3',4'-Dihydroxyflavonol Inhibits Fibrotic Response in a Rabbit Model of Glaucoma Filtration Surgery.

Post-operative fibrosis of the filtering bleb limits the success of glaucoma filtration surgery (GFS). To minimise subconjunctival scarring following GFS, treatment with antimetabolites such as Mitomycin C (MMC) has become standard practice; however, their use is associated with considerable side effects. This study aimed to investigate the anti-scarring properties of 3',4'-dihydroxyflavonol (DiOHF). GFS was performed in New Zealand white rabbits who received eye drops of DiOHF three times daily and vehicle eye drops after surgery (n = 5) or a single intraoperative treatment of MMC (n = 5). Blebs were imaged immediately following surgery and on days 7, 15, 21, and 28 for clinical examination. On day 28, eyes were harvested to assess collagen deposition, expression of α-SMA, oxidative stress, angiogenesis, fibroblast activity, and inflammation in the conjunctiva/Tenon's layer. At 7 and 28 days post-GFS, MMC-treated blebs were more ischaemic than DiOHF- or vehicle-treated blebs. On day 28, DiOHF treatment significantly suppressed collagen accumulation, CD31 expression, Vimentin expression, and CD45 expression compared to the vehicle control. No difference was observed in 3-Nitrotyrosine or αSMA expression between treatment groups. Treatment with DiOHF reduced conjunctival scarring and angiogenesis in rabbits with GFS, which was comparable to MMC. DiOHF may be a safer and more effective wound-modulating agent than conventional antifibrotic therapy in GFS.

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2-3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

Establishment and Characterization of Three Human Ocular Adnexal Sebaceous Carcinoma Cell Lines.

Ocular adnexal sebaceous carcinoma (SebCA) represents one of the most clinically problematic periocular tumors, often requiring aggressive surgical resection. The pathobiology of this tumor remains poorly understood, and few models exist that are suitable for preclinical testing. The aim of this study was to establish new cell lines to serve as models for pathobiological and drug testing. With patient consent, freshly resected tumor tissue was cultured using conditional reprogramming cell conditions. Standard techniques were used to characterize the cell lines in terms of overall growth, clonogenicity, apoptosis, and differentiation in vitro. Additional analyses including Western blotting, short tandem repeat (STR) profiling, and next-generation sequencing (NGS) were performed. Drug screening using mitomycin-C (MMC), 5-fluorouricil (5-FU), and 6-Diazo-5-oxo-L-norleucine (DON) were performed. JHH-SebCA01, JHH-SebCA02, and JHH-SebCA03 cell lines were established from two women and one man undergoing surgical resection of eyelid tumors. At passage 15, they each showed a doubling time of two to three days, and all could form colonies in anchorage-dependent conditions, but not in soft agar. The cells contained cytoplasmic vacuoles consistent with sebaceous differentiation, and adipophilin protein was present in all three lines. STR profiling confirmed that all lines were derived from their respective patients. NGS of the primary tumors and their matched cell lines identified numerous shared mutations, including alterations similar to those previously described in SebCA. Treatment with MMC or 5-FU resulted in dose-dependent growth inhibition and the induction of both apoptosis and differentiation. MYC protein was abundant in all three lines, and the glutamine metabolism inhibitor DON, previously shown to target high MYC tumors, slowed the growth of all our SebCA models. Ocular adnexal SebCA cell lines can be established using conditional reprogramming cell conditions, and our three new models are useful for testing therapies and interrogating the functional role of MYC and other possible molecular drivers. Current topical chemotherapies promote both apoptosis and differentiation in SebCA cells, and these tumors appear sensitive to inhibition or MYC-associated metabolic changes.

Chitinase 3-Like 1 and C-X-C motif chemokine ligand 5 proteins and the hair cycle.

Dermal papilla cells (DPCs) exhibit self-recovery ability, which may be involved in hair growth. Therefore, we tested whether DPCs subjected to temporary growth-inhibiting stress (testosterone, 17ß-estradiol, mitomycin C, or undernutrition) treatments exhibit self-recovery behavior that can activate hair follicle growth, and examined the changes in cell proliferation capacity and gene expression. Related proteins were identified and their relationships with the hair cycle was examined using a mouse model. Recovery-period DPCs (i.e., from day 3 after loading) were subjected to microarray analysis to detect genetic variations common to each stress treatment. Co-culture of recovery-period DPCs and outer root sheath cells (ORSCs) confirmed the promotion of ORSC proliferation, suggesting that the activation of hair follicle growth is promoted via signal transduction. Chitinase 3-like 1 (CHI3L1) and C-X-C motif chemokine 5 (CXCL5) exhibited ORSC proliferation-promoting effects. Measurement of protein content in the skin during each phase of the hair cycle in mice revealed that CHI3L1 and CXCL5 secretion increased immediately after anagen transition. In a hair-loss mouse model treated with testosterone or 17ß-estradiol, CHI3L1 and CXCL5 secretion was lower in treated telogen skin than in untreated skin. Our results suggest that CHI3L1 and CXCL5 secreted by recovery-state DPCs promote hair growth.

