Nader Pourmand

Patterns of Known and Novel Small RNAs in Human Cervical Cancer

Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria.

Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments

Gradients of antibiotics generated in a microfluidic device provoke selection of ciprofloxacin resistance in Escherichia coli. The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.

Multiplex protein assays based on real-time magnetic nanotag sensing

Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.

The relationship between human papillomavirus status and other molecular prognostic markers in head and neck squamous cell carcinomas.

PURPOSE To evaluate the relationship between human papillomavirus (HPV) status and known prognostic makers for head and neck cancers including tumor hypoxia, epidermal growth factor receptor (EGFR) expression and intratumoral T-cell levels and to determine the prognostic impact of these markers by HPV status. METHODS AND MATERIALS HPV status in 82 evaluable head and neck squamous cell carcinomas patients was determined by pyrosequencing and related to p16(INK4a) staining and treatment outcomes. It was correlated with tumor hypoxia (tumor pO(2) and carbonic anhydrase [CAIX] staining), EGFR status, and intratumoral lymphocyte expression (CD3 staining). RESULTS Forty-four percent of evaluable tumors had strong HPV signal by pyrosequencing. There was a significant relationship between strong HPV signal and p16(INK4a) staining as well as oropharynx location. The strong HPV signal group fared significantly better than others, both in time to progression (TTP, p = 0.008) and overall survival (OS, p = 0.004) for all patients and for the oropharyngeal subset. Positive p16(INK4a) staining was associated with better TTP (p = 0.014) and OS (p = 0.00002). There was no relationship between HPV status and tumor pO(2) or CAIX staining. However, HPV status correlated inversely with EGFR reactivity (p = 0.0006) and directly with CD3(+) T-lymphocyte level (p = 0.03). Whereas CAIX and EGFR overexpression were negative prognostic factors regardless of HPV status, CD3(+) T-cell levels was prognostic only in HPV(-) tumors. CONCLUSION HPV status was a prognostic factor for progression and survival. It correlated inversely with EGFR expression and directly with T-cell infiltration. The prognostic effect of CAIX and EGFR expression was not influenced by HPV status, whereas intratumoral T-cell levels was significant only for HPV(-) tumors.

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such ‘Bar-seq’ assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.

One-pot synthesis of monodisperse iron oxide nanoparticles for potential biomedical applications

One-pot reaction of iron(III) acetylacetonate, Fe(acac)3, [or Fe(acac)3 and M(acac)2 where M = Mn and Co], with 1,2-alkanediol, oleic acid, and oleylamine in high boiling organic solvent leads to monodisperse ferrite MFe2O4 nanoparticles. Depending on the concentration of the metal precursors, surfactant-to-metal precursor ratio and the solvent used in the reaction, the particle size from this one-pot reaction can be tuned from 4 to 15 nm. The as-synthesized iron oxide nanoparticles have an inverse spinel structure, and their magnetic properties are controlled by particle size and M in the MFe2O4 structure. The hydrophobic iron oxide nanoparticles are readily transformed into hydrophilic ones by functional phospholipid addition to the as-synthesized particles and as a result, the monodisperse nanoparticles are readily functionalized with biotin, -COOH, -SH, and -NH2, facilitating their link to biomolecules for biomedical applications.

Convergent antibody signatures in human dengue.

Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.

Giant Magnetoresistive Biochip for DNA Detection and HPV Genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10 nM in most cases, but concentrations of 10 pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Whole-transcriptome RNAseq analysis from minute amount of total RNA

RNA sequencing approaches to transcriptome analysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptome analysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500 pg) of total RNA. We compared a single-sample library prepared by this Ovation® RNA-Seq system with two available methods of mRNA enrichment (TruSeq™ poly-A enrichment and RiboMinus™ rRNA depletion). Using the Ovation® preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (<3.5%) while retaining transcriptome sequencing fidelity. We further validated the Ovation® amplification technique by examining the resulting library complexity, reproducibility, evenness of transcript coverage, 5′ and 3′ bias and platform-specific biases. Notably, in this side-by-side comparison, SOLiD sequencing chemistry is biased toward higher GC content of transcriptome and Illumina Genome analyzer IIx is biased away from neutral to lower GC content of the transcriptomics regions.