bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Effects of Mefloquine Enantiomers on a Cholinergic Synapse
Authors: Patel Axay , Patel Vishwendra , Dassonville‐Klimpt Alexandra , Sonnet Pascal , Sultatos Lester , McArdle Joseph
Published: June 2021

Racemic mefloquine activates spontaneous vesicle release (SVR) from motor nerve endings and inhibits endplate acetylcholinesterase (AChE). Such cholinergic effects may reduce mefloquine's safety as an antimalarial drug. We tested the hypothesis that (+) and (‐) mefloquine differentially affect the neuromuscular junction (NMJ). Intracellular recordings at NMJs of mouse Triangularis sterni muscles suggested that 30 min exposure to 10 μM (+) or (‐) enantiomer alters the frequency of miniature endplate potentials (mEPPs). Quantitative comparisons were not possible since (+) reduced mEPP amplitude. Therefore, we recorded miniature endplate currents (mEPCs). (+) and (‐) significantly (p<0.01) increased mEPC frequency to 59.7 ± 20.05 Sec‐1 (mean ± SEM, N=21 NMJs) and 17.2 ± 1.98 Sec‐1 (N=14), respectively, from the control value of 5.6 ± 0.76 Sec‐1 (N=25). (+) also reduced amplitude (p<0.01) to 2.9 ± 0.18 nAmps (N=21) from the control value of 3.4 ± 0.18 nAmps (N=25); (‐) did not affect mEPC amplitude. (+), but not (‐), significantly (p<0.01) prolonged the endplate current decay time constant to 2.5 ± 0.16 mSec (N=12) from the control value of 1.1 ± 0.05 mSec (N=16). Thus, (+) may reduce conductance and prolong open time of the endplate acetylcholine receptor (AChR) as well as activate SVR. Neither (+) nor (‐) altered stimulus‐evoked quantal release. Assay of whole muscle homogenates revealed that while both enantiomers (1 to 100 μM) inhibit AChE, (‐) is more potent. The data suggest that (‐) is a less potent activator of SVR but a more potent inhibitor of AChE than (+) mefloquine. Supported by the Kirby Foundation.
What do we know about fruit and vegetable consumption in the UK? Trends from the National Diet and Nutrition Survey Rolling Programme (NDNS RP)
Authors: Ray Sumantra , Nicholson Sonja , Ziauddeen Nida , Steer Toni , Cole Darren , Solis‐Trapala Ivonne , Amoutzopoulos Birdem , Page Polly
Published: June 2021

Increased fruit and vegetable (FV) consumption is a public health nutrition priority. NDNS provides the only source of representative data on diet, nutrient intakes and nutritional status for the UK population. Combined data from the first 4 years of NDNS RP enables extensive characterisation of FV consumption patterns including distribution by age, sex, time and income. Also, disaggregation of FV from composite dishes provides improved estimates for total intake in relation to the UK's “5‐a‐day” public health target.

We have shown that whilst overall FV consumption falls below 5‐a‐day, the proportion meeting the target increases with age. Exploratory analysis also shows that consumption of 4 portions/day is more frequently achieved (16‐56%), however a large proportion of the population still falls below this. In the under‐65y population, FV consumption is significantly lower in all age/sex groups with the lowest income compared with those with highest income.

This work highlights the relevance of tracking FV consumption in population nutrition surveillance. Identification of groups where consumption is particularly low enables targeted public health nutrition initiatives and continuous monitoring of impact. Disaggregated FV data also provides opportunities to further investigate health effects through more refined information on consumption patterns and eating habits which is important for refining such initiatives. The NDNS datasets offer potential for robust comparisons of the UK's FV consumption with other countries.

Table 1: Comparison of disaggregation and non‐disaggregation methods of calculating “5‐a‐day” FV portions for NDNS Years 1‐4 (2008‐12); comparison with non‐disaggregated results from NDNS of adults 19 to 64 years (2000‐01).
Age group (years) and sex

Boys
Men
Girls
Women

11‐18
19‐64
65+
11‐18
19‐64
65+

NDNS Years 1‐4 (2008‐2012): Disaggregation method
Number of portions
3.0
4.1
4.7
2.8
4.1
4.6

% achieving 5‐a‐day
11
30
40
7
30
41

NDNS Years 1‐4 (2008‐2012): Non‐disaggregation method
Number of portions
2.6
3.4
3.7
2.6
3.5
3.6

% achieving 5‐a‐day
9
18
19
6
17
19

NDNS of adults aged 19‐64 years (2000‐01): Non‐disaggregation method
Number of portions
‐
2.7
‐
‐
2.9
‐

% achieving 5‐a‐day
‐
14
‐
‐
15
‐

NDNS RP methods and headline results are published via web‐based reports and data are available via the UK Data Archive.
Bacterial Translocation and Obesity in Antiretroviral (ARV)‐Naïve HIV+ Adults in Botswana
Authors: Martinez Sabrina , Campa Adriana , Marlink Richard , Makhema Joseph , Huffman Fatma , Williams O. Dale , Baum Marianna
Published: June 2021

Background

Intestinal bacterial products such as lipopolysaccharide (LPS) are a marker of microbial translocation which causes chronic immune activation associated with HIV disease progression. Excessive weight among patients not on ARV is associated with slower HIV disease progression; however the mechanism of action is unknown. We investigated LPS levels in overweight/obese ARV‐naive HIV+ adults.

Methods

Anthropometrics, CD4 count, HIV viral load and bioimpedance were obtained. Overweight/obesity were defined as body mass index (BMI) 蠅25 kg/m2. LPS was determined (Limulus Amebocyte Lysate, Lonza, MD) in 60 participants at baseline and 18 months. Regression analyses were adjusted for age, gender, smoking and body fat mass%.