An evaluation of mitomycin-containing reverse thermal gel for the treatment of low-grade upper tract urothelial carcinoma.

INTRODUCTION: Endoscopic management of upper tract urothelial carcinoma (UTUC) is increasingly relevant with greater detection of low-grade disease and guidelines recommending kidney preservation for low-risk disease. Historically, laser or thermal ablation has served as the primary tool for endoscopic management of UTUC, however, chemoablation is rapidly being developed to serve as a primary or adjuvant treatment option, which warrants review. AREAS COVERED: The current literature was reviewed to compare the outcomes and clinical utility of endoscopic treatment modalities for low-grade UTUC, with a focus on mitomycin-containing reverse thermal gel (UGN-101). EXPERT OPINION: The overall outcomes of mitomycin-containing gel therapy are promising, but adverse effects such as ureteral stricture call for careful consideration when using this treatment. We believe it is reasonable to consider use of mitomycin-containing gel as an adjuvant chemotherapy with endoscopic laser resection of low-grade upper tract urothelial carcinoma.

Anti-Inflammatory Effect of Atorvastatin on Primary Human Endothelial Cells Incubated under Genotoxic Load.

The level of cytokine expression was measured in human coronary artery (HCAEC) and internal thoracic artery (HITAEC) endothelial cells exposed to 500 ng/ml alkylating mutagen mitomycin C (MMC) and 5 µM atorvastatin. It was found that treatment of MMC-exposed HCAEC with atorvastatin decreased secretion of macrophage migration inhibitory factor (MIF), IL-8, and IL8 gene expression, but increased the expression of SERPINE1 gene encoding the PAI-1 protein. In atorvastatin-treated HITAEC, the concentration of MIF protein and the expression of the IL8 and SERPINE1 genes were reduced. We can conclude that atorvastatin prevents proinflammatory activation of endothelial cells cultured under conditions of genotoxic load. However, the molecular mechanisms of this effect require further research.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates.

BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2.

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.

Cellular metabolomics study of the antitumor mechanism of Sijunzi decoction combined with mitomycin C.

Mitomycin C (MMC) has an antitumor effect and is considered as a broad-spectrum antibiotic. Sijunzi Decoction (SJZD), a well-known ancient Chinese prescription, is widely used in the treatment of cancer when combined with chemotherapy drugs. Studies have shown that SJZD can be combined with other drugs to enhance the therapeutic effect against cancer and inhibit the toxicity of chemotherapy drugs, but the specific mechanism is not clear. Thus, we hope to further explore the antitumor mechanism of combined SJZD and MMC. 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry, western blot, 1H NMR and HPLC-MS were used to study the mechanism at the cellular level. The results show that the combined administration can have a more significant effect on inhibiting the proliferation of cancer cells, promoting their apoptosis. Based on metabolomics, 38 biomarkers were found in the MMC group and 43 biomarkers were found in the combined administration group. Among them, 18 unique biomarkers were discovered in the combined administration group. Studies have shown that the antitumor mechanism of combined administration is related to amino acid metabolism, energy metabolism, lipid metabolism and nucleotide metabolism, among which amino acid metabolism is the most important. In addition, SJZD achieves the effect of toxin reduction and efficiency enhancement by improving the body's immunity and improving the oxidative stress environment.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer.

INTRODUCTION: Appendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line. PATIENTS AND METHODS: PTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C with and without bromelain pretreatment. RESULTS: From October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs. CONCLUSIONS: Bromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

Chromosome-specific induction of micronuclei and chromosomal aberrations by mitomycin C: Involvement of human chromosomes 9, 1 and 16.

