bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Characterization of a Novel Role for Rfa2 N‐terminal Hyper‐phosphorylation During Checkpoint Adaptation
Authors: Wilson Timothy M , Baumgartner Trevor A , Senger Barbara L , Miles Nolan M , Haring Stuart J
Published: November 2023

Replication Protein A (RPA) is an essential heterotrimeric complex found in all eukaryotic organisms and has critical roles in DNA metabolism and cell cycle progression. In human cells, RPA is known to be hyper‐phosphorylated, primarily on the N‐terminus of the second subunit (Rpa2), in response to genotoxic stress. The significance and function of Rpa2 N‐terminal (NT) phosphorylation has been the subject of over 25 years of research; however, the physiological function of this modification has yet to be clearly determined. In budding yeast, Rpa2 N‐terminal phosphorylation is detectable upon prolonged exposure to commonly used clastogens or upon generation of an irreparable break at the MAT locus using HO endonuclease. Upon cleavage of the MAT locus, the master checkpoint kinase Rad53 becomes hyper‐phosphorylated, leading to signaling events that arrest yeast cells at the G2/M checkpoint to allow adequate time for DNA repair.

During checkpoint adaptation, cell cycle progression restarts, despite the presence of unrepaired DNA. Checkpoint adaptation affords cells the ability to bypass an established checkpoint, with the potential negative consequence of cellular division with broken chromosomes, leading to mutations and/or chromosome loss. Through the use of functional and phenotypic analysis of yeast containing Rpa2 NT mutations, we have determined that the phospho‐state of the Rpa2 NT may play a key role in mediating checkpoint adaptation. We demonstrate here that the Rpa2 NT is necessary for, and readily hyper‐phosphorylated during, checkpoint adaptation. We have determined the subregions of the Rpa2 NT that promote checkpoint adaptation when phosphorylated, as well as some of the conserved kinases that appear to phosphorylate the Rpa2 NT during checkpoint adaptation. Finally, we propose a model by which Rfa2 NT hyper‐phosphorylation directly leads to dephosphorylation/deactivation of the master checkpoint signaling kinase Rad53 in order to coordinate checkpoint adaptation in yeast. Understanding the mechanistic role the Rpa2 NT plays in coordinated checkpoint exit in the face of persistent damage may shed light on events that lead to deregulation of checkpoint signaling, and subsequently, mechanisms that lead to aberrant cell growth.

Support or Funding Information

This research was supported by NSF‐CAREER 1253723 to SJH
Dissecting the TORC1 Gene Interaction Network by Perturbing Different Subunits
Authors: Gutierrez Cesar Julian Torres , Santos Sean M. , Hartman John L.
Published: November 2023

Target of Rapamycin Complex 1 (TORC1) is a kinase that controls cell growth. Regulation of TORC1 activity contributes to aging and the human orthologous complex mTORC1 often becomes dysregulated in cancer cells. Rapamycin (a TORC1 inhibitor) is an immunosuppressive agent used for patients with organ transplantation or cancer. In Saccharomyces cerevisiae, high Rapamycin concentrations create a nutrient starvation physiology resulting in decreased protein synthesis and entrance into a G0 state. TORC1 is composed of multiple subunits (Kog1/Lst8/Tco89/Tor1/Tor2) that have been extensively studied previously, however their function and interactions with other genes await further characterization. We aim to determine which S. cerevisiae genes have an undiscovered role in the TORC1 complex in relation to decreased expression of Kog1 and to compare these results with previous data from decreased expression of Tor1/Tor2. Tet‐Off‐Kog1 S. cerevisiae (Tetracycline‐Controlled Transcriptional Activation) were grown in different concentrations of Doxycycline (0.5μg/ml‐10μg/ml) and a non‐growth‐inhibitory Rapamycin concentration (5nM) in order to find a concentration that would inhibit cell growth. Tet‐Off‐Kog1 S. cerevisiae were mated with the yeast knockout and knockdown library (YKO/KD) of 5,851 genes using the Synthetic Genetic Array method. The Tet‐Off‐Kog1 YKO/KD was then plated with growth‐inhibiting concentrations of Rapamycin (5nM) and Doxycycline (0.5–1.0–2.0–5.0μg/ml). Cell growth was measured until carrying capacity was reached and analyzed using a quantitative high throughput cell array phenotyping (Q‐HTCP) method. By analyzing the growth of the Tet‐Off‐Kog1 YKO/KD and comparing them to a distribution of references and previous Tor1/Tor2 data, we identified gene deletions buffering cell proliferation against loss of TORC1.

