bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of homology models in assigning enzyme function
Authors: Jacobson Matthew P.
Published: June 2021

Reliable assignment of function to proteins discovered in genome sequencing projects is a major challenge in genomic biology. I will discuss the use of protein homology models to help guide investigations of enzyme‐substrate specificity, and the identification of novel enzymatic functions. We have studied a group of ~100 proteins in the functionally diverse enolase superfamily that are homologous to the characterized Ala‐Glu epimerase in B. subtilis. Among these, several were predicted based on homology modeling and ligand docking to have different substrate specificity, suggesting novel, but as yet unknown, biological functions. Experimental testing (group of John Gerlt, UIUC) confirmed many of these predictions, and assigned novel enzymatic functions of Ala‐Phe epimerase to a protein from T. martima and D‐Ala‐D‐Ala epimerase to a protein from C. hutchinsonii; divergent specificities were also identified in three other enzymes. The annotations for all of these are incorrect in GenBank. In the case of the Ala‐Phe epimerase, crystal structures (group of Steve Almo, Albert Einstein College of Medicine) confirmed the modeling predictions. Finally, I will discuss the strengths, weaknesses, and future prospects for this type of "structure‐based" approach to investigating protein function, and the complementarity with sequence‐based methods (group of Patsy Babbitt, UCSF).
The AP‐4 Complex Mediates Sorting and Processing of the Alzheimer's Disease Amyloid Precursor Protein
Authors: Burgos Patricia V. , Mardones Gonzalo A. , daSilva Luis L. P. , Prabhu Yogikala , Bonifacino Juan S.
Published: June 2021

Proteolytic processing of the transmembrane amyloid precursor protein (APP) to aggregation‐prone amyloid‐beta (Abeta) peptide underlies the development of Alzheimer's disease. Processing is carried out by alpha‐, beta‐, and gamma‐secretases after export of newly‐synthesized APP from the endoplasmic reticulum. We find that the heterotetrameric adaptor protein 4 (AP‐4) complex recognizes an unusual tyrosine‐based sorting signal in the cytosolic tail of APP, leading to APP transport from the trans‐Golgi network to endosomes. This diversion from the secretory pathway decreases gamma‐secretase‐catalyzed production of Abeta. These findings identify AP‐4 as a novel regulator of APP trafficking and processing, and as a protective factor against Alzheimer's disease.
Resolution of metabolic syndrome parameters in subjects following a Mediterranean‐style, low glycemic load diet in conjunction with a soy protein, plant sterol‐(SPS) supplement
Authors: Jones Jennifer , Ackermann Daniela , Comperatore Michael , Leite Jose O , Barona Jacqueline , Ratliff Joseph C , Calle Mariana C , Kim Jung Eun , Ata Shymaa , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

We investigated effects of a Mediterranean‐style low glycemic load diet, with or without a soy protein, plant sterol‐(SPS) supplement, on cardiometabolic risk factors in 8 women with metabolic syndrome (MetS). Subjects had high plasma triglycerides (TG) (163.5 ± 59.1 mg/dL), elevated body mass index (31.0 ± 3.5 kg/m2), total body fat of 39 ± 4.9 kg, total trunk fat of 20.7 ± 2.9 kg, and plasma LDL of 107.1 ± 31.8 mg/dL. The 8‐week intervention diet provided an average energy distribution of 30:45:25 fat:carbohydrate:protein. Five subjects were randomly allocated to the SPS supplement. Compared to baseline, dietary protein intake increased (P<0.005), while added sugar and saturated fat decreased at 8 wks. Glycemic index decreased from 62.1 ± 5.4 to 48.8 ± 8.9 (P < 0.01) and glycemic load from 111.3 ± 34.2 to 50.1 ± 27.8 (P < 0.005). All subjects experienced significant reductions in body weight (5%; P < 0.001), total body fat (9%; P < 0.005), trunk fat (12%; P < 0.005), and in both systolic (P < 0.01) and diastolic (P < 0.05) blood pressure. Waist circumference decreased from 93.5 ± 3.9 to 88.3 ± 3.6 cm (P < 0.01). Plasma TG were reduced by 33% (P < 0.002) and total cholesterol from 203.6 ± 33.6 to 171.0 ± 44.5 mg/dL (P < 0.05). The intervention resulted in significant health benefits on all MetS parameters, mainly by reducing trunk fat and alleviating dyslipidemias. [Supported by Metagenics Inc., Gig Harbor, WA]
Effect of non‐oil seed pulses on glycemic control: a meta‐analysis of randomized controlled experimental trials in humans.
Authors: Sievenpiper John L , Esfahani Amin , Wong Julia MW , Carleton Amanda J , Jiang Henry Y , Bazinet Richard P , Jenkins David JA , Kendall Cyril WC
Published: June 2021

Background

Dietary pulses (beans, lentils, chickpeas, etc.) are a good source of viscous fibre and a valuable means for lowering the glycemic‐index (GI) of the diet.

Objective

To assess the effect of pulses on glycemic control, we conducted a meta‐analysis of experimental trials investigating the effect of pulses alone or in low‐GI or high‐fibre diets on indices of glycemic control.

Methods

We searched MEDLINE, EMBASE, CINAHL, and Cochrane Library for relevant controlled trials of =7d in humans. Two independent reviewers extracted information on study design, participants, treatments, and outcomes. Generic inverse variance models were used for pooled analyses. Heterogeneity was assessed by Chi2 and quantified by I2.

Results

Forty‐four trials were included. Pulses alone (12 trials) lowered fasting blood glucose (FBG) (standardized mean difference [SMD] ‐0.81 [95% CI ‐1.32,‐0.29]) and insulin (‐0.61[‐1.07,‐0.14]). Pulses in low‐GI‐diets (19 trials) lowered glycosylated proteins (GPs) (‐0.28[‐0.42, ‐0.14]). Pulses in high‐fibre diets (12 trials) lowered FBG (‐0.22[‐0.43,‐0.00]) and GPs (‐0.33[‐0.59,‐0.06]). Inter‐study heterogeneity was significant in all cases.

