bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Forces Acting on the Equine Navicular Bone in Normal and Dorsiflexed Positions
Authors: Ruff Kaitlyn C , Osborn Michelle L , Uhl Elizabeth W
Published: November 2023

Navicular disease is a common form of chronic lameness in horses that can be managed but not effectively treated. A pathomechanical approach as is used for human athletes may offer a better explanation of its pathogenesis and provide more effective treatment options. The navicular apparatus is a functional enthesis organ very similar to the human Achilles, and is highly adapted to dissipate forces away from the osteotendinous junction between the deep digital flexor tendon and the third phalanx. Based upon functional analyses of horses with navicular disease, we have hypothesized that chronic overloading of the forelimbs, which induces the habitual and exaggerated dorsiflexion of the foot, changes the balance of forces acting on the navicular apparatus. We tested our hypothesis by comparing the forces acting on the navicular apparatus of an equine foot in a normal versus a dorsiflexed position, using the method of free‐body analysis on 3D models. These models were reconstructed from data of x‐ray CT scans of a horse forelimb. Results indicate that the dorsiflexed position of the foot puts the deep digital flexor tendon under greater tension, thereby creating a larger compressive force on the navicular bone. This compression would explain the bone lesions characteristic of navicular disease. The dorsiflexed position of the foot also pulls taut the impar and suspensory ligaments, thereby increasing tension on their attachments sites on the navicular bone. This increased tension explains the presence of osteophytes along the entheses of these ligaments, which is a common lesion in navicular disease. Thus, rehabilitative therapy focused on decreasing dorsiflexion of the foot by reducing overloading of the forelimb is likely to be an effective treatment for many cases of navicular disease.
Increase in therapeutic effects and therapeutic window of hepatocyte xenotransplantation in hemophilia B mice with a safe pretreatment strategy coupled with genetically engineered donor cells
Authors: Hsu Yu‐Chen , Yang Shu‐Jhu , Chen Kuang‐Yen , Yeh Chiu‐Wen , Huang Yu‐Jen , Yu I‐Shing , Wu Yao‐Ming , Lin Shu‐Wha
Published: November 2023

Xenotransplantation of hepatocytes has extensive value for patients with liver‐based metabolic defects, coagulation disorders and liver failure. Since the low efficiency of integration of transplanted hepatocytes into recipient livers currently limits clinical applications, a safe strategy to improve donor hepatocyte engraftment can be expected to widen the clinical use of cell therapy in liver disease. Hemophilia B is considered to be a good disease model for developing cell therapy because it is caused by a single protein (coagulation factor IX, FIX) deficiency, and the quality of patients’ lives can be dramatically improved through a slight increase in therapeutic coagulation protein. In our previous study, we observed an enhanced engraftment of intrasplenically injected cancer cells into the liver of a specific mouse strain due to its narrowed sinusoids, and therefore we suspected that narrow sinusoids may enhance the efficiency of hepatocyte transplantation. Here, we developed a strategy by pre‐administration of the antibody (anti‐X) to decrease the hepatic sinusoidal diameter in the mice and applied this strategy to improve the therapeutic effect of hepatocyte transplantation for hemophilia B. We observed that the amount of transplanted cells was nearly 2 times more in the anti‐X pretreated mice than in the WT livers at all time points up until one month. Furthermore, we coupled the anti‐X pretreatment strategy with a bioengineered human FIX with augmented specific activity, named FIX‐Triple, to improve the effects and therapeutic window of hepatocyte transplantation in Hemophilia B mice. After intravenous infusion of an adeno‐associated viral vector encoding FIX‐wildtype or FIX‐Triple, the donor hepatocytes were isolated and transplanted into the hemophilia B mice with anti‐X or control IgG pretreatment, then the haemostatic protection of the hepatocyte transplantation was evaluated on day 25 after transplantation. Compared with the control IgG pretreated mice receiving FIX‐wildtype expressing hepatocyte transplantation, the anti‐X pretreatment strategy coupled with FIX‐Triple‐donor increased the FIX clotting activity more than 5‐fold and dramatically decreased the bleeding time and total blood volume loss in the recipient hemophilia B mice. Our results demonstrated that the antibody‐pretreatment strategy coupled with the high activity FIX variant FIX‐Triple resulted in significant improvement in the haemophilic phenotype after hepatocyte transplantation.
Insights into translational utility of rodent models of hepatic fibrosis
Authors: Saravanan Chandrassegar , Judge Joyce , Song Yanli , Dietrich Bill , Wu Jing , Bauer Andreas , Mansfield Keith
Published: November 2023

Activation of hepatic stellate cells (HSCs) has been established as the dominant process in the progression of hepatic fibrosis. A number of phenotypic alterations occur during activation of HSCs, including proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis and retinoid loss. These phenotypic changes could serve as potential targets for anti‐fibrotic therapy. In the past, several rodent models have been used to delineate the mechanism of fibrogenesis. However, the translatability of these animal models for clinical development remains largely questionable. The goal of this study is to compare the number of proliferating HSCs in commonly used mouse models of hepatic fibrosis [carbon tetrachloride (CCl4), bile duct ligation (BDL) and metabolic disease (ob/ob high fat diet)] and human cirrhotic liver samples. Dual immunostaining for Ki67 (proliferative marker) and a marker for activated HSCs (aHSCs) on formalin fixed paraffin embedded liver sections were performed. Whole slide quantitative image analysis on dually immunostained human cirrhotic liver samples (n=5) showed only 1.9% (range 1.3–3%) of aHSCs cells were positive for Ki67. In contrast, BDL, ob/ob high fat diet, and CCl4 mouse models showed approximately 4%, 5%, and 17% of aHSCs were positive for Ki67, respectively. In summary, cognizance of the discrepancy in the number of proliferating HSCs between human cirrhotic samples and mouse models of fibrosis will assist in careful design of preclinical studies.
Loss of Wnt secretion by macrophages promote heptobiliary injury following DDC diet
Authors: Jiang An , Okabe Hirohisa , Preziosi Morgan E , Poddar Minakshi , Monga Satdarshan P , Nagarajan Shanmugam
Published: November 2023

