HeLa Research Articles

Biocompatible fluorinated poly(2-hydroxypropenimine)s for safe and efficient gene transfection

The development of non-viral gene delivery systems often requires balancing transfection efficiency and cytotoxicity, particularly when using cationic polymers. Poly(2-hydroxypropenimine) (PHP), with its hydroxyl groups, presents a more biocompatible alternative to traditional polymers such as polyethyleneimine (PEI). In this study, we explored the potential to enhance PHP’s transfection capabilities and reduce its cytotoxicity through a targeted fluorination strategy. Specifically, fluorinated alkyl groups were grafted onto PHP via an ethylene oxide ring-opening reaction, with the reaction conditions optimized for temperature, solvent, and time to ensure efficiency. Transfection experiments using HEK 293 cells demonstrated that fluorinated PHP variants, especially PHP-FH816, significantly improved transfection efficiency, achieving a 75.1 % gene expression rate at an N/P ratio of 2, compared to 44.0 % for unmodified PHP. The transfection experiments in Hela cells also showed substantial transfection efficiency. The gene expression rate was 53.2 % at an N/P ratio of 2 compared to 31.6 % for unmodified PHP. Notably, the fluorinated polymers exhibited lower cytotoxicity and increased serum stability, which suggests their potential as safer gene delivery systems. This study carefully evaluates the feasibility of using fluorinated cationic polymers as next-generation gene delivery vectors, balancing efficacy and safety. It underscores the strategic application of fluorination technology in developing modified polymer vectors that could lead to more efficient and safer non-viral gene therapy solutions.

Polydimethylsiloxanes - Based Fluorescent Probe for H2S Detection in Living Cells.

The development of fluorescent probes for H2S detection especially in living cells is of great significance due to its fundamental role as signal molecule. A promising scaffold for the development of such probes is polydimethylsiloxanes (PDMS), which is cost-effectiveness, non-toxicity, flexibility, and biocompatibility and easy to post-functionalize. Surprisingly, fluorescent probes for H2S detection based on PDMS have not been investigated. Moreover, 4-nitro - 2,1,3-benzoxadiazole (NBD) derivates provides high fluorescence quantum yield, a large molar absorption coefficient, and sensitivity to environmental changes. Through reasonable design and adjustment of substituents on the NBD group, precise control of its fluorescence properties can be achieved. Herein, a novel H2S fluorescent probe, P-NBD, was designed and synthesized by a one-step aromatic ring nucleophilic substitution of Cl-NBD with a PDMS derivative. Due to the occurrence of the cleavage reaction strategy and the intramolecular charge transfer process, P-NBD can detect H2S via a colorimetric method. The limit of detection is down to 93 nM. P-NBD demonstrated considerable detection capability comparable to other reported types of H2S probes. Moreover, the probe can also be utilized for H2S imaging in HeLa cells. This work provides new insights into the design and synthesis of novel H2S probes while also offering experimental evidence for the application of PDMS in cellular imaging.

Structural Aspects, Binding Interactions, Biological Activity of Co(II)‐ and Ni(II)‐N′‐(2‐fluorobenzoyl)benzo[d]thiazole‐2‐carbohydrazide Chelatess

