bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc supplementation activates Nfe2l2/ARE‐MT2‐antioxidant pathway in a model of diabetic nephropathy
Authors: Ding gangqiang , Hao Mary , Tian Peiyao , Tang Qingping , Zhao Hong , Darzynkiewicz Zbigniew , Ferreri Nicholas R. , Hao Shoujin
Published: November 2023

We previously showed that metallothioneins (MTs) can be induced by zinc supplementation. The contribution of this mechanism to the regulation of MTs was determined both in vitro and in vivo by the activating Nfe2l2/ARE antioxidant pathway with zinc supplementation in this study. Diabetic male mice (age: 12 wk) were induced using streptozotocin (STZ), then divided into 2 groups and treated with zinc or vehicle. Liver and kidney Zn were increased after dietary Zn for 3 weeks; no significant increase in plasma zinc concentration. Systemic and intrarenal oxidative stress was reduced by zinc supplementation by examination of 8‐hydroxyde‐oxyguanosine (8‐OHdG) in the kidney tissues and urine. The expression of nuclear factor erythroid 2‐related factor 2(Nfe2l2) significantly increased for kidney tissues in the zinc‐treated group compared to the vehicle‐treated group. In contrast, protein expression of nuclear factor‐kB p65 in the kidney decreased in the zinc‐treated group compared to the vehicle‐treated group. Interestingly, MT2 gene expression in renal cortical tubules was also found to increase approximately 54% in the zinc‐treated group compared to the vehicle‐treated group, despite no significant increase for other MT isoforms. Pretreatment of primary renal tubular epithelial cells with an activator digitoflavone of Nfe2l2 before H2O2 exposure significantly decreased oxidant generation and cytotoxicity, whereas Nfe2l2 inhibition and gene silencing (shRNA) exacerbated the injury in vitro studies. Anti‐genotoxic effect of MTs was evident in prevention of DNA damage and micronucleus formation after in renal tubular epithelial cells pretreated by adding MTs (mixture of MT1 and MT2). The MT2 gene silencing in renal tubular epithelial cells significantly increased reactive oxygen species generation and provided marked protection against H2O2‐induced cytotoxicity. Thus, the present study shows that Zinc supplementation attenuates oxidative stress via Nfe2l2/ARE‐MT2 pathway in a model of diabetic nephropathy.

Support or Funding Information

973 project, National Natural Science Fund Project, NIH
Comparison of Macro Mineral Contents following Kind of Vegetables produced in Korea
Authors: Hwang Jin Bong , Park Eun‐Joo , Kim Soyoung , Seo Dongwon , Choi Youngmin , Kim Se‐Na , Nam Jin‐Sik , Yang Mi‐Ok
Published: November 2023

Recently, macro minerals (Na, Ca, K Mg and P) have the excellent functions associated with prevention of disease. We estimated an amount of macro minerals in the vegetables produced in Korea in order to build the food nutritional ingredient table. A SRM 1849a purchased from NIST in USA was digested by the microwave and validation of macro minerals in SRM 1849a was carried out by inductively coupled plasma optical emission spectrometer (ICP‐OES). Regression analysis of correlation coefficient (R2) was higher than 0.999 which revealed good linearity. Based on this analysis, the raw vegetables contained 0.20 to 144.98 mg/100 g of sodium, 7.81 to 266.57 mg/100 g of calcium, 230.57 to 812.73 mg/100 g of potassium, 12.16 to 76.28 mg/100 g of magnesium and phosphorus ranged from 35.32 to 123.81 mg/100 g. In addition, the contents of the minerals in the blanched vegetables were 1.40 to 134.75 mg/100 g of sodium, 7.80 to 177.58 mg/100 g of calcium, 150.32 to 651.71 mg/100 g of potassium, 11.17 to 56.63 mg/100 g of magnesium and 36.20 to 136.61 mg/100 g of phosphorus. Hence, it would be expected that the mineral contents could be used to draw up the food nutritional ingredient table.

Support or Funding Information

This work was carried out with the support of “Cooperative Research Program for Agriculture Science &Technology Development (Project No. PJ010846022015)” Rural Development Administration, Republic of Korea.
Immunohistochemical Detection of Autophagy‐Related Proteins in Canine Appendicular Osteosarcoma
Authors: Schott Courtney R. , Wood Geoffrey A.
Published: November 2023

Dogs diagnosed with appendicular osteosarcoma commonly succumb to metastatic disease despite undergoing the standard of care treatment for curative intent: amputation of the affected limb, followed by adjuvant chemotherapy. Post‐surgical chemotherapy improves one‐ and two‐year survival rates, but prognosis is still poor. In response to adverse environmental conditions normal cells are capable of undergoing autophagy, a survival mechanism involving self‐degradation and recycling of cellular components. Neoplastic cells take advantage of this mechanism to enhance their survival during chemotherapy. Autophagy's role in osteosarcoma has been investigated in humans however similar studies in dogs are rare. Our objective was to examine the expression patterns of a select group of autophagy‐related proteins in canine osteosarcoma amputation samples and to identify potential correlations with survival. Immunohistochemistry for beclin‐1, LC3, phospho‐mTOR, phospho‐AKT1, phospho‐S6 ribosomal protein, and p62 was performed on a tissue microarray containing naïve canine appendicular osteosarcoma cores from dogs who began the standard of care treatment (≥1 dose of chemotherapy). For survival analyses, Kaplan‐Meier survival curves for different expression patterns were compared using the log‐rank test and interactions were considered significant if p < 0.05. The median survival time from the date of diagnosis was 250 days and survival time ranged from 67–897 days (n=20, 95% CI: 122–381). Intense phospho‐S6 ribosomal protein labeling was identified most consistently adjacent to osteoid matrix. Phospho‐mTOR labeling was most evident in the nuclear compartment, however cells undergoing mitosis displayed strong cytoplasmic labeling. Canine osteosarcoma cells exhibited a mixture of diffuse and punctate LC3 cytoplasmic labeling, potentially distinguishing between free cytoplasmic LC3‐I and autophagosome anchored LC3‐II, respectively. Western blot analysis of the beclin‐1 antibody produced a 58 kDa band as predicted. Three distinct intensities of cytoplasmic labeling were observed for beclin‐1 in canine osteosarcoma cells. Strong cytoplasmic beclin‐1 labeling in the majority of neoplastic cells was not observed in any cases with greater than one‐year survival (0/6); this pattern was observed only in cases with less than one‐year survival (5/14). Dogs with tumours having light (n=4), moderate (n=9), and strong (n=5) cytoplasmic labeling had median survival times of 294, 301, and 122 days, respectively. Strong cytoplasmic labeling was a significantly correlated with decreased survival time (Chisq=6.9, 2 df, p < 0.05). This preliminary study provides evidence that canine osteosarcoma cells undergo autophagy in vivo, with a similar LC3 labeling pattern to that described in other tumour types in various species. Additionally, it identifies beclin‐1 as a promising candidate biomarker of chemoresponsiveness in canine appendicular osteosarcoma.

