bluuebunny/crossref_metadata_2025 huggingface dataset:
Colonic fermentation of indigestible carbohydrates of a previous evening meal increases tissue glucose uptake
Authors: Priebe Marion G , Wang Hongwei , Preston Tom , Small Alexandra , Vonk Roel
Published: June 2021

Evening meals rich in indigestible carbohydrates have been shown to lower postprandial glucose concentrations after ingestion of a high glycemic index breakfast. This phenomenon is linked to colonic fermentation of indigestible carbohydrates but the underlying mechanism is not fully elucidated. We examined in which way glucose kinetics after the breakfast are changed due to colonic fermentation. In a cross‐over manner 10 healthy male subjects (21 ± 2 years, BMI: 21,4 ± 1 kg/m2) ingested as an evening meal either white wheat bread or cooked barley kernels, rich in indigestible carbohydrates. In the morning the dual isotope technique was applied to determine glucose kinetics after intake of 50 g 13C‐enriched glucose. Plasma insulin and non‐esterified fatty acid concentrations were measured as well as hydrogen excretion in breath. Postprandial glucose response was 29 % lower after the barley evening meal, whereas the concentrations of insulin and non‐esterified fatty acids were the same. Exogenous glucose appearance in the systemic circulation and endogenous glucose production did not differ, but tissue glucose uptake was increased after the barley evening meal. Hydrogen excretion was higher after the barley meal indicating increased colonic fermentation. Factors related to colonic fermentation of indigestible carbohydrate seem to have the ability to increase tissue insulin sensitivity in healthy subjects.
Comparable Dose‐Response Glucose Lowering Effect with Whole vs finely Ground, Novel Omega‐3 rich Grain Salba (Salvia Hispanica L) Baked into White Bread
Authors: Vuksan Vladimir , Dias Andre , Lee Amy , Jovanovski Elena , Rogovik Alex , Jenkins Alexandra
Published: June 2021

Objective

Oily grains such as flax, sesame and poppy are common additions to bakery products. To evaluate the effect of particle size on postprandial glycemia, whole or finely ground omega‐3 rich grain Salba was baked into white bread in a dose response study.

Methods

Using an acute randomized, double‐blind, crossover design, 13 healthy subjects (5M; 8F; BMI 26.3 ± 7.3kg/m2) received, on 8 different occasions, either 7, 15 or 24 g of whole or finely ground Salba baked into white bread cotaining 50g available carbohydrate or bread control given twice. Capillary blood was taken fasting and at 15, 30, 45, 60, 90 and 120 minutes after consumption.

Results

Compared to control, all treatments containing 7,15,or 24g of whole and ground Salba reduced blood glucose iAUC (p=0.03) by 20%, 28% and 35% respectively compared to control (p<0.05), in dose dependent fashion (slope = ‐1.2 ± 0.05). No differences in glucose responses were found between whole vs ground Salba at the same dose levels.

Conclusions

Addition of either whole or finely ground Salba to white bread lowers postprandial glycemia equally in a dose‐dependent manner making this novel omega‐3 grain versatile for consumption. Baking whole Salba in bread may also extend shelf life by minimizing fat oxidation.
Dietary mushrooms reduce mitogenesis and induce apoptosis and cytotoxicity in MCF‐7 human breast cancer cells
Authors: Martin Keith R , Brophy Sara
Published: June 2021

Cancer is a leading cause of death in the U.S. Studies show that people with diets rich in fruits and vegetables have a reduced risk of cancer. Moreover, an accumulating body of evidence also suggests that consumption of dietary mushrooms, or fungi, can protect against many forms of human cancer. In this study, we tested the ability of five commonly consumed mushrooms to modulate the cancer process in MCF‐7 human breast cancer cells. Hot water extracts (5% v/v) of freeze‐dried maitake (MT), crimini (CRIM), portabella (PORT), king oyster (OYS), and white button (WB) mushrooms or water alone were incubated overnight with MCF7 cells. Mitogenesis, measured by BrdU incorporation, was significantly reduced by all mushrooms up to 33% with MT and OYS being the most potent. Cell number, however, did not significantly differ between groups as measured by hemacytometry. Apoptosis, measured by TUNEL, was increased significantly (p<0.05) by 40% after incubation with MT and ~20% (p>0.05) by other test mushrooms. MTT reduction, a mitochondrion‐dependent marker of proliferation, was unchanged. Cytoplasmic LDH release significantly increased after MT and WB, but not after other test mushrooms, indicating cytotoxicity although trypan blue exclusion appeared marginally elevated (95%) compared to control cells (92%) after 24 hours. Overall, dietary mushrooms reduced mitogenesis and induced apoptosis and cytotoxicity in MCF7 cells.
Effects of daily black walnut consumption on blood lipid levels
Authors: Fitschen Peter , Winfrey Michael , Wilson Edward , Maher Margaret
Published: June 2021

