bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased differentiating kidney epithelial cell density induced by changes in fluid flow velocity (1180.12)
Authors: Seetharaman Vikram , Resnick Andrew
Published: June 2021

Mouse cortical collecting duct cells were allowed to differentiate under conditions of no fluid flow (vf = 0; negative control) or constant rapid flow (vf = max; positive control) on orbital shakers. Cells were subjected to Δvf at 3, 5, 10, 20 days post‐differentiation, from vf = 0 to vf = max, or vice versa. At various times post‐Δvf, cells were harvested, nuclei were DAPI stained for cell counting, and RNA/protein extracted. Some cells were fixed and immunostained for the presence of the markers of MCCD cell differentiation E‐cadherin (E‐cad), important in adherens junction formation, and polycystin 1 (PC1), important in cellular mechanosensation. If MCCD’s exposed to Δvf do indeed de‐differentiate, then the relative expression of these 2 proteins should decrease. Although PC1 was absent from all cells sampled before 10 days post‐differentiation and unchanged in cells exposed to 5 days of vf = max followed by 5 days of vf = 0 compared to cell exposed to 10 days of vf = max only, E‐cad expression appeared slightly lower in cells exposed to 5 days of vf = max followed by 4 hours of vf = 0 compared to cells exposed to both 5 days of vf = 0 only and 5 days of vf = max only. Quantitative real time‐polymerase chain reaction and Western blot analyses will subsequently be used to determine concentrations of several mRNA transcripts [cyclin dependent kinase 1 (cdk1)/c‐Myc/cyclin D] and phosphorylated proteins [phospho‐Protein Kinase B (PKB, also known as Akt)/phospho‐mammalian Target of Rapamycin (mTOR)/phospho‐Mitogen Activated Protein Kinase (MAPK, also known as ERK1&2)], respectively, considered markers of cell proliferation. If MCCD’s exposed to Δvf do indeed divide, then all of these mRNA transcripts and phospho‐proteins should increase in concentration except for phospho‐PKB, which should decrease in concentration.

Grant Funding Source: Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716
Hydrogen sulfide activates heterologously expressed human CFTR in Xenopus oocytes (1181.16)
Authors: Althaus Mike , Perniss Alexander , Beuter Liesa
Published: June 2021

Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule. Physiological responses to H2S are often attributed to its impact on various sodium, calcium and potassium channels. However, little is known about the influence of H2S on chloride channels. This study investigated the putative regulation of the chloride channel CFTR (cystic fibrosis transmembrane conductance regulator) by H2S.

Human CFTR was heterologously expressed in Xenopus oocytes and its activity was measured by microelectrode recordings using the two‐electrode voltage‐clamp technique. CFTR expression was verified by the application of forskolin/IBMX which elicited a transmembrane current that was sensitive to the CFTR inhibitor CFTRinh‐172. The H2S‐forming salt Na2S (5‐300 µM) significantly increased transmembrane currents of CFTR‐expressing oocytes but had no effect on native oocytes. The effect of Na2S was dose dependent and blocked by CFTRinh‐172. The slow H2S‐releasing molecule GYY4137 also activated CFTR, although to a smaller extent compared to Na2S. The adenylate cyclase inhibitor MDL‐12330A significantly decreased the activation of CFTR by Na2S. These data indicate that H2S activates CFTR in Xenopus oocytes by the cAMP/adenylate cyclase pathway. In epithelia, an increased CFTR activity may correspond to a secretory response to H2S which may be endogenously produced by the epithelium or a H2S‐generating microflora.

Grant Funding Source: Supported by the German Research Foundation (AL‐1453/1‐2).
An intrinsically disordered region of the transcription factor, NFAT5, becomes more ordered with an increase in osmolality (1182.8)
Authors: DuMond Jenna , Kumar Raj , Ramkissoon Kevin , Izumi Yuichiro , Thompson Brad , Burg Maurice , Ferraris Joan
Published: June 2021

Hypertonicity stimulates NFAT5 nuclear localization and transactivating activity. Many transcription factors are known to contain intrinsically disordered regions (IDRs) which become more structured with local environmental changes such as osmolality, temperature and tonicity. The N‐terminal region of NFAT5 is predicted to contain an IDR. Using far‐UV CD and fluorescence we found that addition of the organic osmolyte trehalose to the purified N‐terminal IDR of NFAT5 caused an increase in the structure of the IDR, consistent with a 2‐state transition, ID to native. To investigate how this increased structure affects cellular protein‐protein interactions, we looked for a correlation between change in structure and association with other proteins. Mass spectrometry identified NFAT5 IDR protein binding partners. We thus have demonstrated that a change in osmolality gives rise to an increase in IDR structure and affects NFAT5 protein‐protein interactions. These data will aid in understanding transcriptional upregulation of NFAT5 under cellular osmotic stress.