ABSTRACTN′‐(2‐Fluorobenzoyl)benzo[d]thiazole‐2‐carbohydrazide (OFBBTCH, HL) and its chelates with Co(II) and Ni(II) have been synthesized and characterized by elemental analysis, liquid chromatography‐mass spectrometry (LC–MS), Fourier transform infrared spectroscopy (FT‐IR), ultra‐violet visible (UV–vis), nuclear magnetic resonance (NMR) spectroscopy, thermogravimetric analysis (TGA), and powder X‐ray diffraction. The conductance measurements reveal that the chelates are non‐electrolytic, and the spectral analysis indicates octahedral geometry of the chelates. The kinetic and thermodynamic parameters were obtained by Coats–Redfern relations, which shows positive Gibbs's free energy and negative entropy for both chelates. The equilibrium studies carried out by pH‐metry revealed the dissociation constant (pKa) of OFBBTCH as 8.13 and the formation of 1:1 and 1:2 Co(II)‐L and Ni(II)‐L complexes in solution. The CT‐DNA binding studies carried out by electron absorption, fluorescence quenching, steady state emissions and viscosity studies revealed hyperchromism and groove binding for HL and chelates. The Kb values calculated from electron absorption studies are 2.4 × 106, 4 × 106, and 9 × 106 M−1, from the fluorescence quenching studies Ksv values are 0.1722, 0.278, and 0.748 M−1, and Kapp values are 5.9 × 104, 1.3 × 105, and 2.9 × 105 M−1 for HL, Co(II)‐OFBBTCH and Ni(II)‐OFBBTCH, respectively. Bovine serum albumin (BSA) and human serum albumin (HSA) binding interactions were carried out by electron absorption and fluorescence quenching studies. The light switching properties of the chelates, the hydrolytic and oxidative cleavage of CT‐DNA, and the antioxidant properties were evaluated. Anti‐microbial studies carried out by pour plate method showed high activity for Ni(II)‐OFBBTCH chelate. Cytotoxicity of the compounds was performed for HeLa and MCF cell lines by MTT assay. Molecular docking studies revealed the docking of HL and Co(II)‐HL at the minor groove, and Ni(II)‐HL was docked at the major groove with hydrogen bonding.

Altered tubulin detyrosination due to SVBP malfunction induces cytokinesis failure and senescence, underlying a complex hereditary spastic paraplegia.

Senescence, marked by permanent cell cycle arrest may contribute to the decline in regenerative potential and neuronal function, thereby promoting neurodegenerative disorders. In this study, we employed whole exome sequencing to identify a previously unreported biallelic missense variant in SVBP (p.Leu49Pro) in six patients from three unrelated families. These affected individuals present with a complex hereditary spastic paraplegia (HSP), peripheral neuropathy, verbal apraxia, and intellectual disability, exhibiting a milder phenotype compared to patients with nonsense SVBP mutations described previously. Consistent with SVBP's primary role as a chaperone necessary for VASH-mediated tubulin detyrosination, both patient fibroblasts with the p.Leu49Pro mutation, and HeLa cells harboring an SVBP knockdown exhibit microtubule dynamic instability and alterations in pericentriolar material (PCM) component trafficking and centrosome cohesion. In patient fibroblasts, structural abnormalities in the centrosome trigger mitotic errors and cellular senescence. Notably, premature senescence characterized by elevated levels of p16INK4, was also observed in patient peripheral blood mononuclear cells (PBMCs). Taken together, our findings underscore the critical role of SVBP in the development and maintenance of the central nervous system, providing novel insights associating cytokinesis failure with cortical motor neuron disease and intellectual disability.

Targeting AGAT gene expression – a drug screening approach for the treatment of GAMT deficiency

ABSTRACT Background Targeting the enzyme L-Arginine:glycine amidinotransferase (AGAT) to reduce the formation of guanidinoacetate (GAA) in patients with guanidinoacetate methyltransferase (GAMT) deficiency, we attempted to identify drugs for repurposing that reduce the expression of AGAT via transcriptional inhibition. Research Design and Methods The authors applied a HeLa cell line stably expressing AGAT promoter and firefly luciferase reporter for high-content screening and secondary screening. For further assessment, the authors integrated Nanoluc luciferase as a reporter into the endogenous AGAT gene in HAP1 cell lines and used the human immortalized cell line RH30 as model of GAMT deficiency. Results Screening 6,000 drugs and drug-like compounds, the authors identified 43 and 34 high-score candidates as inhibitors and inducers of AGAT promoter-reporter expression, respectively. After further deselection considering dose response, drug toxicity, topical formulations, price, and accessibility, the authors assessed seven candidates and found none of them demonstrating efficacy in HAP1 and RH30 cells and warranting further assessment. Conclusion The selection of the test models is crucial for screening of gene repressor drugs. Almost all drugs with an impact on gene expression had off-target effects. It is unlikely to find drugs that are selective inhibitors of AGAT expression, rendering pharmacological AGAT gene repression a risky approach for the treatment of GAMT deficiency.

