Cell Protein Research Articles

Characterization and molecular targeting of CFIm25 (NUDT21/CPSF5) mRNA using miRNAs.

Changes in protein levels of the mammalian cleavage factor, CFIm25, play a role in regulating pathological processes including neural dysfunction, fibrosis, and tumorigenesis. However, despite these effects, little is known about how CFIm25 (NUDT21) expression is regulated at the RNA level. A potential regulator of NUDT21 mRNA are small non-coding microRNAs (miRNAs). In general, miRNAs bind to the 3'untranslated regions (3'UTRs) and can target the bound mRNA for degradation or inhibit translation thus affecting the levels of protein in cells. Interestingly, a mechanism known as alternative polyadenylation (APA) enables mRNAs to escape miRNA regulation by generating mRNAs with 3'UTRs of different sizes. As many miRNA target sites are located within the 3'UTR, shortening the 3'UTR allows mRNAs to evade miRNAs targeting this region. The differences in the lengths and the sequence composition of the 3'UTRs may also impact the mRNA's translatability and subcellular localization. APA has been reported to regulate over 70% of protein coding genes, thus increasing the transcript repertoire. Several proteins, including mammalian cleavage factor, CFIm25 (NUDT21), have been shown to regulate APA. In this study we wanted to determine whether CFIm25 (NUDT21), itself a regulator of APA, undergoes APA to evade miRNA regulation. We used the blood cancer mantle cell lymphoma (MCL) cells as a model and showed that in these cells, NUDT21 is relatively stable with a long half-life. In addition, the NUDT21 pre-mRNA undergoes alternative APA within the same terminal exon. The three different sized NUDT21 mRNAs have different 3'UTR lengths and they each use a different canonical polyadenylation signal, AAUAAA, for 3'end cleavage and polyadenylation. Use of miRNA mimics and inhibitors showed that miR-23a, miR-222, and miR-323a play a significant role in regulating NUDT21 expression. Hence, these results suggest that NUDT21 mRNA is stable and the different 3'UTRs generated through APA of NUDT21 play an important role in evading miRNA regulation and offers insights into how levels of CFIm25 (NUDT21) may be fine-tuned as needed under different physiological and pathological conditions.

Broad-spectrum efficacy of CEACAM6-targeted antibody-drug conjugate with BET protein degrader in colorectal, lung, and breast cancer mouse models.

Despite remarkable advances in cancer treatment, most solid cancers remain difficult to cure. We recently developed an antibody-drug conjugate (ADC, 84-EBET) for pancreatic cancer by using the carcinoembryonic-antigen-related cell-adhesion molecule 6 (CEACAM6) antibody #84.7 and the bromodomain and extra-terminal (BET) protein degrader EBET. Here, we showed the overexpression of CEACAM6 in colorectal, lung, and breast cancers (CRC, LC, BC) and the broad-spectrum efficacy of 84-EBET in mouse models of these cancers. In vitro assays using cancer organoids and cell lines of CRC, LC, and BC revealed that 84-EBET was more potent than ADCs with known approved payloads-DXd, SN38, and monomethyl auristatin E (MMAE)-or standard chemotherapies. In mouse studies, a single injection of 84-EBET induced marked regression of CRC-, LC-, and BC-patient-derived xenograft tumors and cell-line-derived xenograft tumors. Moreover, in mouse syngeneic CRC, LC, and BC models resistant to PD-1 antibody, the combination of 84-EBET and PD-1 antibody induced complete regression of most tumors. Mechanistically, 84-EBET degraded BRD4 protein in both cancer and stromal cells via bystander efficacy. It decreased stromal inflammatory phenotypes and increased activated T-cell numbers in tumors. These results demonstrate that delivering BET protein degraders to tumors and their microenvironments via a CEACAM6-targeted ADC may be effective against a wide range of solid cancers.