Support or Funding Information

This research was partially supported by the UAB Preparation for Graduate and Medical Education (PARAdiGM) Program through a grant from the National Institutes of Health (NIH/NHLBI R25 HL120883) and the RISE Program (NIH Grant #R25GM096955).
Anticancer activity of novel benzimidazole derivatives against MCF‐7 cancer cells
Authors: Özdemir Aysun , Uzunoğlu Sümeyye , Çalışkan Burcu , Banoğlu Erden , Ark Mustafa
Published: November 2023

Cancer takes the third place in the cause of death worldwide after cardiovascular and infectious disease. Benzimidazole derivatives have demonstrated as potential new therapeutics for the treatment of cancer in vitro and in vivo. In this study in order to develop more potent anticancer agents, 27 novel benzimidazole derivatives have been designed and synthesized to evaluate for their in vitro cytotoxic activity in MCF‐7 cells.

Cytotoxic effects of synthesized compounds were evaluated using MTT assay and the most active ones were further studied to determine the mechanism of cytotoxic activity. Four active benzimidazole derivatives, compounds A, 13, 22 and 23, were chosen with the IC50 values of 19.93±1.84, 16.06±7.47, 3.98±0.12, 10.59±1.61 μM in MCF‐7 cells, respectively. Treatment of these compounds at 18 μM for 48 h resulted in rounded and shrunken‐cell shape in MCF‐7 cells. Additionally, change in nuclear morphology after the treatments was determined by DAPI staining of nucleus. We found that selected benzimidazole derivatives significantly increased the nuclear fragmentation comparing to control group in MCF‐7 cells. Morever compounds A, 13, 22 and 23 significantly increased the cleavage of PARP‐1 and led to form an inactive fragment of 89 kDa demonstating that caspases are activated after the treatment of compunds. Taken all together our findings indicate that cytotoxic activities of these benzimidazole‐derived compounds are mediated by apoptotic cell death mechanism. These selected benzimidazole derivatives have the potential in the development of chemotherapeutic agents.

Support or Funding Information

This work was supported by TUBITAK in 115S016 project.
Exploiting Immunogenic Modulation in Chordoma: Sublethal Radiation Increases EGFR Expression and Sensitizes Tumor Cells to Cetuximab
Authors: Griner Jake , Padget Michelle , Hodge James
Published: November 2023

Chordoma is a rare primary spinal neoplasm for which standard therapy consists of surgery and radiation. However, the high dose of radiation required to kill chordoma is not always possible given their proximity to the brain and spinal cord. Cancer cells surviving radiation often have phenotypic changes making them more susceptible to immunotherapy, a process termed immunogenic modulation. Among these phenotypic changes is the upregulation of surface markers for which FDA approved monoclonal antibodies exist, such as trastuzumab (Anti‐HER2), rituximab (anti‐CD20), and cetuximab (anti‐EGFR). Upregulation of monoclonal antibody targets have previously been shown to increase natural killer (NK) cell lysis by antibody dependent cell‐mediated cytotoxicity (ADCC) in both chordoma and other carcinoma types. It is known that approximately 80% of chordomas express Epidermal Growth Factor Receptor (EGFR), though to varying degrees of positivity. We hypothesized that sublethal radiation of chordoma cells would increase EGFR expression and enhance the therapeutic effects of cetuximab. Three distinct chordoma cell lines were exposed to sublethal doses of radiation and changes in surface EGFR expression were monitored via flow cytometry. ADCC assays were then performed using NK cells from normal donors and cetuximab. We show here that chordoma cells surviving low dose ionizing radiation express increased EGFR levels. The functional consequence of this upregulation is significantly decreased proliferation compared to either radiation or cetuximab alone. ADCC assays also show increased killing in cells that have been irradiated compared to those which have not. While both radiotherapy and cetuximab have had mixed results as monotherapies for chordoma, our results suggest that cetuximab in combination with radiation will produce a more complete response than either therapy alone.