Dual-emission carbon dots-based biosensor for polarity/targeting bimodal recognition and mild photothermal therapy of tumor

It is essential to develop a multifunctional nanoplatform for biosensing, tumor diagnosis and treatment simultaneously. Herein, dual-emission fluorescent carbon dots (HA-CDs) were fabricated via a one-pot solvothermal method using spinach powder as carbon source and hyaluronic acid (HA) as targeting agent. The obtained HA-CDs exhibited outstanding optical properties, good targeted tumor and excellent photothermal conversion performance. Interestingly, HA-CDs can sensitively perceive the changes in polar environments attributed to the inherent ratiometric fluorescence characteristics, and combined with the intrinsic targeting tumor ability achieved tumor cell recognition. More importantly, the HA-CDs possess good photothermal conversion efficiency of 21.2 % to be beneficial for photothermal therapy of tumors. The survival rate of HeLa cells incubated with HA-CDs dramatically decreased to 14 % after 660 nm laser irradiation, revealing the significant tumor inhibition of HA-CDs in vitro. Notably, through individual intraperitoneal and intratumoral injection, it was found that HA-CDs demonstrated a similar tumor suppressed effect on 4T1 tumor-bearing mice exposed to laser irradiation, fully uncovering that HA-CDs can efficiently accumulate at tumor site by intraperitoneal injection. Besides, the histopathological analysis of major organs ex vivo revealed a good biosafety profile. Collectively, this strategy of designed HA-CDs provides a new multifunctional nanoplatform for potential clinical application.

Physico-chemical properties and biological evaluation of graphene quantum dots for anticancer drug susceptibility

Graphene quantum dots (GQDs) possess unique optical and biocompatible properties, making them suitable candidates for biomedical and pharmaceutical applications. This study reports the hydrothermal synthesis of pristine-GQD and doped variants: Nitrogen-GQD and Sulfur-GQD. The materials underwent thorough characterization techniques such as UV–vis, fluorescence, XRD, FE-TEM/SEM, EDX, and Raman spectroscopy. The particle sizes of these GQDs range from 2 to 5 nm. We conducted a comprehensive study through MTT assays to evaluate the potential cytotoxic effect of GQD and the doped variants. This study demonstrated their synergistic interactions with an anti-cancer drug, methotrexate (MTX), and also improvement of cytocompatibility in the presence of folic acid (FA). Systematic MD simulations revealed a compacting effect on the dynamic behavior of GQD and its variants in the presence of drugs. Fluorescence spectroscopy and computational modeling suggest non-intercalative surface interactions between GQDs and the drugs. The cytotoxic activity of pristine GQD on HeLa cervical cancer cells is higher than that of N-GQD and S-GQD. When treated with GQD-IC50–MTX-IC50, only 5.6 % of HeLa cells remained viable. The doped variants exhibited bio-compatibility when tested on normal HEK cell lines. Overall, this study emphasizes the potential of GQDs for targeted cancer therapy through an interdisciplinary approach involving material characterization, computational modeling, and biological assays.

Pulsed electromagnetic fields inhibit IL-37 to alleviate CD8+ T cell dysfunction and suppress cervical cancer progression.

Pulsed electromagnetic field (PEMF) therapy is a potential non-invasive treatment to modulate immune responses and inhibit tumor growth. Cervical cancer (CC) is influenced by IL-37-mediated immune regulation, making PEMF therapy a potential strategy to impede CC progression. This study aimed to elucidate the effects of PEMF on IL-37 regulation and its molecular mechanisms in CC. CC cell-xenografted mouse models, including IL-37 transgenic (IL-37tg) mice, were used to assess tumor growth through in vivo fluorescence imaging and analyze CC cell apoptosis via flow cytometry. TCGA-CESC transcriptome and clinical data were analyzed to identify key inflammation and immune-related genes. CD8+ T cell models were stimulated with PEMF, and apoptosis, oxidative stress, and inflammatory factor expression were analyzed through RT-qPCR, Western blot, and flow cytometry. PEMF treatment significantly inhibited IL-37 expression (p < 0.05), promoted inflammatory factor release (TNF-α and IL-6), and activated oxidative stress, leading to increased CC cell apoptosis (p < 0.05). IL-37 interaction with SMAD3 impacted the p38/NF-κB signaling pathway, modulating CD8+ T cell activity and cytotoxicity. Co-culture of Hela cells with CD8+ T cells under PEMF treatment showed reduced proliferation (by 40%), migration, and invasion (p < 0.05). In vivo experiments with CC-bearing mice demonstrated that PEMF treatment downregulated IL-37 expression (p < 0.05), enhanced CD8+ T cell function, and inhibited tumor growth (p < 0.05). These molecular mechanisms were validated through RT-qPCR, Western blot, and immunohistochemistry. Thus, PEMF therapy inhibits CC progression by downregulating IL-37 and improving CD8+ T cell function via the SMAD3/p38/NF-κB signaling pathway.

