Cell Protein Research Articles

Preoperative risk factors for ileostomy-associated kidney injury in colorectal tumor surgery following ileostomy formation

PurposeDiverting ileostomy is related to postoperative high-output stoma (HOS) leading to kidney injury. The purpose of our study was to clarify the risk factors for ileostomy-associated kidney injury, which is kidney injury starting after the first operation to ileostomy closure after colorectal tumor surgery with diverting ileostomy.MethodsBetween January 2013 and December 2020, 442 patients who underwent colorectal tumor surgery (cancer, neuroendocrine tumor, and leiomyosarcoma) following diverting ileostomy formation were included. We used the KDIGO (Kidney Disease Improving Global Outcomes) guidelines, which defines the acute kidney injury (AKI) to classify patients with ileostomy-associated kidney injury. The definition of AKI was (i) serum creatinine (sCr) ≥ 0.3 mg/dL or (ii) sCr ≥1.5-fold the preoperative level. Multivariate analyses were performed to identify the independent risk factors for kidney injury.ResultsKidney injury developed in 99/442 eligible patients (22.4%). Patients in the kidney injury group were older age, male sex, high American Society of Anesthesiologists Physical Status Classification System (ASA-PS) score, hypertension, cardiovascular diseases, diabetes. The preoperative hemoglobin, albumin, prognostic nutritional index (PNI), and creatinine clearance (CCr) were lower, and the maximum wound length was more extended than the non-kidney injury group. The median highest daily stoma output was significantly higher in the kidney injury group. The postoperative white blood cell (WBC) and C-reactive protein (CRP) levels were also high in the kidney injury group. The univariate analysis showed older age, male sex, high ASA-PS score, hypertension, cardiovascular diseases, and diabetes were the risk factors for kidney injury. The multivariate analysis revealed that age 70 or older, ASA-PS III/IV, hypertension, and HOS ≥2000 ml/day were independent risk factors for kidney injury.ConclusionsSurgeons should consider diverting colostomy creation for patients with risk factors such as age 70 or older, ASA-PS III/IV, and hypertension.

Radioprotective potential of pomegranate peel extract against gamma irradiation-induced hazards

BackgroundWhile gamma irradiation’s damaging biological effects are well-established, the natural radioprotective agents from agricultural waste remain an underexplored area of significant potential.Aim of studyThis study was to investigate the novel use of pomegranate peel ethanol extract (PE) as a radioprotective agent against gamma radiation damage.MethodsWe pretreated Wistar rats with PE (100 mg/kg) for 14 days prior to 6 Gy gamma irradiation. We analyzed blood biochemicals, oxidative stress, and inflammatory markers. These included tests of red cell membrane integrity, lipid and protein oxidation, antioxidant enzyme levels, and cytokine profiles.ResultsThe study showed that PE demonstrated remarkable radioprotective effects across multiple parameters. Antioxidants were significantly enhanced, as evidenced by increased glutathione peroxidase activity (87.00 ± 6.11 mg/ml in PE-treated irradiated rats compared to 26.40 ± 1.21 mg/ml in irradiated controls). Oxidative damage was markedly reduced, with MDA levels dropping from 9.59 ± 0.24 nmol/ml in irradiated controls to near-control levels in PE-treated rats. Notably, PE treatment resulted in unprecedented maintenance of red blood cell membrane integrity post-irradiation. Furthermore, PE exhibited novel modulation of inflammatory cytokines, effectively reducing pro-inflammatory markers IL-6 and TNF-α while simultaneously boosting anti-inflammatory IL-4 and IL-10 levels. These multifaceted protective effects highlight PE’s potential as a comprehensive radioprotective agent.ConclusionThis study presents PE as an effective new natural radioprotective agent. Its protective effect is due to its high polyphenol content, which enhances antioxidant defenses, reduces oxidative damage, and prevents inflammation. The findings open new avenues for sustainable, cost-effective radioprotection strategies and demonstrate the potential for repurposing agricultural byproducts for critical health applications.