BIBR1532 inhibits proliferation and metastasis of esophageal squamous cancer cells by inducing telomere dysregulation

BACKGROUND Esophageal squamous cell carcinoma (ESCC) is a malignant tumor with high morbidity and mortality, and easy to develop resistance to chemotherapeutic agents. Telomeres are DNA-protein complexes located at the termini of chromosomes in eukaryotic cells, which are unreplaceable in maintaining the stability and integrity of genome. Telomerase, an RNA-dependent DNA polymerase, play vital role in telomere length maintain, targeting telomerase is a promising therapeutic strategy for cancer. AIM To investigate the efficacy and underlying mechanisms of BIBR1532, a telomerase inhibitor, in ESCC. METHODS KYSE150 and KYSE410 cells were cultured and exposed to various concentrations of BIBR1532. Cell viability was assessed at 48 hours and 72 hours to determine the IC50 values. The effects of BIBR1532 on ESCC cell proliferation, migration, and cellular senescence were evaluated using the cell counting kit-8 assay, plate colony formation assay, scratch assay, transwell assay, and β-galactosidase staining, respectively. Western blotting was performed to detect the expression of proteins in BIBR1532-treated ESCC cells, such as human telomerase reverse transcriptase (hTERT), key molecules involved in DNA damage response (DDR) or cellular senescence, as well as telomere-binding proteins. Additionally, a tumor-bearing nude mouse model was established to evaluate the anti-cancer effect of BIBR1532 in vivo . RESULTS The IC50 values for KYSE150 and KYSE410 cells after 48 hours of BIBR1532 exposure were 48.53 μM and 39.59 μM, respectively. These values decreased to 37.22 μM and 22.71 μM, respectively, following a longer exposure of 72 hours. BIBR1532 exhibited dose-dependent effects on KYSE150 and KYSE410 cells, including decreased hTERT expression, inhibition of proliferation and metastasis, and induction of cellular senescence. Mechanistically, BIBR1532 upregulated the expression of the DDR protein, γ-H2AX, and activated the ataxia telangiectasia and Rad3-related protein (ATR)/ check point kinase 1 (CHK-1) and ataxia-telangiectasia mutated gene (ATM)/CHK2 pathways. BIBR1532 downregulated the expression of telomere-binding proteins, including telomeric-repeat binding factor 1 (TRF1), TRF2, protection of telomeres 1, and TIN2-interacting protein 1. In a nude mouse xenograft model, BIBR1532 significantly suppressed tumor growth, reduced hTERT expression, and increased γ-H2AX protein levels. Hematoxylin and eosin staining of various organs, including the heart, liver, spleen, lungs, and kidneys, revealed no apparent adverse effects. CONCLUSION BIBR1532 exerts anti-cancer effects on ESCC by inducing DDR through the ATR/CHK1 and ATM/CHK2 pathways and downregulating the expression of telomere-binding proteins.

Tuberculous longitudinal extensive transverse myelitis: 2 cases and literature review

Context This study aimed to investigate the characteristics, diagnosis, and management of tuberculous longitudinally extensive transverse myelitis (TB-LETM), a rare manifestation of tuberculosis. Findings We analyzed two rare cases of TB-LETM and discussed their clinical manifestations and imaging findings in the context of the relevant literature. Patient 1, a 23-year-old female, presented with quadriplegia and dysuria, and spinal magnetic resonance imaging (MRI) revealed lesions extending from C1 to T3. Patient 2, an 18-year-old female, reported acute-onset numbness and weakness in both lower limbs, with MRI showing lesions from T1 to T4, along with multiple intracranial leptomeningeal enhancements. Both patients had elevated cerebrospinal fluid (CSF) cell counts and protein levels, and positive blood T-cell spot test (T-SPOT.TB), but no microbiological evidence of Mycobacterium tuberculosis was found. Diagnosis was based on clinical presentation, medical history, chest computed tomography (CT) findings, and CSF analysis. Over the past decade, 14 cases of TB-LETM have been reported. Common symptoms include fever, acute paralysis, sensory deficits in the lower limbs, and dysuria. Elevated CSF protein, lymphocytosis, and decreased glucose levels are essential for differentiating TB-LETM from autoimmune disorders. Conclusion Longitudinally extensive transverse myelitis (LETM), especially when involving the cervical and thoracic cords, should be considered a potential manifestation of tuberculosis infection. Analysis of cerebrospinal fluid, MRI findings, and T-SPOT testing can provide crucial diagnostic insights. A combination of anti-tuberculosis therapy and corticosteroids has proven clinically effective in managing this condition.

