bluuebunny/crossref_metadata_2025 huggingface dataset:
Whole Slide Quantitative Image Analysis Can Demonstrate Bleomycin Induced Dose‐Dependent Increases in Fibrotic Areas Within Histologically Stained Rat Lung Tissue
Authors: Young Alan , McElroy Mary , Naylor Stuart , Anderson Mark , Bigley Alison , McGregor Kieran , Sherry Lorcan
Published: November 2023

Rationale

The accurate classification and quantification of specific morphological and cellular components within the bleomycin (Bleo)‐induced rodent lung tissue model is challenging. This study aimed to employ histology in combination with whole slide digital image analysis to quantify the extent of lesion formation and content within lung tissue from Bleo treated rats.

Methods

Male SD rats, n=5 per group (Gr), were treated with either saline (Gr1) each day for 7 days or 1.5mg/Kg Bleo (Gr2) and 3.0mg/Kg Bleo (Gr3) on one dose occasion or 0.5mg/Kg Bleo (Gr4) and 1.0mg/Kg Bleo (Gr5) each day for 7 days. On day 22, lungs were fixed, paraffin embedded, sectioned and histologically stained using Picrosirius Red. Whole slide images of lung tissue were captured and analysis algorithms developed by OracleBio (using Definiens Tissue Studio® software) to define specific regions of interest (ROI) per section including Parenchyma and Lesion, excluding basal collagen. Collagen content and cell density can then be quantified within the Lesion and Parenchyma ROI, but have not been presented in this abstract. The % Fibrotic Lesion area was calculated relative to the combined Lesion and Parenchyma areas only.

Results

The mean % Lesion area in Gr1 was 1.1% (95% CI: 0.5 to 2.7). This value was attributed as artefact which was classified during the objective application of the analysis algorithm and was excluded from further analysis. There was a clear trend showing an increase in % Lesion area as the Bleo dose increased from 0.5 to 3.0mg/Kg. The mean % Lesion area in Gr2 was 7.0% (95% CI: 4.5 to 9.6), Gr3 was 9.1% (95% CI: 1.7 to 16.5), Gr4 was 10.1% (95% CI: 6.5 to 13.7) and Gr5 was 16.2% (95% CI: 9.4 to 23.1). A significant difference was observed between Gr2 and Gr5 (p = 0.0124).

Conclusion

Quantitative whole slide image analysis can be utilised to accurately classify therapeutically relevant regions of interest. This may provide a new tool to assess and better interpret changes in morphological and cellular tissue parameters within histologically stained lung tissue sections from pre‐clinical bleomycin fibrosis models.

Support or Funding Information

Charles River Laboratories and OracleBio
Inflammasome Inhibition in Influenza A Virus Infected Juvenile Mice Leads to Improved Survival and Outcomes
Authors: Ravindran Nandini , Koch Clarissa M , Ridge Karen M , Coates Bria M
Published: November 2023

Rationale

Influenza A virus (IAV) is a highly contagious respiratory virus that infects up to 40% of children each year. Young children who progress to lower respiratory tract IAV infection are at risk of severe, life‐threatening disease. We previously demonstrated that viral clearance does not predict severity of illness in juvenile mice infected with IAV. Instead, excess activation of the NLRP3 (NLR family pyrin domain containing 3) inflammasome by IAV and the resultant cytokine storm are associated with increased lung injury in juvenile mice. Therefore, we hypothesized that inhibition of the NLRP3 inflammasome would protect juvenile mice from lethal IAV infection.

Methods

Juvenile mice (4 week‐old) were intratracheally infected with IAV (WSN 12.5 PFU). Two, three, and four days following infection, treatment with either an NLRP3 inflammasome inhibitor (MCC950) or saline control was initiated and continued until resolution of infection or death. Mice were evaluated for viral clearance and the development of acute lung injury. In addition, production of pro‐inflammatory cytokines, antiviral interferons and activation of the NLRP3 inflammasome were measured in bronchoalveolar lavage fluid (BALF). The immune cell profile was also evaluated in response to MCC950 treatment.

Results

Inhibition of the NLRP3 inflammasome with MCC950 3 days following infection protected juvenile mice from IAV‐induced lung injury and significantly increased survival. Earlier or later inhibition was not protective. Compared with vehicle‐treated controls, MCC950‐treated mice achieved an equal rate of viral clearance, but secreted decreased amounts of pro‐inflammatory cytokines. Further studies will be conducted to assess the effects on the immune cell population.

Conclusion

Modulation of the juvenile inflammatory response to IAV does not impair viral clearance. Instead, inhibition of excessive inflammation in juvenile IAV infection with a specific NLRP3 inflammasome inhibitor attenuates lung injury and improves survival in IAV‐infected juvenile mice. Our data suggest that therapies directed at the inflammatory response to IAV, and not viral clearance, warrant further investigation for treatment of severe IAV infection in children.