Results

Among 60 ARV‐naïve HIV+ asymptomatic adults, median age was 33 years (IQR: 29‐39) and 76.4% were women. Overweight/obesity was inversely associated with LPS levels at baseline (OR=.79; P=.04) and at 18 months (OR=.85; P=.03). Those with higher fat mass% also had lower LPS at baseline (OR=.85; P=.01). LPS 蠅 the median was associated with higher viral load (OR=2.6; P=.03) and viral load 蠅 4 log10 copies/mL (OR=8.01; P=.01) at baseline, and approached significance at 18 months (OR=3.78; P=.06).

Conclusions

Overweight/obesity was associated with lower levels of LPS, which was directly associated with viral load. These findings indicate that lower LPS may mediate better survival of overweight/obese ARV‐naive HIV+ patients. The inflammatory mechanisms in HIV disease are complex and not entirely understood, which warrants further investigation.
The Brain on a High Fat Diet: The Effects of Beta‐Hydroxybutyrate on Astrocytes
Authors: Haluska Robert , Earp Jacob , White Robin
Published: June 2021

Astrocytes are brain cells that store and transport energy to neurons in the form of glucose. When glucose levels are low or absent, fat is metabolized and ketone bodies are created as an alternative energy source. In recent years, alternative dieting strategies such as low carbohydrate/high fat and intermittent fasting have become popular. Interestingly, these diets have shown therapeutic potential in treatment of diseases including Alzheimer's disease and epilepsy. However, it is unknown how astrocytes are affected by these diets. Ketogenesis results in the production of 3 ketone bodies: beta‐hydroxybutyrate (βHB), acetone and acetoacetate. Previous studies have shown that βHB levels increase significantly in the brain under ketogenic conditions. Thus, we chose to focus on how replacement of glucose with βHB impacts number and morphology of C6 astrocytic cells. Four experimental groups were tested: No Glucose/No βHB, 25mM Glucose (control), 1mM βHB, and 25mM βHB. Following treatment, cell morphology was visualized via actin immunohistochemistry (IHC) and nuclei were labeled using diamindino‐2‐phenylindole (DAPI). Quantification of the DAPI staining revealed a significant decrease in cell number in the No Glucose/No βHB, 1mM βHB and 25mM βHB groups compared to the 25mM Glucose control. Cells in the No Glucose/No βHB and 1mM βHB groups exhibited a rounded shape distinct from the 25mM Glucose and 25mM βHB groups. While previous studies have shown positive effects of a ketone‐rich diet, these results suggest that astrocyte health may be impaired when βHB is substituted for glucose.

This work was supported by the Westfield State University Research Grant.
Coating Polyurethane Surfaces by Electrostatic Charging Followed by Dip Coating/Electrophoretic Deposition
Authors: Bhardwaj Garima , Webster Thomas
Published: June 2021

Aim

Surface associated infections are one of the main causes for failures associated with medical devices. Bacterial localization and biofilm formation may lead to acute and chronic infection. Biofilm on the surface protects the bacteria from the immune system and antibiotic therapy. This needs aggressive treatment of antibiotics. The high doses of antibiotic treatment prevent healthy tissue formation at the site and triggers antibiotic resistance. Thus, to prevent infections, various strategies have been developed besides conventional systemic and local antibiotic treatment. Recently, there is an increasing interest for coating these surfaces to improve bioactivity and prevent infection. The current study aimed to modify the surface of polyurethane endotracheal tubes by charging them electrostatic ally to induce a positive and then coating them with an anti‐ bacterial protein via dip coating(3 times for a minute each) and electrophoretic deposition(using titanium foil as counter and working electrode at a voltage of 20V for 10 minutes).

Results and conclusion

image

(a)

image

(b)

Figure 1: SEM images of the coated surfaces

A thick and uniform coating was achieved on both the inside and outside surfaces of the tubes. Bioactivity of the protein was maintained and the entire process was cost and time efficient.

Acknowledgements

The authors would like to thank N8 Medical devices and Northeastern university for funding and Brigham Young University for providing the endotracheal tubes.
Sevoflurane Causes Long‐lasting Dendritic Spine Head Enlargement without Lasting Effect on Neuritic Extension
Authors: Zimering J , Dong Y , Fang F , Zhang Y , Xie Z
Published: June 2021

Early postnatal anesthesia causes long‐lasting learning and memory impairment in rodents, however, the underlying mechanisms remain largely unknown. We tested a hypothesis that clinically relevant concentrations of the volatile anesthetic sevoflurane alters morphology in developing mouse hippocampal neurons. Mouse fetal day 16 hippocampal neurons were cultured in Neurobasal medium for 7 days in vitro (DIV) prior to a single (4 hour) exposure to 3% sevoflurane in 95% air/5% CO2 or control condition (95% air/5% CO2). Neurons were immediately fixed in 4% paraformaldehyde or cultured for 14 more days prior to staining for filamentous (F)‐actin using Alexa Fluor555‐Phalloidin, or actin‐binding drebrin. Proximal and distal dendritic segments were randomly selected for analysis. Sevoflurane induced acute significant decreases in mean dendritic filopodial length (n=49), compared to control DIV7 neurons (n=105; P < 0.00001). The effect was completely prevented by Y27632 (10 µM), a selective Rho kinase inhibitor consistent with a RhoA/Rho kinase‐mediated mechanism of F‐actin depolymerization. Sevoflurane‐treated neurons recovered to control mean filopodial length by DIV14. On the other hand, early spine enlargement persisted until DIV14 in sevoflurane‐treated neurons (0.83 µm vs 0.61 µm, P = 0.0001, n= 52) associated with significantly (P = 0.047) increased F‐actin concentration. Drebrin immunoreactive spines showed similar results. Sevoflurane‐treated neurons (DIV21) had a higher proportion of large spines (> 8 μm) (18% vs 10%; P = 0.053) compared to control neurons. These data suggest sevoflurane differentially affects F‐actin pools in spine head and neck.
Melatonin‐mediated suppression of early peripheral sensitization in osteoarthritic meniscus
Authors: Hong Yunkyung , Kim Joo‐Heon , Hong Yonggeun
Published: June 2021