Type-H endothelial cell protein Clec14a orchestrates osteoblast activity during trabecular bone formation and patterning

Type-H capillary endothelial cells control bone formation during embryogenesis and postnatal growth but few signalling mechanisms underpinning this influence have been characterised. Here, we identify a highly expressed type-H endothelial cell protein, Clec14a, and explore its role in coordinating osteoblast activity. Expression of Clec14a and its ligand, Mmrn2 are high in murine type-H endothelial cells but absent from osteoblasts. Clec14a−/− mice have premature condensation of the type-H vasculature and expanded distribution of osteoblasts and bone matrix, increased long-bone length and bone density indicative of accelerated skeletal development, and enhanced osteoblast maturation. Antibody-mediated blockade of the Clec14a-Mmrn2 interaction recapitulates the Clec14a−/− phenotype. Endothelial cell expression of Clec14a regulates osteoblast maturation and mineralisation activity during postnatal bone development in mice. This finding underscores the importance of type-H capillary control of osteoblast activity in bone formation and identifies a novel mechanism that mediates this vital cellular crosstalk.

GingisREX: A Complementary Enzyme for the Detection of Bacterial Proteins.

Reliable enzymatic digestion underscores successful mass-spectrometry-based proteomics experiments. In this study, we compare the use of the arginine-specific protease, GingisREX, against a more traditional approach in the identification of Escherichia coli proteins. An increased number of protein identifications were noted when GingisREX was used compared to a trypsin/lys-C mixture. This improvement was attributed to the generation of fewer peptides per protein, resulting in a simpler peptide mixture. Furthermore, GingisREX exhibited increased digestion efficiency, fewer missed cleavages, and improved MS/MS data quality for higher molecular weight peptides. The data here establish GingisREX to be a protease complementary to trypsin for enhanced detection of bacterial proteins. With further optimization, GingisREX could prove to be an effective alternative to trypsin for identifying host cell proteins in biotherapeutics.

CREB3L1 deficiency impairs odontoblastic differentiation and molar dentin deposition partially through the TMEM30B

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.

Clinical features of Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuritis associated with SLE

ObjectiveWe report on the clinical characteristics, treatments and outcomes of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuritis (CIDP) associated with SLE.MethodsPatients treated at Peking Union Medical College Hospital between...

Effect of Gossypol on Gene Expression in Swine Granulosa Cells

Gossypol (GP), a polyphenolic compound in cottonseed, has notable effects on female reproduction and the respiratory system in pigs. This study aimed to discern the alterations in gene expression within swine granulosa cells (GCs) when treated with two concentrations of GP (6.25 and 12.5 µM) for 72 h, in vitro. The analysis revealed significant changes in the expression of numerous genes in the GP-treated groups. A Gene Ontology analysis highlighted that the differentially expressed genes (DEGs) primarily pertained to processes such as the mitotic cell cycle, chromosome organization, centromeric region, and protein binding. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated distinct impacts on various pathways in response to different GP concentrations. Specifically, in the GP6.25 group, pathways related to the cycle oocyte meiosis, progesterone-mediated oocyte maturation, and p53 signaling were prominently affected. Meanwhile, in the GP12.5 group, pathways associated with PI3K-Akt signaling, focal adhesion, HIF-1 signaling, cell cycle, and ECM–receptor interaction showed significant alterations. Notably, genes linked to female reproductive function (CDK1, CCNB1, CPEB1, MMP3), cellular component organization (BIRC5, CYP1A1, TGFB3, COL1A2), and oxidation–reduction processes (PRDX6, MGST1, SOD3) exhibited differential expression in GP-treated groups. These findings offer valuable insights into the changes in GC gene expression in pigs exposed to GP.

Proanthocyanidin Regulates NETosis and Inhibits the Growth and Proliferation of Liver Cancer Cells - In Vivo, In Vitro and In Silico Investigation.

Liver cancer ranks third in global cancer-related mortality, with about 700,000 deaths recorded yearly, making it one of the most common cancers worldwide. Even though prognoses differ according to the severity of the diseases, many patients now exhibit an increased life cycle since the implementation of chemotherapy. In the current study, we investigated the effect of proanthocyanidin ‒a polyphenol molecule found in many plants‒ on the proliferation and invasion of liver cancer cells. In particular, we determined the effect of proanthocyanidin on the serum levels of four strategic liver cancer target, TNFα, IL-6, cfDNA, and IL-1β. Further molecular insight on the inhibitory mechanism of proanthocyanidin against TNFα, IL-6, and IL-1β was obtained via molecular docking, molecular dynamics simulations and binding free energy calculations. Results showed that proanthocyanidin inhibited the growth of HepG2 and HEP3B cells, and effectively reduced clonogenic survival and invasion potential when compared to control cells. Proanthocyanidin was also found to suppress the expression of Bcl-2 (26 kDa) protein in HepG2 cells, while increasing the expression of Bax (21 kDa). Molecular dynamics (MD) and thermodynamic binding free energy calculations showed that proanthocyanidin maintained stable binding within the active site of target proteins across the entire 100 ns MD simulation period, and its binding affinity outscored respective control molecules.In conclusion, the multifaceted analysis showcased in this study demonstrated promising anti-cancer effect of proanthocyanidin on HepG2 and HEP3B cancer cells, highlighting its potential as a viable liver cancer therapeutic alternative.