Support or Funding Information

HL128194 and HL071643
The Effect of Plumbagin on Proinflammatory Cytokines Expression in LPS‐Activated BV‐2 Microglia Cells
Authors: Messeha Samia S , Zarmouh Najla O , Kolta Malak G , Soliman Karam F.
Published: November 2023

In the central nervous system, activated microglial cells produce proinflammatory mediators such as inducible nitric oxide (NO2) and cytokines. Uncontrolled release of these mediators can cause neuronal damage in the brain leading to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Therefore, inhibition of these proinflammatory mediators may provide a neuroprotective strategy in preventing neurodegenerative diseases. the other hand, plumbagin is a biologically active compound in the roots of the Indian medicinal plant Plumbago zeylanica L. which has been used in treating rheumatoid arthritis. The present study was conducted to examine the antiinflammatory effect of the plumbagin in LPS‐activated microglial BV‐2 cell model. The obtained data indicated that plumbagin (IC50 = 0.386 uM) was about 20‐fold more potent than l‐N6‐(1‐iminoethyl) lysine (LNL), the selective inhibitor of inducible nitric oxide synthase (iNOS) (IC50 =7.4 uM). Moreover, our immunofluorescence study and ELISA confirmed the positive relationship between NO2 decrease and iNOS expression inhibition. RayBio AAM‐CYT‐3 and 4 cytokine antibody protein arrays were selected for detecting extracellular proinflammatory cytokines expression changes in LPS–activated BV‐2 cells in the presence of plumbagin. In the activated BV‐2 cells, 2 ums of plumbagin significantly inhibited the release of IL‐6, MCP 1, MCP5, MIP‐1 alpha and gamma, IL‐1 alpha, G‐CSF, IL‐12 p40/70 and MDC. The obtained results of ELISA analysis validated the cytokines array data. Taken all together, we concluded that plumbagin is a potent antiinflammatory agent reduces multiple proinflammatory cytokines and iNOS.

Support or Funding Information

Supported by NIH Grants G12 MD007582 and P20 MD 006738
West‐Nile virus replicon particles infect 293T cells expressing Dendritic Cell‐Specific Intercellular Adhesion Molecule‐3‐Grabbing Non‐Integrin Related.
Authors: Leonard Nathan , Crowson Nicole , Madigan Amanda , Schmitz Madilyn , Alcala Elana , Martinez Osvaldo
Published: November 2023

West‐Nile virus (WNV) is an arthropod‐borne virus that is frequently transmitted to humans through the bite of an infected mosquito. Less than one percent of cases result in a serious neurological illness such as meningitis or encephalitis. To investigate WNV tropism we have established several 293T stable cell lines expressing dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin related (DC‐SIGNR) or DC‐SIGN. Previous studies have suggested that WNV infection efficiency is enhanced by expression of DC‐SIGNR, but not DC‐SIGN on target cells. We demonstrate high levels of DC‐SIGNR expression in selected 293T cells. Replication incompetent virus replicon particles (VRPs) are used to safely and conveniently study viral entry. Successful VRP entry into target cells transfers a mutated incomplete viral genome containing the GFP gene which is expressed from the infected cells. Infections assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta‐lactamase enzyme, show significant infection in DC‐SIGNR expressing, but not control cells that do not express DC‐SIGNR. The establishment of this entry assay system will allow us to safely study virus tropism.

Support or Funding Information

Student Research Grants, Winona State University Winona, MN
RGDSK Peptide Functionalized Helical Rosette Nanotubes (RGDSK‐HRNs) Block Integrin αvβ3 and Inhibit E. coli Adherence to Intestinal Porcine Epithelial 1 Cell Line (IPEC1) in vitro
Authors: Le Nguyen Phuong Khanh , Quach Chi Cuong , Aulakh Gurpreet , Gerdts Volker , Fenniri Hicham , Singh Baljit
Published: November 2023

E. coli infection is a leading cause of intestinal disease and death, and inflicts billions of dollars in economic losses. There is ongoing effort to find ways to reduce intestinal colonization by E. coli. Integrin αvβ3, recognizing arginine‐glycine‐aspartic acid (RGD) sequences, has important functions in cell adhesion, signaling and survival. However, the expression and role of integrin αvβ3 in the adhesion of E. coli on intestinal epithelium remain unknown.

We used porcine jejunum, IPEC1 cells and E. coli K88 to firstly explored the hitherto unknown expression of integrin αvβ3 with light, fluorescent and electron immunocytochemistry, immunoprecipitation and Western blots. We then used RGDSK peptide and RGDSK‐HRNs to test the role of integrin αvβ3 in adhesion of E. coli K88 to IPEC1 cells.

Normal porcine jejunum strongly expresses integrin αvβ3 on the nucleus and apical surface of epithelium and gland cells. The expression of integrin αvβ3 decreased in the epithelium but increased on vascular endothelium of the jejunum infected with E. coli or E. coli associated with Salmonella. Integrin αvβ3 was found on plasma membrane, cytoplasm, and nucleus of IPEC1 cells. The expression of integrin αvβ3 on IPEC1 decreased at 15 minutes but returned to normal after 90 minutes of infection with E. coli K88 (P<0.05). Immuno‐gold quantification showed changes in sub‐cellular expression of the integrin αvβ3 in IPEC1. The light, fluorescent and electron microscopy, and western blots showed novel expression of integrin αvβ3 on E. coli K88. Finally, we found that RGDSK peptides and RGDSK‐HRNs bind integrin αvβ3 on both IPEC1 and E. coli K88. RGDSK peptides block the attachment of E. coli to IPEC1 (5 minutes IPEC1 exposed to 50μM RGDSK, P<0.05), and reduce survival of E. coli (10 minutes IPEC1 exposed to 50μM RGDSK, P<0.05). Dose dependent RGDSK‐HRNs mediates the attachment of E. coli to IPEC1 more efficiently and regularly (10 minutes IPEC1 exposed to HRNs containing equivalent 5μM RGDSK, P<0.001). These are the first data to show a role for the integrin αvβ3 in the attachment of E. coli to the intestinal epithelium, and that novel RGDSK helical rosette nanotubes can inhibit the attachment of E. coli to the epithelium.

