bluuebunny/crossref_metadata_2025 huggingface dataset:
Student Attendance at Pharmacology Lectures and Its Relation to Exam Performance: A Two Year Observational Analysis
Authors: Powers Claud Andrew , Carroll Mairead A.
Published: November 2023

Student lecture attendance is now often voluntary due to lecture‐capture technology that records sessions and posts audiovisual files to on‐line databases for student use. However, it is unclear if lecture recordings promote learning as well as live attendance, and there is limited data on the features of student lecture attendance or its relation to exam performance since the arrival of lecture capture technology. This study reports two years of observational data from the Fall Medical Pharmacology course for 2nd year medical students (~200 students per year) where attendance data was collected from 13 of 40 voluntary sessions by an audience‐response system when students answered questions using wireless response devices. The data enabled characterization of student attendance and analysis of its relation to exam performance. Since all lectures were recorded and posted on‐line, the data also provided a comparison of the learning efficacy of live attendance versus use of on‐line materials. On average, only 25–31% of the class attended any given monitored session and only 12–14% exhibited a high attendance level (>80%). On average, students with high attendance had significantly higher exam scores and there was a significant shift in their scoring distribution to higher ranks coupled with a decrease in lower ranks in both years studied. Students with intermediate (77–50%) or low attendance (44–8%) did not display a scoring advantage relative to students with no attendance and had an erratic attendance pattern compared to students with high attendance. The performance advantage for students with high attendance remained evident 22 weeks after completion of the Fall semester when students took a comprehensive NBME exam in pharmacology. The data show that high lecture attendance was associated with higher exam scores even though all students had access to the identical material in on‐line lecture‐capture recordings that could be viewed whenever and as often as desired. The educational efficacy of live lecture attendance relative to use of on‐line lecture recordings merits further analysis, and it may be prudent to reconsider suggestions for the wide‐scale replacement of live lectures with on‐line recordings.
Use of PC‐NSAIDs to treat 2nd degree burn injury in rodent models
Authors: Lichtenberger Lenard M. , Ono Kaori , Phan Tri , Fang Dexing , Poindexter Brian J. , Bick Roger J. , Dial Elizabeth J.
Published: November 2023

Background

There is a need for drug treatment for 2nd degree burn injury that can be applied quickly to both relieve pain and promote healing. Currently used drugs for chronic pain have substantial side‐effects which limit their usefulness. These include opioids and their addictive potential, gabapentin and its sedation, and coxibs with their risk of cardiovascular failure. Nonsteroidal anti‐inflammatory drugs (NSAIDs) are potent analgesics and provide relief from burn pain, but their use is limited due to side effects related to gastrointestinal (GI) bleeding insusceptible patients.

Objective/Aim

The goal of this project is to develop rodent model systems to evaluate the analgesic efficacy and side‐effects of a novel treatment for 2nd degree burn injury to alleviate pain and promote healing using a new class of NSAIDs that are complexed with the soy phosphatidylcholine (PC) that can be administered as a sterile topical alone, parenterally or in combination.

Methods

Employing two established methods, for the induction of 2nd degree burn injury in rats to either the dorsal skin or the hindlimb we investigated the effects of the PC‐NSAIDs, Ibuprofen‐PC and Indomethacin‐PC vs the unmodified NSAIDs administered (either subcutaneously (sc) or topically to the affected skin) daily over a 10‐day study at a dose of 20 mg/kg and 2mg/kg respectively. We then assessed pain sensation behaviorally, the depth of skin injury by light and fluorescent microscopy and tissue inflammation biochemically and by non‐invasive IVIS imaging.

Results & Conclusion

Both Indomethacin‐PC and Ibuprofen‐PC when sc administered provided moderate hyperalgesia at 3‐ and 5‐days post‐burn injury, which was similar to the unmodified NSAIDs and in some cases more efficacious as analgesic/anti‐inflammatory agents. Topical administration of our test PC‐NSAIDs provided more variable responses using the two rodent model systems, with the most consistent analgesic/anti‐inflammatory effect being recorded with Indomethacin‐PC. Morphological analysis revealed that the depth of skin injury was reduced in rats treated with the PC‐NSAIDs with no evidence of GI bleeding. Supported by a USARMC grant, W81XWH‐14‐2‐0016.

Support or Funding Information

Supported by a USARMC grant, W81XWH‐14‐2‐0016.
Analgesic actions of novel derivatives of the active compound, Incarvillateine, from the Chinese herb (Incarvillateine sinesis) in acute pain
Authors: Priebe Alaini , Hunke Megan , Pattabiraman Mahesh , Chandra Surabhi
Published: November 2023

