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At least 181 records · Page 10

Antigen‐binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones

It has been shown previously that a set of three modifications—termed S1, Crystal Kappa, and elbow—act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions—located near the heavy-chain elbow region—which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions—which complement the S1, Crystal Kappa, and elbow modifications—to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Quantitative DNA Stable Isotope Probing Identifies Active Microorganisms Assimilating Volatile Fatty Acids in Full-Scale Enhanced Biological Phosphorus Removal Processes

Enhanced biological phosphorus removal (EBPR) systems often rely on exogenous carbon sources, such as volatile fatty acids (VFAs), to achieve higher P removal. Here, we employed DNA quantitative stable isotope probing (qSIP) using two VFAs, acetate and propionate, in cyclic anaerobic/aerobic incubations to assess their effects on P cycling and microbial activity with biomass from two full-scale EBPR water resource-recovery facilities that utilize VFA addition. We found that anaerobic VFA uptake preferences differed within known groups of PAOs, such as Candidatus Accumulibacter and Tetrasphaera-affiliated members (e.g., Ca. Phosphoribacter), between the two biomasses. The combination of qSIP with metagenomics identified isotopically labeled phages that were linked to active PAOs, highlighting their potential roles in modulating EBPR community composition and activity. The highest levels of anaerobic labeling from acetate were in genomes belonging to Saccharimonadales and Rickettsiales, which are generally host-associated with bacteria and eukaryotes, respectively. Furthermore, this finding highlights the possibility of cross-feeding between PAO hosts and their parasites or predators, as well as the role of so-far uncharacterized organisms participating in carbon cycling under EBPR conditions. Collectively, these results expand our understanding of the ecological interactions involved in communities anaerobically uptaking VFAs and cycling P that are central to EBPR.

Polyphosphate accumulating organisms↗

Ecology and molecular targets of hypermutation in the global microbiome

Abstract Changes in the sequence of an organism’s genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities.

59 BASIC BIOLOGICAL SCIENCES↗

Matrix Metalloproteinases as Candidate Antigenic Determinants for Anti‐Tumor Autoantibodies in Human Ovarian Cancer: A Post Hoc Analysis

Circulating antibodies in patients with cancer can facilitate the identification of accessible epitopes on autoantigens expressed by tumors. To identify previously unrecognized protein targets in ovarian cancer, we computationally assessed a heptapeptide consensus motif (VPELGHE, flanked by two cysteine residues yielding a cyclic nonapeptide under oxidizing conditions) previously discovered via phage display-based epitope mapping of autoantibodies in patients. Eight proteins associated with ovarian cancer encompass amino acid sequences similar to the consensus motif and were, therefore, considered as candidate native autoantigens. Among these candidate targets, however, matrix metalloproteinase 14 (MMP14) demonstrates gene expression that is both high and negatively correlated with survival in ovarian cancer patient cohorts. MMP14 protein levels are also stable in tumor versus non-tumor tissues. Moreover, the corresponding heptapeptide mimic in MMP14 occurs within an α-helical secondary structural element observed in its catalytic domain. These findings demonstrate that a subset of patient-derived autoantibodies may interact with a previously unknown antigenic epitope found in MMP14 and other MMPs, thereby providing opportunities for the development of new targeted agents.

Biochemistry & Molecular Biology↗

Marine DNA methylation patterns are associated with microbial community composition and inform virus-host dynamics

