bluuebunny/crossref_metadata_2025 huggingface dataset:
When Homotrimeric Enzymes Display a Different Function for Each Chain in Each Turnover: Evidence from The E2 Components of the 2‐Oxoacid Dehydrogenase Complexes
Authors: Jordan Frank , Nemeria Natalia S , Park Yun-Hee , Song Jaeyoung , Wang Junjie , Kumaran Sowmini
Published: June 2021

The superfamily of 2‐oxoacid dehydrogenases is important in metabolic pathways, including human and bacterial. The pyruvate dehydrogenase complex (PDHc) is at the intersection of glucose metabolism and the citric acid cycle and comprises multiple chains of three components: thiamin diphosphate dependent E1, lipoylated E2 and the FAD/NAD+‐dependent dihydrolipoyldehydrogenase E3. We are investigating primarily the E1 and E2 components, the latter produce acylCoenzyme A from the 2‐oxoacid reactants. Among the E2 components, both the octahedral symmetry bacterial enzyme, and the icosahedral human enzyme have multiples of three chains; we are presenting experiments to understand the need for multiples of three chains. Expt. A. We have made two types of E2 constructs of PDHc from Escherichia coli: one defective in posttranslational lipoylation by the ligase, the other defective in the synthesis of acetylCoenzyme A. While neither construct is active by itself, their complementation produces active enzyme. Expt. B. We use a combination of NMR and mass spectrometry to define whether the binding sites on E2 for the E1 and E3 components are identical or non‐identical. The results of both experiments on the Escherichia coli PDHc point to the likelihood that during each turnover, the three chains have different roles. The model can also be readily extrapolated to the human enzymes. Supported by NIH‐GM‐050380.
Cellular confluence and cohesion regulates CXCL11/IP9 expression during keratinocyte re‐epithelialization
Authors: Huen Arthur C. , Wells Alan
Published: June 2021

A role for CXCL11/IP9/I‐TAC in coordinating cellular events to transition the regenerative phase to the remodeling phase of wound healing has been demonstrated. However, the physiologic signals regulating CXCL11 expression remain unknown. Of specific interest are the factors that stimulate and later downregulate expression of this transiently‐expressed chemokine. To investigate keratinocyte‐mediated CXCL11 expression, in vitro cell culture and skin organ culture model systems were used. CXCL11 expression was found to be upregulated soon after cellular confluence was reached. In addition, CXCL11 was not dependent upon calcium‐dependent differentiation as the increased expression was also noted in confluent cultures grown in low calcium conditions. These results suggest that achievement of cellular confluence results in the upregulation of chemokine CXCL11 likely as a paracrine to the underlying dermis that covering re‐epithelialization has occurred.
Examining the Role of Lipoprotein‐Associated Phospholipase A2 in Atherosclerosis
Authors: Gilpatrick Timothy Edward , Tomczak Jonathan , Bahnson Brian J.
Published: June 2021

Interest in the enzyme Lipoprotein Associated Phospholipase A2 (Lp‐PLA2) emerged when it was recognized to be elevated in patients at risk for developing atherosclerosis. Lp‐PLA2 exists in human plasma on the membrane of lipoproteins, and preliminary investigations indicate that the physiological effect of Lp‐PLA2 is dependent on the lipoprotein to which it is coupled— namely, that the enzyme has a protective effect when it is on the surface of High Density Lipoproteins (HDL) and is atherogenic when on the surface of Low Density Lipoproteins (LDL). This project aims to develop and employ an assay for the investigation of factors that perturb the distribution of Lp‐PLA2 between lipoproteins, identifying correlations between its location and its role in human health. To analyze Lp‐PLA2 distribution, lipoproteins are separated using electrophoresis in custom designed non‐denaturing poly‐acrylamide gels. Separation of lipoproteins by native electrophoresis has been done previously, and a modification of this method is employed for a cost‐efficient approach that causes LDL and HDL to form discrete bands, allowing for quantification of Lp‐PLA2 in each. By increasing our understanding of the function of Lp‐PLA2 we have the ability to improve current models, aiding in the future development of improved treatments for atherosclerosis.
Pharmacological evaluation of an analogue of L‐carnitine in obese Zucker‐Lepr fa/fa rats
Authors: Rodríguez-Fragoso Lourdes , Mendoza Brissa , De la Cruz Cordero Ricardo , Vite Odon , Avalos-Soriano Anaguiven , Esparza Jorge Reyes
Published: June 2021

Different diseases such Diabetes mellitus type 2, hypertension, dyslipidemia, among others, are attributed to overweight and obesity, which have become a major health problem worldwide and turn all these factors are the main components of metabolic syndrome (MS). Molecules like L‐carnitine has been shown to be effective in treating a variety of related diseases with SM. Result obtained of this molecule has led to the search for the synthesis of new analogues of L‐carnitine to have new treatment options. Therefore aim of this research was to evaluate the pharmacological effect of different doses of an analogue of L‐carnitine on parameters related to metabolic syndrome in Zucker rats. Animals treated for 5 weeks with L‐carnitine and the doses used were 100 and 250 mg / kg. The results showed that the analogue of L‐carnitine at doses of 100 mg / kg led to the decrease in hepatic glucose, serum triglycerides and liver, hepatic cholesterol and increased liver glycogen levels, populations changed peripheral blood lymphocytes and CD3/CD8 increasing CD3/CD4, CD3 thymus decreased and increased CD3/CD4/CD8 of this population. With doses of 250 mg / kg, only decreased serum triglyceride levels and increased liver glycogen levels, among others effects. In conclusion, the results obtained in this project showed that the analogue of L‐carnitine increased efficacy and potency at a dose of 100 mg / kg. Support from CONACYT 153324.
Association of apolipoprotein A5 gene ‐1131T>C polymorphism and metabolic syndrome in Korean subjects
Authors: Song Kwang Hoon , Yu Sung-Gon , Yu Hyunjoo , Kim Jong Yeol
Published: June 2021

The apolipoprotein A5, APOA5, genetic polymorphisms, ‐ 1131T>C (rs662799) has been reported to be associated with hypertriglyceridemia that is one of the metabolic syndrome components. We assessed the association between the genetic polymorphism of APOA5 ‐1131T>C and lipid parameter and other risk factors of the metabolic syndrome in Korean subjects.

