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.

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.

Mitomycin C-induced effects on aerobic methanotrophs in a landfill cover soil; implications of a viral shunt?

A viral shunt can occur when phages going through a lytic cycle, including lysogenic phages triggered by inducing agents (e.g. mitomycin C), results in host lysis and the release of cell constituents and virions. The impact of a viral shunt on the carbon, including methane cycle in soil systems is poorly understood. Here, we determined the effects of mitomycin C on the aerobic methanotrophs in a landfill cover soil. To an extent, our results support a mitomycin C-induced viral shunt, as indicated by the significantly higher viral-like particle (VLP) counts relative to bacteria, elevated nutrient concentrations (ammonium, succinate), and initially impaired microbial activities (methane uptake and microbial respiration) after mitomycin C addition. The trend in microbial activities at <2 days largely corresponded to the expression of the pmoA and 16S rRNA genes. Thereafter (>11 days), the active bacterial community composition significantly diverged in the mitomycin C-supplemented incubations, suggesting the differential impact of mitomycin C on the bacterial community. Collectively, we provide insight on the effects of mitomycin C, and potentially a viral shunt, on the bacteria in the soil environment.

Contribution of Iron-Transport Systems and ß-Lactamases to Cefiderocol Resistance in Clinical Isolates of Acinetobacter baumannii Endemic to New York City.

The development of resistance to cefiderocol among multidrug resistant Acinetobacter baumannii has been attributed to downregulation in iron transport systems and a variety of ß-lactamases. However, the precise contribution of each in clinical isolates remains to be determined. Sixteen clinical isolates with varying degrees of cefiderocol resistance were investigated. Susceptibility testing was performed with and without the presence of iron and avibactam. Expression of 10 iron transport systems and blaADC and blaOXA-51-type were analyzed by real time RT-PCR. The acquisition of a variety of ß-lactamases was also determined. In 2 isolates the impact of silencing the blaADC gene was achieved using a target specific group II intron. For most resistant isolates, MICS for cefiderocol were similar with or without the presence of iron, and there was an overall decrease in expression of receptors (including pirA and piuA) involved in ferric uptake. However, expression of the ferrous uptake system (faoA) persisted. The addition of avibactam (4 µg/mL) lowered most cefiderocol MICs to 2 to 4 µg/mL. Most isolates possessed ADC-25 or ADC-33. Cefiderocol resistance correlated with over-expression of blaADC; silencing of this ß-lactamase resulted in a ≥ 8-fold decrease in cefiderocol MICs. Over-expression of specific blaADC subtypes, in a background of generalized repression of ferric uptake systems, were consistent features in clinical isolates of cefiderocol-resistant A. baumannii.

The ablative effect of mitomycin reverse thermal gel: Expanding the role for nephron preservation therapy in low grade upper tract urothelial carcinoma.

PURPOSE: Assess the real-world ablative effect of mitomycin reverse thermal gel for low-grade upper tract urothelial carcinoma (UTUC) in patients who undergo biopsy only or partial ablation and evaluate utility of complete ablation prior to UGN-101. MATERIAL AND METHODS: We retrospectively reviewed low-grade UTUC patients treated with UGN-101 from 15 high-volume centers. Patients were categorized based on initial endoscopic ablation (biopsy only, partial ablation, or complete ablation) and by size of remaining tumor (complete ablation, <1cm, 1-3cm, or >3cm) prior to UGN-101. The primary outcome was rendered disease free (RDF) rate at first post-UGN-101 ureteroscopy (URS), defined as complete response or partial response with minimal mechanical ablation to endoscopically clear the upper tract of visible disease. RESULTS: One hundred and sixteen patients were included for analysis after excluding those with high-grade disease. At first post-UGN-101 URS, there were no differences in RDF rates between those who at initial URS (pre-UGN-101) had complete ablation (RDF 77.0%), partial ablation (RDF 55.9%) or biopsy only (RDF 66.7%) (P = 0.14). Similarly, a complimentary analysis focusing on tumor size (completely ablated, <1cm, 1-3cm or >3cm) prior to UGN-101 induction did not demonstrate significant differences in RDF rates (P = 0.17). CONCLUSION: The results of the early real-world experience suggest that UGN-101 may play a role in initial chemo-ablative cytoreduction of larger volume low-grade tumors that may not initially appear to be amenable to renal preservation. Further studies will help to better quantify the chemo-ablative effect and to identify clinical factors for patient selection.