Nuclear TOP1MT Confers Cisplatin Resistance via Pseudogene in HNSCC.

Cisplatin resistance is one of the major causes of treatment failure in head and neck squamous cell carcinoma (HNSCC). There is an urgent need to uncover the underlying mechanism for developing effective treatment strategies. A quantitative proteomics assay was used to identify differential proteins in cisplatin-resistant cells. Mitochondrial topoisomerase I (TOP1MT) localization was determined using laser confocal microscopy and nucleocytoplasmic separation assay. Chromatin immunoprecipitation sequencing, dual-luciferase reporter assay, and RNA immunoprecipitation were used to identify the interaction between pseudogenes, miRNAs, and real genes. In vivo experiments verified the interaction between TOP1MT and pseudogenes on cisplatin resistance. TOP1MT was identified as a driving factor of cisplatin resistance in vitro, in vivo, and in HNSCC patients. Moreover, TOP1MT exceptionally translocated to the nucleus in cisplatin-resistant HNSCC cells in a signal peptide-dependent manner. Nuclear TOP1MT (nTOP1MT) transcriptionally regulated the mitochondrial functional pseudogene MTATP6P1, which bound to miR-137 and miR-491-5p as a competing endogenous RNA (ceRNA) and promoted the expression of MTATP6. An increase in MTATP6 enhanced mitochondrial oxidative phosphorylation (OXPHOS), which conferred cisplatin resistance in HNSCC. Our findings revealed that nTOP1MT transcriptionally activated MTAPT6P1 and increased MTATP6 expression via ceRNA, which facilitated OXPHOS and cisplatin resistance. These results provide novel insight for overcoming cisplatin resistance in HNSCC.

GAP JUNCTION FUNCTION IS ESSENTIAL FOR SURVIVAL OF ACUTE LYMPHOBLASTIC LEUKEMIA CELLS.

Acute lymphoblastic leukemia has an intimate physical relationship with nonmalignant bone marrow stromal cells. We have recently demonstrated that stromal cells contribute to the survival of leukemia cells and that there is a bidirectional transfer of intracellular material between them. Understanding the mechanisms of stromal support of leukemia may provide insights into new therapies. To test the hypothesis that gap junctions are formed between acute lymphoblastic leukemia cells and nonmalignant stromal cells, and that gap junction function is essential for the survival of leukemia cells. We employed a well-characterized in vitro model of human bone marrow stromal cells and primary human B lymphoblastic leukemia cells and measured leukemia cell survival in coculture using flow cytometry. We measured the effects of gap junction antagonist peptides, carbenoxolone (a drug known to interfere with the gap junction function), and several leukemia chemotherapy drugs including methotrexate upon leukemia cell survival. We demonstrated that stromal cells need to be alive and metabolically active to keep leukemia cells alive. Physical contact between stromal and leukemia cells leads to an increase in gap junction proteins in leukemia cells. Gap junction inhibitory peptides impaired leukemia cell survival as did carbenoxolone, a nonpeptide inhibitor of the gap junction function. Stromal cell survival was not affected. We observed a very modest enhancement of methotrexate antileukemia activity by low-dose carbenoxolone but no significant interactions with dexamethasone, vincristine, mercaptopurine, or doxorubicin. These studies demonstrate that acute lymphoblastic cell survival is impaired by interference with the gap junction function. The development of drugs targeting gap junctions may provide a novel approach to the therapy of acute lymphoblastic leukemia.

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.

MHC class I polypeptide-related sequence B shedding modulates pancreatic tumor immunity via the activation of NKG2DLow T cells.