A total of 2,683 participants from 20 oriental medical hospitals in Korea between 2006 and 2011 were enrolled. The APOA5 ‐ 1131T>C genotype was significantly associated with serum high‐density lipoprotein cholesterol levels (effect= −1.516 mg/dL, p = 6.414E‐06) in total study population after adjustment for differences in age and gender. The association of APOA5 ‐ 1131T>C with serum triglyceride was also significant in an additive genetic model (effect = 19.891 mg/dL, p = 3.876E‐17). Multiple logistic regression model adjusted for age and gender revealed that the subjects with CC genotype had an increased risk for metabolic syndrome (OR= 1.951, 95% CI=[1.443–2.637], P = 1.397E‐05).

We demonstrated that APOA5 ‐1131T>C polymorphism was not only associated with serum high‐density lipoprotein cholesterol and triglyceride levels but also was associated with the metabolic syndrome as a result of its remarkable effect on serum triglyceride levels in Korean subjects in the current study.
Correlation between Hemoglobin Oxygen Affinity and Nitrite Reductase Activity
Authors: Olsen Scott , Malavalli Ashok , Vandegriff Kim
Published: June 2021

Plasma hemoglobin (Hb) has been associated with vasoconstriction that has been attributed to nitric oxide (NO) scavenging by heme. Evidence suggests that the reduction of nitrite to NO at deoxygenated hemes dilates blood vessels under hypoxic conditions. This reaction may be why a high oxygen affinity Hb has been shown not to constrict arterioles despite binding NO. We investigated the nitrite reductase activity of several Hb modification chemistries, including maleimidation, which reacts at Hb β93Cys residues to decrease p50. Maleimides tested include the small molecule, N‐ethylmaleimide, and maleimide‐polyethylene glycol (PEG). Using thiolation chemistry, we increased the degree of PEGylation from 2 to 4 to 8 PEGs, progressively decreasing p50. We also tested Hbs cross‐linked between α or β subunits to produce tetrameric Hbs, with varying p50s, and their maleimide‐PEGylated products. The p50 values ranged from 3 mmHg (for PEGylated ββ‐crosslinked Hb) to 33 mmHg (for αα‐crosslinked Hb). We found a direct correlation between p50 and nitrite reductase activity; Hbs with high oxygen affinity (low p50) reduced nitrite to NO at a higher rate than low affinity Hbs. With a 10‐fold excess of nitrite, the maximum rate of nitrite reduction varied from 0.46 μM/s (for PEGylated ββ‐crosslinked Hb) to 0.015 μM/s (for αα‐crosslinked Hb). Importantly, we found that NO produced by this reaction can escape heme capture and be released from Hb, and the rate of NO release is relative to the nitrite reductase activity. Together, these results suggest that oxygen affinity may be important in cell‐free Hb design, not only to target oxygen delivery, but also to optimize nitrite reductase activity.
Characterization of Lipid Droplet and Its Regulation by Caveolin‐1 in Endothelial Cells
Authors: Kuo Andrew , Zhang Xinbo , Harrison Kenneth D. , Sessa William C.
Published: June 2021

Lipid Droplet (LD) is a unique organelle involved in many biological processes such as cellular energy homeostasis and lipid metabolism. One of the LD‐associated proteins reported is Caveolin‐1 (Cav‐1). Cav‐1 is a 21‐kDa integral membrane protein with a putative hairpin structure and localized in caveolae microdomains on the plasma membrane. Recent studies have shown that Cav‐1 can be redirected to the surface of LD, implying a novel function of Cav‐1.

Endothelial cells (EC), as a key cell type segregating plasma lipids from tissue, are highly enriched in Cav‐1, suggesting the potential role of Cav‐1 in regulating LD homeostasis in EC. In this study, we first show that LDs are formed in EC at atheroma, indicating the potential physiological function of LD in EC. We next find that LD formation is significantly reduced in EC isolated from Cav‐1 null mice. Fatty acid uptake is not altered in Cav‐1 deficient EC, suggesting an intracellular regulation of LD by Cav‐1. We therefore measure LD formation and degradation separately in Cav‐1 deficient EC. Interestingly, activity of diacylglycerol acyltransferases is increased in Cav‐1 null EC while breakdown of triglyceride (TG) is not significantly changed. Taken together, our findings showed that LD can be observed in EC in vivo and its formation is regulated by Cav‐1 independently of fatty acid uptake and TG biosynthesis.
Breastmilk cells produce milk proteins in the culture dish
Authors: Hassiotou Foteini , Metzger Philipp , Trengove Naomi , Lai Ching Tat , Filgueira Luis , Hartmann Peter
Published: June 2021

We have recently identified in human breastmilk early‐stage stem cells capable of multi‐lineage differentiation. To elucidate the potential of these cells to differentiate into functional lactocytes in vitro, freshly expressed breastmilk was collected, and cells shown by qPCR to express stem cell markers were cultured in mammary differentiation medium for up to four weeks. qPCR was then used to detect and quantify mRNA of milk proteins (lactoferrin, LF; α‐lactalbumin, LA) in cultured cells, whereas immunofluorescence microscopy was used to detect them at the protein level. Culture supernatants were harvested at regular time points and Western Blotting was used to examine expression of milk proteins and their glycosylation patterns. Plated breastmilk cells attached to the substrate and proliferated, forming epithelial colonies. Cells from the colonies gradually differentiated into functional lactocytes producing LF and LA, which were detected in harvested cultured cells both at the mRNA and protein levels. LF and LA were also secreted into the culture supernatants over the culture period of 4 weeks. Glycosylation of secreted milk proteins was observed at the molecular weights of LF and LA. It is concluded that breastmilk cells are able to differentiate in culture into functional lactocytes producing and secreting milk proteins, a number of which are glycosylated.