High interstitial fluid pressure enhances USP1-dependent KIF11 protein stability to promote hepatocellular carcinoma progression

BackgroundHCC is characterized by a high interstitial fluid pressure (HIFP) environment, which appears to support cancer cell survival. However, the mechanisms behind this phenomenon are not fully understood.MethodsThis study investigates the role of kinesin family member 11 (KIF11) in HCC under HIFP conditions, using both in vivo and in vitro models. In vitro experiments replicated the HIFP environment to observe changes in HCC cell behavior and protein expression, while in vivo studies involved mice portal hypertension to create an orthotopic liver cancer model, allowing for the evaluation of tumor progression. Additionally, clinical samples from HCC patients were analyzed for consistency with the experimental findings.ResultsResults demonstrated that the HIFP environment significantly increased the proliferation, invasion, and metastasis in HCC cells. Omics analysis and subsequent molecular validation revealed that KIF11 protein levels were markedly upregulated under HIFP, despite no significant change in mRNA levels. Further investigation showed that this upregulation was linked to a reduction in KIF11's ubiquitin-mediated degradation, suggesting that the HIFP environment stabilizes KIF11 expression by inhibiting its degradation pathway. Co-immunoprecipitation and proteomic analysis identified ubiquitin-specific peptidase 1 (USP1) as a crucial factor in this process, deubiquitinating KIF11 at the K77 site, thus stabilizing its protein levels. Clinical analysis confirmed that both USP1 and KIF11 were significantly overexpressed in HCC patients with portal hypertension, with a strong correlation between the two. Inhibition of USP1 using ML323 significantly reduced KIF11 protein levels and suppressed tumor progression in the mouse model.ConclusionThese findings suggest that the HIFP environment fosters HCC progression through USP1-mediated stabilization of KIF11, highlighting USP1 as a potential therapeutic target, especially in patients with portal hypertension.

Multiplex genome editing eliminates lactate production without impacting growth rate in mammalian cells.

The Warburg effect, which describes the fermentation of glucose to lactate even in the presence of oxygen, is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production in cells for bioprocessing have failed as lactate dehydrogenase is essential for cell growth. Here, we effectively eliminate lactate production in Chinese hamster ovary and in the human embryonic kidney cell line HEK293 by simultaneous knockout of lactate dehydrogenases and pyruvate dehydrogenase kinases, thereby removing a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. These cells, which we refer to as Warburg-null cells, maintain wild-type growth rates while producing negligible lactate, show a compensatory increase in oxygen consumption, near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. Warburg-null cells remain amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titres and maintaining product glycosylation. The ability to eliminate lactate production may be useful for biotherapeutic production and provides a tool for investigating a common metabolic phenomenon.

Targeting deubiquitinase USP7-mediated stabilization of XPO1 contributes to the anti-myeloma effects of selinexor