Natural killer group 2 member D ligands (NKG2DLs) are expressed as stress response proteins in cancer cells. NKG2DLs induce immune cell activation or tumor escape responses, depending on their expression. Human pancreatic cancer cells, PANC-1, express membrane MHC class I polypeptide-related sequence A/B (mMICA/B), whereas soluble MICB (sMICB) is detected in the culture supernatant. We hypothesized that sMICB saturates NKG2D in NKG2DLow T cells and inhibits the activation signal from mMICB to NKG2D. Knockdown of MICB by siRNA reduced sMICB level, downregulated mMICB expression, maintained NKG2DLow T cell activation, and inhibited NKG2DHigh T cell activation. To maintain mMICB expression and downregulate sMICB expression, we inhibited a disintegrin and metalloproteinase (ADAM), a metalloproteinase that sheds MICB. Subsequently, the shedding of MICB was prevented using ADAM17 inhibitors, and the activation of NKG2DLow T cells was maintained. In vivo xenograft model revealed that NKG2DHigh T cells have superior anti-tumor activity. These results elucidate the mechanism of immune escape via sMICB and show potential for the activation of NKG2DLow T cells within the tumor microenvironment.

Preliminary Study on The Characteristics of Effluent From A Cachoeiro de Itapemirim – ES and Analysis of Possible Biotechnological Treatments

Purpose: The objective of this research was to organize and evaluate physical-chemical parameters of the industrial dairy effluent and to propose viable alternative treatments using biotechnological methods, aiming not only to prepare the effluent for disposal but also to generate value-added products Theoretical framework: With the growth of the population, the food industry sector expands exponentially. Brazil ranks among the top five milk producers globally, with the dairy industry being a significant producer of effluent, generating between 1.1 m³ and 6.8 m³ of effluent per cubic meter of processed milk. Dairy wastewater carries a high pollutant load and requires treatment prior to discharge into water bodies. There is an increasing demand for converting waste into raw materials, prompting research into biotechnological treatments that not only prepare effluent for disposal but also yield products from such waste. Method/design/approach: The data on wastewater characteristics were provided by the dairy itself, and the physicochemical aspects of the effluents over 19 months were interpreted, establishing relationships among key parameters, as well as the ratios BOD/COD, BOD: N and COD: N. The survey of biotechnological treatments was conducted through bibliographic research on Google Scholar, Scielo, Web of Science, and Scopus databases. Results and conclusion: Through the analysis and correlations of the physicochemical characteristics of effluents from the Cachoeiro dairy, it is evident that this type of effluent is rich in low biodegradability organic matter, as the BOD5/COD ratio was less than 0.15 in over 79% of the samples, indicating that physical-chemical treatment is the most appropriate approach for this effluent. The linear correlation coefficient between BOD5 and COD was 0.6, suggesting a weak correlation that does not allow one parameter to be accurately predicted from the other. According to the literature, it is possible to derive biofuels, bioplastics, single cell protein, organic acids, polysaccharides, biosurfactants, and other products from dairy effluent, while also meeting environmental requirements Research implications: Evaluation of optimal treatments based on effluent characteristics and suggestion of alternative treatments aiming to utilize wastewater as a raw material for obtaining secondary products complementary to the industry's focus, in addition to compliance with current disposal regulations. Originality/value: Development of relationships to assist in evaluating effluent treatments and identification of treatments that can reuse this substantial volume of generated waste.

Native Digestion and Shotgun Proteomics for Host Cell Protein Profiling of Adeno-Associated Viruses.

Host cell proteins (HCPs) are contaminants of biotherapeutics produced from engineered living systems; they can influence the product's quality, efficacy, and toxicity. Liquid chromatography coupled to mass spectrometry can detect HCPs thereby mitigating their risks. However, highly abundant biotherapeutics hamper the detection of low-level HCPs. Sample preparation termed native digestion has proven effective to preferentially digest and draw out HCPs from intact antibodies. Here, we adapted native digestion to adeno-associated viruses (AAV), which is a vector gaining popularity for gene therapy. We leveraged quantitative proteomics using capillary-flow liquid chromatography-mass spectrometry (LC-MS) and demonstrated that native digestion was more effective than applying denaturing conditions to extract the HCPs associated with different AAV serotypes.

TMT-Based Quantitative Proteomic Profiling of Human Esophageal Cancer Cells Reveals the Potential Mechanism and Potential Therapeutic Targets Associated With Radioresistance.