Grant Funding Source: Supported by SBC, NHLBI, National Institutes of Health, Intramural Research Program
Associations between diet quality and cognitive control in childhood (124.1)
Authors: Khan Naiman , Raine Lauren , Drollette Eric , Scudder Mark , Cohen Neal , Kramer Arthur , Hillman Charles
Published: June 2021

The influence of diet on specific aspects of cognitive function and brain health in childhood remains unknown. We aimed to investigate the relationship between diet quality and cognitive control in prepubertal children (8‐9‐years). Participants (N=58) completed a modified Eriksen flanker task assessing interference control. Healthy eating index (HEI) scores, based on 3‐day food records, were used to assess diet quality. Percent fat mass (%FM) and aerobic fitness were measured using DXA and VO2max, respectively. Partial correlations between HEI scores and flanker task performance (accuracy and reaction time [RT]) were conducted following adjustment of age, sex, SES, IQ, VO2max and %FM. HEI scores were not correlated with accuracy in the congruent task condition (r=0.12, p=0.20), RT in the congruent (r=0.19, p=0.08), and RT in the incongruent condition (r=0.18, p=0.09). However, HEI scores were positively correlated with accuracy during the incongruent condition (r=0.28, p=0.02) and negatively related to task interference (r=‐0.29, p=0.02). Given that the incongruent, relative to the congruent, task condition requires greater amounts of interference control, these data are among the first to indicate that higher diet quality may be important for upregulation of cognitive control when faced with higher cognitive demands in childhood.

Grant Funding Source: Supported by NIH HD069381
Effect of oral nutritional supplementation on cognitive status in community dwelling elderly with sarcopenia and malnutrition (124.3)
Authors: Tian Min , Hustead Deborah , Mustad Vikkie
Published: June 2021

Past research suggests that age‐related cognitive decline is associated with compromised nutritional and functional status. Much of this literature has focused on either institutionalized elderly or healthy community‐dwelling elderly. The purpose of this study was to determine whether oral nutritional supplementation (ONS) can improve cognitive status in community‐dwelling older adults with malnutrition and sarcopenia.

Older adults (N=285; 65‐95 yrs) from 8 countries across Europe and North America completed a 24‐week ONS intervention trial. Sarcopenia was determined using the European Working Group on Sarcopenia in Older People (EWGSOP) definition. Cognitive status was assessed by Mini‐Mental State Exam (MMSE).

MMSE scores did not change significantly in the overall study population. In a subgroup of malnourished, sarcopenic older adults who began the intervention with cognitive impairment (MMSE score 蠄 26, n = 74), MMSE scores significantly improved from baseline to 12 weeks (p = 0.004) and from baseline to 24 weeks (p = 0.03). These data demonstrated that in community elderly with malnutrition and sarcopenia, ONS maintained cognitive status and even improved cognitive status in the subgroup with cognitive impairment.

Grant Funding Source: Supported by Abbott Nutrition
Nutrient biomarker patterns and cognitive decline in older adults (124.6)
Authors: Bowman Gene , Mattek Nora , Dodge Hiroko , Quinn Joseph , Howieson Diane , Kaye Jeffrey
Published: June 2021

We previously identified distinct nutrient biomarker patterns associated with both psychometric and neuroimaging indices of brain health in a cross‐sectional analysis of older adults. Those results propose that certain nutritional combinations influence global cognitive capacity, but also some relationships were more discrete and mapped to specific domains of cognition. The objective of this study was to determine if attention/working memory, episodic memory, and executive function decline is explained by nutrient biomarker patterns.

Attention/Working memory (Digit Span Forward + Backward), episodic memory (CERAD) and executive function (Trails B + category fluency) Z scores were constructed. Nutrient biomarker pattern analysis of 30 biological markers of diet generated distinct nutrient biomarker patterns. Mixed‐effects estimated cognitive Z‐score change explained by the nutrient biomarker patterns.

Eighty‐five non‐demented adults (53 women) with complete data had a mean age and duration of follow up of 86.4 (±10.4) and 4.6 (±1.8) years, respectively. One nutrient biomarker pattern representing a combination of eicosapentaenoic acid (20:5n‐3), docosahexaenoic acid (22:6n‐3), 25‐hydroxyvitamin D and pyridoxal 5‐phosphate was associated with less attention/working memory (AWM) decline after full adjustment (age, gender, education, ApoE genotype, hypertension, depression, baseline cognitive status, p=0.0099), however, these nutrients in isolation were not significant. Episodic memory and executive decline were not explained by the nutrient biomarker patterns.

NBP analysis is one fruitful strategy for assessing the relationship between nutritional combinations and cognitive aging. These results suggest that long chain n‐3 PUFAs, vitamins D and B6 can jointly, but not independently prevent age‐related AWM decline. These results also highlight that indeed some aspects of cognition appear more sensitive to nutritional influence than others.

Grant Funding Source: Supported by NCCAM AT004777 (GLB) NIA AG043398 (GLB), NIA‐AG08017 (JAK)
Lactation sufficiency is predicted by fasting plasma glucose at 1 month postpartum (131.1)
Authors: Nommsen‐Rivers Laurie , Glueck Charles , Huang Bin , Wang Ping , Dolan Lawrence
Published: June 2021

We examined if postpartum metabolic health predicts lactation sufficiency. N=14 healthy mothers were enrolled during their maternity stay and followed up at 4 days (interview) and 1 month (interview, anthropometry and oral glucose tolerance test). The cohort had strong intentions to exclusively breastfeed but were overweight or obese (BMI, 33 + 5). Milk supply was grouped as: ABUNDANT, N=5, exclusively breastfeeding since birth with no milk supply concerns at either interview; SUFFICIENT, N=5, exclusively breastfeeding at 1 month but reported either delayed lactogenesis or current milk supply concern; and INSUFFICIENT, N=4, despite effort, unable to exclusively breastfeed. BMI, fasting insulin, insulin resistance index, and fasting plasma glucose (FPG) were all significantly lower in the ABUNDANT as compared to the SUFFICIENT or INSUFFICIENT groups (P‐value, 0.0003 to 0.03), but the strongest independent predictor of milk supply was FPG (P<0.0003). None in the abundant or sufficient, but 75% in the insufficient group, had impaired glycemia (FPG, 95 ‐ 125 g/dL) suggesting waning insulin secretion in the face of insulin resistance may be an important contributor to low milk supply.

