Biology subjects

Brown, A. C.

Publications and source records attributed to Brown, A. C..

At least 19 recordsLinked to original sources

bioRxiv2026

REGULATION OF COLONIC MACROPHAGES AND TYPE-17 AND REGULATORY T CELLS IN DSS-COLITIS BY IBD-ASSOCIATED TRANSCRIPTION FACTOR, CREM

BackgroundRecent genome wide association studies (GWAS) performed by our laboratory identified polymorphisms at the locus containing the gene, cAMP-responsive element modulator (CREM), that influence Entamoeba histolytica+ diarrheal disease susceptibility in children. CREM is a cAMP-responsive transcription factor that regulates genetic expression and epigenetic modulation in a context- and cell-specific manner. Polymorphisms at this locus have been previously associated with IBD susceptibility, suggesting CREM regulates enteric inflammation in infectious and autoimmune colitis. MethodsMice were generated with either a tamoxifen-inducible global deletion or an intestinal epithelial cell (IEC)-specific deletion of Crem. Dextran-sodium sulfate (DSS) was administered to chemically induce colitis and mice were assayed for weight loss, clinical score, spectral flow cytometry of colonic lamina propria and mesenteric lymph node white blood cells, and shallow shotgun whole genome sequencing of fecal samples. ResultsTamoxifen-inducible global deletion of Crem significantly ameliorated DSS-colitis severity as measured by clinical scoring and weight loss over the course of disease (p = 2.29 x 10-15, p = 2.24 x 10-21, respectively). Protection was not phenocopied when Crem was deleted exclusively in IECs. When sampled during acute colitis, protection seen in Crem-deleted mice was associated with a significant increase in macrophages, and ROR{gamma}t+ regulatory (pTregs) and T helper (Th17) cells in the colonic lamina propria, along with an increase of T-follicular like helper cells in the mesenteric lymph node. ConclusionsInducible global deletion of Crem reduced the severity of DSS colitis while increasing colonic macrophages, ROR{gamma}t+ regulatory (pTregs) and T helper (Th17) cells. Future work will investigate the aforementioned cell types to determine the mechanism by which CREM aggravates DSS-colitis, thereby defining the immunoregulatory role of CREM in intestinal inflammation with the goal of identifying new therapeutic targets for IBD.

immunology↗

bioRxiv2026

Quantifying Adipose Tissue Thermogenesis Using Highly Sensitive Isothermal Microcalorimetry

Isothermal microcalorimetry enables direct measurement of thermogenic heat production, but optimal conditions for its application to adipose tissue have not been systematically defined. Here, we establish experimental parameters for quantifying thermogenesis using the CalScreener, a high-throughput isothermal microcalorimetry platform, across adipocyte organoids, freshly isolated adipocytes, and intact adipose tissue explants. Heat output scaled with spheroid size within a defined range and increased linearly with spheroid number per well, improving measurement consistency. Freshly isolated adipocytes and intact adipose tissue exhibited robust and depot-specific thermogenic activity that was sensitive to physiological modulation. Notably, intact adipose tissue retained functional thermogenic capacity for at least 6 hours ex vivo when maintained in nutrient-containing medium, enabling batch analysis of large cohorts. In contrast, storage conditions designed to preserve mitochondrial integrity during cold handling suppressed basal heat production and did not maintain integrated thermogenic activity, despite preserved adrenergic responsiveness. Together, these results provide practical guidance for tissue handling, assay design, and interpretation of isothermal microcalorimetry-based measurements of adipose tissue thermogenesis.

cell biology↗

bioRxiv2025

Human immune response to primary cryptosporidiosis parallels murine infection models