Joint pain is classified as a major symptom in most of osteoarthritic (OA) cases. Most common management for OA is to alleviate symptoms through administration with conventional analgesic drugs, but patients continued to experience pain. Therefore, the present study is aimed at determining the effects of melatonin on peripheral nociceptive processing in animals with collagenase‐induced cartilage damage. Melatonin (10 mg/kg) was injected twice daily subcutaneously. There was TrkB upregulation as well as increase of non‐glycosylated NK‐1R despite no difference of its glycosylation in OA meniscus, but melatonin blocked it. To study the effect of melatonin on cells exposed to inflammatory neuropeptides, primary cultured chondrocytes were treated with both TNF‐α and rCGRP. Increase of Bax/Bcl‐2 ratio is accompanied by reduction of cell viability, and there were a loss of type II collagen, increase of active MMP‐13, and nuclear Ca2+ deposition. But melatonin at the nanomolar concentration rescued from damage responses. These molecular results might indicate early anti‐nociceptive effect of melatonin, but behavior of the animals was deemed hypersensitive to innocuous mechanical stimuli. Further experiments are needed to elucidate their inconsistency.

Funding: 2012R1A1A2005089, 2013R1A2A2A01067169, KGM4611512
Centrosome Overduplication in Canine Hemangioma and Hemangiosarcoma
Authors: Durrant Jessica , Kushner Erich
Published: June 2021

Centrosome overduplication (more than 2; >2C) is commonly associated with aneuploidy in many human cancers and is theorized to contribute to malignant transformation via increased chromosomal instability. >2C is not been described in endothelial cell (EC) tumors such as hemangioma (HA) and hemangiosarcoma (HSA). HAs are common, benign canine tumors that may undergo malignant transformation to HSA through unknown cellular mechanisms. To characterize the possible contribution of >2C to the development of HSA, archived formalin‐fixed paraffin‐embedded canine tissue (5‐8 uM thick) with a previous diagnosis of HA (n=4) or HSA (n=6) from the subcutis, spleen or liver were labeled with EC (isolectin) and centrosome (gamma‐tubulin) markers by immunofluorescence. Samples were imaged via confocal microscopy and quantified as % of neoplastic EC with >2C. HSAs had significantly greater >2C compared with HAs (20.1+/‐ 1.4% vs 4.4 +/‐ 2.3%; p<0.001). HA and control tissues did not demonstrate differences in >2C (controls 2.8 +/‐ 1.0%; p=0.24). Percentage of >2C cells in subcutaneous or visceral HSA was not different (18.2 +/‐ 1.1 vs 23.6 +/‐ 2.8; p=0.11). A subset of subcutaneous tissues (n=2) demonstrated regions of both HA and HSA morphology (HSA: increased mitotic rate, pleomorphism, solid growth); within the same tissue, areas of HSA morphology had significantly greater >2C compared with areas of HA morphology (27.6 +/‐ 3.0% vs 5.9 +/‐ 4.9%; p=0.003). These results support an association of >2C with phenotypic malignancy in canine endothelial cell neoplasms; thus, centrosome overduplication may contribute to transformation of neoplastic endothelial cells in canine HSA.
The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c‐met and platelet derived growth factor receptor
Authors: Luo Jianhua , Chen Zhang‐Hui , Yu Yan
Published: June 2021

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and over‐expressed in a variety of human malignancies. In this report, we showed that MCM7 binds SF3B3. The binding motif is located in the N‐terminus (aa221‐248) of MCM7. Knocked‐down of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR) and c‐met. Dramatic drop of reporter gene expression of oxytocin exon1‐intron‐exon2‐EGFP construct was also identified in SF3B3 and MCM7 knocked‐down PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knock‐down of SF3B3 and MCM7 leads to increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c‐met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, and thus give significant new insight into the oncogenic activity of this protein.
Targeting Efferocytic M2 Monocytes and Macrophages Offers Therapeutic Promise in Prostate Cancer Skeletal Metastasis
Authors: Jones Jacqueline , Soki Fabiana , Koh Amy , Shiozawa Yusuke , McCauley Laurie , Morgan Todd , Pienta Kenneth , Hofbauer Lorenz , Roca Hernan , Wang Yugang
Published: June 2021

Macrophages play a vital role in maintaining tissue homeostasis through efferocytosis. However, their functional role and pathophysiological contribution to the bone metastatic microenvironment remains unclear. Therefore, we investigated the efferocytic monocyte/macrophage type and its role in supporting PCa bone metastasis. FACS analysis revealed that M2 bone marrow macrophage polarization resulted in increased efferocytosis and pro‐tumorigenic gene expression. Soluble factors released from macrophage efferocytosis increased the PCa cell growth by 30%. Using tumor mouse models, Trabectedin (a chemotherapeutic drug) treatment diminished M2 like macrophages, resulting in decreased tumor metastasis. Clinically, FACS analysis of circulating monocytes isolated from patients with PCa bone metastasis showed a 20% increase in CD68+/CD14+/CD16+ monocytes. Moreover, these phagocytic monocytes presented a 2.5 fold increase in efferocytosis. Tissue analysis revealed high risk PCa tissue expressed significantly higher levels of CD68, which significantly correlated with a high Gleason score. These findings suggest efferocytic M2 like monocytes and macrophages promote prostate cancer bone metastasis.
Effects of Glucose Deprivation on The Biological Phenotypes of Human Malignant Mesothelial Cells
Authors: Kim Hang‐Ah , Kim Myung‐Chul , Kim Na‐Yon , Seo Yu‐Ri , Kim Yongbaek
Published: June 2021