Conclusions

These data demonstrate that pulses alone or in low‐GI or high‐fibre diets improve markers of glycemic control in humans. Subgroup analyses are planned to explore sources of heterogeneity.

Funding source

Pulse Canada.
Mediterranean diet and incidence and mortality of coronary heart disease and stroke in women
Authors: Fung Teresa , Rexrode Kathryn , Mantzoros Christos , Manson JoAnn , Willett Walter , Hu Frank
Published: June 2021

In this study, we prospectively assessed the association between adherence to the Mediterranean diet and risk of incident coronary heart disease (CHD) and stroke. 74,886 women aged 38‐63 in the Nurses' Health Study, without a history of cardiovascular disease (CVD) and diabetes were followed from 1984 to 2004. We computed an alternate Mediterranean diet score from dietary data collected through validated food frequency questionnaires administered 6 times during follow‐up. Relative risks for incident CHD, stroke, and combined fatal CVD were estimated using Cox proportional hazards models adjusted for CVD risk factors. During 20 years of follow‐up, 2391 incident cases of CHD, 1763 incident cases of stroke, and 1077 CVD deaths (fatal CHD and strokes combined) were ascertained. Women in the top aMed quintile were at lower risk for both CHD and stroke compared with the bottom quintile (RR=0.71 (95%CI=0.62‐0.82; p trend< 0.0001) for CHD; RR=0.87 (95% CI=0.73‐1.02; p trend=0.03) for stroke). CVD mortality was significantly lower among women in the top quintile of the aMed (RR=0.61, 95g% CI=0.49‐0.76, p trend<0.0001).

Conclusion

Greater adherence to the Mediterranean diet was associated with a lower risk of incident CHD and stroke in women. Funded by NIH grants CA87969, HL60712, HL34594, HL88521.
Retinoic Acid Induced nuclear localization of HDAC3
Authors: Persaud Shawna Devi , Ho Ping‐Chih , Wei Li‐Na
Published: June 2021

Histone deacetylase 3 (HDAC3) plays an important role in regulating transcription, cell cycle progression and developmental events through its deacetylase activity on core histones. Recently, it has been shown HDAC3 has functions independent of its deacetylase activity. Specifically, it has been demonstrated that HDAC3 functions as a carrier molecule. HDAC3 nuclear export is regulated by the Chromosome region maintenance 1 (CRM1)/Exportin‐1 pathway. Inhibition of CRM1/Exportin‐1 pathway results in nuclear accumulation of HDAC3. In this study, we found all‐trans retinoic acid (atRA) induced retinoylation of HDAC3. Retinoylation is a post translational modification resulting in acylation of a protein by retinoic acid. We hypothesize that retinoylation of HDAC3 results in nuclear accumulation of the molecule by abrogating interaction with CRM1 necessary for nuclear translocation. This is the first study showing HDAC3 retinoylation and its modification as a nuclear localization signal.
Placental insufficiency and leptin programming in two generations of Sprague Dawley rats
Authors: Anderson Cindy M , Lopez Faye , Sandeen Ashley R , Benoit Joseph N
Published: June 2021

Placental insufficiency (PI) leads to impaired fetal growth and increased risk for hypertension in mothers and their children in later life. Our lab reported hypoleptinemia in pregnant Sprague‐Dawley rat dams with placental insufficiency associated with intrauterine growth restriction. In this study, we investigated the relationships between leptin levels, growth and hypertension, programmed by PI and maternal hypoleptinemia across two generations. Pregnant rat dams had PI induced via reduced utero‐placental perfusion pressure (RUPP) or sham in controls. F1 offspring were bred for second generation (F2). Systolic blood pressure (SBP) was measured weekly (4‐12 weeks). Whole blood was collected from offspring at 6, 9 and 12 weeks. Plasma leptin levels were determined by EIA. Leptin levels were not significantly different in male offspring groups. Weights and leptin levels were increased in female F1 at 12 weeks of age (p<0.05) compared to control and F2. Compared to control, SBP was significantly increased in F1 across age groups (p < 0.05). F2 SBP was increased over control in males at 9 and 12 weeks and across age groups among females (p<0.05). There were no significant correlations with plasma leptin levels and SBP in offspring groups. Our findings indicate that fetal development during maternal hypoleptinemia led to altered growth patterns in females and was not related to hypertension in offspring exposed to PI
The Effect of Sesame Lignan and Flaxseed Lignan and Oil on the Growth of Human Estrogen Receptor‐Positive Breast Tumors (MCF‐7)
Authors: Truan Jennifer , Chen Jian‐min , Thompson Lilian
Published: June 2021

Flaxseed (FS) reduces breast cancer growth perhaps due to its high lignan (secoisolariciresinol diglycoside (SDG)) and α‐linolenic acid (ALA)‐rich oil (FSO) content. Sesame seed (SS), which is also rich in lignans like sesamin (SES) but low in ALA, is tumorigenic. Like SDG, SES is converted to estrogen‐like enterodiol (ED) and enterolactone (EL). Our objective is to determine the effects of SES, SDG and FSO on breast tumor growth to explain the opposing effects of FS and SS. Estrogen‐positive breast tumor cells (MCF‐7) were tested for proliferation in vitro after 4‐day treatment with ALA (50uM), SES (1uM), ED and EL (1uM). Only ALA reduced proliferation (33%, P<0.001). Ovariectomized mice with MCF‐7 tumors were fed for 8 weeks the basal diet (BD; control) or BD supplemented with 0.1% SDG, 0.1% SES, or 4.0% FSO, at levels found in 10% FS or SS diets. Compared to control, SES and FSO reduced palpable tumor area by 23% (P<0.05) and 33% (P<0.01), respectively. FSO reduced tumor volume and weight (both 37%, P<0.05), while SES caused decreases of 21% and 25%, respectively, though not different from control or FSO. SDG had no effect. SES and FSO increased apoptosis by 91% and 110% (P<0.01), respectively, and decreased proliferation by 38% (P<0.01). SDG did not affect apoptosis but reduced proliferation by 37% (P<0.05). Thus, ALA may account for the protectiveness of FS and SES may not account for the tumorigenicity of SS. (Funded by NSERC)
Anti carcinogenic effects of Citrus auraptene in N‐methyl nitrosourea (MNU) induced mammary carcinogenesis in rats
Authors: Krishnan Prasad , Windler David , McLarty Jerry , Yan Karen , Li Benjamin , Kleiner Heather
Published: June 2021