Grant Funding Source: Supported by the Cincinnati Diabetes and Obesity Cener and NIH 8 UL1 TR000077
Creating a roadmap for educational scholarship (14.3)
Authors: O'Sullivan Patricia
Published: June 2021

Creating a Roadmap for Educational Scholarship Planning ahead can help bring educational scholarship to dissemination. The attendees will review the steps and sequence of activities to take an idea from something of interest to one that can be disseminated. Careful attention will be paid to the considerations in the methods that will ensure a robust study. Guidance will be given concerning publication opportunities.

Specific Objectives: At the end of the session, participants will have: 1. Identified the steps necessary to move an idea from inception to dissemination.

2. Considered the different approaches to conducting educational scholarship.
Effect of E. coli outer membrane vesicles on monocyte‐platelet aggregates (145.9)
Authors: Dobrydneva Yuliya , Wahab Kamal , Soult Michael , Lonergan Natalie , Sullivan Claretta
Published: June 2021

Bacterial outer membrane vesicles (OMVs) can initiate the inflammatory response in the transition of an infection to sepsis, and sepsis is a prothrombotic state. The role of OMVs in the development of prothrombotic tendency is unknown. The objective: to determine whether OMVs and endothelial microparticles can induce formation of prothrombotic monocyte‐platelet aggregates (MPA). Donor blood was incubated either with OMVs from clinical isolate of an enterotoxigenic strain of E. coli or with supernatant from HUVEC (Human Umbilical Endothelial Cells) cultured in the presence of OMV for 24 hrs. MPA were measured in whole blood by flow cytometry as CD61+CD14+ cells. In whole blood from healthy human volunteers, MPA level is 19±2% in unstimulated state and 37±8% in ADP‐stimulated state. Incubation of blood with OMVs did not significantly increase MPA neither in unstimulated blood (23±2%), nor in ADP‐stimulated blood (24±7%). However, supernatant from HUVEC cultured with OMVs increased level of MPA in ADP‐stimulated blood (54±9%). Incubation with microparticles‐free HUVEC supernatant decreases MPA in ADP‐stimulated blood to a control level (35±4%). Thus, it is likely that microparticles derived from OMV‐treated HUVEC increase MPA. Therefore OMV may contribute to a prothrombotic state by stimulating microparticles release from activated endothelium and increasing prothrombotic MPA formation.
Inhibition of platelet‐induced NETosis via disruption of CXCL4/CCL5‐heteromerformation ameliorates organ damage after in ventilator‐induced lung injury (146.3)
Authors: Herter Jan , Rossaint Jan , Soehnlein Oliver , Weber Christian , Stadtmann Anika , Block Helena , van Aken Hugo , Zarbock Alexander
Published: June 2021

Platelet leukocyte interactions are a hallmark of inflammation. We here report that platelet induced formation of neutrophils extracellular traps (NETs) is crucial for neutrophil recruitment following ventilator induced lung injury (VILI) as the inhibition of NET formation by DNAse dampens the severity of VILI. Furthermore, administration of MKEY, a peptide reported to disrupt heterodimer formation of platelet derived CXCL4 and CCL5, or blocking antibodies against CXCL4 and/or CCL5 caused a decrease in neutrophil influx and organ damage that was comparable to DNAase treatment or platelet depletion. The combination of platelet‐depletion and MKEY or antibodies against CXCL4/CCL5 did not show additive effects. Taken together, our data indicates that platelet derived CXCL5/CCL5 heterodimer formation is critical for platelet induced NET‐formation contributes crucially to the pathogenesis of VILI.

Grant Funding Source: Supported by DFG: AZ428/3‐1, AZ428/6‐1, SFB1009/A5 to AZ, HE6810/1‐1 to JH, IZKF: SEED01/12 to JR
Focal adhesion kinase (FAK)‐related non‐kinase (FRNK) negatively regulates eosinophil recruitment (146.4)
Authors: Sharma Ritu , Zhang Hong , Stevens Kasia , Colarusso Pina , Patel Kamala
Published: June 2021

Focal adhesion kinase (FAK)‐related non‐kinase (FRNK) is an independently expressed protein containing the C‐terminal domain of FAK. A recent study showed that FRNK negatively regulates lung fibrosis. In this study we asked if FRNK could negatively regulate human eosinophil recruitment in response to IL‐4. Exogenous expression of FRNK blocked >90% of eosinophil firm adhesion and transmigration to IL‐4‐stimulated endothelial cells (HUVEC) under flow conditions. FRNK blocked eosinophil recruitment by preventing the transcription and translation of VCAM‐1 and eotaxin‐3 (CCL26), two proteins we previously showed were critical for eosinophil recruitment. GATA6 has been shown to regulate VCAM‐1 in response to TNF, but its role in IL‐4‐mediated VCAM‐1 expression is not known. We found the IL‐4 increased GATA6 expression and down‐regulating GATA6 with siRNA blocked VCAM‐1 expression and function. Expressing FRNK in HUVEC prevented GATA6 upregulation by IL‐4 suggesting that FRNK blocks VCAM‐1 expression through its actions on GATA6. FRNK can act directly by binding to target molecules or indirectly by displacing FAK at focal adhesions and blocking FAK’s kinase activity. To determine if FRNK was acting through FAK, we down‐regulated FAK with siRNA and found that knocking down FAK had no effect on GATA6, VCAM‐1 or CCL26 expression. These data suggest that FRNK acts independent of FAK to negatively regulate eosinophil recruitment.