Occurrence and genetic diversity of prophage sequences identified in the genomes of L. casei group bacteria.

It is widely believed that microorganisms belonging to L. casei group can have positive effects on the human body. Therefore, these bacteria are used in many industrial processes, including the production of dietary supplements and probiotic preparations. When using live microorganisms in technological processes, it is important to use those without phage sequences within their genomes that can ultimately lead to lysis of the bacteria. It has been shown that many prophages have a benign nature, meaning that they don't directly lead to lysis or inhibit microbial growth. Moreover, the presence of phage sequences in the genomes of these bacteria increases their genetic diversity, which may contribute to easier colonization of new ecological niches. In the 439 analyzed genomes of the L. casei group, 1509 sequences of prophage origin were detected. The average length of intact prophage sequences analyzed was just under 36 kb. GC content of tested sequences was similar for all analyzed species (44.6 ± 0.9%). Analyzing the protein coding sequences collectively, it was found that there was an average of 44 putative ORFs per genome, while the ORF density of all phage genomes varied from 0.5 to 2.1. The average nucleotide identity calculated on sequence alignments for analyzed sequences was 32.7%. Of the 56 L. casei strains used in the next part of the study, 32 did not show culture growth above the OD600 value of 0.5, even at a mitomycin C concentration of 0.25 µg/ml. Primers used for this study allowed for the detection of prophage sequences for over 90% of tested bacterial strains. Finally, prophages of selected strains were induced using mitomycin C, phage particles were isolated and then genomes of viruses obtained were sequenced and analyzed.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation.

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 µM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 µM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 µM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Atorvastatin Can Modulate DNA Damage Repair in Endothelial Cells Exposed to Mitomycin C.

HMG-CoA reductase inhibitors (statins) are widely used in the therapy of atherosclerosis and have a number of pleiotropic effects, including DNA repair regulation. We studied the cytogenetic damage and the expression of DNA repair genes (DDB1, ERCC4, and ERCC5) in human coronary artery (HCAEC) and internal thoracic artery endothelial cells (HITAEC) in vitro exposed to mitomycin C (MMC) (positive control), MMC and atorvastatin (MMC+Atv), MMC followed by atorvastatin treatment (MMC/Atv) and 0.9% NaCl (negative control). MMC/Atv treated HCAEC were characterized by significantly decreased micronuclei (MN) frequency compared to the MMC+Atv group and increased nucleoplasmic bridges (NPBs) frequency compared to both MMC+Atv treated cells and positive control; DDB1, ERCC4, and ERCC5 genes were upregulated in MMC+Atv and MMC/Atv treated HCAEC in comparison with the positive control. MMC+Atv treated HITAEC were characterized by reduced MN frequency compared to positive control and decreased NPBs frequency in comparison with both the positive control and MMC/Atv group. Nuclear buds (NBUDs) frequency was significantly lower in MMC/Atv treated cells than in the positive control. The DDB1 gene was downregulated in the MMC+Atv group compared to the positive control, and the ERCC5 gene was upregulated in MMC/Atv group compared to both the positive control and MMC+Atv group. We propose that atorvastatin can modulate the DNA damage repair response in primary human endothelial cells exposed to MMC in a cell line- and incubation scheme-dependent manner that can be extremely important for understanding the fundamental aspects of pleoitropic action of atorvastatin and can also be used to correct the therapy of patients with atherosclerosis characterized by a high genotoxic load.