BackgroundTargeting exportin1 (XPO1) with Selinexor (SEL) is a promising therapeutic strategy for patients with multiple myeloma (MM). However, intrinsic and acquired drug resistance constitute great challenges. SEL has been reported to promote the degradation of XPO1 protein in tumor cells. Nevertheless, in myeloma, the precise mechanisms underlying SEL-induced XPO1 degradation and its impact on drug responsiveness remain largely undefined.MethodsWe assessed XPO1 protein and mRNA levels using western blotting and RT-qPCR. Cycloheximide (CHX) chase assays and degradation blockade assays were used to determine the pathway of XPO1 degradation induced by SEL. The sensitivity of MM cell lines to SEL was evaluated using CCK8-based cell viability assays and AV-PI staining-based cell apoptosis assays. The subcellular localization of the cargo protein RanBP1 was assessed via immunofluorescence staining. Immunoprecipitation coupled with mass spectrometry (IP-MS), bioinformatics analysis and ubiquitination assays, were employed to identify the molecular targets responsible for SEL-induced degradation of XPO1. shRNA-mediated knockdown assays and small molecule inhibitors of USP7 were utilized to disrupt the function of USP7. The role of USP7 in modulating SEL sensitivity was analyzed in MM cell lines, primary CD138+ cells, and xenograft mouse models.ResultsSEL promotes the degradation of XPO1 in MM cells through the ubiquitin–proteasome pathway. There is a positive correlation between XPO1 degradation and sensitivity to SEL in these cells. Inhibiting XPO1 degradation reduces the functional inhibitory effects of SEL on XPO1, as evidenced by decreased nuclear localization of the cargo protein RanBP1. USP7 stabilizes XPO1 in MM cells via its deubiquitinating activity. SEL accelerates the ubiquitination and subsequent degradation of XPO1 by disrupting the interaction between XPO1 and USP7. The expression of USP7 is negatively correlated with patient prognosis and the sensitivity of MM cells to SEL. Inactivating or knocking down USP7 significantly enhances the anti-myeloma effects of SEL both in vitro and in vivo.ConclusionIn conclusion, our findings underscore the essential role of XPO1 degradation in the anti-myeloma efficacy of SEL and establish a research foundation for targeting USP7 to improve the effectiveness of SEL-based therapies in MM.

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y.

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multifunctional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y Chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X Chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered: by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization-as shown by metabolic labeling of newly transcribed RNA-and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3X gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging.

Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

Distribution characteristics and proteomic analysis of glioma-associated oncogene homolog 1 positive cells during mouse orthodontic tooth movement

Objective: To explore the distribution characteristics of glioma-associated oncogene homolog 1 (Gli1) positive cells during orthodontic tooth movement process and conduct a proteomic analysis of these cells. Methods: Forty Gli1-LacZ transgenic mice were used to establish an in vivo orthodontic tooth movement (OTM) model for labeling Gli1 positive cells in Gli1-LacZ transgenic mice (OTM group) and an unforced control group, with tooth movement distance measured using micro-CT. The spatial relationship and distribution characteristics of Gli1 positive cells and H-type vessels of CD31 and endomucin (EMCN) in periodontal tissues were detected by immunofluorescence staining. Twenty Gli1-membrane-targeted tandem dimer Tomato (mT)/membrane-targeted green fluorescent protein (mG) double-genotype mice were bred and Gli1 positive cells were sorted for proteomic sequencing after tamoxifen induction. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for enrichment analysis. Results: The micro-CT three-dimensional reconstruction results showed that the mesial movement of the maxillary first molar in mice after 7 days of force application was (69±15) μm, indicating the successful establishment of the Gli1-LacZ transgenic mouse OTM model. Immunofluorescence staining showed that the blood vessels in periodontal tissue were mostly H-type vessels of CD31 and EMCN. The blood vessels in the periodontal tissues are predominantly H-type vessels positive for both CD31 and EMCN. The percentage of Gli1 positive cells in the OTM group, expressed as (54.5±13.2)%, and the relative fluorescence intensity, expressed as 2.6±0.9, were both significantly greater than those in the control group, which had a Gli1 positive cell percentage of (36.3±9.1)% (t=3.60, P=0.002) and a relative fluorescence intensity of 1.0±0.3 (t=5.20, P<0.001). In contrast to the control group where only a small number of Gli1 positive cells were consistent with the distribution of H-type vessels, in the OTM group the number of Gli1 positive cells increased on the tension side were closely associated with the spatial distribution of H-type vessels. GO enrichment analysis of biological processes found that a large number of proteins in Gli1 positive cells were enriched in pathways such as angiogenesis and tissue remodeling. KEGG enrichment analysis found that related proteins were mainly enriched in pathways related to angiogenesis and Gli1, such as hypoxia-inducing factor 1 signaling pathway, vascular endothelial growth factor signaling pathway and hedgehog signaling pathway. Conclusions: The number of Gli1 positive cells increased on tension side and were closely related to H-type blood vessels in response to mechanical force during orthodontic tooth movement. This may be related to profile of inducing blood vessel formation and tissue remodeling.

