Traumatic brain injury (TBI) is one of the leading causes of death worldwide. There are currently no effective treatments for TBI, and trauma survivors suffer from a variety of long-lasting health consequences. With nutritional support recently emerging as a vital step in improving TBI patients' outcomes, we sought to evaluate the potential therapeutic benefits of nutritional supplements derived from bovine thymus gland, which can deliver a variety of nutrients and bioactive molecules. In a rat model of controlled cortical impact (CCI), we determined that animals supplemented with a nuclear fraction of bovine thymus (TNF) display greatly improved performance on beam balance and spatial memory tests following CCI. Using RNA-Seq, we identified an array of signaling pathways that are modulated by TNF supplementation in rat hippocampus, including those involved in the process of autophagy. We further show that bovine thymus-derived extracts contain antigens found in neural tissues and that supplementation of rats with thymus extracts induces production of serum IgG antibodies against neuronal and glial antigens, which may explain the enhanced animal recovery following CCI through possible oral tolerance mechanism. Collectively, our data demonstrate, for the first time, the potency of a nutritional supplement containing nuclear fraction of bovine thymus in enhancing the functional recovery from TBI.

To identify and monitor SARS-CoV-2 variant evolution, UNC Charlotte has introduced a surveillance sequencing program for the campus and surrounding county area. We have generated a step by step protocol which has been optimized from the ”High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples” (Pater et al 2021, https://doi.org/10.1101/2021.02.09.430478) protocol for preparing SARS-CoV 2 viral genome libraries for next generation sequencing using the Oxford Nanopore PromethION instrument. The protocol is designed to work in a 96-well format throughout. It is intended to produce sufficient sequence for genome assembly to meet standards required by GISAID and NCBI for SARS-CoV-2 sequence submission. The protocol has been modified to achieve maximum yield and includes modifications to address isolate sequencing failures arising from low viral titer in some clinical samples (Cq values below 30). The protocol provides detailed guidelines and steps for working with high Cq value clinical samples, as well as a protocol variation for working in half-reaction volumes throughout to reduce sequencing costs.

Wastewater based epidemiology (WBE) has drawn significant attention as an early warning tool to detect and predict the trajectory of COVID-19 cases in a community, in conjunction with public health data. This means of monitoring for outbreaks has been used at municipal wastewater treatment centers to analyze COVID-19 trends in entire communities, as well as by universities and other community living environments to monitor COVID-19 spread in buildings. Sample concentration is crucial, especially when viral abundance in raw wastewater is below the threshold of detection by RT-qPCR analysis. We evaluated the performance of a rapid ultrafiltration-based virus concentration method using InnovaPrep Concentrating Pipette (CP) Select and compared this to the established electronegative membrane filtration (EMF) method. We evaluated sensitivity of SARS-CoV-2 quantification, surrogate virus recovery rate, and sample processing time. Results suggest that the CP Select concentrator is more efficient at concentrating SARS-CoV-2 from wastewater compared to the EMF method. About 25% of samples that tested negative when concentrated with the EMF method produced a positive signal with the CP Select protocol. Increased recovery of the surrogate virus control using the CP Select confirms this observation. We optimized the CP Select protocol by adding AVL lysis buffer and sonication, to increase the recovery of virus. Sonication increased Bovine Coronavirus (BCoV) recovery by 19%, which seems to compensate for viral loss during centrifugation. Filtration time decreases by approximately 30% when using the CP Select protocol, making this an optimal choice for building surveillance applications where quick turnaround time is necessary.