Grant Funding Source : Women and Infants Research Foundation, Medela AG
Active transcription factors in the development of the small intestine during early life
Authors: Zhang Man , Liao Yalin , Lonnerdal Bo
Published: June 2021

Transcription factors (TFs) regulate downstream gene expression individually or cooperatively in different cell types and specific tissues. Human milk contains several growth factors that maintain gastrointestinal mucosal homeostasis. We chose two major growth factors in human milk, TGF‐β and IGF‐1 to study global activation of TFs during early intestinal development in IEC‐6 (crypt cell) and FH74 cells (fetal intestine). TGF‐β treatment (10 ng/ml) for 5d significantly induced differentiation (expression of alkaline phosphatase) of IEC‐6 and lead to an abundance of cytoplasm and clear cell boundaries. Optimum proliferation of FHs 74 was found at 30 ng/ml IGF‐1 treatment for 24h (no significant change in morphology). Active transcription factors were detected by Protein‐DNA Array. For IEC‐6 cells, we found EGR1 and YY1 were upregulated; CREB, E2F1, Pax3 and OCT1 were downregulated. By bioinformatics analysis, we found 3 binding sites for EGR1 in the promoter of mir‐146b (−795/−786, −900/−812, −1013/−1005), which has been shown to be upregulated and play a role in TGF‐β signaling. In FHs 74 cells, c‐Myb was upregulated, whereas CREB, EGR1, P53 and Pax3 were downregulated. In conclusion, we showed several transcription factors are regulated by TGF‐β and IGF‐1 during small intestinal development. EGR1 was further found to participate in miRNA regulation of the TGF‐β treatment.

Grant Funding Source : Mead Johnson Nutrition Global Discovery
New culinary uses for pork testicles from immunologically castrated male pigs
Authors: Pucciarelli Deanna L. , Friesen Carol A. , Schroeder Aubrey L.
Published: June 2021

The objectives of this project were to research and develop recipes for foodservice utilizing immunologically castrated male pig testicles; create a pork testicle handling guide for cooks and foodservice managers; identify critical control points to reduce production food safety hazards; and, conduct nutritional analyses on developed recipes. A total of 14 recipes were developed. Sensory evaluation determined that the best uses for the product were in the forms of forcemeat and whole smoked. The pilot study sensory panel determined that the meat texture preference was favorable in a mixed product application. When tasted in tandem with calf testicles, the pig testicles mouth feel was found chewier. Grinding the product improved the texture. Utilizing hand cutting, testis production yield post‐tunica vaginalis removal was 57%; therefore, mechanization to improve yield is desirable. Immunologically castrated male pig testicles have 10.5% protein, 2.9 % fat and 1.3% carbohydrate. With 2mg/100g (uncooked weight) heme iron, the meat is 2.5 times more iron‐rich than pork loin and provides 25% of the USDA daily recommended intake for males over 18y. Trans fatty acids were measured in the non detectable range. Implications for the foodservice industry included potential increased immunologically castrated male pig product utilization. This project was supported by Pfizer Animal Health, Veterinary Medicine Research & Development, Kalamazoo, MI.
Zinc‐metallothionein ratios reflect cellular zinc status
Authors: Killilea David William , Shenvi Swapna V , Holland Tai C , Burke Sarah J , Shigenaga Mark K , Sutherland Barbara , King Janet C
Published: June 2021

Animal and human studies show that intracellular zinc levels are sensitive to changes in dietary zinc. The protein metallothionein (MT) is maintained in proportion to intracellular zinc content, and therefore may be a useful marker of zinc status. Measuring MT levels is difficult due to the unique properties of the protein, so most studies focus on MT gene expression. Our objective was to develop new methods to quantify MT protein and MT‐bound zinc content. Zinc deficiency or excess was modeled in the Jurkat leukocytic cell line using the zinc‐selective chelator TPEN or exogenous zinc sulfate, respectively. Cellular protein was separated using size‐exclusion filtration to isolate an MT‐rich fraction. Then, endogenous zinc content was released, apo‐MT was loaded with cadmium, and MT‐bound cadmium levels were quantified by ICP; this method permitted the determination of cellular MT‐bound zinc content (MBZC) and MT zinc saturation (%MTsat). Gene expression of the major MT and zinc transporter isoforms were measured in parallel by RT‐PCR, which reflected the change in zinc balance in these cells. MBZC and %MTsat levels correlated with intracellular zinc content and changes in MT gene expression, showing bimodal responses to zinc deficiency and zinc excess. These measures of the cellular zinc‐metallothionein ratio may have utility as markers of zinc homeostasis. This work was supported by the HarvestPlus.
Vitamin D2 intake increases 25‐hydroxy vitamin D2 but decreases 25‐hydroxy vitamin D3 concentration in the serum of healthy adults
Authors: Stephensen Charles B. , Zerofsky Melissa , Burnett Dustin J. , Lin Yanping , Hammock Bruce D. , Hall Laura M. , McHugh Tara
Published: June 2021