PARP1 acetylation at K119 is essential in regulating the progression and proliferation of cervical cancer cells.

Cervical cancer, CC, is one of the malignant cancers in women worldwide. Many studies about the genesis and progression of CC have been done at genomic, transcriptional, translational, and epigenetic levels. However, much less is done at post-translational modification (PTM) level. We first used pan-PTM antibodies to compare the pan PTM levels between clinical normal cervical tissues and CC tissues; we then sent the selected samples for label-free identification of acetylation sites. Next, we employed WT or K119A mutant PARP1-EGFP-STREPII plasmid transfection in Hela cells and examined various indexes including colony formation, wound healing, ROS generation, early apoptosis, and immunofluorescence and quantification of proliferation markers (Ki67, PCNA, and p-P53). Last, we examined the levels of multiple important kinases regulating cervical cancer progression. We found that pan-acetylation was the most downregulated in clinical CC samples, whereas the acetylation of PARP1, Poly(ADP-ribose) polymerase-1, was upregulated at K119. Next, we showed that PARP1-WT overexpression significantly suppressed the proliferation and progression in CC cell line Hela, while K119A overexpression didn't show any impact. Finally, PARP1-WT overexpression significantly decreased p-ERK1/2 while didn't affect the phosphorylation levels of other important kinases such as AKT, MTOR, and RPS6. This study discovered a new type of PTM of PARP1 in CC, and showed that PARP1 acetylation at K119 is essential in regulating the proliferation and progression of CC through ERK1/2. Further studies are required to investigate how PARP1 acetylation impact its function.

Novel Tricyclo[4.2.1]nonane and Bicyclo[2.2.1]heptane Derivatives: Synthesis, in vitro Biological Activities and in Silico Studies.

The target compounds benzothiazole derivatives (8a-g) were synthesized starting from the norbornene. The antiproliferative activities of the compounds 6a-g and 8a-g were determined against C6 (rat brain tumor) and HeLa (human cervical carcinoma cells) cell lines using BrdU cell proliferation ELISA assay using 5-fluorouracil (5-FU) as standard. In both series, when compared with 5-FU (IC50=<5 µM for C6 and 16.33µM for HeLa), the most active compounds against C6 cells were 6a and 8g with IC50 values of 14.13 µM and 29.99 µM, respectively, while 6a, 6e, 6f and 8b were the most active compounds against HeLa cells with IC50 values of <5, <5, 19.33 and 1813 µM, respectively. Addition, to predict the physicochemical and AMDE properties of the tested compounds, SwissADME online web tool was used. The results showed that all compounds possess promising predicted physiochemical and pharmacokinetic properties, and they complied with Lipinski's rule of 5 indicating that they are predicted to be orally bioavailable, and they possess a predicted bioavailability score of 0.55. Furthermore, in SwissADME Boiled-Egg chart, all compounds showed high predicted GIT absorption, and while compounds 6a-g showed blood brain barrier (BBB) permeation, the compounds 8a-g did not. Moreover, all compounds are not p-glycoprotein (P-gp) substrates.

The Green Extraction of Blueberry By-Products: An Evaluation of the Bioactive Potential of the Anthocyanin/Polyphenol Fraction.