Microenvironment is important in the maintenance of biological phenotypes such as survival, proliferation, and migration capacity in cancer cells. Decreased concentration of glucose is a common feature of the tumor microenvironment and has been reported to play a critical role in the acquirement of malignancy.Cancer cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. This study was performed to evaluate the effects of glucose‐free on the phenotypes of human malignant mesothelioma (HMM) cells. Glucose free media decreased the cell survival and proliferation of HMM cells. The mobility and invasion capacities were significantly decreased by glucose deprivation. These characteristics of cancer cells were resulted from the promotion of apoptosis. Gene expression level of ABCG2, an efflux transporter of cytotoxic drugs, was increased in glucose free media compared to normal media. The results suggest that deprivation of glucose from cancer cells affect their biological phenotypes due to lack of energy and increase multidrug resistance.
The Discovery of Novel TAAR1 Ligands through the use of a Homology Model and in silico Screening
Authors: Lam Vincent , Rodriguez David , Zhang Thomas , Koh Eun , Carlsson Jens , Salahpour Ali
Published: June 2021

Trace amines (TA) are by‐products of the synthesis of classical neurotransmitters within the brain. TA's exert their effect by activating a class of GPCRs including, trace amine associated receptor 1 (TAAR1) where TAAR1 signalling has been shown to be a negative regulator of dopamine transmission. In this study, we aimed to identify novel selective TAAR1 compounds for TAAR1 for in vivo testing. To this end, a TAAR1 homology model was generated and an in silico screen of 3 million commercially available compounds was conducted. The top 42 compounds, based on predicted affinities, were ordered and screened for TAAR1 activity using a cAMP BRET biosensor. From the 42 compounds tested, a mix of antagonists (seven compounds) and agonists (nine compounds) were found, yielding an initial hit rate of 35%. Of the nine identified agonists, three compounds (8, 16, and 25) displayed high potency for TAAR1 with EC50 values of 18, 1.0, and 52 µM respectively. In order to identify compounds with improved affinity for TAAR1, five analogs of compounds 8 and 16 were tested. Of the five analogs of compound 8 tested, only one compound (8b) displayed agonist activity with no improvements on efficacy or potency compared to compound 8. Two of the five compound 16 analogs (16a and 16b) had a left shift of ~10 fold in their EC50 for activation of cAMP signalling by TAAR. Furthermore the analog 16b had higher efficacy for TAAR1 activation than compound 16. In conclusion we have discovered three unique novel partial TAAR1 agonists with new chemical backbones where compound 16 and its analogs had the most optimal pharmacological profile. The in vivo effects as well as identification of higher affinity analogs of these compounds will be undertaken in the future.
Substance P and IL‐33 Synergistically Stimulate Mast Cells to Release IL‐1β and TNF‐α implicated in psoriasis; inhibition by the flavonoid methoxyluteolin
Authors: Taracanova Alexandra , Theoharides Theoharis
Published: June 2021

Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and inflammation. The pathogenesis of psoriasis appears to involve interactions among keratinocytes, mast cells (MC) and cytokines. MC are immune cells that secrete various cytokines and chemokines including interleukin the (IL)‐1 family of cytokines (IL‐1β, 18, 33), which can stimulate keratinocyte activation. IL‐33 also augments activation of MC with further release of several cytokines such as TNF‐α, IL‐6, and prostaglandin D2. Also, IL‐33 has synergistic effect with a neuropeptide substance P (SP) on VEGF release in MC. Both IL‐33 and SP are upregulated in psoriatic skin. Moreover, SP stimulates keratinocytes to secrete IL‐1β. Our findings show that SP and IL‐33 synergistically stimulate MC to produce and release IL‐1β and TNF‐α, pro‐inflammatory mediators involved in psoriasis pathogenesis. The combination of SP and IL‐33 also significantly up‐regulates IL‐1β and TNF‐α gene expression. The natural flavonoid methoxyluteolin (Metlut) is a potent inhibitor of both keratinocyte and MC activation. Here we report that Metlut significantly inhibits IL‐1β and TNF‐α gene expression, production and release triggered by SP and IL‐33 combination. Therefore, Metlut‐based formulations could be a novel treatment for psoriasis.
Anti‐obesity effect of a small molecule repressor of RORγ
Authors: Chang MiRa , Kamenecka Theodore , Griffin Patrick
Published: June 2021

The orphan nuclear receptor RORγ is a key regulator for TH17 cell differentiation and it regulates metabolic and circadian rhythm genes in peripheral tissues. Previously it was shown that the small molecule inverse agonist of RORγ SR1555 suppressed TH17 differentiation and stimulated iTreg cells and it was shown to regulate expression of a range of metabolic genes in liver cells. Here treatment of cultured pre‐adipocyctes with SR1555 led to increased expression of ROR , FGF21, and adipoQ while expression of RORγ was repressed. Chronic administration of SR1555 to obese diabetic mice resulted in a modest reduction in food intake accompanied with significant reduction in fat mass resulting in reduced body weight and improved insulin sensitivity. Analysis ex vivo of treated mice demonstrates that SR1555 induced expression of the thermogenic gene program in fat depots. Further studies in cultured cells showed that SR1555 inhibited activation of hormone sensitive lipase and increased fatty acid oxidation. Combined, these results suggest that pharmacological repression of ROR may represent a strategy for treatment of obesity by increasing thermogenesis and fatty acid oxidation, while inhibition of hormone sensitive lipase activity results in a reduction of serum free fatty acids leading to improved peripheral insulin sensitivity.
Effects of Chronic Δ9‐Tetrahydrocannabinol During Adolescence on Adult Repeated Acquisition and Performance Behavior and CB1 Receptor Expression in Male and Female Rats
Authors: Weed Peter , Winsauer Peter
Published: June 2021