Our goal was to determine the anticarcinogenic effects of auraptene against breast cancer. In human breast carcinoma, MDA‐MB‐231 cells, auraptene inhibited cell proliferation by 50% at 12 μM and by 85% at 25 μM. In the in vivo study using the N‐methylnitrosourea (MNU) induced rat mammary carcinogenesis model, female Sprague‐Dawley rats were fed control diet, or diets containing auraptene (200 or 500 ppm) , at 42 days of age till the end of the study. Since eIF4E is over expressed in human breast cancer, another group with rapamaycin, an inhibitor of eIF4E was included (3.33 ppm in the diet). Auraptene (500 ppm) significantly delayed median time to tumor by 39 days compared to MNU only group. Analysis of rat mammary glands by HPLC with fluorescence detection indicated auraptene reaching the mammary glands in micromolar concentrations. In the analysis of human breast cancer samples in a tissue micro array (TMA) format, expression of cyclin D1, c‐Myc, and ODC (ornithine decarboxylase)the downstream effector molecules of eIF4E of showed an upregulation in the cancer samples.We studied the expressionof ODC in the mammary tumors in the auraptene‐fed groups and saw a significant reduction (p<0.05) in the expression of ODC. Recent studies in MCF‐7 cells showed that auraptene and rapamycin reduced the expression of cyclin‐D1.These results point to the potential chemopreventive effects of auraptene in mammary carcinogenesis.

This study was supported by NCI: 1K22 CA102005‐01A2
Dietary Vitamin D and Calcium Intake on Prostate Tumor Progression in Athymic Mice: High Calcium Intake Does Not Enhance Prostate Tumor Growth
Authors: Ray Rahul , Abruzahra Hilal , Tannenbaum Andrew , Holick Michael F.
Published: June 2021

The purpose of this study is to investigate the concern that increasing calcium and vitamin D intake may increase risk and progression of prostate cancer. Nu/nu male athymic mice were fed either 1) low (L) calcium (Ca) (0.02%) and vitamin D3 (5,000 IU/kg) (+D), 2) normal (N) Ca (0.9%) + D, 3) a high (H) Ca (2%) + D, 4) NCa and no vitamin D, (‐D) or 5)H Ca ‐D. After one week, DU145 cells were injected subcutaneously in mice. Tumor growth rates were measured at the first appearance of tumor at day ~ 42 and followed for four weeks. Tumor growth rate for mice on a NCa ‐D diet was 627 ± 211% compared to mice on a NCa +D diet 184 ± 49% (P = 0.01). Tumors in mice on a HCa ‐D diet had as growth rate of 276 ± 93% while animals on a NCa +D diet had a growth rate of 298 ± 97%. For mice on a LCa +D diet, the tumor growth rate was 192 ± 59%. Serum Ca determinations revealed that mice fed a LCa +D or NCa +D diet had serum calciums of 10.5 ± 0.5 and 9.41 ± 0.5 mg/dl respectively (P= 0.32). Mice fed a NCa ‐D or HCa ‐D diet had serum calcium levels of 8.0 ± 0.4 and 8.0 ± 0.3 mg/dl respectively. (P= 0.44). Mice fed a LCa +D, NCa+D and HCa+D diet had 25(OH)D levels of 42.6 ± 3.4, 24.9 ± 3 and 24.9 ± 1.6 ng/ml respectively. Vitamin D deficiency promoted prostate tumor growth. Furthermore, a HCa ‐D diet limited tumor progression compared to the mice fed a NCa ‐D diet. This model may provide an insight into the role of dietary calcium and vitamin D in the progression of prostate cancer.
Lack of responsiveness to plant sterols in wild type C57BL/6J mice is due to increased HMG‐CoA reductase transcription and higher fractional cholesterol synthesis rate
Authors: Rideout Todd Christopher , Harding Scott V , Jones Peter JH
Published: June 2021

Although plant sterols (PS) are effective in reducing plasma cholesterol (C) in humans and various animal models, previous work suggests that wild type C57BL/6J mice do not respond to PS therapy despite reductions in intestinal cholesterol absorption. To explain this non‐responsiveness, we examined changes in hepatic HMG‐CoA reductase (HMG‐CoAr) expression and C synthesis in C57BL/6J mice. Mice (n = 10) were fed a high‐fat control diet or the control diet with 2% PS for 30 d in a randomized block design. Mice received an intraperitoneal injection of deuterium oxide 2h prior to sacrifice and whole‐body C synthesis was measured by deuterium incorporation. Although total C and non‐HDL C did not differ between treatments, PS consumption reduced (P<0.05) plasma triglycerides by 29% compared to control (0.68±0.02 vs. 0.91±0.17 mmol/L). PS consumption increased hepatic HMG‐CoAr mRNA expression (2.7 fold of control, P<0.05) but did not affect (P>0.05) HMG‐CoAr protein abundance. In agreement with HMG‐CoAr mRNA expression, PS consumption increased whole‐body fractional C synthesis rate compared to control (2.0±0.16 vs. 1.3±0.43 % day−1). HMG‐CoAr mRNA expression was positively correlated with whole‐body C synthesis (r = 0.61, P<0.05). The lack of response of plasma C to PS consumption in C57BL/6J mice is due to an upregulation of hepatic HMG‐CoAr mRNA expression and a compensatory increase in C synthesis. Funded by NSERC.
Knockout of SOD1 decreased GPX1 activity but not GPX1 protein in murine liver
Authors: Wang Shikui , Lei X G
Published: June 2021

