Limited Metabolomic Overlap between Commensal Bacteria and Marine Sponge Holobionts Revealed by Large Scale Culturing and Mass Spectrometry-Based Metabolomics: An Undergraduate Laboratory Pedagogical Effort at Georgia Tech.

Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.

Surveillance-to-Diagnostic Testing Program for Asymptomatic SARS-CoV-2 Infections on a Large, Urban Campus in Fall 2020.

BACKGROUND: Six months into the COVID-19 pandemic, college campuses faced uncertainty regarding the likely prevalence and spread of disease, necessitating large-scale testing to help guide policy following re-entry. METHODS: A SARS-CoV-2 testing program combining pooled saliva sample surveillance leading to diagnosis and intervention surveyed over 112,000 samples from 18,029 students, staff and faculty, as part of integrative efforts to mitigate transmission at the Georgia Institute of Technology in Fall 2020. RESULTS: Cumulatively, we confirmed 1,508 individuals diagnostically, 62% of these through the surveillance program and the remainder through diagnostic tests of symptomatic individuals administered on or off campus. The total strategy, including intensification of testing given case clusters early in the semester, was associated with reduced transmission following rapid case increases upon entry in Fall semester in August 2020, again in early November 2020, and upon re-entry for Spring semester in January 2021. During the Fall semester daily asymptomatic test positivity initially peaked at 4.1% but fell below 0.5% by mid-semester, averaging 0.84% across the Fall semester, with similar levels of control in Spring 2021. CONCLUSIONS: Owing to broad adoption by the campus community, we estimate that the program protected higher risk staff and faculty while allowing some normalization of education and research activities.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing.

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

Modifying a Commonly Expressed Endocytic Receptor Retargets Nanoparticles in Vivo.

Nanoparticles are often targeted to receptors expressed on specific cells, but few receptors are (i) highly expressed on one cell type and (ii) involved in endocytosis. One unexplored alternative is manipulating an endocytic gene expressed on multiple cell types; an ideal gene would inhibit delivery to cell type A more than cell type B, promoting delivery to cell type B. This would require a commonly expressed endocytic gene to alter nanoparticle delivery in a cell type-dependent manner in vivo; whether this can occur is unknown. Based on its microenvironmental regulation, we hypothesized Caveolin 1 (Cav1) would exert cell type-specific effects on nanoparticle delivery. Fluorescence was not sensitive enough to investigate this question, and as a result, we designed a platform named QUANT to study nanoparticle biodistribution. QUANT is 108× more sensitive than fluorescence and can be multiplexed. By measuring how 226 lipid nanoparticles (LNPs) delivered nucleic acids to multiple cell types in vivo in wild-type and Cav1 knockout mice, we found Cav1 altered delivery in a cell-type specific manner. Cav1 knockout did not alter LNP delivery to lung and kidney macrophages but substantially reduced LNP delivery to Kupffer cells, which are liver-resident macrophages. These data suggest caveolin-mediated endocytosis of nanomedicines by macrophages varies with tissue type. These results suggest manipulating receptors expressed on multiple cell types can tune drug delivery.

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation.

Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.

Development of a pan-tau multivalent nanobody that binds tau aggregation motifs and recognizes pathological tau aggregates.

Alzheimer's disease and other tauopathies are characterized by the misfolding and aggregation of the tau protein into oligomeric and fibrillar structures. Antibodies against tau play an increasingly important role in studying these neurodegenerative diseases and the generation of tools to diagnose and treat them. The development of antibodies that recognize tau protein aggregates, however, is hindered by complex immunization and antibody selection strategies and limitations to antigen presentation. Here, we have taken a facile approach to identify single-domain antibodies, or nanobodies, that bind to many forms of tau by screening a synthetic yeast surface display nanobody library against monomeric tau and creating multivalent versions of our lead nanobody, MT3.1, to increase its avidity for tau aggregates. We demonstrate that MT3.1 binds to tau monomer, oligomers, and fibrils, as well as pathogenic tau from a tauopathy mouse model, despite being identified through screens against monomeric tau. Through epitope mapping, we discovered binding epitopes of MT3.1 contain the key motif VQIXXK which drives tau aggregation. We show that our bivalent and tetravalent versions of MT3.1 have greatly improved binding ability to tau oligomers and fibrils compared to monovalent MT3.1. Our results demonstrate the utility of our nanobody screening and multivalent design approach in developing nanobodies that bind amyloidogenic protein aggregates. This approach can be extended to the generation of multivalent nanobodies that target other amyloid proteins and has the potential to advance the research and treatment of neurodegenerative diseases.

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing.

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Mxyococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display.

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.

Parallel deployment of passive and composite samplers for surveillance and variant profiling of SARS-CoV-2 in sewage.

Wastewater-based epidemiology during the COVID-19 pandemic has proven useful for public health decision-making but is often hampered by sampling methodology constraints, particularly at the building- or neighborhood-level. Time-weighted composite samples are commonly used; however, autosamplers are expensive and can be affected by intermittent flows in sub-sewershed contexts. In this study, we compared time-weighted composite, grab, and passive sampling via Moore swabs, at four locations across a college campus to understand the utility of passive sampling. After optimizing the methods for sample handling and processing for viral RNA extraction, we quantified SARS-CoV-2 N1 and N2, as well as a fecal strength indicator, PMMoV, by ddRT-PCR and applied tiled amplicon sequencing of the SARS-CoV-2 genome. Passive samples compared favorably with composite samples in our study area: for samples collected concurrently, 42 % of the samples agreed between Moore swab and composite samples and 58 % of the samples were positive for SARS-CoV-2 using Moore swabs while composite samples were below the limit of detection. Variant profiles from Moore swabs showed a shift from variant BA.1 to BA.2, consistent with in-person saliva samples. These data have implications for the broader implementation of sewage surveillance without advanced sampling technologies and for the utilization of passive sampling approaches for other emerging pathogens.