Commonly used pain medications, opioids and non‐steroidal anti‐inflammatory drugs (NSAIDs) are highly efficacious for treating chronic pain and post‐surgical pain, but are associated with neurological and abdominal side effects. This reduces patient compliance while also leading to abuse and misuse as non‐prescription medication. Thus, there is a high need for formulating alternative pain treatment strategies to investigate drugs with potent action but minimal side effects. Incarvillateine (INCA), derived from the Chinese herb Incarvillea sinensis, has been widely used in traditional medicine for treating rheumatism and pain. Research on antinociceptivity of INCA in chronic and acute pain models indicate that the diaryl cyclobutane dicarboxylic acid core is essential for its medical property. The small number of tested compounds, despite the larger number of possible INCA analogs, is due to the lack of efficient synthetic route to construct the various regio‐ and stereoisomers of the diphenylcyclobutane‐dicarboxylate core. There are reports that though INCA binds to both opioid and adenosine receptors, its primary analgesic action is through the adenosine receptors, thus having minimal side effects on central nervous system. While INCA is a highly effective natural compound, research on synthetic analogs of this compound has received less attention till date. We hypothesized that novel INCA analogs synthesized in our lab show potent antinociceptive action, and their effect is mediated through adenosine receptor action. Compounds were synthesized using innovative cavitand‐mediated photodimerization (CMP) method, which utilizes a macromolecule (gamma‐cyclodextrin) to control the excited state reactivity of photoactive compounds to yield target tetra‐substituted cyclobutanes (dimers). The dimers generated so far show positive response in suppressing formalin‐induced acute pain in mice hind paw while the monomers were ineffective. The antinociceptive effect of these analogs was observed in the inflammatory phase suggesting a primary non‐opioid mechanism for pain reduction. Our further characterization and selection of INCA analogs (with predominant A3AR ‐ adenosine receptor action) will help us to generate a new class of antinociceptives with precise chemical modifications using CMP methodology. The broad goal of this project is to provide a targeted low‐cost approach towards synthesis of novel analgesics with potent action, high efficiency, and minimal aftereffects.

Support or Funding Information

American Chemical Society‐Petroleum Research Fund (MP) University of Nebraska‐Kearney Start‐up funds (SC)
Highly Predictive Objective Measurement of OA Joint Pain in Rat Using Bioseb Automated Dynamic Weight Bearing System
Authors: Rashid Harun , Cornicelli Joe , Young Alan , Samadfam Rana
Published: November 2023

Background

Osteoarthritis (OA) is often associated with chronic debilitating joint pain. Similar pain has been reported in animal models of OA. However, most pain measuring techniques in animal are subjective in nature where reflex responses upon stimulation with nociceptive stimuli are measured. The purpose of the present study is to provide a highly reproducible objective joint pain measuring technique in the mono‐iodoacetate (MIA)‐induced OA model in rats.

Methods

Joint pain is induced in rat by a single intra‐articular injection of MIA (3mg/25uL) into the right knee joint. Dynamic weight bearing (DWB) patterns by different limbs and tail in the rats are quantified using the BioSeb automated DWB system. The system consists of an arena box with a pressure‐sensitive sensor mat on the bottom and an attached high‐resolution camera on the top. The rat can move freely inside the arena box. A two‐minute recording is done for each rat. Analysis of dynamic weight bearing data is done off‐line using the BioSeb software. The system automatically calculates the weight borne by each limb and the tail. DWB data are calculated by normalizing as percent of total body weight borne by each limb and tail. Joint pain is indicated by reduction in weight bearing by the MIA‐injected right hind limb. Effects of tramadol, dexamethasone, morphine, naproxen and celecoxib on MIA‐induced joint pain were evaluated at different time points post MIA injection.

Results

Baseline DWB data were assessed across a number of studies and very consistent and reproducible baseline weight bearing pattern was observed. The deficit in weight bearing by the MIA‐injected right hind limb was mostly compensated by extra weight bearing in left hind limb although a smaller portion was also compensated by the right front limb. Weight bearing assessment over a period of four weeks showed that MIA‐induced OA joint pain was often more intense during the first few days post MIA injection, which might be due to the acute inflammation of the joint synovium, and pain is also intense in later time points during the third and fourth week, which might be due to the cartilage damage. Of the various standard of care OA pain relieving agents examined in this model, morphine, tramadol and dexamethasone were effective in both early and late phase of the model while naproxen and celecoxib were effective only during the early phase.

Conclusion

Overall, the current results suggest that Bioseb dynamic weight system is reliable for objective measurement of OA joint pain in rats. Moreover, results from the validation studies with standard of care OA pain relieving agents also support the biphasic nature of the MIA‐induced OA joint pain whereby the early phase is more sensitive to NSAIDs than the later phase(s).
A Comparative Study of Two Analgesic Compounds, Fenobam Sulfate and Fenobam Free Base, Using a Model of Inflammatory Nociception
Authors: Cox Abigail , Lax Neil , Welsh William , Peng Youyi , Kolber Benedict
Published: November 2023