We previously reported a 40% decrease of liver glutathione peroxidase 1 (GPX1) activity in copper,zinc superoxide dismutase knockout (SOD1‐/‐) mice. This study determined respective changes in hepatic GPX1 and SOD1 protein expression in the SOD1‐/‐ and GPX1 knockout (GPX1‐/‐) mice. Two experiments were conducted with 6 SOD1‐/‐ and GPX1‐/‐ male mice (8 wk), along with their respective wild‐type (WT) littermates. Liver SOD1 activities were similar between the GPX1‐/‐ and the WT mice (1,073 ± 72 vs. 1,128 ± 81 U/mg protein). In contrast, liver GPX1 activity was lower (P < 0.05) than WT mice (487 ± 56 vs. 822 ± 45 U/mg protein). Western blot analysis of liver homogenates showed no alteration of either enzyme protein amount from the WT by deleting the other enzyme. To explore physical interactions of SOD1 and GPX1 proteins, we performed co‐immunoprecipation (Co‐IP) with liver homogenates and HepG2 cells lysates. Samples were prepared in NP40 buffer, and Co‐IP was conducted with 100 µg protein of homogenates, 1 µg of anti‐SOD1 (Cell Signal, Boston, MA) or anti‐GPX1 (Abfrontier, Seoul, S. Korea) antibody, and 20 µl of A/G‐coupled agarose beads (Abcam, Cambridge, MA). The Western analysis showed no Co‐IP of either enzyme protein by the antibody against the other enzyme. In conclusion, the decreased liver GPX1 activity in the SOD1‐/‐ was likely mediated by factors other than changes of the enzyme protein abundance (NIH DK 53018).
Wound macrophage phenotype is independent of IL‐4 receptor‐alpha
Authors: Daley Jean Marie , Mastrofrancesco Balduino , Henry William L. , Reichner Jonathan S. , Albina Jorge E.
Published: June 2021

Repair macrophages are characterized as alternatively activated macrophages (AAM). Because IL‐4 and IL‐13 induce AAM, we investigated whether these cytokines determine wound macrophage phenotype. Five polyvinyl alcohol sponges were sterilely inserted beneath the dorsal skin of B6D2F1 male mice. Wound cells and wound fluids were isolated from sponges at 1, 3, and 7 days. IL‐4 was measured by ELISA and bioassay; IL‐4 and IL‐13 were also assessed by the capacity of wound fluids to phosphorylate Stat6 in naïve cells. No IL‐4 or IL‐13 was detected in wound fluids. Wound macrophages had functional IL‐4 receptor‐α (IL‐4Rα) but not phosphorylated Stat6, confirming the absence of IL‐4/IL‐13. The lack of IL‐4/IL‐13 in the wound environment suggested that wound macrophage phenotype was not dependent on IL‐4Rα or its ligands. To test that hypothesis, we characterized wound macrophages in IL‐4Rα ko mice. Macrophages from day 7 wounds in IL‐4Rα ko mice were similar to those in wild type mice: dectin‐1, mannose receptor, arginase I, and Ym1 were expressed in macrophages from both IL‐4Rα ko and wild type mice; iNOS was not present. Wound macrophages have characteristics of AAM. Factors other than IL‐4 and IL‐13 may determine the phenotype of macrophages in wounds. Supported by NIH GM 79227, NIH GM 42859, and RI Hospital.
Effects of Glycogen Synthase Kinase‐beta Inhibition in Ischemia‐Reperfusion Injury of the Liver
Authors: Sepodes Bruno Miguel , Rocha João Pedro , Eduardo‐Figueira Maria , Thiemermann Christoph , Mota‐Filipe Helder
Published: June 2021

GSK‐3beta is a serine/threonine protein kinase involved in the modulation of inflammatory response. Here we investigate the effects of two GSK‐3beta inhibitors on the liver injury caused by ischemia‐reperfusion injury, in the rat, using a synthetic inhibitor, TDZD‐8, and the endogenous inhibitor, insulin. Male Wistar rats were randomly allocated as described: (1) Sham Group; (2) I/R Group: rats to which for 45 minutes the blood supply to ¾ of the liver was interrupted, plus by reperfusion for 2 h; (3) TDZD‐8 + I/R Group: rats received TDZD‐8 (1 mg/kg i.v.) 30 minutes prior to liver I/R; (4) Insulin + I/R Group: rats received insulin (1,4 UI/kg i.v.) 30 minutes prior to liver I/R; (5) TDZD‐8 and (6) Insulin Control Groups. Blood samples were obtained at the end of reperfusion and tissue samples for histology. Our results show that both TDZD‐8 and insulin administration reduced the increase on the level of biochemical markers for liver injury when compared to I/R group: AST (1132±61 vs. 541±254 and 555±10 IU/l, for TDZD‐8 and insulin, respectively) and ALT (1941±92 vs. 583±90 and 174±27 IU/l, for TDZD‐8 and insulin, respectively), and the same was observed for LDH and gamma‐GT, and light microscopy examination We demonstrate here that TDZD‐8 and insulin cause a substantial reduction in the ischemia‐reperfusion‐induced increase in the serum levels of hepatic injury markers, providing, to our knowledge for the first time, the evidence that inhibition of GSK‐3beta reduces the liver injury induced by ischemia‐reperfusion, in the rat. We propose that GSK‐3beta inhibitors, such as TDZD‐8 and insulin, may be useful in the therapy of liver injury associated with ischemia‐reperfusion of the organ.
Resolvin E1‐induced Intestinal Alkaline Phosphatase contributes to inflammation resolution via LPS detoxification
Authors: Campbell Eric , Keely Simon , MacManus Christopher , Scully Melanie , Colgan Sean P
Published: June 2021

Inflammatory bowel diseases are disorders of unknown etiology characterized by inappropriate inflammation. There is recent intense interest in understanding mechanisms of inflammatory resolution. Resolvin‐E1 (RvE1) has been demonstrated to promote resolution in numerous disease models. Given the importance of epithelial cells in coordinating mucosal inflammation, we hypothesized that RvE1 elicits an epithelial inflammatory resolution signature.

