Foci Formation Research Articles

Protein O-GlcNAcylation homeostasis regulates facultative heterochromatin to fine-tune sog-Dpp signaling during Drosophila early embryogenesis

Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly shortgastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.

Abstract B074: Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in the US. There is a largely unmet clinical need to identify and develop novel strategies, that work either alone or in concert with AR-directed therapeutics, to combat CRPC. The highly conserved histone acetyltransferases CBP/p300 are potent co-activators for AR, and high p300 expression is associated with locally advanced disease and castration-resistant AR function. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC. By employing clinically relevant PCa models, the clinical significance of CBP/p300 expression in PCa patients as well as mechanistic evaluation of CBP/p300 transcriptional reprogramming and DNA damage response pathways have been undertaken. The molecular response to CBP/p300 inhibition will be assessed to discern novel metrics for precision medicine for PCa patients to improve therapeutic efficacy. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. CCS1477 (inobrodib) is a first-in-class bromodomain inhibitor developed by Cell Centric and targeted to inhibit CBP/p300 mediated bromodomain activity, and thus regulate cell survival. Inhibition of the CBP/p300 bromodomain resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression, as well as inhibition of associated factors such c-MYC and its downstream targets, in multiple PCa models. Transcriptomic analysis indicated that both CBP and p300 expression correlate with expression of genes involved in double strand break (DSB) DNA repair process including homologous recombination (HR) and non-homologous end joining (NHEJ) in both primary PCa and CRPC models. CCS1477 directly impacted DNA damage response and repair dynamics, as shown via delay in time to resolution of DNA damage foci formation, including RAD51 and γH2AX foci. Inhibition of CBP/p300 activity decreased tumor cell proliferation, blocked CRPC xenograft growth in vivo, and decreased proliferation ex vivo in patient-derived prostate tumor explants. Importantly, CBP/p300 expression correlated with HR genes in human prostate tissue samples in different cohorts. Lastly, inhibition of CBP/p300 activity also decreased HR gene expression in patients further supporting the essential role CBP/p300 plays in DNA repair. In sum, CBP/p300 inhibition mediates HR repair and impacts patient outcome. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Combined, these studies have the capacity for significant near-term impact in the prevention and/or management of metastatic disease. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati Chand, Wei Yuan, Denisa Bogdan, Jon Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann DeBono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting CBP/p300 and its downstream transcriptional machinery in advanced PCa [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B074.