Support or Funding Information

Funded by Pet Trust
Ppm1b Negatively Regulates Necroptosis through Dephoshophorylating Rip3
Authors: Jianfeng Wu , wanze Chen , Yangxin Li , Tingting Ai , Jiahuai Han
Published: November 2023

The auto‐phosphorylation of murine receptor‐interacting protein 3 (Rip3) on Thr‐231 and Ser‐232 in the necrosome is required to trigger necroptosis. We identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1brestricts necroptosis in two settings: spontaneous necroptosis caused by Rip3auto‐phosphorylation in resting cells, and tumour necrosis factor (TNF)‐α‐induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain‐like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF‐‐α induced death in a Rip3‐dependent manner, and the role of Ppm1bin inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF‐α treated Ppm1b(d/d) mice. We also found epithelial cells in caecum damaged rapidly after TNF‐α treatment. Cecectomy could reverse TNF‐α‐induced death, but not LPS‐induced death in mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo, and epithelial cell necroptosis in caecum may play important roles in several diseases.

Support or Funding Information

This work was supported by the National Basic Research Program of China (973 Program; 2015CB553800), the National Scientific and Technological Major Project (2013ZX10002‐002), the National Natural Science Foundation of China (91429301, 31420103910, 31330047, 91029304, 31221065, and 31090360), the Hi‐Tech Research and Development Program of China (863 program; 2012AA02A201), the 111 Project (B12001), the Science and Technology Foundation of Xiamen (No. 3502Z20130027), the National Science Foundation of China for Fostering Talents in Basic Research (Grant No.J1310027) and the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University. This research was also partly supported by grants from MOST (2012CB966600) and NIH (R01GM63773, R01AR053591, R01CA108454).
Inhibition of polyamine oxidase reduces hyperoxia‐mediated neurovascular injury in retina
Authors: Narayanan Priya , Patel Chintan , Xu Zhimin , Shosha Esraa , Caldwell Ruth B
Published: November 2023

Retinopathy of prematurity (ROP) is a potentially blinding eye disease that affects premature infants and often leads to lifelong vision impairment. Hyperoxia treatment is shown to induce neurovascular damage in the developing retina. Using an oxygen‐induced retinopathy (OIR) mouse model for ROP, we have demonstrated that deletion of the arginase 2 reduced hyperoxia–induced retinal neurodegeneration through the regulation of polyamine metabolism. The present study was undertaken to study the impact of polyamine oxidase (PAO) inhibitor, MDL 72527, on hyperoxia‐induced retinal neurovascular injury in the OIR retina. Newborn wild‐type (WT) mice pups were exposed to 70% oxygen from postnatal day (P) 7 to P12. Pups were treated daily starting at P6 with either vehicle or MDL 72527 (39 mg/kg of body weight, in saline, i.p.) and sacrificed at different stages of hyperoxia treatment. Retinal cryostat sections were used for neurodegeneration studies using immunolabeling of retinal neurons. Analysis of vaso‐obliteration (N=10–12), vessel sprouting (N=7–9) and microglial activation (N=6) were performed on retinal flat mounts. Mixed rat retinal neuronal cultures (7 DIV) were subjected to normoxia (21%) or hyperoxia (50%) with or without MDL 72527 at different time points (N=6–8). These were processed for cellular signaling analysis using Western blotting. Immunolabeling studies using synaptophysin antibody showed the presence of improved synaptic contacts in MDL 72527‐treated OIR retina (P12) compared to vehicle treated groups. Analysis of retinal flat mounts showed significantly reduced a vascular area at P9 and P12( p<0.05) and increased vascular sprouting at P9 (p<0.01) in MDL 72527‐treated compared to vehicle‐treated OIR retinas after 2 or 5 days of hyperoxia. These results demonstrate reduced vascular injury in OIR retina in response to PAO inhibition. Immunolabeling studies using Iba‐1 antibody showed that activation of microglia was significantly reduced and number of resting microglia were increased in the OIR retina with MDL treatment (p<0.05). Analysis of signaling pathways in retinal neurons in response to hyperoxia showed activation of neuro‐inflammatory signaling studied by increased p‐NFkB, p‐ERK1/2 and cytochrome‐C levels, which were significantly reduced by MDL treatment. Our data suggest that PAO inhibition offers a new therapeutic target towards hyperoxia‐induced retinal neurovascular injury.

