Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139

Erratum: Wunderlich RE, Simons EL, Jungers WL. 1996. New Pedal Remains of Megaladapis and Their Functional Significance. Am J Phys Anthropol 100:115–139.     

Limited Anti-Inflammatory Role for Interleukin-1 Receptor Like 1 (ST2) in the Host Response to Murine Postinfluenza Pneumococcal Pneumonia

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2 ^−/−) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2^−/− mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1β, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2^−/− mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.     

Signaling by estrogens and tamoxifen in the human endometrium

Tamoxifen is used as adjuvant treatment for postmenopausal breast cancer patients. The mechanism of action of tamoxifen in breast cancer patients is that tamoxifen inhibits growth of cancer cells by competitive antagonism for estrogens at the estrogen receptor (ER). In the endometrium, tamoxifen has an effect that varies with the ambient concentration of estrogen: in premenopausal women (high estrogen levels), tamoxifen displays an estrogen-antagonistic effect, while in postmenopausal women (low estrogen levels), tamoxifen displays an estrogen-agonistic mode of action. Here, using microarray technology we have compared estrogen signaling with tamoxifen signaling in the human endometrium. It was observed that on the one hand tamoxifen-treatment results in modulation of expression of specific genes (370 genes) and on the other hand tamoxifen-treatment results in modulation of a set of genes which are also regulated by estrogen treatment (142 genes). Upon focusing on regulation of proliferation, we found that tamoxifen-induced endometrial proliferation is largely accomplished by using the same set of genes as are regulated by estradiol. So, as far as regulation of proliferation goes, tamoxifen seems to act as estrogen agonist. Furthermore, tamoxifen-specific gene regulation may explain why tamoxifen-induced endometrial tumors behave more aggressively than sporadic endometrial tumors.     

Paternal leakage of mitochondrial DNA in experimental crosses of populations of the potato cyst nematode Globodera pallida

Animal mtDNA is typically assumed to be maternally inherited. Paternal mtDNA has been shown to be excluded from entering the egg or eliminated post-fertilization in several animals. However, in the contact zones of hybridizing species and populations, the reproductive barriers between hybridizing organisms may not be as efficient at preventing paternal mtDNA inheritance, resulting in paternal leakage. We assessed paternal mtDNA leakage in experimental crosses of populations of a cyst-forming nematode, Globodera pallida. A UK population, Lindley, was crossed with two South American populations, P5A and P4A. Hybridization of these populations was supported by evidence of nuclear DNA from both the maternal and paternal populations in the progeny. To assess paternal mtDNA leakage, a ~3.4?kb non-coding mtDNA region was analyzed in the parental populations and in the progeny. Paternal mtDNA was evident in the progeny of both crosses involving populations P5A and P4A. Further, paternal mtDNA replaced the maternal mtDNA in 22 and 40?% of the hybrid cysts from these crosses, respectively. These results indicate that under appropriate conditions, paternal leakage occurs in the mtDNA of parasitic nematodes, and supports the hypothesis that hybrid zones facilitate paternal leakage. Thus, assumptions of strictly maternal mtDNA inheritance may be frequently violated, particularly when divergent populations interbreed.     

Promoter hypermethylation using 24-gene array in early head and neck cancer: Better outcome in oral than in oropharyngeal cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.