bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid homeostatic effects of oral potassium loading on the kidney
Authors: Grossmann Solveig , Sorensen Mads Vaarby , Roesinger Marian Andreas , Loffing‐Cueni Dominique , Barmettler Gery , Ziegler Urs , Odermatt Alex , Loffing Johannes
Published: June 2021

A dietary K+ load induces a rapid kaliuresis and natriuresis, which may occur even before plasma K+ and aldosterone (aldo) levels increase. To get insights into underlying molecular mechanisms, we analyzed the effects of K+ loading on plasma ion concentrations, aldo levels, urinary ion excretion, and expression and/or phosphorylation of renal ion transport proteins in mice that received 2% K+ either via their diet or gastric gavage. After high K+ treatment, plasma K+concentrations rose rapidly (15’) followed by a significant rise of plasma aldo (30’). A rapid and sustained kaliuresis as well as rapid but short natriuresis was detectable. Functional changes were accompanied by a rapid and sustained (15’–6h) dephosphorylation of the NaCl co‐transporter (NCC) and a late (6h) up‐regulation of proteolytically activated epithelial sodium channels (ENaC). The rapid effects on NCC were independent from the co‐administered anion. NCC dephosphorylation was also aldo‐independent, as indicated by experiments in aldo‐deficient mice. The observed urinary Na+ loss relates to NCC, since it was markedly diminished in NCC‐deficient mice. Thus, rapid down regulation of NCC and subsequent activation of ENaC are part of the homeostatic adaptation of the kidney to the increased K+ intake. Down‐regulation of NCC likely explains the natriuretic effect of an acute oral K+ load and may improve renal K+ excretion by increasing the amount of intraluminal Na+ that can be exchanged against K+ in the aldo‐sensitive distal nephron.
Ventral respiratory column tonic expiratory neurons: Nodes for convergent afferent modulation of inspiratory drive
Authors: Segers Lauren S. , Nuding Sarah C. , Ott Mackenzie M. , Dean Jay B. , Bolser Donald C. , O'Connor Russell , Morris Kendall F. , Lindsey Bruce G.
Published: June 2021

The medullary ventral respiratory column (VRC) has an essential role in generating the motor pattern for breathing and inspiratory (I) drive. Contemporary models include a rostral‐to‐caudal excitatory I neuron chain extending to premotor populations that also acts upon inhibitory I neurons with recurrent and feed‐forward connections upon other VRC excitatory and inhibitory I neurons and expiratory (E) neurons. Prior work suggests (i) baroreceptor stimulation inhibits inspiratory drive in part via evoked tonic E (t‐E) neuron activity and (ii) central chemoreceptor stimulation enhances I drive via disinhibition through reduced t‐E neuron activity. A recent computational neuromechanical model (O'Connor et al. 2012) predicted that inhibition of t‐E neurons amplifies I drive during coughing ‐ a result supported by in vivo data (Segers et al. 2012). We now report recording sites of pericolumnar t‐E neurons (n=140) and show that selective stimulation of carotid chemoreceptors via close injection of CO2‐saturated saline reduces the I‐phase activity of t‐E cells coincident with increased I drive in vagotomized decerebrate cats. Spike train cross correlation and gravity clustering methods revealed short‐time scale impulse synchrony in 24 t‐E neuron pairs. We conclude that multiple afferent systems tune inspiratory drive via actions of coordinated groups of VRC t‐E neurons. Support: NIH grant NS19814.
Effects of IV remifentanil (Remi) on the discharge of canine pontine respiratory group (PRG) neurons in the parabrachial complex (PB)
Authors: Prkic Ivana , Mustapic Sanda , Radocaj Tomislav , Stucke Astrid G. , Stuth Eckehard A. , Hopp Francis A. , Zuperku Edward J.
Published: June 2021

Mu‐opioids at clinical doses produce bradypnea, which is reversed by microinjection of naloxone into the pontine PB. We studied the effects of the mu‐opioid agonist Remi (IV) on PRG neuron discharge patterns in decerebrate, vagotomized, ventilated, paralyzed dogs. A NeuroNexus 16‐electrode microprobe was used to record from multiple PRG neurons before, during and after IV Remi. Histograms (CTHs) of PRG discharge patterns, triggered from the phrenic neurogram (PNG), were used to quantify Remi effects on ten PRG neuron subtypes. Remi (0.5–0.8 mcg/kg/min) increased TI and TE by 21% and 23% respectively, and decreased peak PNG by 33±3%. PRG neurons (n=197) were located 0.5–2 mm caudal to inferior colliculus, 5–6.5 mm lateral to midline and 2.5–4.5 mm below the dorsal surface. The depression (peak Fn) of all PRG neurons (28±3%; n=197) was similar among 9 of 10 subtypes, but Edec neurons were more depressed (70±6%; n=24; p<0.001). Magnitude of neuronal depression was not related to control Fn, or changes in TI or TE except for Icon neurons and TI (r = −0.72; p<0.001). PRG neurons, except Edec, were less depressed than the peak PNG. Marked depression in PRG Edec neurons may underlie loss of recurrent laryngeal Edec activity. Opioid‐induced depression of PRG subtypes coincident with bradypnea is consistent with reduced excitation to medullary rhythm generation. Supported by VA Merit Review Award: 1 I01 BX000721–01.
Hepoxilin A3 is a key driver of neutrophil migration in a model of acute P. aeruginosa infection.
Authors: Pazos Michael A , Hurley Bryan P
Published: June 2021

