bluuebunny/crossref_metadata_2025 huggingface dataset:
Homeostatic model assessment index 1 and 2 are associated with serum 25‐hydroxyvitamin D among Haitians and African‐Americans living in South Florida
Authors: Ajabshir Sahar , Exebio Joel , Zarini Gustavo , Voccaro Joan , Huffman Fatma
Published: November 2023

Purpose

The purpose of this study was to investigate the association between insulin resistance calculated by homeostatic model assessment index 1 (HOMA1‐IR), homeostatic model assessment index 2 (HOMA2‐IR) and serum 25‐hydroxyvitamin D [25(OH)D] among a group of Haitian‐Americans and African‐Americans living in South Florida.

Significance

Low serum vitamin D level is associated with a wide range of health concerns such as diabetes, osteomalacia, auto‐immune disorders and certain cancers. Several studies indicated higher insulin resistance is associated with increased risk of developing metabolic syndrome and type 2 diabetes. There are currently no studies investigating the relationship between vitamin D and insulin resistance among Haitian‐Americans and African‐Americans.

Participants

The sample for this study included 241 participants without type 2 diabetes, Haitian Americans (n= 124) and African‐Americans (n= 117), ages 35–82 years.

Design and methodology

In a cross‐sectional study participants were recruited from Miami‐Dade and Broward counties, Florida. Serum 25(OH)D, total cholesterol, triglycerides and C‐reactive protein were measured by ELISA method. HOMA1‐IR was calculated by the formula HOMA1‐IR=[fasting plasma insulin (μU/ml) × fasting plasma glucose (mmol/L)]/22.5 and HOMA2‐IR values were determined by an online calculator. Vitamin D and calorie intakes were calculated from a validated food frequency questionnaire. Multi‐linear regression models were run, adjusting for age, gender, ethnicity, serum total cholesterol, triglycerides, C‐reactive protein, systolic blood pressure, vitamin D and daily calorie intakes, smoking and alcohol consumption. HOMA1‐IR and HOMA2‐IR were the dependent variables.

Data analysis and results

The mean age was 52.37±9.96 years. The mean serum 25(OH)D, HOMA1‐IR and HOMA2‐IR were 22.91±8.55 ng/ml, 2.88±2.09 and 1.51±1.02, respectively. Serum 25(OH)D was negatively correlated with HOMA1‐IR (r=−.147, P=.023) and HOMA2‐IR (r=−.143, P=.027). Multi‐linear regression analyses controlling for the covariates indicated a significant negative association between serum 25(OH)D and HOMA1‐IR (B=−.038, P=0.012) as well as HOMA2‐IR (B=−.019, P=0.009). These models explained 20.2% of the variation in HOMA1‐IR and 18% of the variation in HOMA2‐IR.

Conclusion

Lower serum 25(OH)D is associated with higher insulin resistance calculated by both HOMA1‐IR and HOMA2‐IR methods. Presence of insulin resistance may be predictive of vitamin D insufficiency.

Support or Funding Information

Funding source: Funding for this research was provided through a grant from NIH/NIDDK.
3D Printed Anatomical Teaching Material ‐ A Genuine Alternative?
Authors: McMenamin Paul G
Published: November 2023

Whilst many medical schools, universities and teaching institutions charged with the task of teaching anatomy to medical students and allied health professionals have access to human cadaver material some cannot access this resource for a number of reasons. Use of human cadavers in teaching involves special ethical and legal considerations as well as resources or facilities that have an inherent, often hidden cost. In many countries cultural and religious factors also act as an impediment to cadaver‐based anatomy teaching. At Monash University, faced with ever expanding student loads, we were faced with a need for large numbers of anatomy prosections however problems of cost and the lack of our own donor bequest program meant that alternatives had to be considered. To address this issue we developed several methods of capturing surface topography and internal structural features from existing cadaver prosections and converting them into 3D printable files with high fidelity colour and resolution. The ‘Monash 3D Printed Human Anatomy Series’ now consists of over fifty 3D prints that display normal human anatomy ranging from orbital dissections to full dissected torsos. The 3D prints can be scaled up or down in size to suit specific needs. The 3D prints have been successfully integrated into our teaching programs and a double blind randomized controlled trial has shown their effectiveness against cadaveric materials for learning cardiac anatomy. One of the principle motivations for the creation of the 3D printed series was to give institutions that do not have cadaver access an alternative teaching resource beyond the currently available stylized plastic anatomical models and the expensive plastinated cadaver material that had none of the ethical issues associated with the latter. An example of the use of the 3D printed series in a medical school in The Republic of Liberia, West Africa, will be shown to illustrate how laboratory style anatomy teaching can be achieved in a country where exposure to cadavers has very specific health risks for staff and students. Recent research on the use of multicolour/multimaterial 3D printing of anatomical preparations will be illustrated as will preliminary data on the use of 3D printing to reproduce rare human pathological specimens.

