bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThese 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 InformationFunding: NIH (P20 RR021940, T32ES007079‐34, 1R01DK098414), AASLD/ALF Liver Scholar Award
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 InformationFulbright 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 InformationSupported by USPHS Grants R01DA05018, T32DA03111, and K05DA017918.
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 InformationQueen'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

IntroductionCrystallization 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.MethodsPeptides 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.ResultsA 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.ConclusionThe 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 InformationSupported 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 effectsSupport or Funding InformationResearch 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 InformationThis 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 InformationNIH 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 InformationSupported by the Graduate Studies Department of MCPHS University and Saudi Arabian Cultural Mission