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states including acute bacterial pneumonia, cystic fibrosis, ARDS, as well as several others. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown in vitro to mediate the transepithelial passage of neutrophils. Using a murine model of acute infection with Pseudomonas aeruginosa, we demonstrate HXA3 plays a significant role in transepithelial migration of neutrophils in vivo. Here we present data that modulation of HXA3 signaling during bacterial lung infection of mice significantly reduces neutrophil accumulation in the airspace. Using bronchoalveolar lavage, we measure the numbers of neutrophils and quantify lipid‐associated chemotactic bioactivity within the airspace. We target HXA3 through inhibition of the HXA3 biosynthetic pathway, enzymatic degradation of HXA3 chemotactic potential, and competitive inhibition. We conclude that modulation of HXA3 signaling represents a viable therapeutic target for the prevention of neutrophil infiltration and subsequent immunopathology.
Exclusive breastfeeding in the first 24 hours postpartum associated with improved breastfeeding outcomes of low‐income, overweight and obese women
Authors: Chapman Donna , Pérez‐Escamilla Rafael
Published: June 2021

We evaluated the relationship between exclusive breastfeeding (EBF) in the first 24 hours (EBF24) postpartum (pp) and breastfeeding (BF) outcomes of overweight/obese women, using data from a randomized trial evaluating a peer counseling intervention (vs usual care). Demographic, biomedical and infant feeding data were collected at birth and monthly through 6 months pp. Bivariate and Cox survival analyses identified predictors of any and EBF through 6 months pp. 45% of women in both the intervention and control groups EBF24, with no effect of the intervention on BF/EBF rates, thus we did not control for study group. Puerto Ricans (vs. others) were significantly less likely to EBF24 (p<0.01). There were no baseline differences in BF self‐efficacy or other relevant covariates between those who did (n=51) vs did not (n=66) EBF24. Adjusted Cox survival analyses showed those who EBF24 were more likely (HR: 1.82; 95% CI: 1.11–3.0) and Puerto Ricans were less likely (HR: 0.42; 95% CI: 0.26–0.69) to continue any BF in the first 6 months pp. Participants who EBF24 were more likely to report EBF in the past day or week at months 1 – 4 pp (all p<0.05). These preliminary findings suggest that maternity care practices during the first 24 hours pp are significantly associated with BF outcomes.

Funded by Donaghue Medical Research Foundation and NIH/NCMHD grant P20MD001765
Fatty acid bioavailability study of single oral doses of milled chia seed snack clusters or chia seed oil in healthy subjects
Authors: Nieman David Christopher , Gillitt Nicholas D , Knab Amy M. , Cialdella‐Kam Lynn , Jin Fuxia
Published: June 2021

Chia seeds are a rich source of alpha‐linolenic acid (ALA), but the acute plasma ALA response to chia seed or chia seed oil ingestion has not yet been determined. Female subjects (N=16, 20 to 45 y) reported to the lab after an overnight fast on 3 separate occasions and ingested, in random order by visit, a snack cluster (PL), snack cluster with 8 g milled chia seed (CS; 1.3 mg ALA), or chia seed oil (CSO; 1.3 mg ALA). A catheter was placed in an antecubital vein, and blood samples were taken at pre‐ingestion and postingestion (0.5, 1.5, 2.5, 3.5, 4.5, and 6 h) during which the subjects rested and ingested a 550 kcal breakfast at post‐1.75 h. Subjects returned to the lab to provide a 24 h sample. Blood samples were analyzed for ALA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) using GC‐MS. A 3 × 8 repeated measures ANOVA revealed a significant interaction effect for ALA (P<0.001) but not for EPA (P=0.581) or DHA (P=0.445). ALA in CS (↑82%) and CSO (↑91%) increased significantly 2.5 h post‐ingestion and stayed elevated above PL with some attenuation at 3.5 h, dropping to pre‐ingestion levels at 24 h. In conclusion, these bioavailability data indicate that 1.3 mg ALA from CS or CSO nearly doubles plasma ALA levels within 2.5 h ingestion, staying elevated for several hours, and then falling to pre‐ingestion levels without conversion to EPA or DHA within 24 h.

Funded by Dole Food Company, Inc.
Mast cell deficiency induces inflammation and exacerbates inflammatory bowel symptoms in IL10−/− mice
Authors: Zhang Hanying , Xue Yansong , Wang Hui , Huang Yan , McCormick Richard J , Means Warrie J , Du Min , Zhu Meijun
Published: June 2021

Background

The interleukin 10 deficient (IL10−/−) mice has abnormal intestinal immune responses against enteric antigens, leading to sustained inflammation, and develops inflammatory bowel disease spontaneously. Mast cells abundantly present under gut epithelium and are critical for gut immune response. The objective is to test the role of mast cells in gut inflammation and barrier function using IL10−/− mice.

Methods

Mast cell deficient (KitW‐sh/W‐sh) mice were crossbred with IL10−/− mice to obtain double knockout (DKO) mice. Mucosal damage and colitis status were compared between DKO and their IL10−/− littermates.

Results

Mast cell deficiency enhanced gut mucosal damage and colitis as revealed by increased gut permeability and impaired tight junction protein expression, increased pathobiological score, higher myeloperoxidase content and pronounced goblet cell loss in DKO mice. In addition, DKO mice showed splenomegaly and elevated serum TNFα and INF levels, indicating exaggerated systemic inflammation. Consistently, the colon of DKO mice demonstrated enhanced inflammation and oxidative stress.