Support or Funding Information

Monash University and Eric Glasgow Memorial Fund
Stress and Inflammatory Markers: The Impact on Military Physical Performance
Authors: Tingelstad Hans Christian , Martin Julie , Tang Vera , Fillion Lionel G. , Spivock Michael , Haman Francois
Published: November 2023

Military personnel are an occupational group susceptible to high levels of stress. Chronic stress can lead to elevated levels of cortisol and pro‐inflammatory makers, and recently published studies have suggested a potential negative relationship between levels of pro‐inflammatory markers and physical performance. The purpose of this study was to examine if a relationship exists between inflammatory makers and physical performance in a military cohort. Participants were male (n=133, age 36±11 years, height 175.8±6.2cm, weight 85.7±14.6kg, BMI 28±4 kg/m2) and female (n=87, age 37±9 years, height 162.6±5.8cm, weight 69.0±11.8kg, BMI 26±4 kg/m2) Canadian Armed Forces military personnel. A blood sample was collected in the morning between 8–10 am, and analyzed for levels of cortisol, CRP and IL‐18. Performance was evaluated on six military specific tasks; sandbag fortification, escape to cover, picking and digging, pickets and wire carry, stretcher carry and vehicle extrication, and total performance was calculated based on percentile scores of performance on each of the six tasks. A partial correlation was used to correct for BMI and age. In the male group, a negative correlation between CRP levels and total performance was observed (r=−0.25, p<0.01). A weak, but significant negative correlations between CRP levels and performance on single tasks; picking and digging (r= −0.19, p=0.04), stretcher carry (r=−0.23, p=0.01) and sandbag fortification (r=−0.20, p=0.03) was also observed. In the female group, a negative correlation was observed between CRP levels and total performance (r=−0.31, p<0.01). A negative correlation between CRP levels and performance on the picking and digging task (r=−0.23, p=0.05), stretcher carry (r=−0.23, p=0.05) and vehicle extrication (r=−0.24, p=0.04) was also found. To determine the impact of levels of stress and inflammatory markers on performance outcome, a linear regression analysis was performed, including factors of morphology and physical fitness previously recognized to have an effect on performance on these specific tasks. The linear regression analysis showed that none of the stress or inflammatory markers were included in the final model predicting total performance outcome in either the male or female group. Although a correlative relationship between levels of stress and pro‐inflammatory markers and performance on military specific tasks could be observed, we did not find evidence of levels of stress and pro‐inflammatory markers having a negative effect on total performance outcome on six military specific physical performance tasks.
Effect of the Exposure to Progesterone Analogues in the Proliferation and Production of TNF‐alpha and IL‐10 by Circulating Leukocytes
Authors: Contreras Irazu , Ochoa‐Arriaga Fany Guadalupe , Erazo‐Muñoz Maria Carolina , Alvarado‐Guadarrama Michelle , Estrada Jose Antonio
Published: November 2023

Exposure to progesterone analogues is related to physiological alterations, including changes in body weight, behavior and estrous cycles. Use of these drugs for contraceptive purposes is statistically associated with the appearance of gynecologic pathologies, such as breast, uterine, ovarian and endometrial cancer. The main objective of this study was to analyze the effect of the exposure to progesterone analogues contained in oral contraceptives on the proliferation and cytokine production in circulating mononuclear leukocytes.

We sampled 20 clinically healthy women who had not been previously exposed to oral contraceptives. Routine clinical data were collected and a 10 mL blood sample by venipuncture was obtained. Mononuclear leukocytes were isolated by Hypaque‐Ficoll density gradient. 1 X 106 cells were plated in 12 well plates in AIM‐V‐medium and exposed to the oral contraceptive at an equivalent dose to that found in serum (5 mg/mL), as well as a lower and higher dose (2.5 and 10 mg/mL). Cells were cultured for 3 and 24 hrs. Cell supernatants were used to measure TNF‐α and IL‐10 concentrations by ELISA and the remaining cells were harvested and used to estimate cell proliferation by propidium iodide incorporation by flow cytometry.

We observed that administration of progesterone analogs from oral contraceptives increases the concentration of IL‐10 and decreases TNF‐α in cell supernatants after 24 hrs of exposure at a dose of 10 mg/mL, although the only statistical difference was observed with TNF‐α. Regarding cellular proliferation, we found that the same concentration reduced leukocyte proliferation, since we observed decreased incorporation of propidium iodide. Our preliminary data suggests that high concentrations of progesterone analogs may impair the pro‐inflammatory capacity and proliferation of mononuclear cells in circulation, and may increase the susceptibility to infection and cancer.

Support or Funding Information

Consejo Mexiquense de Ciencia y Tecnología
CD36‐Myeloperoxidase Connection and the Activation of Intestinal Fibroblasts
Authors: Anderson‐Thomas Meghan , Nillas Andrea , Reaves Titus A.
Published: November 2023

Intestinal fibroblasts are mesenchymal cells that express CD36, which is a scavenger and membrane‐bound receptor with several functions including an indicator of inflammation, interacts with TLR‐2, 4, 6 and is a receptor for collagen. Recent evidence emphasizes the important role of CD36 in intestinal detection of fatty acids under physiologic conditions. Myeloperoxidase (MPO) is a heme protease predominantly released by leukocytes and functions to destroy pathogens, marks tissue for removal, and can bind high density lipoprotein (HDL). Given that HDL removes very low density lipoprotein (VLDL‐ “bad” cholesterol), having high levels of HDL is beneficial. Thus, we investigate the role of MPO (expressed by fibroblasts) and CD36 in intestinal fibroblasts. Both CD36‐deficient fibroblasts and CD36‐deficient mice appear to display less inflammation. Using immunopurification, MPO and CD36 appear to be connected on the surface of fibroblasts. There is a hereditary MPO deficiency that results in an increased susceptibility to infections (candida albicans). Thus, MPO‐deficient fibroblasts were examined for differences in expression and function of CD36; activity of CD36 is also connected to the activity of MPO. These results highlight the importance and impact of the connection of nutrition and immune function.