Cryptosporidium is a protozoan parasite that causes cryptosporidiosis, an enteric infection associated with diarrhea, malnutrition, and impaired childhood development in low- and middle-income countries. Both humoral and cell-mediated immune responses have been implicated in protection, but the durability and quality of human immune responses in immunocompetent adults remain poorly defined. We investigated the development of immunity in two healthy US adults following primary cryptosporidiosis acquired during travel to Bangladesh. Longitudinal plasma samples were analyzed for antibody responses to Cryptosporidium antigens Cp17 and Cp23 and for circulating cytokine profiles. Circulating antibody peaked at three weeks post-infection but declined rapidly thereafter, approaching baseline within 16 weeks. In contrast, antibody avidity increased steadily over time, consistent with ongoing affinity maturation in germinal centers. While affinity maturation occurred, the composition memory B cells specific to Cryptosporidium antigens was skewed toward IgM+ cells across timepoints suggesting extrafollicular responses dominated and germinal center-derived, class-switched memory was limited. Cytokine profiling revealed an acute Th1-skewed response, with elevations in CXCL9, CXCL10, IL-27, IFN{gamma}, IL-12, and IL-18 during early infection. These signatures mirrored protective pathways identified in murine models, underscoring the importance of type I immunity in parasite clearance. Together, these findings highlight that while antibody responses to Cryptosporidium are short-lived, avidity maturation persists, and Th1-driven cytokine responses dominate during acute infection. This work provides rare longitudinal data on immune responses in naive adults following natural cryptosporidiosis and offers insight into mechanisms that may inform vaccine development and strategies to mitigate recurrent infection in vulnerable populations.

immunology↗

bioRxiv2025

Combined Deletion of miR-27a and miR-27b Enhances Protection Against Diet-Induced Obesity

MicroRNAs (miRNAs) are frequently encoded within polycistronic clusters thought to function as coherent regulatory units, yet whether individual cluster members act cooperatively or exert distinct physiological effects remains unclear. miR-27a and miR-27b are closely related miRNAs located within separate paralogous miR-23/27/24 clusters, but their in vivo relationship is unresolved. Using isoform-specific knockout models generated by precise CRISPR-based excision of individual miRNA hairpins, we defined how miR-27a and miR-27b regulate adipose thermogenesis and systemic metabolism. High-fat feeding increased miR-27a/b expression and suppressed thermogenic gene programs in subcutaneous white adipose tissue. In primary beige and brown adipocytes, loss of either isoform produced modest activation of thermogenic programs, whereas combined deletion caused additive increases in Ucp1 expression and mitochondrial DNA content. Ex vivo heat production was significantly elevated in double-knockout adipose explants relative to wild-type and single-knockout controls. In vivo, double-knockout mice exhibited increased energy expenditure and, under high-fat diet conditions, reduced adiposity with improved glucose homeostasis compared with wild-type and single-knockout mice. In contrast to metabolic dysfunction reported following whole-cluster deletion, selective miR-27 loss reveals a protective thermogenic program. These findings demonstrate that miRNA clusters need not act as coherent regulatory modules and identify miR-27a/b as a cooperative thermogenic checkpoint with therapeutic implications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/666011v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@7d6b74org.highwire.dtl.DTLVardef@199d56org.highwire.dtl.DTLVardef@1a87e48org.highwire.dtl.DTLVardef@fab51a_HPS_FORMAT_FIGEXP M_FIG C_FIG * CRISPR isoform-specific knockouts isolate miR-27a/b contributions to metabolism. *Dual miR-27a/b loss drives cooperative thermogenic activation. *Double knockouts enhance energy expenditure and metabolic fitness in diet-induced obesity. *miR-27a/b deletion reveals that clustered miRNAs can exert non-coherent regulatory roles.