The anti-scar effect of tyrosine-kinase inhibitor nintedanib in experimental glaucoma filtration surgery in rabbits.

PURPOSE: To investigate the efficacy of nintedanib on preventing postoperative scar in formation following glaucoma filtering surgery (GFC) in rabbits in comparison with Mitomycin-C (MMC). DESIGN: Experimental Animal Study. METHODS: 24 New Zealand rabbits were divided randomly into 3 groups as Sham, Nindetanib and MMC(n = 8). Limbal-based trabeculectomy was performed on the right eyes of the rabbits. Left eyes that did'nt undergo surgery were included in the control group (n = 8). Following surgery, Intraocular pressures (IOP), postoperative complications and morphological changes in the bleb were evaluated. On the 28th day, eight eyes from each group were enucleated and histologically and immunohistochemically analyzed. Matrix metalloproteinase-2 (MMP-2), Transforming Growth Factor-1 (TGF-B1) and alpha-smooth muscle actin (a-SMA) were evaluated. RESULTS: It was observed that nintedanib has no side effects and reduces subconjunctival fibrosis. Postoperative IOP values in the Nindetanib group were lower than the other groups (p < 0.05). The longest bleb survival was observed in the Nintedanib group and the shortest in the Sham group (p < 0.001). Conjunctival vascularity and inflammation was reduced in the Nintedanib group compared to the Sham group (p < 0.05). The highest subconjunctival fibrosis was observed in the Sham group and the least in the Nintedanib group (p < 0.05). Although the fibrosis score was found lower in the Nintedanib group compared to the MMC(p > 0.05). α-SMA TGF-ß1, MMP-2 expressions were similar in Nintedanib and MMC groups (p > 0.05), however, it was observed that significantly decreased in both groups compared to Sham group (p < 0.05). CONCLUSION: It has been observed that Nindetanib suppress fibroblast proliferation Thus, It may be a drug that can prevent subconjunctival fibrosis in GFC.

Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes.

Antifungal drug tolerance is a response distinct from resistance, in which cells grow slowly above the MIC. Here, we found that the majority (69.2%) of 133 Candida albicans clinical isolates, including standard lab strain SC5314, exhibited temperature-enhanced tolerance at 37°C and 39°C, and were not tolerant at 30°C. Other isolates were either always tolerant (23.3%) or never tolerant (7.5%) at these three temperatures, suggesting that tolerance requires different physiological processes in different isolates. At supra-MIC fluconazole concentrations (8 to 128 µg/mL), tolerant colonies emerged rapidly at a frequency of ~10-3. In liquid passages over a broader range of fluconazole concentrations (0.25 to 128 µg/mL), tolerance emerged rapidly (within one passage) at supra-MICs. In contrast, resistance appeared at sub-MICs after 5 or more passages. Of 155 adaptors that evolved higher tolerance, all carried one of several recurrent aneuploid chromosomes, often including chromosome R, alone or in combination with other chromosomes. Furthermore, loss of these recurrent aneuploidies was associated with a loss of acquired tolerance, indicating that specific aneuploidies confer fluconazole tolerance. Thus, genetic background and physiology and the degree of drug stress (above or below the MIC) influence the evolutionary trajectories and dynamics with which antifungal drug resistance or tolerance emerges. IMPORTANCE Antifungal drug tolerance differs from drug resistance: tolerant cells grow slowly in drug, while resistant cells usually grow well, due to mutations in a few known genes. More than half of Candida albicans clinical isolates have higher tolerance at body temperature than they do at the lower temperatures used for most lab experiments. This implies that different isolates achieve drug tolerance via several cellular processes. When we evolved different strains at a range of high drug concentrations above inhibitory levels, tolerance emerged rapidly and at high frequency (one in 1,000 cells) while resistance appeared only later at very low drug concentrations. An extra copy of all or part of chromosome R was associated with tolerance, while point mutations or different aneuploidies were seen with resistance. Thus, genetic background and physiology, temperature, and drug concentration all influence how drug tolerance or resistance evolves.