Support or Funding Information

These studies are supported by AHA 11SDG7440088, Culver VDI Pilot Grant Award and NEI‐R01‐EY011766.
Role of Hepatocyte Nuclear Factor 4 Alpha (HNF4α) in Hepatocyte Regeneration
Authors: Huck Ian , Manley Michael W. , Apte Udayan
Published: November 2023

Hepatocyte nuclear factor 4 alpha (HNF4α) is the master regulator of hepatic differentiation because it regulates over 60% of genes involved in hepatocyte specific function. Recent studies from our laboratory have revealed a novel role of HNF4α in regulation of hepatocyte quiescence. These data indicate that HNF4α can suppress proliferation of mature hepatocytes and is down regulated in hepatocellular carcinoma. However, the role of HNF4α in liver regeneration is not known. We examined HNF4α expression and function in liver regeneration after partial hepatectomy (PH) using three‐month‐old male C57BL/6 mice over a two‐week time course. Whereas no change in HNF4α mRNA were detected in regenerating livers, western blot analysis showed a rapid decline in nuclear HNF4α protein levels within 1 hour after PH, which continued until 6 hours. Nuclear HNF4α protein levels began increasing 12 hours post‐PH but remained significantly lower than normal till 3 days post‐PH and recovered to pre‐PH levels at 5 days after PH. Cytoplasmic HNF4α protein levels mimicked the nuclear expression pattern. Expression of several positive target genes of HNF4a involved in hepatocyte differentiation declined and expression of negative target genes of HNF4α involved in cell proliferation increased consisting with HNF4α protein changes. We further studied the role of HNF4α in liver regeneration after PH using conditional hepatocyte‐specific HNF4α knockout mice (HNF4α‐KO). Deletion of HNF4α resulted in high levels of proliferation at 24 hours and sustained higher levels 7 days post‐PH when compared with wild‐type mice. HNF4α‐KO mice exhibited earlier initiation of regeneration and exhibited defect in termination of regeneration. Finally, we investigated the role of HNF4α in HGF and EGF‐stimulated growth of primary rodent hepatocytes using a chemically defined media. Mitogen treatment stimulated hepatocyte proliferation, which coincides with accelerated loss of HNF4α when compared with untreated hepatocytes. Taken together, these data indicate that loss of HNF4α expression and activity is one of the earliest events in initiation of cell cycle after PH. Similarly, re‐expression of HNF4α and restoration of its activity is essential for termination of liver regeneration. These studies, for the first time, demonstrate that HNF4α reprogramming is a critical step in liver regeneration and could have potential therapeutic benefits.

Support or Funding Information

Funding: NIH (P20 RR021940, T32ES007079‐34, 1R01DK098414), AASLD/ALF Liver Scholar Award
Group A Streptococcus Streptococcal Collagen‐like Protein (Scl‐1) Enhances Bacterial Resistance to Neutrophil Serine Proteases
Authors: Kim Jessica , Etesami Neelou , Dohrmann Simon , Nizet Victor , Okumura Cheryl
Published: November 2023

The streptococcal collagen‐like protein (Scl‐1) of the gram‐positive Group A Streptococcus (GAS) is thought to contribute to bacterial resistance to the killing mechanisms of cells of the innate immune system. Our previous work has shown that the Scl‐1 protein was found to enhance bacterial survival in the presence of phagocytic cells to allow persistence at the site of infection. Specifically, Scl‐1 prevented bacterial killing by preventing binding of the antimicrobial peptide LL‐37, which is a contributor to the bactericidal activity of neutrophil extracellular traps (NETs). In addition to the role of LL‐37 in extracellular killing, LL‐37 is deployed intracellularly in neutrophils along with neutrophil serine proteases found in azurophilic and specific granules. In our current study, we examine the protective effects of Scl‐1 when bacteria are intracellular, such as when they are phagocytosed by neutrophils and macrophages. A mutant strain of GAS lacking the Scl‐1 gene (ΔScl‐1) exhibited significantly decreased survival in vitro with the neutrophil serine proteases cathepsin G, elastase and lysozyme compared to wild‐type and plasmid‐complemented strains. In addition to the direct antimicrobial activity of these enzymes, the results suggest that in the context of a cell, digestion of surface molecules by these enzymes may subsequently render the bacterial more susceptible to LL‐37. Scl‐1 would thus serve as a molecular “shield” when other cell wall components are not present. We are currently investigating the synergy between the granular enzymes and LL‐37. The results suggest that Scl‐1 is critical in suppressing the effects of LL‐37 both intracellularly and extracellularly, prolonging the survival of GAS in the face of the innate immune defense system.
Antihyperalgesic and antiallodynic effects of 3a,4,5,9b‐Tetrahydro‐4‐(1‐naphthalenyl)‐3H‐cyclopentan[c]quinoline‐8‐sulfonamide in an animal model of neuroinflammatory pain
Authors: Abbas Muzaffar , Rahman Shafiqur
Published: November 2023

Neuroinflammatory pain is a chronic neurological disorder characterized by allodynia and hyperalgesia. We have shown that microglial alpha‐7 nicotinic acetylcholine receptors (α7 nAChRs) in the hippocampus is involved in pain production and maintenance. The objective of the present study was to determine the effects of 3a,4,5,9b‐Tetrahydro‐4‐(1‐naphthalenyl)‐3H‐cyclopentan[c]quinoline‐8‐sulfonamide (TQS), an alpha7 nAChR positive allosteric modulator, and methyllycaconitine (MLA), an α7 nAChR antagonist, on lipopolysaccharide (LPS)‐induced neuroinflammatory pain using von Frey hair test, a widely used method for the quantification of mechanical allodynia and hot plate test, a widely used technique for hyperalgesia assessment in mice. In addition, we examined the effects of TQS (1 or 4 mg.kg) on ionized calcium‐binding adapter molecule‐1 (Iba‐1), a microglial activation marker. Acute administration of LPS (1 mg/kg, i.p.) increased (p<0.05) the sensitivity to pain (allodynia and hyperalgesia) after 6 hours. TQS (0.25, 1 or 4 mg/kg, i.p.) decreased LPS‐induced allodynic or hyperalgesic response (p<0.05). Pretreatment of MLA reversed TQS‐induced antiallodynic and antihypergesic effects. TQS (1 or 4 mg/kg, i.p.) also reduced the expression of Iba‐1 in the hippocampus. Taken together, these results suggest that TQS decreases LPS‐induced neuroinflammatory pain by reducing hippocampal microglial activation through modulating α7 nAChR allosteric site.