In the context of a circular economy, this study explores the valorization of blueberry pomace (BP) as a source of bioactive compounds using sustainable extraction methods. Microwave-assisted extraction (MAE) and microwave-assisted subcritical water extraction (MASWE) were employed to obtain two distinct fractions: MAE 1° and MASWE 2°. The first extract, MAE 1°, obtained at 80 °C, had a high total anthocyanin content (21.96 mgCya-3-glu/gextract), making it suitable as a natural pigment. Additionally, MAE 1° exhibited significant enzyme inhibition, particularly against α-amylase and β-glucosidase, suggesting potential anti-diabetic and anti-viral applications. The second extract, MASWE 2°, obtained at 150 °C, contained a higher total phenolic content (211.73 mgGAE/gextract) and demonstrated stronger antioxidant activity. MASWE 2° showed greater inhibition of acetylcholinesterase and tyrosinase, indicating its potential for use in Alzheimer's treatment, skincare, or as a food preservative. MASWE 2° exhibited cytotoxicity against HeLa cells and effectively mitigated H2O2-induced oxidative stress in HaCat cells, with MAE 1° showing similar but less pronounced effects. A tested formulation combining MAE 1° and MASWE 2° extracts in a 3:2 ratio effectively enhanced anthocyanin stability, demonstrating its potential as a heat-stable pigment. The extract characteristics were compared with a conventional method (MeOH-HCl in reflux condition), and the protocol's sustainability was assessed using several green metric tools, which provided insights into its environmental impact and efficiency.

Integrated strategy for high-confident global profiling of the histidine phosphoproteome

BackgroundHistidine phosphorylation (pHis) plays a key role in signal transduction in prokaryotes and regulates tumour initiation and progression in mammals. However, the pHis substrates and their functions are rarely known due to the lack of effective analytical strategies. ResultsHerein, we provide a strategy for unbiased enrichment and assignment of the pHis peptides. First, the entire procedure was designed under alkaline conditions to maintain the stability of the N–P bond of pHis and high-pH reverse-phase chromatography was used to efficiently separate the pHis peptides. Second, exploiting the coelution benefits of diethyl labelling, the ratios of light- and heavy-labelled peptides were accurately quantified, and the sites of phosphorylated histidine were assigned. Finally, Cu-IDA bead enrichment and data-independent acquisition mass spectrometry analysis were used to improve the coverage of the histidine phosphoproteome. With this novel strategy, 768 and 1125 potential pHis peptides were identified from lysates of E. coli and HeLa cells, respectively. And these values represent the highest coverage of the histidine phosphoproteome for both cell types. SignificanceThese data strongly support the presumption that pHis modifications are widely present in bacteria. The study provides an efficient strategy and can lead to a better understanding of pHis-modified substrates and their biological functions.

Cap-Specific m6Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m6A Methyltransferase Activity.

Chemical modifications of cellular RNAs play key roles in gene expression and host defense. The cap-adjacent N6,2'-O-dimethyladenosine (m6Am) is a prevalent modification of vertebrate and viral mRNAs and is catalyzed by the newly discovered N6 methyltransferase PCIF1. However, its role in gene expression remains unclear due to conflicting reports on its effects on mRNA stability and translation. In this study, we investigated the impact of siRNA-mediated transient suppression of PCIF1 on global mRNA expression in HeLa cells. We identified a subset of differentially expressed genes (DEGs) that exhibited minimal overlap with previously reported DEGs. Subsequent validation revealed that PCIF1 positively and negatively regulates RAB23 and CNOT6 expression, respectively, at both the mRNA and protein levels. Mechanistic analyses demonstrated that PCIF1 regulates the stability of these target mRNAs rather than their transcription, and rescue experiments confirmed the requirement of PCIF1's methyltransferase activity for these regulations. Furthermore, MeRIP-qPCR analysis showed that PCIF1 suppression significantly reduced the m6A levels of RAB23 and CNOT6 mRNAs. These findings suggest that PCIF1 regulates the stability of specific mRNAs in opposite ways through m6A modification, providing new insights into the role of m6Am in the regulation of gene expression.