The transcription factor TCF4 regulates the miR-494-3p/THBS1 axis in the fibrosis of pathologic scars.

The fibrosis of pathologic scar (PS) is formed by the excessive deposition of extracellular matrix, resulting in an abnormal scar. Recent clinical tests have indicated that the regulation of PS fibroblast cells (PSF cells) proliferation can serve as an intervention measure for PS. Our work aimed to elucidate the specific mechanism of action of TCF4 on the progression of PS fibrosis. Our study used qRT-PCR and Western blot to search for the expression of key proteins in PS clinical samples and cells. Transwell, CCK-8, and wound scratch assays were employed to analyze the proliferation and migration of PSF cells. CHIP, dual-luciferase reporter experiments, and bio-informatics analysis were used to analyze the interactions between molecules. The analysis of PS clinical samples confirmed a positive correlation between TCF4 and miR-494-3p. This regulatory mechanism was related to the progression of PS. We verified that the overexpression of miR-494-3p or the knockdown of THBS1 both suppressed the proliferation and migration of PSF cells. Furthermore, we also confirmed the binding relationships between TCF4, miR-494-3p, and THBS1. Simultaneously, we verified the existence of the TCF4/miR-494-3p/THBS1 regulatory network in PS. This regulatory process affects the development of PS fibrosis. Our study results indicate that TCF4, miR-494-3p, and THBS1 are abnormally expressed in PS. TCF4 increases the proliferation and migration ability of PSF cells through the miR-494-3p/THBS1 signaling pathway, which promotes the fibrosis of PS.

Royal Jelly's Strong Selective Cytotoxicity Against Lung Malignant Cells and Macromolecular Alterations in Cells Observed by FTIR Spectroscopy.

Several nutraceuticals, food, and cosmetic products can be developed using royal jelly. It is known for its potential health benefits, including its ability to boost the immune system and reduce inflammation. It is rich in vitamins, minerals, and antioxidants, which can improve general health. Royal jelly (RJ) is also being studied as a potential therapeutic agent for cancer and other chronic diseases. It is effective in reducing tumor growth and stimulating immunity. In this study, we investigated the effects of royal jelly on cancerous A549 cells and healthy MRC-5 cells at various doses ranging from 1.25 to 10 mg/ml. Royal jelly's anti-proliferative effect was evaluated by MTT and SRB assay for 48 h. The induction of necrosis and apoptosis was assessed by flow cytometry as well. The relative amounts of major molecules in Royal jelly were determined by FTIR spectroscopy to identify key functional groups and molecular structures. In addition, this technique was used for the first time to detect changes in the macromolecular composition of lung cells treated with royal jelly. Thus, it provided insights into the relative abundance of proteins, lipids, and carbohydrates, which could correlate with their bioactive properties. The antiproliferative effect of Royal jelly was found to be selective on A549 cells in a dose-dependent manner with an IC50 of 9.26 mg/mL, with no cytotoxic effects on normal MRC-5 cells. Moreover, Royal jelly induced predominantly necrotic cell death in A549 cells, %39.10 at 4 mg/ml and %57.88 at 10 mg/ml concentrations. However, the necrosis rate in MRC-5 cells was quite low, at 9.16% and 20.44% at the same doses. Royal jelly showed dose-dependent selective cytotoxicity toward A549 cells, whereas it exhibited no apparent cytotoxicity in MRC-5 cells. In order to identify the biomolecular changes induced by royal jelly, we used two unsupervised chemometric pattern recognition algorithms (PCA and HCA) on the preprocessed sample FTIR spectra to determine the effects of royal jelly on cell biochemistry. According to PCA and HCA results, RJ treatments especially affected biomolecules in A549 cells. The total spectral band variances in the PCA loading spectra were calculated for understanding biomolecular alterations. These plots revealed profound changes in the lipid, protein, and nucleic acid content of RJ-applied lung cells, primarily identifying RJ and H2O2 treated groups for A549 cells. Ultimately, the selective cytotoxicity of royal jelly toward A549 cancerous cells suggests that royal jelly may be a promising therapeutic agent for identifying innovative lung cancer treatment strategies. Additionally, understanding the molecular alterations induced by royal jelly could guide the development of novel cancer treatments that exploit its bioactive properties. This could lead to more effective and safer therapies.