English walnuts (EW) have been marketed for cardiovascular benefits including favorable changes in blood lipid profile, attributed to their fatty acid content. In contrast, black walnuts (BW) have not yet been studied for such benefits. The purpose of our research was to compare black versus English walnut effects on blood lipid levels. Thirty‐six subjects (female=20, male=16), ages 62±12 years, were recruited and randomly assigned to either EW or BW groups (18 per group). Venous blood lipid levels were analyzed following a 12‐hour fast before and after 28‐30 days of walnut consumption. Subjects consumed 30 g of provided pre‐weighed nuts daily with their usual diet. They reported usual food intake and medications and were weighed before and after each nut treatment. After a 12‐week washout period, 29 subjects (female=16, male=13) crossed over and consumed the other type of walnut for 28‐30 days (BW=13, EW=16). Response to EW consumption in both genders was largely consistent with previous studies. A significant reduction in LDL, HDL and total cholesterol was found in men who consumed BW; however, a significant increase in total cholesterol, HDL, and LDL was found in women who consumed BW. Consumption of either walnut type did not result in weight gain. Walnuts may benefit health, but black walnut benefits may be affected by gender. This research was funded by UW‐L internal grants and BW were supplied by Hammons Products Co.
Ellagic acid prevents ultraviolet radiation‐induced chronic skin damage of skin cells and in the hairless mice
Authors: Bae Ji‐Young , Kim Jung Lye , Kang Young‐Hee
Published: June 2021

Ellagic acid, a polyphenol compound present in berries and nuts, has received attention as an agent that may have potential bioactivities preventing chronic diseases. UV radiation led to collagen degradation mediated by MMP has been suggested to cause skin wrinkling. In this study, we examined the photoprotective effect of ellagic acid on collagen degradation and inflammation responses in UV‐B irradiated human skin cells. Ellagic acid attenuated the UV‐B‐induced toxicity of skin cells, evidenced by MTT assay. Western blot analysis revealed that ellagic acid markedly prevented collagen degradation through blocking production of MMP in UV‐B‐exposed dermal fibroblasts. In addition, ellagic acid mitigated expression of inflammatory ICAM‐1 in UV‐B‐irradiated HaCaT cells. Anti‐wrinkle activity of topical ellagic acid was further investigated in vivo in hairless mice exposed to UV‐B for 8 weeks. Topical application of 10 µM ellagic acid on the skin surface of hairless mice inhibited UV‐B‐triggered skin wrinkle formation and increase in epidermal thickness. Additionally, ellagic acid attenuated production of pro‐inflammatory cytokines IL‐1β and IL‐6 in mice. These results demonstrate that ellagic acid prevents UV‐B‐irradiated wrinkle formation and photoaging caused by collagen destruction and inflammatory responses. Supported by grant (KRF F00072) from Korea Research Foundation and Brain Korea 21.
Alteration of the autophagy‐lysosomal system in models of Parkinson's disease
Authors: Pivtoraiko Violetta Nikolaevna , Schacka John , Roth Kevin
Published: June 2021

In Parkinson's disease (PD), increasing evidence suggests that neuron death may be related to alterations in the autophagy‐lysosomal pathway (ALP). To further investigate the role of the ALP in neuron death regulation in PD models, differentiated SH‐SY5Y human neuroblastoma cells were treated with either chloroquine (CQ), known to induce autophagic neuronal death, morphologically defined by the aberrant accumulation of autophagic vacuoles (AVs) concomitant with markers of apoptosis and/or necrosis, or rotenone (Rt), a selective inhibitor of complex I of the mitochondrial electron transport chain. CQ induced AV accumulation and inhibited cathepsin D processing (lysosomal aspartic protease), suggesting a CQ‐induced compromise of the ALP. CQ promoted α‐synuclein aggregation, one of the principal hallmarks of PD, emphasizing the potential for altered lysosome function in PD. CQ‐induced an increase in caspase‐3‐like enzymatic activity. Rt‐induced cell death was also accompanied by AV accumulation and increased caspase‐3‐like enzymatic activity. However, pharmacological inhibition of caspase activity did not significantly attenuate CQ or Rt‐induced cell death. These observations suggest that alteration of the ALP may facilitate accumulation of aggregated protein and contribute to the induction of multiple types of neuron cell death in PD.

This work was supported by NS57098, NS 47466, and NS35107
Retinoid signaling pathway protein alterations in patients with amyotrophic lateral sclerosis
Authors: Kolarcik Christi , Bowser Robert
Published: June 2021

The mechanisms underlying the motor neuron (MN) degeneration that occurs with amyotrophic lateral sclerosis (ALS) are poorly understood. Multiple studies have described the differential expression of retinoid signaling components in ALS patients and transgenic animal models. We investigated this pathway in human lumbar spinal cord tissue of patients with sporadic (SALS) and familial (FALS) ALS as well as controls with immunohistochemistry, immunoblotting, co‐immunoprecipitation and DNA‐binding assays. In SALS MNs, cellular retinoic acid binding protein (CRABP)‐II, the protein responsible for facilitating nuclear receptor interactions, was concentrated to the nucleus while FALS and control MNs had a diffuse cytoplasmic pattern. Nuclear retinoic acid receptor (RAR) βexhibited increased immunoreactivity in MN nuclei of individuals with SALS although both controls and individuals with FALS lacked this nuclear immunostaining. For MNs with RARβ nuclear staining, activated caspase‐3 was negative. Our results indicate that retinoid signaling is altered in ALS. Differences with respect to RARβ suggest that retinoid signaling impacts only SALS and that this is a pro‐survival response.

