Functional recovery following traumatic brain injury in rats is enhanced by oral supplementation with bovine thymus extract.

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.

Performance evaluation of virus concentration methods for implementing SARS-CoV-2 wastewater based epidemiology emphasizing quick data turnaround.

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.

Characterization of Environmental and Cultivable Antibiotic-Resistant Microbial Communities Associated with Wastewater Treatment.

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.

Implementing building-level SARS-CoV-2 wastewater surveillance on a university campus.

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.

Transcript Polymorphism Rates in Soybean Seed Tissue Are Increased in a Single Transformant of Glycine max.

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.

CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

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.

A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

BACKGROUND: 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. METHODS: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting.  Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing.  Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks.  Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO. RESULTS: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected. CONCLUSIONS: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.

Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.

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.