bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optogenetic Stimulation of BLA Cholinergic Terminals Induces a Hypophagic Phenotype
Authors: Addison Angela Patricia , Ortiz‐Guzman Joshua , Arenkiel Benjamin Russell 
Published: November 2023

Eating disorders in the United States have a morbidity rate of over 7,000 lives a year, thus highlighting the need for new treatment therapies. Towards this goal, a more complete understanding of the neuronal circuitry underlying feeding behavior is required. To date, feeding behavior has been largely attributed to the neural circuits that reside in the hypothalamus. However, a recent study from our lab suggests that acetylcholine (ACh) signaling and associated cholinergic circuits play a critical role in feeding behavior.Alongside the hypothalamus, the limbic system influences autonomic functions that dictate many behaviors. Interestingly, we have recently found that cholinergic neurons that populate the Diagonal Band of Broca (DBB) innervate both feeding and limbic associated structures. Through immunohistochemical and genetic tracing studies, we identified cholinergic terminals from the DBB in both the hypothalamus and Basolateral Amygdala (BLA). Moreover, we found that ablation of DBB cholinergic neurons that project to the BLA resulted in hyperphagic mice that developed overt and rapid obesity. Strikingly, these mice were up to 2.5 times larger than their control counterparts. Although we found that ablating these cholinergic neurons resulted in an obese phenotype, it was unknown if the ACh release in the BLA was directly related to eating. Therefore, we sought to test the hypothesis that: DBB cholinergic neurons through ACh release in the BLA regulate feeding behaviors.To investigate this question, we targeted DBB cholinergic neurons for cell type‐specific activity manipulations. To increase cholinergic signaling to the BLA, we stereotaxically delivered an adeno‐associated virus (AAV) engineered to express the light‐gated ion channel, Channelrhodopsin‐2 (ChR‐2) to DBB cholinergic neurons, and implanted an optic fiber into the BLA. These fiber optic implants provided cell type‐specific and spatially‐restricted stimulation through controlled cholinergic release from BLA projecting cholinergic terminals. Baseline food intake, prior to ChR2‐mediated photostimulation, was found to be approximately 3.5 grams. Following an overnight fast, cholinergic terminals were acutely stimulated the next day. After stimulating the terminals for an hour, food was presented to the subject and subsequently measured for consumption. During stimulation, the experimental mice ate significantly less food than their controls. Notably, when the light was turned off, the mice resumed normal eating patterns. Together, these data indicate that stimulation of the BLA cholinergic terminals, and the subsequent release of ACh, results in a hypophagic phenotype.
Measuring Nestin Expression in RA‐BDNF Differentiated SH‐SY5Y Human Neuroblastoma Cells by Flow Cytometry
Authors: Graham Jessica , Train Tonya Laakko 
Published: November 2023

The SH‐SY5Y human neuroblastoma cell line acquires many of the characteristics of primary neuronal cultures when sequentially treated with all‐trans‐retinoic acid (RA) and brain‐derived neurotrophic factor (BDNF). Therefore, this cell line can be used as an in vitro model for human neurotoxicity and neurodegenerative disease studies. Some toxins may affect the differentiation process and methods including western blot, qRT‐PCR, and immunocytochemistry are commonly used to measure the expression of neuronal specific markers in order to quantify differentiation. Flow cytometry is more rapid and quantitative in comparison making it a useful tool in studying neuronal differentiation and the modulation of neuronal biomarkers. Nestin is a cytoskeletal protein that is downregulated upon neuronal differentiation. Here we investigated differentiation by measuring nestin expression by flow cytometry. SH‐SY5Y cells were induced to undergo differentiation by treatment with retinoic acid for five days and BDNF for an additional three days. Cells were fixed, permeabilized and labeled with fluorochrome conjugated anti‐nestin antibody. Differentiated cells yielded a 55% reduction in mean nestin fluorescence. Imaging studies additionally confirmed neuronal differentiation. These data suggest that flow cytometry can be used to assess nestin expression and differentiation in SH‐SY5Y cells.Support or Funding InformationElon College Fellows Program and Lumen Scholars Program
Neural Stem Cells Promote Nerve Regeneration through IL12‐induced Schwann cell Differentiation
Authors: Chiu Ing‐Ming , Lee Don‐Ching , Chen Jong‐Hang 
Published: November 2023

Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting analyses. Levels of IL12p80 in these conduits are 1.6‐fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5‐fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and, in turn, enhance the functional recovery and increase the diameter of regenerated nerves in a mouse sciatic nerve injury model.Support or Funding InformationNational Health Research Institutes, Taiwan; Ministry of Science and Technology, Taiwan
The Role of Rho/ROCK Pathway on the Sacral Neural Crest Cell Migration in the Mouse Gut
Authors: Zhang Xiaoting , Chan Wood Yee 
Published: November 2023

The enteric nervous system (ENS) originates from neural crest cells (NCCs) at two different levels along the neuraxis: vagal (somites 1–7) and sacral (caudal to somite 24 in the mouse) levels. While the development of vagal NCCs has been widely studied, little has been known about sacral NCCs, particularly in mammals. Our objective was to test whether inhibition of small GTPases such as ROCK (an effector of Rho) affects sacral NCC migration in the mouse gut.First, cultured sacral NCCs from pelvic ganglia were treated with different concentrations of Y‐27632 to suppress active ROCK in vitro, and results showed that inhibition of ROCK significantly decreased neurite outgrowths and migration distances of sacral NCCs. Immunofluorescent localization demonstrated that the expression of cofilin which is a downstream protein of Rho/ROCK pathway remained unchanged while its phosphorylated form p‐cofilin (inactive state) was increased significantly. Expression of phalloidin which is a marker of stress fibers decreased after Y‐27632 treatment. Time‐lapse live cell imaging also showed that the speed and persistence of sacral NCC migration decreased, and the directional migration was disrupted after Y‐27632 treatment. Interestingly, expression of both phalloidin and p‐cofilin in sacral NCCs was restored to normal levels by Rho activator treatment.Next, we established an embryonic gut explant culture, where a pelvic ganglion (serving as a source of sacral NCCs) was recombined with a recipient hindgut segment ex vivo. Whole‐mount preparation and cryosection staining showed that the migration and differentiation of endogenous vagal NCCs in the hindgut explant cultured ex vivo was similar to those in the hindgut developed from E12.5 to E16.5 in vivo. Hence the gut culture explant system could support the normal development of the hindgut explant ex vivo. The recipient hindgut segment into which sacral NCCs from the pelvic ganglion had migrated 2 day after cultured ex vivo was then treated with different concentrations of Y‐27632. It was found that Y‐27632 reduced the migration distance, cell number and axonal extension of sacral NCCs within the hindgut segment. Moreover, time‐lapse live cell imaging also suggested that the speed and persistence of axonal extension of sacral NCCs decreased, and directional migration was disrupted after Y‐27632 treatment.In summary, these observations provide evidence that Rho/ROCK pathway regulates stress fiber formation and directional migration during sacral NCC migration.Support or Funding InformationThe work was supported by grants from RGC General Research Fund (Project Reference: 14102214) and RGC Theme‐based Research Scheme (Project Reference: T12C‐714/14‐R).
Prion ‐ like protein polymerisation underlies signal transduction in innate immunity: The emergence of a universal mechanism ?
Authors: O'Carroll Ailis , Ve Thomas , Moustaqil Mehdi , Giles Nichole , Bhumkar Akshay , Hunter Dominic , Kobe Bostjan , Sierecki Emma , Gambin Yann 
Published: November 2023

