James C. Alwine

We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strips. The RNA is coupled covalently to diazobenzyloxymethyl groups on the paper. After transfer and appropriate treatment of the paper to destroy remaining diazo groups, specific RNA bands can be detected by hybridization with 32P-labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA bands with high sensitivity and low background.

Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2alpha kinase PERK; however, the amount of phosphorylated eIF2alpha was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2alpha phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing alpha-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.

A dysbiotic microbiome can potentially contribute to the pathogenesis of many different diseases including cancer. Breast cancer is the second leading cause of cancer death in women. Thus, we investigated the diversity of the microbiome in the four major types of breast cancer: endocrine receptor (ER) positive, triple positive, Her2 positive and triple negative breast cancers. Using a whole genome and transcriptome amplification and a pan-pathogen microarray (PathoChip) strategy, we detected unique and common viral, bacterial, fungal and parasitic signatures for each of the breast cancer types. These were validated by PCR and Sanger sequencing. Hierarchical cluster analysis of the breast cancer samples, based on their detected microbial signatures, showed distinct patterns for the triple negative and triple positive samples, while the ER positive and Her2 positive samples shared similar microbial signatures. These signatures, unique or common to the different breast cancer types, provide a new line of investigation to gain further insights into prognosis, treatment strategies and clinical outcome, as well as better understanding of the role of the micro-organisms in the development and progression of breast cancer.