A diverse array of chronic pain conditions exist, each with varying causative mechanisms and multiple hypothesized receptor targets for treatment. One potentially effective target for analgesia is the metabotropic glutamate receptor subtype 5 (mGluR5). A selective antagonist of mGluR5, fenobam free base, has been shown to provide pain relief in preclinical models. Due, in part, to fenobam free base's poor solubility and short half‐life, however, it carries limitations for efficacy in human conditions. A new salt formulation of the compound, fenobam sulfate, has improved solubility and a longer half‐life, which may contribute to extended analgesic effects. A comparative study of fenobam sulfate and fenobam free base was performed in male and female mice using the spontaneous formalin pain model. The main objective was to determine if fenobam sulfate shares similar analgesic properties with its free base counterpart. The two compounds were each dissolved in two separate vehicles, DMSO and BCD. Fenobam sulfate produced analgesic effects similar to those of free base when dissolved in either vehicle. Both compounds dissolved in DMSO, however, produced greater analgesic effects compared to BCD. The duration of time in which fenobam sulfate had an effect was also characterized through mechanical hypersensitivity tests two and three hours following formalin injection. Fenobam sulfate in BCD induced analgesia in female mice for at least two hours, but not in male mice. As fenobam free base has been shown to increase locomotion, the impact of fenobam sulfate on locomotion was investigated using an open field assay. Both compounds were found to induce similar increases in locomotive activity. Overall, these results suggest that fenobam sulfate may be a more effective analgesic compound than fenobam free base. In the future, it would be valuable to analyze sex differences that may contribute to the observed prolonged analgesia in female mice.

Support or Funding Information

Supported by the American Physiological Society Undergraduate Summer Research Fellowship, the Duquesne University Undergraduate Research Program, and the Duquesne University Pain Undergraduate Research Experience.
Conditional deletion of L‐type calcium channels in oligodendrocyte progenitor cells affects remyelination in mice
Authors: Gonzalez Diara A Santiago , Cheli Veronica T , Spreuer Vilma , Paez Pablo M
Published: November 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the CNS. Demyelination impairs axon conduction and can lead to deficits in motor, sensory and/or cognitive function depending on the location of the affected axons. Remyelination occurs in many MS lesions, but becomes increasingly less effective over time and eventually fails. Our findings indicate a role for L‐type voltage‐operated calcium channels (L‐VOCCs) as potential modulators of oligodendrocyte progenitor cell (OPC) development in the postnatal mouse brain. To determine whether L‐VOCCs are required for OPC development and remyelination, we have generated an inducible conditional knockout mouse in which the L‐VOCC isoform Cav1.2 was deleted in NG2 positive OPCs. Using the cuprizone model of demyelination we establish that Cav1.2 deletion in OPCs lead to less efficient remyelination of the adult brain. We have found that Cav1.2 deficient OPCs are less effective in remyelinating several structures of the adult brain including the corpus callosum and cortex. Specifically, Cav1.2 KO OPCs mature slower and produce less myelin than control oligodendrocytes after two and four weeks of recovery following the cuprizone intoxication. Furthermore, during the recovery period of the cuprizone model the corpus callosum of Cav1.2 KO animals presented an important decrease in the percentage of myelinated axons and a substantial increase in the mean g‐ratio of myelinated axons compare to controls. These results suggest that Ca++ influx mediated by L‐VOCCs in oligodendroglial progenitor cells is necessary for normal remyelination and is an essential calcium channel for OPC maturation during the remyelination process.

Support or Funding Information

NIH/NINDS grant 1R01NS078041‐03A1 and National Multiple Sclerosis Society Grant RG4554‐A‐2.
Antidepressant‐like Effects of Ketamine in Serotonin Transporter Knockout Mice and Organic Cation Transporter 3 Knockout Mice
Authors: Bowman Melodi Ann , Vitela Melissa , Owens W. Anthony , Koek Wouter , Daws Lynette
Published: November 2023

Twenty percent of adults are diagnosed with major depressive disorder, which is typically treated with selective serotonin reuptake inhibitors. However, this type of medication takes approximately six weeks to produce therapeutic effects. Recently, low doses of ketamine, a noncompetitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist, have been shown to produce rapid and long‐lasting antidepressant effects. Studies that have begun to examine the mechanisms underlying these effects have focused on intracellular changes mediated by NMDA and/or AMPA receptors. However, the traditional view of the etiology of depression involves the need for an increase in extracellular serotonin to regulate mood. Surprisingly, there appears to be little research into the effects of ketamine on serotonin. One study found an increase in extracellular serotonin while another found an inhibition of serotonin uptake following ketamine treatment. Together, these studies suggest a role for serotonin in the antidepressant‐like effects of ketamine, and putatively one involving the serotonin transporter (SERT). In order to investigate this, we performed in vivo chronoamperometry. A nafion coated carbon fiber electrode and four barrel micropipette was inserted into the CA3 region of the hippocampus and serotonin, ketamine, or vehicle was applied exogenously. Our results showed a decrease in clearance rate of serotonin after application of ketamine as well as an increase in signal amplitude. To further elucidate this effect, we examined antidepressant‐like effects of ketamine in SERT knockout mice bred on a C57BL/6J background. SERT knockout and wildtype adult mice were treated with vehicle or ketamine and tested in the tail suspension test. Ketamine had antidepressant‐like effects only in SERT knockout mice. Taken together with published findings that SERT knockout mice have increased expression of organic cation transporter 3 (OCT3) relative to wildtype littermates, and that blockade of OCT3 produces antidepressant‐like effects and inhibits serotonin uptake, we next examined the antidepressant‐like effects of ketamine in OCT3 knockout mice using the forced swim test. Our results indicate that the antidepressant‐like effect of ketamine is attenuated in the OCT3 knockout mice. Ongoing studies are investigating the dose‐response relationship of these effects in SERT and OCT3 knockout mice to investigate a potential role for OCT3 in antidepressant‐like effects of ketamine.

