Biology subjects

Segal, E.

Publications and source records attributed to Segal, E..

28 records · Page 2Linked to original sources

bioRxiv2019

Antifungal susceptibility testing of Aspergillus niger on silicon microwells by intensity-based reflectometric interference spectroscopy

The increasing number of invasive fungal infections among immunocompromised patients and the emergence of antifungal resistant pathogens has resulted in the need for rapid and reliable antifungal susceptibility testing (AFST). Accelerating antifungal susceptibility testing allows for advanced treatment decisions and the reduction in future instances of antifungal resistance. In this work, we demonstrate the application of a silicon phase grating as sensor for the detection of growth of Aspergillus niger (A. niger) by intensity-based reflectometric interference spectroscopy and its use as an antifungal susceptibility test. The silicon gratings provide a solid-liquid interface to capture micron-sized Aspergillus conidia within microwell arrays. Fungal growth is optically tracked and detected by the reduction in the intensity of reflected light from the silicon grating. The growth of A. niger in the presence of various concentrations of the antifungal agents voriconazole and amphotericin B is investigated by intensity-based reflectometric interference spectroscopy and used for the determination of the minimal inhibitory concentrations (MIC), which are compared to standard broth microdilution testing. This assay allows for expedited detection of fungal growth and provides a label-free alternative to standard antifungal susceptibility testing methods, such as broth microdilution and agar diffusion methods.

bioengineering↗

bioRxiv2019

Sticky Situations: Bacterial Attachment Deciphered by Interferometry of Silicon Microstructures

The peculiarities of surface-bound bacterial cells are often overshadowed by the study of planktonic cells in clinical microbiology. Thus, we employ phase-shift reflectometric interference spectroscopic measurements to observe the interactions between bacterial cells and abiotic, microstructured material surfaces in a label-free, real-time manner. Both material characteristics (i.e., substrate surface charge and wettability) and characteristics of the bacterial cells (i.e., motility, cell charge, biofilm formation, and physiology) drive bacteria to adhere to a particular surface. We conclude that the attachment of bacterial cells to a surface is determined by the culmination of numerous factors. When specific characteristics of the bacteria are met with factors of the surface, enhanced cell attachment and biofilm formation occur. Such knowledge can be exploited to predict antibiotic efficacy, biofilm development, enhance biosensor development, as well as prevent biofouling.

microbiology↗

bioRxiv2018

Disruption of IRE1α through its Kinase Domain Attenuates Multiple Myeloma

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1 supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1-XBP1s pathway; however, the validity of IRE1 as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1 or XBP1s, or pharmacologic IRE1 kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1 perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1 kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1 inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1 with kinase-based small-molecule inhibitors in MM.\n\nSignificance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1 mitigates ER stress by expanding the ERs protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1 elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1 as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1-XBP1s pathway plays a critical role in MM growth. We further show that IRE1s kinase domain is an effective and safe potential small-molecule target for MM therapy.

cancer biology↗

bioRxiv2018

Extensive programmed ribosomal frameshifting in human as revealed by a massively parallel reporter assay

Programmed ribosomal frameshifting is the controlled slippage of the translating ribosome to an alternative frame. This tightly regulated process is widely employed by human viruses such as HIV and SARS coronavirus and is critical for their life cycle and virulence. It is also utilized from yeast to human to implement a feedback control mechanism to regulate polyamine levels. Here, we developed a high-throughput, fluorescence-based approach to assess the frameshifting potential of a sequence. We designed and tested >12,000 sequences based on 15 viral and human frameshifting events, allowing us to elucidate the rules governing ribosomal frameshifting in a systematic way and to discover novel regulatory inputs based on amino acid properties and tRNA availability. We assessed the natural variation in HIV gag-pol frameshifting rates by testing >500 clinical isolates and identified subtype-specific differences as well as associations between viral load in patients and the optimality of gag-pol frameshifting rates. We further devised computational models that accurately predict frameshifting potential (up to auROC=0.93) and frameshifting rates (up to Pearson r=0.81) of novel variants, including subtle differences between HIV clinical isolates (r=0.60). Taken together, this systematic approach can contribute to the development of antiviral agents acting on programmed ribosomal frameshifting.

genomics↗

bioRxiv2018

Molecular anatomy and plasticity of the long noncoding RNA HOTAIR

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure-function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe the hypothetical molecular anatomy of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) inferred from direct, high-resolution visualization by atomic force microscopy (AFM) in nucleus-like conditions at 37 degrees. Our observations reveal that HOTAIR has a distinct anatomy with a high degree of plasticity. Fast AFM scanning enabled the quantification of this plasticity, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides the first biologically-plausible hypothetical description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.

biophysics↗

bioRxiv2018

Specific detection of cell-free DNA derived from intestinal epithelial cells using methylation patterns