Support or Funding Information

Saskatchewan Agriculture Development Fund and NSERC
Development of an in vitro Bladder Epithelial Cell Culture System
Authors: Kim Seongmi
Published: November 2023

Urinary tract infections (UTIs) are very common and highly recurrent. Our laboratory has developed a murine model of the recurrent bladder infection: C3H/HeN mice with a history of UTI that was cleared with antibiotic therapy become “sensitized” to severe recurrent UTI upon subsequent UPEC challenge, while “resolved” mice are resistant to challenge infection relative to age‐matched “naïve” mice. Quantitative proteomics has found evidence of bladder epithelial (urothelial) cell remodeling in sensitized mice. To study these remodeling changes ex vivo, we have adopted a primary epithelial cell culture method for use with urothelial cells. Urothelial cells isolated from naive, resolved and sensitized mice were passaged in matrigel‐suspended “spheroid” culture with conditioned media from L‐cells expressing Wnt, R‐spondin and noggin to promote proliferation, then cultured on transwells, allowing them to differentiate into polarized urothelial layers with terminal differentiation markers. We observed sensitized urothelium had characteristic differences in its morphology and cytokine response to UPEC infection that were similar to differences observed in vivo. Serum biomarkers that predict chronic cystitis and predisposition to rUTI in human and mice, such as IL‐6, G‐CSF, and CXCL1 (KC), showed increased expression patterns in sensitized urothelia. Interestingly, baseline chemokine RANTES expression is suppressed in sensitized urothelia. These results indicate that mucosal remodeling is happening at the urothelial cell level, and thus there is potential to study these changes in great detail in vitro. This primary culturing system represents a significant advance in the study of bladder mucosal responses and has great potential for translation to human clinical studies.

Support or Funding Information

Mucosal Immunology Studies Team consortium U01 AI095542
Addition of Retinoic Acid‐Inducible Gene 1 to Enhance Ebola Virus‐Like Particle Vaccine
Authors: Oredola Emmanuel O , Pottebaum Ethan , Zell Maxwell , Martinez Osvaldo
Published: November 2023

Ebola virus (EBOV), a filovirus family member, is a highly pathogenic virus that causes Ebola hemorrhagic fever resulting in documented mortality rates in humans as high as 50%. Currently, the basic EBOV virus‐like particle (VLP) vaccine contains the Ebola virus (EBOV) matrix VP40 and attachment glycoprotein (GP). VLPs are morphologically and biochemically similar to parental virus, yet because they lack a genome and cannot replicate, are safe enough to be used as vaccines. We hypothesize that addition of a constitutionally active retinoic acid‐inducible gene 1 (RIG‐I) would enhance the ability of the vaccine to induce interferon‐dependent immune functions yielding an improved vaccine. Expression of EBOV VP40 in 293T cells induces the spontaneous production of VLPs into the media supernatant and if expressed with EBOV GP, will produce VLPs studded with the attachment GP. Recombinant chimeric constitutively active (ca)RIG‐I‐VP40 matrix and a nonfunctional mutant L58A (mu)RIG‐I‐VP40 matrix genes were constructed to produce VLPs containing constitutively active and nonfunctional RIG‐I. Supernatant from 293Ts transfected with caRIG‐I‐VP40, muRIG‐I‐VP40 or VP40 along with GP expression plasmids were tested for the presence of VLPs. Western blotting of purified VLPs confirmed the presence of RIG‐I in caRIG‐I‐VP40 and muRIG‐I‐VP40, but not VP40 containing VLPs. Monocyte‐like and PMA‐differentiated macrophage‐like THP‐1 Dual cells were treated with nothing, VP40+GP, caRIG‐I‐VP40+GP and muRIG‐I‐VP40+GP VLPs as well as LPS control and tested for induction of NF‐kappaB or interferon signaling. CaRIG‐I containing, but not muRIG‐I containing VLPs induced interferon signaling from macrophages, but not monocytes. Future studies will be required to determine caRIG‐I containing VLP vaccine efficacy
The Effect of Cardiolipin on Microglial Activation and Microglia‐Mediated Neuronal Death
Authors: Pointer Caitlin , Klegeris Andis
Published: November 2023

Microglia, the immune cells of the brain, become activated in response to a variety of stimuli. Under normal physiological conditions, microglial activation is advantageous, as it produces an acute inflammatory response that helps rid the brain of the noxious stimuli through the release of pro‐inflammatory mediators and cytotoxins, including reactive oxygen species (ROS) and reactive nitrogen species (RNS). When microglial activation is sustained, however, the persistent and non‐specific release of these cytotoxic molecules results in damage to surrounding cells, including neurons and other microglia. Such state of chronic neuroinflammation is a characteristic feature of Alzheimer's disease (AD). Cardiolipin (CL), a phospholipid located in the inner mitochondrial membrane, is significantly reduced in AD brains. The remaining CL is exposed to the high levels of ROS present in AD brains, which modifies the structure of CL and hinders its ability to regulate mitochondrial processes. This results in the further production of ROS and extensive neuron death. The intracellular role of CL is well defined; however, the effects of extracellular CL on microglial activation, and subsequently on neuronal viability, have not been described.

We utilized cell culture techniques to study the effects of extracellular CL on the activation of microglia, and their subsequent toxicity towards neurons. BV‐2 murine microglia, as well as human HL‐60 promyelocytic and THP‐1 monocytic cells (microglia models), were exposed to CL in the presence of noxious stimuli for 24 or 48 hours. Pro‐inflammatory mediators including tumor necrosis factor (TNF)‐alpha, monocyte chemoattractant protein (MCP)‐1, ROS and RNS in supernatant were quantified to determine the effect CL has on the secretory profile of activated microglia. Additionally, THP‐1 cell supernatants were transferred onto human SH‐SY5Y neuroblastoma cells (neuron model) to determine whether CL regulates microglia‐mediated neurotoxicity.