Combining lavendustin C and 5-arylidenethiazolin-4-one-based pharmacophores toward multitarget anticancer hybrids

Lavendustin C, a natural-product derived anticancer lead compound, was modified at its carboxylic group by esterification or amidation (compounds 6–10) and at its amino group by introducing 5-arylidenethiazolin-4-ones (14a–c to 17a–c, 18a and 18b). Two strategies were used to combine these moieties and to optimize the yield. These new compounds were evaluated for their antiproliferative activities against a panel of nine cancer cell lines. The results clearly show that 5-arylidenethiazolin-4-one moiety contributes substantially to the activity. Also, methyl esters are more potent than amides, while N-ethylamides are the most potent among amides. 14b showed the highest potency against all tested cancer cell lines with IC50 1.4–2.5 µM, while against normal cell line IC50 > 50 µM. It showed arrest of HeLa cells at G0/G1, S phases and reduction of the percent of cells in G2/M. Moreover, 14b triggered death of HeLa cancer cells via apoptosis induction. EGFR inhibitory potency of 14b was found to be comparable to that of erlotinib. Computational docking and in silico pharmacokinetic studies were performed and discussed. In conclusion, 14b might serve as a multitarget lead compound for further development of anticancer agents.

A synthetic curcumin-like diarylpentanoid analog inhibits rhinovirus infection in H1 hela cells via multiple antiviral mechanisms.

Rhinovirus (RV) infection is a major cause of common colds and asthma exacerbations, with no antiviral drug available. Curcumin exhibits broad-spectrum antiviral activities, but its therapeutic effect is limited by a poor pharmacokinetics profile. Curcumin-like diarylpentanoid analogs, particularly 2-benzoyl-6-(3,4-dihydroxybenzylidene)cyclohexen-1-ol (BDHBC) and 5-(3,4-dihydroxyphenyl)-3-hydroxy-1-(2-hydroxyphenyl)penta-2,4-dien-1-one (DHHPD), have better solubility and stability compared to curcumin. Therefore, this study aims to evaluate and compare the antiviral effects of curcumin, BDHBC, and DHHPD in an in vitro model of RV infection. The inhibitory effects on RV-16 infection in H1 HeLa cells were assessed using cytopathic effect (CPE) reduction assay, virus yield reduction assay, RT-qPCR, and Western blot. Antiviral effects in different modes of treatment (pre-, co-, and post-treatment) were also compared. Additionally, intercellular adhesion molecule 1 (ICAM-1) expression, RV binding, and infectivity were measured with Western blot, flow cytometry, and virucidal assay, respectively. When used as a post-treatment, BDHBC (EC50: 4.19µM; SI: 8.32) demonstrated stronger antiviral potential on RV-16 compared to DHHPD (EC50: 18.24µM; SI: 1.82) and curcumin (less than 50% inhibition). BDHBC also showed the strongest inhibitory effect on RV-induced CPE, virus yield, vRNA, and viral proteins (P1, VP0, and VP2). Furthermore, BDHBC pre-treatment has a prophylactic effect against RV infection, which was attributed to reduced basal expression of ICAM-1. However, it did not affect virus binding, but exerted virucidal activity on RV-16, contributing to its antiviral effect during co-treatment. BDHBC exhibits multiple antiviral mechanisms against RV infection and thus could be a potential antiviral agent for RV.

Impact of Protein Coronas on Lipid Nanoparticle Uptake and Endocytic Pathways in Cells.

Lipid nanoparticles (LNPs), widely used in disease diagnosis and drug delivery, face the challenge of being surrounded by biological macromolecules such as proteins upon entering the human body. These molecules compete for binding sites on the nanoparticle surfaces, forming a protein corona. The impact of different types of protein coronas on LNP delivery remains unclear. In this study, we employed a newly developed, highly sensitive LNP labeling platform and analyzed the endocytosis of HeLa cells under different nutritional conditions using proteomics to address this critical issue. Our research found that under conditions of complete medium and amino acid starvation, most DNA-FITC vesicles in HeLa cells were located in the perinuclear region 4 h after transfection. In contrast, under serum starvation conditions, only a small portion of DNA-FITC vesicles were in the perinuclear region. On the other hand, through proteomics, we discovered that cells that were enriched in amino acids and complete medium contained more proteins, whereas those under serum starvation had relatively fewer enriched proteins. Through KEGG pathway enrichment analysis, we identified the phagosome and endocytosis pathways as particularly important. Lastly, differential analysis of proteins in these pathways revealed that proteins such as F-actin, Coronin, vATPase, TUBA, TUBB, MHCII, and TSP may have significant impacts on cellular endocytosis. Our research findings indicate that it is necessary to regulate cellular endocytosis based on different protein coronas to achieve optimal cytoplasmic release.