Conclusions

Mast cell deficiency induced systemic inflammation, and inflammation and oxidative stress in the colon, which might explain the impaired gut barrier function and severe colitis in DKO mice. NIH 1R15HD073864; INBRE P20RR016474; USDA 2008–35203‐19084; 2009–65203‐05716
Investigating Novel Roles for Exogenous and Endogenous Galectin‐3 in Controlling Vascular Inflammation
Authors: Gittens Beatrice , Perretti Mauro , Cooper Dianne
Published: June 2021

Studies in Galectin‐3 (Gal‐3) null mice indicate a role for this β‐ galactoside‐binding lectin in β2integrin‐independent neutrophil recruitment, whilst the recombinant protein induces L‐Selectin shedding and CD11b upregulation in naïve and primed neutrophils. However, its role in leukocyte trafficking in β2‐dependent models has not been systematically addressed.

Intravital microscopy of the cremaster muscle was performed in C57BL/6 (WT) or Gal‐3 null mice, following intrascrotal injection of PBS, IL‐1β (30ng), TNF‐α (300ng) or Gal‐3 (200–1000ng) and intravenous administration of anti‐Ly6G to label neutrophils.

Treatment with IL‐1β and TNF‐α significantly reduced leukocyte rolling velocities in WT mice, an effect that was absent in Gal‐3 null mice. In addition, a significant reduction in cell emigration was observed in the Gal‐3 null mice treated with IL‐1β. Administration of Gal‐3 to WT mice resulted in a significant reduction in rolling velocity associated with increased numbers of adherent and emigrated leukocytes, approximately 50% of which were Ly6G‐positive neutrophils.

These data suggest that Gal‐3 positively regulates leukocyte recruitment in a stimulus dependent fashion in the inflamed microcirculation. This study unveils novel biology for exogenous and endogenous Gal‐3 in controlling vascular inflammation.

Supported by British Heart Foundation Studentship FS/10/009/28166
Avidity regulation of the leukocyte integrin LFA‐1
Authors: Lefort Craig T. , Ley Klaus
Published: June 2021

Leukocyte‐endothelial cell interactions are mediated by a diverse set of adhesion molecules, including heterodimeric integrin receptor binding to immunoglobulin superfamily glycoproteins. In neutrophils, LFA‐1 is the predominant integrin receptor involved in the transition from a rolling to an arrested phenotype. The function of LFA‐1 in regulating leukocyte interactions with the inflamed vascular wall may involve changes in its affinity for ligand as well as increased valency, or clustering, within the plasma membrane. We have recently found that talin‐1 and Kindlin‐3, two cytoplasmic proteins that associate with LFA‐1, have distinct roles in regulating its structural conformation and ligand binding affinity. In the current study, we have tested whether talin‐1 and Kindlin‐3 are involved beyond affinity regulation in modulating the valency of LFA‐1/ICAM‐1 bonds using functional and ligand binding assays. Our data indicate that artificial enhancement of LFA‐1 affinity results in leukocyte arrest, even in the absence of talin‐1 and Kindlin‐3. Supported by the American Heart Association 12SDG12080281.
Integrin Crosstalk Regulation of Human Neutrophils Adhered to Fibronectin and Beta‐glucan
Authors: Johnson Courtney Michele , Byrd Angel , Reichner Jonathan
Published: June 2021

Complement Receptor 3 (CR3), an integrin found on neutrophils, plays an important role in fungal recognition. Co‐ligation of CR3 with fibronectin (Fn) at the I‐domain and β‐glucan (B) at the lectin‐like domain is possible due to spatially distinct locations. Dual ligation of CR3 with Fn+B induces homotypic aggregation of primed neutrophils, a response representative of the immune cells encounter with fungi within tissues. To understand the molecular mechanisms involved in this response, we explored the role of other integrins and intercellular signaling molecules. Antibody blockade of VLA3, the canonical laminin receptor, showed abrogation of aggregate formation on immobilized Fn+B. VLA3 did not show an effect on either substrate alone and was specific as blockade of other β1 integrin family members failed to prevent aggregation including VLA5, the canonical Fn receptor. Western blotting analysis showed a significant increase in ERK phosphorylation on Fn+B as compared to either substrate alone. VLA3 inhibition on Fn+B showed a decrease in p‐ERK while VLA5 inhibition showed a complete loss of p‐ERK. Findings indicate integrins may communicate via crosstalk pathways involving p‐ERK. NIH GM‐066194
HS1 regulates chemokine‐induced Rap‐1 activation via PKA in neutrophils to facilitate extravasation
Authors: Schnoor Michael , Latasiewicz Joanna , Jing Ding , Vestweber Dietmar
Published: June 2021

Neutrophil extravasation is a critical step in innate immunity in response to tissue injury or invading pathogens. Inflammatory signals activate β2‐integrins and facilitate neutrophil adhesion onto the endothelial apical surface and intraluminal crawling to the site of diapedesis. Hematopoietic cell‐specific lyn substrate (HS1) regulates actin dynamics at cell contacts and may thus be important for extravasation. Indeed, intravital microscopy of the cremaster revealed an increased rolling velocity and an inhibition of neutrophil adhesion and transmigration in HS1‐deficient mice. This effect can be explained by disturbed Rap1 activation causing reduced neutrophil adhesion in response to chemokine treatment. Interestingly, this process is dependent on PKA activation since PKA inhibition blocked chemokine‐induced Rap1 activation. The importance of PKA for HS1‐mediated support of extravasation is corroborated by the fact that PKA activation increases whereas inhibition reduces transmigration of WT neutrophils but not of HS1‐KO neutrophils. However, HS1 is not a direct substrate of PKA but it interacts with phosphorylated VASP. This interaction is also inhibited after PKA inhibition and may thus provide an important scaffold for Rap1 activation. Our results establish HS1 and PKA as critical signal mediators that coordinate the molecules required for Rap1 activation and neutrophil transmigration.
Developmental mechanisms for morphological evolution
Authors: Abzhanov Arkhat
Published: June 2021