Microarray profiling of epithelial cells exposed to RvE1, revealed transcriptional induction of numerous inflammatory response genes. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and enhanced epithelial ALPI enzymatic activity. ALPI was recently demonstrated to detoxify bacterial LPS. In our studies, both purified ALPI and "conditioned" LPS‐containing media derived from RvE1‐exposed epithelia were demonstrated to detoxify LPS. To define these features in vivo, we employed a murine DSS model of colitis. Compared to vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices. RvE1 treatment resulted in significantly increased ALPI expression in murine intestinal epithelial cells. Moreover, disease severity inversely correlated with ALPI expression. Taken together, these data implicate a previously unappreciated role for ALPI in RvE1 mediated inflammatory resolution.
Estrogen receptor (ER) β modulates natural T regulatory cells (nTregs) and experimental Crohn's disease (CD).
Authors: Garg Rekha Rani , Sachedina Muhammad A , Cominelli Fabio , Reuter Brian K , Rissman Emilie F , Pizarro Theresa T
Published: June 2021

CD4+CD25+FoxP3+ nTregs are critical for modulating immune responses in chronic inflammatory disorders, such as CD. Estrogen has recently been shown to augment Treg function. This study investigates the role of estrogen and its receptors, ERα and β, in the SAMP1/YitFc (SAMP) model of CD ileitis. Histologic evaluation of ileitis showed significant gender differences in early disease (age 6 wks) with increased severity in F‐ vs M‐SAMP (N=20/12; 10.8±1.0 vs 6.9±0.8, P<0.05), which continued up to 20 wks (N=17/18; 15.8±0.7 vs 12.7±0.8, P<0.05), with no inflammation in AKR controls. 17‐β estradiol significantly reduced ileitis in gonadectomized (GDX) M‐SAMP (N=6; 9.0±0.7 vs 17.5±0.5 (cont), P<0.01), with no effect on GDX F‐SAMP (N=6; 15.3±1.2 vs 16.5±1.0 (cont), ns). Western blots showed reduced ERβ in MLN cells from F‐ vs M‐SAMP and F‐AKR, while FACS revealed significant expression of CD25+FoxP3hi nTregs in MLN from M‐ vs F‐SAMP (N=12/12; 10±0.7 vs 6.2±0.4%, P<0.01). Stimulation of sorted MLN Tregs with ERα (PPT) and β (DPN)‐specific agonists showed DPN‐specific upregulation of FoxP3 and TGFβ in M‐ vs F‐SAMP (N=7/6; 15.3±0.7 vs 4.9±1.9%, P,<0.01 and 548.8±54.4 vs 320.5±12.7 pg/ml, P<0.01). Taken together, these data support a role for estrogen/ERβ‐dependent regulation of nTregs in modulating the severity of disease in experimental CD. Supported by the NIH (DK056762 & DK57880/TTP) and CCFA (Research Fellowship/ RRG).
Materializing the Potential of Nanomedicine via a Tumor‐Targeting Nanodelivery Platform
Authors: Chang Esther H
Published: June 2021

A platform nanodelivery system has been developed comprising a self assembled, biodegradable, cationic liposomal nanoparticle, which bears targeting molecules that home to the surface of tumor cells. When systematically administered, this nanocomplex can efficiently and selectively deliver nucleic acid‐based molecular therapeutics, diagnostic MRI contrast agents, and small molecules to not only primary tumors, but also metastases in animal models of a number of human cancers. The nanodelivery of imaging agents results in a marked improvement in the sensitivity and resolution in detecting minute metastatic lesions. The tumor targeting delivery of various molecular therapeutics have also been shown to dramatically synergize with conventional radio‐ and chemotherapies. This approach is now entering clinical trials. There is ongoing collaborative effort to correlate integrated high‐resolution imaging and physical characteristics with the biological activity of these nanoparticles to accelerate in vivo optimization of the nanodelivery system.
Notch signaling in vascular smooth muscle regulates angiogenesis
Authors: Proweller Aaron , Banerjee Suhanti , Yang Ke
Published: June 2021

Notch signals in endothelial cells (ECs) are critical for early angiogenic remodeling of immature vascular networks, yet much less is known of their role within vascular smooth muscle cells (VSMCs). Our previous characterization of a mouse model engineered to suppress canonical Notch signaling in VSMCs revealed unprecedented cerebral arterial patterning abnormalities suggesting Notch signals in VSMCs influence vessel formation and stereotypic patterning. To investigate the molecular basis of the vascular phenotype, we have employed post‐natal angiogenic assays including aortic ring explantation and demonstrate that vascular tissue from mice with deficient smooth muscle Notch signaling yields markedly diminished angiogenic responses to growth factors including VEGF and PDGF. Taken together, these findings support a model wherein Notch signals in VSMCs provide instructive cues to neighboring ECs important for efficient tubulogenesis and neovessel formation. Furthermore, we are determining whether these instructions are conveyed through Notch ligand‐receptor interactions by heterotypic contact between VSMCs and ECs in vessels undergoing sprouting angiogenesis. This research is supported by NIH HL079072 to A.P.
CaMKII‐dependent regulation of HDAC4/5 and gene transcription in vascular smooth muscle cells
Authors: Ginnan Roman Gregory , Sun Li Yan , Singer Harold A
Published: June 2021