Loss of DDB2 in type II diabetes mellitus induces dysregulated ubiquitination of KMT2A in lipid metabolism disorders.

The disorders of glucose and lipid metabolism contribute to severe diseases, including cardiovascular disease, diabetes, and fatty liver. Here, we identified DNA damage-binding protein 2 (DDB2), an E3 ubiquitin ligase, as a pivotal regulator of lipid metabolism disorders in type II diabetes mellitus (T2DM). A mouse model of T2DM and primary mouse hepatocytes with steatosis were induced. DDB2 overexpression alone or in combination with lysine N-methyltransferase 2A (KMT2A) overexpression vectors were delivered into db/db mice and in vitro hepatocytes. DDB2 was expressed highly, while KMT2A was expressed poorly in liver tissues and primary hepatocytes of db/db mice. DDB2 ameliorated glucose intolerance and insulin resistance, decreased liver/body weight ratio, downregulated expression of lipogenesis-associated proteins (SREBP1, FASN, and SCD1) and gluconeogenesis-related proteins (PEPCK and G6Pase) in liver tissues and cells, and decreased triglyceride and total cholesterol levels in steatotic hepatocytes. DDB2 reduced KMT2A expression through ubiquitination modification. Overexpression of KMT2A promoted glucose intolerance, and alleviated insulin resistance, while promoting lipogenesis and lipid deposition, inhibiting glycogen accumulation in the presence of DDB2. Overall, our data demonstrate that DDB2 alleviates hepatic lipogenesis and lipid deposition via degradation of KMT2A, thereby repressing lipid metabolism disorders in T2DM.

Anti-neuroinflammatory and Neuroprotective Effects of T-006 on Alzheimer's Disease Models by Modulating TLR4-Mediated MyD88/ NF-κB Signaling.

Neuroinflammation derived from the activation of the microglia is considered a vital pathogenic factor of Alzheimer's Disease (AD). T-006, a tetramethylpyrazine derivative, has been found to alleviate cognitive deficits via inhibiting tau expression and phosphorylation in AD transgenic mouse models. Recently, T-006 has been proven to dramatically decrease the levels of total Amyloid β (Aβ) peptide and Glial Fibrillary Acidic Protein (GFAP) and suppress the expression of ionized calcium binding adaptor molecule-1 (Iba-1) in APP/PS1 mice. Therefore, we have further investigated the effects of T-006 on neuroinflammation in AD-like pathology. The anti-inflammatory effects of T-006 and its underlying mechanisms were evaluated in Lipopolysaccharide (LPS)-induced AD rats. The potential protective effects against LPS-activated microglia-mediated neurotoxicity were also measured. T-006 significantly improved the cognitive impairment in LPS-induced AD rats by inhibiting the microglia/astrocyte activation. Further cellular assays found that T-006 significantly reserved the anomalous elevation of inflammatory cytokines in LPS-induced BV2 microglial cells in a concentration-dependent manner, while T-006 treatment alone showed no effects on the normal cultured cells. T-006 also reduced the levels of Toll-like Receptor 4 (TLR4)/Myeloid Differentiation protein-88 (MyD88)/NF-κB signaling-related proteins in BV2 cells exposed to LPS stimulation. TAK242, which selectively inhibits TLR4, slightly lessened the effects of T-006 in LPS-treatment BV2 cells without significance. Importantly, T-006 protected neurons against LPS-induced neuroinflammation by inhibiting the Reactive Oxygen Species (ROS) production and maintaining mitochondrial function. T-006 inhibited TLR4-mediated MyD88/NF-κB signaling pathways to suppress neuroinflammation in the LPS-induced AD rat model.