Support or Funding Information

This project was supported by NIH training grant T32 NS082145 funded by the Jointly Sponsored NIH Predoctoral Training Program in Neurosciences and by R01 MH093320 and R01 MH106978
Modulating the balance of synaptic and extrasynaptic NMDA receptors (NMDARs) shows priority over non‐selective NMDAR antagonists against beta‐amyloid induced neurotoxicity
Authors: Huang Yan , Shen Wei , Liu Gang , Zhou Wen‐xia , Zhang Yong‐xiang
Published: November 2023

Alzheimer's disease (AD) patients suffer a disturbance in the balance between synaptic NMDARs (NR2A, mediating the protective pathway) and extrasynaptic NMDARs (NR2B, mediating the excitotoxic pathway), and restoring the balance of NR2A and NR2B should be beneficial for AD. Aβ treated hippocampal slices and Aβ icv injection animals were used as AD models in this study. Long‐term potentiation (LTP) and behavior test were used to evaluate the effects of drugs and signal pathways were analyzed by Western Blotting. Specific activation of NR2A and inhibition of NR2B showed the best protective effect against Aβ‐induced long‐term potentiation (LTP) deficits. In Aβ icv injection animals, the combination of ifenprodil and D‐cycloserine (a co‐activator of NMDRs similar to D‐serine) showed better protective effects against Aβ‐induced cognitive deficits (nest building, novel object recognition and Morris water maze) than ifenprodil or D‐cycloserine alone. The ratio of NR2A to NR2B, TORC dephosphorylation and ERK1/2 activation (which could be initiated by NR2A) decreased in the hippocampal tissues of Aβ‐treated animals. As a result, the activation of CREB and the content of brain‐derived BDNF decreased. The combination of ifenprodil and D‐cycloserine showed the best protective effects, indicating that Aβ‐induced toxicology was mediated both by inhibiting NR2A and enhancing NR2B. These results indicate that enhancing synaptic NMDARs and inhibiting extrasynaptic NMDARs concurrently showed good effects against Aβ‐induced neurotoxicity, suggesting that modulation of the balance between NR2A and NR2B could be a potential strategy for AD drug development and therapy.

Support or Funding Information

Project supported by the National Natural Science Foundation of China (81202505)
Fumaric acid esters as a new therapeutic target for traumatic brain injury
Authors: Esposito Emanuela , Campolo Michela , Casili Giovanna , D'amico Ramona , Lanza Marika , Cuzzocrea Salvatore
Published: November 2023

Traumatic brain injury (TBI) provokes secondary injury mechanisms, including dynamic interplay between ischemic, inflammatory and cytotoxic processes. Moreover, impact induces massive cell death and outcomes in extensive dendrite degeneration leading to persistent cognitive, sensory and motor dysfunction. Fumaric acid esters (FAEs) were showed beneficial effects in preclinical models of neuro‐inflammation, neurodegeneration, and toxic oxidative stress but its precise mechanism of action remains unclear. Therefore, the aim of the present work was to evaluate the potential beneficial effects of dimethyl fumarate (DMF) and monomethyl fumarate (MMF), the most pharmacologically effective molecules among the FAEs, in a murine model of TBI.

TBI was induced in mice by the controlled cortical impact (CCI), one of the most common models of TBI; mice were orally administered with DMF and MMF (both at 30mg/Kg) 1h and 4h after trauma. We observed that mice treated with DMF exhibited a significantly and sustained recovery of motor function. FAEs significantly reduced the severity of inflammation by a modulation of proinflammatory cytokines and apoptosis factors, and increased neurotrophic factors such as BDNF, GDNF and NT‐3. Furthermore, DMF treatment up‐regulated Nrf‐2 pathway, increasing NeuN+/Nrf‐2+ cells in the cortex and inducing activation of manganese superoxide dismutase (Mn‐SOD) and heme‐oxygenase‐1 (HO‐1), regulating glutathione levels too. Our results showed important protective effects of DMF in an animal model of TBI by reducing the secondary inflammation and tissue injury that characterize this model. DMF may constitute a promising target for future TBI therapies.
Neonatal Erythropoietin Reverses Cognitive Deficits in a Preclinical Model of Encephalopathy of Prematurity
Authors: Jantzie Lauren , Robinson Shenandoah
Published: November 2023