Exposure of mice to a hepatotoxic diet containing 3,5‐diethoxycarbonyl‐1, 4‐dihydrocollidine (DDC) induces biliary damage followed by hepatocyte injury mimicking human cholestatic liver disease. Hepatobiliary injury is repaired through two known phenomena ‐ atypical ductular proliferation that contributes to ductular repair and hepatocyte proliferation that assist in replacing lost hepatocytes. Also, hepatocyte trans differentiation to biliary cells has been reported in this model as a repair mechanism. Using liver‐specific β‐catenin conditional knockout (KO) mice, previously we reported lack of b‐catenin in hepatocytes and cholangiocytes worsens the hepatic injury after DDC treatment. However, the source of Wnt, specifically Wnt secreted by macrophages contributing to DDC‐diet induced hepatobiliary repair has not been studied. In this report, we investigated the role for macrophage‐specific Wntless (Wls), a critical regulator of Wnt secretion, in DDC‐induced hepatobiliary injury and regeneration. We generated macrophage specific Wls knockout mice, which lack the ability of macrophages to secrete Wnts. Mac‐Wls male KO (n=5) and littermate controls (n=5) were fed a DDC diet for 14 days. Mac‐Wls KO mice on the DDC diet showed larger liver weight to body weight ratios than control. Steatosis was observed in both groups. We observed increased serum levels of ALT, ALP, and bilirubin in Mac‐Wls KO mice compared to wild type littermates. Interestingly, Mac‐Wls KO mice after DDC diet had notably more inflammation compared to control. In addition, F4/80 positive macrophages were more abundant in Mac‐Wls KO mice after DDC treatment. Wild type macrophages polarized to M1 showed upregulation of pro‐inflammatory cytokines and iNOS2 expression. Interestingly, M1 polarized macrophages showed reduced Wls expression, while M2 polarization with IL‐4 did not alter Wls expression. These findings suggest a possibility that lack of Wnt secretion by macrophages under inflammatory conditions may lead to increased inflammation by inflammatory cells including macrophages and dendritic cells. Taken together, these results suggest macrophage specific wls‐deficient mice are more prone to injury, inflammation in response to DDC‐induced injury.
Group A Streptococcus Persists in the Lysosomes of Phagocytic Cells
Authors: Zheng Kelvin , Snipper Joshua , No Hyewon , Till Andreas , Okumura Cheryl
Published: November 2023

Group A Streptococcus (GAS) is a gram‐positive bacterium that is responsible for invasive diseases such as necrotizing fasciitis and streptococcal toxic shock‐like syndrome. Normally, phagocytic cells of the innate immune system such as macrophages rid the body of pathogens by ingestion and digestion of pathogenic microbes. Pathogens are internalized into structures called phagosomes, which then fuse with lysosomes, facilitating the degradation of the pathogen. Acidification of the phagolysosome/lysosome compartment is an important part of the process to activate the proteolytic enzymes of the lysosome. To visualize GAS as it is trafficked through this pathway, we transformed bacteria with a pH‐sensitive green fluorescent protein, mWASABI. Continued mWASABI fluorescence and persistence of GAS within the lysosomal compartment suggests that GAS survives phagolysosomal degradation by preventing acidification of the compartment. We verified that the mWASABI protein expressed by GAS is appropriately quenched in low pH environments and that GAS is not acid‐tolerant. We hypothesize that streptolysin O (SLO), a GAS secreted protein capable of creating pores, contributes to GAS prevention of acidification. To study this, a mutant strain of GAS lacking the SLO gene (ΔSLO) was transformed to express mWASABI. Surprisingly, ΔSLO mutants also persisted in the lysosomal compartment and were equally fluorescent to wild type bacteria. We are currently exploring whether overexpression of mWASABI may be masking the quenching of the pH‐sensitive probe. We are also exploring whether other pore‐forming toxins contribute to preventing lysosomal acidification. Determining the mechanism for how GAS prevents lysosomal acidification will be important in the development of therapeutics aimed at enhancing the ability of phagocytic cells to destroy GAS.
Role of the Human Parainfluenza Virus Type 3 Fusion Machinery in Viral Growth in the Natural Host
Authors: Jusino Shirley , Palermo Laura M. , Moscona Anne
Published: November 2023

