HeLa Research Articles

A rapid near-infrared turn on fluorescence probe for the detection of sulfite in food and its application in biological imaging.

A near-infrared fluorescent "turn on" probe DTMI featuring simple skeleton was constructed easily. It undergoes a structure transformation from an A-π-A to a D-π-A framework towards SO32-. Besides, DTMI is capable of distinctive sensing sulfite with a fast response and a significant Stokes shift as well as with high sensitivity, excellent selectivity, long-term stability of fluorescence signals, and good anti-interference ability. The detection limit (LOD) of DTMI for sulfite within the linear concentration range of 0.5-10μM is 27.39nM. More importantly, DTMI has been favorably utilized for detecting sulfite in food samples such as red wine and vermicelli. Based on its low biotoxicity, DTMI has been successfully applied in imaging experiments involving HeLa cells, onion inner epidermal cells, and zebrafish. Therefore, the results show that the presented probe possesses potential sensing activity towards sulfite in complex biological system and food samples.

Loureirin B inhibits Cervical Cancer Development by Blocking PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

Loureirin B (LB) is an iconic component of Chinese dragon's blood that presents anti-cancer effects in gastric cancer and liver cancer. Although LB has shown benefits in treating several disorders such as cardiac fibrosis, cerebral ischemia/reperfusion, and osteoporosis, its effect on cervical cancer remains unknown. This study aimed to investigate the effects and mechanisms of LB on treating cervical cancer. A CCK-8 assay was conducted to determine the influence of LB on the viability of HeLa cells. Colony formation assay was performed to verify the impact of LB on HeLa cell proliferation. Cell cycle and apoptosis were detected by flow cytometry and western blot. The scratch assay, Transwell assay and western blot were used to examine the migration and invasion capacity of HeLa cells. The potential targets and signaling pathways of LB treating cervical cancer were predicted by network pharmacology analysis and subsequently validated in vitro. The results showed that the HeLa cell viability gradually declined to 64.83% for 12h, 53.17% for 24h, and 42.38% for 48h after treatment with 5-80μg/mL LB. Treatment with 20μg/mL LB decreased cell colonies from 156.7 ± 11.7 to 102.7 ± 5.7. LB arrested cell cycle by reducing the expressions of Ki-67 and PCNA. Compared to the cell apoptosis rate of 2.63% in control group, LB increased it to 6.59% via upregulating Bax and suppressing Bcl-2 expressions. Additionally, LB reduced the invasion and migration capacity of HeLa cells by decreasing MMP-2 and MMP-9 levels. Network pharmacology analysis revealed that LB might suppress the PI3K/AKT signaling pathway to exert the aforementioned effects, as evidenced by a PI3K agonist attenuating the effects of LB on HeLa cells. In conclusion, this study demonstrated that LB inhibited the proliferation of cervical cancer cells, induced its apoptosis, and reduced its invasion and migration via targeting the PI3K/AKT signaling pathway.

Synthesis, Characterization, and Cytotoxicity Evaluation of Novel Water-Soluble Cationic Platinum(II) Organometallic Complexes with Phenanthroline and Imidazolic Ligands.

Platinum-based chemotherapeutic agents are widely used in the treatment of cancer. However, their effectiveness is limited by severe adverse reactions, drug resistance, and poor water solubility. This study focuses on the synthesis and characterization of new water-soluble cationic monofunctional platinum(II) complexes starting from the [PtCl(η1-C2H4OEt)(phen)] (1, phen=1,10-phenanthroline) precursor, specifically [Pt(NH3)(η1-C2H4OEt)(phen)]Cl (2), [Pt(1-hexyl-1H-imidazole)(η1-C2H4OEt)(phen)]Cl (3), and [Pt(1-hexyl-1H-benzo[d]imidazole)(η1-C2H4OEt)(phen)]Cl (4), which deviate from traditional requirements for antitumor activity. These complexes were evaluated for their cytotoxic effects in comparison to cisplatin, using immortalized cervical adenocarcinoma cells (HeLa), human renal carcinoma cells (Caki-1), and normal human renal cells (HK-2). While complex 2 showed minimal effects on the cell lines, complexes 3 and 4 demonstrated higher cytotoxicity than cisplatin. Notably, complex 4 displayed the highest cytotoxicity in both cancer and normal cell lines. However, complex 3 exhibited the highest selectivity for renal tumor cells (Caki-1) among the tested complexes, compared to healthy cells (HK-2). This resulted in a significantly higher selectivity than that of cisplatin and complex 4. Therefore, complex 3 shows potential as a leading candidate for the development of a new generation of platinum-based anticancer drugs, utilizing biocompatible imidazole ligands while demonstrating promising anticancer properties.