FKBP Prolyl Isomerase 11: A Novel Oncogene Interacting With SRSF1 in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the main subtypes of esophageal carcinoma with high morbidity. This study aimed to explore the role of FKBP prolyl isomerase 11 (FKBP11) in ESCC and investigate the underlying mechanism. FKBP11 levels in ESCC tumor tissues and cell lines were measured. Cell function assays were conducted to evaluate the role of FKBP11 in ESCC cells. The xenograft mouse model was established to validate the effect of FKBP11 on ESCC tumorigenesis in vivo. The co-immunoprecipitation assay was performed to determine the FKBP11-interacting proteins. Obvious upregulations in FKBP11 expression were found in ESCC tumor tissues and cell lines. In vitro, FKBP11 knockdown weakened cell proliferation, migration, and invasion capacities and reinforced cell apoptosis in ESCC cells. In vivo, FKBP11 knockdown slowed ESCC tumorigenesis. The following mechanism investigation determined serine and arginine-rich splicing factor 1 (SRSF1) as the FKBP11-interacting protein in ESCC cells. FKBP11 directly bound to SRSF1 and FKBP11 knockdown decreased SRSF1 mRNA level. SRSF1 overexpression abrogated the inhibitory effect of FKBP11 knockdown on the proliferation and migration of ESCC cells. KBP11 functions as an oncogene in ESCC by targeting SRSF1.

An Entropy-Based Approach to Model Selection with Application to Single-Cell Time-Stamped Snapshot Data.

Recent single-cell experiments that measure copy numbers of over 40 proteins in individual cells at different time points [time-stamped snapshot (TSS) data] exhibit cell-to-cell variability. Because the same cells cannot be tracked over time, TSS data provide key information about the time-evolution of protein abundances that could yield mechanisms that underlie signaling kinetics. We recently developed a generalized method of moments (GMM) based approach that estimates parameters of mechanistic models using TSS data. However, when multiple mechanistic models potentially explain the same TSS data, selecting the best model (i.e., model selection) is often challenging. Popular approaches like Kullback-Leibler divergence and Akaike's Information Criterion are difficult to implement because the distribution that gave rise to the "noisy" data is only known numerically and approximately. To perform model selection in this situation, we introduce an entropy-based approach that incorporates our GMM based parameter estimation and commonly used estimators in kernel density estimation. Using simulated TSS data, we show that our approach can select the "ground truth" from a set of competing mechanistic models. Furthermore, we use a bootstrap procedure to compute model selection probabilities, which can be useful when measuring the relative support of a candidate model.

Leishmania mexicana N-Acetyltransferease 10 Is Important for Polysome Formation and Cell Cycle Progression.

Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post-transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4-acetylcytidine (ac4C) catalyzed by the enzyme N-acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana, finding that the enzyme exhibits all the conserved acetyltransferase domains although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisiae. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single-knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote-amastigote differentiation. Additionally, invivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.

C-Myc-targeted therapy in breast cancer: A review of fundamentals and pharmacological Insights.

The oncoprotein c-Myc is expressed in all breast cancer subtypes, but its expression is higher in triple-negative breast cancer (TNBC) compared to estrogen receptor (ER+), progesterone receptor (PR+), or human epidermal growth factor receptor 2 (HER2+) positive tumors. The c-Myc gene is crucial for tumor progression and therapy resistance, impacting cell proliferation, differentiation, senescence, angiogenesis, immune evasion, metabolism, invasion, autophagy, apoptosis, chromosomal instability, and protein biosynthesis. Targeting c-Myc has emerged as a potential therapeutic strategy for TNBC, a highly aggressive and deadly breast cancer form. This review highlights c-Myc as a pharmacological target, discussing antitumor compounds in preclinical and clinical trials. Notably, the c-Myc inhibitor OMO-103 has shown promise in a Phase II clinical trial for advanced cancer patients. Further research is needed to develop new drugs targeting this gene, protein, or its pathways, and additional studies on cancer patients are encouraged.

CAVIN2 attenuates ventilator-induced lung injury in rats by MAPK/ERK1/2 signaling pathway.