Abstract B060: Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities

Abstract Background: CDK12 expression is lost in ~5% of metastatic castration-resistant prostate cancer (mCRPC) and associates with aggressive disease. Previous literature, based on acute CDK12-loss models, proposes a mechanism where CDK12 loss leads to a homologous recombination deficiency (HRD) phenotype via premature intronic polyadenylation of classic HR pathway genes, including BRCA1 and BRCA2. Cyclin K is an essential gene and functions in a heterodimer with CDK12 or CDK13. We sought to test if CDK12 loss: (1) confers HRD and a corresponding sensitivity to platinum and poly-ADP ribose polymerase (PARP) inhibitors and/or (2) confers synthetic lethality to other cellular targets and pathways. Methods: To investigate the role of CDK12 loss we used a combination of acute (Tet-inducible shRNA, CDK12/13 inhibitors) and adapted (naturally mutant LuCaP 189.4, CRISPR KO clones) models. DNA repair gene expression and polyadenylation site usage was measured by RNA-seq, qPCR, and western blot while HR function was measured by immunofluorescence for RAD51 foci following irradiation. Stable CDK12-negative (CDK12-neg) cell lines were treated with sgRNAs, PARP inhibitors, carboplatin, and CDK inhibitors to test for potential targeted vulnerabilities. Results: Acute CDK12 inhibition (6h, SR4835) confirmed the reported mechanism of 3’ transcript loss, but the effects were minimal in long term shCDK12 or CRISRP KO lines as measured by RNA-seq, qPCR, and western blot. CDK12-neg lines showed slight decreases in ATR and ATM protein but minimal changes in BRCA1 or BRCA2 and no decrease in RAD51 foci formation. Furthermore, these lines did not show increased sensitivity to carboplatin and only partial sensitivity to PARPi. Human tumor data were analyzed and showed that CDK12-neg tumors lack genomic signatures of HRD and do not show significant de-enrichment of long genes. However, CDK12-neg cell lines did show sensitivity to CDK13 KO or pharmacologic inhibition with CDK12/13 inhibitors (SR4835, THZ531, dinaciclib). In vivo treatment of LuCaP xenograft lines showed that 189.4 responded to SR4835, while two CDK12-intact lines (35 and 136) did not. Conclusions: Our data support a model where HRD is primarily a result of acute CDK12 loss, while the downregulation of long DNA repair genes is largely compensated for (via currently unknown mechanisms) in cells that have adapted to CDK12 loss. We suspect that the partial PARPi sensitivity observed in CDK12-neg lines is due to other (non-HRD) DNA repair deficiencies and potential mechanisms are under investigation. CDK12-neg cells exhibited dependency on CDK13, which can be targeted with semi-selective pharmacologic compounds, though our data support the development of specific CDK13 inhibitors to maximize synthetic lethality and minimize off-target effects. Finally, additional work is ongoing with CRISPR and drug screens to identify additional vulnerabilities in adapted CDK12-neg models and provide future clinical targets for this aggressive subset of mCRPC. Citation Format: Sander Frank, Ilsa Coleman, Navonil De Sarkar, Dmytro Rudoy, Valeri Vasioukhin, Pete Nelson. Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B060.

DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing m6A binding

N6-methyladenosine (m6A) is the most prevalent internal RNA modification, and its regulators include writers, readers and erasers. m6A is under stringent control and takes part in many biological events, but it is not known whether there is an interplay between m6A and glycosylation. Here we investigated an m6A reader, YTHDC1, which has been shown to be recruited to the DNA-RNA hybrid at DNA damage sites and regulate homologous recombination (HR) during DNA damage repair. We found that YTHDC1 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification at Ser396 upon DNA damage, which is pivotal for YTHDC1 chromatin binding and ionization radiation induced focus (IRIF) formation. RNA immunoprecipitation (RIP) and molecular dynamics (MD) simulations indicate that O-GlcNAcylation is vital for YTHDC1 to bind with m6A RNA. Fluorescence recovery after photo bleaching (FRAP) analysis revealed that YTHDC1 O-GlcNAcylation is essential for DNA damage-induced YTHDC1-m6A condensate formation. We further demonstrate that YTHDC1 O-GlcNAcylation promotes HR-mediated DNA damage repair and cell survival, probably through recruitment of Rad51 to the damage sites. We propose that YTHDC1 O-GlcNAcylation is instrumental for HR.

A phase II, randomized, double-blind study of the use of rucaparib vs placebo maintenance therapy in metastatic and recurrent endometrial cancer.