Grant Funding Source: Supported by the Canadian Institutes of Health Research
CD99 signals through soluble adenylyl cyclase to regulate leukocyte transendothelial migration (146.6)
Authors: Watson Richard , Muller William
Published: June 2021

Transendothelial migration (TEM) is the process by which leukocytes extravasate out of the vasculature into the interstitium and is one the key steps of the inflammatory response. PECAM and CD99 are two critical proteins at the endothelial cell border that function sequentially to regulate TEM (PECAM upstream of CD99). These proteins partially reside in a novel, intracellular endothelial cell compartment, known as the Lateral Border Recycling Compartment (LBRC), which supplies the extravasating leukocyte with unligated proteins and membrane surface area to facilitate its movement across the endothelium, in a process called targeted recycling (TR). While much is known regarding the function of PECAM during TEM, very little is understood regarding how CD99 signals. The C‐terminal tail of CD99 is short (39AA) and contains no known signaling motifs. Blocking homophilic interaction of leukocyte CD99 with endothelial cell CD99 arrests leukocytes part way through the junctions; cross‐linking endothelial CD99 overcomes this block. Here we show that engagement of leukocyte CD99 with endothelial cell CD99 (or crosslinking CD99 on endothelial cells to mimic this) activates PKA via a soluble form of adenylyl cyclase, ADCY10 (sAC). Blocking either PKA or sAC, but not transmembrane adenylyl cyclase (tmAC), recapitulates the phenotype of CD99 arrest on TEM and also prevents crosslinking‐mediated restoration of CD99 blockade. Activation of PKA restores TR of LBRC membrane to the arrested leukocytes to overcome the CD99 blockade. Current research is focused on identifying the mediators downstream of PKA as well as the signaling events taking place between the cytoplasmic domain of CD99 and sAC.

This research was supported by F30 HL116100‐01A1 to RLW and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.

Grant Funding Source: NIH NRSA F30 HL116100‐01A1
Vesamicol, an inhibitor of vesicular acetylcholine transporter, causes apoptosis in human lung adenocarcinomas (151.3)
Authors: Dom Aaron , Brown Kathleen , Lau Jamie , Thornhill Brent , Crabtree Clayton , Witte Theodore , Hardman W , McNees Christopher , Stover Cody , Carpenter A , Chen Y , Dasgupta Piyali
Published: June 2021

Clinical studies show that the development of lung adenocarcinomas (LACs) is correlated with smoking habits. Nicotine, the addictive component of cigarettes, accelerates human LAC proliferation through nicotinic acetylcholine receptors (nAChRs). Human LACs contain all of the cholinergic system proteins, including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT) and nAChRs. In the present study we show that nicotine upregulates VAChT and ChAT. Therefore, we hypothesized that VAChT antagonists, such as vesamicol, may abrogate the growth of human LACs. Vesamicol induced potent apoptosis of nicotine‐treated human LACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or IGF‐II‐induced growth of human LAC’s. The ablation of VAChT levels by siRNA methodology reversed the apoptotic activity of vesamicol. We also observed that vesamicol caused cell death by inhibiting Akt phosphorylation. Taken together, our data shows that inhibition of nicotine‐induced cholinergic pathway by agents such as vesamicol may be useful for the treatment of human LACs.

Grant Funding Source: Supported by an NIH R15‐Area Grant, a FAMRI YCSA Grant, and WV INBRE
Translational and post‐translational regulation of XIAP by eIF2α and ATF4 promotes ER stress‐induced cell death during the unfolded protein response (151.5)
Authors: Hiramatsu Nobuhiko , Messah Carissa , Han Jaeseok , LaVail Matthew , Kaufman Randal , Lin Jonathan
Published: June 2021

Chronic endoplasmic reticulum (ER) stress causes cell death and contributes to disease pathogenesis and progression. ER stress activates intracellular signaling pathways termed the Unfolded Protein Response (UPR). The mechanisms by which chronic ER stress and UPR promote cell death are poorly understood. The PERK arm of the UPR has been implicated in the induction of cell death through its upregulation of proapoptotic CHOP transcription factor. Chop‐/‐ cells are only partially resistant to ER stress‐induced apoptosis, and CHOP overexpression alone does not induce apoptosis. These findings strongly suggest that additional mechanisms are involved in regulating cell death downstream of PERK. Here, we find chronic ER stress dramatically suppresses XIAP protein levels. We find that PERK signaling is responsible for suppressing XIAP by eIF2a‐dependent translational attenuation and ATF4‐mediated proteasomal degradation. PERK’s down‐regulation of XIAP occurs independently of CHOP. Loss of XIAP leads to increased cell death, while XIAP overexpression significantly enhances resistance to ER stress‐induced cell death, even in the absence of CHOP. We propose a “two‐hit” model of ER stress‐induced apoptosis involving concomitant CHOP upregulation and XIAP down‐regulation both regulated by PERK.
Jaw muscle reconstruction and vector analysis in ornithischian dinosaurs (17.4)
Authors: Nabavizadeh Ali
Published: June 2021