Support or Funding Information

Fulbright Foundation, USA
Controlling for Individual Differences in Temperature Sensitivity Reduces Potency Differences Among Subjects in a Warm Water Tail Withdrawal Procedure in Rhesus Monkeys
Authors: Weed Peter F , Gerak Lisa R , Maguire David R , France Charles P
Published: November 2023

Warm water tail withdrawal is a widely used procedure for assessing the antinociceptive effects of drugs, including opioids. Traditionally, this assay uses the same predetermined temperature(s) to measure tail withdrawal latencies for all subjects in a group; however, under some conditions, the potency of drugs for increasing tail withdrawal latencies varies markedly among subjects. That variance might result from individual differences in sensitivity to drug effects and/or individual differences in sensitivity to the thermal stimuli. This study tested the hypothesis that variance in the antinociceptive potency of opioids is due, in part, to baseline differences in sensitivity to thermal stimuli among individual subjects. Four rhesus monkeys were tested using a warm water tail withdrawal assay under two conditions. In the first condition, the antinociceptive effects of morphine (0.56–5.6 mg/kg, s.c.) and fentanyl (0.0056–0.056 mg/kg, s.c.) were assessed with a single water temperature, 54°C. Both morphine and fentanyl dose dependently increased tail withdrawal latencies; however, for both drugs, there was a greater than 3‐fold difference in dose that produced a full effect across subjects. In the second condition, temperature‐effect curves were first determined for individual subjects by varying the water temperature from 40 to 54°C. Tail withdrawal latency decreased in all subjects in a temperature‐dependent manner; however, the lowest temperature that significantly decreased tail withdrawal latency varied depending on the subject. The temperatures that reduced tail withdrawal latency to less than four seconds ranged from 48ºC to 52ºC. When the antinociceptive effects of morphine and fentanyl were redetermined using these temperatures that were functionally equivalent among monkeys, rather than the same temperature (54°C), the previous between‐subject differences in potency were reduced such that effective doses of either morphine or fentanyl were the same among subjects. These results demonstrate that differences among individual subjects in the antinociceptive potency of opioids can be minimized by using functionally equivalent temperatures in a warm water tail withdrawal assay to accommodate for differences in sensitivity to a thermal stimulus. Manipulations that alter drug sensitivity or effectiveness, such as administering drugs in combination, can be assessed in a more quantifiably rigorous manner by controlling for individual variability in temperature sensitivity.

Support or Funding Information

Supported by USPHS Grants R01DA05018, T32DA03111, and K05DA017918.
Expression and Pharmacological Modulation of Visceral Pain‐Induced Conditioned Place Aversion in Mice
Authors: AlSharari Shakir , Bagdas Deniz , Muldoon Pretal , Carroll Ivy , Negus Stevens , Damaj Imad
Published: November 2023

Since pain is an unpleasant sensory and emotional experience, to evaluate the negative affective component of visceral pain induced by intraperitoneal (i.p.) noxious acetic acid (AA) injection is important as much as measuring the nociceptive behaviors as seen in stretching. Recent studies showed that i.p. AA resulted conditioned place aversion (CPA) in rats. In this study we first characterized the AA‐CPA in mice, and then we compared the affective and sensory components of pain using well‐known analgesic drugs. AA‐received mice showed CPA in respect to sensorial results which also determined by number of stretches. The nonsteroidal anti‐inflammatory drugs (NSAIDs) ketoprofen, the opioid receptor agonist morphine, and the k‐opioid receptor agonist U50,488H inhibited the AA‐induced stretching. Ketoprofen and morphine also blocked the AA‐induced CPA but not the U50,488H. The reversal ability of analgesic drugs on CPA is unique to pain‐stimulated place aversion. In respect, ketoprofen and morphine failed to reduce non‐noxious LiCl‐induced CPA. In conclusion, AA‐induced CPA may serve a measure of affective pain sign as pain‐related place aversion, and possible novel analgesic drugs can be test for their effectiveness on affective component of pain in mice.
Responses of Central Microglial Cells and Ganglionic Macrophages to Peripheral Injury and Disease
Authors: Kawaja Michael
Published: November 2023