Vitamin D deficiency is common in the U.S. Few foods contain vitamin D but treatment of mushrooms with ultraviolet (UV) light increases vitamin D2 content and could provide an additional dietary source of vitamin D. We evaluated the impact of consuming UV‐treated white button mushrooms (Agaricus bisporus) on the vitamin D status of healthy adults. Thirty‐eight volunteers were randomized to four treatments taken with a standard lunch for 6 wk: untreated mushrooms providing 34 IU/d vitamin D2 (n = 10), UV‐treated mushrooms providing 352 IU/d (n = 10) or 686 IU/d (n = 9), and untreated mushrooms plus a vitamin D2 capsule providing 1,131 IU/d (n = 9). Serum total 25(OH)D at baseline was 83 ± 38 nmol/L. Serum 25(OH)D2 was 2.4 ± 2.0 nmol/L and increased in the three treatment groups (p < 0.05). Mean increases were 1.2 ± 5.2, 13.8 ± 7.3, 12.7 ± 3.7 and 32.8 ± 3.3 nmol/L, respectively. Serum 25(OH)D3 decreased proportionally in the same groups (p < 0.05). Mean decreases were −3.9 ± 16.3, −10.4 ± 6.4, −20.6 ± 14.6 and −29.5 ± 15.9 nmol/L, respectively. Serum 24,25(OH)2D3 did not differ by group. Total serum 25(OH)D decreased in the treatment groups but concentrations never differed significantly from the control group. In summary, vitamin D2 from these treatments was absorbed and metabolized to 25(OH)D2 but did not affect vitamin D status because serum 25(OH)D3 decreased proportionally. Funding was provided by The Mushroom Council and ARS project 5306‐51530‐018.
Reproducibility and validity of a food frequency questionnaire for the assessment of vitamin D intake in Canadian lactating women
Authors: Phan Anna , Gallo Sina , Vanstone Catherine , Rodd Celia , Weiler Hope
Published: June 2021

Valid dietary assessment tools to capture vitamin D intake from foods are required to facilitate research regarding the relationships among intake and status. To our knowledge, no prior study has validated a food frequency questionnaire (FFQ) for assessment of vitamin D intake in lactating women. The objective of this study was to validate the Willett FFQ, adapted for Canadians, for assessing vitamin D intake in lactating women. Healthy women (n=42) from Montreal completed a FFQ at 4 months postpartum and 24 hour dietary recalls at 1, 2.5, and 4 months postpartum. Fasted venous blood was collected for analysis of plasma 25‐ hydroxyvitamin D (25(OH)D) by LIAISON® (Diasorin, Stillwater, MN). A subsample (n=7) completed the FFQ at baseline for reproducibility testing. Mean vitamin D intake was similar between FFQ and recalls (p>0.05). There was a significant correlation between FFQ and recalls for dietary and total vitamin D (p<0.01) but not between FFQ and 25(OH)D. Bland‐Altman analyses indicated a mean difference of −10 IU/d (LoA: 359, −380 IU) between dietary methods. Based on total vitamin D intake, 69% were classified into the same tertile with KW=0.63 between dietary methods and 45.2% were classified into the same tertile with KW=0.14 between FFQ and 25(OH)D. These findings suggest that the Canadian adapted Willet FFQ may be a valid tool for the assessment of vitamin D intake among lactating women. (Supported by CFDR).
Season, adiposity, and baseline 25‐hydroxyvitamin D are predictors of maternal change in vitamin D status from 1 to 4 months postpartum
Authors: Phan Anna , Gallo Sina , Vanstone Catherine , Agellon Sherry , Rodd Celia , Weiler Hope
Published: June 2021

There is limited information on the vitamin D status of lactating women and the potential known predictors that contribute to vitamin D status during lactation. The objectives of this study were to determine vitamin D status of lactating women and identify key predictors of vitamin D status at 4 mo and change since 1 mo postpartum. Healthy, lactating mothers (n=44) in Montreal were recruited between May 2010 to April 2011. Fasted venous blood samples were collected at 1 and 4 mo postpartum for analyses of plasma 25‐hydroxyvitamin D (25(OH)D) by LIAISON® (Diasorin). Demographics, sun exposure, skin pigmentation, anthropometry, body composition, supplement use, and nutrition information were collected to identify predictors of 25(OH)D. From 1 to 4 mo postpartum, there was a significant decrease in 25(OH)D from 73.0 ± 21.6 nmol/L to 62.4 ± 18.3 nmol/L (p<0.001). At 1 mo, 7 (15.9%) mothers had 25(OH)D <50 nmol/L and 26 (59.1%) had 25(OH)D <75 nmol/L. At 4 mo, 9 (21.4%) mothers had 25(OH)D <50 nmol/L and 30 (71.4%) had 25(OH)D <75 nmol/L. Predictors of vitamin D status at 4 mo postpartum included weeks in the synthesizing period, vitamin D intake, and % change in body fat and predictors of change in 25(OH)D included baseline 25(OH)D, weeks in the synthesizing period, and % change in weight. These predictors are important to consider to prevent declines in vitamin D status during lactation.

(Supported by CFDR).
Response of dorsal root ganglion tissue to chronically stimulated electrodes
Authors: Kolarcik Christi , Rost Erika , Albrecht Ingrid , Luo Xiliang , Catt Kasey , Weber Douglas J. , Cui X. Tracy
Published: June 2021

