Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

Background: eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. Methods: A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (ß-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. Results: The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. Conclusion: The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Knowledge of stroke and the window period for thrombolytic therapy in ischemic stroke among South Indians: A hospital-based survey with educational intervention.

Objectives: The objective of this study was to determine the awareness of stroke in regards to the risk factors, warning symptoms, and knowledge of the therapeutic window period among varied strata of non-medical people attending a tertiary care center. Materials and Methods: The interventional study involved the collection of data regarding awareness of stroke using a structured questionnaire with a total score of 16. Pre-intervention assessment was followed by intervention in the form of education regarding awareness of stroke administered one-on-one for personalized and effective comprehension by subjects. Then, subjects were asked to recall the information that was delivered to them and were scored accordingly. Results: Among the 500 subjects included, 51% were female. About 76.8% of participants were young (age <50 years), and 83.4% were literate. Only 25.4% of participants were aware of the brain as the site of stroke. About 32.2% of candidates were aware of a few risk factors for stroke. Among them, the majority of participants were aware of hypertension (24%) as a risk factor. The most known warning symptom was "Numbness" or weakness of arm. The majority of the subjects (97.8%) were unaware of a therapeutic window period for stroke being 4.5 h or below. The mean pre-intervention score was 2.52 ± 1.65 while the mean post-intervention score was 15.10 ± 1.79 (P < 0.0001). Conclusion: The study showed that even among literate participants, only a meager number of subjects were aware of the golden window period of intravenous thrombolysis. Educational intervention by means of an in-person and one-on-one explanation achieved significant levels of understanding of stroke. The study could be used to formulate large-scale educational programs that focus on spreading awareness of symptoms and risk factors while also instilling the importance of timely medical intervention for efficient thrombolytic therapy.

Nurses' Attitudes Concerning Analgesia Administration for Pediatric Patients with Sickle Cell Disease in Jordan: A Cross-Sectional Study.

BACKGROUND: Pain management of vaso-occlusive crises is a fundamental priority in the lifelong care of children and adolescents with sickle cell disease. AIM: This study examined nurses' attitudes towards caring for children with sickle cell disease (SCD) and SCD pain management in those with vaso-occlusive pain. METHOD: A structured, self-reporting survey was provided to a convenience sample of 298 nurses across 10 hospitals serving Jordan's northern and middle regions. Descriptive and inferential statistics were applied for data analysis. RESULTS: Most nurses (77%) perceived their experience caring for children with SCD as positive. Many nurses (65%) felt frustrated about caring for these children during painful episodes. Participants identified workload and inadequate time as limiting their ability to address the analgesic needs of children with SCD. Receiving structured education specialized in pain management and more years of experience in nursing significantly predicted less hesitancy in administering opioid-based analgesia. CONCLUSIONS: The results of this study provided further insight into factors that potentially contribute to vaso-occlusive pain crises frequently being poorly managed and inadequately addressed among pediatric patients. Nurses' attitudes and understanding of SCD pain management must be addressed to advance the clinical practice of managing pain in children with SCD.

Nurses' beliefs and perceptions regarding family-centered care services in acute pediatric healthcare settings.

OBJECTIVES: To describe pediatrics nurses' beliefs about family-centered services (FCS) as a model of providing healthcare to children in acute care settings in Jordan. DESIGN AND METHODS: This is a cross-sectional descriptive study. Nurses who provide direct acute care to children (n = 246) completed the 'Measure of Beliefs about Participation in Family-Centered Service' questionnaire. Descriptive statistics were used to describe nurses' beliefs about participation, practical feasibility, implementation self-efficacy, principles, and potential adverse outcomes of FCS. RESULTS: Many nurses feel confident (70%) about their ability to work with others in providing FCS and perceive having the ability to operate according to family-centered care (FCC) principles (68%). Many (75%) nurses believed parents should be encouraged to decide how much they want to be involved in the child's care. However, only 46% of the nurses valued attending to family priorities if the health decisions made by the family differed from the healthcare providers' priorities. Many nurses (70%) believed that healthcare professionals' competencies and capacities to work utilizing FCC are more important than their personal preferences and opinion. CONCLUSIONS: The findings of this study clearly indicate that nurses positively viewed providing children's care within a FCS. This supports the efforts to reasonably integrate FCC as an operating model in the pediatric healthcare settings in Jordan. PRACTICE IMPLICATIONS: FCS is a complex task requiring integrating multidisciplinary effort and healthcare providers' positive attitudes toward families as care partners. Steps should help maximize the organizational resources to facilitate family presence and create opportunities for professional-families partnerships for children's care.