TPS5626 Background: There are currently no approved maintenance therapies for metastatic and recurrent endometrial cancer, which carries a dismal long-term prognosis. The PARP inhibitor drug class has demonstrated significant clinical benefit as a maintenance therapy for patients with germline/somatic BRCA mutations or homologous recombination deficiency (HRD) in ovarian cancers. However, BRCA mutations are not common in endometrial cancer. Loss of function of the tumor suppressor gene PTEN has been demonstrated in greater than 80% of endometrioid endometrial cancers 1. PTEN loss of function is well known to lead to activation of the PI3K-AKT-mTOR pathway but has also been shown to lead to defects in homologous recombination (HR) DNA repair of double strand breaks 2. Specifically, PTEN silencing leads to decreased RAD51 foci formation. Based on this mechanism, in vitro data on endometrial cancer cell lines has demonstrated sensitivity to PARP inhibitors. Notably, sensitivity of PARP inhibition in PTEN loss of function is independent of microsatellite instability (MSI) 3. Methods: This is a multi-center phase II, placebo controlled, double-blinded study of the use of rucaparib as maintenance therapy in patients with metastatic or recurrent endometrial cancer. A maximum of 138 patients will be randomized 1:1 to receive rucaparib at starting dose of 600mg BID dosing vs. placebo. Standard dose reductions of known toxicities will be followed based on FDA approved indications of rucaparib. Patients will remain on therapy until progression of disease or toxicity induced discontinuation. Eligible patients will have Stage III/IV or recurrent endometrial cancer and must have received one or two prior lines of chemotherapy. All histologic subtypes, including carcinosarcoma will be included. Treatment arms will be stratified on histology, number of lines of prior therapy, and complete vs partial response to prior line of therapy. The primary endpoint is progression free survival (PFS). Secondary endpoints include overall survival (OS), overall response rate (ORR), and toxicity. To date, 79 patients have been enrolled. 1. Mutter GL, Lin MC, Fitzgerald JT, et al. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst. 2000;92(11):924-930. 2. Shen WH, Balajee AS, Wang J, et al. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell. 2007;128(1):157-170. 3. Dedes KJ, Wetterskog D, Mendes-Pereira AM, et al. PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors. Sci Transl Med. 2010;2(53):53ra75. Clinical trial information: NCT03617679 .

Alliance A092104: A randomized phase 2/3 study of olaparib plus temozolomide versus investigator’s choice for the treatment of patients with advanced uterine leiomyosarcoma after progression on prior chemotherapy.

TPS11584 Background: Uterine leiomyosarcoma (uLMS) is an aggressive sarcoma subtype with frequent metastatic relapse. Advanced uLMS is treated with gemcitabine/docetaxel or anthracycline-based regimens. After failure of initial chemotherapy, remaining options provide limited benefit (trabectedin: objective response rate (ORR) 11%, median progression-free survival (mPFS) 4.0 months (mo); pazopanib: ORR 11%, mPFS 3.0 mo; dacarbazine: ORR 9%, mPFS 1.5 mo). 18-23% of uLMS harbor deleterious alterations in homologous recombination (HR) DNA repair genes. HR-deficient cancers are unable to effectively repair double-stranded DNA breaks and may be sensitive to poly ADP-ribose polymerase (PARP) inhibitor-based strategies. In preclinical studies, the combination of temozolomide (T), an alkylating agent, and olaparib (O), a PARP inhibitor, markedly suppressed proliferation of uLMS models. Among 22 uLMS patients with median 3 prior lines, O+T demonstrated mPFS 6.9 mo and ORR 27% (Ingham M. et al. ASCO 2021: #11506). In correlative analysis, alterations in HR genes including PALB2 and RAD51B or absence of RAD51 foci formation by a functional assay were observed in patients with prolonged PFS (Bose S. et al. ASCO 2022: #11509). Based on these results, we designed a randomized phase 2/3 study to further evaluate O+T in advanced, pretreated uLMS. Methods: Alliance for Clinical Trials in Oncology A092104 is a randomized, open-label phase 2/3 clinical trial of O+T versus investigator’s choice in advanced uLMS. Eligible patients have ECOG performance status ≤2, progression on ≥ 2 prior treatment lines and measurable disease. Patients are randomized 1:1 to receive T 75 mg/m2 orally daily + O 200 mg orally twice daily on days 1-7 in 21-day cycles (Arm 1) or investigator’s choice of trabectedin 1.5 mg/m2 over 24 hours every 21 days or pazopanib 400-600 mg orally daily (Arm 2). For phase 2, the primary endpoint is PFS. The design evaluates for an improvement in PFS from 4 mo to 8 mo, requires 70 patients, and yields 90% power and 1-sided alpha 0.10. If phase 2 is positive, phase 3 will enroll. For phase 3, the primary endpoint is overall survival (OS). The design evaluates for an improvement in OS from 13 mo to 23 mo, requires 165 patients (including 70 from phase 2) and yields 90% power with 1-sided alpha 0.025. Secondary endpoints include ORR, duration of response, safety and patient-reported outcomes. Archival tissue will be collected and evaluated with a RAD51 foci formation assay. Genomic sequencing results will be obtained where available. The study opened to accrual in 1/2023. Support: U10CA180821, U10CA180882, U24 CA196171; https://acknowledgments.alliancefound.org . Clinical trial information: NCT05633381 .

Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells.

APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and in vitro deamination. Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities in vitro. Notably, in nuclear lysates, the in vitro deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that in vitro deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.

Insight into the Toxicological and Pathophysiological Effects of Moroccan Vipers' Venom: Assessing the Efficacy of Commercial Antivenom for Neutralization.

Morocco is one of the richest countries in biodiversity in the Mediterranean region, especially in its ophidian fauna. In total, there are eight species of venomous snakes, with seven belonging to the Viperidae family, responsible for 67.2% of severe envenomation cases in the country. Cerastes cerastes, Daboia mauritanica and Bitis arietans are considered among the most venomous vipers whose bites cause high levels of morbidity, disability or mortality. Despite their wide distribution in the kingdom, the incidence of these snakebites remains poorly understood and largely underestimated. Moreover, intraspecific variations in the venom composition significantly affect the effectiveness of antivenoms. Due to the unavailability of locally produced antivenoms, we evaluated the efficacy of Inoserp-MENA, the only available antivenom in Morocco, against C. cerastes, D. mauritanica and B. arietans. First, we conducted a comprehensive characterization of these venoms, including an LD50 test to examine their toxicity and SDS-PAGE as a technique to analyze the enzymes responsible for biological activities, such as hemorrhagic and edematous activities and myotoxicity, which generate physiopathological effects in the skin, paws and muscles of envenomed mice. Then, we assessed the ability of Inoserp-MENA antivenom to neutralize the toxic activities of Moroccan vipers. Our results indicate that the venom of C. cerastes, D. mauritanica and B. arietans are toxic, causing severe alterations such as edema, myotoxicity, myonecrosis and significant hemorrhages with the formation of hemorrhagic foci. C. cerastes venom is more dangerous in terms of lethality and hemorrhages, while B. arietans venom is more edematous. The effects of C. cerastes venom were effectively neutralized, but Inoserp-MENA antivenom failed to protect mice against the toxic effects induced by B. arietans and D. mauritanica venom. The study reveals alarming shortcomings in the effectiveness of the current commercially available antivenom's dosage and neutralization capabilities, highlighting the urgent need to develop a region-specific viper envenomation therapy.

The E3 Ubiquitin Ligase SCF Cyclin F Promotes Sequestosome-1/p62 Insolubility and Foci Formation and is Dysregulated in ALS and FTD Pathogenesis

Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCFcyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1/p62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCFcyclinF complex. We found that SCFcyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.

Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis

Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth invitro and invivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

Actin nucleators safeguard replication forks by limiting nascent strand degradation.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that β-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing β-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing β-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

Protection against Microglia Senescence by the Dietary Supplement Dekosilhue® in BV2 Cells: A New Perspective for Obesity and Related Complications