Support or Funding Information

National Science Foundation RII
Maturation of intestinal barrier function by Glial cell line‐derived neurotrophic factor (GDNF) is mediated by inactivation of p38 MAPK
Authors: Meir Michael , Flemming Sven , Burkard Natalie , Germer Christoph‐Thomas , Schlegel Nicolas
Published: November 2023

Recent data demonstrate that the glial cell line‐derived neurotrophic factor (GDNF) is involved in intestinal epithelial barrier differentiation. In the present study we analysed the mechanism underlying GDNF‐induced intestinal epithelial barrier maturation.

As demonstrated previously application of recombinant GDNF on immature intestinal epithelial monolayers (Caco2 and HT29B6 cells) for 24h resulted in increased transepithelial electric resistance (TER) and a reduced permeability of 4 kDa FITC‐Dextran in a transwell filter system. This confirmed that GDNF contributes to intestinal epithelial barrier maturation. Barrier differentiation by GDNF was associated with a significant reduction of phosphorylated p38MAP kinase. Inactivation of p38 MAP kinase by incubation of immature Caco2 and HT29B6 monolayers with SB‐202190 mimicked GDNF‐induced barrier maturation. Vice versa, activation of p38 MAPK by Anisomycin led to augmented epithelial permeability. Furthermore, p38 MAPK activation by Anisomycin blocked GDNF‐induced barrier maturation. As revealed by immunostaining GDNF‐induced barrier maturation was paralleled by augmented staining patterns of tight junction proteins claudin‐1, claudin‐5, occludin and ZO‐1. In preliminary experiments the desmosomal protein Desmoglein2, which is regulated by p38 MAPK was upregulated in Western Blot analysis. Accordingly, in immunostaining Dsg2 was distributed to the cell borders following GDNF application indicating a maturation of desmosomal adhesion.

In summary our present data show that GDNF contributes to epithelial barrier maturation by inactivation of p38 MAPK signaling which appears to affect Dsg2 integrity and thereby may stabilize tight junction stability.
The Roles of Matrix Metalloproteinases in Anterior Regeneration in Aeolosoma viride
Authors: Tseng Tzu Lun
Published: November 2023

Matrix metalloproteinases (MMPs) play key roles in turnover of extracellular matrix (ECM) and serve as important regulators of cell‐ECM interaction during development and regeneration. In previous studies, MMP‐2, MMP‐9 and MMP‐14 are found involved in regenerative processes in vertebrates. However, in invertebrates, no MMP‐2 or MMP‐9 has been found. So, the function of MMPs in the regenerative ability of invertebrates is an interesting question. In this research, a fresh water annelid, Aeolosoma viride, with high regenerative ability, was used to solve out this question. In the NGS transcriptome data of A. viride, only four matrix metalloproteinases, named as AviMMP1, AviMMP2, AviMTMMP1 and AviMTMMP2, were found. GM6001, MMPs inhibitor, had been treated during regeneration in A. viride and found that inhibiting MMPs activities at the early stage of anterior regeneration could delay the whole regenerative process, which means MMPs play some roles at the early stage of regeneration. The sequences of MMPs had been cloned and, in phylogenetic tree, they grouped with MMPs of other species. The qPCR data of blastema at different regenerative time point showed that Avimmp2 and Avimtmmp2 expressed highly at the early stage of anterior regeneration, but Avimmp1 and Avimtmmp1 showed no differences in expression during anterior regeneration. In situ hybridization analysis also indicated that high expression of Avimmp2 in blastema at 12, 24 and 48 hours post amputation (hpa). These data suggested that the AviMMP2 might be involved in the early stage of regeneration. On the other hand, SDS‐PAGE gelatin zymography showed the highest protease activity at 24 hpa blastema. These data suggested that MMPs have some effects on early stage of anterior regeneration, and AviMTMMP2 and AviMMP2 might be the key factors in the processes. The relationship between MMPs and the regulation of MMPs need further research to resolve.
Aberrant Modulation of the BRCA1 and G1/S Cell Cycle Pathways in Alcoholic Hepatitis Patients with Mallory Denk Bodies Present Revealed by RNA Sequencing
Authors: LIU HUI
Published: November 2023

Mallory‐Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Liver injury from alcohol administration causes balloon hepatocytes and MDB formation impeding liver regeneration. By comparing AH livers where MDBs had formed with normal liver transcriptomes obtained by RNA sequencing (RNA‐Seq), there was significant up regulation of BRCA1‐mediated signaling and G1/S cell cycle checkpoint pathways. The transcriptional architecture of differentially expressed genes from AH patients reflected step‐wise transcriptional changes progressing to AH. Key molecules such as BRCA1, p15 and p21 involved in BRCA1 signaling and G1/S cell cycle checkpoints were significantly up regulated both in patients’ livers and in the livers of the DDC re‐fed mice model where MDBs had formed. MiR‐483‐3p was down regulated in AH livers. The down regulation of miR‐483‐3p may be the mechanism for the up regulation of BRCA1 since miR‐483‐3p is an inhibitor of BRCA1 expression. The hyperactivation of G1/S cell cycle checkpoint inhibitors p15 and p21 results in cell cycle arrest and liver regeneration inhibition.

Conclusions

Provided here for the first time is the RNA‐Seq data that represents the fully annotated catalogue of the expression of mRNAs. The most prominent alterations observed were the changes in BRCA1‐mediated signaling and G1/S cell cycle checkpoint pathways. These new findings expand previous and related knowledge in the search for gene changes that might be critical in the understanding of the underlying progression to the development of AH.