Epithelial cells of the intestine undergo rapid turnover and are thought to be cleared via stool. Disruption of tissue architecture, as occurs in colorectal cancer (CRC), results in the release of material from dying intestinal epithelial cells to blood. This phenomenon could be utilized for diagnosis and monitoring of intestinal diseases, if circulating cell-free DNA (cfDNA) derived from intestinal cells could be specifically identified. Here we describe two genomic loci that are unmethylated specifically in intestinal epithelial cells, allowing for sensitive and specific detection of DNA derived from such cells. As expected, intestinal DNA is found in stool, but not in plasma, of healthy individuals. Patients with inflammatory bowel disease (IBD) have minimal amounts of intestinal cfDNA in the plasma, whereas patients with advanced CRC show a strong signal. The intestinal markers are not elevated in plasma samples from patients with pancreatic ductal adenocarcinoma (PDAC), and a combination of intestine- and pancreas-specific markers allowed for robust differentiation between plasma cfDNA derived from CRC and PDAC patients. Intestinal DNA markers provide a mutation-independent tool for monitoring intestinal dynamics in health and disease.

developmental biology↗

bioRxiv2018

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Most human genes are alternatively spliced, allowing for a large expansion of the proteome.The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematicsequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate bothcause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75-0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel,single-cell perspective on splicing regulation.

genomics↗

bioRxiv2017

Deciphering Transcriptional Regulation of Human Core Promoters

Despite its pivotal role in regulating transcription, our understanding of core promoter function, architecture, and cis-regulatory elements is lacking. Here, we devised a highthroughput assay to quantify the activity of [~]15,000 fully designed core promoters that we integrated and expressed from a fixed location within the human genome. We find that core promoters drive transcription unidirectionally, and that sequences originating from promoters exhibit stronger activity than sequences originating from enhancers. Testing multiple combinations and distances of core promoter elements, we observe a positive effect of TATA and Initiator, a negative effect of BREu and BREd, and a 10bp periodicity in the optimal distance between the TATA and the Initiator. By comprehensively screening TF binding-sites, we show that site orientation has little effect, that the effect of binding site number on expression is factor-specific, and that there is a striking agreement between the effect of binding site multiplicity in our assay and the tendency of the TF to appear in homotypic clusters throughout the genome. Overall, our results systematically assay the elements that drive expression in core- and proximal-promoter regions and shed light on organization principles of regulatory regions in the human genome.

genomics↗

bioRxiv2017

Environmental factors dominate over host genetics in shaping human gut microbiota composition

Human gut microbiome composition is shaped by multiple host intrinsic and extrinsic factors, but the relative contribution of host genetic compared to environmental factors remains elusive. Here, we genotyped a cohort of 696 healthy individuals from several distinct ancestral origins and a relatively common environment, and demonstrate that there is no statistically significant association between microbiome composition and ethnicity, single nucleotide polymorphisms (SNPs), or overall genetic similarity, and that only 5 of 211 (2.4%) previously reported microbiome-SNP associations replicate in our cohort. In contrast, we find similarities in the microbiome composition of genetically unrelated individuals who share a household. We define the term biome-explainability as the variance of a host phenotype explained by the microbiome after accounting for the contribution of human genetics. Consistent with our finding that microbiome and host genetics are largely independent, we find significant biome-explainability levels of 16-33% for body mass index (BMI), fasting glucose, high-density lipoprotein (HDL) cholesterol, waist circumference, waist-hip ratio (WHR), and lactose consumption. We further show that several human phenotypes can be predicted substantially more accurately when adding microbiome data to host genetics data, and that the contribution of both data sources to prediction accuracy is largely additive. Overall, our results suggest that human microbiome composition is dominated by environmental factors rather than by host genetics.

genetics↗

bioRxiv2017

Demonstration of de novo chemotaxis in E. coli using a real-time, quantitative, and digital-like approach

Chemotaxis is the movement of an organism in response to an external chemical stimulus. This system enables bacteria to sense their immediate environment and adapt to changes in its chemical composition. Bacterial chemotaxis is mediated by chemoreceptors, membrane proteins that bind an effector and transduce the signal to the downstream proteins. From a synthetic biology perspective, the natural chemotactic repertoire is of little use since bacterial chemoreceptors have evolved to sense specific ligands that either benefit or harm the cell. Here we demonstrate that using a combined computational design approach together with a quantitative, real-time, and digital detection approach, we can rapidly design, manufacture, and characterize a synthetic chemoreceptor in E. coli for histamine (a ligand for which there are no known chemoreceptors). First, we employed a computational protocol that uses the Rosetta bioinformatics software together with high threshold filters to design mutational variants to the native Tar ligand binding domain that target histamine. Second, we tested different ligand-chemoreceptors pairs with a novel chemotaxis assay, based on optical reflectance interferometry of porous silicon (PSi) optical transducers, enabling label-free quantification of chemotaxis by monitoring real-time changes in the optical readout (expressed as the effective optical thickness, EOT). We found that different ligands can be characterized by an individual set of fingerprints in our assay. Namely, a binary, digital-like response in EOT change (i.e. positive or negative) that differentiates between attractants and repellants, the amplitude of change of EOT response, and the rate by which steady state in EOT change is reached. Using this assay, we were able to positively identify and characterize a single mutational chemoreceptor variant for histamine that mediated chemotaxis comparably to the natural Tar-aspartate system. Our results demonstrate the possibility of not only expanding the natural chemotaxis repertoire, but also provide a new quantitative assay by which to characterize the efficacy of the chemotactic response.

synthetic biology↗