Microglial cells in the brain and spinal cord are exquisitely sensitive to injury and disease, responding by activation that results in changes of morphology and function. These cells are also responsive to trauma/inflammation in peripheral targets, such that injury or disease of motor, sensory and/or autonomic fibers can result in robust activation of microglia centrally. This activation of microglia triggers the presentation of phenotypes that resembles cells with macrophage‐like properties (i.e., increased cytokine production). Curiously, resident macrophages in peripheral neural tissues (e.g., nerves and ganglia) display many similar morphological and functional properties of central microglia. We have used a number of injury paradigms to assess the activation of central microglial cells and peripheral macrophages, as a consequence of direct or indirect neural damage. These experimental models include i) unilateral partial sciatic nerve ligation (a model of peripheral nerve damage), ii) unilateral injection of monosodium‐iodoacetate (MIA) into the hind limb footpad (a model of cutaneous/joint inflammation), and dextran sodium sulfate (DSS) in the drinking water (a model of colonic inflammation). We have also assessed the activation of central microglial cells and peripheral macrophages in aging models (i.e., NGF transgenic mice and TgCRND8 mice). The transmembrane p75 neurotrophin receptor (p75NTR) binds with comparable affinity to neurotrophins (e.g., nerve growth factor, NGF) and cytokines (e.g., tumor necrosis factor alpha, TNFα). Both neurotrophins and cytokines have been implicated in the pathophysiological features of sensory neuron dysfunction as a consequence of injury, disease, and aging. For instance, following peripheral nerve damage, macrophages (i.e., resident and/or infiltrating blood‐borne) in the associated dorsal root ganglia (DRG) display increased process extension, which is indicative of activation. Central microglial cells act in kind. Our studies have sought to determine whether the expression of p75NTR in adult mice affects the activation of microglial cells in the spinal cord and macrophages in sensory ganglia after peripheral nerve/tissue injury. Effects we have measured in p75NTR‐deficient mice are curious because central microglial cells and peripheral macrophages (damaged or otherwise) do not express p75NTR. Since target tissue damage and/or inflammation can stimulate increased p75NTR levels by ganglionic glial cells (i.e., satellite and Schwann cells), we have speculated that such glial expression of p75NTR plays a role in the activation of central microglial cells and peripheral macrophages. One possible mechanism may be through p75NTR‐mediated sequestration of increased levels of neurotrophins and/or cytokines by glial cells, thereby minimizing microglia/macrophage activation in mice following injury and inflammation.

Support or Funding Information

Queen's University
Structure‐Activity Relationship of Peptide Inhibitors of the Wnt/frizzled interaction
Authors: Blankesteijn W. Matthijs , Hermans Kevin CM , Fernandez‐Llamazares Ana Iris , Kriek Nicole , Timmerman Peter
Published: November 2023

Introduction

Crystallization of the Xenopus Wnt8 in complex with the cysteine rich domain (CRD) of Frizzled8 has shown that the interaction of Wnt and frizzled resembles the outline of a hand with a thumb and index finger grasping the CRD at two opposing sites. Previous experiments from our group have shown that both the thumb and the index finger fragments can interfere with the Wnt/frizzled interaction at micromolar concentrations. In this follow‐up study, we investigated the structure‐activity relationships of these fragments by changing their size and composition. Moreover, we studied the contribution of the disulfide bonds that are formed between the cysteine residues present in both fragments.

Methods

Peptides resembling the thumb and index finger of Wnt3a and Wnt5a were synthesized and tested in HEK293 and 3T3 cells expressing the TOPFlash reporter. Canonical Wnt signaling was induced by addition of Wnt3a conditioned medium.

Results

A gradual decrease of the length of the thumb‐derived sequence from 22 to 13 amino acids resulted in a 40% loss of antagonistic effect at 1.10−5 M. Palmitoylation of the thumb was found to be essential for biological function. For the index finger‐derived peptide, antagonistic properties remained constant during gradual reduction from 23 to 13 amino acids, but a further reduction resulted in complete loss of antagonistic activity. For both fragments the disulfide bonds most proximal to the tip were important for antagonistic activity whereas the distal disulfide bonds were dispensable. Scrambled peptides did not have antagonistic properties.

Conclusion

The antagonistic properties of peptide fragments of the thumb and index finger of Wnt peptides depend on the length and proper tertiary structure.

Support or Funding Information

Supported by the Dutch Heart Foundation (2010B196) and Cyttron (FES0908)
Management of Fluoride Toxicity on Adult Male Wistar Rats’ Fecundity Using Some Selected Antioxidants
Authors: Anacletus Francis Chukwuma , Onyegeme‐Okerenta Blessing Minaopunye , Iheka Chidiebere Uchenna
Published: November 2023

Management of fluoride toxicity using the ameliorative antioxidant potentials of some selected antioxidants on adult male Wistar rats’ fecundity were investigated. Thirty‐six adult male albino rats were divided into 6 groups. Group I (positive control) received oral dose of normal feed and water for 4 weeks and 6 weeks, Group II (negative control) sodium fluoride (10mg/kg), Group III received sodium fluoride (10mg/kg) + selenium(0.5mg/kg), Group IV sodium fluoride (10mg/kg) + zinc (14.8mg/kg), Group V sodium fluoride (10mg/kg) + ginseng (10mg/kg), Group VI sodium fluoride (10mg/kg) + vitamin E (100mg/kg). At the end of the 4th and 6th week, hormone assay (testosterone, prolactin, luteinizing hormone and follicle stimulating hormone, sperm quality analysis: morphology, motility, abnormality, sluggishness, deadness and sperm count) were analysed. Result showed that Group I had a significant decrease (p<0.05) in the sperm quality parameters and a significant decrease (p<0.05) in the hormone parameters compared to the control group. However, there was no significant difference in the sperm sluggishness parameter after 4 weeks and 6 weeks and also no significant difference in the sperm abnormality parameter after 4 weeks. Testicular examination showed necrosis, epithelial apoptosis, and atrophied seminiferous tubules. Concomitant administration of sodium fluoride +selenium, sodium fluoride+ zinc, sodium fluoride +ginseng, and sodium fluoride + vitamin E respectively showed a significant reversal of the toxic effect of NaF on the treated animals and this corroborated with the histology results of the testes. The result showed that sodium fluoride induced severe toxic changes in the male reproductive system while these antioxidants provided partial and complete amelioration against these toxic effects