Support or Funding Information

NIH (AAU01021898‐04).
DNA Damage Response Plays Critical Role In Liver Regeneration After Acetaminophen Overdose
Authors: Borude Prachi C. , Bhushan Bharat , Apte Udayan
Published: November 2023

Acetaminophen (APAP) overdose is a leading cause of Acute Liver Failure (ALF). Previous studies have shown that liver regeneration followed by APAP overdose plays a critical role in survival. Here we show that significant DNA double strand break leading to inhibited DNA repair results in prolonged activation of cell cycle checkpoint delaying liver regeneration. We used a novel incremental dose model developed in our laboratory using a regenerating (300 mg/kg, APAP300) and a non‐regenerating (600 mg/kg, APAP600) dose of APAP to identify pathways involved in regeneration after APAP overdose. Marker of liver injury, regeneration and microarray‐based global gene expression changes were studied in male C57BL6 mice over time course of 0 to 72 hr following treatment with either APAP300 or APAP600. The ingenuity pathway analysis of microarray data revealed significant differences in DNA damage, replication and checkpoint related pathways between both doses of APAP. Western blot and immunofluorescence staining of pH2AX Ser139, a hallmark of DNA double strand break, showed prolonged phosphorylation of histone H2AX in mice treated with APAP600 than APAP300. DNA repair proteins involved in Non Homologous End Joining (NHEJ) pathway showed a rapid induction in APAP300 but were significantly suppressed in APAP600, indicating delay or lack of DNA repair. Further, extensive stabilization and activation of p53, the DNA damage Response (DDR) effector protein, was observed in APAP600 as compared to APAP300. p53 stabilization was accompanied by significantly higher and prolonged activation of several p53 target genes such as GADD45α,PAI1, BAX, p21 in APAP600 treated mice as compared to APAP300 treated mice. Furthermore, studies in p53KO mice showed significantly higher injury than WT mice after APAP300 treatment at initial time points. Interestingly, despite the higher injury, p53KO mice recovered similarly as the WT mice due to faster cell cycle resulting in much higher liver regeneration. These data illustrate that lack of prompt DSB repair response after APAP overdose leads to p53‐mediated prolonged growth arrest and replicative senescence. These results suggest that improving DNA repair process to rapidly decrease cell cycle checkpoint alert resulting in faster initiation of liver regeneration following APAP overdose may have therapeutic benefit.

Support or Funding Information

These studies were supported by NIH‐COBRE (P20 RR021940‐03), NIEHS Toxicology Training Grant (T32ES007079‐34) and NIDDK R0198414
Sequence Variation in Hepatitis B Virus Variants of Chronically Infected Saudi Patients and Their Interaction with Host Genetic Factors
Authors: Almajhdi Fahad N , Al‐Qudari Ahmed , Amer Haitham M , Abdo Ayman A
Published: November 2023

Hepatitis B virus (HBV) continues to be o‐‐‐‐‐‐‐‐‐ne of the most important viral pathogens in humans. Approximately 350–400 million individuals worldwide are estimated to be chronically infected with HBV. In Saudi Arabia, the prevalence and dynamics of HBV infection is far from being unambiguously clear. The current study was designed to explore: i) the epidemiology of HBV in Riyadh and the different risk factors’ impact, ii) the genetic diversity among circulating Saudi variants, iii) the interaction between mutant proteins of Saudi HBV variants and host genetic factors. A total of 123 blood samples were collected from HBV infected patients (114 at chronic stage and 9 at cirrhotic stage) at Riyadh during 2012–2013. Clinical and biochemical parameters, serological markers and HBV viral load were evaluated in all patients. HBV Pre‐core/Core (P/C), HBsAg (S) and X gene mutations were determined in different disease categories by sequence and phylogenetic analysis. Two representative mutant open reading frames were cloned for both P/C and X genes. Cloned genes were transfected in Huh7 cells and the expression of different host factors including uPA, TIMP‐2, MMP‐2, TGF‐β1and CXCR4 was in‐vitro evaluated as compared to wild‐type genes on both transcriptional level (by real‐time RT‐PCR) and translational level (by ELISA). All clinical samples were tested positive for HBV infection using HBsAg ELISA and real‐time PCR. Of these samples, 28.5% were positive for HBeAg and negative for anti‐HBeAg antibodies. The rate of HBV infection and viral load was higher in males (68.3% and 8.5×106 IU/mL, respectively) than in females (31.7% and 1.9×104 IU/mL, respectively). Sequence analysis of Saudi HBV variants has identified a collective number of 33, 21 and 5 unique mutations in the P/C, S and X genes, respectively. Phylogenetic analysis revealed that all Saudi variants are members of genotype D, with the majority located in subgenotypes D1 and D3. Studying the effect of mutant genes on the expression of host factors demonstrated that both mutant and wild‐type forms have the same effect. HBx gene upregulated the expression of all host genetic factors, whereas P/C gene only up‐regulated MMP‐2 and slightly TGF‐β1. HBV is circulating in Saudi Arabia in males more than females with no correlation between the viral load and disease severity. This study is the first to classify Saudi HBV variants as members of genotype D. The positive regulatory effect of mutant and wild‐type genes on host genetic factors necessitates a series of comprehensive studies to characterize the nature of this protein‐protein interaction.
Group A Streptococcus Prevents the Acidification of Phagolysosomes in Macrophages
Authors: Franscioni Henry , Snipper Joshua , No Hyewon , Okumura Cheryl
Published: November 2023

