Metabolic targeting of NRF2 potentiates the efficacy of the TRAP1 inhibitor G-TPP through reduction of ROS detoxification in colorectal cancer.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial homolog of HSP90 chaperones. It plays an important role in protection against oxidative stress and apoptosis by regulating reactive oxidative species (ROS). To further elucidate the mechanistic role of TRAP1 in regulating tumor cell survival, we used gamitrinib-triphenylphosphonium (G-TPP) to inhibit TRAP1 signaling pathways in colon cancer. Inhibition of TRAP1 by G-TPP disrupted redox homeostasis and induced cell death. However, colon cancers show a wide range of responses to G-TPP treatment through the induction of variable ER stress responses and ROS accumulation. Interestingly, a strong inverse correlation was observed between the expression of TRAP1 and antioxidant genes in colon tumor tissues using the GSE106582 database. Using a luciferase reporter assay, we detected increased transcriptional activation of antioxidant response elements (AREs) in G-TPP-treated DLD1 and RKO cells but not in SW48 cells. We found that G-TPP induced upregulation of GRP78, CHOP and PARP cleavage in G-TPP-sensitive cells (SW48). In contrast, G-TPP treatment of G-TPP-resistant cells (DLD1 and RKO) resulted in excessive activation of the antioxidant gene NRF2, leading to ROS detoxification and improved cell survival. The NRF2 target genes HO1 and NQO1 were upregulated in G-TPP-treated DLD1 cells, making the cells more resistant to G-TPP treatment. Furthermore, treatment with both a NRF2 inhibitor and a TRAP1 inhibitor led to excessive ROS production and exacerbated G-TPP-induced cell death in G-TPP-resistant cells. Taken together, dual targeting of TRAP1 and NRF2 may potentially overcome colon cancer resistance by raising cellular ROS levels above the cytotoxic threshold.

Epithelial Inclusions in Gallbladder Specimens Mimic Parasite Infection: Histologic and Molecular Examination of Reported Cystoisospora belli Infection in Gallbladders of Immunocompetent Patients.

Recent publications have described epithelial cytoplasmic vacuoles and inclusions incidentally noted within gallbladder epithelium and concluded that they represent coccidian parasite infection, in particular, Cystoisospora belli. We identified 8 gallbladder specimens from our institution in the past 3 years in which this diagnosis was suggested or in which similar epithelial alterations were prominent. Molecular analysis was performed on the 8 gallbladder specimens and on 3 positive control specimens: small bowel biopsies from acquired immunodeficiency syndrome patients with diarrhea. Polymerase chain reaction using primers designed to amplify an internal transcribed spacer (ITS2) in the C. belli ribosomal gene cluster was performed on the DNA samples. All 8 gallbladder specimens were negative for amplification, while a product consistent with C. belli was amplified from all 3 positive controls. Histologically, the gallbladder cytoplasmic inclusions stained diffusely positive for Grocott-Gomori's methenamine silver and Periodic acid-Schiff with diastase. In contrast, sections from a positive control small bowel biopsy demonstrated organisms that were negative for Grocott-Gomori's methenamine silver and showed a distinct capsular and punctate internal staining on Periodic acid-Schiff with diastase in various parasite forms. Together, the lack of molecular evidence of C. belli and the distinct morphologic and special staining patterns in these gallbladders compared with positive control small bowel suggest that these epithelial changes do not represent true C. belli infection. Our results suggest that gallbladders of immunocompetent patients may occasionally show epithelial changes that can morphologically mimic C. belli infection. Pathologists should be aware of this histologic variant to minimize unnecessary treatment, testing, and patient anxiety.

A Survey of the Prevalence and Impact of Reporting Guideline Endorsement in Pathology Journals.

OBJECTIVES: To determine the prevalence of reporting guideline endorsement in pathology journals and to estimate the impact of guideline endorsement. METHODS: We compared the quality of reporting in two sets of studies: (1) studies published in journals that explicitly mentioned a guideline vs studies published in journals that did not and (2) studies that cited a guideline vs studies that did not. The quality of reporting in prognostic biomarker studies was assessed using the REporting recommendations for tumor MARKer prognostic studies (REMARK) guideline. RESULTS: We found that six (10%) of the 59 leading pathology journals explicitly mention reporting guidelines in the instructions to authors. Only one journal required authors to submit a checklist. There was significant variation in the rate at which various REMARK items were reported (P < .001). Journal endorsement was associated with more complete reporting (P = .04). Studies that cited REMARK had greater adherence to the REMARK reporting guidelines than studies that did not (P = .02). CONCLUSIONS: The prevalence of guideline endorsement is relatively low in pathology journals, but guideline endorsement may improve the quality of reporting.