physiology↗

bioRxiv2025

Exosome Trafficking Is a Key Regulator of Adipocyte Thermogenesis

Activation of beige adipocytes enhances energy expenditure and promotes metabolic health, presenting a promising approach for combating obesity and diabetes. As part of this process, thermogenesis, fueled in part by uncoupled mitochondrial respiration, plays a central role in converting calories into thermal energy, thereby preventing their storage as fat. Here, we identify a role for exosome trafficking as an intrinsic regulator of beige adipocyte thermogenesis. Exosomes are small extracellular vesicles that mediate cell-cell and intracellular communication by transporting regulatory cargo, including microRNAs, proteins, and lipids. Using both human cells and mouse models, we show that thermogenic activation of beige adipocytes promotes the rapid release of exosomes enriched in microRNAs known to suppress thermogenic programs. Genetic or pharmacological blockade of exosome secretion attenuates thermogenesis, whereas enhancing exosome release amplifies thermogenic output. Mice deficient in the exosome secretion regulator Rab27a exhibit reduced energy expenditure in response to both cold exposure and {beta}3-adrenergic stimulation. These findings establish exosome trafficking as a key contributor to beige adipocyte thermogenic capacity, highlighting an intracellular mechanism that may be leveraged to enhance energy expenditure and treat obesity-related metabolic diseases. Significance StatementThermogenic adipocytes, including beige fat cells, help maintain energy balance by converting excess nutrients into heat, thereby reducing fat storage and supporting metabolic function. Although these cells are known to promote energy expenditure, the intracellular processes that enable their full thermogenic response are not well defined. Here, we show that exosome secretion is required for beige adipocytes to reach their full thermogenic potential. Blocking exosome release dampens this response, while boosting it amplifies thermogenic output. These findings point to exosome release as an essential part of thermogenic regulation and a potential target for improving metabolic health.

cell biology↗

bioRxiv2025

Evaluation of Enterobacterales carrying Acinetobacter-associated blaOXA genes--United States, 2017-2022

Through their ability to hydrolyze carbapenems, Ambler class D beta-lactamases endanger patients by limiting the clinical efficacy of beta-lactam antimicrobials. Further, plasmid-mediated transmission can increase mobility of carbapenemase genes between bacteria and facilitate their spread between patients. In the United States and elsewhere, the plasmid-mediated Ambler class D carbapenemase genes blaOXA-23-like, blaOXA-24/40-like, and blaOXA-58-like are commonly associated with Acinetobacter species and have rarely been reported outside of this genus. However, multiple recent international reports indicate detection of Enterobacterales isolates carrying Acinetobacter-associated class D carbapenemase genes. This evaluation aimed to provide insight into whether Enterobacterales harboring these class D genes may be circulating undetected in the United States, thereby signaling a need for adapting testing strategies to prioritize the detection of these potentially emerging public health threats. We analyzed whole-genome sequencing data generated through multiple Centers for Disease Control and Prevention (CDC) activities, including testing conducted across the Antimicrobial Resistance Laboratory Network, to determine whether any Enterobacterales isolates sequenced from 2017-2022 harbored Acinetobacter-associated class D carbapenemase genes. Among [~]10,000 predominantly carbapenem-resistant Enterobacterales isolates, we identified only a single Enterobacterales isolate harboring an Acinetobacter-associated class D gene - a Klebsiella pneumoniae isolate harboring a blaOXA-23-like gene. Our findings suggest that blaOXA-23-like, blaOXA-24/40-like, and blaOXA-58-like genes are rare among Enterobacterales isolates sequenced through CDC public health activities in the United States and do not warrant changes to current testing priorities at this time.

microbiology↗

bioRxiv2025

Crucial role for iron metabolism in mediating influenza A virus infection and associated disease