Vascular smooth muscle is well known for phenotype plasticity and upon injury or in response to disease it modulates from a contractile phenotype to a sythetic phenotype that contributes to vascular remodeling. We have demonstrated that increased expression of CaMKII is associated with modulation of VSM to the synthetic phenotype but there is very little direct evidence indicating a function for the kinase in regulating VSM cell gene transcription. Herein, we report that the transcriptional co‐repressors HDAC4/5 are phosphorylated in a CaMKII‐dependent manner . Furthermore, effective suppression of CaMKII expression resulted in decreased HDAC5 phosphorylation in response to PDGF and AngII. Nur77 is an immediate response gene shown to be regulated in a Mef2/HDAC‐dependent manner. Our initial studies in cultured VSM indicate robust activation of nur77 in response to PDGF. Inhibition or suppression of CaMKII resulted in the decrease of Nur77 expression. Taken together, these results lead us to hypothesize that Ca2+ signaling, via CaMKII, regulates VSM gene transcription and may be a determinant of VSM phenotype.
Overview of limbic system anatomy and organization
Authors: Walker James J
Published: June 2021

The limbic system, as originally defined by Broca (1878), is an anatomically‐related group of medial brain structures between the neocortex and diencephalon. Learning the organization of this system has traditionally been challenging because of its seemingly endless connectivity across diverse neural centers. For some, the limbic system, lacking clarity in definition, doesn't even exist (LeDoux, 1996). What is clear however is that the limbic components comprise phylogenetically old parts of the brain subserving important functions dealing with adaptation and survival. This talk will focus on introducing the audience to basic limbic anatomy and organization and will explore some of the more controversial themes related to the functional localization of the emotional (visceral) brain.
Microvascular response to ischemia in mouse spinotrapezius muscle: lessons for human vascular variability
Authors: Mac Gabhann Feilim , Bailey Alexander M , Skalak Thomas C , Peirce Shayn M
Published: June 2021

One of the great promises of Systems Biology is the refinement of therapeutic strategies through identification of vulnerable or responsive patient subpopulations. Ultimately, this leads to personalized medicine: interpersonal variability can influence both the severity of disease and the therapies that will be effective for each individual. Inbred mouse strains allow us to reproducibly mimic some of these variable physiologies and pathologies. Other groups have previously identified the remodeling responses of mouse strains to ischemic ligation of hindlimb arteries. Here we induce arterial ischemia in the mouse spinotrapezius, a model system that allows for the whole‐mount, en‐face visualization of the entire muscle microvasculature. We show that mice can be divided into two groups: ischemia‐vulnerable, characterized by a dendritic arteriolar structure, and ischemia‐protected, characterized by arteriole‐to‐arteriole connections. These groups ‐ even from different strains ‐ consistently respond differently to ischemic insult, and we identify potential for therapeutic pre‐treatment that can create an ischemia‐protected vasculature. Support provided by: NIH‐HL082838‐02 and NIH‐T32‐HL007284.
Overpressure‐induced brain injury at the cellular level
Authors: Leung Lai Yee , VandeVord Pamela
Published: June 2021

Veterans returning from wars exposed to blast with no visible wounds were diagnosed with mild traumatic brain injury (Warden 2006). Animal studies revealed the effects of primary blast wave on central nervous system. These studies mainly focused on systemic effect (Kaur et al 1995‐97; Cernak et al 2001). The molecular mechanism of blast wave‐induced brain injury is still unclear. We conducted a series of in vitro experiments to examine the cellular responses of astrocytes and neurons after exposing to overpressure (OP). A barochamber was designed and fabricated to generate a pressure with magnitude ranged from 17 to 58 psi and duration of 10 msec. Individual cell types were cultured on Petri dish, sealed and then exposed to OP. Viability assays and total RNA extraction for quantitative real‐time PCR were carried out at 24, 48 and 72 hours. Results of astrocytic response were published, which showed that astrocytes survived the OP and became reactive (VandeVord et al 2008). In rat cortical neuron cultures, Bcl‐2/BAX expression ratios were less than 1 (indicating apoptosis) in all groups except controls at 24 hour post‐exposure, followed by a significant increase (ratios>1, indicating survival) at 48 hour. The expression level of apoptosis‐related gene Caspase‐8 reached the peak at 72 hour. Neurons exhibited both apoptotic and survival processes after OP insult.

Grant Funding Source

VA RR&D Career Development Award
A model for the investigation of regenerative or degenerative motoneurons and neuropil: Relevance to injury and ALS
Authors: Mesnard Nichole A , Haulcomb Melissa M , Sanders Virginia M , Jones Kathryn J
Published: June 2021

Amyotrophic lateral sclerosis (ALS) involves progressively fatal neurodegeneration, specifically targeting motoneurons (MN). In the SOD1 transgenic mouse model of ALS, recent evidence has shown that disease progression results in two populations of MN, one compensatory and one degenerative. Using facial nerve axotomy to investigate MN survival, we have also discovered two populations of MN, one regenerative and one degenerative, resulting from an uneven distribution of survival across the six facial nucleus subnuclei in wild‐type (WT) mice. Facial nerve axotomy is used in this study to examine the injury‐induced molecular response of diseased MN in pre‐symptomatic SOD1 mice compared to WT. The regenerative ventromedial (VM) subnucleus maintains the highest level of MN survival following axotomy in WT (100%) and SOD1 (60%), while the degenerative ventrolateral (VL) elicits a significant MN survival decrease in WT (70%) and SOD1 (20%). WT and SOD1 subnuclei were characterized via laser microdissection with RT PCR (PACAP, GAP‐43, ?II tubulin, BDNF, Hn1, CX3CL1, GFAP, caspase 8, TNFá, IFNã, IL‐10, IL‐4), and in situ hybridization. The axotomized WT and SOD1 neuronal response is similar. However, differences in the SOD1 neuropil may lead to the increased cell death observed in the VM and VL compared to WT.

Supported by the Les Turner ALS Foundation (NAM and KJJ).