Preterm infants are prone to numerous neurological deficits including cerebral palsy, epilepsy, sensory processing impairment and intellectual disability. Among the most impactful repercussions of preterm birth, cognitive deficits pose a significant barrier to achieving independent adult living. Touchscreen testing is a novel paradigm for assessing cognitive domains in rodents after brain injury. Previously, we have shown gait deficits and impairment in social interaction observed in adult rats following prenatal transient systemic hypoxia‐ischemia (TSHI) normalized to sham levels following high‐dose erythropoietin (EPO) treatment in the postnatal period. Here, we hypothesized that EPO would induce recovery of chronic cognitive deficits often seen in children born extremely preterm, including executive function, congruent with its beneficial mechanism of action on developing oligodendrocytes and neurons. Accordingly, pregnant rats underwent laparotomy on embryonic day 18, and TSHI was induced via uterine artery occlusion for 60 minutes. Shams had anesthesia and laparotomy for the same duration without arterial clamping. On postnatal day 1 (P1), TSHI pups were randomized to EPO (2000U/kg/dose) or vehicle treatment (sterile saline). Beginning on P35, a touchscreen platform was applied to assess multiple cognitive domains. Specifically, rats were trained on a touchscreen operant platform by blinded investigators, and following successful training, rats performed visual discrimination (VD) and reversal tasks. Data were compared using two‐way ANOVA with posthoc Bonferroni correction, or Kruskal‐Wallis, with p<0.05 as significant. Results indicate that TSHI‐veh, TSHI‐EPO and sham rats meet visual discrimination criteria, showing adequate visual‐cognitive capacity. TSHI‐veh rats showed impaired cognitive flexibility, an executive function also impaired in children born preterm, compared to shams and TSHI‐EPO rats. Specifically, when assessing reversal learning, only 30% of TSHI‐veh animals successfully passed criteria compared to 58% of TSHI animals treated with EPO. Notably, TSHI animals committed significantly more errors and required more correction trials (642±62) compared to Sham (504±41) and CCI‐EPO (441±51, p<0.05 for all), concomitant with increased perseveration (all p<0.05). This is the first time that highly translational touchscreen platforms utilized in both humans and rodents have been successfully used in perinatal brain injury to test repair cognitive and executive function. Further testing with this platform will allow dissection of deficits in distinct pillars of cognition following in utero injury, and the response to novel emerging therapeutic strategies for preterm infants. These data also validate EPO treatment for the mitigation of cognitive disorders following perinatal brain injury.

Support or Funding Information

University of New Mexico Johns Hopkins University NIH NINDS
PLC‐Dependent Hydrolysis of Phosphatidylinositol 4‐Phosphate is Responsible for the Majority of Total Inositol Phosphate Production and PKC/D Activation Downstream of Multiple GPCRs
Authors: de Rubio Rafael Gil , Malik Sundeep , Smrcka Alan V
Published: November 2023

Receptor activation of phospholipase C (PLC) is important for many physiological processes, including neurotransmission and cell proliferation. PLC enzymes are 6 families of proteins that hydrolyze the plasma membrane lipid, phosphatidylinositol 4,5‐bisphosphate (PIP2) into inositol trisphosphate and diacylglycerol (DAG). Our group identified a novel pathway in cardiac cells, where G protein coupled receptor (GPCR) stimulation leads to PLC‐dependent hydrolysis of the PIP2‐precursor, phosphatidylinositol 4‐phosphate (PI4P) on intracellular Golgi membranes. We proposed that this localized DAG production was essential for nuclear processes involved in cardiac hypertrophy.

Our hypothesis is that receptor‐stimulated PI4P hydrolysis expands beyond cardiac cells as a mechanism for DAG production without calcium signaling. We measured PI4P in various cell types using PI4P specific binding domains fused to GFP and found that PI4P was depleted both at the Golgi and plasma membrane (PM) in response to GPCR‐stimulation. Since PI4P is the precursor of PIP2, one could argue that depletion of PI4P happens to replenish PIP2 used by PLC and not as an actual substrate. To test this, we depleted PIP2 prior to GPCR stimulation. We used the PM‐targeted FRB/FKBP‐5‐phosphatase rapamycin‐induced dimerization system. After rapamycin addition, the PI‐5‐phosphatase is recruited to the PM where it removes the 5 phosphate from the head group of PIP2. As predicted, depletion of PIP2 did not inhibit GPCR‐stimulated PM or Golgi PI4P depletion, total IP production or PKD activation. In contrast, pharmacological inhibition of PI4‐kinases to deplete PI4P specifically at the PM, without affecting PIP2 levels, completely blocked GPCR‐dependent total IP production and PKD activation. These data strongly suggest that PI4P hydrolysis is the major reaction catalyzed by PLCs in terms of phosphoinositide mass and is a key source of DAG for regulation of protein kinases.

While we have identified PI4P hydrolysis in various cell types, it is not clear which PLC isoforms catalyze this reaction. PLC epsilon performs this function at the Golgi‐Nuclear Envelope interface, but all PLC isoforms hydrolyze PI4P in vitro and may participate in PI4P hydrolysis in different cell types. To determine the identity of PLC isoforms responsible for PI4P hydrolysis, we used CRISPR technology to knockout individual PLC isoforms and will assess their roles in total IP production and PKD activation. Ultimately, finding which PLC isoforms are involved may provide new and more specific therapeutic targets for diseases.