Human parainfluenza virus type 3 (HPIV3) is a single‐stranded, negative‐sense RNA enveloped virus that causes severe respiratory diseases in children; including croup, bronchiolitis and pneumonia. HPIV3 infects its target cells by the coordinated action of the receptor binding protein hemagglutinin‐neuraminidase (HN) and the fusion (F) envelope glycoprotein, which together comprise the molecular fusion machinery. Previous studies from our lab show that viruses bearing HN‐F machinery that confer excellent growth in monolayer cultures cannot thrive in vivo. In turn, viruses that efficiently replicate in vivo‐ for example clinical isolates (CIs) ‐ bear HN‐F machinery that cannot cope with in vitro cell culture. Our aim is to characterize the fusion machinery properties of lung adapted HPIV3 virions. We hypothesized that circulating HPIV3 viruses (CIs) bear HN‐F fusion machineries well suited to the natural environment. In order to characterize the different functions of the CIs HNs and F HPIV3, viral RNA was extracted from CIs and the genomic sequences were analyzed by deep sequencing techniques. The cDNA encoding the CIs viral glycoproteins were inserted in expression vectors and assayed for expression levels by a biotinlyation assay. Additionally, neuraminidase activity was tested for all CIs HNs. Results were expressed as relative percentage of lab adapted ‐HPIV3 HN. Preliminary results suggest that HPIV3 CIs have a higher neuraminidase activity in comparison to lab adapted ‐HPIV3 HN and low fusogenicity. Overall, our results suggest that the fusion machinery of CIs is more stable and less readily activated than viruses adapted to growth in culture.

Support or Funding Information

Grant: 5R01AI031917‐23

Grant: NIH NIGMS R25GM096955
Identification of Protein Kinase R and Tissue Distribution in Channel Catfish
Authors: Ball Jenna , Vides Melissa , Kobayashi Yass
Published: November 2023

Protein kinase R (PKR) is one enzyme responsible for the cessation of translation during stress exposure in mammals by inactivating the eukaryotic translation initiation factor 2 through phosphorylation. PKR is also considered a viral sensor as activity of PKR increases in response to the presence of double‐stranded RNA within the cell during viral infection. Consequently, activation of PKR leads to the inhibition of cell proliferation and apoptosis, which prevents generation of new viral particles. Channel catfish have been considered an excellent model fish species in basic and comparative physiological studies. However, little is known about the immune response of channel catfish against viral infection, and the underlying mechanism(s) associated with improved disease response has not been well‐defined. Given that PKR is an integral component of mammalian innate immunity, this study with PKR in channel catfish may serve as a potential model to investigate human immune response to viral infections. The objective of this study was to identify PKR mRNA in channel catfish and to examine the tissue distribution. The gene encoding the PKR was identified by comparing channel catfish EST database with zebra fish PKR mRNA. Expression of PKR mRNA was examined in the cDNA of brain, liver, muscle, spleen, trunk kidney, and heart using RT‐PCR. The PKR mRNA was detectable in all tissues examined at varying degrees. Nucleotide sequence of the amplicon (399 bp) was identical to the corresponding channel catfish EST (100%), but not with any other PKR mRNA. Currently, this study is investigating expression of PKR mRNA in relation to long‐term stress associated with food deprivation.

Support or Funding Information

This study is supported by the Kansas IDeA Network of Biomedical Research Excellence (Grant number 20GM103418).
Substitutions in the Proximity of the Active Site of Peptidylarginine Deiminase from P. gingivalis ‐ Is There a Correlation with Enzyme Activity?
Authors: Bereta Grzegorz , Gawron Katarzyna , Łazarz‐Bartyzel Katarzyna , Łazarz Mieczysław , Stożek Karol , Naskalska Antonina , Burmistrz Michał , Pyrć Krzysztof , Chomyszyn‐Gajewska Maria , Potempa Jan
Published: November 2023

Background

Periodontitis (PD) is a chronic inflammatory disease leading to tooth loss, which affects over 30 % of adults worldwide. A major causative agent of the disease is an anaerobic bacterium, Porphyromonas gingivalis (Pg). Recently, considerable interest has been focused on peptidylarginine deiminase (PPAD) from Pg, an enzyme which catalyzes conversion of arginine to citrulline. Our recent results imply PPAD activity as a critical factor in gingival cell infection and prostaglandin E2 induction. Those findings encouraged us to analyze polymorphisms of the PPAD gene and enzyme activity among Pg clinical strains.

Objective

Analysis of PPAD gene polymorphisms and enzyme activity in clinical isolates of Pg from patients with chronic periodontal disease (CPD), reference strains (RS) and healthy volunteers (HV).

Material and methods

Gingival crevicular fluid was collected from 9 CPD and 4 HV, seeded on blood agar and grown under anaerobic conditions. Pg was confirmed by 16S rRNA and PPAD PCR, followed by genomic DNA purification. Next, PPAD coding sequences were cloned into a pTZ57R vector, amplified and sequenced. The sequences from CPD and HV were aligned with the PPAD sequences deposited in the GenBank database, i.e. 2 of RS (ATCC33277, W83), 4 strains of PD patients (F0568, F0569, F0570, SJD2), and 5 other strains (F0185, F0566, JCVI SC001, W50, W4087). Further, PPAD activity from CPD, RS and HV was analyzed using colorimetric method.

Results

In total, mutations were identified in 50 nucleotide positions in the PPAD sequence from all clinical isolates and those deposited in GenBank. One sequence was identical to the ATCC33277, but varied from the W83 strain, while 4 sequences were identical to the W83 strain. One isolate harboured 7 nucleotide substitutions, which resulted in a change of 3 amino acid residues in the proximity of the PPAD active site. This isolate displayed significantly increased PPAD activity in comparison to W83 strain. The identical nucleotide substitutions were noted in PPAD of strains from patients with PD found in USA and China.