The innate immune system uses a multitude of receptors such as Toll‐like Receptors and Nod‐like Receptors to sense pathogens and danger signals. Upon their activation, a signalling cascade is initiated culminating in the transcription and translation of cytokines and other proteins vital for host protection. Recent studies have discovered that an array of these intracellular signalling proteins assemble into oligomeric signalling platforms, consequently amplifying the transmission of the signal from the stimulated receptor to the activation of relevant transcription machinery.Unexpectedly, it was also discovered that some of these proteins present in innate immune signalling such as the inflammasome adaptor protein ASC and the mitochondrial membrane‐associated protein MAVS, can form fibrils. These fibrils can elongate in a prion‐like manner to create super‐sized signalling platforms.In this study, we screened over 100 innate immune intracellular signalling proteins using single molecule fluorescence. We defined the oligomerisation and aggregation propensity of these proteins and track the specific signature of polymerisation. We identified multiple candidates forming super‐sized protein aggregates; through protein seeding experiments, we were able to conclude that ~20 of these proteins could self‐propagate aggregation as found in prion‐like mechanisms.These innate immune cytosolic proteins are not isolated to one signalling pathway but transverse most, if not all, signalling pathways of innate immune defence, inflammation and cell death. Intriguingly, some of these proteins could act to regulate and even inhibit the signalling cascade through their prion‐like protein propagation. Through focusing in on each pathway, their protein‐protein interactions and their disease‐associated point mutations, we are delineating the diverse networks of signalling proteins that drive and regulate inflammation and innate immune defence as well as cell death. Thus, aiding in elucidating the etiology of the diseases associated with these point mutations such as those of cancer‐causing mutations in TLR signalling adaptor proteins.Together these findings reveal a universal mechanism where the creation of polymer‐like platforms increases the number of binding sites and dramatically amplifies the signal. Our observations suggest that prion‐like polymerization underlies the backbone of intracellular communication and drives the complex interplay that exists among the major signalling cascades of the innate immune system. Our disease‐associated mutagenesis data highlights the crucial functions that these large signalling platforms have in the optimal execution of defence against pathogens and intracellular homeostasis and will contribute to the development of therapies aimed at aiding and regulating this critical first line of defence.
Recombinant Adenoviral Vaccine Carrying the S1 Subunit of the Middle East Respiratory Syndrome Coronavirus (MER‐CoV) Spike Gene Can Elicit Cellular Immune Response in Mice
Authors: Ababneh Mustafa 
Published: November 2023

Middle East respiratory syndrome (MERS‐CoV) coronavirus has distributed in a vast area of Middle East countries since 2012. Then the virustransmitted to other continents with high morbidity and mortality rates. Neither drug nor vaccine is currently available against MERS. Spike protein is the main immunogenic part of the MERS‐CoV. Its role in viral entry and replication has been established. As well, it contains the S1 subunit which has the receptor binding domain (RBD). The latest has a prominent role in binding with the host DPP4 and elicit protective immunity. In this study, a recombinant adenoviral‐based vaccine carrying the S1 subunit of MERS‐CoV genome was constructed. Experimental mice were immunized intramuscularly with the vaccine and boosted intranasally three weeks later. Experimental animals were distributed randomly into three groups; group 1 received the recombinant vector vaccine; group 2 received only adenovirus vector and the last group served as control group that were injected with phosphate buffer saline (PBS). The expression of the S1 protein in kidneys and lungs was detected using conventional PCR method targeting 1142 bp region within the S1 subunit at week 3 and 5 post vaccination as well as by Immunohistochemistry technique at week 6. Immune response was evaluated in cell culture obtained from different experimental groups after in vitro stimulation with a specific 9‐mer epitope within the S1 protein (CYSSLILDY). Cytokines such as IFN‐γ, IL‐12 and IL‐4 were analyzed in cell culture supernatant by enzyme linked immunosorbant assay (ELISA) and by qRT‐PCR for only IFN‐γ and IL‐4 in culture cell pellet. In addition, specific antibody response in serum of the experimental animals was also investigated at week 4 and 6 after vaccination. It was revealed that the S1 protein was present in lung and kidney of the vaccinated animals. In Immunohistochemistry technique the S1 protein was clearly expressed in the proximal tubules of mouse kidney of vaccinated animals. This vaccine elicited specific S1‐IgG antibody response which was detected in the sera of the vaccinated mice at week 4 and 6. The results also revealed a significant increase in the amount of Th1‐related cytokines (IFN‐γ and IL‐12), and significant decrease in the Th2‐related cytokine (IL‐4) in the splenocytes cell culture of the vaccinated group was noticed in comparing with those in control groups (vector and PBS). Similar results were shown in the qRT‐PCR analysis to the IFN‐γ and IL‐4 genes expression. These findings reveal that the recombinant adenovirus vaccine carrying the MERS‐S1 subunit can elicit Th1‐immune response (cellular immunity) in mice, and give a promise to develop an effective vaccine that can control and might prevent MERS‐CoV human infection in the futureSupport or Funding InformationJUST deanship grant No. 20140275
Novel Mechanism That Regulates Innate immunity
Authors: Frolov Andrey , Csepeggi Chad , Yang Lihua , Jiang Min , Cook Erik , Crofford Leslie J 
Published: November 2023