Natural-Target-Mimicking Translocation-Based Fluorescent Sensor for Detection of SARS-CoV-2 PLpro Protease Activity and Virus Infection in Living Cells.

Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins-red mScarlet I and green mNeonGreen-separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.

Synthesis of Azahelicenes via Friedel‐Crafts‐Type Ring‐Closing Reaction of Axially Chiral Compounds

AbstractAza[6]helicene and aza[7]helicene were synthesized by Fridel‐Crafts‐type cyclization of axially chiral compounds. From the axially chiral compounds prepared by Fischer indole synthesis, aza[6]helicene and aza[7]helicene were synthesized. Aza[7]helicene was also synthesized from an axially chiral pyrrole compound prepared by Piloty‐Robinson‐type pyrrole synthesis through simultaneous cyclization on both sides of the pyrrole ring. Aza[6]helicene isomerized readily at room temperature, while aza[7]helicene isomerization at room temperature was less likely to proceed. The cell growth inhibitory activity of each enantiomer of N‐Me‐aza[7]helicene in HeLa cells was examined. Differences in the strength of activity between the enantiomers were observed.

Bright ESIPT emission from 2,6-di(thiazol/oxazol/imidazol-2-yl)phenol derivatives in solution, aggregation and solid states

Most luminophores often suffer from the problem of aggregation-caused quenching (ACQ) or fluorescence disappearance in dilute solution. It is significant to bridge the gap between ACQ and AIE. In this work, a facile but effective strategy was proposed for the fabrication of always-on luminophores based on the excited state intramolecular proton transfer (ESIPT) mechanism, and six luminophores emitting bright fluorescence in solution, aggregation and solid states were synthesized from 5-tert-butyl-2-hydroxyisophthalaldehyde. All these ESIPT systems show only keto emission owing to their congested structures which block the breakage of intramolecular hydrogen bond (O–H⋯N) by solvation, and subsequently make enol emission impossible. Three of these luminophores are prone to convert into the corresponding phenolate anions emitting blue-shifted emission, which enable them to sense pH variation in the weakly basic range. Furthermore, white-light emission was achieved by combining two of them which show complementary-color fluorescence, and one of them was utilized for bioimaging of living Hela cells and the high-resolution image was obtained.

Smartphone-based sensing and in vivo and in vitro imaging of Mn(VII) based on nitrogen-doped red fluorescent carbon dots.

Smartphone-assisted visual assay platform provides novel insight for the real-time in-field quantitation of intended analytes in resource-insufficient areas. Herein, nitrogen-doped red fluorescent (FL) carbon dots (R-CDs) were developed for the timely on-site quantitation of Mn(VII) using the smartphone-assisted assay platform. R-CDs, possessing a desirable bright red FL at 616 nm under a 470 nm excitation, were fabricated through hydrothermal treatment adopting passion fruit and neutral red as precursors. Interestingly, bright red FL at 616 nm are gradually quenched upon introducing Mn(VII) based on the inner filter effect, concurrently accompanying with significant FL color variation from bright red to dark red. Inspired by the above-mentioned phenomena, hue-saturation-values (HSV) of real-time captured images could be precisely quantified through a color recognition APP within the smartphone, of which the V/S values could be employed to quantify Mn(VII) with a linear range of 50-400 μM. Furthermore, confocal fluorescence imaging of HeLa cells and zebrafish larvae demonstrates that R-CDs could be employed for the visual determination of Mn(VII) in vivo and in vitro, illustrating that R-CDs possess powerful practical application prospect in biosystem.

Cyclosporin A-Based PROTACs Can Deplete Abundant Cellular Cyclophilin A without Suppressing T Cell Activation.

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

GuiErBai: a potent inhibitor, exhibiting broadly antitumor effect against cervical cancer in vitro and in vivo.

Introduction: Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and A. dahurica alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. Methods: HeLa and SiHa cell lines were utilized for in vitro experiments and treated with 5mg/mL and 10mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the in vivo experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10mg/mL), and low-dose GEB (5mg/mL) groups. Results: In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased (p < 0.001), while the G2/M phase increased notably (p < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines (p < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated (p < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased (p < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. Discussion: GEB exhibits a robust antitumor effect against cervical cancer, both in vitro and in vivo, in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.

An easily accessible probe for H2S based on AIE mechanism and its application in cell imaging

Hydrogen sulfide (H2S) is a crucial endogenous gas transmitter, maintaining the redox homeostasis in biological systems. Thus, a real-time and specific H2S detection approach is urgently necessary to study the attack and management of disorders linked to H2S. In this paper, a novel fluorescent probe HMP based on AIE property for the detection of H2S was elaborately conducted. In PBS buffer solution HMP exhibited high photostability, low detection limit (0.82 μM), significant anti-interference ability and fast response time and specificity to H2S. What’s more, it could be employed for the monitoring and luminescent imaging of H2S with Hela cells, thereby broadening the prospective capacity for diagnosing associated diseases.