New naphtho[2,1-b]furan-2,5-dione and isocoumarin derivatives from bambusicolous fungus Gelatinomyces siamensis

Two new compounds, 6-hydroxy-1-(4-hydroxyphenyl)naphtho[2,1-b]furan-2,5-dion (1) and 3-methyl-6-α-L-rhamnopyranosyl-2H-chromen-2-one (2), along with three known compounds (3-5) were isolated from the rare bambusicolous fungus Gelatinomyces siamensis. The structures were elucidated through the analysis of IR, NMR and HRESIMS spectrographic data. Compound 1 exhibited cytotoxicity against HeLa, HT29, MCF-7 and PC-3 cells, but was not toxic against normal Vero cells. The most abundant metabolite, oosporein (5), exhibited significant cytotoxic effects against all tested cancer cells.

Multicolor iLIFE (m-iLIFE) volume cytometry for high-throughput imaging of multiple organelles

To be able to resolve multiple organelles at high throughput is an incredible achievement. This will have immediate implications in a range of fields ranging from fundamental cell biology to translational medicine. To realize such a high-throughput multicolor interrogation modality, we have developed a light-sheet based flow imaging system that is capable of visualizing multiple sub-cellular components with organelle-level resolution. This is possible due to the unique optical design that combines an illumination system comprising two collinear light sheets illuminating the flowing cells and a dedicated dual-color 4f-detection, enabling simultaneous recording of multiple organelles. The system PSF sections up to 4 parallel microfluidic channels through which cells are flowing, and multicolor images of cell cross-sections are recorded. The data is then computationally processed (filtered using ML algorithm, shift-corrected, and merged) and combined to reconstruct the 3D multicolor volume. System testing is conducted using multicolor fluorescent nano-beads (size ∼175\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 175$$\\end{document} nm) and flow-based imaging parameters (PSF size, motion-blur, flow rate, frame rate, and number of cell-sections) are determined for quality imaging. Drug treatment studies were carried out for healthy and cancerous HeLa cells to check the performance of the proposed system. The cells were treated with a drug (Vincristine, which is known to promote mitochondrial fission in cells), and the same is compared with untreated control cells. The proposed multicolor iLIFE system could screen ∼800\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 800$$\\end{document} cells/min (at a flow speed of 2490μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$2490 ~\\upmu$$\\end{document}m/s), and the drug treatment studies were carried out up to 24 h. Studies showed the disintegration of mitochondrial network and dysfunctional lysosomes and their accumulation at the cell membrane, which is a clear indication of cell apoptosis. Compared to control cells (untreated), the mortality is highest at a concentration of 500 nM post 12 h of drug treatment. With the capability of multiorganelle interrogation and organelle-level resolution, the multicolor iLIFE cytometry system is suitably placed to assist optical imaging and biomedical research.

Antimicrobial, Antibiofilm and Anticancer Potentials of Glycine and Glycyl-Glycine; an in vitro study

Globally, there is a huge demand for novel agents capable of providing protection against both pathogen microorganisms and tumor cells. In this study, the antimicrobial, biofilm inhibitory, and anticancer effects of glycine and glycyl-glycine were investigated. The antimicrobial effects were determined using the broth dilution method, while the biofilm inhibitory effects were assessed through the crystal violet binding assay. Cytotoxic effects on HeLa cell viability were measured using the MTT assay. Our results indicate that, although 100 mg/mL of glycine only inhibited S. epidermidis W17 among the three tested isolates, 400 mg/mL of glycyl-glycine inhibited both S. epidermidis W17 and P. mirabilis U15 strains. Additionally, sub-MICs (concentrations below the Minimum Inhibition Concentration) of glycine inhibited biofilm formation by more than 70% in all tested clinical isolates and exhibited significantly more biofilm inhibition against P. mirabilis U15 and S. epidermidis W17 strains (p

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.