Growing evidence indicates chronic low-grade systemic inflammation as a major pathophysiological mechanism of obesity. Systemic inflammation provokes an immune response in the brain through the activation of microglia that results in the development of neuroinflammation, cellular senescence, and occurrence of neurological dysfunction. In the efforts to identify an innovative intervention with potential efficacy on obesity and associated complications, our aim was to study the capability of the dietary supplement Dekosilhue® (DKS), successfully used for improving the control of body weight, to attenuate microglia senescent phenotype. Microglia senescence was induced by intermittent stimulation of BV2 cells with LPS 500 ng/mL every 72 h for 4 h/day, over a period of 10 days. DKS (100 µg/mL) treatment reduced ß-galactosidase activity and expression, the formation of senescence-associated heterochromatin foci to control levels, and increased cell viability of senescent BV2 (2 folds of control). DSK reduced the expression of Nuclear Factor-kB (NF-kB) (20% lower than control), a key molecule involved in the acquisition of the senescence-associated secretory phenotype (SASP). DKS promoted a neuroprotective effect by increasing the cell viability of SH-SY5Y neuronal cells exposed to the senescent BV2 conditioned medium to values of non-senescent cells. In conclusion, DKS attenuated the senescent microglia phenotype, showing senotherapeutic activity that might be further investigated as adjunctive intervention for obesity and obesity-related neurological disturbances.

The TCF4 Trinucleotide Repeat Expansion of Fuchs' Endothelial Corneal Dystrophy: Implications for the Anterior Segment of the Eye.

In the United States, 70% of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact. We examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium. CUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5' end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium. Expression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.

Optical focusing inside scattering media with iterative time-reversed ultrasonically encoded near-infrared light.

Focusing light inside scattering media is a long-sought goal in optics. Time-reversed ultrasonically encoded (TRUE) focusing, which combines the advantages of biological transparency of the ultrasound and the high efficiency of digital optical phase conjugation (DOPC) based wavefront shaping, has been proposed to tackle this problem. By invoking repeated acousto-optic interactions, iterative TRUE (iTRUE) focusing can further break the resolution barrier imposed by the acoustic diffraction limit, showing great potential for deep-tissue biomedical applications. However, stringent requirements on system alignment prohibit the practical use of iTRUE focusing, especially for biomedical applications at the near-infrared spectral window. In this work, we fill this blank by developing an alignment protocol that is suitable for iTRUE focusing with a near-infrared light source. This protocol mainly contains three steps, including rough alignment with manual adjustment, fine-tuning with a high-precision motorized stage, and digital compensation through Zernike polynomials. Using this protocol, an optical focus with a peak-to-background ratio (PBR) of up to 70% of the theoretical value can be achieved. By using a 5-MHz ultrasonic transducer, we demonstrated the first iTRUE focusing using near-infrared light at 1053 nm, enabling the formation of an optical focus inside a scattering medium composed of stacked scattering films and a mirror. Quantitatively, the size of the focus decreased from roughly 1 mm to 160 µm within a few consecutive iterations and a PBR up to 70 was finally achieved. We anticipate that the capability of focusing near-infrared light inside scattering media, along with the reported alignment protocol, can be beneficial to a variety of applications in biomedical optics.

Benzo(ghi)perylene (BgP) a black tattoo ingredient induced skin toxicity via direct and indirect mode of DNA damage under UVA irradiation

Tattooing is a very common fashion trend across all the ages and gender of the society worldwide. Although skin inflammatory diseases are very frequent among tattoo users because of the active chemical ingredients used in tattoo ink, yet no ingredient-specific toxicity study has been performed. Benzo(ghi)perylene (BgP) is one of the PAHs and an important ingredient of black tattoo ink that shows strong absorption in UVA and UVB radiation of sunlight. Therefore, understanding the hazardous potential of BgP especially under UVA exposure is important for the safety of skin of tattoo users by considering the fact that penetration of UVA is in the dermis region where tattoo ingredients reside. To evaluate the hazardous potential of BgP on human skin under UVA exposure, different experimental tools i.e., in-chemico, in-silico and in-vitro were utilized. Our results illustrated that BgP photosensitized under UVA (1.5 mW/cm2) irradiation shows a degradation pattern till 4 h exposure. Photosensitized BgP reduced significant cell viability (%) at 1 μg/ml concentration. However, the pretreatment of singlet and hydroxyl radical quenchers, restoration of cell viability observed, confirmed the role of type-I and type-II photodynamic reactions in phototoxicity of BgP. Further, intracellular uptake of BgP in HaCaT cells was estimated and confirmed by UHPLC analysis. Molecular docking of BgP with DNA and formation of γ-H2AX foci demonstrated the DNA intercalation and double-stranded DNA damaging potential of BgP. Furthermore, acridine orange and ethidium bromide (AO/EB) dual staining showed apoptotic cell death via photosensitized BgP under UVA irradiation. The above findings suggest that BgP reached the human skin cell and induced dermal toxicity via direct and indirect mode of DNA damage under UVA exposure finally promoting the skin cell death. Thus, BgP-containing tattoo ink may be hazardous and may induce skin damage and diseases, especially in presence of UVA radiation of sunlight. To minimize the risk of skin diseases from synthetic ingredients in tattoo ink, the study highlights the importance of developing eco-friendly and skin-friendly tattoo ingredients by companies.