Bacterial resistance to antibiotics is a growing global concern, threatening human and environmental health, particularly among urban populations. Wastewater treatment plants (WWTPs) are thought to be “hotspots” for antibiotic resistance dissemination. The conditions of WWTPs, in conjunction with the persistence of commonly used antibiotics, may favor the selection and transfer of resistance genes among bacterial populations. WWTPs provide an important ecological niche to examine the spread of antibiotic resistance. We used heterotrophic plate count methods to identify phenotypically resistant cultivable portions of these bacterial communities and characterized the composition of the culturable subset of these populations. Resistant taxa were more abundant in raw sewage and wastewater before the biological aeration treatment stage. While some antibiotic-resistant bacteria (ARB) were detectable downstream of treated wastewater release, these organisms are not enriched relative to effluent-free upstream water, indicating efficient removal during treatment. Combined culture-dependent and -independent analyses revealed a stark difference in community composition between culturable fractions and the environmental source material, irrespective of culturing conditions. Higher proportions of the environmental populations were recovered than predicted by the widely accepted 1% culturability paradigm. These results represent baseline abundance and compositional data for ARB communities for reference in future studies addressing the dissemination of antibiotic resistance associated with urban wastewater treatment ecosystems.

The COVID-19 pandemic has been a source of ongoing challenges and presents an increased risk of illness in group environments, including jails, long-term care facilities, schools, and residential college campuses. Early reports that the SARS-CoV-2 virus was detectable in wastewater in advance of confirmed cases sparked widespread interest in wastewater-based epidemiology (WBE) as a tool for mitigation of COVID-19 outbreaks. One hypothesis was that wastewater surveillance might provide a cost-effective alternative to other more expensive approaches such as pooled and random testing of groups. In this paper, we report the outcomes of a wastewater surveillance pilot program at the University of North Carolina at Charlotte, a large urban university with a substantial population of students living in on-campus dormitories. Surveillance was conducted at the building level on a thrice-weekly schedule throughout the university's fall residential semester. In multiple cases, wastewater surveillance enabled the identification of asymptomatic COVID-19 cases that were not detected by other components of the campus monitoring program, which also included in-house contact tracing, symptomatic testing, scheduled testing of student athletes, and daily symptom reporting. In the context of all cluster events reported to the University community during the fall semester, wastewater-based testing events resulted in the identification of smaller clusters than were reported in other types of cluster events. Wastewater surveillance was able to detect single asymptomatic individuals in dorms with resident populations of 150-200. While the strategy described was developed for COVID-19, it is likely to be applicable to mitigation of future pandemics in universities and other group-living environments.

Urban streams are heavily influenced by human activity. One way that this occurs is through the reintroduction of treated effluent from wastewater treatment plants. We measured the microbial community composition of water, sediment, and soil at sites upstream and downstream from two Charlotte treatment facilities. We performed 16S rRNA gene sequencing to assay the microbial community composition at each site at four time points between the late winter and mid-summer of 2016. Despite the location of these streams in an urban area with many influences and disruptions, the streams maintain distinct water, sediment, and soil microbial profiles. While there is an overlap of microbial species in upstream and downstream sites, there are several taxa that differentiate these sites. Some taxa characteristics of human-associated microbial communities appear elevated in the downstream sediment communities. In the wastewater treatment plant and to a lesser extent in the downstream community, there are high abundance amplicon sequence variants (ASVs) which are less than 97% similar to any sequence in reference databases, suggesting that these environments contain an unexplored biological novelty. Taken together, these results suggest a need to more fully characterize the microbial communities associated with urban streams, and to integrate information about microbial community composition with mechanistic models.

This special issue on Antimicrobial Resistance in Environmental Waters features 11 articles on monitoring and surveillance of antimicrobial resistance (AMR) in natural aquatic systems (reservoirs, rivers), and effluent discharge from water treatment plants to assess the effectiveness of AMR removal and resulting loads in treated waters. The occurrence and distribution of antimicrobials, antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) was determined by utilizing a variety of techniques including liquid chromatography—mass spectrometry in tandem (LC-MS/MS), traditional culturing, antibiotic susceptibility testing (AST), molecular and OMIC approaches. Some of the key elements of AMR studies presented in this special issue highlight the underlying drivers of AMR contamination in the environment and evaluation of the hazard imposed on aquatic organisms in receiving environments through ecological risk assessments. As described in this issue, screening antimicrobial peptide (AMP) libraries for biofilm disruption and antimicrobial candidates are promising avenues for the development of new treatment options to eradicate resistance. This editorial puts into perspective the current AMR problem in the environment and potential new methods which could be applied to surveillance and monitoring efforts.