We discovered that CL decreased the secretion of TNF‐alpha, MCP‐1, ROS and RNS from activated microglial cells. Additionally, CL reduced the cytotoxicity of activated THP‐1 cells towards SH‐SY5Y neuronal cells.

These findings indicate that CL may act as a novel intercellular signaling molecule that is capable of reducing microglial activation, as well as neuron death. Further studies will explore glial signaling pathways engaged by CL and reveal its role in neuron‐microglia communication.
High Fat Diet Increases Cognitive Decline and Neuroinflammation in a Model of Orexin Loss
Authors: Duffy Cayla Marie , Nixon Joshua P , Butterick Tammy A
Published: November 2023

Midlife obesity is a risk factor for cognitive decline and is associated with the earlier onset of Alzheimer's disease (AD). Diets high in saturated fat can potentiate the onset of obesity, microglial activation, and neuroinflammation. Signaling deficiencies in the hypothalamic peptide orexin A (OXA) and/or orexin fiber loss are linked to neurodegeneration in humans and animal models. Prior studies in orexin/ataxin‐3 (O/A3) mice, a transgenic mouse model of orexin neurodegeneration, demonstrate that OXA treatment improves cognitive processes. In separate studies we have demonstrate that OXA can suppress neuroinflammation and is neuroprotective against the saturated fatty acid palmitate. Our overall hypothesis is that OXA treatment attenuates hippocampal neuroinflammation and prevents cognitive decline in obesity during high fat diet (HFD) feeding. Our short term goal for this project is to determine if OXA loss increases neuroinflammation and impairs cognition during HFD feeding. To examine this, we tested male O/A3 mice (7–8 mo. of age) in a two‐way active avoidance (TWAA) hippocampus‐dependent memory task. We tested whether 1) OXA loss impaired cognitive function; 2) HFD worsened cognitive impairment; and 3) HFD increased microglial activation and neuroinflammation. We show that O/A3 mice have significant impairments in TWAA task learning vs. wild type (WT) mice (increased escapes p<0.05, reduced avoidances p<0.0001). Mice were then placed on HFD (45% total fat, 31.4% saturated fat) or remained on normal chow (NC; 4% total fat and 1% saturated fat), and TWAA was retested at 2 and 4 weeks. Learning impairment was evident at both 2 and 4 weeks in O/A3 mice fed HFD for following diet exposure vs. WT mice on normal chow or HFD (increased escapes, reduced avoidances p<0.05). Additionally, O/A3 mice had increased gene expression of the microglial activation marker, Iba‐1 (measured via qRT‐PCR, p<0.001). Characterization of the microglial immune response (M1 neurotoxic vs. M2 protective phenotypes) is ongoing and will include gene and protein markers (such as TNF‐α, iNOS, UCP2, and Arg1). Collectively, our results indicate that OXA loss impairs memory, and that HFD accelerates hippocampus‐dependent learning deficits and the onset of neuroinflammation. Future studies will focus on restoration of OXA within hippocampal circuits to reduce neuroinflammation and improve cognitive function

Support or Funding Information

This work was funded by the US Department of Veterans Affairs BLR&D IK2 BX001686 and IS1 BX003083A (to TAB), the University of Minnesota Healthy Foods, Healthy Lives Institute (to JPN and TAB), MnDRIVE Brain Conditions Fellowship in Neuromodulation (to CMD), and the Minnesota Veterans Medical Research & Education Foundation (to TAB).
Inhibition of Stroke‐Induced Injury by Phlebotomy
Authors: Tsai Yung‐Hsu , Liou Houng‐Chi , Tang Sung‐Chun , Liou Horng‐Huei , Jeng Jiann‐Shing , Fu Wen‐Mei
Published: November 2023

Stroke is the second most common death and a leading cause of long‐term disability worldwide, yet the therapeutic options are still limited. Hemodilution has been reported to reduce brain infarction in experimental stroke model. Phlebotomy causes hypovolemic‐hemodilting effect, but whether it offers protection after ischemic stroke remains unclear. Using permanent occlusion model, we aimed to assess the effect of phlebotomy on ischemic stroke.

Male spontaneously hypertensive rats (SHR) were subjected to permanent MCA occlusion (pMCAO) to produce cerebral ischemia. Phlebotomy was conducted by drawing 0.8 ml of whole blood after the pMCAO. In another set of experiment, macrophage migration inhibitory factor (MIF, 10 or 20μg/kg, i.v.) or its antagonist, ISO‐1 (3 mg/kg, s.c.), were administered 3 h after pMCAO. Brain infarction, neurological function and protein expression were analyzed 24 h after pMCAO. Cerebrovascular integrity was analyzed 7 h after pMCAO. Analysis of serum by proteome profiler array and ELISA were performed at 3 h, 6 h and 24 h after pMCAO.

Our results show that phlebotomy at 3 h after MCA occlusion significantly reduced stroke‐induced infarction and brain edema and shows an improvement in behavioral deficit. Inflammatory responses were decreased in phlebotomy‐3h group as the expression of inflammation‐related mediators were reduced. Analysis of the cytokine profile in serum led us to presume Macrophage migration inhibitory factor (MIF) as a possible mediator of the protection by phlebotomy. MIF aggravated stroke injury with a larger infarction and a more severe cerebrovascular disruption was observed, whereas, its antagonist ISO‐1 inhibited stroke‐induced infarction. In conclusion, phlebotomy offers protection when conducted 3 h after stroke onset and may exert its protective effect through the suppression of inflammatory cascade by interfering the balance of inflammatory mediators in serum.

