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.

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.

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.

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.

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.

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.

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.

A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

SN-38, an active metabolite of irinotecan, inhibits transcription of nuclear factor erythroid 2-related factor 2 and enhances drug sensitivity of colorectal cancer cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) significantly contributes to drug resistance of cancer cells, and Nrf2 inhibitors have been vigorously pursued. Repurposing of existing drugs, especially anticancer drugs, is a straightforward and promising strategy to find clinically available Nrf2 inhibitors and effective drug combinations. Topoisomerase inhibitors SN-38 (an active metabolite of irinotecan), topotecan, mitoxantrone, and epirubicin were found to significantly suppress Nrf2 transcriptional activity in cancer cells. SN-38, the most potent one among them, significantly inhibited the transcription of Nrf2, as indicated by decreased mRNA level and binding of RNA polymerase II to NFE2L2 gene, while no impact on Nrf2 protein or mRNA degradation was observed. SN-38 synergized with Nrf2-sensitive anticancer drugs such as mitomycin C in killing colorectal cancer cells, and irinotecan and mitomycin C synergistically inhibited the growth of SW480 xenografts in nude mice. Our study identified SN-38 and three other topoisomerase inhibitors as Nrf2 inhibitors, revealed the Nrf2-inhibitory mechanism of SN-38, and indicate that clinically feasible drug combinations could be designed based on their interactions with Nrf2 signaling.

Efficacy of anti-vascular endothelial growth factor and mitomycin C on wound healing after trabeculectomy in glaucoma patients: A meta-analysis.

Trabeculectomy is the main surgical treatment for glaucoma, but scar formation during wound healing may lead to surgical failure. In this study, we evaluated the efficacy of anti-vascular endothelial growth factor (anti-VEGF) and mitomycin C (MMC) on wound healing after glaucoma surgery. We have been looking for Pubmed, Embase and other databases. The last time we looked at an electronic database was August 2023. A case control study was conducted to compare the use of anti-VEGF and mitomycin C for the treatment of glaucoma. We used the Cochrane standard methodology for collecting and analysing the data. Based on the criteria of inclusion, we have determined 369 related papers and selected seven eligible trials for data analysis. Three hundred and twenty-six cases were treated with trabeculectomy, of which 166 were injected with anti-VEGF and 160 were given MMC for trabeculectomy. In six trials, anti-VEGF and MMC were not found to have any statistical significance on postoperative wound leakage after surgery (OR, 1.55; 95% CI, 0.71, 3.35 p = 0.27). The three trials showed that anti-VEGF and MMC did not differ in terms of reducing postoperative wound hypotony after surgery (OR, 0.78; 95% CI, 0.20, 3.11 p = 0.73). Five trials demonstrated that anti-VEGF and MMC were not associated with a lower incidence of shallow anterior chamber (OR, 1.17; 95% CI, 0.5, 2.76 p = 0.71). There is no significant difference in the effect of anti-VEGF and MMC on wound healing after glaucoma surgery. A multicentre randomized controlled trial with a larger sample size is needed to confirm this study.

A new PK/PD target for assessing efficacy of micafungin against Candida parapsilosis.

BACKGROUND: Pharmacokinetic/pharmacodynamic (PK/PD) targets of echinocandins failed to support current clinical breakpoints of Candida parapsilosis as the PTA is low for susceptible isolates despite the good clinical efficacy of echinocandins against these infections. We therefore investigated the effect of micafungin against C. parapsilosis using an in vitro PK/PD in the presence of 10% human serum. METHODS: Three susceptible (MIC = 0.5-2 mg/L) and one resistant (MIC > 8 mg/L) C. parapsilosis sensu stricto isolates were tested at two different inocula (104 and 103 cfu/mL) simulating micafungin human exposures in RPMI and in RPMI + 10% pooled human serum. The exposure-effect relationship tAUC0-24/MIC was described and different PK/PD targets were determined in order to calculate the PTA for the standard 100 mg IV q24h dose. RESULTS: A maximal effect was found at fCmax ≥ 4 mg/L in RPMI and tCmax ≥ 64 mg/L (fCmax = 0.08 mg/L) in the presence of serum for which in vitro PK/PD targets were 50 times lower. Stasis in the presence of serum was found at 272-240 tAUC0-24/MIC, close to the clinical PK/PD target (285 tAUC/MIC), validating the in vitro model. However, the PTA was low for susceptible isolates with EUCAST/CLSI MICs ≤ 2 mg/L. Among the different PK/PD targets investigated, the PK/PD target 28 tAUC/MIC associated with 10% of maximal effect with the low inoculum resulted in PTAs ≥ 95% for susceptible isolates with EUCAST/CLSI MICs ≤ 2 mg/L. CONCLUSIONS: A new PK/PD target was found for micafungin and C. parapsilosis that supports the current clinical breakpoint. This target could be used for assessing echinocandin efficacy against C. parapsilosis.