Research Support: NIH grant ES013469 (RB); NIH T32 EB001026 (CK); NIH T32 05 TL1 RR024155‐02 (CK)
Role of FRNK tyrosine phosphorylation in vascular remodeling following balloon angioplasty
Authors: Koshman Yevgeniya Emre , Engman Steven J , Iyengar Rehka , Kim Taehoon , Paul Jennifer T , Henderson Kyle K , Samarel Allen Mark
Published: June 2021

FRNK (FAK‐Related Non‐Kinase) expression is greatly increased in remodeled arteries, and may serve to limit neointimal formation via inhibition of smooth muscle cell (SMC) migration and proliferation. To determine the role of tyrosine phosphorylation of FRNK, rat carotid arteries were balloon‐injured and FRNK expression and phosphorylation were examined by immunoprecipitation and Western blotting with phosphospecific antibodies. FRNK expression increased 3‐4 fold in injured, but not in contralateral, control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. A 5‐6 fold increase in total FAK levels was also detected. Furthermore, endogenously expressed FRNK was phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to Ang II treatment of cultured A7r5 cells. When adenovirally overexpressed in cultured A7r5 cells and rat aortic SMC, GFP‐wtFRNK was phosphorylated at residues Y168 and Y232, and GFP‐wtFRNK inhibited cell spreading. GFP‐FRNK mutated at Y168 abrogated FRNK‐mediated inhibition of cell spreading. We conclude that FRNK phosphorylation at Y168 plays an important role in inhibition of FAK‐dependent signal transduction. Supported by the Falk Medical Research Trust and an AHA Predoctoral Fellowship.
Pericytes facilitate leukocyte transmigration in vivo
Authors: Proebstl Doris , Voisin Mathieu‐Benoit , Nourshargh Sussan
Published: June 2021

Venular walls are composed of endothelial cells, pericytes, and the vascular basement membrane (BM). Very little is known about the role of pericytes in leukocyte transmigration through venular walls. We have previously found that gaps between adjacent pericytes are co‐localised with matrix protein low expression regions (LERs) in the BM of murine cremasteric venules, regions that are preferentially used by transmigrating leukocytes to penetrate venular walls (Wang et al., J Exp Med., 2006). We now show that pericyte coverage determines the LERs expression profile in multiple other vascular beds, eg mesentery and skin. Furthermore, analysis of IL‐1b‐ or TNFa‐stimulated mouse cremaster muscles by immunofluorescence and confocal microscopy indicated the ability of these cytokines to induce contraction of pericytes in vivo (eg TNFa induced a 97% increase at 2h in the size of gaps between adjacent pericytes; p<0.001). Time‐course studies indicated that TNFa‐induced pericyte shape change preceded leukocyte transmigration. This response occurred independently of PMNs as it was also noted in PMN depleted mice. Collectively, the results demonstrate that pericytes facilitate leukocyte transmigration by governing the existence of leukocyte permissive regions in the vascular BM (ie LERs) and through active shape change at sites of inflammation. Funded by Barts & The London School of Medicine and The Wellcome Trust
Effect of soluble JAM‐C on leukocyte transmigration in models of ischemia/reperfusion injury
Authors: Colom Bartomeu , Scheiermann Christoph , Woodfin Abigail , Meda paolo , Patel Nimesh , Marrelli Allessandra , Pitzalis Costantino , Thiemermann Chris , Aurrands‐Lions Michel , Imhof Beat , Nourshargh Sussan
Published: June 2021

Numerous in vitro studies have implicated the adhesion molecule JAM‐C in leukocyte transendothelial cell migration but the role of JAM‐C in leukocyte migration in vivo requires further investigations. Here we used soluble JAM‐C (sJAM‐C) as a blocker of JAM‐C‐mediated reactions to investigate the role of this molecule in leukocyte transmigration in vivo as induced by ischemia/reperfusion (I/R). The study employed a murine model of kidney I/R injury and investigated I/R‐induced leukocyte migration by intravital microscopy in mouse cremasteric venules. In the kidney model, mice were subjected to an acute bi‐lateral renal ischemia (30 min) followed by a 24hr reperfusion period after which kidneys were removed and PMN infiltration was quantified in tissue sections by immunohistochemistry. Kidneys subjected to I/R injury exhibited a significant PMN infiltration response as compared to sham‐operated kidneys (~22 fold increase) and mice pre‐treated with sJAM‐C (3mg/kg, i.v.) showed a significant reduction in PMN infiltration (49.6% inhibition, p<0.05). In cremasteric venules, sJAM‐C suppressed I/R‐induced (30min ischemia/120 min reperfusion) leukocyte adhesion and transmigration. The results demonstrate a role for JAM‐C in PMN migration as induced by I/R injury.

Funded by The Wellcome Trust and The British Heart Foundation.
Protein‐coding and MicroRNA Biomarker Gene Panels Predictive of Clinical Recurrence in Prostate Cancer
Authors: Moreno Carlos S , Lai Yu‐Heng , Seth Arun , Osunkoya Adeboye O , Zhou Wei , Petros John A , Johnson Brent A , Nam Robert K , Barwick Benjamin G , Abramovitz Mark , Bouzyk Mark , Leyland‐Jones Brian R
Published: June 2021

An important challenge in prostate cancer research is to develop effective predictors of tumor recurrence following surgery. To identify predictive biomarkers, we performed DASL expression profiling with a custom‐designed panel of 522 prostate cancer relevant genes on 71 FFPE radical prostatectomy specimens with long‐term outcome. We identified a panel of eight genes that could be used to predict recurrence (p = 0.001). Furthermore, this gene panel could separate patients with and without recurrence in the subset of 46 patients with Gleason 7 tumors (p = 0.022) for whom the decision regarding adjuvant therapy is particularly important and difficult. We also performed comprehensive miRNA profiling of these samples, and identified six miRNAs predictive of recurrence (p=0.001) for all 71 patients and for the subset of 46 Gleason 7 patients (p=0.032). Following promoter analysis of these miRNA genes, we determined that four of them are directly bound by SOX4 in chromatin immunoprecipitations, suggesting that SOX4 may regulate their expression. Among the potential downstream targets of these miRNA genes are several tumor suppressors involved in cell‐cycle arrest and apoptosis. It may therefore now be possible to increase the accuracy of prognostication for individual patients following radical prostatectomy using universally available archived specimens.
Atorvastatin Attenuates Lrp5 Mediated Calcification in the Hypercholesterolemic Aortic Valves from ApoE mice
Authors: Kruger Bryan Mathew , Stock Stuart R , Rajamannan Nalini M
Published: June 2021

Background

Calcification in the cardiovascular system is becoming an important prognostic marker for valvular disease. The cellular mechanisms of the calcification process are not well known.