Wastewater treatment plants (WWTPs) are thought to be potential incubators of antibiotic resistance. Persistence of commonly used antibiotics in wastewater may increase the potential for selection of resistance genes transferred between bacterial populations, some of which might pose a threat to human health. In this study, we measured the concentrations of ten antibiotics in wastewater plant influents and effluents, and in surface waters up- and downstream from two Charlotte area treatment facilities. We performed Illumina shotgun sequencing to assay the microbial community and resistome compositions at each site across four time points from late winter to mid-summer of 2016. Antibiotics are present throughout wastewater treatment, and elevated concentrations of multiple antibiotics are maintained in moving stream water downstream of effluent release. While some human gut and activated sludge associated taxa are detectable downstream, these seem to attenuate with distance while the core microbial community of the stream remains fairly consistent. We observe the slight suppression of functional pathways in the downstream microbial communities, including amino acid, carbohydrate, and nucleic acid metabolism, as well as nucleotide and amino acid scavenging. Nearly all antibiotic resistance genes (ARGs) and potentially pathogenic taxa are removed in the treatment process, though a few ARG markers are elevated downstream of effluent release. Taken together, these results represent baseline measurements that future studies can utilize to help to determine which factors control the movement of antibiotics and resistance genes through aquatic urban ecosystems before, during, and after wastewater treatment.

Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered.

Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene.

Presently, human thyroglobulin is extracted from cadaver or surgically resected thyroid tissue. Such reliance on human organs for recovery and purification of proteins required for diagnostic applications complicates the manufacturing process from acquisition, to purification and quality control. These limitations have defeated efforts to establish a universally recognized standard, preventing normalization of assays to detect circulating thyroglobulin or antibodies against this protein. Unfortunately, producing intact, recombinant human thyroglobulin in protein expression systems is a daunting task that, until recently, has not been successful. We have achieved expression of full-length human thyroglobulin in stable lines of soy, and have been able to purify this protein from transgenic soybean seeds. This accomplishment provides several manufacturing advantages, as well as some potential solutions for the problems encountered when using thyroid-extracted thyroglobulin. The uniformity of recombinant protein is especially attractive when one considers the numerous post-translational modifications occurring in thyroid cells. The ease of production and purification of soybean-derived thyroglobulin is cost effective, but also allows for manufacturing flexibility in that protein expression can be separated in time and geography from the final purification of this protein in soy powders. Taken together, soybean-derived thyroglobulin has characteristics consistent with those necessary and desired for an industry standard. The availability of a renewable source of recombinant human thyroglobulin also has implications for solving some of the open-ended problems in diagnostic assays which The license for this PDF is unlimited except that no part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services. Kenneth L. Bost, Kevin C. Lambirth, Laura C. Hudson et al. 24 employ this protein. Immobilization or radiolabeling heterogeneous lots of thyroidderived thyroglobulin are variables which should be minimized with more homogenous starting material. Furthermore, the ability to remove interfering anti-thyroglobulin antibodies from patients‟ sera or tissue homogenates could also be accomplished using devices constructed from a plentiful source of renewable thyroglobulin. Our ability to express full length human thyroglobulin in transgenic soybean seeds provides, for the first time, a practical solution for the many limitations inherent in present day diagnostic assays that currently must rely on a protein isolated from primary thyroid tissue.

Today biotechnology and the process of genetic modification is emerging and advancing worldwide. At the forefront of this technology is soybean, which has become a popular sub‐ ject of genetic modification due to its versatility and economical importance as a crop plant. Over 15 years ago the first herbicide resistant soybeans were introduced into the market. By 1997, approximately 8% of all soybeans cultivated for commercial use in the United States were genetically modified. This trend has grown exponentially and by 2011 the percentage of genetically modified soybean rose to 94% in the United States and 81% worldwide. The technology to genetically modify soybean has not only had a huge impact on the commer‐ cial agricultural market, but has paved the way to an onset of both traditional and nontradi‐ tional uses for soybean as well as opening up many new potential applications for this important crop plant. Soybean has become a popular subject of genetic modification over the past two decades and with the advancement of plant transformation technology, it is now possible to manipulate and or add various traits to soybean.

Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.