There is evidence that marijuana use during adolescence can result in long‐term learning deficits, and that these deficits may occur in a sex‐specific manner. To examine these effects further, male and female adolescent rats received daily injections of saline or 5.6 mg/kg of Δ9‐tetrahydrocannabinol (THC) for 40 days. This yielded four treatment groups with respect to sex and adolescent treatment (female/saline, female/THC, male/saline, and male/THC). Rats were then trained to respond under a multiple schedule of repeated acquisition and performance of response sequences, and subsequently administered THC acutely to determine adult sensitivity. Brain tissue was collected at two time points and analyzed for CB1 receptor (CB1R) levels by western blot. With respect to the behavioral effects, THC produced both rate‐decreasing and error‐increasing effects in both sexes; however, males were less sensitive than females and there was an equipotent disruption of response rate and percent errors. In females, response rate was disrupted more potently than percent errors. THC administration during adolescence did not alter adult sensitivity for either sex, but it did affect adult expression levels in the hippocampus and striatum despite the lack of CB1R downregulation following chronic treatment. More specifically, adult female subjects had long‐term increases in CB1R levels in both brain regions, whereas the expression pattern in males varied depending on the region. In summary, THC during adolescence was unable to alter the sex‐dependent effects of acute THC on a complex operant task, but it was able to produce long‐term changes in CB1R expression in the adult brain.
Somatostatin Receptor Subtype‐4 Agonist Enhances Memory in Apo‐E4 knock‐in Mice
Authors: Witt Ken , Farr Susan , Crider A. , Wooten Joshua , Sandoval Karin
Published: June 2021

This study assessed the impact of somatostatin receptor subtype‐4 (SSTR4) agonist NNC 26‐9100 on learning, memory, cortical neprilysin activity, and blood chemistries in ApoE3 and ApoE4 knock‐in mice. Twelve‐month old ApoE3 and E4 mice were treated with NNC 26‐9100 (10 mg/kg, i.p. daily for 28 days) with all comparisons made to respective vehicle control groups (n=7‐8/group). Statistical significance set at P<0.05. ApoE4 mice, but not ApoE3 mice, spent more time with the novel object (object recognition memory test, day‐28) with NNC 26‐9100 treatment. T‐maze evaluation identified no significant change in learning (day‐21) or memory (day‐28) in either ApoE3 or ApoE4 mice with NNC 26‐9100 treatment. An enhancement in neprilysin activity was found in ApoE3 mice, but not ApoE4 mice, treated with NNC 26‐9100 treatment. Blood chemistries showed enhanced high density lipoprotein (HDL) levels and reduced non‐HDL levels in ApoE4 mice, but not ApoE3 mice, with NNC 26‐9100 treatment. No changes in glucose, insulin, total cholesterol, or triglycerides in either ApoE3 or E4 mice with NNC 26‐9100 treatment.These data show SSTR4 agonist NNC 26‐9100 can mitigate some forms of memory deficit associated with the ApoE4 genotype, but may do so independently of neprilysin activity. Moreover, SSTR4 agonist action enhanced the beneficial HDL levels and reduced the detrimental non‐HDL levels. This work was supported by Southern Illinois University Edwardsville STEP grant.
Clinical Evaluation of Gamma Hydroxybutyric Acid (GHB) and its Glucuronide in Biological Matrices of Succinic Semi Aldehyde Dehydrogenase Deficient Patients
Authors: Ainslie Garrett , Vogel Kara , Jansen Erwin , Salomons Gajja , Pedersen Daniel , Gibson K. Michael
Published: June 2021

Gamma hydroxybutyric acid (GHB), an endogenous metabolite, is an agonist towards both GHB and GABAB receptors. GHB is elevated during illicit drug use and in patients with heritable succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder of GABA degradation. Previous reports have postulated that the glucuronide conjugate (GHB‐gluc) may represent a urinary biomarker for GHB intoxication. The objective of the present work was to examine the level of GHB‐gluc in SSADH‐deficient patients and its applicability as a potential clinical biomarker. Urine was quantified for GHB and GHB‐gluc on an AB Sciex 6500 mass spectrometer. Semi validation of the urine method was conducted using plasma and cerebrospinal fluid (CSF). SSADH patient urine, plasma and CSF and healthy volunteer control urine and plasma were collected and quantified for GHB and GHB‐gluc using LC‐MS/MS. Absolute quantification of GHB and GHB‐gluc in patient urine samples revealed an inverse correlation between GHB and GHB‐gluc (r2, 0.6; p value<0.05). Relative to control samples, the GHB‐gluc was only moderately elevated in patient samples (~15%). The GHB‐gluc was below the limit of quantification (25 ng/ml) in patient CSF and was not discernible between groups in plasma specimens. This work indicates that GHB‐conjugation may occur primarily in the kidney, or that a fraction of the GHB‐gluc is excreted in the bile.

Acknowledgement: The generous support of the SSADH Association (www.ssadh.net) is gratefully acknowledged.
Dexmedetomidine enhances cerebral cortex GIRK1 protein expression and attenuates Acute Postoperative Pain in rats
Authors: Wang Lingzhi , Wu Zhikun , Liu Shiming , Li Haobo , Xia Zhengyuan , Huang Huansen
Published: June 2021