Peripheral sites including the dorsal root ganglion (DRG) are target implantation regions for neural prosthetics that restore function/provide therapy. To improve long‐term performance, conductive polymers and carbon nanotubes (CNTs) can be coated on the electrode surface and doped with anti‐inflammatory drugs. We compared the performance of and tissue damage associated with chronically‐stimulated modified microelectrodes. Following partial laminectomy, uncoated, PEDOT/CNT and dexamethasone‐doped PEDOT/CNT coated dual microelectrodes were implanted into the DRG of thirteen adult male rats. For chronic stimulation, cathodic pulses (20 μAmps, 200 μs) were followed by anodic pulses (10 μAmps, 400 μs) for 1 h/d for 10 of 14 d. Hematoxylin and eosin staining along with the following were used: Iba1 (microglia/macrophages), NF200 (neuronal axons), NeuN (neurons), GFAP (astrocytes), S100 (Schwann cells), vimentin (fibroblasts) and caspase‐3 (cell death). Quantitative image analyses were performed using MATLAB and electrode performance assessed. Our results indicate that coated microelectrodes perform better and cause less tissue damage than uncoated controls suggesting that these coatings may be valuable modifications for in vivo applications. Funding for this work was provided in part by the Department of Defense TATRC grant WB1XWH‐07‐1‐0716 and the National Institute of Health R01NS062019.
New method for systemic sclerosis, systemic lupus erythematosus and vascular angiopathy diagnosis
Authors: Cattaruzzi Giacomo , Vitrani Francesco , Moretti Massimo , Scorza Raffaella , Curcio Francesco
Published: June 2021

Systemic sclerosis (SSc) is characterized by skin thickening and abnormalities of many visceral organs. Vascular damage, immune activation and extracellular matrix excessive synthesis and deposition are SSc features. Serum autoantibodies are strong predictors of disease outcome and organ complication pattern but their pathogenetic role is unclear, although there is evidence that they also have a role in pathogenesis. Angiogenesis is strongly disturbed: as demonstrated by capillaroscopy, vessels damage evolves progressively and is characterized by different morphological aspects. Almost all patients develop Raynaud's phenomenon which, together with structural vasculopathy, results in ulceration and critical digital ischemia.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies to cell nucleus and a diverse array of clinical manifestations. The primary pathological findings in patients with SLE are inflammation, immune complex deposition and vasculopathy.

We developed a cell‐extract based method which is capable of identify SSc and SLE patients. The results of a study involving 340 individuals indicated that our method is highly specific. Our method can identify, within SSc (limited cutaneous and diffuse forms), subjects showing vascular damages at early stages; it also correlates with the Raynaud's phenomenon in SLE subjects.
Putting the More back in Morphology: Quantitating Multiple Protein Expressions in Intact Tissue Sections
Authors: Mansfield James Robert , Hoyt Clifford
Published: June 2021

Biological systems are complex; multiparameter detection methods such as expression arrays and flow cytometry make this apparent. In tissues it is important not just to measure the average expression of molecules, but also their spatial distribution, while preserving architectural features. Such high‐resolution molecular imaging is challenging, especially when signals are co‐localized. In fluorescence, sensitivity and quantitation can be compromised by autofluorescence, which is commonly present in formalin‐fixed tissues. Imaging challenges can be addressed using multispectral imaging. On a microscope, at least 6 markers can be simultaneously detected and imaged, and sensitivity can be increased by two orders of magnitude, depending on relative levels of specific label and autofluorescence. For analysis, we have developed a train‐by‐example software platform that can be trained to separate image regions into appropriate classes (“cancer”, “inflammation”, e.g.) and to accurately quantitate their areas. When combined with multispectral imaging and cellular segmentation tools to extract data automatically from cellular and sub‐cellular compartments it can provide the information necessary for designing and testing targeted diagnostic and therapeutic reagents.
The effect of PKMζ inhibition on cocaine‐induced place preference and locomotor sensitization
Authors: Howell Kristin , Carmack Stephanie A , Anagnostaras Stephan G
Published: June 2021

While many molecules have been implicated in the induction of a memory, less is known about the molecular mechanisms underlying the persistence of a memory. Protein kinase M ζ (PKMζ) has been demonstrated to be necessary for the maintenance of LTP, spatial memory and conditioned taste aversion. This study investigated whether PKMζ is also necessary for the maintenance of two addiction‐related memories, cocaine‐induced conditioned place preference (CPP) and locomotor sensitization. The role of PKMζ is typically examined by giving a single, region‐specific application of the PKMζ inhibitor, ζ inhibitory peptide (ZIP). The current study examined the effects of chronic PKMζ inhibition on the development of CPP and sensitization induced by cocaine (15 mg/kg, ip). ZIP was continuously delivered i.c.v. using an osmotic minipump throughout the six training sessions. Mice receiving ZIP during training showed reduced locomotor activity as compared to control mice. However, a sensitization test given while animals were not receiving ZIP revealed only a minor deficit that was not significant. Further, memory for CPP, tested off‐ZIP was not impaired. This suggests, while on‐ZIP, development and expression of sensitization are impaired, but off‐ZIP there was no deficit in memory for sensitization and place preference. This research was supported by NSF Graduate Research Fellowship (KKH), NIH grant DA020041 (SGA).
Effects of cocaine self‐administration on cognition in monkeys and evaluation of cognitive enhancement as a therapeutic strategy
Authors: Gould Robert Warren , Nader Michael A
Published: June 2021

Cocaine use is associated with impaired executive function, thus cognitive enhancement may aid relapse prevention. Four adult male rhesus monkeys with extensive cocaine self‐administration (SA) histories (> 5 year, >1.5 g/kg lifetime intake) and five age‐matched, cocaine‐naive monkeys were trained in morning sessions to perform cognitive tasks measuring learning, memory, response inhibition and behavioral flexibility; afternoon sessions consisted of cocaine SA (1.5 mg/kg/day) or food reinforcement (cocaine‐naive). Monkeys self‐administering cocaine in afternoon sessions showed impairments in reversal learning and set shifting in subsequent morning test sessions. FDG‐PET imaging showed lower glucose utilization in areas associated with error‐detection, memory, and reward compared to cocaine‐naive monkeys during set shifting, a measure of behavioral flexibility, demonstrating neurobiological and cognitive effects of cocaine. Working memory performance was disrupted on days following high dose cocaine SA. During periods of abstinence, nicotinic acetylcholine receptor (nAChR) stimulation via nicotine, the α4β2*‐ and α7‐subtype‐selective nAChR agonists varenicline and PNU‐282987 each improved working memory in cocaine‐experienced and cocaine‐naive monkeys, supporting nAChR stimulation for cognitive enhancement in cocaine‐dependent populations.
Nanometer precise vertical localization of single particles with standard fluorescent microscope in biological samples
Authors: Marki Alex , Ermilov Eugeny , Koller Akos , Secomb Timothy , Pries Axel
Published: June 2021