Support or Funding Information

This work was supported by the Ministry of Science and Technology grants, 104‐2321‐B‐002‐033, Taiwan.
1,2,3,4,6 Penta‐O‐Galloyl‐β‐D‐Glucose attenuates LPS/IFNγ‐induced MCP‐5 and Pro‐MMP‐9 Cytokines Release in BV‐2 Microglia Cells by Inhibiting NFkB and MAPK Signaling Proteins
Authors: Mendonca Patricia F , Taka Equar F , Bauer David F , Soliman Karam F
Published: November 2023

Neurodegenerative diseases are characterized by an inflammatory process and activation of microglia cells that produce pro‐inflammatory cytokines, which are hardly evident in the normal brain. Many natural compounds derived from plants have shown anti‐inflammatory activity, such as 1,2,3,4,6 Penta‐O‐Galloyl‐β‐D‐Glucose (PGG), which is a naturally occurring polyphenolic compound highly enriched in some medicinal herbs such as Rhus chinensis Mill and Paeonia suffruticosa. PGG has been shown to exhibit an antiinflammatory effect through the inhibition of many cytokines in different experimental models, but not in microglia cells. In the present study, we investigated the role of PGG as an antiinflammatory agent in LPS/IFNγ‐activated BV‐2 microglia cells. Mouse cytokine antibody arrays were used to assess the effect of PGG in the release of proinflammatory cytokines, and ELISA experiments were performed to validate the results from the arrays. PGG signaling pathway was evaluated with NFκB and MAP Kinase PCR assays for the target genes, and the effect of PGG on protein expression was evaluated by Western blot. The results obtained showed that PGG decreased the expression of monocyte chemotactic protein‐5 (MCP‐5) and pro‐matrix metalloproteinase 9 (Pro‐MMP‐9). Both cytokines are upregulated during the inflammatory process and have been shown to be involved in brain injury, inflammation, and neurodegeneration. PGG also exhibited an inhibitory effect on several signaling proteins involved in the NFκB (IRAK1, NFKB1, NOD1) and MAP Kinase signaling pathways (CDK2), which were validated by mRNA and protein expression. In summary, these findings show that PGG modulatory effects on the release of MCP‐5 and Pro‐MMP‐9 cytokines may be through the downregulation of proteins involved in the NFκB and MAP Kinase pathways.

Support or Funding Information

Supported by NIH Grants G12 MD007582 and P20 MD 006738
Naturally, Occurring Neurotrophic Factor from Mu Bie Zi (Momordica cochinchinensis) seeds
Authors: Mazzio Elizabeth A , Georges Beatrice , Soliman Karam F
Published: November 2023

The discovery of nerve growth factor (NGF) mimetics could have the potential application for Alzheimer's disease prevention and therapy. Previously, we have conducted a high‐throughput (HTP) screening of over 1100 nutraceutical products (herbs, vitamins, plant‐based phytochemicals, etc.) to determine if any dietary based substance could induce neurite outgrowth similar to 7S NGF at <200ug/mL in PC‐12 cells. Only a single biologically active with compelling NGF mimetic capacity in the seed of Mu Bie Zi, Momordica cochinchinensis (bitter cucumber) (MCS). The objective of the current study is to identify the bioactive substances within the ground seed pit by testing for NGF biological activity in the crude seed solvent extractions and purified proteins. The active protein fraction was subjected to one‐dimensional gel electrophoresis (native), gel staining, sectioning / excision, electro‐elution back into solution and re‐evaluation for NGF mimetic effects. Results obtained show that the NGF mimetic resided in a low abundant visually absent gel section at around 17 kD. The 17kD gel slice was excised, digested and run on a UPLC‐MS/MS –with a Q Exactive Hybrid Quadrupole ‐ Orbitrap Mass Spectrometer. The data were evaluated using X! Tandem, OMS, X Hunter and K‐score algorithms. Proteomic evaluation of the 17kD band was conducted showing the presence of 11S globulin subunit beta, napin, oleosin 18.2 kDa, trypsin inhibitor 1 MCoTI‐I /II (known to contain growth factor cystine knots) and Two Inhibitor Peptide Topologies TIPTOPs (isoforms 1,2,3,5,6), all with taxonomical identities inherent to the cucumber family (Momordica cochinchinensis, macrophylla, charantia, Cucumis sativus).

Support or Funding Information

This research was supported by the National Institute for Minority Health and Health Disparities of the National Institutes of Health through Grant Number G12 MD007582 and Grant Number P20 MD006738
A Team‐based Assignment to Integrate Basic Science and Pharmacotherapeutic Principles for Anticancer Agents
Authors: Jungsuwadee Paiboon , Sakharkar Prashant , Kurup Sonali
Published: November 2023

Objective

To implement and evaluate an active‐learning, team‐based assignment centered on anticancer agents for the integration of basic science and pharmacotherapeutic principles.

Design

Students enrolled in the integrated hematologic and oncologic disorders course were divided into 5–6 students/group. Each group was assigned a specific anticancer agent and was expected to answer a series of questions designed to measure students learning abilities and competencies, followed by a presentation to the class.

Assessment

Each assignment was assessed using a grading rubric that was mapped to the 2013 Center for the Advancement of Pharmacy Education (CAPE) outcomes to assess whether expected abilities and competencies are being met within a PharmD curriculum. Student learning was evaluated using performance on the assignment and examination questions. Student perceptions of the assignment were assessed using a short survey.