Support or Funding Information

National Cancer Institute
Connections between NF‐κB Misregulation and Carcinogenesis
Authors: Link Tina , Chyong Donian , Naderi Aurian , Lee Chase , Arpornsuksant Ty , Lu Allison , Dilip Megha , Zhang Margaret
Published: November 2023

In this review poster, we researched the function, regulation, and structure of NF‐κB as it relates to carcinogenesis. Found in almost all animal cells, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) is a transcription factor that plays various roles in cellular proliferation, cell survival, inflammation, and T cell activation. There are two different NF‐κB signaling pathways: the canonical pathway and the non‐canonical pathway. NF‐κB forms a p50/Rel A heterodimer in the canonical pathway and a p52/Rel B heterodimer in the non‐canonical pathway. NF‐κB protein is normally sequestered in the cytoplasm as an inactive complex with a κB inhibitor (IκB) protein. Outside stimuli such as reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF‐α) activate cell surface receptors such as Toll‐Like Receptors (TLRs) and Receptor Activators of NF‐κB (RANK). These surface receptors, in turn, activate IκB kinase (IKK). IKK phosphorylates IκB, which causes ubiquitination of IκB, signaling proteasomes to degrade it. NF‐κB then enters the nucleus through a nuclear pore and forms a transcription complex with coactivators and RNA polymerase. NF‐κB binds to specific DNA‐binding sites to induce transcription. One of the target genes of NF‐κB encodes IκBs, so NF‐κB regulates its own activity through a negative feedback loop. Because the cell maintains storage of the signaling protein NF‐κB in the cytoplasm, NF‐κB allows the cell to respond to sudden stimuli by activating quick changes in gene expression. Because proteins that promote cellular proliferation and cell survival are mainly transcribed through NF‐κB activation, uncontrolled overactivation of this transcription factor has major implications in carcinogenesis. Many types of cancer cells have high levels of constitutively active NF‐κB. Mutations in the genes for NF‐κB itself or its inhibitors such as IκBs often found in cancer cells can cause cells to accelerate their proliferation or confer resistance to apoptosis. Some types of cancer cells secrete signaling molecules, inducing improper NF‐κB activity. Because of the tumor promoting characteristics of NF‐κB activation, a focus of current research is to find methods of suppressing NF‐κB to kill cancer cells or slow their growth. We designed a 3D‐printed model of the p50/RelA NF‐κB protein using Jmol to highlight the relationship between its structure and function.

Support or Funding Information

Diane Munzenmaier, Ph.D., Center for Biomolecular Modeling, Milwaukee School of

Engineering
Characterizing the role of the Na+‐ H+ Exchanger Isoform 1 (NHE1) in cell proliferation and migration in ovarian cancer cells.
Authors: Manzella Taylor M , Provost Joseph J , Wallert Mark A.
Published: November 2023