The recurrence of esophageal squamous cell carcinoma (ESCC) in radiation therapy treatment presents a complex challenge due to its resistance to radiation. However, the mechanism underlying the development of radioresistance in ESCC remains unclear. In this study, we aim to uncover the mechanisms underlying radioresistance in ESCC cells and identify potential targets for radiosensitization. We established two radio-resistant cell lines, TE-1R and KYSE-150R, from the parental ESCC cell lines TE-1 and KYSE-150 through fractionated irradiation. A TMT-based quantitative proteomic profiling approach was applied to identify changes in protein expression patterns. Cell Counting Kit-8, colony formation, γH2AX foci immunofluorescence and comet assays were utilized to validate our findings. The downstream effectors of the DNA repair pathway were confirmed using an HR/NHEJ reporter assay and Western blot analysis. Furthermore, we evaluated the expression of potential targets in ESCC tissues through immunohistochemistry combined with mass spectrometry. Over 2,000 proteins were quantitatively identified in the ESCC cell lysates. A comparison with radio-sensitive cells revealed 61 up-regulated and 14 down-regulated proteins in the radio-resistant cells. Additionally, radiation treatment induced 24 up-regulated and 12 down-regulated proteins in the radio-sensitive ESCC cells. Among the differentially expressed proteins, S100 calcium binding protein A6 (S100A6), glutamine gamma-glutamyltransferase 2 (TGM2), glycogen phosphorylase, brain form (PYGB), and Thymosin Beta 10 (TMSB10) were selected for further validation studies as they were found to be over-expressed in the accumulated radio-resistant ESCC cells and radio-resistant cells. Importantly, high S100A6 expression showed a positive correlation with cancer recurrence in ESCC patients. Our results suggest that several key proteins, including S100A6, TGM2, and PYGB, play a role in the development of radioresistance in ESCC. Our results revealed that several proteins including Protein S100-A6 (S100A6), Protein-glutamine gamma-glutamyltransferase 2 (TGM2), Glycogen phosphorylase, brain form (PYGB) were involved in radio-resistance development. These proteins could potentially serve as biomarkers for ESCC radio-resistance and as therapeutic targets to treat radio-resistant ESCC cells.

Regulatory Mechanisms Governing the Autophagy-Initiating VPS34 Complex and Its inhibitors.

VPS34 is a crucial protein in cells, essential for handling cellular stress through its involvement in autophagy and endocytosis. This protein functions as a Class III phosphatidylinositol 3-kinase, producing phosphatidylinositol 3-phosphate, which is necessary for autophagy and vesicle trafficking. Additionally, VPS34 forms two mutually exclusive complexes, each playing a vital role in autophagy and endocytic sorting. These complexes share common subunits, including VPS15, VPS34, and Beclin 1, with complex I having ATG14 as a specific subunit. Due to its association with various human diseases, regulation of the VPS34 complex I has garnered significant interest, emerging as a potential therapeutic target for drug discovery. Summaries of the structure, function of VPS34 complexes, and developed VPS34 inhibitors have been provided, along with discussions on the regulation mechanism of VPS34, particularly in relation to the initiation complex I of autophagy. This offers valuable insights for treating autophagy-related diseases.

Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Transcript errors generate amyloid-like proteins in huwman cells

Aging is characterized by the accumulation of proteins that display amyloid-like behavior. However, the molecular mechanisms by which these proteins arise remain unclear. Here, we demonstrate that amyloid-like proteins are produced in a variety of human cell types, including stem cells, brain organoids and fully differentiated neurons by mistakes that occur in messenger RNA molecules. Some of these mistakes generate mutant proteins already known to cause disease, while others generate proteins that have not been observed before. Moreover, we show that these mistakes increase when cells are exposed to DNA damage, a major hallmark of human aging. When taken together, these experiments suggest a mechanistic link between the normal aging process and age-related diseases.