Our aim was to establish and validate an easy‐to‐use application for standard fluorescence microscopy, which allows vertical localization of single fluorescent particles with nanometer precision. Vertical localization of single fluorescent particles can be performed with precision beyond the resolution limit by decoding the information contained in out‐of‐focus images of the particle because the size of the images is proportional to out‐of‐focus distance. Images of single fluorescent particles at defined defocus distances were used to calibrate the approach using whole diffraction patterns (WP) and outermost ring diameters (OR). Precision and validity of the WP‐ and OR‐approach were determined by in house software using independent images at defined defocus distances. In addition, Brownian motion tracking and near‐wall particle image velocymetry were performed. WP and OR yielded similar precision (6.3 nm vs. 7.4 nm) for optimal imaging conditions, underexposed images or images with overlapping patterns. WP compared to OR proved to be more robust and precise in the presence of cells and image distortions.
T‐cell dependent IL‐6 signaling mediates angiotensin II‐enhanced microvascular thrombosis
Authors: Senchenkova Elena Y. , Granger D. Neil
Published: June 2021

Hypertension is associated with a prothrombotic and hypercoagulable state. Angiotensin II (AngII) is a key mediator of the thrombotic abnormalities that accompany hypertension. However, the mechanisms that underlie the prothrombotic actions of AngII remain poorly understood. We have previously demonstrated that AngII enhances thrombus development in the microvasculature and that T‐lymphocytes contribute to the AngII‐enhanced microvascular thrombosis. We have examined the role of interleukin‐6 (IL‐6) in the AngII‐enhanced thrombosis response and assessed whether/how the actions of IL‐6 are linked to T‐cells. Thrombus formation in cremaster arterioles was induced using the light/dye endothelial cell injury model. Wild type (WT) mice, IL‐6−/−mice, and immunodeficient Rag‐1−/− injected with T cells from WT or IL‐6−/− mice were infused (14 d) with AngII (1 μg/kg/min). Some AngII infused WT mice were treated (24 hrs before the thrombus formation) with a blocking antibody directed against either the alpha or beta (gp130) chain of the IL‐6 receptor (IL‐6r). We found that IL‐6−/− mice, Rag‐1−/− mice reconstituted with IL‐6−/− (but not WT) T cells, and WT mice treated with either IL‐6r antibody were significantly protected against the prothrombotic actions of chronic AngII administration. These findings link IL‐6 signaling to the T‐cell dependent prothrombotic effects of AngII. (Supported by HL26441)
Instigation of NALP3 Inflammasome Activation and Glomerular Injury in Mice on the High Fat Diet: Role of Acid Sphingomyelinase Gene
Authors: Boini Krishna M , Xia Min , Abais Justine A , Li Pin-Lan
Published: June 2021

Ceramide has been reported to initiate inflammasome formation and activation in obesity and different pathological conditions. The present study was performed to explore the role of acid sphingomyelinase (Asm) in the development of high fat diet (HFD)‐induced inflammasome formation and activation and consequent glomerular injury. Asm knockout (Asm−/−) and wild type (Asm+/+) mice were fed a HFD or normal chow for 6 weeks to produce obesity and associated glomerular injury. HFD significantly increased the body weight, renal Asm activity, O2.− production, and ceramide production in Asm+/+mice than in control diet‐fed mice. However, such HFD‐induced increases in body weight, Asm activity, O2− production and ceramide production was attenuated in asm−/− mice. Confocal fluorescent microscopy showed that HFD enhanced the colocalization of NALP3 with ASC or Caspase‐1, the essential components of NALP3 inflammasomes, in glomeruli of Asm+/+ than in Asm−/− mice. In consistency with decreased inflammasome formation, the caspase‐1 activity and IL‐1β production was significantly attenuated in Asm−/− mice fed a HFD. Morphological examinations showed that HFD induced profound injury in glomeruli of Asm+/+ mice which was markedly attenuated in Asm−/− mice(the glomerular damage index (GDI) in Asm+/+: 2.1 ± 0.05 vs. Asm−/−: 1.05 ± 0.04). The decreased GDI in Asm−/− mice was accompanied by attenuated proteinuria and albuminuria. Fluorescent immunohistochemical examinations using podocin as a podocyte marker showed that the inflammasome formation induced by the HFD were mostly located in podocytes as demonstrated by co‐localization of podocin with NALP3 or Caspase‐1. In conclusion, these observations disclose a pivotal role of Asm in the HFD‐induced inflammasome formation and consequent glomerular inflammation and injury (supported by NIH grants DK54927, HL‐091464 and HL‐75316).
Oxidation of SNAP25‐Syntaxin complex reduce its stability and prevents refolding
Authors: Ogawa Nozomi , Baba Tomonori , Taylor Ryan , Prince John T , Woodbury Dixon J
Published: June 2021