Result

Assessments for student learning abilities and competencies were performed based on the team‐based assignment and presentation as well as three examinations, in which the first exam was taken place prior to the team‐based assignment and in‐class activities. Overall mean score on examination 1 was 75%, examination 2 and examination 3 were 81% and 77%, respectively. When only questions related to drugs covered in the assignment were analyzed, we found that students performed better on examination 2 and 3 that were completed after the team‐based assignment vs. examination 1. Student performance on 14 of the 53 questions on examination 2 that were related to the drugs covered as part of the assignment was 93.7%. Students performed better on assignment related questions on examination 2 compared to unrelated questions. Student performance on 21 of the 61 questions on examination 3 that were related to the drugs covered as part of the assignment was 85.9%. Although, student performance was better on assignment related questions on examinations 2 and 3 compared to unrelated questions, this differences were not significant (p=0.253). Finally, Student perceptions of the effectiveness of the team‐based assignment were also positive and majority of the students (83%) agree or strongly agreed that the assignment improved their knowledge and understanding of anticancer agents and should.

Conclusion

The assignment provided a focused review of basic science and pharmacotherapeutic principles and enabled integration of concepts relating to the therapeutic application of anticancer agents and management of anticancer agent mediated adverse effects. The assignment could contribute toward preparing students for the evolving role of the pharmacist in the management of cancer.
Promoting the Active Learning of Pharmacology and Clinical Therapeutics Utilizing Team‐Based Learning (TBL) Methods in Second Year Systems Modules
Authors: Gorman Laurel
Published: November 2023

Curricular reforms in pharmacology teaching have stressed a movement toward more active learning methods that support both higher order critical thinking and collaborative learning to develop teamwork skills essential to resolving complex problems. Secondly, evidence suggests that teaching pharmacology using clinically relevant scenarios encourages retention, motivation, and may facilitate transfer from foundational knowledge to clinical application as medical learners progress from their second year (M2) into clerkship. However, limited curricular and faculty resources can make the transition to more active learning methods challenging. To stimulate higher order clinical reasoning about complex pharmacotherapeutics in our curriculum, pharmacology concepts were incorporated into large classroom team‐based active learning sessions (TBLs) addressing the diagnosis and treatment of diseases in cardiopulmonary and neurological systems second year modules. Six TBLs incorporating cardiopulmonary pharmacology were implemented at the beginning of the M2 year while 7 sessions incorporating neuropharmacology were implemented at the end of M2 year. High yield categories incorporated into TBL learning included: antihypertensives, antiarrhythmics, heart failure therapeutics, bronchodilators, antidepressants, antipsychotics, anxiolytics, anticonvulsants, opioid analgesics, and mood stabilizers. Preparatory materials included pharmacology interactive didactics, electronic self‐learning module narrated tutorials (SLMs) with interactive games and quizzes, and/or text book readings. Individual preparation was assessed by individual readiness assurance exams (IRAT) and the learners worked in TBL‐assigned diverse groups of 6–7 to answer questions covering the diagnosis and treatment of diseases. TBLs designed to integrate multi‐therapeutics covered topics such as chronic pain, medication‐induced delirium, and heart failure. Ambiguous questions addressing treatment choices encouraged more debate, small group engagement, and large classroom discussion between teams. Outcomes data support effectiveness of learning using this method, as mean performance on assessment items covered in TBL has been above the pharmacology mean of 86% and learners have evaluated the neuropharmacology TBLs positively for promoting clinical relevance and integration of psychiatry with pharmacology. Additionally, learners value TBLs on topics like chronic pain because multi‐therapeutic drugs are reviewed and disorders covered in previous systems, like diabetic neuropathy, are integrated to provide an end of year review preceding USMLE Step 1 exams. Further, TBL's can be implemented using few faculty and limited resources. In summary, incorporation of pharmacology into TBLs supports active learning, critical thinking, and clinical integration of foundational knowledge using large classroom collaborative learning processes.
Tools enhance learning of Medical Pharmacology
Authors: Zhong Qing
Published: November 2023

Objective

Medical pharmacology learning is challenging in a Caribbean Medical School. The medical pharmacology curriculum only has 12 weeks. We created extra‐lecture activities (tools) to help pharmacology learning.

Methods

We introduced practice of baroreflex and volume‐pressure loop by applying drugs to enhance cardiovascular pharmacology learning, and tables to help chemotherapy learning. We had pre‐lecture and post lecture quiz, clinical case study, and team‐based learning (TBL) throughout the whole pharmacology course. We did a questionnaire to investigate how helpful of these tools in 2015 and 2016 after students finished the course. Students were asked to rate the tools on a 1–5 scale. Scale 3 represented neutral response, and scale 4 and 5 were helpful and very helpful to their learning pharmacology, respectively. Frequency was expressed as a percentage.

Results

Total 91 responses were collected from 110 students. When combining scale 4 and 5 together which represented the helpful and very helpful, post‐lecture quiz was rated the most helpful (76.9%), followed by making table for antibiotics (72.5%). Practice of baroreflex and volume‐pressure loop were rated positive by 65% of students. TBL was rated fairly positive, with 59.3% at scale 4 and 5, and 54% student liked pre‐lecture quiz, and only 29.9% of students thought the case‐study beneficial.

Conclusions

Post lecture quiz, making tables for antibiotics, and practicing of baroreflex and volume‐pressure loop are regarded very helpful to enhance students' learning of Medical Pharmacology.