To sustain proliferative growth, cancer cells exhibit disrupted signaling pathways that compromise the ability of the cell to regulate homeostasis. The Na+‐ H+ Exchanger Isoform 1 (NHE1) is a transmembrane protein activated in cancer cells that plays a role in sustaining proliferative growth. In this study, three ovarian adenocarcinoma lines, SKOV‐3, OVCAR‐3, and CAOV‐3 have been characterized to determine the role that NHE1 plays in their growth and progression. We have used proliferation assays to demonstrate NHE1 involvement in the regulation of cell growth in ovarian cancer cell lines. Cells growth is stimulated when cells are cultured in 10% serum compared to 0.5% serum and this growth stimulation is blocked by the NHE1 inhibitor Cariporide. Additionally, we investigated the effects of four inhibitors that block specific kinases that phosphorylate NHE1 to evaluate their impact on cell proliferation. Specific inhibitors, MK2206 (AKT), Sch772984 (ERK), Y27632 (Rock), and B‐ID1870 (RSK) were evaluated in the three ovarian cell lines to determine key pathways in regulating NHE1. Amongst the SKOV‐3 and CAOV‐3 cell lines, the RSK site inhibitor, B‐ID1870 demonstrated the largest impact of inhibition while in the OVCAR‐3 cell line, the AKT site inhibitor, MK2206, demonstrated the greatest impact. In addition to serum stimulation we are evaluating the ability of lysophosphatidic acid (LPA) and urokinase‐type plasminogen activator (uPA) to activate NHE1 and stimulate proliferation in these cells. Further investigation of the role of NHE1 will include preparing an NHE1 knock out cell line using CRISPR‐CAS9 gene editing technology as well as the evaluation of the role of NHE1 in invasion and metastasis using ECIS migration assays to define cell growth.
Salicylates Promote ROS‐induced Endoplasmic Reticulum Stress Triggering Apoptosis on B16F10 Melanoma
Authors: Ausina Priscila , Albanese Jamille Mansur , Leandro João Gabriel Bernardo , Mendonça Ana Paula Miranda , Palhano Fernando , Foguel Débora , Oliveira Marcus Fernandes , Zancan Patricia , Sola‐Penna Mauro
Published: November 2023

Endoplasmic reticulum stress (ERS) and activation response to this stress have been documented in several types of cancer, suggesting that these cells activate the stress response as a survival strategy. Thus, drugs that cause an imbalance in this response may impair cancer cell viability. Salicylates have proven preventive and anti‐tumor effects in various types of cancer. On regard of ERS, it has been reported to act as potent inhibitor of protein synthesis in rat islets and in the gastric mucosa of guinea pigs. Salicylates also activate ERS through PERK activation in human promonocytic cells. In addition, the treatment with salicylates is correlated to increased reactive oxygen species (ROS) production, probably due to the direct inhibition of catalase activity. Nevertheless, there is still no correlation among salicylates, ERS and cancer. The aim of the present work is to investigate a new mechanism for salicylic acid (SA) and acetylsalicylic acid (ASA) related to their antitumoral effects, which involves the induction of ERS by ROS, and corroborate the new perspectives for the use of these drugs. Melanoma B16F10 cell line was used in all experiments and treated with 10mM SA or ASA. We tested cell viability and apoptosis using Anexin V assay, and ROS production using DCFDA, both by cell sorting. Unfolded protein response (UPR) markers were evaluated by qPCR and Western blot analysis. The treatment of the cells with SA or ASA for 1 or 2 hours promoted an increase in ROS production, as well as in the mRNA levels of the UPR markers ATF4 and CHOP. Increased protein levels of other UPR markers, such as PERK, IRE1, ATF6, cleaved ATF6, BiP and CHOP, are detected within 24 hours of treatment with both drugs. Finally, the treatment triggers apoptosis, which was detected on its early stage after 24 hours. Taken together, our data indicate that treatment of melanoma B16F10 cells with SA or ASA reduces cell viability thought a ROS‐induced ERS mechanism.

Support or Funding Information

Financial support: FAPERJ, CAPES, CNPq
TG‐interacting Factor Can Elicit Hedgehog Pathway to Increase Resistance to Gemcitabine in Urothelial Carcinoma
Authors: Huang Huei‐Sheng , Huang Yue‐Jhu
Published: November 2023