One of the chief aims of modern biology is to understand the causes and mechanisms of morphological evolution. Multicellular animals display a stunning diversity of shapes and sizes of their bodies and individual structures, much of it important to their survival. What is the most efficient way to study the evolution of morphological diversity? The old‐new field of evolutionary developmental biology (evo‐devo) can be particularly useful for understanding the origins of animal forms, as it aims to consolidate advances from disparate fields such as phylogenetics, genomics, morphometrics and developmental biology. We use examples of our work on developmental mechanisms for small‐ and large‐scale morphological evolution to suggest an efficient strategy that uses highly interdependent components: (a) morphometrics, (b) identification of candidate mechanisms, and (c) functional experiments. Our studies illustrate how multifarious evo‐devo approaches taken within the three‐winged evo‐devo research program can explain developmental mechanisms for morphological evolution across different phylogenetic scales.
A novel mechanism of chondrocyte column formation in growth plate cartilage
Authors: Dudley Andrew T
Published: June 2021

Proper morphogenesis of the long bones depends in part on the specific arrangement of chondrocytes in the growth plate cartilage. Within this tissue, actions of secreted signaling molecules regulate spatial and temporal aspects of chondrocyte maturation. One crucial step is the transition from elliptical cells that are arbitrarily arranged in the extracellular matrix to columns of discoid cells that are stacked like coins. Previously, we presented evidence that columnar organization results from convergent extension cell movements that are regulated by the planar cell polarity pathway. Using new assays, we refine this model and show a link between the signaling pathways that regulate chondrocyte maturation and the control of tissue architecture.
Anatomy as a garden of educational delights: Medical education, professional formation, and the hidden curriculum
Authors: Hafferty Frederic W
Published: June 2021

Because of its symbolic and practical connection to the human body, buttressed by its historical practice of cadaver dissection, the teaching and learning of anatomy has proven to be a veritable cornucopia of pedagogical opportunities for medical students and faculty alike. In this presentation, I will examine some of these opportunities beginning with the author's own explorations of anatomy lab during the early 1970s as an under appreciated site of emotional socialization. In turn, examining these more tacit forms of learning helped to fuel my later work on the hidden curriculum of medical education, along with more recent efforts to explore the social networks of anatomy small group learning. To this end, I will explore the range of approaches used by anatomists in teaching this most fundamental of the basic sciences. Finally, we will examine how instruction in anatomy might stand as a pedagogical beacon for the other basic sciences.
Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Within 3D Bioprinted Neotissues
Authors: Evinger Albert J , Jeyakumar Jey M , Hook Lilian A , Choo Yen , Shepherd Benjamin R , Presnell Sharon C
Published: June 2021

Mesenchymal stromal/stem cells (MSC) have been studied extensively for their potential use in the generation of connective tissues for both therapeutics and biomedical research. Here, using the NovoGen™bioprinting platform and osteogenic differentiation media discovered with the high throughput combinatorial platform CombiCult®, we report the rapid fabrication of, and efficient osteogenesis in, 3D, scaffold‐free, bioprinted tissue constructs containing adipose‐derived MSC (aMSC) or aMSC and endothelial cells (EC). Constructs were bioprinted in multi‐well tissue culture vessels enabling rapid generation of replicate tissues for interrogation with standard laboratory assays. Bioprinted constructs cultured in media used for aMSC propagation showed no signs of osteogenesis. Evaluation of bioprinted 3D tissue constructs maintained in CombiCult® (OsteoMax‐XF™, EMD Millipore) differentiation media revealed marked evidence of osteogenic differentiation. By 5d post‐printing, aMSC‐containing tissue constructs exposed to CombiCult® were found to contain osteopontin‐ and alkaline phosphatase‐positive cells and mineralization was confirmed by Alizarin Red S staining. Osteogenesis was associated with elevated levels of IL‐1α, IL‐6, IL‐8, CCL2 and CXCL1. Experiments were repeated with multiple lots of commercially‐sourced aMSC implicating utility of this model for a variety of MSC sources.
Biogenesis of transport carriers
Authors: Malhotra Vivek
Published: June 2021

A chemical called Ilimaquinone (IQ) was identified based on its ability to convert the mammalian Golgi apparatus into small uniform size vesicles. We reasoned IQ hyper activates a membrane fission reaction and thus suitable for understanding this process. This reaction required a serine/threonine kinase called PKD. Surprisingly, PKD was found to control the biogenesis of transport carriers at the Trans Golgi Network (TGN) that are destined to cell surface. Other exit routes from the Golgi membranes were PKD independent. PKD binds diacylglycerol (DAG) and Arf1, and promotes the production of phosphatidylinositol‐4‐phosphate (PI4P). PI4P in turn recruits ceramide transfer protein (CERT) and the oxysterol binding protein (OSBP). The binding of Arfaptin1 to the TGN also requires PI4P. Arfaptin 1 contains a domain that has the ability to assemble into an amphipathic helix; PKD phosphorylates within this region and dissociates Arfaptin 1 from the TGN. PKD thus regulates membrane curvature by controlling the dynamics of the BAR domain containing Arfpatin 1 at the TGN. PKD also regulates the association of CERT and OSBP by phosphorylation. Altogether this suggests that PKD regulates the levels of PI4P, ceramide and sterols at the TGN, and important for transport carrier biogenesis. We have tested this hypothesis directly by affecting the levels of the ceramide product Sphingomyelin at the TGN. Perturbing the balance of Sphingomyelin inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. These findings highlight the role of lipid homeostasis in transport carrier formation. Altogether, the discovery of IQ as a Golgi vesiculating compound has been valuable for revealing the mechanism of transport carrier biogenesis during protein secretion.
Exocytic and Endocytic Mechanisms: Discovering a role for membrane curvature and curvature stress
Authors: McMahon Harvey T
Published: June 2021