BackgroundWe have recently identified Niemann‐Pick type C2 (NPC2) protein as important regulator of somatic cell plasticity and inflammation (1,2). The latter unfolds in the setting of NPC2‐deficiency and is characterized by significant increase in production and secretion of inflammatory mediators and immune modulators including IL‐1β, IL‐6 and prostaglandin E2 (1–3). Here we probed involvement of NPC2 in regulation of innate immunity by examining its role in macrophage differentiation, polarization and in the response of human fibroblasts to the immunogenic stimuli.MethodsNPC2‐null human skin fibroblasts were derived from patients with NPC2 disease (2). The human monocytic cell line THP‐1 and human cryopreserved peripheral blood mononuclear cells (PBMC) were obtained respectively from ATCC and C.T.L. sources. THP‐1 monocyte‐derived macrophages were obtained through PMA stimulation (4). PBMC macrophage differentiation was performed according to C.T.L. protocol. PBMC macrophage polarization was achieved according to (5). Real‐time RT‐PCR analysis was conducted as described (1).ResultsImmunostaining of the lesional area of human atherosclerotic aorta (provided by Dr. N. Weintraub) revealed increased expression of NPC2 protein and its strong association with inflammatory cells. Real‐time RT‐PCR data demonstrated robust and gradual upregulation of NPC2 expression during macrophage differentiation reaching 4.2±0.3 at 24 h and 7.1±1.0 fold at 48 h after PMA stimulation. Silencing NPC2 gene in THP‐1 monocytes with shRNA triggered their spontaneous differentiation into macrophages as evidenced by the upregulated expression of specific markers CD11b (3.2±0.6), MSR1 (2.0±0.05) and IL‐6 (62.6±5.0). NPC2 protein (gift from Dr. C. Heegaard), when it was added to M2 macrophage polarization medium at 6 or 60 nM, skewed macrophage polarization efficiently and dose‐dependently toward M1. Finally, NPC2‐null fibroblasts demonstrated dramatically muted response to IFN‐γ (~ 100‐fold) and to the TLR2, TLR3, TLR4 and MDA‐5 specific ligands (~ 15‐fold) despite significant (up to 5‐fold) increase in the TLRs gene and protein expression.ConclusionThis work identifies a novel, NPC2‐mediated multifaceted mechanism that regulates innate immunity at the levels of macrophage differentiation, polarization, and cell response to immunogenic stimuli.Support or Funding InformationNIH RO1AR049010 and American Heart Association Grant SDG 023017N
Detecting Pal Lipoprotein in Gram‐Negative Sepsis Patients
Authors: Farquharson Kara , Novick Bethany , Snyder Emma , Pichichero Michael , Hellman Judith , Michel Lea 
Published: November 2023