Methods

In this study we tested the effect on lipids with and with atorvastatin in the ApoE mouse model to determine the localization of calcification and the regulation of the Lrp5 receptor in the valves. ApoE‐/‐ mice (n=60). Group I (n=20) normal diet, Group II (n=20) 0.25% chol diet (w/w), and Group III (n=20) 0.25% (w/w) chol diet+atorv for the development of calcification over 24 weeks. The aortic valve (AVA) was examined for calcification, Lrp5 and bone matrix markers. Bone formation was assessed by micro Computed Tomography (microCT), Lrp5, cbfa‐1 and osteopontin expression.

Results

The cholesterol diet induced complex bone formations in the calcified AV with an increase in osteopontin, cbfa‐1 expression and Lrp5 receptor expression as compared to the Control(>3‐fold(p<05). Atorvastatin attenuated these markers. Echocardiography demonstrated a mild increase in the peak jet velocity across the aortic valve but not statisically significant.

Conclusion

These results demonstrate that Lrp5 mediated bone formation in the hypercholesterolemic aortic valves is attenuated with atorvastatin. Providing evidence in that statins play a role in the treatment of this disease.
Hepatocytes express stem cell markers under the influence of growth factors in vitro and during transdifferentiation to billiary epithelial cells in vivo
Authors: Bhave Vishakha Sudhir , Bowen William C , Paranjpe Shirish G , Michalopoulos George K
Published: June 2021

The objective of our present work was to test if hepatocytes express stem cell markers during regeneration or transdifferentiation. An in vitro model of primary rat hepatocytes and an in vivo model of transdifferentiation were used. Primary rat hepatocytes were cultured hepatocyte growth factor and epidermal growth factor. Plates were harvested at 0, 2, 4, 6, 8, and 10 days for mRNA, protein, and immunocytochemistry. The RE1‐silencing transcription factor that has previously been shown to regulate the expression of other stem cell markers was upregulated along with stem cell markers like Oct3/4, Nanog, cMyc, Klf4 in the presence of growth factors. Studies from our laboratory have shown that hepatocytes transdifferentiate to billiary epithelial (BE) cells in vivo in case of impaired billiary regeneration following bile duct ligation (BDL) and subsequent administration of billiary toxicant methylene dianiline (DAPM). We found that one week after BDL? treatment to rats, there was an upregulated expression of stem cell markers by hepatocytes. The levels reverted back by week 4. The fact that hepatocytes transiently express stem cell markers (responsible for maintaining pluripotency in embryonic stem cells) is interesting and could be a mechanism that facilitates their transdifferentiation to BE. This work is supported by 5R01CA103958‐05 titled HGF and signaling pathways in hepatic tissue assembly.
Association of Breast Cancer Resistance Protein/ABCG2 Phenotypes and Novel Promoter and Intron 1 Single Nucleotide Polymorphisms
Authors: Poonkuzhali B. , Lamba J. , Strom S. , Sparreboom S. , Thummel K. , Watkins P. , Schuetz E.
Published: June 2021

The hypothesis was tested that sequence diversity in breast cancer resistance protein (BCRP)'s cis‐regulatory region is a significant determinant of BCRP expression. The BCRP promoter and intron 1 were resequenced in lymphoblast DNA from the polymorphism discovery resource (PDR) 44 subset. BCRP single nucleotide polymorphisms (SNPs) were genotyped in donor human livers, intestines, and lymphoblasts quantitatively phenotyped for BCRP mRNA expression. Carriers of the ‐15622C>T SNP had lower BCRP expression in multiple tissues. The intron 1 SNP 16702C>T was associated with high expression in livers; 1143G>A was associated with low expression in intestine; 12283T>C was associated with higher expression in the PDR44 and White livers. The ‐15994C>T promoter SNP was significantly associated with higher BCRP expression in multiple tissues. Patients with the ‐15994C>T genotype had substantially higher clearance of p.o. imatinib. Liver polymorphically expressed an alternatively spliced mRNA [splice variant (SV) 1] skipping exon 2. Although SV1+ livers did not uniformly carry the exon 2 G34A allele, 90% of G34A livers expressed SV1. BCRP mRNA was significantly lower among Hispanic livers with the G34A variant genotype and may be due, in part, to polymorphic exon 2 splicing. Allele expression imbalance and use of multiple promoters were seen in BCRP mRNA. In conclusion, BCRP expression in lymphoblasts, liver, and intestine is associated with novel promoter and intron 1 SNPs.
Hemangiopericytoma with chromosomal aberrations on chromosomes 8 and 10: Evidence for a distict brain tumor entity
Authors: Schober Ralf , Ahnert Peter , Krupp Wolfgang , Holland Heidrun , Ogunlade Vera , Livrea Michela , Meixensberger Jürgen
Published: June 2021