Cell shape is adapted to function. Organelle shape and local membrane architectures are likewise optimised for the processes that take place on and within these microenvironments. We focus on the dynamic regulation of membrane shape, which can occur by the interplay between the transient and regulated insertion of membrane bending motifs and the detection and stabilisation of membrane shape. This approach has allowed us not only to describe the biophysics of membrane shape changes but also to take a fresh look at membrane dynamics in physiological processes like exocytosis and endocytosis. In doing so we have noted that proteins with amphipathic helices or hydrophobic membraneinserting loops are likely to effect or respond to curvature and that the membrane interaction surfaces of proteins can sense shape (like proteins of the BAR Superfamily). This molecular view has allowed us to ascribe novel cell‐biological functions to proteins (e.g. the mechanistic affect of synaptotagmin in membrane fusion) and to give a more insightful view of how these processes work. Thus we can now go from the biophysics of a molecule, to better understanding of known pathways and to the molecular characterisation of novel cellular trafficking pathways both of endocytosis and exocytosis. See: http://www.endocytosis.org/
Novel Secreted Protein Kinases
Authors: Dixon Jack E.
Published: June 2021

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated but the kinases responsible for these phosphorylations are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. A member of this protein kinase family, Fam20C, appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S‐x‐E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization. Mutations in Fam20C cause an osteoscierotic bone dysplasia in humans known as Raine syndrome. Fam20C phosphorylates dozens of other substrates as well, and is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.
Homeostatic control of cell growth and differentiation by Golgi proofreading
Authors: Demetriou Michael , Mkhikian Haik
Published: June 2021

Homeostatic proofreading is an essential feature of critical biological systems. Here we describe a homeostatic mechanism within the Golgi that controls cell growth and differentiation. Galectins bind N‐acetyllactosamine (LacNAc: Galâ1,4GlcNAc) in branched N‐glycans attached to surface glycoproteins, forming a molecular lattice that controls cell growth and differentiation by regulating receptor clustering/surface‐retention/signaling. LacNAc density and galectin avidity for N‐glycans increase proportional to the number of branches produced by the Golgi branching enzymes Mgat1, 2, 4, and 5. Mgat5 deficiency marginally reduces LacNAc content by limiting N‐glycans to three branches, yet results in Tcell hyperactivity and autoimmunity. Restricting N‐glycans to a single branch via Mgat2 deficiency or mannosidase II inhibition does not produce a more hyperactive state. Rather, a marked increase in poly‐LacNAc extension maintains galectin binding to N‐glycans, thereby preventing further enhancement of T cell activity; phenotypes reversed by deficiency of the poly‐LacNAc extension enzyme B3GnT2 or complete blockade of branching. Homeostatic maintenance of LacNAc levels is also present in epithelial and stem cells. These data define Golgi proofreading and LacNAc content, rather than unique N‐glycan structures, as critical regulators of the galectin lattice and cell homeostasis.
Mechanism of Dynamin‐Catalyzed Membrane Fission
Authors: Schmid Sandra L. , Neumann Sylvia , Liu Ya‐Wen , Mattila Juha‐Pekka
Published: June 2021

Dynamin, best studied for its role in clathrin‐mediated endocytosis, is the prototypical member of a family of multi‐domain GTPases involved in fission and remodeling of multiple organelles. Dynamin alone can catalyze fission of membrane tubules and vesicle formation from planar lipid templates. We recently proposed a two‐stage model for dynamin‐catalyzed fission (Frolov and Schmid, Ann. Rev. Cell and Dev. Biol. 2011. 27: 79). In stage one, mechanochemical activities of assembled dynamin helices induce localized curvature stress. In stage two the tightly packed lipid‐interacting pleckstrin homology domains insert hydrophobic wedges into the bilayer to create a catalytic center that guides lipid remodeling and drives membrane fission through hemi‐fission intermediates. We have been using site‐directed fluorescent labeling of dynamin to the study nucleotide‐dependent conformational changes required for dynamin‐catalyzed fission. Our data suggests that a concerted conformational change and coordinated GTP hydrolysis are required for dynamin‐catalyzed fission. We are also studying the role of BAR domain‐containing proteins as dynamin partners in membrane fission.
Adaptation of the Nutrition Environment Measures Survey ‐ Stores (NEMS‐S) to assess a Mexican/Mexican‐American nutrition environment
Authors: Winham Donna M , Quiroga Seline Szkupinski
Published: June 2021

The Nutrition Environment Measurement Survey‐Stores (NEMS‐S) is a robust and valid assessment tool for the quality, cost and healthiness of food sold in retail outlets for the mainstream US population. However, the type of food items and the type of food retail outlets included are not culturally appropriate to accurately evaluate a Latino nutrition environment. Two‐day dietary recall food consumption data from the 2003–2004 and 2005–2006 National Health and Nutrition Examination Surveys were used to determine the most frequently consumed foods for adults who reported Hispanic ethnicity. Qualitative and quantitative research in predominately Mexican/Mexican‐American neighborhoods was used to validate food items from the NHANES list. The adapted NEMS‐Mex includes healthy culturally preferred items such as corn tortillas, tropical fruits like mango and papaya, and vegetables like chayote and chilies. Inter‐rater reliability for 34 stores using the adapted instrument was high. Latino retail outlets assessed with the NEMS‐Mex had more “healthful” ratings than with the original NEMS‐S.
Effects of Garlic (Allium sativum) on indicators of diabetic nephropathy
Authors: Thomson Martha , Al‐Qattan Khaled , Mansour Mohamed H. , Ali Muslim
Published: June 2021