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Prevalence of multidrug-resistant and extended-spectrum ß-lactamase-producing Escherichia coli from chicken farms in Egypt.

Background and Aim: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains exhibit antibiotic resistance and are known to infect humans worldwide. This study assessed the phenotypic and genotypic prevalence of ESBL-resistant E. coli isolates recovered from the respiratory tracts of chickens in El-Sharkia Governorate, Egypt. Materials and Methods: We obtained 250 lung samples (one lung/bird) from 50 chicken farms (5 chickens/farm) to isolate, identify, and serotype E. coli. Antimicrobial resistance susceptibility was determined using the disk diffusion method, while the ESBL phenotype was identified using double disk synergy. We detected the ß-lactamase genes, blaTEM, and blaSHV, using a polymerase chain reaction. Results: The results showed that 140/250 (56%) were infected with E. coli. All the serogroups of isolated E. coli exhibited high multi-antimicrobial resistance index values (>0.2), and 65.7% were confirmed to have ESBL. Among the isolates with the ESBL phenotypes, 55 (60%) and 32 (35%) contained the blaTEM and blaSHV genes, respectively. Conclusion: The widespread distribution of multidrug-resistant and ESBL-producing E. coli among poultry farms is a significant human health hazard. These results will help the Egyptian authorities to implement a national one-health approach to combat the antimicrobial resistance problem.

Hemoglobin Levels, Anemia, and Their Associations with Body Mass Index among Pregnant Women in Hail Maternity Hospital, Saudi Arabia: A Cross-Sectional Study.

The level of association between hemoglobin level/anemia and obesity during pregnancy is not yet fully understood. A cross-sectional study was conducted in Hail Maternity Hospital in northern Saudi Arabia from March to May 2023 to assess the associations between hemoglobin levels, anemia, and obesity among pregnant women. Reproductive history was gathered through a questionnaire. Body mass index (BMI) was calculated/computed from the women's weight and height. Linear and binary regression analyses were performed. A total of 334 pregnant women were enrolled. The median (interquartile range (IQR)) age and parity were 33.2 (27.4-37.8) years and 3 (1-5), respectively. Of the 334 women, 52 (15.6%) were normal weight, while 87 (26.0%) were overweight and 195 (58.4%) were obese. In the multiple linear regression, parity (coefficient = -0.121, p = 0.001) and BMI (coefficient = 0.030, p = 0.006) were negatively associated with the hemoglobin level. Ninety-nine (26.9%) women had anemia. In the multivariate analysis, primiparity (adjusted odds ratio (AOR) = 0.54, 95% confidence interval (CI) = 0.30-0.97), increasing BMI (AOR = 0.93, 95% CI = 0.89-0.97), and obesity (AOR = 0.31, 95% CI = 0.16-0.61) were associated with decreased ORs of anemia. Increasing parity was associated with increased ORs of anemia (AOR = 1.18, 95% CI = 1.05-1.32). Being overweight was not associated with anemia (AOR = 0.56, 95% CI = 0.27-1.16). In the current study, a high hemoglobin level and lower prevalence of anemia were observed among obese pregnant women.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway.

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.

The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Mft1, identified from a genome-wide screen of the yeast haploid mutants, mediates cell cycle arrest to counteract quinoxaline-induced toxicity.

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.

Epidemiological Profile of Urinary and Intestinal Schistosomiasis in the Kingdom of Saudi Arabia: A Seven-Year Retrospective Study.