The clinical condition of sepsis often results from a bacterial infection, such as Escherichia coli (E. coli), which then leads to a systemic hyper‐inflammatory response. During E. coli sepsis, a lipoprotein, Pal, has been shown to be released from the bacterial cell and is thought to contribute to the inflammatory response in human cells. Evidence suggests that Pal is implicated in the pathogenesis of E. coli sepsis, but Pal has not been detected in sepsis patients. The goal of this work was to detect released Pal in the human serum and/or urine of E. coli sepsis patients to further confirm Pal's role in sepsis. We used purified recombinant Pal to determine accessible detection levels in healthy human serum and urine, and we employed SDS‐PAGE and Western Blot techniques to detect Pal in E. coli sepsis patient samples. Our preliminary results suggest that we can detect a protein of similar molecular weight to Pal, using a monoclonal anti‐Pal antibody, in the urine of several E. coli sepsis patients.Support or Funding InformationThe Camille and Henry Dreyfus Foundation: Special Grant Program in the Chemical Sciences (L. Michel)
Protection from Influenza A Virus Infection by Modulating Nucleotide‐Binding Oligomerization Domain Containing 2 (NOD2) Signaling
Authors: Wiese Kristin Marie , Koch Clarissa M , Coates Bria , Ridge Karen M 
Published: November 2023

RationaleSeasonal influenza A virus (IAV) infection is a highly contagious respiratory illness that results in over 200,000 hospitalizations per year in the United States. Nucleotide‐binding oligomerization domain containing 2 (NOD2) is an important component of the innate immune response, the body's first line of defense against viral infection. Its activation results in the production of type I interferons which promote pro‐inflammatory cytokines and restriction of viral replication. NOD2 deficient mice are more susceptible to IAV infection, but interestingly amplified expression of NOD2 has also been associated with increased lethality. This suggests that tight regulation of NOD2 signaling is required to adequately control infection without producing immunopathology. We hypothesize that modulation of the NOD2 signaling pathway will result in improved outcomes following IAV infection.MethodsA549 cells were infected with PR8 influenza and NOD2 mRNA expression was measured via quantitative real‐time PCR (qRT‐PCR). NOD2 knockdown (NOD2KD) of human THP‐1 macrophages was accomplished via anti‐NOD siRNA. These cells were subsequently infected with IAV or treated with 5′pppRNA, a NOD2 agonist, and IFN‐β levels were assessed with an ELISA. In vivo, forty plaque‐forming units of WSN Influenza A virus was instilled intratracheally in 8–10 week‐old wild type (WT), NOD2 knock‐out (NOD2−/−), and NOD2 heterozygous knock‐out (NOD2+/−) mice and a death curve was obtained. In a separate experiment, the animals were sacrificed at set time points post‐infection, total cell number, protein concentration and cytokines (IFN‐α, IFN‐β, IL‐18) in bronchoalveolar lavage fluid (BALF) were measured. Lung tissue was collected and used for viral plaque assay and western blot analysis.ResultsInduction of NOD2 signaling following IAV infection was confirmed via qRT‐PCR in vitro. NOD2KD cells infected with IAV or 5′pppRNA failed to stimulate the production of IFN‐β. In vivo, WT and NOD2−/− mice infected with IAV had 100% mortality at 10 days post‐infection. In contrast 50% of the NOD2+/− were alive at this time point. Complete knockdown of NOD2 resulted in the absence of the downstream type I interferon products in BALF. The NOD2+/− mice demonstrated a diminished BALF interferon response when compared to WT.ConclusionsType I interferons are a critical component to influenza A recovery, but in excess can contribute to immunopathology and increased lethality. Modulation of type I interferon production via a heterozygous knock‐out mouse model improved IAV mortality. These data suggest that regulating the activity of NOD2 may allow the advantageous inflammatory aspects of its signaling to be preserved while limiting the likelihood of immunopathology.Support or Funding InformationKristin M. Wiese is supported in part by NIH/NHLBI training grant 2T32HL076139‐12 and also through funding from HL071643