The anti‐diabetic, antioxidant and anti‐nephropathic effects of an aqueous extract of raw garlic (RGE) were studied in diabetic rats. Diabetes was induced by IP injection of streptozotocin (STZ) and a fasting blood sugar >;300 mg/dl indicated diabetes. Diabetic rats were divided into 2 groups; control and garlic‐treated. RGE was administered daily for 8 weeks. Blood glucose and serum creatinine and urea were elevated in diabetic rats, and lowered by RGE treatment. In contrast, serum and kidney protein and antioxidants, and serum albumin levels were decreased in diabetic rats, and increased in RGE‐treated diabetic rats. Serum insulin levels were decreased over 10 fold in diabetic rats and increased by RGE treatment. In addition, total urine creatinine, protein and albumin were elevated in diabetic animals and decreased by RGE treatment. In contrast, urine urea was decreased in diabetic rats and elevated by RGE treatment. Plasma and kidney antioxidant enzymes (GR, GPx, catalase) were lower in diabetic rats with RGE‐treated diabetic rats having increased activities. Lipid peroxidation (MDA levels) were increased in kidneys of diabetic rats and lowered in RGE‐treated diabetic rats. Urine enzymes (LDH, ALP and NAG) were increased in diabetic controls and decreased in RGE‐treated diabetic rats. Immunochemical analysis of angiotensin receptor expression and localization in kidney tissues indicated that AT1 receptors were up‐regulated and AT2 receptors were down‐regulated in kidneys of diabetic rats, while these changes were reversed in RGE‐treated diabetic rats. Thus, RGE relieved oxidative stress and nephropathy in diabetic rats.

The study was supported by KU grant #SL 09/10.
Artificial sweetener consumption differentially affects the gut microbiota‐host metabolic interactions
Authors: Cowan Theresa E , Palmnas Marie , Reimer Raylene , Ardell Kendra , Yang Jaeun Jane , Vogel Hans , Shearer Jane
Published: June 2021

While there is general agreement on the safety and potential uses of artificial sweeteners, the literature is still lacking a consensus on their long‐term effects on gut microbiota and host‐microbe interactions. To this end, we examined the impact of chronic sweetener consumption on the gut microbiome in lean and diet‐induced obese rats. Male Sprague‐Dawley rats (n=40) were randomized into two dietary groups; chow (CH, 12% kcal fat) and high fat (HF, 60% kcal fat). Each dietary group was further divided into groups consuming water or artificially sweetened water (0.4g/100mL EQUAL®, aspartame) for 8wk (n=10/treatment). Animals consumed food and fluids ad libitum. CH and HF animals showed a difference in weight gain and body fat, with the HF rats becoming obese and glucose intolerant (OGTT). Analysis of insulin tolerance (ITT) demonstrated sweetener consumption to reduce insulin sensitivity in both CH and HF. Analysis of the gut microbiome showed marked differences between both diet and fluid treatment. Total Eubacteria was reduced by HF, but maintained with sweetener consumption. Reductions in Lactobacillus were observed in CH sweetener and both HF groups. Bacteroides was reduced in both HF groups but not CH sweetener compared to water controls. In conclusion, results show sweetener to alter host‐microbe interactions in both chow and diet‐induced obese animals.
Do babies eat what mothers eat? The relationship between maternal and child dietary diversity in Bangladesh, Vietnam and Ethiopia
Authors: Nguyen Phuong H. , Avula Rasmi , Saha Kuntal K. , Ali Disha , Tran Lan M. , Frongillo Edward A. , Rawat Rahul , Ruel Marie T. , Menon Purnima
Published: June 2021

Diverse diets are linked to better child growth from 6 mo onwards. Interventions typically focus on educating mothers about the importance and ways of improving child dietary diversity (DD). However, new evidence suggests that maternal and child DD are associated. Data from surveys on mother and child (6–24 mo) dyads in Bangladesh (BD), Vietnam (VN) and Ethiopia (ET) were used to explore the concordance and strength of the association between maternal and child DD and determine if maternal DD can predict child's minimum DD. DD scores were derived from 24‐hour recall of intake of foods from 7 groups. Fair mother/child concordance (kappa = 0.23–0.40) was found for intake of legumes, dairy and eggs in BD and ET; animal‐source foods (0.26) and eggs (0.27) in VN; and vitamin A‐rich vegetables in ET (0.23). Multivariate regression analysis showed a strong association between maternal and child DD in all 3 countries, suggesting that education should focus on improving both maternal and child DD, rather than child DD only, to maximize benefits for both mother and child. The area under the curve values from receiver operating characteristic analysis were greater than 0.7 and significant in all 3 countries, indicating that maternal DD could predict child DD. However, because a standard maternal DD cut‐off point could not be identified, both indicators should be measured in large surveys to accurately estimate mother and child DD.

Funding support from Bill & Melinda Gates Foundation, through Alive & Thrive, managed by FHI360
Genome‐wide DNA methylation and gene expression profiles analysis show novel regulatory pathways in alcohol‐related hepatocellular carcinoma
Authors: Udali Silvia , Guarini Patrizia , Ruzzenente Andrea , Guglielmi Alfredo , Ferrarini Alberto , Tononi Paola , Pattini Patrizia , Moruzzi Sara , Delledonne Massimo , Choi Sang Woon , Friso Simonetta
Published: June 2021

Chronic alcohol consumption is a dietary habit associated to cancer and in particular to hepatocellular carcinoma (HCC) but the mechanisms of alcohol‐related liver carcinogenesis are still incompletely understood. Alcohol is known to interfere with onecarbon metabolism, the methyl transfer reactions occurring in the liver, and consequently with DNA methylation, one of the main epigenetic features related to cancer development.