Expression vectors for Acinetobacter baylyi ADP1.

Acinetobacter baylyi ADP1 is naturally competent and proficient at homologous recombination, so it can be transformed without restriction digests or ligation reactions. Expression vectors for this system, however, are not yet widely available. Here we describe the construction and characterization of inducible expression vectors that replicate as plasmids in A. baylyi or integrate into a nonessential part of its chromosome. These tools will facilitate the engineering of genes and genomes in this promising model organism.

Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies.

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nM-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.

Ribonucleotide incorporation in yeast genomic DNA shows preference for cytosine and guanosine preceded by deoxyadenosine.

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Barcoding chemical modifications into nucleic acids improves drug stability in vivo.

The efficacy of nucleic acid therapies can be limited by unwanted degradation. Chemical modifications are known to improve nucleic acid stability, but the (i) types, (ii) positions, and (iii) numbers of modifications all matter, making chemically optimizing nucleic acids a combinatorial problem. As a result, in vivo studies of nucleic acid stability are time consuming and expensive. We reasoned that DNA barcodes could simultaneously study how chemical modification patterns affect nucleic acid stability, saving time and resources. We confirmed that rationally designed DNA barcodes can elucidate the role of specific chemical modifications in serum, in vitro and in vivo; we also identified a modification pattern that enhanced stability. This approach to screening chemical modifications in vivo can efficiently optimize nucleic acid structure, which will improve biomaterial-based nucleic acid drugs.

Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery.

Lipid nanoparticles (LNPs) are formulated using unmodified cholesterol. However, cholesterol is naturally esterified and oxidized in vivo, and these cholesterol variants are differentially trafficked in vivo via lipoproteins including LDL and VLDL. We hypothesized that incorporating the same cholesterol variants into LNPs-which can be structurally similar to LDL and VLDL-would alter nanoparticle targeting in vivo. To test this hypothesis, we quantified how >100 LNPs made with six cholesterol variants delivered DNA barcodes to 18 cell types in wild-type, LDLR-/-, and VLDLR-/- mice that were both age-matched and female. By analyzing ∼2000 in vivo drug delivery data points, we found that LNPs formulated with esterified cholesterol delivered nucleic acids more efficiently than LNPs formulated with regular or oxidized cholesterol when compared across all tested cell types in the mouse. We also identified an LNP containing cholesteryl oleate that efficiently delivered siRNA and sgRNA to liver endothelial cells in vivo. Delivery was as-or more-efficient as the same LNP made with unmodified cholesterol. Moreover, delivery to liver endothelial cells was 3 times more efficient than delivery to hepatocytes, distinguishing this oleate LNP from hepatocyte-targeting LNPs. RNA delivery can be improved by rationally selecting cholesterol variants, allowing optimization of nanoparticle targeting.

Bacteria-Based Materials for Stem Cell Engineering.

Materials can be engineered to deliver specific biological cues that control stem cell growth and differentiation. However, current materials are still limited for stem cell engineering as stem cells are regulated by a complex biological milieu that requires spatiotemporal control. Here a new approach of using materials that incorporate designed bacteria as units that can be engineered to control human mesenchymal stem cells (hMSCs), in a highly dynamic-temporal manner, is presented. Engineered Lactococcus lactis spontaneously colonizes a variety of material surfaces (e.g., polymers, metals, and ceramics) and is able to maintain growth and induce differentiation of hMSCs in 2D/3D surfaces and hydrogels. Controlled, dynamic, expression of fibronectin fragments supports stem cell growth, whereas inducible-temporal regulation of secreted bone morphogenetic protein-2 drives osteogenesis in an on-demand manner. This approach enables stem cell technologies using material systems that host symbiotic interactions between eukaryotic and prokaryotic cells.

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously.

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.

Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase.

We have previously shown that an antisense, effective in the knock down of cytosolic phospholipase A2 (cPLA2), localizes mainly in the nucleus of human endothelial cells and monocytes and that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in its nuclear localization. In this study, we clarify how GAPDH participates in the nuclear localization of this antisense oligodeoxynucleotide (ODN) directed against cPLA2 mRNA. A central TAAAT motif providing specificity and high affinity binding was assumed to interact with the enzyme Rossmann fold region on the basis of competition to this site by NAD+. To asses whether the TAAAT motif interacts directly with the enzyme Rossmann fold region, we evaluated the binding to GAPDH of different oligonucleotides and the effect of competitors such as NAD+, NADH, mononucleotides, DNA, polyribonucleic acids and polyanions. We found that the dissociation constant for TAAAT containing oligonucleotides was three--to fivefold higher with respect to oligo not containing this motif. By covalently linking 32P-labeled cPLA2p(N)16 to GAPDH and after executing hydrolysis with hydroxylamine, the labeling was exclusively found in the C-terminal domain (aa 286-334). These results indicate that the antisense oligonucleotide interacts with a site not having a defined function but which can be negatively allosterically regulated when NAD+ or polynucleotides are bound to Rossmann fold.

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition.

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.