A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis.

The Burkholderia pseudomallei complex classically consisted of B. mallei, B. pseudomallei, and B. thailandensis, but has now expanded to include B. oklahomensis, B. humptydooensis, and three unassigned Burkholderia clades. Methods for detecting and differentiating the B. pseudomallei complex has been the topic of recent research due to phenotypic and genotypic similarities of these species. B. mallei and B. pseudomallei are recognized as CDC Tier 1 select agents, and are the causative agents of glanders and melioidosis, respectively. Although B. thailandensis and B. oklahomensis are generally avirulent, both display similar phenotypic characteristics to that of B. pseudomallei. B. humptydooensis and the Burkholderia clades are genetically similar to the B. pseudomallei complex, and are not associated with disease. Optimal identification of these species remains problematic, and PCR-based methods can resolve issues with B. pseudomallei complex detection and differentiation. Currently, no PCR assay is available that detects the major species of the B. pseudomallei complex. A real-time PCR assay in a multiplex single-tube format was developed to simultaneously detect and differentiate B. mallei, B. pseudomallei, and B. thailandensis, and a common sequence found in B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis. A total of 309 Burkholderia isolates and 5 other bacterial species were evaluated. The assay was 100% sensitive and specific, demonstrated sensitivity beyond culture and GC methods for the isolates tested, and is completed in about an hour with a detection limit between 2.6pg and 48.9pg of gDNA. Bioinformatic analyses also showed the assay is likely 100% specific and sensitive for all 84 fully sequenced B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis strains currently available in GenBank. For these reasons, this assay could be a rapid and sensitive tool in the detection and differentiation for those species of the B. pseudomallei complex with recognized clinical and practical significance.

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking.

PCR-based Methodologies Used to Detect and Differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis.

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.

The differential susceptibility of spores from virulent and attenuated Bacillus anthracis strains to aldehyde- and hypochlorite-based disinfectants.

This study compared the sensitivity of spores from virulent and attenuated Bacillus anthracis strains in suspension to inactivation by various chemical disinfectants. Spore suspensions from two virulent strains (A0256 and A0372) and two attenuated strains (Sterne and A0141) of B. anthracis were tested against two aldehyde-based disinfectants and one hypochlorite-based disinfectant. A novel statistical model was used to estimate 4-log(10) reduction times for each disinfectant/strain combination. Reduction times were compared statistically using approximate Z and χ(2) tests. Although there was no consistent correlation between virulence and increased sporicidal resistance across all three disinfectants, spores from the two virulent and two attenuated strains did display significantly different susceptibilities to different disinfectants. Significant disinfectant-strain interactions were observed for two of the three disinfectants evaluated. The comparative results suggest that the use of surrogate organisms to model the inactivation kinetics of virulent B. anthracis spores may be misleading. The accuracy of such extrapolations is disinfectant dependent and must be used with caution.

Authentic active learning activities demonstrating the use of serial dilutions and plate counts.

Serial dilution and plate counting is often taught in courses for both microbiology and allied health students. Lecture examples and examination questions addressing how the method is used can sometimes be contrived: artificial data sets may have little or no meaning other than to have students perform a calculation. Here we provide a set of activities employing data sets acquired from the primary literature. Our objective was to have the students think critically about a real scenario in which serial dilution and plate count was used. Each activity requires students to read a paragraph describing the study, predict the results, perform the appropriate calculations, and then evaluate the results in light of their predictions. To test the efficacy of these activities, a pretest quiz was given to approximately 100 students in an allied health/general microbiology course. After a lecture on how microbes are enumerated, students were given a different quiz. The class was then divided randomly into groups of three or four students and assigned one of the activities. A postactivity quiz was also administered. Approximately two weeks later, a serial dilution/plate count question was used on an examination and served as a final posttest. Standardized learning gains were calculated for the quiz administered after each learning activity. Even though learning gains were significantly higher after the lecture, there was also a significant improvement between the lecture and the activity. Using an exercise based on an authentic set of data significantly improved student learning gains, and is a useful practice for teaching microbiology.