Jaw muscle reconstruction has long been utilized for inferences in the paleobiology of ornithischian dinosaur feeding mechanisms. Previous studies in dinosaur jaw musculature have used extant phylogenetic bracketing methods to qualitatively compare muscle origins and insertions in a select few ornithischian genera. The current study uses criteria from previous jaw muscle studies, as well as more in‐depth case‐by‐case analyses, to reconstruct musculature and measure vector angles of the m. adductor mandibulae externus (MAME) complex as well as m. adductor mandibulae posterior (MAMP) in a large diversity of ornithischian taxa spanning all subclades. Results of both MAME and MAMP vectors show general trends from a mid‐range caudodorsal orientation in basal ornithischians and most armored thyreophorans to a decrease in vector angles indicating more caudally oriented jaw movements convergently in other taxa (e.g., derived thyreophorans, basal ornithopods, lambeosaurines, pachycephalosaurs, and derived ceratopsids). The extensive amount of variation in muscle vectors, both between and among subclades, however, suggests highly variable feeding mechanisms among genera and shows that feeding mechanisms of an entire subclade should not be based only on a single taxon, as has been previously done.
Individual and group performance during an interactive lecture: anatomy of a session (211.2)
Authors: Pizzimenti Marc
Published: June 2021

Interactive lecturing (IL) techniques attempt to shift the focus of the lecture toward student engagement in learning. These techniques, combined with effective activities, can be applied to large groups (> 100 students), yet work equally well for smaller class sizes. The current work investigated the temporal and performance data of students during an interactive lecture to determine the positive impact of such a session. In the session, students were engaged in answering questions individually, group discussions coupled with elaboration, and whole class debriefing exercises. Details from a repeated (8X) IL session in Gross Anatomy were analyzed and compared student performance on a series of questions (individual vs. group) and the amount of time spent in each of the various components of the lecture. Individual performance on questions (69.9%) was greatly enhanced after group discussion (88.2%) of the anatomical concept inherent in the question, and this discussion/elaboration likely contributed to this improved performance (p < 0.001). Moreover, the time that students spent in discussion and debriefing was highly correlated with the difficulty of the concept (as indicated by larger pre/post differences) being investigated. On average, the amount of time students were engaged in activities that were likely to promote learning was similar to that that spent in the whole group debriefing. Students felt that the individual and group involvement helped them to better understand relevant anatomical concepts of the autonomic nervous system. These data suggest that planned activities can serve to promote student learning in large/small class settings.
Unexpected genotype‐phenotype associations in bone dysplasias driven by whole exome sequencing (213.2)
Authors: Lee Brendan
Published: June 2021

Over the past three decade the study of Mendelian forms of skeletal dysplasias best represented by Osteogenesis Imperfecta (OI) has led to the identification of disease mechanisms that affect both the quality and quantity of bone. This has been revolutionized by next generation sequencing and whole exome analysis. They have led to the discovery of enormous genetic heterogeneity that is becoming the rule for genetic phenotypes. In the case of OI, structural mutations in the most abundant components of matrix type I collagen, protein complexes that modify and chaperone collagen, and most recently components of signaling pathways have all been implicated. Together they underscore the factors the regulate mineralization of extracellular matrix and the differentiation and function of osteoblasts, osteoclasts, and osteocytes. In so doing they have also pointed to novel therapeutic strategies that may also need to be customized based on nature and consequence of mutation. Finally, the most recent data on the mutations that affect matrix‐cell signaling and cell‐cell signaling point to overlap with early onset osteoporosis phenotypes.
Specialized ribosomes: a new frontier in gene expression and organismal development (213.3)
Authors: Barna Maria
Published: June 2021

The regulatory logic for how the one‐dimensional genetic code is translated into three‐dimensional morphology in a multicellular organism poses one of the greatest challenges to modern biology. Notably, the prevailing dogma has been that the ribosome is an integral but passive participant in directing how the genome is functionally expressed. Our findings unexpectedly reveal that fundamental aspects of gene regulation and mammalian development are instead controlled by “specialized ribosomes,” harboring a unique composition or activity, which direct where and when specific proteins are made. For example, we have identified that RPL38, one of the approximately 80 core ribosomal proteins (RPs), acts to establish the mammalian body plan by selectively regulating the translation of Homeobox mRNAs, key master regulators of animal development. These findings suggest newfound specificity to how the genomic template is decoded into proteins to instruct key cell fate decisions. Collectively, our ongoing work seeks to define the regulatory basis by which “specialized ribosomes” control gene expression. I will present our recent findings that identify novel cis‐acting RNA elements within mammalian 5’ UTRs, which act as regulatory filters that interface with specialized ribosomes to regulate spatial‐temporal gene expression in vivo. We have also carried out state‐of‐the‐art mass spectrometry to delineate for the first time ribosome heterogeneity during cellular differentiation and to define the repertoire of transcripts that rely on specialized ribosome components for cell fate specification and differentiation. Together, these studies reveal that specialized translational machinery in conjunction with unique cis‐acting RNA elements within the 5’ UTRs of mammalian mRNAs provide a new layer of regulatory control to gene expression that guides evolution and organismal development.
Cognitive effects of methamphetamine in humans: separating fact from hysteria (222.3)
Authors: Hart Carl
Published: June 2021