Cytotoxicity Screening of Endophytic Fungi from Phaleria macrocarpa (Scheff) Boerl) Collected in Yogyakarta District

Cancer is a disease that is a global problem with a high prevalence and mortality rate. Indonesia has biodiversity with enormous potential as a source of medicinal ingredients. Phaleria macrocarpa (Scheff) Boerl) It is known to contain active metabolites, potentially an anticancer. Some endophytes are reported to be able to produce secondary metabolites similar to their parent plants. This study aimed to obtain endophytic fungi from P. macrocarpa capable of producing compounds with anticancer potential. Endophytes producing anticancer compounds were explored by screening endophytic fungi producing secondary metabolites from parts of the plant P. macrocarpa collected in the Yogyakarta area. The anticancer bioassay (MTT assay) was performed in vitro using cancer cells T47D, MCF7, HeLa, and normal Vero cells. Potential extracts were traced for their mechanism of cell cycle modulation and apoptosis using Flowcytometry. First, fungal identification was carried out on fungi producing potential cytotoxic extracts. Then the compound content was analyzed using LC-HRMS. The results from the six extracts obtained from fruit, leaf, and stem parts showed that the endophytic extract from the leaves (code PC-L2) had the best cytotoxicity compared to other endophytes and showed the best result on MCF-7 cells with an IC50 value of 110.66 µg/mL. The producing PC-L2 extract fungi are closely associated with Clonostachys wenpingii strain DUCC5606. This extract induced apoptosis but not through the mechanism of cell cycle modulation.

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction subunits have been linked to inherited human diseases. Functional studies of human gap junction (GJ) channels are essential for revealing mechanistic insights on the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

Magnetic‐Driven Hydrogel Spiral PVA/Fe3O4 Micromotors for Targeted Photothermal Therapy

AbstractPhototherapy has emerged as a promising technique for cancer treatment, offering high efficiency and minimal side effects. Nevertheless, conventional phototherapy agents suffer from low targetability and inefficient delivery. Here, a novel approach is proposed using helical hydrogel motors fabricated by combining polyvinyl alcohol, melamine, water, and Fe3O4 nanoparticles. These magnetic motors are capable of precise therapy on tumor cells driven by an external magnetic field. By converting light into heat upon near‐infrared illumination, the motors elevate the temperature of the targeted region, resulting in a significant therapeutic effect, with over 60% eradication of HeLa cells achieve under 808 nm near‐ infrared light (1.5 W cm−2). These helical micromotors exhibit excellent movement performance and biocompatibility, facilitating their in vivo applications and contributing to the advancement of biomedicine.

Phytochemical composition and antitumor activity of a new arctic lichen Anamylopsora pakistanica

For the first-time, chemical composition and in vitro antitumor activity was investigated of a newly described lichen Anamylopsora pakistanica Usman & Khalid from the second highest plateau of the world (Deosai Plains, Pakistan). HPLC–UV method was used for identification of secondary metabolites and the acetone extract had higher values of TPC (41.90 mg GA/g) and TFC (75.37 mg RE/g) as compared to methanol extract. As chemical constituents 5,7-dihydroxy-6-methylphthalide, haematommic acid and alectorialic acid, were identified as major compounds. Atranol, alectorialin, gyrophoric acid and usnic acid were detected as minor substances. Acetone and methanol extracts induced a dose-dependent and time-dependent decrease in the viability of three types of tumour cells HeLa, HCT116 and MDA-MB-231. This lichen extract can induce S phase arrest in HeLa as compared to the untreated cells. Extract of this unique lichen, A. pakistanica, can be used safely as a significant source of biologically active compounds.

Synthesis, molecular modeling simulations and anticancer activity of some new Imidazo[2,1-b]thiazole analogues as EGFR/HER2 and DHFR inhibitors

New imidazo[2,1-b]thiazole analogs were designed, synthesized, and biologically evaluated as anticancer agents. In vitro biological evaluation of the anticancer properties of the compounds was performed against different cancer cell lines. Compounds 23 and 39 showed remarkable broad −spectrum cytotoxic potency on most of the tested cell lines. Compounds 23 and 39 exhibited potent activity against the MCF-7 breast cancer cell line, with IC50 values of 1.81 and 4.95 μM, respectively, compared to DOX and SOR (IC50 values of 4.17 and 7.26 μM, respectively). An enzyme inhibition assay was carried out to clarify the possible mode of action of the tested compounds. Compounds 23 and 39 were identified as possible EGFR, HER-2, and DHFR inhibitors. Cell cycle arrest results indicated that compound 23 caused cell cycle arrest at the G0/G1 phase in the MCF-7 cells and at the G2/M phase in the Hep G2 cells. Compound 39 induced cell cycle arrest at the G2/M phase in Hela cells. In vivo testing of the anticancer activity of the two most promising molecules in this study was conducted, and the results indicated that they possess considerable in vivo anticancer activity in mice. Data obtained from the molecular modeling simulation study were consistent with the biological evaluation results.