Conclusions

Our analyses shown variations in nucleotide and amino acid sequence of PPAD among clinical and reference strains. The presence of specific, identical amino acid substitutions in clinical isolates from Poland, USA and China in comparison to the RS along to the variation of enzyme activity may suggest an impact of PPAD on the virulence of Pg. This observation deserves further elucidation of functional and clinical significance of this mutation in the pathogenesis of PD.

Support or Funding Information

National Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424‐329/10, J.P.)
Efficacy Related Modulation of Behavioral Effects of Opioids During Chronic Buprenorphine Treatment in Squirrel Monkeys
Authors: Withey Sarah Louise , Paronis Carol Anne , Bergman Jack
Published: November 2023

Opioid addiction is characterized as a chronic relapsing disorder in which renewed drug‐seeking behavior during abstinence can be provoked by exposure to an opioid or opioid‐associated cues. In laboratory subjects, drug‐seeking behavior similarly can be reinstated by priming with the drug or drug‐related stimuli. Buprenorphine (BUP), a partial agonist at the μ opioid receptor, is commonly prescribed for the management of opioid addiction but its ability to reduce reinstatement behavior in laboratory subjects is not well understood. In the present studies, squirrel monkeys (n=4) were trained to respond under a drug/food choice procedure (i.e., concurrent FR schedules of i.v. oxycodone and milk delivery). Next, the priming strength (i.e., % injection lever responding when the choice was between i.v. saline or milk) of different opioids was determined before and during chronic BUP treatment (0.1 or 0.32 mg/kg/day). Results thus far indicate that: 1) despite decreased potency, indicative of tolerance, full agonists (oxycodone, methadone) maintain priming strength during chronic treatment whereas 2) the priming strength of partial agonists (nalbuphine, butorphanol) is dramatically decreased. These results suggest a reduction in the ability of lower efficacy, but not high efficacy agonists to provoke relapse in BUP‐maintained people. The extent to which these findings may reflect efficacy‐related differences in other opioid endpoints (e.g. analgesia) during BUP maintenance remains to be determined.

Support or Funding Information

Funded by NIH/NIDA: DA035857
Novel inhibitors of the soluble epoxide hydrolase block pain in multiple models
Authors: Wagner Karen , Lee Kin Sing Stephen , Hwang Sung Hee , Hammock Bruce D.
Published: November 2023

The soluble epoxide hydrolase (sEH) is a master regulatory enzyme downstream in the cytochrome P450 (CYP450) branch of the arachidonic acid (ARA) cascade. The sEH enzyme is the principal route of degradation for the bioactive epoxidized metabolites of long chain fatty acids formed by CYP450 activity. The most studied of these are the epoxyeicosatrienoic acids (EETs) but the epoxidized metabolites of both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) the epoxydocosapentanoic acids (EDPs) and eicosatetraenoic acids (EEQs) respectively are known substrates of sEH. Targeting the sEH to inhibit the metabolism of these epoxy fatty acids (EpFAs) has been shown to be antiinflammatory, antihypertensive and to delay seizure. Important to the focus of this work, inhibiting sEH has also been efficacious against both inflammatory and neuropathic pain. Here we use newly designed inhibitors based on a N, N′ disubstituted urea pharmacophore guided by crystal structures which have exhibited improved drug‐like properties, pharmacokinetic profiles and potency in both equilibrium binding and drug residence time on the recombinant sEH of several species. This increased potency and druggability has transferred into better efficacy against modeled pain in vivo. We have tested the efficacy of these inhibitors in models of murine inflammatory pain and induced diabetic neuropathic pain. Newly we have extended this testing to the Akita mouse model which has a genetic mutation in the insulin receptor resulting in a loss of function and eventual type I diabetes. The inhibitors were potent and efficacious in these models using both withdrawal and operant nociceptive assays. Additionally, the inhibitors show synergy with both coxibs and fatty acid amide hydrolase inhibitors and the absence of any reward side effects which relate with addiction potential as seen with narcotics. Thus, targeting sEH is a promising strategy to alleviate pain and holds potential for impacting human health with translation to the clinic.

Support or Funding Information

NIEHS R01 ES002710, NIEHS/Superfund P42 ES004699, NIH/NIDDK U24 DK097154, NIDDK R01DK090492, KW is supported by 5 T32L086350‐08.
Effects of MIA‐induced osteoarthritis on hind limb weight bearing and bone biology endpoints in rats with or without access to running wheels
Authors: Cone Katherine , Krivitsky Rebecca , Warner Emily , Imbert Ian , Allen Joshua , Henderson Terry , Rosen Clifford J. , Bilsky Edward J. , King Tamara , Stevenson Glenn W
Published: November 2023

Our laboratories have been characterizing the interactions between exercise and pain‐like states in rodents. The present set of experiments characterized the effects of monosodium iodoacetate (MIA) on hind limb weight bearing in rats that were either sedentary or had access to running wheels. Rats were given intra‐articular saline or MIA (3.2 mg) into the left hind knee. Rats were divided into two groups. Group 1 had 24 hr voluntary access to running wheels for 42 consecutive days (21 days acquisition followed by 21 days post‐MIA). Group 2 had no access to running wheels. Weight bearing was measured on post‐MIA days 3, 7, and 21. In sedentary rats, MIA produced shifts onto the uninjured hind limb on PD 3, 7 only. In rats with access to running wheels, MIA produced weight asymmetry on PD 3, 7 and 21. To determine potential causal mechanisms behind the differences between sedentary and exercised rats, ex vivo microCT imaging analysis was performed to assess architectural changes to hind limb distal femur/proximal tibia and knee. Data indicated that MIA runners had greater lateral (but not medial) subchondral total volume loss relative to MIA non‐runners. Data for trabecular region are currently being analyzed. These results suggest that within the specific parameters of this study, voluntary wheel running exacerbated the effects of MIA‐induced OA on both behavioral and bone biology measures.