SNAP25 plays a critical role in neuronal exocytosis. The SNAP25 linker region contains a cysteine‐rich domain which is the site of various post‐translational modifications such as palmitoylation and possibly oxidation. Palmitoylation anchors SNAP25 to the membrane, while the level of oxidation may regulate the extent of transmitter release. Using tandem mass spectrometry (LTQ Orbitrap), we characterize the modification of the four cysteine residues in the SNAP25B linker. We verified that oxidation of SNAP25B leads to disulfide bond formation within the cysteine‐rich domain, the predominant form containing two disulfide bonds. Analysis of oxidized SNAP25B‐syntaxin complex shows reduced stability. In addition, oxidized SNAP25B does not refold and re‐complex with syxntain. This suggests that oxidation has a regulatory role during synaptic transmission. Through additional experimentation we hope to elucidate the effects of palmitoylation and oxidative stress on SNARE complex formation and exocytosis.
Aeroallergen‐induced hypoxic bradycardia in spontaneously breathing house dust mite‐sensitive nonhuman primates
Authors: Wardle Robert L , Ellis Stuart R , Gaddis J Leslie , Olmstead Stephen G , Putnam Benjamin D , Reece Shaun P , Fick Robert B , Van Scott Michael R
Published: June 2021

RATIONALE

A small number of house dust mite (HDM)‐sensitive nonhuman primates (NHP) exhibit life‐threatening bradycardia after aero‐HDM challenge. Retrospective analysis was performed to elucidate conditions associated with the adverse cardiac response.

METHODS

Data recorded for 71 NHP over 4 years was screened for a >25% decrease in HR and minimum HR <100 bpm following HDM challenge. Prior to data acquisition, NHP were anesthetized, intubated, and instrumented to record SpO2 & HR, lung resistance (RL), and dynamic compliance (Cdyn).

RESULTS

Hypoxic bradycardia was observed in 10 of 71 NHP. It was associated with airway sensitivity to HDM and was preceded by rapid‐shallow breathing and often apnea. HR decreased 50% to 76±6 bpm and was associated with an SpO2 of 71±4%, increase in RL of 139±37%, and decrease in Cdyn of 59±5%. Bradycardia was also observed during methacholine provocation performed 24 hours after HDM challenge. Bradycardia was reversed by mechanical ventilation or atropine.

CONCLUSIONS

Hypoxic bradycardia during respiratory stasis has been described in humans and canines. Our data indicate that HDM‐allergy can lower O2 and tidal volume thresholds for the reflex in susceptible subjects.
Expression of ROS catabolic enzymes in the medial nucleus tractus solitarii (nTS) of rats and upregulation during acute hypoxia
Authors: Ostrowski Tim Daniel , Barr Stacy L , Dantzler Heather A , Hasser Eileen M , Kline David D , Heesch Cheryl M
Published: June 2021

Reactive oxygen species (ROS) increase in the nTS following acute hypoxia and likely contribute to hypoxia‐induced changes in cardiorespiratory function. H2O2 is a diffusible signaling molecule that is inactivated by catalase (CAT) and glutathione peroxidase (GPx). Immunohistochemistry in untreated rats identified CAT throughout the nTS. In NeuN‐identified neurons, CAT was located perinuclear to a dense continuous network that extended into the cellular processes, presumably the endoplasmic reticulum. In microglia‐like cells, CAT was identified in the cytosol and membrane. CAT was not localized to GFAP‐identified astrocytes. Strong GPx‐1 staining was located primarily cytosolic in scattered neurons and in the nuclei of all identified astrocytes throughout the nTS. mRNA for the ROS inactivating enzymes superoxide dismutase (SOD) 1, SOD 2, CAT, Gpx1, and Gpx4 was expressed in tissue punches of the medial nTS of male rats exposed to 2 hr. of normoxia (21% O2, n = 6) or hypoxia (10% O2, n = 6). Only relative expression of Gpx1 was increased (P< 0.02) in the nTS of hypoxic (1.8 ± 0.3) versus normoxic rats (1.0 ± 0.1). Thus, enzymes responsible for catabolism of ROS are present in the nTS and upregulation of GPx‐1 may be an early compensatory response to a hypoxic challenge. Support: NIH Multi‐Investigator RO1 HL098602.
Central Leptin‐glutamate Signaling Contributes to the Exaggerated Sympatho‐excitation in Rats with Type 2 Diabetes
Authors: Zheng Hong , Liu Xuefei , Patel Kaushik P
Published: June 2021

Accumulated evidence indicates that type 2 diabetes (T2D) is associated with enhanced sympathetic activation. The present study was conducted to investigate the role for leptin‐glutamate signaling in centrally mediated sympathetic response in rats with T2D. 12–14 weeks combination of high‐fat diet (HFD) and low‐dose strepzotocin (STZ, 30mg/kg) induced hyperglycemia, hyperleptinemia and insulin resistance in rats. In anaesthetized condition, microinjections of leptin (5ng~100ng) into the arcuate nucleus (ARCN) and paraventricular nucleus (PVN) induced increases in renal sympathetic nerve activity (RSNA) and blood pressure in control and T2D rats. These effects of leptin were significantly blunted in T2D rats (n=8/group). It is suggesting that in T2D rats, hyperleptinemic condition might be implicated in generating the elevation of RSNA and blood pressure, resulting in hyposensitivity to exogenous leptin. Moreover, we found that pre‐microinjections of NMDA receptor antagonist AP5 (16pmol) into the ARCN and PVN can reduce leptin‐induced RSNA and blood pressure (n=8/group). By confocal calciumimage, in cultured neuronal cell NG108, pre‐incubation with leptin for 24hrs induced a robust increase of intracellular calcium green fluorescence when the cells were challenged with glutamate. Taken together, these data suggest that within the hypothalamic nuclei, leptin‐induced sympatho‐excitation maybe mediated through an activation of glutamate receptors.
The effect of Vivo‐Morpholino targeting Vmat2 on daily physical activity in mouse soleus compared to scrambledmorpholino control
Authors: Ferguson David P , Schmitt Emily E , Lightfoot J Timothy
Published: June 2021