Support or Funding Information

Research was funded by the authors
Establishing the Competitive Counterflow Assay for OATP1B1 and OATP1B3
Authors: Katuwal Miki , Schnegelberger Regina , Wityk Elliott , Ruggiero Melissa , Hagenbuch Bruno
Published: November 2023

OATP1B1 and OATP1B3 are two important drug uptake transporters expressed in human hepatocytes. They mediate the uptake of numerous drugs and other compounds and multiple inhibitors have been identified. However, it has also been suggested that these inhibitors may act as substrates for OATPs. Therefore, we wanted to establish the competitive counterflow assay described by Harper and Wright (Am J Physiol Renal Physiol 2013; 304:F56–67) for OATP1B1 and OATP1B3 with the model substrate estradiol‐17β‐glucuronide (E17βG). This assay should allow to identify compounds that are able to exchange for an OATP substrate such as [3H]‐E17βG that was used to preload OATP expressing CHO cells. CHO cells stably expressing either OATP1B1 or OATP1B3 were used and time dependent uptake of 10nM [3H]‐E17βG was determined on 96‐well plates. Uptake reached a steady‐state plateau at 2 hours and addition of unlabeled E17βG to the extracellular medium at this time triggered time and concentration dependent efflux of [3H]‐E17βG from the preloaded cells, indicating that extracellular E17βG exchanged for intracellular [3H]‐E17βG. This assay was then used to test whether several known OATP substrates would also lead to [3H]‐E17βG efflux, indicating that they are exchanged for the intracellular substrate. After preloading the cells with [3H]‐E17βG, addition of known substrates such as E17βG, estrone‐3‐sulfate, bromosulfophthalein, taurocholate, and rifampicin to the extracellular medium indeed resulted in [3H]‐E17βG efflux, confirming that OATP1B1 and OATP1B3 handle their substrates as exchangers. As negative controls we used glucose, glutamic acid, ouabain and metformin which did not result in [3H]‐E17βG efflux. We then began testing known OATP inhibitors to see whether they would trigger [3H]‐E17βG efflux, and thus would be OATP substrates. Among the tested inhibitors, many resulted in [3H]‐E17βG efflux from both OATP1B1 and OATP1B3 expressing CHO cells, and thus seem to be substrates. These included bromocryptine, cyclosporine A, DIDS, indomethacin, ketoconazole, reserpine, and salinomycin. A few, including berberine, erythromycin, gliquidone, nifedipine, and paranitrosaniline seem to be substrates only for OATP1B1. Thus, because the tested substrates do not need to be radiolabeled and the CCF assay can be performed on 96‐well plates, this is a good screening tool to identify OATP inhibitors that are transported substrates.

Support or Funding Information

This work was supported by the National Institute of Health Grants R01‐GM077335 and P20‐GM103549‐07.
Synergism between bioengineered miR‐34a prodrug and doxorubicin in suppressing osteosarcoma cell proliferation and xenograft tumor growth
Authors: Tu Meijuan , Zhao Yong , Yu Yifeng , Wang Weipeng , Chen Qiuxia , Qiu Jingxin , Yu Aixi , Yu Aiming
Published: November 2023

Osteosarcoma (OS) is a common form of malignant bone tumor and it is prevalent among children and young adolescent adults. Recently we have established a novel approach to bioengineering large quantity of microRNA‐34a (miR‐34a) prodrug for miRNA replacement therapy. In this study, we aimed to evaluate combination therapy with miR‐34a prodrug and doxorubicin, which may synergistically reppress human OS cell growth via RNA interference and DNA intercalation. Synergism was indeed obvious between miR‐34a prodrug and doxorubicin in suppressing OS cell proliferation, as revealed by Chou‐Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was attributable to much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest, and associated with an elevated miR‐34a level and reduced protein levels of miR‐34a targeted (proto‐)oncogenes including SIRT1, c‐MET, and CDK6. Furthermore, orthotopic OS xenograft tumor growth was inhibited to a significantly greater degree in mouse models when miR‐34a prodrug and doxorubicin were co‐administered intravenously. In addition, multiple doses of miR‐34a prodrug and doxorubicin showed no or minimal effects on mouse blood chemistry profiles. These results demonstrate that combination therapy with doxorubicin and miR‐34a prodrug produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism‐based combination therapy to combat OS and bioengineered miR‐34a prodrug represents a new natural miRNA agent.

Support or Funding Information

NIH U01CA175315
Synthesis and Studies of N, N‐(Dimethylamino) Methylene Prodrugs of Gemcitabine, Ara‐A and Ara‐G
Authors: Tawfeeq Nada , Kerr Stephen
Published: November 2023

There is a need for developing prodrugs of nucleoside analogs to overcome limitations of permeability and bioavailability. One approach to nucleoside modification is by replacing the exocyclic amino groups on the nucleoside bases of adenosine, guanosine and cytidine with N, N‐(dialkylamino) methylene side chains to form their analogous amidine prodrug derivatives to improve their lipophilicity. In this study, the N, N‐(dimethylamino) methylene prodrugs of gemcitabine, 9‐β‐D‐arabinofuranosyladenine (Ara‐A), and 9‐β‐D‐arabinofuranosyl guanine (Ara‐G) were synthesized and their log P and hydrolysis rate constants were compared to 2′‐deoxycytidine and Ara‐C. Partition coefficients between 1‐octanol and the phosphate buffer saline (PBS) for all compounds were measured using the shake‐flask procedure. The lipophilicity of the prodrugs ranged from 1 to 1.5 fold more than the parent unmodified nucleoside. The hydrolytic pseudo‐first order rate constants were determined spectrophotometrically in PBS at 37 deg C. The hydrolytic studies indicated conversion of the prodrugs to the parent drugs was spontaneous with corresponding half‐lives of the prodrug ranging from 3 to more than 14 h. The dimethylamino‐methylene pro‐drug derivatives of Ara–G and Ara‐A were found to be more stable than the corresponding cytosine analogs. The Ara‐G prodrug derivative was the most stable derivative in terms of hydrolysis to its parent nucleoside. Cytotoxicity studies in an MCF‐7 cell line showed limited cytotoxic activity. These studies provide a basis to modulate the lipophilicity and hydrolytic stabilities of such nucleosides by varying the N,N‐dialkyl substitution to give derivatives with greater lipophilicity and stability.