Support or Funding Information

Funded by NIAMS/NIH grant AR054975‐02A1 to G.W.S.
The Lysophosphatidic Acid LPA1 Receptor as a Novel Molecular Target of Antidepressants in Neuronal Cells
Authors: Olianas Maria C. , Dedoni Simona , Onali Pierluigi
Published: November 2023

Neuronal death and atrophy of brain limbic structures have been associated with stress and depression, and antidepressants have been reported to protect against neuronal cell loss induced by different neurotoxic insults. However, the molecular mechanisms mediating the neuroprotective effects of antidepressants have not been completely elucidated. Several studies have shown that the bioactive phospholipid lysophosphatidic acid (LPA) affects neurogenesis, synaptic plasticity and anxiety‐related behaviour through specific G‐protein coupled receptors, termed LPA1–6, which are expressed in the developing and adult brain. We have recently reported that different antidepressants induce growth factor receptor transactivation and cell proliferation in Chinese hamster ovary cells (Olianas et. al., Biochem. Pharmacol, 95, 311–323, 2015). In the present study, we show that in immortalized mouse hippocampal HT22 cells the tricyclic antidepressants amitriptyline and imipramine and the atypical antidepressants mianserin and mirtazapine trigger ERK1/2 phosphorylation and protect against apoptosis through the LPA1 receptor. ERK1/2 phosphorylation induced by acute exposure to the antidepressants or LPA was inhibited by cell pre‐treatment with pertussis toxin, indicating the involvement of G proteins of the Gi/o family. Antidepressants and LPA also promoted the phosphorylation of the transcription factor CREB, a downstream effector of ERK1/2 signaling. Cell treatment with either AM966, a selective LPA1 receptor antagonist, or Ki16425, which preferentially blocks LPA1 and LPA3 receptors, significantly inhibited ERK1/2 and CREB phosphorylation elicited by the antidepressants. HT22 hippocampal cells underwent apoptosis following serum deprivation. Antidepressants and LPA markedly attenuated neuronal apoptosis, as indexed by increased cell viability, decreased percentage of annexin V‐positive cells, inhibition of pro‐caspase activation and poly‐(ADP ribose) polymerase cleavage. AM966 and Ki16425 reduced the protective effects of antidepressants and LPA against HT22 hippocampal cell apoptosis. These data demonstrate the involvement of the LPA1 receptor in the neuroprotective action of different classes of antidepressants.

Support or Funding Information

The study was supported by Regione Autonoma della Sardegna, Italy, L.R. n. 7/2007‐CRP10810/2012.
Citalopram's mechanism for decreasing immobility time in the Tail Suspension Test
Authors: Kingston Rody , Pan Hope , Carneiro Ana
Published: November 2023

Different antidepressants often target common serotonergic and dopaminergic systems. However, antidepressants are not 100% effective suggesting that there are still multiple cellular mechanisms contributing to depression. Antidepressant responses by novel compounds are typically identified in preclinical models using the Tail Suspension Test (TST), a behavioral stress model that measures immobility time after exposure to an inescapable environment. The TST can predict chronic antidepressant efficacy after acute administration, where effective antidepressants decrease immobility time. Although the predictive validity of the TST is well established, the cellular mechanisms underlying the antidepressant‐driven immobility responses remain unknown. The purpose of this study is to identify the central nervous system cellular pathways that respond to the TST. Two antidepressants will be used in this study: olanzapine, and citalopram, which target the GABA and serotonin systems, respectively. Common pathways mediating the immobility effects of these antidepressants will be identified by immunohistological analysis using phosphorylated extracellular signal‐related kinases as markers of neuronal activity. What was found was that the phosphorylation of extracellular regulated kinases was a mediating step to citalopram's mechanism in decreasing immobility time. The implications of this study include the identification of cellular pathways activated by acute antidepressant treatment, the minimum cellular pathways that are necessary for efficacious therapeutic effects, and facilitating the synthesis of compounds that properly combat depression.
Effects of Ketamine in the Forced Swim Test in Female Rats
Authors: Felger Rachel , Carreno Flavia , Frazer Alan
Published: November 2023

Depression affects around 350 million people in the world, according to the World Health Organization. There are traditional pharmacological treatments that work for many, but not all who suffer. In fact, 10–30% of depression patients can be considered treatment resistant, as they do not improve significantly after treatment with traditional antidepressants. Ketamine, a noncompetitive N‐methyl‐D‐aspartate (NMDA) glutamate receptor antagonist, has been shown to produce antidepressant‐like effects in male rat models when administered at a low, sub‐anesthetic, dosage. Ovariectomized (OVX) and normal cycling female rats were used in this study to determine ketamine's effects on behavior in the Forced Swim Test (FST). Results show that OVX females exhibit decreased immobility in the first 24 hours after ketamine administration (p = 0.001), a typical antidepressant‐like effect, but this effect on immobility disappears by 7 days post‐treatment. Conversely, normal cycling females exhibit this antidepressant‐like effect overall at both the 24 hour and 7 day (p = 0.03) time period, and do not display an increase in locomotor activity during the first five minutes of the locomotor activity test. The results indicate the females respond in a similar manner to males, producing antidepressant‐like effects in both normally cycling and OVX females. However, further research is needed to clarify the mechanisms contributing to the shorter duration of response observed in the OVX rats.