Group A Streptococcus (GAS) is a gram‐positive bacterium that can cause a variety of ailments ranging from pharyngitis to necrosis fasciitis. Phagocytes are important first responders to GAS infection, and typically clear the pathogen by phagocytosis and subsequent degradation by fusing the phagosome with the lysosome. Critical to this process is the acidification of the lysosome by the multi‐subunit vacuolar ATPase (V‐ATPase), which pumps hydrogen ions into the lysosomal compartment that activates the degradative enzymes of the lysosome. Studies in our lab have shown that GAS is able to prevent the acidification of the phagolysosome, possibly by using the pore‐forming toxin streptolysin O (SLO), which would allow leakage of protons. SLO binds to cholesterol, a component found in lipid raft structures that facilitate protein assembly, including the V‐ATPase complex. Here, we examined whether SLO prevents phagolysosome acidification by inhibiting the recruitment and assembly of V‐ATPase on the phagolysosome. We used immunofluorescence to probe macrophages infected with both the GAS wild type (M1WT) and an isogenic SLO mutant strain (M1ΔSLO) with an antibody for one of the V‐ATPase subunits. Cells infected with either strain appeared to have no differences in recruitment of V‐ATPase. We also fractionated cells to isolate phagolysosomes in order to determine whether the entire V‐ATPase complex is present in phagolysosomes by mass spectrometry. Purity of the cell fractions was verified by Western blotting using antibodies for EEA‐1 (early endosome/phagosome marker) and LAMP‐2 (lysosome marker). We further probed the phagolysosome fraction with antibodies for 2 V‐ATPase subunits and concluded that SLO does not appear to affect the recruitment of either of these subunits. Preliminary mass spectrometry data indicate that the remaining V‐ATPase subunits appear to be present in phagolysosomes containing wild type GAS. These results indicate that V‐ATPase recruitment is not significantly altered in GAS‐infected phagosomes. Testing the activity of the V‐ATPase is ongoing. These results should shed light on the mechanism by which GAS prevents phagolysosomal acidification.
Cellular and Proteomic Characterization of the Innate Immune Response in Bat Stars (Patiria miniata)
Authors: Andrus Avery Kay , Livingston Brian Thomas
Published: November 2023

Bat star populations on the west coast have been declining due to the Sea Star Wasting Syndrome. This tissue‐decaying epidemic involves a viral infection and skin lesions leading to disintegration in sea stars. The mechanisms causing decay in the symptomatic individuals are still unknown. We considered the immune response in bat stars after the appearance of skin lesions to determine how immune cells react and which proteins are expressed throughout the viral infection and tissue disintegration. We hypothesized that the viral infection is met with an increase in immune cell concentration and phagocytic activity, and an increase in the secretion of immune‐ and stress‐response related proteins into the coelomic fluid in activated defensive pathways. The morphology, behavior, and concentration of the immune cells per mL of coelomic fluid in healthy and wasting bat stars were compared by extracting coelomic fluid and staining nuclei, photographing the cells, and counting them. Immune cells were extracted at healthy and middle time points with no visible lesions, and at an endpoint once visible lesions were present; the cells were counted and incubated with fluorescent bacteria. Phagocytes, cells that engulf invading pathogenic cells, were found in higher cell concentrations after viral infection and the appearance of lesions, showing higher levels of activity and consuming more fluorescent bacteria. The reactions of the cells to these pathogens were observed in coelomic fluid from healthy and wasting sea stars. From the coelomic fluid samples taken throughout the progression of the disease in several individual sea stars, LC/MS/MS analysis and NCBI BLAST characterization of the proteins present showed dynamic abundance patterns in secreted proteins related to cell signaling, stress, and immune response. We are attempting a more thorough characterization of the proteins involved in the immune reaction to better define the defense mechanisms occurring in the bat stars during the response to the viral challenge. The changes in abundance of certain proteins may give clues about the patterns of disintegration occurring at the cellular level and the pathways involved in the tissue disintegration. We may also be able to identify novel antimicrobial or anti‐viral molecules of pathways, with potential for future use in tissue preservation.

Support or Funding Information

COAST Grant, CSULB 2015 Graduate Summer Research Fellowship.
Group A Streptococcus Prevents Initiation of the Oxidative Burst Response in Immune Cells
Authors: Yuen Isabelle , McLain Maya , Franscioni Henry , Etesami Neelou , Okumura Cheryl
Published: November 2023