Hemangiopericytoma is not universally recognized as a tumor entity, and there is a trend to reassign it piecemeal to other tumor categories. However, it has been included in the new WHO classification of brain tumors and has been distinguished from both meningioma and fibrous tumor. Since there are few genetic studies in existence, we have performed a comprehensive cytogenetic analysis of an infratentorial hemangiopericytoma in a 55 y‐old female. It was originally classified as a grade II tumor but recurred as a grade III tumor with a proliferation index of 20 %. By use of trypsin‐Giemsa staining (GTG‐banding) and multicolor fluorescence in situ hybridization (M‐FISH) we could confirm the loss of chromosomal material 10q that has been previously described in hemangiopericytoma, and furthermore we identified de novo chromosomal aberrations on chromosomes 8. Applying high‐density single nucleotide polymorphism array (SNP‐A) karyotyping, we detected several regions suggestive of partial uniparental disomy and clonal deletions as well as segments with loss or gain. These findings together with the results of immunohistochemical investigations seem to justify the nosological separation of hemangiopericytoma as a biologically different entity and should be verified in larger patient series.
Aging and Vascular Endothelial Dysfunction
Authors: Seals Douglas R.
Published: June 2021

Older age is a major risk factor for cardiovascular diseases (CVD). This is believed to be attributable in part to the development of vascular endothelial dysfunction, as most commonly demonstrated by impaired endothelium‐dependent dilation (EDD). EDD is reduced in older adults without clinical disease or major risk factors for disease, as well as in animal models resistant to the development of CVD, suggesting a direct effect of aging processes. Several biological and lifestyle factors influence EDD with aging in adult humans including habitual aerobic exercise, body fatness and its distribution, and circulating concentrations of low‐density lipoprotein cholesterol. The integrative physiological mechanisms mediating age‐associated impairments in EDD involve oxidative stress‐associated reductions in nitric oxide bioavailability. The development of vascular inflammation also appears to play a role, likely interacting with oxidative stress via mechanisms that are incompletely understood. Recent work explores the cellular and molecular events that contribute to impaired EDD with aging, including altered posttranslational modification of proteins and signaling. Efforts also are focusing on promising "anti‐aging" pharmacological interventions that may slow or prevent the development of vascular endothelial dysfunction and other clinically important expressions of arterial aging.
Interaction between Rgs and the Wnt Signaling Network during Zebrafish Organogenesis
Authors: Freisinger Christina M , Griesbach Hilary L , Lin Shengda , Slusarski Diane C
Published: June 2021

We have identified calcium release as an output of Wnt signaling network and are investigating the mechanisms and context by which this Wnt signal mediates its biological effect on the developing embryo. We have determined that the early calcium transients are, in part, modulated by Wnt5 and require G‐protein signaling. To identify potential intracellular regulators of calcium release dynamics, we are characterizing members of the Regulators of G‐protein Signaling (RGS) protein family.

We performed a comprehensive analysis of the role of RGS proteins utilizing the experimentally amenable zebrafish (Danio rerio). We cloned and characterized the expression pattern of rgs3, 4, and 5 and determined the developmental consequences of loss‐of‐function by gene knockdown studies. Developmental defects were identified by morphology, marker gene expression analyses and alteration to vascular outgrowth in transgenic zebrafish lines.

One RGS family member is co‐expressed in similar tissues as Wnt5. Preliminary analyses suggest synergistic interaction. Structural/functional analyses were performed to determine if direct binding to Gα subunits is required for RGS developmental actions. To this end, we evaluated the ability of wild‐type and mutant RGS cDNAs to rescue knockdown phenotypes. We also evaluated the impact of RGS mis‐expression on calcium signaling in the embryo.

Grant Funding Source

March of Dimes (5‐FY99‐806); NIH (RO3 HD045488‐01), RO1 (GM067881‐01); and American Heart Association Predoctoral Fellowship
The meaning of emptiness: Sinuses and sacs from land to sea
Authors: Marquez Samuel , Reidenberg Joy S.
Published: June 2021

The processes and patterns of skull pneumatization have intrigued anatomists for over two thousand years. The paranasal sinuses, in particular, have been observed in an array of animals including: amphibians, reptiles, birds, and terrestrial mammals. Sinus function has eluded investigators, leading some to conclude that they may be non‐functional in nature (vestigial? spandrels?). This is supported by their reduction/absence in some derived vertebrates (e.g., marine mammals). Investigators searching for the purpose of the paranasal sinuses have classified them in three broad categories of function/existence: 1) architectural ‐ e.g., distribution of weight/musculoskeletal forces, 2) physiological ‐ e.g., respiratory/vocal functions, or 3) vestigial ‐ the biological roles these spaces had in their phylogenetic history is no longer relevant. In diving mammals, the constraints of changing pressures made it impractical, and even dangerous, to maintain rigid sinuses with fixed volumes. However, sinus disappearance in diving mammals does not seem merely related to the gradual reduction of a vestigial structure. Rather, many terrestrial sinus functions were preserved and transferred to a novel set of structures: flexible walled, extracranial diverticulae called air sacs. The preservation of air spaces indicates that while the sinuses/air sacs may be "empty," they are still full of function.
A never‐ending pain in the ...: That annoying appendix
Authors: Fisher Rebecca E. , Smith Heather F. , Bollinger R. Randal , Parker William
Published: June 2021