Support or Funding Information

Department of Veterans Affairs and ASPET
Chronic isolation stress alters antidepressant‐like behaviors in animals lacking RGS4
Authors: Talbot Jeffery N , Leon Bernice Poncede , Nielsen Cienna L , Benhatzel Christopher M , Alp Deniz , Kirsh Robert , Traynor John R
Published: November 2023

Conventional treatments for depression increase serotonin (5‐HT) levels with selective serotonin reuptake inhibitors (SSRIs). Although superior to previous treatments, SSRIs cause unpleasant side effects, are ineffective in many patients, and take weeks to months for therapeutic benefit pointing to the need for improved understanding of mechanisms regulating mood and antidepressant drug action. Evidence suggests that antidepressant behaviors and more specifically the antidepressant actions of serotonin (5‐HT) are modulated by endogenous regulators of G protein signaling (RGS) proteins. In the current study, the effects of chronic isolation stress on mood‐related behaviors of mice lacking RGS4 were tested. Genetic deletion of RGS4 (RGS4(−/−)) had no effect on basal antidepressant‐like behaviors or generalized locomotor activity in mice. However, when subjected to chronic isolation stress, RGS4(−/−) mice developed antidepressant‐like phenotypes whereas unstressed RGS4(−/−) animals exhibited basal behaviors similar to stressed and unstressed wild‐type (RGS4(+/+)) littermates. Moreover, chronically‐stressed RGS4(−/−) animals exhibited increased phosphorylation of neuronal GSK3β, a putative biological marker of antidepressant‐like activity. Importantly, RGS4 deletion suppressed SSRI‐induced antidepressant activity but had no effect on basal or drug‐induced locomotor activity. Overall, these data suggest chronic stress differentially influences antidepressant‐like behaviors in an RGS4‐dependant manner and point to a role for RGS4 in the regulation of mood‐related behavior in vivo.

Support or Funding Information

Supported by MH0907012.
Beta‐arrestin1 and testosterone levels in women with depression during premenstrual dysphoric disorder
Authors: Smith Keisha , Alam Farzana , Ansah Twum , Archibong Anthony , Nayyar Tultul
Published: November 2023

OBJECTIVE

Women with premenstrual dysphoric disorder (PMDD), a severe form of premenstrual syndrome (PMS), have been suggested to have greater risk for future depression during pregnancy, post‐partum period and perimenopause. Currently no physical or laboratory test can diagnose PMDD. Reduction in beta‐arrestin1 (β‐AR1) protein and increased levels of testosterone are evident during depression. This study examined the levels of β‐AR‐1 protein in peripheral blood mononuclear leukocytes (PBMC) and plasma testosterone in women with PMDD as diagnosed by objective assessment from questionnaires, which may aid in the diagnosis and treatment for PMDD.

DESIGN METHODS

Study participants (n=25) were non‐pregnant women between 18–42 years of age with the symptoms of PMS/PMDD, but not taking any antidepressants/therapy and at the luteal phase of menstruation. The protein levels of beta‐arrestin1 in the PBMC were determined by ELISA and the testosterone levels in plasma were measured by radioimmuno assay. The participants were evaluated with Neuropsychiatric Interview (DSMIV‐TR). The severity of depression were determined by the Hamilton Depression Rating Scale scores (HRSD).

RESULTS

The magnitude of the different parameters for Axis 1 mental disorders and the plasma testosterone levels were significantly higher and beta arrestin1 protein levels in mononuclear leukocytes were significantly lower in women with depression (PMDD) as compared to PMS women.

CONCLUSION

These findings support that reduction in beta‐arrestin1 protein and associated increased levels of testosterone are implicated in the pathophysiology of depression in women with PMDD.

Support or Funding Information

Clinical Research Education and Career Development in Minority R25RR17577 (NCRR/NIMHD) and U54 MD007593 (NIMHD) and U54 RR026140 (NCRR) at NIH
Role of Serotonin 2A and 2C Receptors in Ketamine‐induced Hypothermia
Authors: Murphy Tyler Joseph , Murnane Kevin Sean
Published: November 2023

Anesthesia‐Induced Hypothermia (AIH) has been reported to cause many postoperative adverse effects. These effects include increased mortality, immunosuppression, increased risk of cardiac events, and a higher prevalence of surgical wound infections. Currently, there are few effective ways of preventing AIH besides pre‐warming fluids and gases for the patients during surgery, or relying on forced‐air heating systems. Although it is established that serotonin has a role in thermoregulation, the receptors responsible for such effects remain unclear. Here, we investigated the role of serotonin receptors in AIH using the 5‐HT2A receptor antagonist M100907 and the 5‐HT2C receptor agonist WAY‐163909. Core body temperature was determined through the use of a rectal probe and hypothermia was induced by ketamine in male, young adult Swiss‐Webster mice. We found that dose‐dependently and reliably ketamine induced hypothermia. Moreover, WAY‐163909 attenuated ketamine‐induced hypothermia. In contrast, ketamine‐induced hypothermia was exacerbated by M100907. These findings suggest a new clinical use for 5‐HT2C receptor agonists, specifically to maintain body temperature during surgery. We will build upon these studies by conducting mechanistic pharmacological and neurobiological experiments, including immunohistochemical assays to determine the effects of these drugs on activity in the preoptic area of the anterior hypothalamus, the thermoregulatory center of the brain.