Group A Streptococcus (GAS) is a gram‐positive bacterium that produces infections ranging from mild self‐limiting infections to more severe infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. In response to pathogens such as GAS, the host innate immune system produces antimicrobial factors such as reactive oxygen species that target microbes directly via oxidative stress, or indirectly via the initiation of other innate immune responses. Some bacteria, such as Lactococcus lactis and Staphylococcus aureus, contain pigment or catalase to detoxify these oxygen radicals. While GAS lacks these enzymes, it is still able to survive in innate immune cells, indicating that GAS may inhibit the oxidative burst response altogether. To examine this possibility, we infected macrophages with M1 and M49 serotype GAS, Staphylococcus aureus, Lactococcus lactis, and Group B Streptococcus and measured hydrogen peroxide production. Cells infected with either GAS serotypes produced significantly less peroxide compared with cells infected with other bacterial species, indicating that GAS suppresses the oxidative burst response. The GAS pore‐forming toxin streptolysin O (SLO) has similarity to listerolysin O (LLO), which prevents the assembly of the multi‐subunit NADPH oxidase on phagosome membranes. NADPH oxidase assembly is necessary to initiate the oxidative burst response. Peroxide generation was restored when cells were infected with GAS mutant bacteria of both M1 and M49 serotypes lacking SLO. We are currently examining the mechanism by which GAS uses SLO to prevents the oxidative burst response. Activation of the NADPH oxidase p47 subunit was verified Western blot for phosphorylated p47. Assembly of the NADPH oxidase is currently being assessed by cell fractionation of phagosomes and mass spectrometry. Preliminary data indicate that several NADPH oxidase subunits are missing from the phagosome. Thus, similar to LLO, SLO may inhibit the proper assembly of the NADPH oxidase, and prevent the generation of harmful reactive oxygen species.
Abuse Liability and Anti‐Addiction Potential of the Atypical Mu Opioid Receptor Agonist IBNtxA
Authors: Keck Thomas M , Verchio Vincent , Wu Chun , Moura‐Letts Gustavo
Published: November 2023

OBJECTIVE

IBNtxA is a novel μ opioid receptor (MOR) agonist, structurally related to the classical MOR antagonist naltrexone, that preferentially signals through truncated MOR splice variants. This unique pharmacological profile produces potent analgesia with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. The purpose of this study is to 1) evaluate a range of IBNtxA doses to more fully assess its abuse liability and 2) determine the effects of IBNtxA on morphine CPP expression and reinstatement of morphine CPP.

METHODS

IBNtxA was synthesized and compared to morphine in standard CPP expression assays. Following morphine CPP training, IBNtxA was tested for its effects in inducing CPP reinstatement on its own or attenuating morphine‐primed reinstatement.

RESULTS and CONCLUSIONS

IBNtxA is an intriguing preclinical drug that opens the door to new MOR splice variant‐selective pharmacotherapeutics with effective analgesic properties but reduced side effects. IBNtxA may have utility as an adjunct therapy in agonist replacement strategies (e.g., methadone). Current collaborative efforts are aimed at developing novel analogues of IBNtxA for further analysis and understanding ligand‐receptor interactions across MOR splice variants using molecular modeling.

Support or Funding Information

Rowan University Seed Fund; travel support from Dean of College of Science and Mathematics and the Rowan University Assistant Professor and Instructor Travel Fund.
Differential role of G protein signaling regulator RGS7 on rewarding effects of opioids and psychostimulants
Authors: Sutton Laurie , Ostrovskaya Olga , Dao Maria , Smith Roy , Martemyanov Kirill
Published: November 2023

The endogenous opioid system is critical for natural reward responses that drive positive behavioral reinforcement, as well as the reinforcing effects associated with addictive substances, such as morphine. The rewarding and reinforcing properties of many drugs of abuse depend on the activation of the m‐opioid receptor (MOR). MOR is a G‐protein coupled receptor whose signaling efficiency is controlled by the regulator of G‐protein signaling (RGS) proteins. In vitro studies have demonstrated that RGS7, a member of the R7 family of RGS proteins, negatively regulates MOR signaling via inhibitory G‐proteins of the Gi/o class. Co‐labeling studies performed in the CNS show that RGS7 and MOR are co‐expressed in the same neuronal population. However, the impact of RGS7 on MOR signaling in vivo is unknown. This study utilizes both genetic and viral approaches to ablate RGS7 both globally and in specific neuronal populations to determine the contribution of RGS7 in reward‐related behaviors. Global RGS7 knockout mice display enhanced morphine‐induced conditional place preference (CPP) and increased locomotor activity in response to morphine. Conditional elimination of RGS7 in striatal neurons results in similar enhancement of morphine‐induced reward in the CPP test. Using an operant self‐administration task these conditional RGS7 knockout mice also displayed increase active lever pressing, as well as a vertically shifted dose‐response profile of morphine‐intake. Together this suggests that RGS7 modulates the reinforcing properties of morphine. To investigate the impact RGS7 has on morphine‐induced alteration in neuronal excitability and plasticity of glutamatergic synapse, electrophysiology approaches were applied to those mice that underwent morphine self‐administration. It was found that RGS7 exerted its effects by controlling morphine‐induced changes in excitability of medium spiny neurons in the nucleus accumbens (NAc) and gating the compositional plasticity of AMPA and NMDA receptors. We further studied the role of RGS7 in reward‐related behaviors in response to other drugs of abuse, such as psychostimulants. Interestingly, mice lacking RGS7 KO exhibit similar responses to cocaine‐induced CPP as their wild‐type littermates. Overall, the results show that RGS7 in the NAc specifically control the rewarding and reinforcing effects of opioids but not of psychostimulants.