Although the first description of the human appendix was published in 1521, its function remains the subject of debate. The human appendix has variably been classified as a vestige and as a synapomorphy uniting hominoids. The vestigial hypothesis was largely based on the lack of a known function for this structure, and the observation that patients are able to thrive after an appendectomy. However, recent studies indicate the mammalian appendix functions to maintain commensal bacteria in the gut, particularly after bouts of intestinal distress. This function may no longer be readily apparent due to modern medical care and hygienic environments. In fact, a comparative anatomical analysis suggests appendices have evolved independently in a number of mammalian groups, including marsupials, lagomorphs, rodents, and primates. Among the primates, appendices have been documented in a number of strepsirrhines, platyrrhines, and cercopithecoids, as well as all hominoids. The distribution of the appendix in primates suggests that this structure may have arisen early in this lineage, likely as a safe house for symbiotic microbial biofilms. Thus, the presence of an appendix in primates, including humans, is a derived character state, and may represent an adaptation or an exaptation in these species. Funded by the Fannie E. Rippel Foundation, Duke Cancer Center, and the NIH (RO1 HL60232‐03 and P30 DK34987).
Blind and naked, but oh so cool: The subterranean world of the naked mole‐rat
Authors: Park Thomas J , Smith Ewan St J , Lewin Gary R , Smith Timothy D
Published: June 2021

Are big ears, tails, and eyes simply anatomical "junk" to subterranean mammals? These animals show remarkable convergent evolution for reducing external ears, tails, and eyes. The naked mole‐rat (NMR, Heterocephalus glaber) displays these features, as well as unusual characteristics even among subterraneans (cold‐blooded and completely furless). Whereas visual and auditory spatial cues are of diminished importance, NMRs are specialized for alternative modalities (primarily via mechanoceptors). We are studying yet other unusual characteristics, which include two structures that may be heading for the anatomical junk pile. First, the NMR is the only mammal with a vomeronasal organ (VNO) that shows no post‐natal growth and sexual suppression is not mediated by urinary signals as in other rodents. Second, the nerve network for processing pain from chemicals (acid and capsaicin) is reduced and lacks neuropeptides that usually signal pain. NMRs are the only mammals that feel no pain from these substances. These features may be a result of this species' unusual combination of lifestyle traits (subterranean + hundreds of individuals + extremely social), resulting in CO2 levels high enough to cause acidosis and chemical pain. Coincidentally, the neuropeptides that are lacking from the pain system also mediate the VNO's vascular pump. NMRs provide unique insights into sensory specialization and evolutionary loss.
Short sleep, poor sleep: novel risk factors for type 2 diabetes
Authors: Van Cauter Eve
Published: June 2021

Sleep curtailment has become a very common behavior in industrialized countries. The aging of the population is also associated with an increased prevalence of sleep disturbances. There is rapidly accumulating evidence to indicate that chronic partial sleep loss and decreased sleep quality may increase the risk of obesity and diabetes. Laboratory studies have shown that sleep restriction (4‐6 h bedtimes) is associated with a decrease in Insulin without adequate compensation in beta cell function, resulting in an elevated risk of diabetes. Reduced sleep quality, without change in sleep duration, is also associated with an increased risk of diabetes. Indeed, selective suppression of slow‐wave sleep rapidly results in a marked reduction in insulin sensitivity and disposition index. Prospective epidemiologic studies in both children and adults are consistent with a causative role of sleep disturbances in the increased risk of diabetes. Sleep curtailment is also associated with a dysregulation of the neuroendocrine control of appetite. Under conditions of controlled caloric intake and energy expenditure, there is a robust negative relationship between leptin levels and sleep duration. Ghrelin levels increase with short sleep. Thus, sleep loss may alter the ability of leptin and ghrelin to accurately signal caloric need. The adverse impact of sleep deprivation on appetite regulation is likely to be driven by increased activity orexinergic. Consistent with the laboratory evidence, multiple epidemiologic studies have shown an association between short sleep and higher BMI after controlling for a variety of possible confounders. Taken together, the current evidence suggest that chronic partial sleep curtailment, a novel behavior that appears to have developed with the advent of the 24‐h society, and reduced sleep quality may be involved in the current epidemic of obesity and diabetes.
Sheet‐based Tissue Engineering: Autologous Blood Vessels and Beyond
Authors: L'Heureux Nicolas
Published: June 2021

Sheet‐Based Tissue Engineering (SBTE) is a method that allows the production of completely biological and living autologous tissues that have remarkable mechanical strength without the inclusion of any exogenous scaffolds. The introduction of this approach marks a paradigm shift away from the "off‐the‐shelf" approaches that have repeatedly failed in past, towards a more realistic approach where the patients own cells produce the basic organ structure in vitro. The first construct produced through SBTE is a small diameter blood vessel. In an initial clinical trial, blood vessels were produced for patients (age up to 89 y) suffering from end‐stage renal disease. Vessels displayed artery‐like burst pressures and satisfactory suture retention strength. Vessels were implanted in 10 patients with primary patency up to 22 months at time of submission. Ultrasound examinations demonstrated an increase in graft compliance to achieve physiological levels. Limited histological samples indicated a remodeling of the vessel wall with expression of smooth muscle cell markers. This study establishes for the first time that completely biological and autologous blood vessels produced in vitro have the potential for long‐term patency.
Evaluation of a biodegradable scaffold‐ and stem cell‐based tissue‐engineered vascular graft in rat and pig models
Authors: Vorp David Alan , Nieponice Alejandro , Soletti Lorenzo , Gharaibeh Burhan , Hong Yi , Huard Johnny , Wagner William
Published: June 2021