Urothelial carcinoma (UC) of the bladder is the 4th most commonly diagnosed cancer and the 8th most common cancer‐related death in male in the United States in 2016. Using gemcitabine/cisplatin as a chemotherapy after surgery of patients with UC is a common treatment. However, the drug‐resistance to the therapy and high recurrence of UC patients usually occurred. Previously, we have demonstrated that TG‐interacting factor (TGIF) is involved in the migration/invasion, poor prognosis, and gemcitabine‐resistance of UC via PI3K/AKT pathway. It also has been proven that the aberrant hedgehog pathway is associated with many cancers and drug‐resistance. Therefore, whether the hedgehog pathway was involved in the gemcitabine‐resistance of UC was addressed. In the present study, we observed that expression of TGIF, pAKTser473, pGSK3βser9 and hedgehog components (Gli2, Smo, and Ptch) was increased in gemcitabine‐resistant NTUB1 (NGR) cells. Overexpression of TGIF or AKT1 in NTUB1 cells increased activity of GLI‐luciferase reporter and Gli2 expression. Furthermore, overexpression of AKT1 could phosphorylate pGSK3βser9 to inactivate its kinase activity and induce activation of hedgehog pathway. In addition, overexpression of GSKS9D could inactivate GSK3β kinase and increase activity of hedgehog pathway. However, overexpression of GSKS9A could activate GSK3β kinase to suppress hedgehog pathway activation. Finally, overexpression of Gli2 increased resistance to gemcitabine in NTUB1 cells, whereas, knockdown of Gli2 reduced resistance to gemcitabine in NGR cells. Accordingly, we suggest that gemcitabine‐induced TGIF expression can activate AKT/GSK3β signaling to elicit hedgehog pathway, which results in the gemcitabine resistance of UC.

Support or Funding Information

This work was supported by grants from the National Science Council (Taipei, Taiwan; NSC102‐2321‐B‐006‐019‐MY2; MOST104‐2314‐B‐006‐055‐MY3)
The chemosensory bitter taste receptors (T2Rs) are involved in proliferation and migration of breast cancer
Authors: Singh Nisha , Shaik Feroz Ahmed , Bhullar Rajinder Pal , Chelikani Prashen
Published: November 2023

Background and Objective

Breast cancer is a very complex disease and involves interactions between many proteins. The human bitter taste receptors (T2Rs) are a group of 25 chemosensory proteins that belong to the G protein‐coupled receptor family. T2Rs mediate signal transduction in response to stimulation by a wide variety of bitter compounds. Earlier studies showed that bitter agonists such as quinidine and chloroquine triggered apoptosis in MCF‐7 breast cancer cells, and bitter melon extract inhibited breast cancer cell proliferation by modulating cell cycle regulatory genes and promoting apoptosis. However, the cell surface receptor (T2R) targets for these bitter compounds, and their expression and characterization in the cancer cells is not yet elucidated.

Methods

To delineate the expression profile of T2Rs in breast cancer MDA‐MB‐231 and MCF‐7 cells and non‐cancerous MCF‐10A cell line, quantitative PCR (qPCR) and flow cytometry analysis was done. Next, analysis of T2R4 and T2R49 in human breast cancer tissue panel was performed using qPCR array (Origene Inc.). Calcium mobilization experiment was pursued to analyze the functional response after stimulation of these cells using bitter compounds. Cell proliferation was pursued using MTT assay and migration analysis by RTCA xCELLigence cell system after stimulating the cells with T2R ligands.

Results

Our results suggest differential expression of T2Rs is breast cancer cells. Stimulation of endogenous T2Rs with bitter agonists leads to increase in intracellular calcium release. Breast cancer cells treated with T2R4 agonist show decrease in cell proliferation and migration compared to non‐cancerous cells. This effect was reversed upon T2R4 antagonist treatment. Furthermore, knockdown and/or overexpression studies of T2R4 in these cells suggest its role in inhibition of cell proliferation and migration of breast cancer cells.

Conclusions

Our results suggest that T2Rs are involved in decrease of breast cancer cell proliferation and migration. Investigating the mechanistic role of these chemosensory T2Rs in breast cancer would aid in developing strategies to prevent and/or treat breast cancer.

Support or Funding Information

The Natural Sciences and Engineering Research Council of Canada (NSERC)
Analysis of Telomere Length in Aging and Age ‐Related Illness
Authors: Connon Scott M , Einstein George P , Tulp Orien L
Published: November 2023