Support or Funding Information

This work was supported by the National Institutes of Health Grants DA026405, DA036082, and DA036596 and by the Canadian Institutes of Health Research (CIHR) Fellowship.
The Effect of Ventral Tegmental Area Rictor Knockout on Susceptibility to Chronic Social Defeat Stress and Stress‐induced Changes in Morphine Reward
Authors: Kaska Sophia , Mazei‐Robison Michelle
Published: November 2023

There is significant co‐morbidity of addiction and depression and this may be due to dysregulation of common brain circuits. For example, similar alterations in signaling in the ventral tegmental area (VTA) have been identified in response to both chronic morphine and chronic social defeat stress (CSDS), a rodent model of depression. We are particularly interested in target of rapamycin complex 2 (TORC2) signaling in the VTA as it has been established to play a critical role in morphine reward and morphine‐induced neuroadaptations. However, whether it plays a similar role in stress‐induced changes in morphine reward, or stress susceptibility itself, is unknown. To address these questions, we used viral and genetic approaches to decrease VTA TORC2 signaling by knocking out the expression of Rictor, a TORC2 component protein necessary for TORC2 function. Floxed‐Rictor mice were either crossed with a TH‐Cre mouse line to eliminate TORC2 signaling in all catecholaminergic neurons or underwent stereotaxic surgery to specifically decrease TORC2 signaling in the VTA via AAV‐Cre infusion. The mice were then subjected to chronic physical or emotional CSDS and their susceptibility to stress was measured using the social interaction (SI) test. Following SI testing, mice underwent a two‐bottle choice assay to assess voluntary morphine preference and consumption. In contrast to previous data that found that decreasing AKT signaling was sufficient to increase susceptibility to stress, we found that decreased VTA TORC2 signaling did not alter stress susceptibility. However, we did observe a difference in morphine consumption, as Rictor knockout mice (both TH‐Cre positive and AAV‐Cre) that underwent stress consumed significantly more morphine than stressed controls (intact TORC2 signaling). Additionally, following stress, Rictor knock‐out mice also exhibited an increase in water and sucrose consumption, suggestive of an overall increase in consummative drive. Importantly, these changes were only evident in mice following stress, suggesting a novel role for VTA TORC2 signaling in stress‐mediated changes in consumption.

Support or Funding Information

This work was supported by NIH T32 GM092715 (SK) and NIDA DA037426 (MMR).
Slick (Kcnt2) sodium‐activated potassium channels limit nociceptor excitability and pain
Authors: Tomasello Danielle Leigh
Published: November 2023

The physiological functions of the large conductance Slick sodium‐activated potassium (KNa) channels have yet to be identified. Immunolabeling of Slick channels in murine dorsal root ganglia (DRG) and trigeminal ganglia indicated localization exclusively to proinflammatory peptide calcitonin‐gene related peptide (CGRP) containing small and medium sized neurons, indicating a possible function in nociception. Staining was found within the spinal cord and at the nerve endings in the paw suggesting a role in both pain detection and neurotransmitter release. Strikingly, we found Slick channels localized to large dense core vesicles (LDCV) containing CGRP in DRG neurons. We then studied the phenotypic consequences of Slick deletion using a novel Slick‐LacZ reporter knockout (KO) mouse line. First, we tested the electrophysiological properties of peptidergic DRG neurons from KO and WT mice. We found a decrease in the total outward potassium current, a decrease in action potential rheobase, and an increase in the height of action potential from DRG neurons isolated from Slick KO mice compared to wildtype (WT) neurons, indicating that Slick channels regulate nociceptor excitability. Utilizing models for neuropathic and inflammatory pain, we then tested thermal hyperalgesia on KO and WT littermates. Prior to nerve injury, Slick KO mice exhibited sensitized basal nociception reinforcing our electrophysiology data, and following injury KO mice had heightened hyperalgesia compared to WT mice. In the inflammatory model, injection of complete Freund's adjuvant into the hindpaw caused prolonged but reversible thermal hyperalgesia in WT mice. Hyperalgesia was exacerbated in Slick KO mice and did not exhibit recovery during the time course tested suggesting that the recruitment of Slick channels to the membrane is important for pain recovery. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP positive LDCV translocated to the neuronal membrane. We then assessed the consequence of Slick deletion after CGRP stimulated release and found Slick KO neurons were more excitable compared to WT neurons in both control and stimulated release. Our data indicate that Slick channels limit the excitability of specifically CGRP peptidergic neurons and reduce pain behavior after injury and inflammation. Given the specificity of Slick expression to CGRP positive neurons only, Slick channels are desirable pharmacologic targets for the treatment of pain and inflammation.

Support or Funding Information

The research was supported by the National Institute of Health, Grant NS078184 (to A.B.). Travel Award to EB 2016 provided by Hello Bio, Bristol UK.
Adelmidrol, a Palmitoylethanolamide Analogue, as a New Pharmacological Treatment for the Management of Inflammatory Pain
Authors: Cuzzocrea Salvatore , Impellizzeri Daniela , Di Paola Rosanna , Cordaro Marika , Casili Giovanna , Esposito Emanuela
Published: November 2023

Palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N‐acylethanolamines (NAEs) and some of its analogues, have shown great efficacy in the treatment of pain and inflammation. Adelmidrol is one of these analogues. The aim of this study was to investigate the effect of Adelmidrol on the modulation of the inflammatory response in a mouse model of collagen‐induced arthritis (CIA). CIA was induced by an intradermally injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen (CII) and complete Freund adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice subjected to CIA were administered intraperitoneally with Adelmidrol (10 mg/kg). Mice developed erosive hind‐paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hindpaws. The incidence of CIA was 100% by day 28 in the CII‐challenged mice, and the severity of CIA progressed over a 35‐day period with a resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with Adelmidrol ameliorated the clinical signs at days 26 to 35 and improved histologic status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in Adelmidrol ‐treated mice, as indicated by nitrotyrosine and malondialdehyde (MDA) levels. Plasma levels of the proinflammatory cytokines and chemokines were significantly reduced by Adelmidrol treatment. We demonstrated that Adelmidrol exerts an antiinflammatory effect during chronic inflammation such as CIA.
Depression of Home Cage Wheel Running: A Novel Method to Assess Spontaneous Migraine Pain
Authors: Kandasamy Ram , Calsbeek Jonas J , Morgan Michael M
Published: November 2023