Chemotherapy and cryopreservation affects DNA repair foci in lymphocytes of breast cancer patients

Purpose Although breast cancer (BC) patients benefit from radiotherapy (RT), some radiosensitive (RS) patients suffer from side effects caused by ionizing radiation in healthy tissues. It is thought that RS is underlaid by a deficiency in the repair of DNA double-strand breaks (DSB). DNA repair proteins such as p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γH2AX), form DNA repair foci at the DSB locations and thus serve as DSB biomarkers. Peripheral blood lymphocytes (PBL) are commonly believed to be an appropriate cell system for RS assessment using DNA repair foci. The amount of DSB may also be influenced by chemotherapy (CHT), which is often chosen as the first treatment modality before RT. As it is not always possible to analyze blood samples immediately after collection, there is a need for cryopreservation of PBL in liquid nitrogen. However, cryopreservation may potentially affect the number of DNA repair foci. In this work, we studied the effect of cryopreservation and CHT on the amount of DNA repair foci in PBL of BC patients undergoing radiotherapy. Materials and methods The effect of cryopreservation was studied by immunofluorescence analysis of 53BP1 and γH2AX proteins at different time intervals after in vitro irradiation. The effect of chemotherapy was analyzed by fluorescent labelling of 53BP1 and γH2AX proteins in PBL collected before, during, and after RT. Results Higher number of primary 53BP1/γH2AX foci was observed in frozen cells indicating that cryopreservation affects the formation of DNA repair foci in PBL of BC patients. In CHT-treated patients, a higher number of foci were found before RT, but no differences were observed during and after the RT. Conclusions Cryopreservation is the method of choice for analyzing DNA repair residual foci, but only similarly treated and preserved cells should be used for comparison of primary foci. CHT induces DNA repair foci in PBL of BC patients, but this effect disappears during radiotherapy.

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

Investigation into the Potential Role of Propionibacterium freudenreichii in Prevention of Colorectal Cancer and Its Effects on the Diversity of Gut Microbiota in Rats.

Colorectal cancer (CRC) accounts for 10% of all cancer diagnoses and cancer-related deaths worldwide. Over the past two decades, several studies have demonstrated the clinical benefits of probiotic supplementation and some studies have shown that certain probiotics can modulate immunity and strengthen gut microbiota diversity. This study aims to assess the impact of the Propionibacterium freudenreichii (PF) probiotic against CRC induced by azoxymethane (AOM), and to investigate its effects on gut microbiota diversity in rats, as well as to evaluate the anti-proliferative activities of PF in HCT116 CRC cells. This experiment was performed using four groups of SD rats: normal control, AOM group, PF group (1 × 109 CFU/mL), and standard drug control (5-fluorouracil, 35 mg/kg). Methylene blue staining of colon tissues showed that the administration of PF significantly reduced the formation of colonic aberrant crypt foci (ACF) compared to the AOM control group. In addition, treated rats had lower levels of malondialdehyde in their colon tissue homogenates, indicating that lipid peroxidation was suppressed by PF supplementation. Furthermore, 16S rRNA gene analysis revealed that probiotic treatment enhanced the diversity of gut microbiota in rats. In vitro study showed that the viability of HCT116 cells was inhibited by the probiotic cell-free supernatant with an IC50 value of 13.3 ± 0.133. In conclusion, these results reveal that consuming PF as probiotic supplements modulates gut microbiota, inhibits the carcinogenic effects of AOM, and exerts anti-proliferative activity against CRC cells. Further studies are required to elucidate the role of PF on the immune response during the development and growth of CRC.