Support or Funding Information

This work was supported by institution funds supplied by the Mercer University College of Pharmacy, an honors thesis award supplied by Oglethorpe University, and a Summer Undergraduate Research Fellowship supplied by the American Society for Pharmacology and Experimental Therapeutics.
Synthesis of chitosan nanoparticles of flavonoids extracted from black bean (Phaseolus vulgaris L.) seed coats to enhance the intestinal absorption and increase plasma residence time
Authors: Sanchez‐Ramirez Carlos , Chavez‐Santoscoy Rocio Alejandra , Cornejo‐Bravo Jose Manuel
Published: November 2023

Beans exert hypolipidemic activity, and it has been attributed to flavonoids and saponins from seed coats, by attenuating lipogenesis and stimulating fatty acid oxidation and cholesterol biliary excretion through the differentially regulation of LXR probably mediated by AMPK phosphorylation. However, their therapeutic potential is limited by their low oral bioavailability, attributed to poor stability and intestinal absorption. Chitosan (CS) is a natural biodegradable, biocompatible and non‐toxic biopolymer extracted from the shells of crustaceans. Chitosan and its derivatives have been examined extensively for medical and pharmaceutical applications such as targeted drug delivery, drug transport, and so on. The chitosan tripolyphosphate (CS/TPP) nanoparticles have been used as an alternative to encapsulate bioactive flavonoids from black bean seed coats. The particle size measurements of the nanoparticles were measured by the Zetasizer Nano and the cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fibroblasts from Mus musculus (NIH/3T3 cell line). Nanoparticles size was approximately 150 nm, the encapsulation efficiency of CS/TPP nanoparticles was found to be 59.9%, and they were not cytotoxic at a concentration of 500mg/mL in fibroblast cells. CS/TPP naanoparticles are an interesting alternative to develop a delivery systems for flavonoids and natural bioactive compounds.

Support or Funding Information

This research was supported by the Research Chair Funds for Full Time Professors (2014) of Autonomous University of Baja California (UABC), program Number 3888. The authors would like to acknowledge Consejo Nacional de Ciencia y Tecnología (CONACYT) in Mexico.
Induction of Nrf2 Activity by Ethyl Acetate Extract of Lagenaria Breviflora
Authors: Oridupa Olayinka Ayotunde , Ritchie Kenneth J
Published: November 2023

The field of cancer chemoprevention is based upon the ability of chemical compounds from any source (synthetic and natural) to prevent cancer. Phytochemicals with the ability to increase the activity of the transcription factor Nrf2 (NF‐E2‐related factor 2) are central to this field. Nrf2 has been reported to modify the expression of up to 200 genes, many of which are involved in cellular defence against toxic insult including heme‐oxygenase 1 (HO‐1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S‐transferases (GSTs) [1]. Interestingly many plant species which are known possess direct antioxidant activity through mechanisms such as free radical scavenging have not been investigated for their ability to modify the expression of such crucial cellular defence genes. This study was consequently designed to investigate the Nrf2 induction capability of the medicinal plant Lagenaria breviflora Roberty (LB) which is documented to possess both in vivo and in vitro antioxidant and anti‐inflammatory activities [2].

Nrf2 induction was investigated using the AREc32 cell line, (Nrf2 luciferase assay reporter cell line) and by activation of the Nrf2 regulated gene NQO1 in HepG2 cells by Western blot. Exposure of AREc32 cells to graded concentrations of LB ethyl acetate extract (4mg/ml, 6mg/ml, 8mg/ml and 12mg/ml) significantly increased luciferase activity by 3 – 7.5 fold relative to control. Higher concentrations (8mg/ml and 12mg/ml) were however toxic to the cells (by MTT assay). Kinetic studies revealed significant induction of luciferase activity in AREc32 cells treated with 6mg/ml of the extract for 24 hours (4 fold) and 72 hours (3 fold). LB extract (6mg/ml) also significantly increased the expression of NQO1 protein in HepG2 cells, relative to control, following 24 hour exposure. These results indicate that the ethyl acetate extract of LB contains Nrf2 modulating activity and as such indicates that LB is worthy of further investigation into its potential to act as a cancer chemopreventative agent.

Support or Funding Information

This research was supported by TETFund Staff Development Grant (Nigeria) Awarded to Dr. Olayinka A. Oridupa
Characterization of Nitrogen Mustard‐Induced Lung Injury Using Molecular Imaging Techniques
Authors: Venosa Alessandro , Schumacher Justin , Adler Derek , Laskin Jeffrey , Yurkow Edward , Gow Andrew , Laskin Debra
Published: November 2023

Nitrogen mustard (NM) is an alkylating agent known to cause acute lung injury, which progresses to fibrosis. High definition magnetic resonance imaging (MRI) and computed tomography (CT) were used to follow the progression of NM‐induced injury. Male rats (8–10 wk) were treated i.t. with 0.125 mg/kg NM or control. Gradient (GRE) and fast spin echo (FSE) MRI, and CT scans were performed 1–28 d later. Image processing was used to analyze and quantify MRI scans for areas of lung injury. NM exposure was associated with an increase in lung injury volume which was evident within 1–3 d; this coincided with acute injury in histologic sections. Whereas histology and bronchoalveolar lavage fluid analysis indicated a time related decrease in extravascular lung fluids, MRI imaging showed low‐density shadows, indicative of fluid buildup, up to 28 d. CT scans were used to generate a high definition 3D reconstruction of the lung. Based on pixel intensity, lung images were segmented and the volume of air, dynamic and consolidated lung tissue identified. Within 3 d of NM dynamic lung volume was significantly decreased, when compared to controls, while the volume of consolidated lung tissue increased with time. These data show that MRI and CT imaging effectively track the progression of NM‐induced lung injury and have the potential to be used to identify a signature of acute injury and fibrosis.