Bi‐layered, synthetic, biodegradable, elastomeric tubular scaffolds comprised of a highly porous medial layer and an external, reinforcing fibrous layer were bulk‐seeded with allogeneic (rat studies) or autologous (pig studies), LacZ+ muscle‐derived stem cells (MDSCs) using a previously described rotational vacuum seeding device, then placed in spinner flask culture for 24 hours. Cellular integration was assessed via nuclear and cytoskeletal staining. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks or carotid arteries of pigs for 30 days. Angiogram, histology, immunostaining and SEM were performed at time of explant. In the rat studies, cell‐seeded constructs showed a higher patency rate than the unseeded controls (63% vs. 0%). A smooth muscle‐like layer of cells was observed near the luminal surface of the constructs. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of vWF‐positive cells was observed in the lumen of seeded constructs, whereas controls showed thrombosis. In the pig studies to date, all TEVGs were patent, though intimal hyperplasia was observed. We have demonstrated the successful integration of allogeneic stem cell‐based TEVGs in a rat model and the feasibility of a completely autologous approach in a pig model. Support from NIH BRP #R01 HL069368.
Tissue engineering of an artificial nerve for injury repair
Authors: Terenghi Giorgio
Published: June 2021

Currently peripheral nerve gap injuries are repaired with an autologous nerve graft, which provides regenerating axons with natural guidance channels, Schwann cells and extracellular matrix molecules. Biocompatible nerve conduits are an alternative for nerve reconstruction, as their surface micro‐geometry may be combined with cultured cells, growth factors and extracellular matrix molecules (ECM) to form a tissue engineered artificial nerve.

The addition of cultured Schwann cells within the conduit has proved to have a crucial role, and we have been able to demonstrate their active participation in the regenerative process. Further improvements are provided by coating the conduits with ECM, which increase Schwann cells proliferation and axonal regeneration.

More recently, we have been able to differentiate adult bone marrow mesenchymal stem cells towards expressing phenotypic and functional characteristics of Schwann cells, and their transplantation in bioengineered nerve conduits has proved beneficial in promoting enhanced nerve regeneration. We have also demonstrated similar differentiation capacity for adipose tissue derived stem cells, which offer a tremendous potential for clinical application due to their easy accessibility. These results indicate that a bioengineering nerve implant is becoming closer to clinical reality.
Insulation and Specificity in Bacterial Two‐Component Signaling Systems
Authors: Goulian Mark
Published: June 2021

Two‐component systems are one of the major modes of signal transduction in bacteria. In their simplest forms, these regulatory circuits are characterized by two proteins ‐ an upstream sensor kinase and a downstream response regulator. They mediate responses to diverse environmental signals and play a central role in regulating many aspects of microbial physiology. Two‐component circuits have been uncovered in remarkable numbers within individual organisms and across different bacterial species. This raises interesting questions regarding the mechanisms that these systems use to minimize inappropriate cross‐talk between homologous circuits. I will present recent work in which we have explored design aspects of the simplest examples of these circuits in E. coli. I will primarily focus on mechanisms that maintain signal strength and provide insulation against cross‐talk.
The relative contribution of 'internal' vs. 'external' sensing to the regulation of gene expression in the budding yeast
Authors: Barkai Naama , Levy Sagi
Published: June 2021

Cells monitor their external environment using membrane receptors, modulating their gene expression pattern in response to external cues. In addition, cells monitor the level of internal metabolites, energy availability, and growth rate, and these internal characters also feedback to tune gene expression. We examined the relative contribution of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression in yeast. Since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis, coordination of ribosomal biogenesis with the requirement for protein synthesis is of particular importance. To distinguish between 'external' and 'internal' sensing, we subjected S. cerevisiae cells to 'confusing' conditions which decoupled the actual vs. environmentally‐expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in continuous cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer‐term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmentally‐mediated changes in cell growth presented a major selection force in the evolution of yeast, and perhaps also of other micro‐organisms.
A Complex Involved in the DNA Damage Response Network of Fanconi Anemia and Breast Cancer
Authors: Yan Zhijiang , Xu Dongyi , Guo Rong , Xue Yutong , Ling Chen , Li Yongjiang , Wang Weidong
Published: June 2021

Fanconi anemia (FA) has recently become an attractive model to study breast cancer susceptibility (BRCA) genes, as three FA genes, FANCD1, FANCN, and FANCJ, are found to be identical to BRCA genes BRCA2, PALB2, and BRIP1. Thirteen FA proteins have been identified, and they function in a DNA damage response network consisting of ATR, BLM, and BRCA1.

Our group has purified the FA core complex, and shown that it contains 8 FA proteins, together with two new FA‐associated polypeptides, FAAP100 and FAAP24. This complex contains FANCL, an ubiquitin ligase essential for monoubiquitination of FANCD2 and FANCI. It also contains FANCM, an enzyme capable of translocating and remodeling branched DNA structures, such as replication forks and recombination intermediates.

We have also purified the BLM helicase complex, and found that it contains two novel proteins, named RMI1 and RMI2. The latter two proteins constitute a novel OB‐fold complex which is essential for BLM to suppress sister‐chromatid exchange. Interestingly, the FA core complex and BLM complex constitute a larger multiprotein complex, termed BRAFT (for BLM, RPA, FA, and Topoisomerase IIIƒÑ). The fact that FA and BLM proteins are in the same complex suggests that they work together in a genome maintenance pathway.
Microbial Rhodopsins: Receptors, Channels, and Pumps from a Single Design
Authors: Spudich John L , Spudich Elena N.
Published: June 2021