Mechanical ventilation is an important treatment in medical treatment, but it may cause or aggravate lung injury, which is called ventilator-induced lung injury (VILI). Studies have shown that CAVIN2 plays an important role in regulating inflammatory responses and cell death. However, its functional mechanism in VILI remains unclear. This study explores the potential role and mechanisms of CAVIN2 in the pathogenesis of VILI. We constructed rat and cell models of VILI. Real-time quantitative polymerase chain reactions (qRT-PCR), Western blot (WB), immunohistochemistry (IHC) and immunofluorescence (IF) were used to detect CAVIN2, ERK1/2, p-ERK1/2, autophagy-associated marker proteins LC3II/I, Beclin1 and P62, and the expression level of pro-inflammatory factors IL-1β and IL-6. Hematoxylin and eosin (H&E) staining were used to evaluate the degree of pathological injury of lung tissue, and the lung permeability was evaluated by measuring the wet-dry ratio of lung tissue and the total protein content in bronchoalveolar lavage fluid (BALF). Molecular docking, co-immunoprecipitation and immunofluorescence were used to verify the binding of CAVIN2 and ERK1/2, and the regulatory mechanisms of both were investigated by rescue experiments. CAVIN2 expression was downregulated in VILI rat lung tissues and ATII cells, whereas p-ERK1/2 expression was up-regulated. Overexpressing CAVIN2 alleviated pathological damage in VILI rat lung tissues and reduced the expression of pro-inflammatory factors and autophagy-related marker proteins in both lung tissues and ATII cells. Conversely, knockdown of CAVIN2 led to increased expression of pro-inflammatory factors and autophagy-related marker proteins in ATII cells. Further mechanism studies showed that CAVIN2 binds to ERK1/2 and inhibits ERK1/2 phosphorylation. Conversely when treated with the p-ERK1/2 agonist Ro67-7476, the protective, anti-inflammatory, and anti-autophagic effects of overexpressing CAVIN2 in VILI rats and ATII cells were reversed. CAVIN2 can bind to ERK1/2 and inhibit the activation of MAPK/ERK1/2 signaling pathway to reduce inflammatory response and autophagy in VILI, thereby reducing lung injury. Therefore, CAVIN2 may be a potential intervention target to provide a new strategy for the treatment of VILI.

Chlorophyllides repress gain-of-function p53 mutated HNSCC cell proliferation via activation of p73 and repression of p53 aggregation in vitro and in vivo.

Head and neck squamous cell carcinoma (HNSCC) cells have a high p53 mutation rate, but there were rare reported about the p53 gain of function through the prion-like aggregated form in p53 mutated HNSCC cells. Thioflavin T (ThT) is used to stain prion-like proteins in cells. Previously, we found that ThT and p53 staining were co-localized in HNSCC cells (Detroit 562 cells) with homozygous p53 R175H mutation. NAMPT inhibitor can repress ThT staining in Detroit 562 cells. In our previous study, co-treatment with p73 activator NSC59984 and NAMPT inhibitor FK886 synergistically repressed Detroit 562 cell proliferation. In this study, we found that two heterozygous p53-R280T mutation HNSCC cell lines, TW01 and HONE-1, also have the ThT staining signal. Treatment with chlorophyllides and p73 activator or NAMPT inhibitor did not synergistically repress cell proliferation in either Detroit 562 or HONE-1 cells. Chlorophyllides reduced the ThT aggregation signal in both Detroit 562 and HONE-1 cells. Chlorophyllides also induced p73 and caspase 3/7 expression and repressed NAMPT expression in both Detroit 562 and HONE-1 cells. Chlorophyllides reduced tumor size in vivo in Detroit 562 cells injected into a xenograft nude mice model, but this in vivo tumor repression effect was not found in p73 knockdown Detroit 562 cells. Moreover, NAMPT was repressed by chlorophyllides independent of p73 status in vivo. We thus concluded that chlorophyllides have a dual anticancer function when applied to HNSCC cells with p53 gain-of-function mutation, via activation of p73 and repression of p53 aggregation.