BACKGROUND: Despite the marked decline of schistosomiasis in Saudi Arabia in recent years, it is still reported in several regions. This study investigates the epidemiology of schistosomiasis in Saudi Arabia over seven years (2014-2020). METHODOLOGY: A retrospective study was retrieved from the annual reports of the Ministry of Health. A Geographic Information System GIS, Chi-square, and logistic regression were used to analyze the data. RESULTS: Out of the 4,371,481 tested, 680 cases were positive for schistosomiasis, with a cumulative incidence rate of 2.155/100,000 population. This number showed significant variation over the study period (p value < 0.001). The highest number of cases detected in 2015 was almost 2-fold (OR = 1.93; 95%CI: 1.36-2.74) higher than in 2020. Both clinical forms (urinary and intestinal schistosomiasis) exist in Saudi Arabia (79.6% and 20.4% of all schistosomiasis cases, respectively). Schistosomiasis was reported in seven out of thirteen regions. Among them, Mecca has a relatively high number of cases (OR = 5.57; 95%CI: 2.49-12.47). Conversely, the Eastern Province has a low number of cases (OR = 0.09; 95%CI: 0.02-0.39) when compared to the Najran region (p value > 0.001). Regarding the distribution of schistosomiasis cases by gender and nationality, we noticed that most of the positive cases were found among males (70.6%) and expatriates (83.6%). CONCLUSIONS: The persistence of schistosomiasis and the disparity in the demographic factors underscores the imperative for intensified and integrative One Health interventions to combat this disease in Saudi Arabia.

The synbiotic mixture of Bacillus licheniformis and Saccharomyces cerevisiae extract aggravates dextran sulfate sodium induced colitis in rats.

BACKGROUND: Uncertain effects of probiotics and/or prebiotics have been reported in experimental and clinical colitis. This study aims to examine the effects of a synbiotic combination comprising Bacillus licheniformis DSM 17236 and Saccharomyces cerevisiae cell wall extract on dextran sulfate sodium (DSS)-induced colitis in Sprague Dawley rats. METHODS: Acute colitis was induced in rats by oral administration of DSS 3.5% for 7 days. Fifty rats were divided equally into five groups; one control group and the other groups were induced with colitis and treated with or without the tested synbiotic, mixed with diet, for 28 days and sulfasalazine (100 mg/kg) via intragastric tube once daily for 14 days. RESULTS: Symptomatically, the synbiotic administration raised the disease activity index (DAI) to comparable scores of the DSS group, specially from the 2nd to 7th days post DSS intoxication. It also induced a significant (p < 0.05) amplification of WBCs, myeloperoxidase (MPO), malondialdehyde (MDA), nuclear factor kappa B (NF-kB) expression and proinflammatory cytokines tumor necrosis factor alpha (TNFα), interferon gamma (INFγ), and interleukin-1 beta (IL-1ß) while depressed the antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) when compared with the DSS and control groups. The DSS intoxicated and Synbiotic+DSS groups showed desquamations of the covering epithelium, noticeable diffuse leukocytic infiltrations, sever catarrhal enteritis, ischemic colitis with diffuse coagulative necrosis of the entire colonic mucosa. Contrarily, sulfasalazine proved to be effective in the reduction of the tested inflammatory markers and the pathological degenerative changes of the DSS ulcerative colitis. CONCLUSION: The examined synbiotic did not ameliorate but aggravated the DSS-induced colitis, so it should be subjected to intensive experimental and clinical testing before their use in animals and human.

Correlates of parental knowledge about smartphone exposure among young children.

BACKGROUND: Unhealthy smartphone habits use can impair the health of young children. Smartphone use among young children is affected by several factors of which the most important is parental knowledge pertains to smartphone exposure among young children. In Jordan, no studies have focused on this factor. METHOD: A nation-wide cross-sectional survey was distributed to parents of children aged <6 years to assess parental knowledge of smartphone exposure among young children and examine its correlates in Jordan. An online self-reporting questionnaire was administered via Survey Monkey and posted on social media platforms in June 2020. RESULTS: A total of 2,781 Jordanian parents completed the survey. During the pandemic the daily hours of Smartphone use among children were significantly higher than prior the coronavirus-2019 (COVID-19) pandemic (2.12 vs. 1.7 h). More than half the children exceeded the recommended daily hours of use. These results were positively associated with increased smartphone use among parents. Overall, the parents proved knowledgeable regarding the effects of excessive smartphone exposure on the health of their children. However, a knowledge deficit was evident in two areas: parental controls on smartphone and safe levels of infant smartphone exposure. The mothers possessed a significantly higher level of knowledge than the fathers. CONCLUSION: The results suggest that, in the context Jordan, increasing parental knowledge has the potential to reduce smartphone exposure among young children. Awareness campaigns are needed to enhance parental knowledge of smartphone exposure among young children and the use of parental controls on smartphones.