The microbial rhodopsin family is comprised of ~4800 homologous proteins containing 7 transmembrane helices forming a pocket for the chromophore retinal. Most are light‐driven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). Since we identified the first sensory rhodopsin in 1982, homologous photosensors have been found to be widely distributed in archaea, eubacteria, and unicellular eukaryotes, and to signal with remarkably diverse mechanisms. Some of the best studied are the phototaxis receptors in haloarchaeal prokaryotes (SRI and SRII) and in eukaryotic algae (channelrhodopsins). SRI and SRII transmit signals by protein‐protein interaction to control a cytoplasmic phosphorylation cascade that modulates the prokaryotic flagellar motors. Mediating algal phototaxis, channelrhodopsins are light‐gated cation channels that depolarize the membrane to trigger Ca++‐influx into the eukaryotic flagellar axoneme. Crystallography, spectroscopy, and genetic engineering have begun to clarify how modifications of the same architecture enable the microbial rhodopsins to carry out their distinctly different molecular functions. Interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions.
Targeted and Pharmacologic Inhibition of DNMT3b Sensitizes Breast Cancer Cells to Chemotherapy
Authors: Sandhu Rupninder , Roll J Devon , Rivenbark Ashley G , Coleman William B
Published: June 2021

Many basal breast cancers express a hypermethylation defect related to DNMT3b overexpression. This feature may contribute to chemotherapeutic resistance and provides a target for development of new treatment strategies. To determine the influence of DNMT3b status on the sensitivity of hypermethylator breast cancer cells (MDA‐MB‐453) to doxorubicin hydrochloride (DOX), we studied the effects of RNAi‐mediated DNMT3b knockdown (KD) and/or demethylating agent 5‐aza. RNAi‐mediated DNMT3b KD increases the effectiveness of DOX, reducing the IC50 from 0.07 µM to 0.049 µM and increasing cell kill by 16% in response to 0.06 µM DOX. Pretreatment of cells with 250 nM 5‐aza for 3d and 7d or 500 nM 5‐aza for 3d did not increase DOX sensitivity. However, 7d pretreatment with 500 nM 5‐aza sensitized cells by reducing the IC50 to 0.039 µM and increased cell kill by 29% at 0.06 µM DOX. 5‐aza treatment of KD cells reduced IC50 to 0.038 µM and increased cell kill by 27% at 0.06 µM DOX. These results show: (i) RNAi‐mediated DNMT3b KD sensitizes hypermethylator breast cancer cells to DOX, (ii) 5‐aza treatment sensitizes hypermethylator cells to DOX as a function of dose and duration of exposure, and (iii) DNMT3b KD and 5‐aza treatment increase the effectiveness of DOX, individually and in combination. These results strongly suggest that targeting DNMT3b can be exploited to improve the efficacy of breast cancer chemotherapy.

Support: NIH CA78343
Extracellular matrix degradation in SIV encephalitis induces IRF‐1 signaling in astrocytes
Authors: Bonneh‐Barkay Dafna , Starkey Adam , Wang Wanda , Wiley Clayton A
Published: June 2021

The neuropathological changes associated with HIV encephalitis (HIVE) include activated and infected macrophages, astrocytosis and loss of perineuronal nets (PNNs) indicating of extracellular matrix (ECM) disruption. The aim of this study was to explore the kinetics of ECM degradation, identify the ECM degrading enzymes involved, study whether IRF‐1 and its target genes are induced by ECM degradation and whether they play a role in nerurodegeneration. Longitudinal analysis of SIV‐infected pigtailed macaques showed an increase in CSF ECM degradation products. Consistent with these findings, the ECM degrading enzyme hyaluronidase and heparinase were upregulated in brain homogenates commensurate with the severity of encephalitis. Microarray analysis showed induction of the transcription factor IRF‐1 in encephalitic animals that was confirmed by semi‐quantitative RT‐PCR. In situ hybridization combined with immunohistochemistry showed that IRF‐1 was induced in astrocytes. Primary human astrocyte cultures exposed to hyaluronidase and heparitinase showed induction of IRF‐1 as well as toll‐like receptor 4 (TLR4) mRNA. Initial screening of the target genes that are induced by ECM degrading enzymes include ADAM8, caspase 1 & 3. We conclude that ECM degradation in SIVE results in the formation of HA and HS fragments that can activate TLR signaling and subsequent IRF‐1 induction in astrocytes.
Probiotic bacteria generate ROS in intestinal epithelial cells
Authors: Kwon Young Man , Kumar Amrita , Neish Andrew Scott
Published: June 2021

The mammalian host maintains a mutually beneficial coexistence with a diverse intestinal microbiota, which can affect many aspects of gut homeostasis. How this bacterial community can modulate host processes is largely unknown. Recently, we reported that commensal bacteria can induce reactive oxygen species (ROS) in gut epithelial cells and influence signaling by transient oxidative inactivation of key pathways such as NF‐kappaB. Here we evaluated the relative capacity of gut bacteria to induce ROS generation in cultured intestinal epithelial cells. Commensal bacteria of the Lactobacillus species were potent ROS inducers, but not other commensals or pathogens. Bacterial ROS stimulating activity was fully recapitulated in cell wall homogenates but not culture supernatant, cytoplasmic extracts, or heat‐treated cell wall preparations, suggesting ROS activation is mediated by an integral membrane protein. ROS generation was inhibited with NAPDH inhibitors and BAPTA (a selective Ca2+ chelator) suggesting the calcium sensitive NADPH isoform Duox was involved. Bacterial populations (and cell wall preparations) isolated from the ceca of mice fed Lactobacilli for nine days showed increased ROS inducing activity in cultured model epithelia. These data indicate that certain members of the microbiota (and commonly used probiotics) have a selective ability to induce ROS mediated epithelial signaling.