This talk provides a critical review of findings from recent research investigating the impact of recreational methamphetamine (MA) use on human cognition. Included in the discussion are findings from studies that have assessed the acute and long‐term effects of MA on several domains of cognition. In addition, relevant neuroimaging data are reviewed in an effort to better understand neural mechanisms underlying MA‐related effects on cognitive functioning. In general, the data on acute effects show that MA improves cognitive performance in selected domains. Regarding long‐term effects on cognitive performance and brain‐imaging measures, statistically significant differences between MA users and control participants have been observed on a minority of measures. More importantly, however, the clinical significance of these findings may be limited because cognitive functioning overwhelmingly falls within the normal range when compared against normative data. Despite this, there seems to be a propensity to interpret any cognitive and/or brain difference as a clinically significant abnormality. The implications of this situation are multiple.
GDE2 downregulates Notch signaling to promote neurogenesis through GPI‐anchor cleavage (223.1)
Authors: Sockanathan Shanthini
Published: June 2021

The six‐transmembrane protein GDE2 induces the differentiation of postmitotic motor neurons by downregulating Notch in adjacent motor neuron progenitors. GDE2 function depends upon the integrity of its extracellular enzymatic domain that is homologous to bacterial glycerophosphodiester phosphodiesterases (GDPD). GDPDs metabolize glycerophosphodiesters into glycerol‐3‐phosphate and their corresponding alcohols, but it is unclear if and how GDE2 utilizes this mechanism to inhibit Notch activation. In this talk, I will discuss our recent observations that GDE2 regulates Notch signaling non cell autonomously, and that it does so through novel enzymatic mechanisms that control the surface availability of the Notch ligand Dll1.
Gene expression genomics in T cells (225.2)
Authors: Teichmann Sarah
Published: June 2021

T helper cells are central to mammalian adaptive immunity, as the different subtypes modulate the immune system by either activating or repressing it. These cells are easily experimentally accessible as they are non‐adherent, and can be studied ex vivo or in vitro. We have used this system to study basic principles of the global regulation of gene expression using next generation sequencing technologies, both at the level of populations of cells (Hebenstreit et al., 2011, Mol Sys Biol; Hebenstreit et al., 2011, Nucleic Acids Res) and at the level of single cells (Brennecke et al., Nature Methods, 2013). Our bulk and single cell RNA‐sequencing data has also directed our attention to a potential new signalling system in T helper cells based on steroid production by these cells, illustrating the power of this genomic approach for providing specific biological insights.
Exploring the mechanism of protein birth (226.1)
Authors: Cavagnero Sylvia
Published: June 2021

Despite much progress over the last six decades, we are still far from understanding the mechanism of protein folding in vitro and, most importantly, in the cell. This lack of information poses tremendous challenges to progress in many areas of fundamental biology, biotechnology, protein design, drug production and neurodegenerative‐disease research. Most soluble cellular proteins fold co‐ and immediately post‐translationally and the ribosome serves as an obligatory host for this process. A compelling unanswered question is whether the ribosome plays any active role in protein folding in the cellular environment. This lecture will discuss how the ribosomal surface and its peculiar electrostatic properties, together with the vectorial nature of protein synthesis, modulate the balance between protein folding and aggregation during the crucial stages of protein birth.
Mechanisms of cellular proteostasis: insights from single molecule approaches (226.3)
Authors: Bustamante Carlos , Kaiser Chrisian , Tinoco Ignacio , Goldman Dan
Published: June 2021

Cells employ a variety of strategies to maintain the proteome homeostasis. Beginning during protein biogenesis, the ribosome itself and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP‐dependent proteases. In this talk I will describe how the application of single‐molecule methods, in particular optical tweezers, is shedding new light on the molecular mechanisms of quality control during the life cycle of proteins.
Searching for conformationally‐selective small molecule therapeutics using ion mobility‐mass spectrometry (227.1)
Authors: Ruotolo Brandon
Published: June 2021

Mass Spectrometry (MS) plays a number of key roles in the discovery and development phases for modern pharmaceutical compounds. Historically, however, MS has had a relatively limited role the drug discovery process in comparison to high‐throughput fluorescence and radiometric screens. This picture may be changing, however, as many presumptive protein targets are coupled to human disease pathways through specific protein‐protein interactions and protein conformations, rather than through enzyme activities. This fact is driving the development of high‐throughput analytical tools that put a stronger emphasis on protein structure information.

We have been actively developing nano‐electrospray ionization coupled to ion mobility‐mass spectrometry (IM‐MS) into such a tool. Our IM‐MS protocols include the collision induced unfolding (CIU) of proteins and complexes, which functions as a highly‐sensitive and selective analog of isothermal calorimetry for gas‐phase protein‐ligand complexes, able to characterize protein stabilities at low concentrations within mixtures. Here, we will present our latest results where we demonstrate the ability of this technology to detect activation state‐selective kinase inhibitors, evaluate key conformational changes within amyloid protein‐ligand complexes, and identify signatures associated with cooperative ligand binding within multiprotein assemblies.
The beginning of the end: how the Atg1 scaffold nucleates autophagosome biogenesis (237.2)
Authors: Hurley James , Stanley Robin , Ragusa Michael
Published: June 2021