Preparation and Evaluation of a Novel 99mTc-Labeled Niraparib Isonitrile Complex as a Potential PARP-1 Imaging Agent.

Poly ADP-ribose polymerase (PARP) plays an important role in the DNA repair process and has become an attractive target for cancer therapy in recent years. Given that niraparib has good clinical efficacy as a PARP inhibitor, this study aimed to develop radiolabeled niraparib derivatives for tumor imaging to detect PARP expression and improve the accuracy of stratified patient therapy. The niraparib isonitrile derivative (CNPN) was designed, synthesized, and radiolabeled to obtain the [99mTc]Tc-CNPN complex with high radiochemical purity (>95%). It was lipophilic and stable in vitro. In HeLa cell experiments, the uptake of [99mTc]Tc-CNPN was effectively inhibited by the ligand CNPN, indicating the binding affinity for PARP. According to the biodistribution studies of HeLa tumor-bearing mice, [99mTc]Tc-CNPN has moderate tumor uptake and can be effectively inhibited, demonstrating its specificity for targeting PARP. The SPECT imaging results showed that [99mTc]Tc-CNPN had tumor uptake at 2 h postinjection. All of the results of this study indicated that [99mTc]Tc-CNPN is a promising tumor imaging agent that targets PARP.

Chondroitin sulfate-functionalized selenium nanoparticle-induced S-phase cell cycle arrest and apoptosis in HeLa Cells.

This study aimed to evaluate the anti-cervical cancer activity of chondroitin sulfate-functionalized selenium nanoparticles (SeCS) and to elucidate their action mechanism. Cytotoxic effect of SeCS on HeLa cells was assessed by MTT assay. Further molecular mechanism of SeCS was analyzed by flow cytometric assay and western blotting. The results showed that treatment with SeCS resulted in a dose- and time-dependent inhibition in the proliferation of HeLa cells. The data obtained from flow cytometry demonstrated that SeCS inhibited HeLa cell growth via the induction of S-phase arrest and cell apoptosis. Further mechanism analysis found that SeCS down-regulated expression levels of cyclin A and CDK2 and up-regulated p21 expression, which contributed to S arrest. Moreover, SeCS increased the level of Bax and decreased the expression of Bcl-2, resulting in the release of cytochrome C from mitochondria and activating caspase-3/8/9 for caspase-dependent apoptosis. Meanwhile, intracellular reactive oxygen species (ROS) levels were elevated after SeCS treatment, suggesting that ROS might be upstream of SeCS-induced S-phase arrest and cell apoptosis. These data show that SeCS has anti-tumor effects and possesses the potential to become a new therapeutic agent or adjuvant therapy for cancer patients. PRACTICAL APPLICATION: In our previous study, we used chondroitin sulfate to stabilize nano-selenium to obtain SeCS to improve the bioactivity and stability of nano-selenium. We found that it possessed an inhibitory effect on HeLa cells. However, the molecular mechanism remains unclear. This study elucidated the mechanism of SeCS damage to HeLa cells. SeCS has the potential to become a new therapeutic agent or adjuvant therapy for cancer patients.

Biotinylated Theranostic Amphiphilic Polyurethane for Targeted Drug Delivery.

In the area of drug delivery aided by stimuli-responsive polymers, the biodegradability of nanocarriers is one of the major challenges that needs to be addressed with the utmost sincerity. Herein, a hydrogen sulfide (H2S) responsive hydrophobic dansyl-based trigger molecule is custom designed and successfully incorporated into the water-soluble polyurethane backbone, which is made of esterase enzyme susceptible urethane bonds. The amphiphilic polyurethanes, PUx (x = 2 and 3) with a biotin chain end, formed self-assembled nanoaggregates. A hemolysis and cytotoxicity profile of doxorubicin (DOX)-loaded biotinylated PU3 nanocarriers revealed that it is nonhemolytic and has excellent selectivity toward HeLa cells (biotin receptor-positive cell lines) causing ∼60% cell death while maintaining almost 100% cell viability for HEK 293T cells (biotin receptor-negative cell lines). Furthermore, better cellular internalization of DOX-loaded fluorescent nanocarriers in HeLa cells than in HEK 293T cells confirmed receptor-mediated endocytosis. Thus, this work ensures that the synthesized polymers serve as biodegradable nanocarriers for anticancer therapeutics.

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

LncRNA CDKN2B-AS1 regulates collagen expression.

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.