Grant Funding Source

Les Turner ALS Foundation & NIH
Fibulin‐1 is a matricellular regulator of the RAS/mitogen activated protein kinase (MAPK)‐mediated signal transduction pathway
Authors: Argraves W. Scott , Cooley Marion A. , Harikrishnan Keerthi , Twal Waleed O. , Fresco Victor M. , Pruitt Matthew W. , Barth Jeremy L.
Published: June 2021

Fibulin‐1 (Fbln1) is a glycoprotein found in extracellular matrices of the embryo and adult. In tissues of vertebrate embryos, Fbln1 is found associated with fibronectin and versican. In postembryonic vertebrate tissues, Fbln1 is found associated with elastin‐containing fibers including those in blood vessels, lungs and skin. Targeted deletion of the Fbln1 gene in mice reveals a critical developmental role for Fbln1 in the directed migration and growth of cranial neural crest cells contributing to the development of pharyngeal glands, craniofacial skeleton, cranial nerves, aortic arch arteries, cardiac outflow tract and cephalic blood vessels. However, Fbln1 deficiency does not lead to overt abnormalities in the formation of elastin‐containing fibers. Therefore, the elastin fiber association of Fbln1 may not be reflective of a role for Fbln1 in the assembly and maintenance of elastin fibers, but of a matricellular function in which it serves to modulate cell functions. Findings from several avenues of investigation point to Fbln1 as being a regulator of the RAS/MAPK‐mediated signal transduction pathway. Many of developmental defects observed in Fbln1‐deficient mouse embryos are consistent with disorders associated with mutations in molecules in the RAS/MAPK cascade. Indeed, we find that Fbln1 influences the activity of several growth factors that stimulate the RAS/MAPK pathway.
Integration of histology and pathology in an electronic curriculum
Authors: Klein Robert M , Karr Michael D , Paolo Anthony , Fishback James L
Published: June 2021

In 2006, the University of Kansas, School of Medicine introduced an integrated, Tablet PC‐based, preclinical undergraduate medical curriculum to the 2010 class. The curriculum began with Foundations of Medicine, Genetics & Neoplasia, and Immunity & Inflammation, followed by systems modules running through year two. Histology and pathology laboratories were merged and offered in each module, using the Aperio(tm) digital slide system. The electronic inking features of the Tablet PC were essential for students to construct individualized histopathology atlases. The classes of 2010 and 2011 used a "follow the leader" approach. For the 2012 class, students are required to preview material using detailed protocols with digital slide links and short videos summarizing each slide. Lab is focused on answering student questions in depth, replacing the superficial review of cases. Following discussion, students take an online quiz with randomized items. Student feedback shows statistically significant improvement in effective use of lab time (p=0.03), compared to traditional microscope labs. Summative exam scores for histopathology items are high in both the initial and current virtual lab formats. Our approach has allowed us to breathe new life into learning histology and histopathology, to increase active learning, and to help students develop the lifelong skills needed to practice medicine in a digital world.
Allosteric Mechanisms in Receptor Tyrosine Kinase Activation
Authors: Kuriyan John
Published: June 2021

The control mechanisms that govern the response of receptor tyrosine kinases to input signals are multi‐layered and complex. Our studies on the epidermal growth factor receptor (EGFR) and the downstream Ras‐specific guanine exchange factor (Son of Sevenless, SOS) will be presented. For EGFR, we have defined a specific asymmetric interaction between kinase domains that is necessary for activity. Potential mechanisms that couple this activating dimerization to the transmembrane segments of the receptor will be discussed. The activation of SOS by EGFR and other tyrosine kinases involves the recruitment of SOS to the membrane, where it encounters Ras. We have uncovered an unexpected mechanism in which Ras itself is capable of tethering SOS to membranes in a way that makes SOS responsive to the presence of PIP2 in the membrane, as well as the surface density of Ras. The implications of these finding for the activation of Ras will be discussed.
Structural and functional analysis of the anaphase‐promoting complex
Authors: Gould Kathleen L. , McLean Janel R. , Andrews Carolyn , Feoktistova Anna , Chambers Melissa , Chen Jun‐Song , Ohi Melanie
Published: June 2021

E3 ubiquitin ligases vary widely in size, composition, and enzymology reflecting their regulatory role in substrate recognition. The conserved E3 required for ubiquitination and degradation of cyclin, securin, and other proteins during mitosis and G1 phase is called the anaphase‐promoting complex or cyclosome (APC/C). APC/C activity is therefore required for chromosome segregation. The APC/C is composed of multiple core subunits (13 in yeast). However, it is not known what most subunits do to facilitate the ubiquitination reaction. To provide insight into this question, we are using single particle cryo‐electron microscopy, chemical crosslinking combined with mass spectrometric analysis, and functional analysis. At the resolution of our current density map (~27Å), the activated APC is a triangular‐shaped structure, ~19 x 17 x 15 nm (H x W x D) in size, with a deep internal cavity and a prominent horn‐like protrusion emanating from a lip of the cavity. Antibody labeling and mutant analysis allowed us to localize the position of 12 core components and the Slp1 activator, enabling us to propose a model for APC/C organization. Current studies are focused on determining the mechanism of checkpoint protein binding, E2 enzyme interaction, and defining the interfaces involved in substrate associations.
Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation
Authors: Deshaies Raymond , Saha Anjanabha
Published: June 2021

We seek to develop quantitative assays of individual steps that underlie substrate ubiquitination, to gain a detailed understanding of the enzymological mechanism by which E2 and E3 collaborate to specify the synthesis of an ubiquitin chain upon substrate. We have directed our initial efforts to understanding how conjugation of Nedd8 influences the activity of the archetypal cullin‐RING ligase (CRL) SCF. Conjugation of ubiquitin‐like protein Nedd8 to cullin (i.e. neddylation) is known to be essential for the function of CRLs. Our data show that neddylation acts through multiple channels to stimulate SCF. First, it stimulates recruitment of ubiquitin‐conjugating enzyme (E2) esterified with ubiquitin (E2~Ub). Second, it helps bridge the ~50 Ågap between E2 and substrate bound to SCF to enable their reaction. Third, it facilitates formation of amide bonds in E2's active site. Combined together, these effects potently stimulate transfer of the initial ubiquitin to substrate. We propose that the initiator ubiquitin partly spans the 50 Ågap, and the impact of neddylation on transfer of subsequent ubiquitins by the E2 Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires =4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation. Given the major functional impact of Nedd8 conjugation upon SCF activity, we sugget that the autoubiquitination often exhibited by other E3s has the potential to have major functional consequences on E3 activity.
Structure, regulation and dynamics of mammalian nitric oxide synthase
Authors: Getzoff Elizabeth D
Published: June 2021