Chronic tympanic membrane perforation repair with a collagen-based scaffold: An in vivo model.

OBJECTIVE: The aim of this study was to assess the in vivo efficacy of a novel regenerative collagen-based scaffold developed by the Royal College of Surgeons in Ireland in a chronic tympanic membrane perforation (TMP) using a chinchilla model. METHODS: Bilateral TMPs were induced in 17 mixed gender chinchillas using tympanic membrane resection followed by a mixture of topical Mitomycin C and dexamethasone for 3 days. These were monitored with weekly otoscopy for 8 weeks. Animals were excluded if signs of infection developed in the follow up period (n = 8). At 8 weeks, intervention began and 18 TMPs were assigned to either treatment with the collagen-based scaffold (treated group) or spontaneous healing (control group). Animals were euthanized 6 weeks post-intervention. Otoscopic imaging and auditory brain response (ABR) were conducted at baseline, 8 weeks post-TMP induction and 6 weeks post-intervention. All TMPs were then evaluated at 6 weeks post-intervention and bullae underwent histologic evaluation. RESULTS: At 6 weeks post-intervention, otoscopic imaging demonstrated various degrees of healing in the treated ears. The treated group was noted to have an increased rate of healing when compared to the control group. Histologic evaluation demonstrated a variation in the degree of perforation healing within groups, with some animals in the treated group showing high levels of perforation healing. At 8 weeks after the TMP procedure, most of the animals had worsened hearing response. At 6-week post the collagen-based scaffold treatment, about 50 % (4/8) of the treated ears had improved in hearing response as compared to those of non-treated ears. CONCLUSION: Given the initial histologic evidence of partial healing in scaffold-treated ears, the post-intervention period should be extended to monitor the potential for complete healing. Given the overall positive findings related to healing with the scaffold-treated ears, this material warrants further investigation.

Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran.

Antimicrobial disinfectants have been extensively used to control hospital-acquired infections worldwide. Prolonged exposure to bacteria could promote resistance to antimicrobial disinfectants. This study evaluated the antimicrobial activity of four commonly used disinfectants; triclosan, chlorhexidine digluconate, benzalkonium chloride, and formaldehyde against Acinetobacter baumannii clinical isolates. This study also determined the prevalence and association of efflux pumps encoding genes qacE, qacED1, emrA, and aceI with tolerance to disinfectants. A total of 100 A. baumannii isolates were included in the current study. The antimicrobial disinfectants' minimum inhibitory concentration (MIC) was determined using an agar dilution method. Genes involved in resistance to disinfectants were investigated by PCR method. The benzalkonium chloride MICs ranged between 32 and 128 µg mL-1, chlorhexidine digluconate 8-64 µg mL-1, triclosan 1-32 µg mL-1, and formaldehyde 128 µg mL-1. Overall, the highest MIC90 value was identified for formaldehyde (128 µg mL-1), followed by benzalkonium chloride and chlorhexidine digluconate (64 µg mL-1, each one) and triclosan (4 µg mL-1). In the present study, the qacE, qacED1, emrA, and aceI genes were found in 91%, 55%, 100%, and 88% of isolates, respectively. The qacG gene was not identified in our A. baumannii isolates. The qacED1 gene was associated with higher MICs for all disinfectants tested (P < 0.05), while the qacE and aceI genes were associated with higher MICs for benzalkonium chloride and chlorhexidine. This study indicated that triclosan is the most effective disinfectant against A. baumannii isolates.

RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks.

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

Effects of cyclophosphamide and mitomycin C on radiation-induced transcriptional biomarkers in human lymphoblastoid cells.