Morin Sensitizes MDA-MB-231 Triple-Negative Breast Cancer Cells to Doxorubicin Cytotoxicity by Suppressing FOXM1 and Attenuating EGFR/STAT3 Signaling Pathways.

Considerable emphasis is being placed on combinatorial chemotherapeutic/natural treatments for breast cancer. This study reveals the synergistic anti-tumor activity of morin and Doxorubicin (Dox) co-treatment on MDA-MB-231 triple-negative breast cancer (TNBC) cell proliferation. Morin/Dox treatment promoted Dox uptake and induced DNA damage and formation of nuclear foci of p-H2A.X. Furthermore, DNA repair proteins, RAD51 and survivin, and cell cycle proteins, cyclin B1 and forkhead Box M1 (FOXM1), were induced by Dox alone but attenuated by morin/Dox co-treatment. In addition, Annexin V/7-AAD analysis revealed that necrotic cell death after co-treatment and apoptotic cell death by Dox alone were associated with the induction of cleaved PARP and caspase-7 without Bcl-2 family involvement. FOXM1 inhibition by thiostrepton showed that co-treatment caused FOXM1-mediated cell death. Furthermore, co-treatment downregulated the phosphorylation of EGFR and STAT3. Flow cytometry showed that the accumulation of cells in the G2/M and S phases might be linked to cellular Dox uptake, p21 upregulation, and cyclin D1 downregulation. Taken together, our study shows that the anti-tumor effect of morin/Dox co-treatment is due to the suppression of FOXM1 and attenuation of EGFR/STAT3 signaling pathways in MDA-MB-231 TNBC cells, which suggests that morin offers a means of improving therapeutic efficacy in TNBC patients.

Methodical Approach to fMRI Assessment of Motor Connectome in Patients After Severe Traumatic Brain Injury

The aim of the study. To identify alterations of motor connectome in patients with varying degrees of hemi-paresis after severe traumatic brain injury (TBI) versus healthy volunteers.Material and methods. The study included 29 patients with TBI aged 18 to 35 years and 23 healthy volunteers aged 20 to 32 years. Participants underwent a comprehensive clinical and neuroimaging study. Motor impairment was evaluated via muscle strength assessment using a five-score scale. The fMRI data were processed using a dedicated CONN software package. Anatomical 3-D connection masks of the whole brain motor functional system in the predetermined regions of interest (ROIs) were used for the assessment. Then the group indicators of functional connectivity (statistical significance of the connection) were computed.Results. It was established that the structure of connections in healthy individuals performing active movement with the right (leading) hand is determined by formation of focus in the cortical and subcortical ROIs in the contralateral hemisphere. With passive movement of the right hand the pale ball becomes functionally active in addition to the activated areas. The striopallidar system structures became active on both sides, and connectivity with the additional motor cortex and the motor cortex of the ipsilateral hemisphere emerged as the paresis increased during active movement. The focus of motor activity during passive movement was determined in the motor cortex and putamen, which makes it possible to use a passive test in patients with gross motor disorders or unconsciousness for a full assessment of the entire structural and functional brain connectome.Conclusion. As hemiparesis increased in patients after severe traumatic brain injury, a decrease in the total number of connection appeared; simultaneous engagement of ancient primordial structures, such as bilateral activation of pale globes, demonstrated neuroplasticity.