Nitric oxide synthase (NOS) enzymes control, by synthesis, the activities of their product nitric oxide (NO) as a signal and defensive cytotoxin. In mammals, three very similar, but differently regulated, NOS isozymes direct NO to distinct roles in neurotransmission, vascular homeostasis and host defense, thereby offering important therapeutic targets. Our mutational, biochemical and biophysical investigations of NOS define structures of its component catalytic oxygenase and electron‐supplying reductase modules, and Ca2+/calmodulin‐bound linker; characterize holo‐enzyme assembly and dynamics; and address current mechanistic questions regarding catalysis, regulation and isozyme‐selective inhibition. Our analyses suggest a large scale swinging motion of the entire FMN‐binding domain of NOS reductase to deliver electrons to NOS oxygenase for catalysis.1 Our inhibitor‐bound NOS oxygenase crystal structures, along with biochemical and mutagenesis results, revealed that plasticity of distant isozyme‐specific second‐ and third‐shell residues modulates conformational changes of invariant first‐shell residues to determine inhibitor selectivity.2 To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues.1E.D. Garcin et al., (2004) J. Biol. Chem. 279: 37918; 2E.D. Garcin et al. (2008) Nat. Chem. Biol. 4: 700. (Supported by NIH R01 HL58883 and the Skaggs Institute for Chemical Biology).
Mechanisms of ATP‐dependent Chromatin Remodeling Enzymes
Authors: Narlikar Geeta J , Racki Lisa , Yang Janet , Cheng Yifan , Naber Nariman , Cooke Roger
Published: June 2021

Compaction of eukaryotic DNA in the form of chromatin represents a major organizational feat allowing the packaging of nearly 2 meters of DNA into a space of the order of a few microns. Despite the dense packaging, a eukaryotic cell responds to extra‐cellular signals within minutes by turning on multiple specific genes from within the large mass of compacted DNA. This rapid response is achieved in large part by enzymatic complexes that use the energy of ATP to reorganize chromatin structure. The mechanisms by which these machines alter chromatin structure are not known. We are using biophysical and structural approaches to understand how these enzymes move nucleosomes and how they couple the energy of ATP to do so.
Heterochromatin Protein 1a stimulates histone H3 lysine 36 demethylation by the Drosophila KDM4A demethylase
Authors: Workman Jerry L , Lin Chia‐Hui , LI Bing , Swanson Selene , Zhang Ying , Florens Laurence , Washburn Michael , Abmayr Susan M
Published: June 2021

Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. However, mechanisms governing the targeting and regulation of histone demethylation remain elusive. Here we report that a Drosophila melanogaster JmjC domain‐containing protein, dKDM4A, is a histone H3K36 demethylase. dKDM4A specifically demethylates H3K36me2 and me3 both in vitro and in vivo. Affinity purification and mass spectrometry analysis revealed that Heterochromatin Protein 1a (HP1a) associates with dKDMA4A. We found that the chromoshadow domain of HP1a and a HP1‐interacting motif of dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A and this stimulation depends on the H3K9me binding motif of HP1a. Finally, we provide in vivo evidence suggesting that HP1a and dKDM4A interact with each other and loss of HP1a leads to increased level of histone H3K36me3. Collectively, these results suggest a function of HP1a in transcription facilitating H3K36 demethylation at transcribed and/or heterochromatin regions.
Ribozymes and riboswitches: modulation of RNA function by small molecules
Authors: Ferre‐D'Amare Adrian R
Published: June 2021

Riboswitches are mRNA domains that directly bind small‐molecule metabolites and control expression of the mRNA they are part of in a ligand‐dependent manner. The glmS riboswitch is thus far unique in that it is also a catalytic RNA, or ribozyme. The self‐cleaving activity of this RNA from Gram‐positive bacteria requires binding of glucosamine‐6‐phosphate (GlcN6P), an essential precursor to the bacterial cell wall. The ribozyme is part of the mRNA for the enzyme GlcN6P synthetase. Self‐cleavage leads to mRNA destruction, and therefore, negative‐feedback regulation. Crystallographic analysis of the glmS riboswitch reveals a remarkably rigid RNA and binding of GlcN6P in contact with the scissile phosphate. This and the inactivity of the ribozyme in the absence of GlcN6P indicate that the metabolite functions as a true coenzyme. However, the RNA is not simply an inert structural scaffold. Mutational, kinetic and structural analyses reveal that an active‐site guanosine and GlcN6P mutually modulate the ability of each other to function as general acid‐base catalysts. Supported by the Damon Runyon Cancer Research Foundation, the Howard Hughes Medical Institute, the NIH and the W.M. Keck Foundation.
Small molecule and genomic approaches to bacterial pathogenesis
Authors: Hung Deborah T.
Published: June 2021

Infectious diseases account for almost 1/3 of total deaths worldwide. A better understanding of how bacterial pathogens cause disease will lead to new paradigms for treatment and management of such diseases. Small molecules can be valuable tools to dissect the pathogenic interactions between the host and bacteria, both in vitro and in vivo. In addition, the smaller genomes of bacteria allow comprehensive genomic approaches to be developed and utilized to identify the targets of active small molecules, to understand the mechanisms of these small molecule probes and to elucidate the fundamental biology of infection. We will illustrate several examples of genomic approaches to target identification in the study of Mycobacterium tuberculosis.