Aim of the present study was to define a possible role for DNA methylation in alcohol‐related HCC in the absence of confounding factors, i.e. mainly hepatitis infections (HBV and HCV).

We performed array‐based genome‐wide promoter DNA methylation and gene expression profiles in HCC tissue compared to homologous tumor‐free liver tissue in eight patients undergoing curative surgery. The merging of DNA methylation and gene expression data allowed the detection of 160 hypermethylated‐repressed, 31 hypomethylated ‐induced, 50 hypermethylated‐induced and 56 hypomethylated‐repressed genes. Among the hypermethylated‐repressed genes we identified several candidate tumor‐suppressor genes, six retinol metabolism genes (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22 and RDH16) and SHMT1 a key gene of one‐carbon metabolism.

In alcohol‐related HCC DNA methylation at promoter site appears to regulate the expression of genes involved in retinol and onecarbon metabolism.
Carbohydrate co‐ingestion with protein delays dietary protein digestion and absorption but does not modulate postprandial muscle protein accretion
Authors: Gorissen Stefan H , Burd Nicholas A , Hamer Henrike M , Gijsen Annemie P , van Loon Luc JC
Published: June 2021

We determined the impact of carbohydrate co‐ingestion with protein on dietary protein digestion and absorption kinetics and postprandial muscle protein accretion in healthy men. Twenty‐four young (age: 21±1 y, BMI: 21.8±0.5 kg/m2) and 24 older (age: 75±1 y, BMI: 25.4±0.6 kg/m2) men received a primed continuous L‐[ring‐2H5]‐phenylalanine infusion and ingested 20 g L‐[1‐13C]‐ phenylalanine‐labeled protein with (PRO+CHO) or without (PRO) 60 g carbohydrate. Biopsies were collected from the vastus lateralis. Carbohydrate co‐ingestion delayed the rise in exogenous phenylalanine appearance rate (P<0.05). Dietary protein‐derived phenylalanine availability over the postprandial period was lower in the old (60±2%) when compared with the young (72±2%; P<0.05), with no differences between the PRO and PRO+CHO condition (P=0.42). Carbohydrate co‐ingestion did not modulate postprandial muscle protein synthesis rates (0.035±0.003 vs 0.043±0.004 and 0.033±0.002 vs 0.035±0.003 %/h following PRO vs PRO+CHO in the young and old, respectively). In accordance, no differences in muscle protein‐bound L‐[1‐13C]‐phenylalanine enrichments were observed between groups (0.020±0.003 vs 0.018±0.003 and 0.017±0.003 vs 0.022±0.004 MPE following PRO vs PRO+CHO in the young and old, respectively). We conclude that carbohydrate co‐ingestion does not modulate postprandial muscle protein accretion in young or older men.
Fundamental Role for HIF‐2‐regulated Creatine Kinase Pathway in Epithelial Apical Junctional Integrity
Authors: Glover Louise Elaine , Saeedi Bejan , Ehrentraut Stefan , Campbell Eric , Kelly Caleb , Kominsky Douglas , Colgan Sean
Published: June 2021

Intestinal epithelia of the lower GI tract are subjected to frequent, pronounced fluctuations in oxygen tension, particularly in inflammation. Adaptive responses to hypoxia are driven largely by hypoxia‐inducible factor (HIF). Given the co‐expression of HIF‐1α and HIF‐2α in intestinal epithelial cells (IECs), we sought to comprehensively define the contribution of HIF‐1/‐2 transactivation to IEC homeostasis. A ChIP‐on‐chip screen of HIF‐1/‐2‐enriched chromatin identified creatine kinase (CK) isozymes and the creatine transporter as novel HIF‐2 targets. CK enzymes temporally regulate ATP regeneration through transfer of high‐energy phosphate from phosphocreatine (PCr) to ADP. Promoter‐reporter and ChIP‐qPCR analyses confirmed HIF‐2 CK binding and transactivation. Immunolocalization studies revealed that CK's localize to cell and tissue adherens junctions (AJ). Pharmacological inhibition of CK significantly attenuated AJ assembly and functionally attenuated epithelial barrier. In vivo, dietary Cr supplementation in two murine models of colitis significantly ameliorated disease. Further analyses in human IBD tissues revealed reduced expression of CK isoforms. These results support a prominent role for HIF‐2‐regulated CK in IEC junctional homeostasis, and implicate a fundamental link between cellular energy and junctional integrity. (Funding: NIH, CCFA)
Differences in the catalytic properties of CYP2B6s between common marmoset and human
Authors: Narimatsu Shizuo , Mayumi Kei , Hanioka Nobumitsu , Masuda Kazufumi , Naito Shinsaku , Miyata Atsuro
Published: June 2021