C. elegans ribosomal protein S3 protects against H2O2-induced DNA damage and suppresses spontaneous mutations in yeast.

Carcinogenicity and cytotoxicity are severe consequences of DNA damage. Base Excision Repair (BER) is a conserved DNA repair pathway that replaces many damaged bases caused by oxidation. Aberrations in BER are associated with carcinogenesis, neurodegeneration, and aging. The nematode C. elegans is an attractive model system for studying BER. However, in this organism, the complete pathway is not fully delineated. To further explore the BER process in C. elegans, we used affinity tag chromatography and mass spectrometry to identify the interactome of uracil DNA glycosylase-1 (CeUNG-1), an enzyme that acts during the first step of the BER pathway. Our analysis identified that CeUNG-1 is associated with the 40 S ribosomal protein S3 (CeRPS-3), homologs of which have been shown to process 8-oxoguanine and abasic site lesions in other organisms. We report a strong in silico association between CeUNG-1 and CeRPS-3 and confirmed this interaction using the yeast two-hybrid system. Downregulation of the Cerps-3 gene reduced the viability of wild-type worms upon exposure to the chemical oxidant hydrogen peroxide. Further analysis shows that Cerps-3 knockdown significantly sensitized the AP endonuclease APN-1-deficient strain, apn-1, but to a lesser extent exo-3, to the lethal effects of hydrogen peroxide. A cross-species complementation experiment reveals that the expression of CeRPS-3 rescued the hydrogen peroxide sensitivity, and suppressed the high mutation frequency of the yeast AP endonuclease-deficient strain lacking Apn1 and Apn2. We propose that CeRPS-3 may function as an auxiliary DNA repair enzyme in C. elegans to process oxidative DNA lesions.

Can Cisplatin Therapy Be Improved? Pathways That Can Be Targeted.

Cisplatin (cis-diamminedichloroplatinum (II)) is the oldest known chemotherapeutic agent. Since the identification of its anti-tumour activity, it earned a remarkable place as a treatment of choice for several cancer types. It remains effective against testicular, bladder, lung, head and neck, ovarian, and other cancers. Cisplatin treatment triggers different cellular responses. However, it exerts its cytotoxic effects by generating inter-strand and intra-strand crosslinks in DNA. Tumour cells often develop tolerance mechanisms by effectively repairing cisplatin-induced DNA lesions or tolerate the damage by adopting translesion DNA synthesis. Cisplatin-associated nephrotoxicity is also a huge challenge for effective therapy. Several preclinical and clinical studies attempted to understand the major limitations associated with cisplatin therapy, and so far, there is no definitive solution. As such, a more comprehensive molecular and genetic profiling of patients is needed to identify those individuals that can benefit from platinum therapy. Additionally, the treatment regimen can be improved by combining cisplatin with certain molecular targeted therapies to achieve a balance between tumour toxicity and tolerance mechanisms. In this review, we discuss the importance of various biological processes that contribute to the resistance of cisplatin and its derivatives. We aim to highlight the processes that can be modulated to suppress cisplatin resistance and provide an insight into the role of uptake transporters in enhancing drug efficacy.

Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers.

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

The histone H2B Arg95 residue links the pheromone response pathway to rapamycin-induced G1 arrest in yeast.

Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. In yeast, rapamycin inhibits the TORC1 kinase signaling pathway causing rapid alteration in gene expression and ultimately cell cycle arrest in G1 through mechanisms that are not fully understood. Herein, we screened a histone mutant collection and report that one of the mutants, H2B R95A, is strikingly resistant to rapamycin due to a defective cell cycle arrest. We show that the H2B R95A causes defects in the expression of a subset of genes of the pheromone pathway required for α factor-induced G1 arrest. The expression of the STE5 gene and its encoded scaffold protein Ste5, required for the sequential activation of the MAPKs of the pheromone pathway, is greatly reduced in the H2B R95A mutant. Similar to the H2B R95A mutant, cells devoid of Ste5 are also resistant to rapamycin. Rapamycin-induced G1 arrest does not involve detectable phosphorylation of the MAPKs, Kss1, and Fus3, as reported for α factor-induced G1 arrest. However, we observed a sharp induction of the G1 cyclin Cln2 (~ 3- to 4-fold) in the ste5Δ mutant within 30 min of exposure to rapamycin. Our data provide a new insight whereby rapamycin signaling via the Torc1 kinase may exploit the pheromone pathway to arrest cells in the G1 phase.

A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.

Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.