Support or Funding Information

NIH AR055073, HL096426, ES004738, ES005022
Altered Ion Transport in Normal Human Bronchial Epithelial (NHBE) Cells Following Exposure to Metal Welding Particles
Authors: Fedan Jeffrey S , Thompson Janet A , Meighan Terence G , Erdely Patti C , Antonini James M
Published: November 2023

Inhalation of welding fumes is associated with adverse effects in the pulmonary system, including susceptibility to infection. Several types of welding processes occur, giving rise to fumes of different compositions. In this study we investigated the effects of particles derived from fumes generated from manual metal arc‐stainless steel (MMA‐SS) and gas metalarc‐mild steel (GMA‐MS) welding on ion transport in NHBE in culture. The toxicities and the composition of the particles generated during MMA‐SS and GMA‐MS processes are different. MMA‐SS particles are more soluble and contain Cr, Ni, Fe and Mn, whereas GMA‐MS particles are less soluble and contain Fe and Mn. Ion transport is critical to the maintenance of the airway surface liquid. We hypothesized that epithelial cells are targets of inhaled welding fumetoxicity. NHBE (Lonza) were cultured in submersion culture for 3 days followed by 21 d in air‐interface on permeable inserts; cells with transepithelial resistance (Rt) >1000 Ω·cm2were used. Generated MMA‐SS or GMA‐MS particles collected on filters were suspended in medium and applied (50 μl) to the apical chamber. The doses of both types of particles ranged from 0.0167 to166.7 μg/cm2. After 18 h of incubation the inserts were placed in Ussing chambers for the measurement of transepithelial potential difference (Vt), short circuit current (Isc) and Rt. The effects of fume exposure on Na+ and Cl− channels, the Na+,K+,2Cl−‐cotransporter and the Na+,K+‐pump, were evaluated by applying the following inhibitors, respectively: amiloride(A; apical; 3×10−5 M), NPPB (N; apical; 10−4 M), bumetanide (B; 10−4 M) and ouabain (O; basolateral; 10−4M). Changes in lactate dehydrogenase(LDH) levels in apical medium also were assessed after exposure. Following exposure, 0.0167–16.7 μg/cm2MMA increased basal Vt. Only16.7 μg/cm2 GMA‐MS increased basal Vt. MMA‐SS exposure potentiated Isc responses (decreases in Isc) to A and B, while not affecting those to N and O. Likewise, exposure to GMA‐MS potentiated Isc responses to A and B, but to a lesser degree than observed after MMA‐SS exposure. Variable effects on Rt were observed in response to A, B and O. Generally, MMA‐SS was more potent in altering responses to A and B than GMA‐MS. MMA‐SS evoked greater increases in LDH release than GMA‐MS, but bioelectric changes occurred in the absence of increased LDH release. Our findings indicate that both apical Na+ transport and basolateral Na+,K+,2Cl−‐cotransportin airway epithelium is affected by fume particle exposure. The greater effect of MMA‐MS compared to GMA‐MS might reflect the actions of soluble metals. Enhanced Na+ absorption and decreased airway surface liquid could compromise lung defenses against infection.

Support or Funding Information

Supported by NIOSH
Evaluation of the embryotoxic and teratogenic effect of the β‐HPC in ovo
Authors: Mendoza‐Rivera Brissa , Reyes‐Esparza Jorge , De la Cruz‐Cordero Ricardo , Rodriguez‐Fragoso Lourdes
Published: November 2023

Exposure of drugs during the pregnancy may have different effects on the development of the embryo, depending on the period of the exhibition. Injuries during the division of the zygote and implementation that can have effects such as malformations, dysfunctions, alterations in growth, stillbirth and altered postnatal functions are called embryotoxicity. On the other hand, irreversible lesions that are compatible with life, but which may result in structural or functional abnormalities in the born products, it is called teratogenicity. The objective of this study was to evaluate whether the β‐HPC produces embryotoxic and teratogenic effects in ovo. Eggs were incubated for 24 h at 37°C and 60% humidity. We included the following treatment groups (n=5): control, negative control, positive control (caffeine 10 mg/mL, for embryotoxicity test) and 60, 120, 240 and 480 μg/mL of β‐HPC. 1 mL of albumin was removed and was replaced by 1 mL of treatment, and sealed with paraffin. The embryos were obtained after 48 hours of incubation with the treatment. They were fixed, dehydrated and were stained for observation. For teratogenicity study, has followed the same procedure, only the embryos developed fully until hatch. Once outside of the hatched chickens were analyzed to verify the presence of abnormalities. Embryotoxicity results showed that β‐HPC different concentrations did not induce structural malformations in embryos. However, the highest dose induced a higher stage compared to the control. In the results of teratogenicity was observed that β‐HPC, did not induce malformations in the development of the chicken. However, it was observed that the development of the chickens was lower, concentration‐dependent. The highest concentration (480 mg/mL) induced a smaller stadium. In conclusion the β‐HPC did not induce effects embryotoxic and teratogenic effects, however, can detain the development of chicken, concentration‐dependent.