Rationale and ObjectivesIron availability and metabolism are important in the pathogenesis of bacterial infections. More recently, links have been reported between iron and the severity of viral infections. In this study, we characterize a crucial relationship between iron metabolism and IAV infection and disease. MethodsIron-related gene expression was assessed in human airway epithelial cells (AEC) infected with IAV. AECs were cultured with ferric iron, iron-loaded transferrin, or iron chelator, deferoxamine (DFO), prior to infection with IAV. Mice were placed on a high iron diet for 8 weeks prior to infection with IAV or treated with anti-transferrin receptor-1 (TFR1) antibody during IAV infection. The effects of iron modulation and depletion of TFR1-mediated responses on IAV infection were assessed. Measurements and main resultsIron-related gene expression and metabolism are altered systemically and in lung tissues and AECs during IAV infections. Increasing iron availability increases viral titer in AECs, while DFO protects against iron-induced increased susceptibility to infection. Increasing systemic iron loading, which increases iron levels in the lung, increases viral titer, proinflammatory responses, airway inflammation, and worsens IAV-induced disease in terms of lung function and weight loss in vivo. Inhibition of TFR1 protects against IAV-induced disease in vivo. ConclusionIAV infections remain a major threat to human health and global economies. Strategies that boost protective, or reduce pathogenic, host responses may provide broadly effective, long-term therapeutic options. We have identified a key role for iron metabolism in modifying host responses to IAV that can be harnessed to protect against disease. Key MessagesO_LIIron metabolism is altered systemically, and in airway epithelial cells and lung tissues, during IAV infection. C_LIO_LIIncreased iron availability increases viral titer both in vitro and in vivo C_LIO_LISystemic iron loading worsens IAV-induced inflammation and disease outcomes in vivo, highlighting iron as a crucial factor for modulating IAV infections and disease. C_LIO_LIHost epithelial cells and lung tissues reduce TFRC gene expression, whilst the number and proportion of TFR1hi expressing cells increase, in response to IAV infection. Neutralising TFR1 protects against IAV-induced disease in vivo, highlighting TFR1 as a potential therapeutic target for IAV infections. C_LI

immunology↗

bioRxiv2025

Escherichia coli outer membrane vesicles encapsulating small molecule antibiotics improve drug function by facilitating transport

The development of novel antimicrobial agents that are effective against Gram-negative bacteria is hindered by the dual membrane cell wall structure of these bacteria. To reach their intracellular targets, most antibiotics must therefore pass through protein channels called porins; however, a common mechanism of acquired resistance is decreased expression of these proteins. Inspired by the ability of outer membrane vesicles (OMVs) to deliver cargo to the bacterial cytosol, we hypothesized that encapsulation of small molecule antibiotics within OMVs would improve the activity of the drugs by facilitating uptake. To test this, we investigated the ability of antibiotic- encapsulated OMVs to inhibit the growth of both Gram-negative and Gram-positive bacteria, including multidrug-resistant clinical isolates. We also demonstrated that this mechanism of delivery does not require porin expression. Together, our results demonstrate the potential of OMVs as novel antibiotic delivery vehicles to treat antibiotic-resistant bacterial infections.

bioengineering↗

bioRxiv2023

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

bioengineering↗

bioRxiv2023

Simple Design for Membrane-Free Microphysiological Systems to Model the Blood-Tissue Barriers

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ{middle dot}cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations. HIGHLIGHTSO_LIBarrier-type organs-on-a-chip are popular due to their mimicry of a variety of tissue constructs and interfaces. C_LIO_LITypical barrier-type organs-on-a-chip rely upon microperforated membranes and complex assembly, which limits both ease of fabrication the desired barrier performance. C_LIO_LIA membrane-free barrier-type organ-on-a-chip is designed, which uses simple Y-channel microfluidics and photopolymerization to form a precise "blood-tissue interface." C_LIO_LIFabrication of the membrane-free design can be easily parallelized and scaled-up. C_LI

bioengineering↗

bioRxiv2023

Toxin-Triggered Liposomes for the Controlled Release of Antibiotics to Treat Infections Associated with Gram-Negative Bacteria

Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin- triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N- methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA- expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.