Autophagy is a conserved mechanism that is essential for cell survival in starvation. Moreover, autophagy maintains cellular health by clearing unneeded or harmful materials from cells. Autophagy proceeds by the engulfment of bulk cytosol and organelles by a cup‐shaped double membrane sheet known as the phagophore. The phagophore closes upon itself to form the autophagosome, which delivers its contents to the vacuole or lysosome for degradation. A multiprotein complex consisting of the protein kinase Atg1 together with Atg13, Atg17, Atg29, and Atg31 (ULK1, ATG13, FIP200, and ATG101 in humans) has a pivotal role in the earliest steps of this process. In yeast, the phagophore nucleates from a cluster of 20‐30 nm diameter Atg9‐containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). We determined the crystal structure of a 2:2:2 complex of the earliest‐acting PAS proteins, Atg17, Atg29, and Atg31, at 3.05 Å resolution. Atg17 is crescent‐shaped with a 10 nm radius of curvature. Dimerization of the Atg17‐Atg31‐Atg29 complex is critical for both PAS formation and autophagy, and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17‐Atg31‐Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C‐terminal EAT domain of Atg1 sense membrane curvature, dimerizes, and tethers lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.
Urine caffeine and caffeine metabolites as indicators of dietary caffeine exposure and metabolism phenotypes in population studies (245.1)
Authors: Rybak Michael , Pao Ching‐I , Sternberg Maya , Pfeiffer Christine
Published: June 2021

Caffeine has been examined as a risk factor for various diseases and conditions, with dietary intake data almost always being used to quantify caffeine exposure. Caffeine and its metabolites are found at detectable levels in the urine, and the measurement of these compounds as a means of assessing caffeine exposure has been proposed. In addition to determining dietary caffeine exposure, the use of urine measures of caffeine and its metabolites have been used to assess differences in CYP 1A2 and NAT2 activity in situations where uncontrolled dietary consumption of caffeine is present. Our laboratory has developed an LC‐MS/MS method capable of determining urine caffeine and 14 caffeine metabolites at concentrations as low as 50‐100 nmol/L depending on the analyte. We applied method to the analysis of a convenience sample set (n=115) of urine samples from anonymous donors, and were able to detect a majority of the analytes in 蠅99 % of samples as well as calculate phenotyping ratios for CYP 1A2 and NAT2. Our method is the first of its kind to use stable isotope labeled internal standards for all analytes, and was capable of method imprecision that was <7 % CV when analyte concentrations were 蠅 1 µmol/L. We have successfully applied this method to the analysis of urine specimens from the NHANES 2009‐2010.
Plasma high‐resolution metabolomic profiling reveals upregulation of specific resolvins in patients with pulmonary tuberculosis (248.3)
Authors: Frediani Jennifer , Jones Dean , Tukvadze Nestan , Sanikidze Eka , Kipiani Maia , Uppal Karan , Kurani Shaheen , Hebbar Gautam , Colas Romain , Dalli Jesmond , Serhan Charles , Tangpricha Vin , Blumberg Henry , Ziegler Thomas
Published: June 2021

Background: Characterization of metabolites altered by tuberculosis (TB) disease may identify pathophysiologic pathways. Resolvins are lipid mediators derived from endogenous EPA and DHA critical for resolution of inflammation.

Methods: Plasma samples from 17 patients (35±12 yrs old) with new pulmonary TB and 17 matched household contacts without TB (controls) were analyzed using high‐resolution LC‐MS. False discovery rate (FDR; q=0.05) and hierarchal cluster analysis was used to distinguish accurate mass ions (metabolites) significantly different between groups. The METLIN Metabolite Database was used to match ions to known metabolites. Identification of specific D‐series resolvins (RvD1 and RvD2) was confirmed by LC‐UV‐MS/MS.

Results: Over 23,000 metabolites were detected in untargeted metabolomic analysis, of which 61 were significantly different between the two groups by FDR. Eight metabolite clusters were evident containing several metabolites quantitatively upregulated in patients with TB (e.g. glutamate, choline derivatives, anti‐TB drug metabolites and several lipids, including likely M. tb cell wall lipids and resolvins RvD1 and RvD2.

Conclusions: This pilot study revealed metabolites that are upregulated during newly diagnosed TB potentially involved in pathogenesis. Upregulation of specific resolvins may mediate recovery from inflammation in TB disease.

Grant Funding Source: Supported by grants from the National Institutes of Health Fogarty grants D43 TW007124
Alpha‐tocopherol supplementation in lactating women impacts stereoisomer distribution but not total tocopherol content (250.1)
Authors: Sherry Christina , Oliver Jeffery , Marriage Barbara
Published: June 2021

Eighty‐eight lactating women were randomized in 3 groups: Synthetic, 101.2 IU dl‐alpha‐tocopherol; Mixed, 30 IU d‐alpha‐tocopherol + 50.6 IU dl‐alpha‐tocopherol; or Natural, 60 IU d‐alpha‐tocopherol to assess the effect of supplementation on lactating women. Breast milk and plasma tocopherols were assessed at the beginning and end of the study by HPLC. There were no differences in alpha, gamma, and delta‐tocopherol in breast milk and plasma or vitamin E intake. There was a significant correlation between breast milk and plasma gamma‐tocopherol (r=0.52, P<0.0001). RRR was the most abundant stereoisomer in breast milk and plasma and increased over time. The natural supplement had a 1.4 and 1.2‐fold (breast milk) and 1.5 and 1.2‐fold (plasma) increase in RRR (percent of total stereoisomers) compared to the synthetic and mixed groups, respectively (P<0.05). There was a 2.4 and 3.1‐fold decrease in the percent of synthetic stereoisomers (2S, RSS, RRS and RSR) in the natural group compared to the synthetic group, in breast milk and plasma, respectively, and a 1.3 and 1.5‐fold decrease in breast milk and plasma compared to the mixed group (P<0.05). Taken together, alpha‐tocopherol stereoisomers in breast milk and plasma of lactating women change according to the type of supplement consumed, and RRR had a greater impact on blunting synthetic stereoisomer distribution likely due to the high percentage of RRR in all groups.