We hypothesized that inhibiting Vmat2 would inhibit daily physical activity. We used Vivo‐Morpholinos targeting Vmat2 (Vmat2‐M) and compared those results versus a scrambled morpholino control (SMC) and saline control. All aspects of this study were conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. At 9 weeks old 18 male C57Bl/6J mice were randomly assigned to a saline (n=6), SMC (n=6), or a Vmat2‐M (n=6) group. After a week of baseline wheel running the mice received an injection of Vmat2‐M (11mg/kg, 110 ul) or saline/SMC(110 ul). After an additional week of wheel running half the cohort was sacrificed to determine Vmat2 knock‐down using western blot. The remaining mice ran for an additional week. There were no differences between saline and SMC in Vmat2 expression (p=0.051). In the Vmat2‐M group, Vmat2 was significantly knocked down compared to the control groups (p=0.018) one week after injection and returned to baseline levels two weeks post injection. Mice receiving Vmat2 injection saw a decrease (p=0.02) in physical activity compared to baseline data. There were no differences in physical activity compared to the control groups. The use of saline or SMC had similar results in this model. Transiently inhibiting Vmat2 in soleus decreased physical activity suggesting a role of Vmat2 in the regulation of physical activity.
Biomarker of whole grain wheat intake associated lower BMI in older adults
Authors: Ma Jiantao , Ross Alastair , Bruce Stephen J , Jacques Paul , Lichtenstein Alice H , Saltzman Edward , Booth Sarah , McKeown Nicola
Published: June 2021

Alkylresorcinols (AR) are phenolic lipids in the bran fraction of some whole grains (wheat, rye and barley). Plasma AR reflect recent intake of these whole grains. We examined the cross‐sectional associations between plasma AR (measured by LCMS/MS), whole wheat intake, and body mass index (BMI) in 407 free‐living older adults (166 men and 241 women; 60–81y; mean BMI 27.9 kg/m2). After adjustment for total cholesterol and triglyceride, median plasma AR across quartile categories of AR were 5, 13, 28, and 62 nmol/L, respectively. Whole grain wheat (servings/d) and bran (g/d) intakes were estimated using a semi‐quantitative FFQ. Spearman correlation coefficients between plasma AR and whole grain wheat and bran were 0.34 and 0.28, respectively. Age and sex adjusted geometric mean daily whole wheat intakes in the lowest and highest quartile categories of plasma AR were 0.4 and 1.1 servings (P‐trend <0.0001). Without inclusion of added bran, whole grain intake (g/d) was weakly correlated with plasma AR (r = 0.12, P<0.0001). After adjustment for lifestyle and dietary covariates, mean BMI values in the lowest and highest quartile category of plasma AR were 27.8 and 26.7 kg/m2, respectively (P‐trend 0.03). Our study confirms the observed inverse relationship between whole grain intake and BMI using an independent biomarker of whole grain wheat intake.

Grant Funding Source: U.S. Department of Agriculture, Agricultural Research Service, under Cooperative Agreement 58‐1950‐7‐707, the National Institutes of Health (AG14759 and HL69272)
Body composition in achondroplasia
Authors: Schulze Kerry , Henry Bobbie , Koerner Celide , Alade Yewande , McGready John , Collop Nancy , Silber Harry , Germain-Lee Emily , Hoover-Fong Julie
Published: June 2021

Background

The major components of body composition are fat and lean mass, quantifiable via dual energy X‐ray absorptiometry (DXA), bioelectrical impedance (BIA) and anthropometry. Average stature normative body composition values are not applicable because of extreme disproportionate short stature in achondroplasia. Obesity is a major concern in the general population, yet little has been done to validate methods for determining body composition in short stature skeletal dysplasias.

Methods

Body composition was determined by DXA, BIA and anthropometry in a cross‐sectional study of 20 adults with achondroplasia.

Results

Mean % body fat by DXA was 33.1%(F 42.7%, M 25.2%) and by BIA was 33.4% (F 43.2%, M 25.4%), r=0.94(p<0.0001). Mean estimated arm fat area was 35.3 cm2 (F 51.1 cm2, M 22.4 cm2). DXA % body fat was highly correlated with umbilical waist (M r=0.93, F r=0.72), maximal hip (M r=0.85, F r=0.77), and neck circumferences (M r=0.89, F r=0.73) and estimated arm fat area(M r=0.84, F r=0.86); all p<0.05 except the female umbilical. BIA % body fat was highly correlated with the same measures, all p<0.05 except female neck.

Discussion

Basic anthropometry is highly correlated to DXA and BIA and more accessible for future investigation of health outcomes. Supported by Greenberg Center for Skeletal Dysplasias
Age associated change in biomarkers of bone metabolism in cats
Authors: Yu Shiguang
Published: June 2021

Age associated change in bone metabolism in cats was investigated by measuring biomarkers of bone metabolism in a cross sectional study. Serum and urine samples were collected from 96 healthy American domestic shorthair cats (29 males and 67 females) with ages ranging from 1.7 to 15.9 years old after they were fed a dry cat food for at least 3 weeks. Following biomarkers were measured: procollagen type II C‐propeptide (PIICP), C‐terminal cleavage products of type II collagen (C2C), C‐terminal telopeptide of type II collagen fragments (CTX II), Type IIA collagen N‐propeptide (PIIANP), N‐terminal cross‐linked telopeptide of type I collegen (NTx), keratan sulfate (KS), chondroitin sulfate epitope 846 (CS846), and bone specific alkaline phosphatase (BAP). Linear correlation coefficient between age and measured biomarkers was calculated.

Serum BAP (r=−0.19, p=0.01) and urine CTX II (r=−0.47, p<0.01) were negatively and serum C2C (r=0.37, p<0.01) and KS (r=0.35, p<0.01) were positively associated with age. Both serum BAP and urine CTX II were significantly higher in cats < 2 years old, suggesting that bone metabolism does not attain maturity in cats until they are 2 years old. No association between age and other measured biomarkers was observed in cats > 1 year old.