Common marmoset (M) is easy in breeding and handling, having a possibility as a useful animal model in the safety evaluation of drug candidates. CYP2B6 is one of important CYP enzymes responsible for drug metabolism. In this study, we cloned a cDNA encoding a novel CYP2B6 from a total RNA of M liver, expressed its protein in insect cells and characterized its enzymatic property. The deduced amino acid sequence of M‐CYP2B6 showed 86.1% identity to that of H‐CYP2B6. Its enzymatic function was examined using two H‐CYP2B6 substrates, bupropion (BUP) and efavirenz (EFV). M‐CYP2B6 did not show any detectable activities for BUP or EFV under the conditions used, while H‐CYP2B6 clearly exhibited activities for the both substrates. Docking simulation indicated two amino acids (X and Z) located in the active‐site cavity of M‐CYP2B6 protein as key residues affecting the substrate specificity. We thus replaced these amino acids with those of H‐CYP2B6 by the site‐directed mutagenesis method. The substitution of X and Z of M‐CYP2B6 with human‐type amino acid residues markedly increased velocities comparing the wild type. Possible mechanisms will be discussed.
Active Site Gating Controls Substrate Selectivity in Cytosolic Sulfotransferases
Authors: Cook Ian Thomas , Wang Ting , Falany Charles N , Leyh Thomas
Published: June 2021

Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl‐moiety (‐SO3) from activated sulfate (PAPS, 3′‐phosphoadenosine 5‐phosphosulfate) to the hydroxyls and primary amines of metabolites and xenobiotics. The addition of the sulfuryl‐group radically alters the acceptor‐target interactions. SULT1A1 and 2A1, which comprise ~90% of the liver SULTs by mass, serve two critical functions in the liver. First is regulating signaling hormones to maintain homeostasis. The homeostasis function requires a high affinity to a set of conserved substrates. The second function is protecting the body from xenobiotics that can disrupt receptor signaling. The protective function requires a broader substrate specificity to handle the diversity of compounds in the environment. In this work we expand on our proposed mechanism for substrate selectivity where the binding of PAPS induces an isomerization that opens and closes the “cap” of the active site. This mechanism allows the SULT to sulfate a wide range of substrates, while maintaining homeostatic function. Computer modeling identified three interactions that regulate the gating. These interactions were tested using mutagenesis. The mutants were examined using equilibrium and pre‐steady state binding assays to measure the influence to gating. All of the mutants resulted in the loss of gating and substrate selectivity, confirming the model.
Incorporating Public Outreach into Existing Curriculum as a Solution to Bridge the Perceived Gap between Classroom and Real World Biochemistry
Authors: Cheng Catherine Yanan , Vardar‐Ulu Didem
Published: June 2021

There is a perceived gap in society between biochemistry as covered in the classroom and that pertinent to real societal concerns. Most traditional outreach efforts remain disconnected from the curriculum and exhibit a unidirectional information flow, from experts to society, widening the gap between “scientists” and “the public.” Here, we present a case study on how an explicit outreach component can be effectively incorporated into an existing upper‐level undergraduate biochemistry course. In the spring of 2012, 5 seniors and 6 juniors enrolled in Biochemistry II were given a choice of disseminating their understanding of a health related biochemical problem to a general audience via i) a layman's abstract or a letter to a senator, ii) an article for the Molecular Anatomy Project initiative at the Protein Data Bank, or iii) a lesson for a non‐science majors chemistry class, in addition to the existing formal research proposal write‐up. Student, audience, and expert feedback on this more student‐ and society‐centered course format were largely positive, pointing to student growth in a diverse set of transferable skills including independent content learning. This outreach‐focused implementation of an existing assessment tool in a terminal course ensures that majors graduate not only with a well‐founded understanding of the field, but also with the ability to effectively communicate it to a diverse audience.
Imaging the connectome
Authors: Lichtman Jeff W.
Published: June 2021

Connectional maps of the brain may have value in developing models of both how the brain works and how it fails when subsets of neurons or synapses are missing or misconnected. Such maps might also provide detailed information about how brain circuits develop and age. I am eager to obtain such maps in neonatal animals because of a longstanding interest in the ways neuromuscular circuitry is modified during early postnatal life as axonal input to muscle fibers is pruned. Work in my laboratory has focused on obtaining complete wiring diagrams (“connectomes”) of the projections of motor neuron axons in young and adult muscles. Each data set is large and typically made up of hundreds of confocal microscopy stacks of images which tile the 3dimensional volume of a muscle. As a first step to analyze these data sets we developed computer assisted segmentation approaches and to make this task easier, have developed second generation “Brainbow” transgenic mice that in essence segment each axon by a unique fluorescent spectral hue. Once the axons are segmented, we have been able to graph the connectivity matrices that result. This effort has led to new insights into the developmental processes which help the mammalian nervous system mold itself based on experience. Analysis of these complete muscle connectomes show a striking single axis gradient of connectovity that we think is related to the ordered ranking of neural activity in axons (the “size principle” of Henneman). In brain however, as opposed to muscle, the high density of neuropil is overwhelming, which has precluded using the confocal optical approaches that have worked in the peripheral nervous system because there are too many neural processes in each optical section. We have thus developed of lossless automated physical sectioning strategy that generates thousands of ultra thin (~25 nm) sections on a firm plastic tape. We have developed a thin‐section scanning electron microscopy approach to visualize these sections at 3nm lateral resolution. This method makes large scale serial microscopic analysis of brain volumes more routine. We are now focused on developing an automated pipeline to trace out neural circuits in brains using this technique.
Structural insights into the dynamic process of G‐protein– coupled receptor activation
Authors: Kobilka Brian
Published: June 2021

GPCRs conduct the majority of transmembrane responses to hormones and neurotransmitters, and mediate the senses of sight, smell and taste. The β2AR adrenoceptor (β2AR) is a prototypical Family A GPCR that mediates physiologic responses to adrenaline and noradrenaline. We have obtained three‐dimensional structures of the β2AR in inactive and active conformations, as well as a structure of the β2AR in complex with the G protein Gs. We have also used fluorescence, EPR and NMR spectroscopy to study the dynamic properties of the receptor, and to map ligand‐specific conformational changes. I will discuss what we these studies have taught us about the structural basis of GPCR function.