Dexmedetomidine (DEX), a highly selective α2‐adrenoceptor agonist, produces both sedative and antinociceptive action. However, the mechanism for DEX analgesia remains unclear. Opening of G protein‐activated inwardly rectifying K+ (GIRK) channels at spinal sites are involved in antinociception induced by opioid and no‐opioid receptor agonists (Pain. 2013;154(12):2853‐9). We investigated the antihyperalgesic effects induced by intravenous administration of DEX in acute incisional pain model in rats and its potential effect on the expression of GIRK1 at the supraspinal sites. The hyperalgesic effects induced by the plantar incision were verified by a significant and transient decrease in the mechanical paw withdrawal threshold for 24 hours (h) after hindpaw incision. The decreases of paw withdrawal threshold at 0.5h, 1h, 2h, 4h after incision were significantly suppressed by intravenous pre‐administration of 5 or 10 ug/kg of DEX. Furthermore, pretreatment with DEX increased the expression of GIRK1 in the cerebral cortex at 2h and 4h after plantar incision as assessed by immunofluorescence and western blotting. It is concluded that administration of DEX before surgery induces antihyperalgesic effects in rats suffering from acute incision pain and that stimulation of GIRK1 expression in the cerebral cortex may represent a mechanism whereby DEX confers antinociception in acute postoperative pain.
Studying α7 nicotinic receptor anti‐inflammatory signaling
Authors: Loring Ralph , Garg Brijesh
Published: June 2021

α7 nicotinic receptors (α7 AChRs) reportedly play anti‐inflammatory roles in innate immune function, in part by blocking the transcription factor NFkB. We tested whether heterologously expressed α7 AChRs directly interact with NFkB signaling in the rat pituitary cell line GH3. GH3 cells show significant 125I‐α‐bungarotoxin (BT) binding when transfected with rat α7 nAChR, but in our hands, commercially available α7 antibodies do not detect α7 AChR in these transfected cells. For instance, α7 AChR‐GFP chimeras transfected into cells show BT binding, and anti‐GFP staining, but not anti‐α7 AChR staining in Western blots. Upon transfection with an NFkB promoter driving secreted alkaline phosphatase (NFkB‐SEAP), GH3 cells show up to 30 fold increases in SEAP secretion following 1‐100 ng/ml rat tumor necrosis factor (TNF) treatment. Nicotine (100 ‐1000 μM) and the selective α7 AChR agonist PNU282987 (PNU, 50‐300 μM) blocked TNF‐stimulated SEAP secretion in GH3 cells transfected with NFkB‐SEAP alone, while GTS21 did not. This suggests some nicotinic compounds have direct effects on NFkB signaling in non‐immune cells, since transfection with α7 had no further effect. To understand whether reported α7 AChR anti‐inflammatory effects are cell‐type dependent, we investigated α7 AChR drug effects in macrophage‐like cells (RAW 264.7 cells) using PNU, GTS21 and nicotine following lipopolysaccharide (LPS) stimulation (10 ng/ml). GTS21 (50‐200 µM) blocked LPS‐induced TNF release from RAW cells, but nicotine and PNU did not. Our findings suggest that α7 AChR do not directly interact with and subsequently block NFKB signaling, and that additional factors are required in heterologous cells to mimic the reported α7 AChR‐mediated anti‐inflammatory effects in immune cells.
THE 18 kDa SIGMA‐2 Receptor and PGRMC1 are Derived From Separate Genes
Authors: Chu Uyen , Mavlyutov Timur , Chu Ming‐Liang , Yang Huan , Mesangeau Christophe , McCurdy Christopher , Guo Lian‐Wang , Ruoho Arnold
Published: June 2021

The sigma‐2 receptor (S2R) is a unique binding site expressed in many mammalian tissues. Due to high expression in tumors, the S2R is a promising target for cancer diagnostic imaging. However, the sequence of the S2R has remained a matter of debate. Historically the identity of S2R has been determined by these criteria: (1) the S2R binds with high affinity to 1,3‐di‐o‐tolylguanidine (DTG) and haloperidol but not the selective sigma‐1 receptor ligand, (+)‐pentazocine; (2) specific photolabeling with [3H]Azido‐DTG, and [125I]‐iodoazido‐fenpropimorph ([125I]IAF) has shown the apparent molecular size of S2R to be 18‐21kDa. The progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, has been recently reported to be the S2R. To establish the identity of the S2R, we created a PGRMC1 knockout NSC34 cell line using CRISPR/Cas9 technology. We found that in NSC34 cells devoid of PGRMC1, the maximum binding (Bmax) of haloperidol protectable [3H]‐DTG binding was similar to that of wild‐type control cells. Moreover, in both control and PGRMC1‐KO cells, [125I]IAF selectively photolabeled a DTG protectable S2R‐18kDa protein. A series of previously reported highly selective S2R compounds showed protection of [125I]‐IAF photolabeling of the S2R‐18kDa in PC12 cell membranes that contain both PGRMC1 and S2R‐18kDa. These results support the conclusion that PGRMC1 and S2R‐18kDa are separate proteins derived from two different genes. Cloning and characterization of the S2R‐18kDa will provide further clarification.
Antiangiogenic Activities of the Trigonella foenum‐graecum Seed Extracts
Authors: Hammad Hana , Habib Zina , Afifi Fatma , Zihlif Malek
Published: June 2021

The role of angiogenesis for sustained tumor growth and metastasis formation has been recognized. Therefore, the inhibition of angiogenesis has become a vital therapeutic target for the treatment of cancer patients. This study aims to assess the antiangiogenic activity of fenugreek. Different fractions of fenugreek crude extracts were prepared and their antiangiogenic properties were assessed using the rat aortic ring assay and the chicken embryo chorioallantoic membrane (CAM) assay. Extracts were investigated for their direct cytotoxic activity in the MCF7 breast cancer cell line using the methylthiazol tetrazolium (MTT) assay. Ethanol extract showed dose‐dependent inhibition of blood vessel outgrowth from primary tissue explants in the rat aortic ring assay with 100% inhibition at a concentration of 100µg/ml (p<0.001) while other extracts did not show significant activity. The CAM assay coincided with the results of the aortic ring assay with ethanol showing significant inhibition (p<0.001). Ethyl acetate extract also showed inhibition (p<0.05). The extracts only showed antiproliferative activity at a concentration of 400µg/ml towards MCF7 cells. Since the rat aortic ring assay and the CAM assay confirm that the ethanol extract inhibited vascularization significantly, further studies on the ethanol extract would be beneficial in isolating the active ingredient responsible for the inhibition.