Migraine is the most common neurological disorder in the world, yet current treatments have limited efficacy and repeated administration can cause medication‐overuse headache. Although new treatments are desperately needed, the difficulty of assessing migraine pain in laboratory animals has limited drug development. Most animal studies of migraine assess mechanical allodynia in the face and hindpaw of the rodent. Although useful, this measure does not assess spontaneous, unevoked headache pain – a key characteristic of migraine. The objective of the present study is to test the hypothesis that home cage wheel running is a sensitive and objective method to assess spontaneous migraine pain in the rat – an approach that mimics the decrease in activity used to diagnose migraine in humans. Adult male and female rats were implanted with a cranial cannula aimed at the periosteal space above the dura mater. Rats were allowed to recover for 8 days in their home cage with unlimited access to a running wheel. Migraine pain was induced by microinjecting 10μL of 10% allyl isothiocyanate (AITC) onto the dura. Control rats were injected with mineral oil. Rats were injected with the anti‐migraine agent sumatriptan (1 mg/kg, s.c.) or saline 30 min following the intracranial injection. Administration of AITC into the periosteal space caused a decrease in running in both male and female rats that lasted 4–7 hrs. Administration of sumatriptan prevented this depression of wheel running. The fact that sumatriptan prevented migraine pain‐induced depression of home cage wheel‐running demonstrates that home cage wheel running is a sensitive and reliable measure of spontaneous headache pain in rats.

Support or Funding Information

The study was supported by State of Washington Initiative Measure No. 171 and Washington State University.
A Novel Activator of Canonical Wnt Signaling
Authors: Baruah Jugajyoti , Wary Kishore K , Hitzman Ryan , Zhang Jane
Published: November 2023

Rationale

As many competitive inhibitors of Glycogen Synthase Kinase‐3 (GSK‐3)β exhibit off‐target effects, we investigated the ability of a newly identified small molecule allosteric inhibitor of GSK‐3β, called Tideglusib (TDG), to activate canonical Wnt signaling.

Methods and Results

The treatment of cultured endothelial cells (ECs) with Wnt3a or TDG stabilized β‐catenin polypeptide species in a dose‐dependent manner. A time‐course analyses showed increased stabilization of β‐catenin in ECs in response to TDG. The addition of TDG in ECs induced increased expression of not only transcription factor Nanog, but also Cyclin‐D1 and Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) at both protein and mRNA levels. Microscopic analyses of ECs stained with anti‐VE‐cadherin antibody provided evidence of the disappearance of VE‐cadherin from the adherens junctions, while increased accumulation of β‐catenin in the nucleus. Importantly, in a TOP‐Flash Luciferase assay, TDG showed significant activation of Wnt pathway in both ECs and 293T cells, also accompanied by increased nuclear β‐catenin. Pretreatment of ECs with TDG followed by Propidium iodide (PI) and Annexin‐V labeling showed increased survival and decreased cell death (p<0.05 vs control groups). Importantly, TDG treatment significantly increased the incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) in ECs. In addition, in a scratch assay and Boyden‐chamber migration experiments indicated the ability of TDG to induce migration of ECs. Furthermore, in an aortic ring assay TDG induced robust neovessel formation.

Our results demonstrate that an allosteric inhibition of GSK‐3β stabilizes β‐catenin, thereby upregulating Cyclin‐D1, Nanog, and VEGFR‐2 that are critical regulators of cell proliferation and stem cell self‐renewal.

Support or Funding Information

American Heart Association (AHA) Mid‐west affiliate Predoctoral Fellowship 2015–2016
Optimizing the analysis of bias to produce more accurate results
Authors: Stahl Edward , Ehlert Frederick J , Bohn Laura M
Published: November 2023

Seven transmembrane receptors interact with a number of intracellular targets including multiple heterotrimeric G proteins, G protein receptor kinases, and the scaffolding protein β‐arrestin2. For more than a decade, a body of evidence has supported the presence of specific ligand‐receptor complexes that can preferentially stimulate a subset of intracellular targets. This has led to screening and development of ligands, for a number of different receptors, that demonstrate preferential signaling. An example of this is the discovery of the kappa opioid receptor (KOR) agonist 6′‐guanidinonaltrindole (6′‐GNTI). This agonist stabilizes an active receptor state of the receptor that prefers to stimulate G protein activity over the recruitment of β‐arrestin2. We have recently published a novel, competitive, model that was well suited to analyze the bias of 6′‐GNTI.

In the studies presented here, we demonstrate a Schild‐type model we have constructed to quantify ligand bias. This method is based on the type of experimental design commonly represented in a Schild plot. Using experimental evidence from the KOR and 6′‐GNTI, as well as data from Monte Carlo simulations, we compare the Schild‐type model with two previously defined models: the standard model and our competitive model. We demonstrate that the Schild‐type model produces a reduction in the error of the parameter estimates when analyzing both the simulated and experimental data (compared to the error produced using the standard model). We are able conclude that, although the cost of employing the Schild‐type model or the competitive model may be greater than for the standard model, these models provide the benefit of producing more accurate and precise bias estimates. From these findings we then introduce a spectrum of rigor for evaluating the bias of competitive ligands.

Support or Funding Information

This work is supported by a grant from the National Institute on Drug Abuse (R01DA033073 LMB).