Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer.

Resistance to aromatase inhibitor (AI) treatment and combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) are crucial clinical challenges in treating estrogen receptor-positive (ER+) breast cancer. Understanding the resistance mechanisms and identifying reliable predictive biomarkers and novel treatment combinations to overcome resistance are urgently needed. Herein, we show that upregulation of CDK6, p-CDK2, and/or cyclin E1 is associated with adaptation and resistance to AI-monotherapy and combined CDK4/6i and ET in ER+ advanced breast cancer. Importantly, co-targeting CDK2 and CDK4/6 with ET synergistically impairs cellular growth, induces cell cycle arrest and apoptosis, and delays progression in AI-resistant and combined CDK4/6i and fulvestrant-resistant cell models and in an AI-resistant autocrine breast tumor in a postmenopausal xenograft model. Analysis of CDK6, p-CDK2, and/or cyclin E1 expression as a combined biomarker in metastatic lesions of ER+ advanced breast cancer patients treated with AI-monotherapy or combined CDK4/6i and ET revealed a correlation between high biomarker expression and shorter progression-free survival (PFS), and the biomarker combination was an independent prognostic factor in both patients cohorts. Our study supports the clinical development of therapeutic strategies co-targeting ER, CDK4/6 and CDK2 following progression on AI-monotherapy or combined CDK4/6i and ET to improve survival of patients exhibiting high tumor levels of CDK6, p-CDK2, and/or cyclin E1.

Detection of low numbers of Bacillus anthracis spores in three soils using five commercial DNA extraction methods with and without an enrichment step.

AIMS: To (i) compare the limits of detection of Bacillus anthracis spores in three soils (one Florida, one Texas, and one a commercial Garden product) by PCR using DNA extracted with five commercial extraction kits and (ii) examine if removing organic acids or adding an enrichment step utilizing a growth medium will improve the detection limits. METHODS AND RESULTS: Bacillus anthracis spores were added to soil aliquots and used immediately with a DNA extraction kit or pretreated to remove organics or incubated overnight in a selective growth medium before the DNA extraction was performed. Using hybridization and PCR assays for capC, pag and lef genes, 10(5) -10(6) B. anthracis spores were detected in untreated Florida soil, 10(4) -10(7) spores in untreated Texas soil and 10(6) -10(7) in Garden soil. Pretreatment did not reliably improve detection. DNA from untreated and pretreated soils was suitable for hybridization but not always for PCR. When 10(1) -10(2) spores were added to the soils and allowed to amplify in a growth medium selective for B. anthracis, DNA extracted using four methods reliably produced PCR acceptable DNA positive for the B. anthracis genes. CONCLUSIONS: The quality of DNA extracted with commercial kits appears to be influenced by the soil type and pretreatment. Yet, with an enrichment step added, four of five extraction methods produced PCR suitable DNA and detected ≤10(2) spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment step could enhance the detection of B. anthracis spores in soils and small samples contaminated with soil.

Procurement of spore-free Bacillus anthracis for molecular typing outside of BSL3 environment.

AIMS: To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint analysis and pulse field gel electrophoresis (PFGE). METHODS AND RESULTS: Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45 degrees C under normal air, enhanced CO(2), microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80 degrees C for 30 min and processed for and analysed by either Riboprinte or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. CONCLUSIONS: This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter and PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.

The inactivation and removal of airborne Bacillus atrophaeus endospores from air circulation systems using UVC and HEPA filters.

AIMS: To (i) evaluate the UV radiation in the 'C' band/high efficient particulate air (UVC/HEPA) instrument's potential to inactivate spores of Bacillus atrophaeus and selected Bacillus species and (ii) test whether a titanium dioxide coating inside the cylindrical HEPA filter improves the system's efficacy. METHODS AND RESULTS: Known amounts of dried spore preparations of B. atrophaeus, Bacillus cereus, Bacillus megaterium, Bacillus stearothermophilus and Bacillus thuringiensis were exposed to the UVC lamp within a cylindrical HEPA filter for different time lengths (30 min to 48 h) and with different air flow speeds (0-235 l s(-1)). The log(10) reduction (range 5-16 logs) of colony forming units for spores exposed to the UVC compared with the unexposed spores was significant (P < 0.0001). The addition of a titanium dioxide (TiO(2)) veneer to the interior surface of the HEPA filter significantly increased the inactivation of spores (P < 0.0001). CONCLUSIONS: The UVC/HEPA unit could inactivate spores of B. atrophaeus, B. cereus, B. megaterium, B. stearothermophilus and B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC/HEPA unit represents an effective method of decontaminating circulating air within an air-duct system as found in a building.

Identification of the conjugative mef gene in clinical Acinetobacter junii and Neisseria gonorrhoeae isolates.

The mef gene, originally described for gram-positive organisms and coding for an efflux pump, has been identified in clinical isolates of Acinetobacter junii and Neisseria gonorrhoeae. These strains could transfer the mef gene at frequencies ranging from 10(-6) to 10(-9) into one or more of the following recipients: gram-negative Moraxella catarrhalis, Neisseria perflava/sicca and Neisseria mucosa and gram-positive Enterococcus faecalis. Three Streptococcus pneumoniae strains could transfer the mef gene into Eikenella corrodens, Haemophilus influenzae, Kingella denitrificans, M. catarrhalis, Neisseria meningitidis, N. perflava/sicca, and N. mucosa at similar frequencies. The mef gene can thus be transferred to and expressed in a variety of gram-negative recipients.