Support or Funding Information

None
Effects of clavulanic acid on ethanol intake and glial glutamate transporters expression in alcohol preferring rats
Authors: Hakami Alqassem Y. , Sari Youssef
Published: November 2023

Studies from our lab showed that upregulation of glutamate transporter 1 (GLT‐1) and cystine‐glutamate exchanger (xCT) expression with ceftriaxone, β‐lactam antibiotic, in central reward brain regions was associated with attenuation in ethanol consumption and reinstatement to cocaine‐seeking behavior. In this study, we tested clavulanic acid (CA), non‐antibiotic β‐lactamase inhibitor. This drug has the central β‐lactam pharmacophore, which is very critical for the upregulation of GLT‐1 and xCT expression in the brain. Therefore, we examined the effects of CA on alcohol consumption as well as GLT‐1, xCT and GLAST expression in adult male alcohol‐preferring (P) rats. We found that CA administered at a dose of 5 mg/kg (i.p.) significantly attenuated ethanol consumption in P rats as compared to the vehicle‐treated group. Importantly, we found that CA induced a significant upregulation of GLT‐1 and xCT expression in nucleus accumbens (NAc). There was no effect of CA on GLAST expression in the NAc. These findings revealed that CA at 20‐fold lower dose than ceftriaxone can attenuate ethanol consumption, in part through upregulation of GLT‐1 and xCT in mesolimbic system. Thus, we suggest that CA might be used as an alternative option to ceftriaxone for the treatment of drugs of abuse with hyperglutamatergic state.

Support or Funding Information

This work was supported by the National Institutes on Alcohol Abuse and Alcoholism (Award Number R01AA019458 to Y.S.).
Learning and memory deficits in a novel mouse model of Alzheimer's disease
Authors: Murnane Kevin , Daphney Cedrick
Published: November 2023

Alzheimer's disease (AD) is responsible for major financial and health‐care burdens to society, burdens that are likely to grow as our population ages. Decades of research have associated the development of AD with the production of toxic forms of the Aβ peptide, through proteolysis of the Amyloid Precursor Protein (APP), that contribute to the development of brain plaques. Traditional mouse models of AD use massive overexpression of APP. This approach has produced many therapeutics that are effective in animal models but has not resulted in notable clinical success, likely because of the nonspecific effects of APP overexpression. Moreover, this process of generating plaques is very distinct from the processes seen in human AD patients. Therefore, in the present study we evaluated the learning and memory in a state‐of‐the‐art triple knock‐in mouse model of AD. We assessed learning and memory using a variety of behavioral tasks and we have established the time‐course of the progression of learning and memory deficits in this novel model of AD. Moreover, we found clear differences between male and female mice from this genetic lineage, a finding of interest as AD disproportionately affects women. If future studies, we will use sophisticated and sensitive operant techniques to further characterize the deficits observed in this new mouse model of AD.
Effects of Self‐Administered Oxycodone on Learning and Cognitive Flexibility in Nonhuman Primates
Authors: Kangas Brian D , Porter Erica N. , Bergman Jack
Published: November 2023

Over the past decade, prescription opioid abuse has become a major public health concern. Despite increased prevalence, however, the effects of chronic opioid abuse on cognition‐related behavior remains poorly understood. Previous studies in our laboratory have shown that self‐administration of other abused drugs can have adverse effects on cognitive behavior. Therefore, the present studies employed similar techniques to examine the effects of daily intravenous self‐administered oxycodone. Touchscreen‐based repeated acquisition and discrimination reversal tasks were designed to assay basic features relevant to learning and cognitive flexibility, respectively. Four squirrel monkeys were initially trained to self‐administer oxycodone. A range of doses was evaluated in each subject over 1, 2, and 3‐hour sessions until stable intake was observed. A peak dose of 0.1 mg/kg/inj during 3‐hr sessions yielded maximum daily intake and was used for subsequent conditions. Following 30 self‐administration sessions, subjects were then introduced to the touchscreen tasks. Subjects were placed in a touchscreen chamber immediately following oxycodone self‐administration and learned to discriminate between two novel stimuli (acquisition) for a palatable food reward. Once discrimination acquisition was mastered, subjects then re‐learned the discrimination under conditions where the consequences were switched (reversal). Dose‐response functions revealed orderly increases in oxycodone intake as a function of dose. In addition, extending session durations increased daily intake at all doses examined. Stable levels of oxycodone intake using the peak dose of 0.1 mg/kg/inj were maintained over extended periods of self‐administration (>100 sessions). Results from touchscreen sessions indicated dramatic deleterious effects on learning and reversal in some subjects; however, in other subjects less profound effects were observed. Future studies will investigate the effects of abrupt discontinuation of opioid self‐administration (withdrawal) on discrimination learning and reversal.

Support or Funding Information

This research was supported by grant K01‐DA035974 (BDK) from the National Institute on Drug Abuse.
Comparative studies of AMPA receptor positive allosteric modulators PF‐04958242 and PF‐04531686
Authors: Stolz Jacob Robert , Ishii Toshiyuki , Swanson Geoffrey
Published: November 2023

Fast excitatory transmission in the central nervous system is mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors responding to the endogenous neurotransmitter L‐glutamate. Dysfunction of AMPA receptors has been associated with various neurological and neuropsychiatric disorders, but therapeutic targeting of these channels has proved difficult due to the preponderance of side effects associated with AMPA receptor agonists. Positive allosteric modulators (PAMs) of AMPA receptors are thus conceptually attractive therapeutics because they rely on endogenous glutamate signaling to potentiate excitatory signaling. The objective of this work is to study the effects of two PAMs, PF‐04958242 and PF‐04531686, on the biophysical properties of recombinant and native AMPA receptors, with the expectation that the comparative data will prove informative for pro‐cognitive preclinical or clinical studies with these small molecules.