Grant Funding Source: Supported by Abbott Nutrition
Ketoisocaproic acid suppresses insulin‐stimulated glucose transport in parallel with activation of mTORC1 in muscle cells (258.1)
Authors: Moghei Mahshid , Tavajohi‐Fini Pegah , Adegoke Olasunkanmi
Published: June 2021

High protein diets are sometimes prescribed in weight management interventions; however these diets and amino acids (AA) have been linked to insulin resistance. Much of the attention has been on the branched‐chain amino acid (BCAA) leucine because of its ability to activate the mammalian target of rapamycin complex 1 (mTORC1). This complex and its substrate (S6K1) are implicated in AA‐induced insulin resistance due to their inhibitory serine phosphorylation of insulin receptor substrate 1 (IRS1). Since AAs other than leucine are present in dietary proteins, we examined their roles. Valine suppressed insulin stimulated glucose uptake (Glu‐trans) in L6 myotubes by 47% (P<0.05) compared to a suppression of 22% observed with leucine. The effect was not due to a greater intracellular accumulation of valine. However, valine and leucine, but not isoleucine, increased phosphorylation of S6K1 (thr389) and IRS1 (ser612 (P<0.05)). This is consistent with a lack of effect of isoleucine on Glu‐trans. We also examined the effect of 2 metabolites of leucine, ketoisocaproic acid (KIC) and isovaleryl CoA. KIC, but not isovaleryl CoA, suppressed Glu‐trans by 25% (P<0.05) and increased S6K1 and IRS1 phosphorylation. Strikingly, AA other than the BCAA, especially arginine and glutamine, also suppressed Glu‐trans by 22‐47% (P<0.05). Inhibition of Glu‐trans was not always linked to mTORC1/S6K1 and IRS1 phosphorylation. We conclude that KIC may mediate the suppressive effects of leucine on insulin stimulated glucose transport, and that amino acids other than leucine can induce insulin resistance of glucose transport.

Grant Funding Source: Supported by NSERC and Faculty of Health, York University
Remethylation with dietary methyl donors contributes a significant proportion of methionine for the synthesis of protein and creatine in neonatal piglets (258.3)
Authors: Robinson Jason , McBreairty Laura , Harding Scott , Randell Edward , Brunton Janet , Bertolo Robert
Published: June 2021

Methionine is an essential amino acid that is in high demand in neonates for both protein synthesis and as the primary methyl donor for transmethylation (TM) reactions. The vast majority of methyl groups are used for synthesis of creatine (CR) and phosphatidylcholine (PC). The demethylated methionine can be remethylated by dietary methyl donors such as folate, choline and betaine, but to what extent is unclear. We hypothesized that these methyl donors provide a nutritionally significant proportion of available methionine for TM and protein synthesis. 4‐8 d old piglets were fed a diet devoid (n=8) or replete (n=7) of methyl donors at 80% of the methionine requirement; methionine and phenylalanine isotopes were infused to calculate flux to products. Whole body protein synthesis and breakdown were respectively, 15% (p<0.05) and 20% (p<0.04) lower during methyl deficiency, corresponding to a ~70% reduction in fractional protein synthesis (Ks) in muscle (p<0.03) and unchanged Ks in liver. Methyl deficiency also led to 25% lower hepatic TM to creatine (p<0.02), which was offset by a 2‐fold greater TM to PC (p<0.03), due to choline deficiency which increases PC synthesis via TM. In conclusion, dietary methyl donors can re‐synthesize a substantial proportion of methionine that is used primarily for protein synthesis and potentially growth.

Grant Funding Source: Supported by CIHR
Anthocyanin degradation in red Norland potatoes (259.2)
Authors: Carlson Justin , Roe Mikel , McManimon Theresa , Hegeman Adrian , Tong Cindy
Published: June 2021

Anthocyanins are water‐soluble, polyphenolic compounds responsible for coloration in fruits and vegetables, and are known to display antioxidant, anti‐carcinogenic and prebiotic properties in human subjects. This study was conducted to analyze the chemical degradation of anthocyanins in Red Norland potatoes based on colorimetric, spectrophotometric and mass spectrometry analysis. Tuber color and anthocyanin chemistry of fresh and stored (at 4ºC for 90 days) tubers were measured. Anthocyanins were also extracted in acidified methanol from peel tissue and their mean absorbance was measured at 513 nm. Colorimetric analysis of light, chroma and hue angle indicated that tubers darkened over storage time and became less intense in color; storage trends indicate loss of anthocyanins, although not significant due to high variation amongst tubers. Chemical changes in the six most abundant anthocyanins were analyzed between the fresh and stored samples via high performance liquid chromatography ‐ mass spectrometry. The six most abundant anthocyanins varied between a 2‐fold and 8‐fold decrease in concentration in the stored compared to fresh tubers. Based on results, for optimal pro‐health effects of anthocyanins, Red Norland potatoes should be consumed closest to harvest as possible.