Support or Funding Information

Supported by the Graduate Studies Department of MCPHS University and Saudi Arabian Cultural Mission
Contribution of NADPH Oxidase (Nox)‐derived Reactive Oxygen Species (ROS) to Doxorubicin‐induced Cardiomyopathy Mediated by Regulator of G protein Signaling 6 (RGS6)
Authors: Chakravarti Bandana , Yang Jianqi , Luo Zili , Ahlers Katelin E.
Published: November 2023

Oxidative stress, defined as an excess of reactive oxygen species (ROS) compared to antioxidant defenses, is implicated in the pathophysiology of heart failure. ROS play a critical role in regulation of cardiac myocyte apoptosis through control of redox‐sensitive signaling cascades and can also activate hypertrophic kinases, transcription factors and proteins involved in extracellular matrix remodeling. Nox proteins have emerged as major sources of ROS in the pathogenesis of myopathic changes associated with heart failure. RGS6 is a member of the RGS protein family of heterotrimeric G protein regulators. Our recent studies have identified an entirely novel signaling action of RGS6 as an upstream modulator of ROS‐dependent apoptosis in the heart. Mice lacking RGS6 are almost completely protected from ventricular ROS generation, cardiac myocyte apoptosis and loss of ventricular function in response to doxorubicin (Dox). We hypothesized that RGS6 is required for Nox‐dependent ROS production in cardiac myocytes induced by Dox and that it promotes apoptosis by a Nox‐ROS‐dependent mechanism. Nox2 and Nox4 are the major isoforms expressed in heart and are found in distinct subcellular locations. Plasma membrane bound Nox2 is critical for cardiac remodeling and contractile dysfunction in response to Dox. Here, we show increased Nox activity and a trend for increases in Nox 2 and Nox4 mRNA in hearts of wild type (WT) mice 5 days following Dox administration, as reported previously. These responses to Dox were not observed in mice lacking RGS6 suggesting that RGS6 is required for Dox‐induced Nox activation and possibly for increases in Nox mRNA expression in heart. In light of these findings, we examined the effects of Dox on cell death, caspase 3 activity and Nox activity in adult cardiomyocytes derived from WT, RGS6−/−, p47−/− (Nox2 subunit) and RGS6−/−; p47−/− double knockout mice. Individual loss of RGS6 or p47 blocked Dox‐induced increases in cell death, caspase 3 activity and Nox activity while their combined loss had no effect. These results provide new evidence that RGS6 is a critical upstream modulator of Nox‐derived ROS contributing to Dox‐induced cardiomyopathy. The mechanism(s) by which RGS6 modulates Nox activity in heart and the importance of the RGS6‐Nox‐ROS signaling cascade in oxidative stress‐induced cardiomyopathies are currently under investigation.

Support or Funding Information

Supported by NIH CA161882, AHA 14GRNT20460208 (RAF) and AHA 11SDG7580008 (JY).
Smooth Muscle/Endothelial KIR Channels Modulate Cell‐to‐cell Communication in Cerebral Arteries
Authors: Gonzalez Maria Sancho , Samson Nina , Hald Bjorn Olav , Hashad Ahmed , Marrelli Sean , Brett Suzanne , Welsh Donald Gordon
Published: November 2023

Global blood flow control is enabled by the conduction of charge along arteries. The distance over which electrical phenomena spread is governed by 3 factors, one being the ionic properties of vascular cells. In this study, we determined the role of inward rectifying K+ channels (KIR) in setting membrane conductance and charge spread along the arterial wall. Small middle cerebral arteries (~100 μm diameter) from hamster were probed with a standard conduction protocol. A focal KCl stimulus elicited a constrictor response that conducted robustly, with a decay constant of ~0.4 μm/100μm vessel length. Selective inhibition each K+ channel class had no effect on conduction, the exception being Ba2+ blockade of KIR, which facilitated decay (~1.6 μm/100μm vessel length). Patch clamp electrophysiology and Q‐PCR highlighted the presence of a KIR current in smooth muscle comprised of KIR2.1/2.2 subunits. The incorporation of this current into an electrical model revealed that it was too small to account for the change in conduction decay; consequently another KIR current must be present. Electrophysiology and Q‐PCR confirmed a second KIR current in the endothelium. Computational modeling subsequently confirmed that inhibiting both currents had a greater effect on conduction decay, the result of a sizable voltage shift increasing feedback from voltage dependent‐ and Ca2+ activated K+ channels. In summary, our observations indicate that KIR channels are present in endothelial and smooth muscle cells, and together these K+ conductances can tune electrical communication.

Support or Funding Information

Supported by CIHR
Estimating Blood Flow in Skeletal Muscle Arteriolar Trees Reconstructed from In Vivo Data
Authors: Goldman Daniel , Saleem Amani H. , Al‐Khazraji Baraa K. , Jackson Dwayne N.
Published: November 2023

Background

Our group uses both computational modeling and intravital videomicroscopy to study the regulation of blood flow in the microcirculation of skeletal muscle. Although we are able to obtain nearly complete arteriolar network structure from in vivo experiments, obtaining complete hemodynamic information is much more difficult and time‐consuming. It is also difficult to obtain the full boundary data needed to directly calculate microvascular blood flow and hematocrit distributions. Therefore, the objective of the present work was to develop a computational model that could accurately predict blood flow in skeletal muscle arteriolar trees in the absence of complete boundary data.