Support or Funding Information

Support from NIH, RO1 grants GM053536 and GM111735 (A.V.S.) and NRSA predoctoral grant F31 GM116557 (R.G.).
Structural Studies of the RhoGEF Trio as a Target in Uveal Melanoma
Authors: Bandekar Sumit , Barton Brenna L. , Tully Ena S. , Tesmer John J.G.
Published: November 2023

The C‐terminal Guanine Exchange Factor (GEF) domain of the RhoGEF Trio (TrioC) has recently been implicated in highly metastatic cancers including uveal melanoma and adult T‐cell leukemia. Upon aberrant activation, TrioC transduces growth and motility signals through the small GTPase RhoA. The regulation of TrioC is not understood on a molecular level and this presents a barrier to future inhibitor design efforts. Preliminary biochemical studies have shown that TrioC is regulated via an autoinhibitory constraint that is released upon the binding of the heterotrimeric G‐protein Gαq. I have solved the 2.7Å crystal structure of TrioC in its basal state, revealing high‐resolution detail of the autoinhibition mechanism. Analysis of this structure in comparison to the activated state of a closely related protein, p63RhoGEF reveals a dramatically different orientation of key regulatory regions. Mutational analysis of these regulatory regions are underway using FRET‐based activity assays and differential scanning fluorimetry. Analysis of current data supports that residues in the α6‐αN linker helix and β3‐β4 loop regions are indeed responsible for stabilizing the autoinhibited state of TrioC. A Trio inhibitory peptide (TRIPα) represents the only known inhibitor of TrioC. I have expressed the peptide as an MBP fusion and am attempting to determine a co‐crystal structure of TRIPα in complex with TrioC.

Support or Funding Information

5T32GM007767‐38, Rackham pre‐doctoral fellowship awarded to S. Bandekar UROP Summer fellowship awarded to B. Barton
Electrostatic Regulation of Phospholipase C β
Authors: Esquina Candi , Rugema Ngango Yvon , Garland‐Kuntz Elisabeth , Lyon Angeline
Published: November 2023

Phospholipase C (PLC) is an enzyme class that cleaves phosphatindylinositol lipids from cellular membranes to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). The PLCβ subfamily is among the best characterized of the PLC super family and is crucial for normal cardiovascular function. Under basal conditions, PLCβ has very low basal activity and is partitioned between the plasma membrane and the cytoplasm. However, it is still unclear how this low activity is maintained from a molecular standpoint. One known autoinhibitory element is the X–Y linker, a loop within the catalytic domain that forms a lid over the active site and participates in interfacial activation. Autoinhibition is thought to require a highly conserved acidic stretch within the linker, as deletions encompassing this region, or the entire linker, result in increased basal activity, decreased efficacy of G protein activation, and decreased thermal stability. These results, together with previous structural studies, suggest that the acidic stretch may regulate activity in part through interactions with conserved basic residues on adjacent surfaces of the catalytic domain. We are using site‐directed mutagenesis, functional assays, and structural studies to establish the contribution of electrostatic interactions to understand how these interactions play a role in the regulation of PLCβ.

Support or Funding Information

Purdue University start‐up funds
ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production
Authors: Collier Justin Brandon , Whitaker Ryan , Schnellmann Rick G
Published: November 2023

Background

Because substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.

Methods

Mouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.

Results

HU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.

Conclusion

We have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.

Support or Funding Information

GM084147 ‐ RGS

F31DK105782 ‐ JBC
Novel Regulation of IGF1R by Ubiquilin1
Authors: Kurlawala Zimple , Gosney Julie , Ceresa Brian P , Beverly Levi
Published: November 2023

Background

Insulin‐like Growth Factor‐1 Receptor (IGF1R) is a receptor tyrosine kinase ubiquitously expressed on cell surfaces of all tissues and is activated upon binding to its ligands (IGF1, IGF2, insulin). Dysregulation of the IGF/IGF1R axis has been implicated in disorders of metabolism, growth and development, progression of cancers like lung and breast and even development of resistance to chemotherapy. Currently, this axis is a major target of research for chemotherapy and other metabolic disorders. However, pharmacological antagonists of IGF1R have failed to show significant benefit in overall patient population. One of the causes is that IGF1R expression and activity does not always correlate with disease progression and response to therapy. This highlights the crucial role of cellular and extracellular factors that modulate activity of IGF1R directly or indirectly. We have identified Ubiquilin1 as a novel interacting partner and regulator of IGF1R. Ubiquilins (UBQLN) are newly discovered proteins and are studied largely in the context of neurodegenerative disorders. Our laboratory recently showed that UBQLN1 is lost in about 50% of non‐small cell lung cancers, indicating a link between UBQLN1 expression and tumorigenesis.

Hypothesis

UBQLN1 regulates expression and activity of IGF1R.

Methods

We characterized regulation of IGF1R by UBQLN1 using the following methods – co‐immunoprecipitation, sub‐cellular fractionation, immunofluorescence, radioligand saturation binding, qRT‐PCR and Western Blot analyses.