bioengineering↗

bioRxiv2023

SARS-CoV-2 Delta Variant Remains Viable in Environmental Biofilms found in Meat Packaging Plants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus that directly infects human airway epithelial cells and caused the COVID-19 pandemic. At the start of the pandemic in 2020, meat-packaging plants saw a surge in SARS-CoV-2 cases, which forced many to temporarily close. To determine why SARS-CoV-2 appears to thrive specifically well in meat packaging plants, we used SARS-CoV-2 Delta variant and meat packaging plant drain samples to develop mixed-species biofilms on materials commonly found within meat packaging plants (stainless steel (SS), PVC, and ceramic tile). Our data provides evidence that SARS-CoV-2 Delta variant remained viable on all the surfaces tested with and without an environmental biofilm. We observed that SARS-CoV-2 Delta variant was able to remain infectious with each of the environmental biofilms, however, we detected a significant reduction in viability post-exposure to Plant B biofilm on SS, PVC, and on ceramic tile chips, and to Plant C biofilm on SS and PVC chips. The numbers of viable SARS-CoV-2 Delta viral particles was 1.81 - 4.57-fold high than the viral inoculum incubated with the Plant B and Plant C environmental biofilm on SS, and PVC chips. We did not detect a significant difference in viability when SARS-CoV-2 Delta variant was incubated with the biofilm obtained from Plant A on any of the materials tested and SARS-CoV-2 Delta variant had higher plaque numbers when inoculated with Plant C biofilm on tile chips, with a 2.75-fold difference compared to SARS-CoV-2 Delta variant on tile chips by itself. In addition, we detected an increase in the biofilm biovolume in response to SARS-CoV-2 Delta variant which is also a concern for food safety due to the potential for foodborne pathogens to respond likewise when they come into contact with the virus. These results indicate a complex virus-environmental biofilm interaction which correlates to the different bacteria found in each biofilm. Our results also indicate that there is the potential for biofilms to protect SARS-CoV-2 from disinfecting agents and remaining prevalent in meat packaging plants. With the highly infectious nature of some SARS-CoV-2 variants such as Delta, and more so with the Omicron variant, even a minimal amount of virus could have serious health implications for the spread and reoccurrence of SARS-CoV-2 outbreaks in meat packaging plants.

ecology↗

bioRxiv2023

Identifying size-dependent toxin sorting in bacterial outer membrane vesicles

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.

biophysics↗

bioRxiv2022

Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with periodontal disease tend to produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is secreted via a type 1 secretion system, from the bacterial cytosol across both the inner and outer membranes in a single step into the supernatant, where it is able to interact with host cells. Upon secretion, some of the toxin associates with the bacterial cell membrane, enabling its release in association with the surface of outer membrane vesicles (OMVs), small, spherical vesicles derived from the outer membrane. We have previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs, a highly abundant population of small (<100 nm in diameter) OMVs, and a less abundant population of large (>300 nm in diameter) OMVs. Here, we have investigated the association of LtxA with the two populations of OMVs varying in size. Our results indicate that surface-associated DNA drives the selective sorting of LtxA to large OMVs.

microbiology↗

bioRxiv2021

Using de novo assembly to identify structural variation of complex immune system gene regions

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data; application of these methods to larger samples would provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

bioinformatics↗

bioRxiv2020

Enhanced Anticoagulation Activity of Functional RNA Origami

Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side-effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target-specific anticoagulant, is described. Improved design and construction techniques for self-folding, single-molecule RNA origami as a platform for displaying pre-selected RNA aptamers with precise orientational and spatial control, are reported. Nuclease resistance is added using 2-fluoro-modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA-linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for ssDNA and ssPNA (peptide nucleic acid) antidotes show restoration of 75% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami was freeze-dried, and stored at room temperature. Freshly produced and freeze-dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami-based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications.

bioengineering↗

bioRxiv2020

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

genomics↗

bioRxiv2019

Evaluating the ALERT algorithm for local outbreak onset detection in seasonal infectious disease surveillance data

Estimation of epidemic onset timing is an important component of controlling the spread of seasonal infectious dis-eases within community healthcare sites. The Above Local Elevated Respiratory Illness Threshold (ALERT) algorithm uses a threshold-based approach to suggest incidence levels that historically have indicated the transition from endemic to epidemic activity. In this paper, we present the first detailed overview of the computational approach underlying the algorithm. In the motivating example section, we evaluate the performance of ALERT in determining the onset of increased respiratory virus incidence using laboratory testing data from the Childrens Hospital of Colorado. At a threshold of 10 cases per week, ALERT-selected intervention periods performed better than the observed hospital site periods (2004/2005-2012/2013) and a CUSUM method. Additional simulation studies show how data properties may effect ALERT performance on novel data. We found that the conditions under which ALERT showed ideal performance generally included high seasonality and low off-season incidence.

epidemiology↗