audience may well want to learn more about our particular journey.
Adenosine A2A Receptor Driven Impulsivity Promotes Cue‐Dependent Reward Seeking Behavior and Increases Hippocampal Immature Neurons
Authors: Oliveros Alfredo , Cho Chang Hoon , Choi Sun , Jang Mi‐Hyeon , Choi Doo‐Sup
Published: November 2023

Mice lacking the ethanol‐sensitive equilibrative nucleoside adenosine transporter 1 (ENT1–/–) display enhanced goal‐directed behaviors due to decreased adenosine A2AR function. However, the role of A2AR signaling in the regulation of impulsivity remains unknown. Using Pavlovian conditioning, second‐order schedule discrimination, and the 5‐choice serial reaction time task (5‐CSRTT), which incorporates differential‐reward of low rate (DRL) schedules, we reveal that deficits in A2AR function promote impulsivity. ENT1–/– mice display exaggerated impulsive responses and faster reaction times to retrieve reward during conditioning and extinction. Interestingly, our results show that basal ERK1/2 phosphorylation is reduced in the dorsal hippocampus (dHPC) of ENT1–/– mice and that impulsive Pavlovian conditioning significantly increases ERK1/2 phosphorylation. Subsequently, we validate that inhibition of A2AR promotes impulsivity during Pavlovian conditioning and the 5‐CSRTT. In addition to increased ERK1/2 phosphorylation, mice demonstrating exaggerated impulsivity as a result of A2AR inhibition exhibit enhanced expression of doublecortin and increased incorporation of BrdU within the HPC. Taken together, these findings indicate that impaired ENT1/A2AR signaling promotes impulsivity during cue‐induced conditioning and contributes to reward seeking during extinction. More importantly, these results demonstrate that impulsive behavior is associated with aberrant hippocampal neurogenesis, which may contribute to the development of maladaptive reward‐seeking behaviors.

Support or Funding Information

NIH R01 AA018779