Results

1) UBQLN1 interacts with both phosphorylated and non‐phosphorylated IGF1R. 2) UBQLN1's primary association with IGF1R is through its chaperone‐like region (STI domains) and there exists a secondary association through its UBA (ubiquitin‐associated) domain, which leads to stabilization of IGF1R. 3) Following loss of UBQLN1 in lung adenocarcinoma cells, there is decreased expression of IGF1R at the mRNA and protein levels. 4) Fewer receptors are available for ligand binding on plasma membrane of UBQLN1 deficient cells. 5) The ratio of active: inactive (phosphorylated: non‐phosphorylated) IGF1R is significantly higher in cells that have loss of UBQLN1 expression.

Conclusions

We conclude that UBQLN1 is critical in normal regulation of IGF1R. Loss of UBQLN1 leads to persistent stimulation of IGF1R which may contribute to transforming events in UBQLN1 deficient cells. Understanding the role of UBQLN1 in regulating receptor tyrosine kinase activity can facilitate development of pharmacological drugs targeting novel pathways in cancers as well as metabolic diseases.

Support or Funding Information

R01CA193220
Flow cytometry‐based quantification of cyclic AMP in primary human neutrophils.
Authors: Chinn Amy , Michkov Alexander , Insel Paul A , Corriden Ross
Published: November 2023

Cyclic 3′, 5′adenosine monophosphate (cyclic AMP, cAMP), a ubiquitous second messenger molecule, plays a central role in signal transduction, a large number of cellular functions and drug responses. Although several methods are used to quantify cellular cAMP, such approaches often lack sensitivity and can be difficult to apply to primary cells. Furthermore, currently available assays often require cell lysis and/or large cell numbers to generate readily detectable signals. Here, we describe a fluorescence‐based flow cytometry approach that enables quantification of cAMP even at low cell numbers. In this method, cells are fixed and permeabilized prior to staining using a primary antibody against cAMP and a fluorescent secondary antibody. Use of this method can quantify cAMP levels in freshly isolated human neutrophils, key mediators of the innate immune response and the most abundant phagocytic leukocytes in circulation. The method reproducibly detects and quantifies basal cAMP levels, in addition to cAMP stimulated by forskolin or agonists/antagonist of several Gs/Gi‐linked G protein‐coupled receptors. This approach, in addition to providing a rapid and simple new strategy for the quantification of cAMP, has the potential to facilitate receptor occupancy‐response relationships in terms of second messenger levels and other readouts that can be detected by flow cytometry.

Support or Funding Information

Supported by NIH grants R56AI110505, T32HL098062 and T32GM007752.
Derisking Renal Tubular Toxicity Identified During Early Compound Screening
Authors: Mantena Srinivasa Raju , Buck Wayne , Peterson Richard , Liguori Michael
Published: November 2023

Renal toxicity is common cause of attrition during non‐clinical development. Renal toxicity was identified in multiple series during the early toxicology studies in rodent species. The toxicity was characterized by basophilia of tubular epithelial cells and karyomegaly upon exposure to the parent molecule. Interestingly the tubular injury was restricted primarily to the S1 and S2 segments of the proximal convoluted tubules. Electron microscopic examination of tubular epithelial cells indicated mitochondrial injury as likely mechanism for tubular injury, as evidenced by mitophagy and mitochondrial swelling. We hypothesized that the segment specificity was due to interaction of the compound with transporters (OAT1) that were specifically expressed in these tubules. To test this hypothesis rat were infused with one of the major metabolite which was determined to be the cause of tubular injury alone or in combination with an OAT1 inhibitor, Probenecid. Rats infused with the major metabolite of the drug had moderate to severe tubular injury. In rats infused with major metabolites for seven days along with probenecid partial protection was observed, indicating that transporter mediated uptake was at least partially responsible for the tubular injury. Transporter facilitated tubular epithelial accumulation likely contributed to the mitochondrial injury noted upon electron microscopic examination. A role for transporters should be considered especially when the injury is segment specific and if a transporter role is confirmed there is potential to derisk successfully as evidenced by successful application of this approach with cidofovir and adefovir.

Support or Funding Information

All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
In Vitro Nephrotoxicity Induced by Dibromoanilines in Isolated Renal Cortical Cells from Male Fischer 344 Rats
Authors: Rankin Gary O'Neal , Tyree Connor , Peters Bunni , Norwood Jalen , Anestis Dianne
Published: November 2023