We expressed a wide variety of AMPA receptor constructs with and without auxiliary subunits in HEK293T/17 cells, and characterized the effects of PAMs on currents elicited by fast‐application of glutamate. Both PAMs increased glutamate‐evoked current amplitudes and completely eliminated desensitization of all AMPA receptors containing the flip splice isoform, whereas desensitization of currents from flop‐containing receptors was slowed to differing degrees. At saturating PAM concentrations, PF‐04531686 slowed deactivation of GluA1 and GluA4 receptors to a greater degree than PF‐04958242.

Comparable effects were seen on deactivation of excitatory synaptic currents (EPSCs) recorded from hippocampal CA1 neurons in slice preparations from wildtype mice. Application (1 mM) of PF‐04531686 slowed both evoked and spontaneous EPSC decays by 3‐fold; in contrast, PF‐04958242 slowed EPSCs 1.5‐fold. At a lower concentration (300 nM), EPSCs were also slowed by PF‐04531686, but not PF‐04958242. There was no effect on paired‐pulse ratio at any concentration of either PAM. These results show that both PAMs slow the decay of EPSCs, resulting from their pharmacological actions on AMPA receptor decay rates. We predict from these data that both PAMs will impact cognition, but that PF‐04958242 might have less risk of unwanted mechanism‐mediated side‐effects due to its more modest modulatory action on synaptic AMPA receptor function.

Support or Funding Information

This project was supported by a contract from Pfizer Neuroscience.
The in vivo pharmacodynamic and biopharmaceutical characterization of a novel dual mechanism, rapid‐acting antidepressant
Authors: Talbot Jeffery N , Rawlins David B , Movahedi Fereshteh , Zhudeva Mariya Y , Vo Annh , Phan Julie , Dorigatti Jonathan , Velazquez Gabriel , Kirsh Robert D , Yellepeddi Venkatta K
Published: November 2023

Rational design of lead compounds targeting monoamine transporters is critical to developing novel therapeutics for treating psychiatric and substance abuse disorders. A 3‐D dopamine transporter (DAT) computer model was used to virtually screen a small molecule library for high DAT affinity drug‐like compounds. One hit, coded “MI‐4,” inhibited human dopamine, norepinephrine, and serotonin transporters in vitro. In vivo administration in mice (i.p.) induced robust, dose‐dependent antidepressant‐like effects in behavioral models of mood responsive to acute administration (tail suspension and forced swim tests) and chronic administration (novelty‐induced hypophagia test, chronic social‐defeat stress). Importantly, MI‐4 exhibited minimal abuse liability in behavioral and neurological models (conditioned place preference and dopamine in vivo microdialysis). To facilitate pharmacokinetic analysis, a rapid and highly selective reversed‐phase HPLC method for of MI‐4 was developed and validated according to ICH guidelines. Analysis in human, rat and mouse plasma resulted in a distinct single peak of MI‐4 with USP tailing factor < 2.0 using the MI‐4 analog ifenprodil as an internal standard. The stability of MI‐4 incubated in simulated fasted and fed‐state gastric fluid was 98–100% after 3 hrs and 91–99% after 6 hrs. The Papp value of MI‐4 (0.5 mM) across caco‐2 monolayers after one hour was 33.2×10−6 cm/s, indicating high membrane permeability. Moreover, oral administration (p.o.) of MI‐4 in mice resulted in dose‐ and time‐dependent reductions in tail suspension test immobility scores comparable to the established antidepressants fluoxetine and desipramine, indicating oral efficacy for antidepressant‐like effects. MI‐4 was found to be Ro‐25‐6981, an ifenprodil analog and reputed NMDA antagonist. These data suggest that Ro‐25‐6981, previously known for rapid‐acting antidepressant activity, may also functionally inhibit monoamine reuptake and produce sustained antidepressant effects in vivo. This demonstrates, as proof of principle, the viability of combining glutamatergic and monoaminergic mechanisms to produce rapid and sustained antidepressant‐like effects.
The Rho‐kinase inhibitor, fasudil, has antidepressant‐like efficacy in adolescent mice
Authors: Shapiro Lauren , Gourley Shannon
Published: November 2023

Adolescence represents a critical period of neurodevelopment, defined by structural reorganization and synaptic maturation within the prefrontal cortex. Although these processes are critical for the transition to adulthood, structural instability may open a window of vulnerability to neuropsychiatric disorders including depression. Interventions that facilitate activity‐dependent neural remodeling, as occurs during adolescence, may be advantageous. Here we evaluated the therapeutic‐like potential of Rho‐kinase (ROCK) inhibition, which can expedite activity‐dependent dendritic spine plasticity. The brain‐penetrant ROCK inhibitor fasudil had antidepressant‐like effects in the forced swim test in adolescent mice and was comparable to ketamine and fluoxetine. Fasudil also decreased the latency to approach a palatable food in the novelty suppressed feeding task, a rapid antidepressant‐like effect. Within the adolescent ventromedial prefrontal cortex (vmPFC), fasudil increased levels of the post‐synaptic marker PSD‐95, while pruning dendritic spines, resulting in adult‐like spine densities. Fasudil stimulated several neurotrophin‐related signaling factors in the vmPFC, including increasing the ratio of full‐length:truncated tyrosine kinase receptor B (TrkB). Subsequent experiments utilizing viral vector‐based and pharmacological manipulations indicated, however, that the antidepressant‐like actions of fasudil are nonetheless attributable to the inhibition of the neuronal ROCK isoform, ROCK2.

Support or Funding Information

This work was supported by F31MH109208, Children's Healthcare of Atlanta, the Molecular and Systems Pharmacology Training Grant 5T32GM008602‐17, the Katherine Deschner Family NARSAD Award and a BRAINS Award: MH101477. The Yerkes Center is supported by the Office of Research Infrastructure Programs/OD P51OD011132. The Emory Viral Vector Core is supported by P30NS055077.