A novel multiresistant Streptococcus pneumoniae serogroup 19 clone from Washington State identified by pulsed-field gel electrophoresis and restriction fragment length patterns.

In 1997, a cluster of multiresistant invasive serogroup 19 pneumococcus infections, including two fatalities, was reported in Washington State. Further investigation identified other cases. Fourteen Washington Streptococcus pneumoniae isolates, four from Alaska, and eight isolates from eastern Canada with reduced penicillin susceptibility (MIC of > or =1 microg/ml) were included in the study. Pulsed-field gel electrophoresis (PFGE) with ApaI, SacII, and SmaI restriction enzymes and IS1167 and mef restriction fragment length polymorphism (RFLP) pattern analysis were performed. Twenty of the 26 isolates had identical or related PFGE patterns, with two or all three enzymes, and identical or related IS1167 RFLP patterns, indicating that they were genetically related. These 20 isolates contained the mef gene conferring erythromycin resistance and had identical mef RFLP patterns. The PFGE and RFLP patterns were distinct from those of six multiresistant clones previously described and suggest that a new multiresistant clone has appeared in Washington, Alaska, and eastern Canada. This newly characterized clone should be included in the Pneumococcal Molecular Epidemiology Network.

A variety of gram-positive bacteria carry mobile mef genes.

The mefE gene codes for a membrane bound efflux protein, which confers resistance to macrolides, and has been identified in Streptococcus pneumoniae. A variety of gram-positive organisms were examined. Twenty-six isolates of S. pneumoniae carried mefE and were resistant to erythromycin (MIC of 2-16 mg/L). Two additional isolates of Emr S. pneumoniae carried both ermB and mefE(MIC of 16-128 mg/L). One Micrococcus luteus, one Corynebacterium jeikeium, three Corynebacterium spp., two viridans streptococci and seven Enterocccus spp. also carried mef genes. It was possible to move the mef gene from all 11 S. pneumoniae tested to susceptible S. pneumoniae and/or Enterococcus faecalis recipients. The addition of DNase (1 g/L) did not affect the gene transfer. It was also possible to move the mef gene from donor Enterococcus spp., viridans streptococci, M. luteus, C. jeikeium and Corynebacterium spp. to E. faecalis recipients. Transconjugant isolates were resistant to erythromycin (MIC = 16 mg/L). Hybridization with a labelled mef oligonucleotide probe against Southern blots and bacterial dot blots confirmed the presence of the mef genes. This is the first time that a mobile mef gene has been identified in four different genera, from three distinct geographical locations.

The presence of the tetO gene in a variety of tetracycline-resistant Streptococcus pneumoniae serotypes from Washington State.

Seventy tetracycline-resistant Streptococcus pneumoniae were tested for the presence of tetracycline resistance genes, tetM and tetO, using a polymerase chain reaction (PCR) assay and DNA-DNA hybridization. Seven isolates representing five serotypes (12, 22, 6A, 19F and 23) carried the tetO gene. Five of the isolates were genetically unrelated as judged using pulsed field gel electrophoresis (PFGE) analysis. Two 19F isolates came from the same patient, carried both tetM and tetO genes and had the same PFGE pattern. The other 63 isolates carried only the tetM gene. DNA sequences from three of the tetO-carrying isolates were determined; they showed 91-95% nucleotide sequence identity over 300 nucleotides, and 93-95% amino acid sequence identity over 100 amino acids. The isolates carrying both tetO and tetM genes could transfer the tetM gene into both Enterococcus faecalis and S. pneumoniae recipients, but not the tetO gene. There was no detectable transfer of the tetO gene, by conjugation, from the other five isolates.

Use of the fluorochrome calcofluor white in the screening of stool specimens for spores of microsporidia.

Microsporidia are obligate intracellular protozoal pathogens associated with chronic diarrhea in individuals infected with HIV. Direct detection methods for microsporidial spores in stool include chromotrope-based, fluorochrome, and immunofluorescent stains. The authors compared the ability to detect microsporidial spores in 168 stool specimens using two stains: a chromotrope-based modified trichrome stain and a fluorochrome stain, calcofluor white (Cellufluor, Polysciences, Warrington, PA). In addition to being faster and easier to perform, the calcofluor white stain was found to be more sensitive than the chromotrope-based stain, as 6 of 24 specimens positive by calcofluor white were negative by the chromotrope-based stain on initial smear evaluation. Repeat examination confirmed these six as being positive. To evaluate the specificity of the calcofluor white stain, 20 formalin-fixed stool specimens (5 positive and 15 negative for microsporidial spores) were evaluated in blinded fashion by two affiliated clinical laboratories using their own formulations of calcofluor white. A single discrepant result (falsely positive) was reported from one laboratory. The use of the calcofluor white stain is recommended as a simple and highly sensitive screening procedure for the detection of microsporidial spores in stool specimens.