Dibromoanilines (DBAs) are used as chemical intermediates in the manufacture of dyes, pesticides, drugs and other organic chemicals. Two DBAs, 3,4‐DBA and 3,5‐DBA, also possess direct fungicidal activity. We previously found that among the 3,5‐dihaloanilines, 3,5‐DBA was the most potent nephrotoxicant in vitro using renal cortical slices from male Fischer 344 rats as the animal model. However, the potential nephrotoxicity of the other five DBA isomers is unknown. The purpose of this study was to determine the comparative nephrotoxic potential of the six DBA isomers using freshly isolated renal cortical cells (IRCC) from male Fischer 344 rats. Briefly, IRCC were isolated using a collagenase perfusion technique, and IRCC (~4.1 million cells/ml; 3 ml) were exposed to a DBA isomer (final bath concentrations of 0.25, 0.5 or 1.0 mM) or vehicle (DMSO, 30 μl) for 30 or 60 min. Cytotoxicity was determined by lactate dehydrogenase (LDH) release assays, with LDH release reported as percent of control. At a 30 min exposure, 3,5‐DBA was the most potent nephrotoxicant among the six DBA isomers, inducing an increase in LDH release at 0.5 mM (21 ± 2% treated vs 9 ± 1% control). LDH release was also increased at 30 min by 2,6‐DBA 0.5 mM (14 ± 2% vs 8 ± 1% control). Other DBA isomers required a bath concentration of 1.0 mM or higher to induce cytotoxicity. The largest increase in LDH release at 60 min was also induced by 3,5‐DBA at 0.5 (35 ± 1%) and 1.0 mM (38 ± 1%) vs the control group (14 ± 1%). While 2,6‐DBA 0.25 mM significantly induced LDH release at 60 min, the elevation in LDH release was not different from the LDH release induced by 3,5‐DBA 0.25 mM. Thus, these results demonstrate that 3,5‐DBA is the most potent nephrotoxicant in vitro in IRCC among the six DBA isomers.

Support or Funding Information

This work was supported in part by NIH grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.
Effect of Aqueous Extract and Anthocyanins of Calyces of Hibiscus sabdariffa L. (Malvaceae) in Rats with Adenine – Induced Chronic Kidney Disease
Authors: Ali Badreldin H. , Cahliková Lucie , Opletal Lubomir , Karaca Turan , Al Suleimani Yousuf , Al Za'abi Mohammed H , Nemmar Abderrahim , Blunden Gerald
Published: November 2023

Hibiscus sabdariffa L. calyces (HS) can mitigate several multi‐organ injuries, one of which is chronic kidney disease (CKD). Here, we tested the effect of water extract of HS and a concentrated extract of its isolated anthocyanins on experimental adenine ‐ induced CKD. Rats were orally given, for 28 consecutive days, either adenine, alone or together with either HS aqueous extract or anthocyanins. For comparative purposes, two groups of rats were given lisinopril (10 mg/Kg). One day after the end of the treatment period, blood pressure was measured, followed by collection of urine, plasma and kidney tissues. Adenine ‐ induced CKD was characterized by significant body weight reduction, renomegaly and hypertension, as well as by elevations in plasma creatinine, urea, neutrophil gelatinase‐associated lipocalin activity, urinary albumin, and N‐acetyl‐β‐D‐glucosaminidase activity, and decrease in creatinine clearance. Adenine also significantly increased the concentrations of several inflammatory cytokines and decreased those of oxidative stress indices. Renal tissue sections of adenine–fed rats showed significant increase in renal damage and markers of inflammation and fibrosis. HS aqueous extracts and isolated anthocyanins lessened, mostly in a dose – dependent manner, the severity of CKD to a similar extent to that of lisinopril. HS and its anthocyanins could be considered as possible promising safe dietary agents that could be used to attenuate the progression of human CKD.

Support or Funding Information

Financially supported from a grant from SQU (IG/SQU/MED/01/2014)
The Potential Role of Acetaminophen and Alcohol in Early Stage Renal Disease: A Bayesian Perspective
Authors: Ndetan Harrison T , Einstein George P , Tulp Orien L
Published: November 2023

In earlier studies, both acetaminophen [AAM] and ethanol [EtOH] have been implicated in renal dysfunction when ingested in variable quantities over a prolonged duration. To determine the potential role of AAM and EtOH on Early Stage Renal Disease [ErSRD], the contribution of threshold dosages of AAM and of light to moderate intakes of EtOH were investigated, and the results analyzed by Bayesian Statistical Methods based on Baye's Theorem. The results of information obtained from the US National Health Examination Survey [2003–3004] were examined, and the data updated with data from a newly designed hospital based case control study conducted in the sub‐Saharan African Region. The results of this study found that renal disease, as determined by measurements of serum creatinine [CTNE], blood urea nitrogen [BUN], Glomerular Filtration Rate [GFR], albumin/creatinine ratios [A/CTNE ratio] values of 0.70 mg/dl, 10.0 mg/dl, 17.0 mg/g, and 135.0 ml/minute/1.73m2, respectively, may occur, marked by statistically significant odds ratios among those who concomitantly ingested therapeutic doses of AAM and ingested light to moderate amounts of EtOH when compared to those who did not ingest the above agents. While light to moderate ingestion of EtOH may not by itself play a significant role, the combination of the two agents appeared to have a synergistic effect on the early development of renal disease in his population. This this study provided both methodological and non‐methodological factors to be considered in the design and implementation of larger scale epidemiologic studies on the interaction of AAM and EtOH on the development of ErSRD.

Support or Funding Information

Supported by Institutional Resources of USAT College of Medicine and the Einstein Institute