This work was supported by a grant of the Deanship of Academic Research, The University of Jordan.
Identifying Synergistic Interactions Between the Rexinoid and Adrenergic Signaling Pathways
Authors: Uray Ivan , Zsolczai David , Yao Linfang , Brown Powel , Bond Richard , Yang Peiying
Published: June 2021

The RXR‐selective retinoid bexarotene is a potent anti‐proliferative agent that effectively suppresses mammary tumorigenesis, yet chronic treatment at effective doses is associated with adverse effects. To identify compounds that synergistically enhance the anti‐proliferative activity of bexarotene, we conducted high throughput screens using a library of clinically tested drugs and small molecules in premalignant human breast epithelial cells. The dual b/a1‐adrenergic receptor antagonist carvedilol showed strong synergy with bexarotene in both anchorage‐dependent and colony formation assays. Long‐term treatment of MMTV‐ErbB2 mice with low‐dose carvedilol (20 mg/kg/d) and bexarotene (20 mg/kg/d) as single agents delayed de novo tumor formation, while their combination effectively inhibited the development of ER‐negative breast cancer. Carvedilol administration was also associated with slower tumor growth compared to control mice.

Most beta‐blockers tested had no effect on cell growth, and did not enhance the anti‐proliferative effect of bexarotene in vitro. However, another antagonist with a beta‐arrestin bias, alprenolol, was also synergistic with bexarotene, suggesting that long‐term modulation of downstream MAPK signaling pathways may play a role in the cancer preventive action of the combination of carvedilol and bexarotene.

Carvedilol's favorable toxicity profile makes it a promising candidate for drug 'repurposing' as an anti‐cancer agent.

Supported by the Duncan Family Institute Seed Funding Research Program and RO3 CA180550 (to I.P.U.).
Impaired Peripheral Dopaminergic System in Patients with Ulcerative Colitis
Authors: Kernychnyi Vitalii , Dziubenko Natalia , Prysiazhniuk Alona , Tolstanova Ganna
Published: June 2021

Ulcerative colitis (UC) is the chronic inflammatory disease unknown etiology. Little is known on the role of dopaminergic system in the pathogenesis of UC, despite the evidence that dopaminergic hypoactivity may result in excessive inflammation. We demonstrated a diminished risk for UC in patients with schizophrenia (hyper‐dopaminergic system) (Tolstanova G. et al. Gastroenterology 2010;138 (5):S‐264). In this study we tested the hypothesize that patients with chronic UC is characterized by the disturbance of peripheral dopaminergic system.

Methods

Colonic biopsy samples from 7 patients with chronic UC with recurrence stage and 7 ‐ from healthy colon were included in the study. Expressions and localization of D2, D3 dopamine receptors, DAT (responsible for dopamine re‐uptake) and tyrosine hydroxylase (TH, rate‐limiting enzyme of dopamine synthesize) were investigated with immunohistochemistry.

Results

Profound expression of DАТ, D2R, D3R and ТН was found in apical membrane of colonocytes and basal membrane of Goblet cells of healthy colonic mucosa. Inflamed colonic mucosa of patients with UC was characterized by 25% decrease in levels of D2R and D3R; 75% decrease levels of TH, mostly in colonocytes. Levels and localization of DAT was not significantly changed.

Conclusion

Recurrence of chronic UC is associated with the downregulation of dopamine synthesize, D2R and D3R expression in colonic mucosa. Restitution of dopaminergic system may be new molecular targets for the UC therapy. Study was supported by the grant 01BF‐03601 MES of Ukraine.
Hungry Like PACAP Man ‐ The Role of PACAP and PACAP6‐38 in Eating Behaviors
Authors: Fangmann J , Rochon D , Wallner J , Shaik T , Emkay P , Kelly K , Cavins K , Deleon‐Camacho F , Alphin D , Braatz A , Groth E , Piotrowski J , Granlund L , Krenz V , Choi S
Published: June 2021

According to the CDC, 34.9% of United States adults are obese, which is linked to premature death, heart disease, cancer, respiratory disorders, fertility problems, Type 2 diabetes, and stroke. Over‐ and under‐eating are related to brain chemistry. A 38 amino acid peptide hormone in the hypothalamus, pituitary adenylate cyclase‐activating peptide (PACAP), may be linked to eating disorders. PACAP binds to PACAP type 1 receptor (PAC1R), a G‐protein coupled receptor. Seven hydrophobic transmembrane (TM) domains hold PAC1R in hypothalamic cell membranes. PAC1R's extracellular domain (ECD) contains a ligand binding site. PAC1R's many negative residues attract PACAP's many positive ECD residues. PACAP's V19, K20, and L27 affect PACAP binding to PAC1R. K20 forms a possible salt bridge with PAC1R's G104, allowing PACAP to align parallel to PAC1R so PACAP's N‐terminus interacts with PAC1R's TM domains. This activates PAC1R, sending a signal inside the cell. Too much PACAP may cause a person to stop eating and lead to eating disorders. PACAP6‐38 is an antagonist formed when a protease removes the first five PACAP residues. When PACAP6‐38 binds to PAC1R, eating increases, possibly leading to obesity. SuJean Choi, PhD wants to determine how ratios of PACAP and PACA6‐38 are regulated. The Greenfield SMART (Students Modeling A Research Topic) Team modeled PAC1R's ECD and its two ligands, PACAP and PACAP6‐38, using 3D printing technology to investigate their relationships. Studying PACAP and PACAP6‐38 regulation and brain chemistry involved in eating behaviors could improve people's lives and decrease obesity‐related US medical costs. Program supported by NIH‐CTSA.
The Reduced Uterine Perfusion Pressure (RUPP) Model of Preeclampsia Causes Decreased Capillary Perfusion in Skeletal Muscle
Authors: Fraser Graham , Morton Jude , Schmidt Sydney , Bourque Stephane , Davidge Sandra , Davenport Margie , Steinback Craig
Published: June 2021