Methods

We used arteriolar trees in the rat gluteus maximus muscle (GM) that were reconstructed from montages obtained via intravital videomicroscopy, and incorporated a recently published method for approximating unknown boundary conditions into our existing steady‐state model of two‐phase blood flow. For varying numbers of unknown boundary conditions, we used the new flow model and GM arteriolar geometry to approximately match red blood cell (RBC) flows corresponding to experimental measurements.

Results

We showed that this method gives errors that decrease as the number of unknown boundary conditions decreases. We also showed that specifying total blood flow into the arteriolar tree decreases the mean RBC flow error and its variance. By varying target values of pressure and wall shear stress required by the model, we showed that results are less sensitive to the target pressure, and we developed a method for estimating the optimal target shear stress.

Conclusion

We have developed and validated a computational method that can accurately estimate RBC flow distribution in arteriolar trees in the absence of complete boundary data.

Support or Funding Information

Funding: Natural Sciences and Engineering Research Council (NSERC) grant #R4081A03 awarded to DG, NSERC grant #R4218A03 awarded to DNJ, and NSERC CGS‐D Scholarship awarded to BKA.
Interventions Blunting Systemic Inflammation and Oxidant Stress Improve Depressive Symptoms and Vascular Function in a Model of Metabolic Syndrome
Authors: Sheets Whitney Jordan , Brooks Steven D , Chantler Paul , Frisbee Jefferson
Published: November 2023

Metabolic syndrome (MetS) is a combination of physical ailments such as diabetes, hypertension, hyperglycemia, and hypertriglyceridemia. MetS is associated with increased likelihood for developing cardiovascular disease (CVD), the leading cause of death in the United States. A growing body of evidence has established CVD and MetS as risk factors for developing depression, raising questions about the physiological links between these highly prevalent conditions. This comorbidity has been observed preclinically in obese Zucker rats (OZR), which concurrently develop MetSyn and symptoms of depression with age. While several studies have sought to evaluate possible cardiovascular benefits of common antidepressants, such as SSRIs, little is known as to whether treating constituent pathologies of Metabolic Syndrome could have beneficial effects on depressive symptoms. The purpose of this study was to evaluate the efficacy of several commonly prescribed therapeutic agents towards improving vascular function and reducing depressive behaviors in adult OZR, and to identify potential mechanisms defining these effects. At 7–8 weeks of age, male OZR and lean control (LZR) animals received one of the following therapies for 9 weeks: anti‐hypertensive (captopril, hydralazine), anti‐diabetic (metformin, rosiglitazone), anti‐dyslipidemic (atorvastatin, gemfibrozil), anti‐oxidant (TEMPOL), or daily treadmill exercise training; at the conclusion of treatment, depressive symptoms, ex vivo arteriolar vascular reactivity, NO bioavailability, and plasma biomarkers of stress and inflammation were assessed in each animal. Of all treatments, exercise, atorvastatin, and TEMPOL were found to significantly improve depressive symptoms in OZR and restore vascular function in skeletal muscle arterioles. Further analysis revealed that these three interventions all reduced levels of plasma TNF‐α, nitrotyrosine, corticosterone, and thromboxane, in addition to improving NO bioavailability and vascular reactivity. Each of the other treatments improved at least their primary pathological target, but did so without improving depressive symptoms. These findings suggest that the pleiotropic benefits of atorvastatin, TEMPOL, and exercise‐ specifically, anti‐inflammatory and anti‐oxidant effects‐ may contribute to improving depressive symptoms. Additionally, these results imply a possible cardiovascular etiology for depression, and raise the possibility that coupling antidepressant therapy with a drug such as atorvastatin could lead to improved outcomes for patients with depression.

Support or Funding Information

AHA

WVU CCRS
Contractile Pericytes Determine the Direction of Blood Flow at Capillary Bifurcations
Authors: Gonzales Albert L , Longden Tom A , Shui Bo , Kotlikoff Michael I , Nelson Mark T
Published: November 2023

Pericytes cover most of the surface area of brain capillaries, and yet their role in controlling cerebral blood flow is controversial. Although pericytes have a distinct morphology from vascular smooth muscle, pericytes at the first and second capillary junctions downstream of the terminal arteriole exhibit many of the same excitation‐contraction coupling molecules as smooth muscle, e.g. smooth muscle a‐actin, Cav1.2 and TRPM4 (from immunohistochemistry). We tested the hypothesis that junctional pericytes are capable of controlling the direction of flow in the capillary bed, insuring efficient delivery of blood to downstream neurons. Using a novel transgenic mouse expressing a genetically encoded Ca2+ biosensor in contractile pericytes and smooth muscle (acta2‐GCaMP5‐mCherry), we observed IP3R‐ and Cav1.2‐, but not ryanodine receptor‐mediated, Ca2+ events. Using in vivo multiphoton microscopy, we observed a higher frequency of Ca2+ events in pericyte projections covering the branch with lower red blood cell (RBC) flux at capillary bifurcation. The thromboxane receptor agonist, U46619, deferentially increased Ca2+ signaling in the pericytes covering the two branches such that less contracted branch increased its Ca2+ signaling and RBC flux was reduced to the level of other branch, i.e. flux was normalized in two branches. These data support the concept that pericytes play a role in controlling the directional flow of blood within the capillary bed.

Support or Funding Information

Totman Medical Research Trust, Foundation Leducq, AHA 14POST20480144, NIH T32HL‐007594, P01HL‐095488, R37DK‐053832, R01HL‐121706, and R24HL‐120847