PURPOSE: Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced transcriptional biomarkers. METHODS: The human B lymphoblastoid cells (AHH-1) were exposed to 0, 20, 50, 100, 200 and 500 µg/ml CP or 0, 0.025, 0.05, 0.1 and 1 µg/ml MMC, respectively. The appropriate concentrations of CP and MMC were added for 1 h before irradiation with 0, 2, 4 and 6 Gy of 60Co γ-rays at a dose rate of 1 Gy/min. Cell viability was evaluated by CCK-8 assay. The gene expression responses of 18 radiation-induced transcriptional biomarkers were examined at 24 h after exposures to CP and MMC, respectively. The expression levels of five crucial DNA interstrand crosslinks (ICLs) repair genes were also evaluated. The biodosimetry models were established based on the specific radiation-responsive gene combinations. RESULTS: The baseline transcriptional levels of the 18 selected genes were slightly affected by CP treatment in the absence of IR, while the transcript responses to IR could be inhibited as the concentration of CP up to 50 µg/ml. MMC treatment up-regulated the background levels in most radiation-responsive gene expressions. Of 18 genes, only the relative mRNA expression levels of CDKN1A and BBC3 were repressed after treatment with IR and MMC in combination. The relative mRNA level of RAD51 was significantly up-regulated after exposure to CP, while the expression of FANCD2, RAD51 and BLM showed an overall increase in response to MMC treatment. After irradiation, the relative mRNA expression levels of FANCD2, BRCA2 and RAD51 exhibited dose-dependent increases in IR alone and MMC treatment groups. In addition, the biodosimetry models were established using 2-4 radiation-responsive genes based on different radiation exposure scenarios. CONCLUSION: Our findings suggested that IR-induced gene expression changes were slightly affected after exposure to a relatively low concentration of CP and MMC. Gene expression combinations might improve the broad applicability of transcriptional biodosimetry across diverse radiation exposures.

Ceftolozane-Tazobactam against Pseudomonas aeruginosa Cystic Fibrosis Clinical Isolates in the Hollow-Fiber Infection Model: Challenges Imposed by Hypermutability and Heteroresistance.

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC, algO, and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF.

Mito-TEMPO mitigates 5-fluorouracil-induced intestinal injury via attenuating mitochondrial oxidative stress, inflammation, and apoptosis: an in vivo study.

BACKGROUND: Recent evidences highlight role of mitochondria in the development of 5-fluorouracil (5-FU)-induced intestinal toxicity. Mitochondria-targeted antioxidants are well-known for their protective effects in mitochondrial oxidative stress- mediated diseases. In the present study, we investigated protective effect of Mito-TEMPO in 5-FU-induced intestinal toxicity. METHODS: Mito-TEMPO (0.1 mg/kg b.w.) was administered intraperitoneally to male BALB/c mice for 7 days, followed by co-administration of 5-FU for next 4 days (intraperitoneal 12 mg/kg b.w.). Protective effect of Mito-TEMPO on intestinal toxicity was assessed in terms of histopathological alterations, modulation in inflammatory markers, apoptotic cell death, expression of 8-OhDG, mitochondrial functional status and oxidative stress. RESULTS: 5-FU administered animals showed altered intestinal histoarchitecture wherein a shortening and atrophy of the villi was observed. The crypts were disorganized and inflammatory cell infiltration was noted. Mito-TEMPO pre-protected animals demonstrated improved histoarchitecture with normalization of villus height, better organized crypts and reduced inflammatory cell infiltration. The inflammatory markers and myeloperoxidase activity were normalized in mito-TEMPO protected group. A significant reduction in intestinal apoptotic cell death and expression of 8-OhDG was also observed in mito-TEMPO group as compared to 5-FU group. Further, mtROS, mtLPO and mitochondrial antioxidant defense status were improved by mito-TEMPO. CONCLUSION: Mito-TEMPO exerted significant protective effect against 5-FU-induced intestinal toxicity. Therefore, it may be used as an adjuvant in 5-FU chemotherapy.

Codelivery of TRAIL and Mitomycin C via Liposomes Shows Improved Antitumor Effect on TRAIL-Resistant Tumors.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) constitutes a promising antitumor drug, tumor resistance to TRAIL has become a major obstacle in its clinical application. Mitomycin C (MMC) is an effective TRAIL-resistant tumor sensitizer, which indicates a potential utility of combination therapy. However, the efficacy of this combination therapy is limited owing to its short half-life and the cumulative toxicity of MMC. To address these issues, we successfully developed a multifunctional liposome (MTLPs) with human TRAIL protein on the surface and MMC encapsulated in the internal aqueous phase to codeliver TRAIL and MMC. MTLPs are uniform spherical particles that exhibit efficient cellular uptake by HT-29 TRAIL-resistant tumor cells, thereby inducing a stronger killing effect compared with control groups. In vivo assays revealed that MTLPs efficiently accumulated in tumors and safely achieved 97.8% tumor suppression via the synergistic effect of TRAIL and MMC in an HT-29 tumor xenograft model while ensuring biosafety. These results suggest that the liposomal codelivery of TRAIL and MMC provides a novel approach to overcome TRAIL-resistant tumors.