Cytogenetic studies have shown that human chromosomes 1, 9, and 16, with a large heterochromatic region of highly methylated classical satellite DNA, are prone to induction of chromatid breaks and interchanges by mitomycin C (MMC). A couple of studies have indicated that material from chromosome 9, and possibly also from chromosomes 1 and 16, are preferentially micronucleated by MMC. Here, we further examined the chromosome-specific induction of micronuclei (MN; with and without cytochalasin B) and chromosomal aberrations (CAs) by MMC. Cultures of isolated human lymphocytes from two male donors were treated (at 48 h of culture, for 24 h) with MMC (500 ng/ml), and the induced MN were examined by a pancentromeric DNA probe and paint probe for chromosome 9, and by paint probes for chromosomes 1 and 16. MMC increased the total frequency of MN by 6-8-fold but the frequency of chromosome 9 -positive (9+) MN by 29-30-fold and the frequency of chromosome 1 -positive (1+) MN and chromosome 16 -positive (16+) MN by 12-16-fold and 10-17-fold, respectively. After treatment with MMC, 34-47 % of all MN were 9+, 17-20 % 1+, and 3-4 % 16+. The majority (94-96 %) of the 9+ MN contained no centromere and thus harboured acentric fragments. When MMC-induced CAs aberrations were characterized by using the pancentromeric DNA probe and probes for the classical satellite region and long- and short- arm telomeres of chromosome 9, a high proportion of chromosomal breaks (31 %) and interchanges (41 %) concerned chromosome 9. In 83 % of cases, the breakpoint in chromosome 9 was just below the region (9cen-q12) labelled by the classical satellite probe. Our results indicate that MMC specifically induces MN harbouring fragments of chromosome 9, 1, and 16. CAs of chromosome 9 are highly overrepresented in metaphases of MMC-treated lymphocytes. The preferential breakpoint is below the region 9q12.

Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin.

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5 µM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

Proteomic Profiling of Endothelial Cells Exposed to Mitomycin C: Key Proteins and Pathways Underlying Genotoxic Stress-Induced Endothelial Dysfunction.

Mitomycin C (MMC)-induced genotoxic stress can be considered to be a novel trigger of endothelial dysfunction and atherosclerosis-a leading cause of cardiovascular morbidity and mortality worldwide. Given the increasing genotoxic load on the human organism, the decryption of the molecular pathways underlying genotoxic stress-induced endothelial dysfunction could improve our understanding of the role of genotoxic stress in atherogenesis. Here, we performed a proteomic profiling of human coronary artery endothelial cells (HCAECs) and human internal thoracic endothelial cells (HITAECs) in vitro that were exposed to MMC to identify the biochemical pathways and proteins underlying genotoxic stress-induced endothelial dysfunction. We denoted 198 and 71 unique, differentially expressed proteins (DEPs) in the MMC-treated HCAECs and HITAECs, respectively; only 4 DEPs were identified in both the HCAECs and HITAECs. In the MMC-treated HCAECs, 44.5% of the DEPs were upregulated and 55.5% of the DEPs were downregulated, while in HITAECs, these percentages were 72% and 28%, respectively. The denoted DEPs are involved in the processes of nucleotides and RNA metabolism, vesicle-mediated transport, post-translation protein modification, cell cycle control, the transport of small molecules, transcription and signal transduction. The obtained results could improve our understanding of the fundamental basis of atherogenesis and help in the justification of genotoxic stress as a risk factor for atherosclerosis.

Mitomycin C and its analog trigger cytotoxicity in MCF-7 and K562 cancer cells through the regulation of RAS and MAPK/ERK pathways.

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Using a Novel, Subconjunctival, Sustained-Release Mitomycin C Formulation in a Rabbit Model of Filtration Surgery with Gel Stent Implantation.

Purpose: To investigate gel stent implantation with and without intraoperative sustained-release mitomycin C (MMC SR) in a rabbit model for gel stent implantation, and to examine aqueous humor outflow (AHO) postimplantation. Methods: Four groups of rabbits were included. Group 1 was untreated (control). Groups 2, 3, and 4 received the gel stent without MMC, with MMC solution (subconjunctival injection), and with MMC SR (subconjunctival injection), respectively. Intraocular pressure (IOP) and AHO were assessed via tonometry and indocyanine green-based angiography, respectively. The main efficacy measure was change in IOP from baseline. Results: Following gel stent implantation, Groups 2, 3, and 4 maintained ≥20% IOP reduction (response) for a median duration of 1 week, 6.5 weeks, and 30 weeks, respectively. Angiography showed normal aqueous humor drainage (Group 1) beginning at the perilimbal trabecular plexus and continuing posteriorly to episcleral outflow vessels. Following implantation, drainage occurred preferentially and directly into the subconjunctival bleb. Conclusions: Gel stent implantation with MMC SR was most effective in achieving sustained, long-term IOP reduction in the rabbit model, compared with implantation with or without MMC solution. Bleb presence and the postimplantation aqueous angiography results indicated redirection of the AHO to the subconjunctival vasculature and presumed lymphatics, suggesting efficient glaucoma filtration to lower IOP in this model. This rabbit model and aqueous angiography may help refine understanding of the mechanism of action of minimally invasive glaucoma surgeries and ultimately translate to improved surgical devices and procedures for patients with glaucoma.