The purpose of this study was to create a detailed analysis of the factual concepts and theoretical principles of telomeres and their application in aging and disease. The analysis is based on the current genetic knowledge of the telomere and its main enzyme telomerase as well considerayion and evaluation of theoretical applications of telomere status. The assessment of telomere lengths were the main concepts being analyzed in this thesis. This area of genetics is being studied to explore its many benefits to understanding key functions of the human chromosome as it relates to wellness and disease during the lifespan. There are many challenges facing the advancement of telomere studies and its application to disease and wellness. For the purposes of this analysis the telomere length and its role in the immune system and aging were the focus of the meta analysis. A strong immune system and ability to aid in the prevention and proliferation of disease can theoretically be associated with the telomere. Free radical and oxidative stress of the cell may also be comprising factors. Cancer is one of the largest chalenges facing modern medicine today. There are many theoretical correlations between the cancer cell mortality and the length of the telomere. Additionally, the amount of telomerase can influence the life span of the cancer cell and making the cell immortal. A greater understanding of this aspect of the chromosome will encourage more research and testing of the telomere. Published data from meta‐analysis and research have shown interesting results in the correlation of decreasing telomere lengths with aging diseases and cancers. Further research may be extremely valuable to patients suffering from age associated disease and tumor related cancers.

Support or Funding Information

Supported by Institutional Resources of USAT College of Graduate Studies and Einstein Medical Institute
Follicle Stimulating Hormone and Reproductive Aging
Authors: Pardue Avery , Dvoretz Illay , Wright Collette , Custard Alaura , Barbara Ella , Langat Diana
Published: November 2023

Follicle‐stimulating hormone (FSH) is a member of the glycoprotein hormone (GPH) family. It is secreted by gonadotropes, which are found in the anterior pituitary gland. FSH is an important hormone for the production and development of gametes in both men and women. The hormone is a part of a cascade that begins with the release of gonadotropin‐releasing hormone that is released from the hypothalamus and stimulates gonadotropes in the pituitary gland. These stimulated gonadotropes release FSH and the luteinizing hormone (LH) into the bloodstream where they travel through the bloodstream to the gonads. In males, FSH stimulate the Sertoli cells to produce a ABP, or androgen‐binding protein, and inhibin. ABP keeps testicular testosterone concentrations elevated for spermatogenesis, while inhibin feeds back to the anterior pituitary to cease production of FSH. LH stimulates testicular Leydig cells to synthesize and secrete testosterone. In females, FSH stimulates ovarian follicular granulosa cells to continue to grow and divide, secrete estradiol, and become responsive to LH stimulation. LH stimulates ovarian theca cells to produce the androgen precursor converted to estradiol by granulosa cells. The LH surge triggers ovulation and LH stimulates progesterone synthesis and release by the corpus luteum during the luteal phase of the menstrual cycle. FSH binds to the FSH receptor (FSHR) to stimulate cellular responses. The FSHR is a G protein‐coupled receptor (GPCR) that possesses an exceptionally large N‐terminal ectodomain composed of leucine‐rich repeats. Glycoprotein hormones, which FSH is member, are identified by a common alpha‐ and hormone‐specific beta‐subunit heterodimeric structure. The alpha‐subunit peptide sequence and disulfide bond pattern are consistent throughout all glycoprotein hormones. The beta‐subunit is what dictates the function of the hormone and dictates binding to the cognate receptor. The alpha and beta subunits are folded into non‐globular, cystine knot structures with three loops extending from the core motif, which consists of three disulfide bridges. An additional peptide emerges from the beta subunit and wraps around the L2 loop of the alpha subunit in a manner similar to a seatbelt. The seatbelt loop possesses the hormone‐specific determinants of the FSH beta‐subunit. FSH binds to the FSHR. Current research is focused on the effect of FSH glycosylation variations on the activation of the receptor.

Support or Funding Information

This is a SMART Team project supported through the contributions of Dr. George R. Bousfield of Wichita State University, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.
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 Information

Elon 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 Information

National 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 Information

The 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 future

Support or Funding Information

JUST 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

Background

We 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.

Methods

NPC2‐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).

Results

Immunostaining 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.

Conclusion

This 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 Information

NIH 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 Information

The 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

Rationale

Seasonal 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.

Methods

A549 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.

Results

Induction 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.

Conclusions

Type 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 Information

Kristin M. Wiese is supported in part by NIH/NHLBI training grant 2T32HL076139‐12 and also through funding from HL071643