12237 Monosynaptic Gabaergic Signaling From Suprachiasmatic Nucleus Neuromedin S Neurons To Preoptic Area Kisspeptin Neurons In Mice

Abstract Disclosure: W.I. Abdulmajeed: None. B.B. Jamieson: None. S.X. Thomas: None. Y. Rim: None. A.G. Novak: None. R.E. Campbell: None. R. Piet: None. In female rodents, projections from suprachiasmatic nucleus (SCN) neurons relay timing cues to the gonadotropin-releasing hormone (GnRH) neuronal network for the preovulatory surge. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) are thought to integrate these cues with estradiol positive feedback to drive GnRH neuron activity, the gonadotropin surge and ovulation. We recently reported that SCN neuron projections release arginine vasopressin (AVP), thereby stimulating female RP3VKiss[1] neuron action potential firing in an estrous cycle stage-dependent manner. In the SCN, AVP is expressed in a subset of neuromedin S (NMS) neurons, a neuronal population essential for circadian rhythms. Moreover, a previous report indicates that exogenous NMS stimulates LH secretion in female rodents. SCNNMS neurons might, therefore, play a role in timing the preovulatory surge. However, whether SCNNMS neurons project to and regulate the activity of RP3VKISS[1] neurons is currently unknown. To address this question, we combined anterograde viral tract-tracing and channelrhodopsin-2 (ChR2)- assisted circuit mapping. Female mice expressing Cre recombinase in NMS cells (NMS-Cre) received stereotaxic injections in the SCN of adeno-associated viral vectors (AAVs) carrying the Cre-dependent sequence for mCherry. Fluorescence immunohistochemistry revealed that mCherry-expressing fibers come in close apposition with the great majority (>80%) of KISS1-immunoreactive neurons in the RP3V, suggesting a SCNNMS-to-RP3VKISS[1] neuron circuit. To interrogate this circuit, we next injected AAVs carrying Cre-dependent channelrhodopsin (ChR2) in the SCN of female NMS-Cre mice that also express the green fluorescent protein in KISS1 cells. Blue LED light (460-487 nm) activation of ChR2-expressing SCNNMS neuronal fibers in the RP3V evoked postsynaptic currents (PSCs) in most KISS1 neurons recorded in whole-cell voltage clamp (-60 mV) in brain slices. Evoked PSCs had short latencies and were blocked by bath application of 0.5 µM tetrodotoxin (TTX), indicating that they were generated by action potential-dependent release. Addition of 100 µM 4-Aminopyridine (4-AP, a potassium channel blocker) to TTX rescued these responses, indicating the existence of a monosynaptic SCNNMS-to-RP3VKISS[1] connection. In addition, evoked PSCs were suppressed by 5 µM gabazine, a GABAA receptor antagonist, but not by 20 µM NBQX and 50 µM D-AP5, AMPA- and NMDA-type glutamate receptor antagonists, respectively, revealing that SCNNMS projections release GABA onto RP3VKISS[1] neurons. Together, our findings are consistent with SCNNMS neurons extending monosynaptic GABAergic projections to RP3VKISS[1] neurons. Further studies are needed to determine the impact of these projections on RP3VKISS[1] neuron activity and the role they might play in the preovulatory surge. Presentation: 6/1/2024

Hiding in plain sight: Optimizing topoisomerase IIα inhibitors into Hsp90β selective binders

Due to their impact on several oncogenic client proteins, the Hsp90 family of chaperones has been widely studied for the development of potential anticancer agents. Although several Hsp90 inhibitors have entered clinical trials, most were unsuccessful because they induced a heat shock response (HSR). This issue can be circumvented by using isoform-selective inhibitors, but the high similarity in the ATP-binding sites between the isoforms presents a challenge. Given that Hsp90 shares a conserved Bergerat fold with bacterial DNA gyrase B and human topoisomerase IIα, we repurposed our ATP-competitive inhibitors of these two proteins for Hsp90 inhibition. We virtually screened a library of in-house inhibitors and identified eleven hits for evaluation of Hsp90 binding. Among these, compound 11 displayed low micromolar affinity for Hsp90 and demonstrated a 12-fold selectivity for Hsp90β over its closest isoform, Hsp90α. Out of 29 prepared analogs, 16 showed a preference for Hsp90β over Hsp90α. Furthermore, eleven of these compounds inhibited the growth of several cancer cell lines in vitro. Notably, compound 24e reduced intracellular levels of Hsp90 client proteins in MCF-7 cells, leading to cell cycle arrest in the G0/G1 phase without inducing HSR. This inhibitor exhibited at least a 27-fold preference for Hsp90β and was selective against topoisomerase IIα, a panel of 22 representative protein kinases, and proved to be non-toxic in a zebrafish larvae toxicology model. Finally, molecular modeling, corroborated by STD NMR studies, and the binding of 24e to the S52A mutant of Hsp90α confirmed that the serine to alanine switch drives the selectivity between the two cytoplasmic isoforms.