The Reduced Uterine Perfusion Pressure (RUPP) model of preeclampsia exhibits angiogenic imbalance, endothelial dysfunction, increased vascular resistance and hypertension. We hypothesized the RUPP model would also exhibit decreased functional capillary density and perfusion in skeletal muscle.
Female Sprague‐Dawley rats (n=30) were randomized to nonpregnancy (NP) or breeding (Preg) at 12 weeks of age and again to RUPP or SHAM surgeries on gestational day (GD) 14 (or equivalent age in NP rats). On GD 20 (or equivalent), capillary structure and perfusion of the extensor digitorum longus was imaged using digital intravital video microscopy. Functional videos were analyzed by a blinded observer. Capillary density (CD) was expressed as capillaries/millimeter intersecting 3 staggered reference lines (150µm). Flow was scored as the percentage of capillaries having: 1) continuous, 2) intermittent, or 3) stopped flow.
Total CD was not different between groups. The RUPP model decreased continuous flow vessels (main effect of surgery, P<0.01) and increased stopped flow (main effect, p<0.01) both which were more pronounced in Preg animals (Continuous: Preg‐SHAM = 80.1±7.8% vs Preg‐RUPP = 67.8±11.2%, p<0.05) (Stopped: Preg‐SHAM = 8.7±3.2% vs Preg‐RUPP = 17.9±5.7% p<0.01).
Our results demonstrate that the RUPP model of preeclampsia (and not just general hypoperfusion) is associated with a decreased capillary perfusion in skeletal muscle.
Supported by AIHS, Olympus and Q‐Imaging
Renin‐angiotensin‐aldosterone system blockade ameliorates myocardial and ventricular function of rats with diabetes mellitus
Authors: Pagan Luana , Gimenes Rodrigo , Damatto Ricardo , Cezar Marcelo , Rosa Camila , Nunes Marcela , Damatto Felipe , Campos Dijon , Gomes Mariana , Bonomo Camila , Okoshi Marina , Okoshi Katashi
Published: June 2021

The objective of this study was to evaluate the influence of renin‐angiotensin‐aldosterone system (RAAS) blockade with losartan (LOS) on cardiac structure and function of rats with diabetes mellitus (DM).

Methods

Male Wistar rats with 300 g body weight were divided into four groups: control (CTL, n=20), CTL+LOS (n=22), diabetic (DM, n=24), and DM+LOS (n=18). DM was induced by streptozotocin; LOS was diluted in drinking water and administered for 8 weeks. Arterial pressure (AP) was measured by tail‐cuff method. Echocardiogram (Echo) was performed to evaluate in vivo cardiac structures and left ventricular (LV) function. In vitro myocardial function was analyzed in LV papillary muscle. Statistics: Variables were compared by ANOVA (p<0.05).

Results

AP was lower in DM+LOS compared to the DM group (103±15 vs 120±15 mmHg). The in vitro functional study showed better myocardial performance in DM+LOS vs. DM group (developed tension ‐ DT: 7.32±2.00 vs. 5.90±1.29 g/mm2;p<0.05). Echo showed greater LV diastolic diameter (CTL: 8.05±0.54; CTL+LOS: 8.11±0.46; DM: 8.42±0.31; DM+LOS: 8.14±0.42 mm) and left atrial (LA) diameter (LA/aorta ‐ CTL: 1.34±0.09; CTL+LOS: 1.33±0.11; DM: 1.50±0.11; DM+LOS:1.51±0.13) in DM groups, and lower LV diastolic diameter in DM+LOS vs. DM group. LV systolic (mitral annular velocity by tissue Doppler) and diastolic function (isovolumic relaxation time and tissue Doppler Em/Am ratio) was impaired in DM group and improved by LOS.

Conclusion

The RAAS blockade improves myocardial and ventricular function in diabetes mellitus rats. Support: CNPq and CAPES.
Day‐to‐day Variability in Arterial Diameter and Brachial Artery Flow‐Mediated Dilation in Sedentary Young Men and Women
Authors: Shenouda Ninette , Skelly Lauren , Gibala Martin , MacDonald Maureen
Published: June 2021

Brachial artery flow‐mediated dilation (FMD), an index of endothelial function, is predictive of cardiovascular disease risk. A wide range of FMD day‐to‐day variability has been reported, but whether this variability differs between men and women is unknown. We examined potential sex‐based differences in FMD and its day‐to‐day variability. Sedentary but otherwise healthy men (N=10, age: 22±3 years) and women (N=7, age: 21±2 years) completed two brachial FMD tests 24 hours apart. Women were tested during the mid‐follicular phase of their menstrual cycle (days 7‐12). Baseline arterial diameter (ADBL) and maximum reactive hyperemia diameter (ADRH) were determined, and relative FMD was calculated as FMD% = (ADRH–ADBL)/ADBL. Day 1 measures and between‐day differences were assessed with independent t‐tests. Day‐to‐day variability was assessed with the coefficient of variation (CV) and intraclass correlation coefficient (ICC). Compared to men, women had smaller arterial diameters (p<0.05), but a similar FMD response (p=0.828). No sex‐differences were observed in between‐day differences for any measures (p>0.05). Day‐to‐day variability was low for AD in both sexes (CV of 3‐5%), but high for FMD (men: 43%, women: 55%). Similarly, ICCs were substantial to almost perfect for AD in both sexes (ICC of 0.77‐0.92), but poor for FMD (men: 0.01, women: 0.07). These findings suggest that arterial diameter exhibits better day‐to‐day repeatability than relative FMD in both men and women. Despite women having greater FMD variability than men, between‐day differences were not statistically significant. A larger sample size may be required to detect potential sex‐differences.

Funded by NSERC, OGS and GSSI.