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Anahtar, Melis

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Anahtar

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Melis

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Anahtar, Melis
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Now showing 1 - 4 of 4
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    Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
    (MyJove Corporation, 2016) Anahtar, Melis; Bowman, Brittany A.; Kwon, Douglas
    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.
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    Rapid and Comprehensive Bacterial Identification and Antimicrobial Resistance Determination Using Microfluidic Enrichment and Whole-Genome Sequencing
    (2017-05-12) Anahtar, Melis
    Sepsis and other severe bacterial infections affect over 750,000 Americans annually, resulting in a 28% mortality rate and $16 billion in health care costs due to delays in proper diagnosis. The current gold-standard method for determining the species identification (ID) and antimicrobial resistance (AMR) profile of a clinical pathogen relies on the century-old method of growing bacteria in culture media, which takes 2-5 days, and frequently fails to cultivate the causative pathogen. In the absence of microbiologic data, physicians are forced to treat suspected infections with broad-spectrum empiric antibiotics that can have significant toxicity to the patient, are not always effective due to the rise of drug resistant pathogens, and can be expensive. Here we describe a process to perform bacterial ID and AMR determination directly from patient samples with a novel enrichment process combined with downstream agnostic DNA sequencing and real-time analysis with a MinION sequencer. By combining a microfluidic cell sorter and immunomagnetic depletion, we achieve at least 2.5x10^4-fold depletion of leukocytes, 10^2-fold depletion of erythrocytes, and 10^2-fold depletion of platelets, while retaining an average of 75% of bacterial cells. With the MinION sequencer, we can achieve ten-fold genome coverage of a bacterial isolate in 30 minutes of sequencing time, even with sample multiplexing. Overall, we show that this approach is feasible when combined with additional human cell and DNA depletion steps, and has the potential to improve clinical care and antibiotic stewardship.
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    The Contribution of Cervicovaginal Microbiota and Hormonal Contraceptives to Genital Inflammation and HIV Acquisition Risk
    (2015-05-17) Anahtar, Melis; Garrett, Wendy; Pillai, Shiv; Anderson, Deborah; Carroll, Michael; Cardozo, David L.
    The HIV epidemic persists in many parts of the world, with the majority of new infections occurring through the female genital tract (FGT). The permissiveness of the genital mucosa to HIV is modulated by the integrity of the epithelial barrier, the presence of pro-inflammatory cytokines, and the frequency of CCR5+CD4+ T cell targets. Here we focus on two biological perturbations: endogenous alterations in cervicovaginal bacteria and exogenous injectable progestin-only contraceptives (IPCs). We examine their effects on the genital mucosal environment and their link to HIV susceptibility in a cohort of young South African women. We first sought to determine how genital microbiota modulate host inflammatory responses. The existing paradigm is that vaginal monocolonization by Lactobacillus is normal, and encroachment by other bacteria is pathologic. By characterizing cervicovaginal bacterial communities in 94 South African women using 16S rRNA and shotgun sequencing, we found that the majority of participants had low Lactobacillus abundance and high ecological diversity. One diverse Prevotella-containing community type strongly correlated with increased concentrations of multiple genital pro-inflammatory cytokines in vivo. We found that these cytokines were produced by epithelial cells and antigen presenting cells via different bacterial sensing mechanisms. Our results identify specific bacterial species that alter the inflammatory state of the FGT and may more broadly impact reproductive health in women. We also investigated the immunological effects of IPCs, the most common form of birth control in sub-Saharan Africa. Although highly effective as a contraceptive, IPCs are controversially associated with increased HIV susceptibility by an unclear mechanism. We found that IPC users had a 5.5-fold higher risk of acquiring HIV than women not using family planning (p=0.0031, 95% CI: 1.733 – 16.80). Phenotypic cellular analysis revealed that IPC users also had a significantly higher frequency of activated HIV target cells in the cervix. Since the availability of target cells in the genital mucosa enables early viral replication, recruitment or retention of these cells by IPCs may explain the observed increased HIV acquisition rates. Furthermore, IPC use was not associated with differences in genital cytokine levels, indicating that cervicovaginal bacteria and exogenous progesterone increase HIV susceptibility by unique pathways.
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    Silicon Nanoparticles as Hyperpolarized Magnetic Resonance Imaging Agents
    (American Chemical Society, 2009) Aptekar, Jacob W.; Cassidy, Maja Clare; Johnson, Alexander C.; Barton, Robert A.; Lee, Menyoung; Ogier, Alexander C.; Vo, Chinh; Anahtar, Melis; Yin, Ren; Bhatia, Sangeeta; Ramanathan, Chandrasekhar; Cory, David G.; Hill, Alison; Mair, Ross; Rosen, Matthew; Walsworth, Ronald; Marcus, C
    Magnetic resonance imaging of hyperpolarized nuclei provides high image contrast with little or no background signal. To date, in vivo applications of prehyperpolarized materials have been limited by relatively short nuclear spin relaxation times. Here, we investigate silicon nanoparticles as a new type of hyperpolarized magnetic resonance imaging agent. Nuclear spin relaxation times for a variety of Si nanoparticles are found to be remarkably long, ranging from many minutes to hours at room temperature, allowing hyperpolarized nanoparticles to be transported, administered, and imaged on practical time scales. Additionally, we demonstrate that Si nanoparticles can be surface functionalized using techniques common to other biologically targeted nanoparticle systems. These results suggest that Si nanoparticles can be used as a targetable, hyperpolarized magnetic resonance imaging agent with a large range of potential applications.