Background: DNA methylation in prokaryotes is involved in many different cellular processes including cell cycle regulation and defense against viruses. To date, most prokaryotic methylation systems have been studied in culturable microorganisms, resulting in a limited understanding of DNA methylation from a microbial ecology perspective. Here, we analyze the distribution patterns of several microbial epigenetics marks in the ocean microbiome through genome-centric metagenomics across all domains of life. Results: We reconstructed 15,056 viral, 252 prokaryotic, 56 giant viral, and 6 eukaryotic metagenome-assembled genomes from northwest Pacific Ocean seawater samples using short- and long-read sequencing approaches. These metagenome-derived genomes mostly represented novel taxa, and recruited a majority of reads. Thanks to single-molecule real-time (SMRT) sequencing technology, base modification could also be detected for these genomes. This showed that DNA methylation can readily be detected across dominant oceanic bacterial, archaeal, and viral populations, and microbial epigenetic changes correlate with population differentiation. Furthermore, our genome-wide epigenetic analysis of Pelagibacter suggests that GANTC, a DNA methyltransferase target motif, is related to the cell cycle and is affected by environmental conditions. Yet, the presence of this motif also partitions the phylogeny of the Pelagibacter phages, possibly hinting at a competitive co-evolutionary history and multiple effects of a single methylation mark. Conclusions: Overall, this study elucidates that DNA methylation patterns are associated with ecological changes and virus-host dynamics in the ocean microbiome.

59 BASIC BIOLOGICAL SCIENCES↗

Cryo-EM visualization of viruses from partially irrigated soils

Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. The method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.

cryo-EM↗

Discovering novel therapeutic V H Hs for emerging viruses: perspectives from VEEV selection strategies

Introduction: Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V H Hs). Methods: In vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of V H Hs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus. Results: Here we report four novel “human” V H Hs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V H Hs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V H Hs. Discussion: Though non-neutralizing, these V H Hs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V H Hs to perform viral “sponging” which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.

59 BASIC BIOLOGICAL SCIENCES↗

Utilizing Amino Acid Composition and Entropy of Potential Open Reading Frames to Identify Protein-Coding Genes

One of the main steps in gene-finding in prokaryotes is determining which open reading frames encode for a protein, and which occur by chance alone. There are many different methods to differentiate the two; the most prevalent approach is using shared homology with a database of known genes. This method presents many pitfalls, most notably the catch that you only find genes that you have seen before. The four most popular prokaryotic gene-prediction programs (GeneMark, Glimmer, Prodigal, Phanotate) all use a protein-coding training model to predict protein-coding genes, with the latter three allowing for the training model to be created ab initio from the input genome. Different methods are available for creating the training model, and to increase the accuracy of such tools, we present here GOODORFS, a method for identifying protein-coding genes within a set of all possible open reading frames (ORFS). Our workflow begins with taking the amino acid frequencies of each ORF, calculating an entropy density profile (EDP), using KMeans to cluster the EDPs, and then selecting the cluster with the lowest variation as the coding ORFs. To test the efficacy of our method, we ran GOODORFS on 14,179 annotated phage genomes, and compared our results to the initial training-set creation step of four other similar methods (Glimmer, MED2, PHANOTATE, Prodigal). We found that GOODORFS was the most accurate (0.94) and had the best F1-score (0.85), while Glimmer had the highest precision (0.92) and PHANOTATE had the highest recall (0.96).

59 BASIC BIOLOGICAL SCIENCES↗

In Vitro Selection of Antibodies Targeting Yersinia pestis Membrane Lipids Using Nanodisc-Based Antigen Presentation

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity.

59 BASIC BIOLOGICAL SCIENCES↗

Permeability-Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems

In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is challenging. Here, a facile strategy is presented to build permeability-configurable compartments, and marked advantages of such compartmentalization are shown in reconstituting sustained synthetic biology systems in vitro. Through microfluidics, the strategy produces micrometer-sized layered microgels whose shell layer serves as a sieving structure for biomolecules and particles. In this configuration, the transport of DNAs, proteins, and bacteriophages across the compartments can be controlled an guided by a physical model. Through permeability engineering, a compartmentalized cell-free protein synthesis system sustains multicycle protein production; ≈100 000 compartments are repeatedly used in a five-cycle synthesis, featuring a yield of 2.2 mg mL -1 . Further, the engineered bacteria-enclosing compartments possess near-perfect phage resistance and enhanced environmental fitness. In a complex river silt environment, compartmentalized whole-cell biosensors show maintained activity throughout the 32 h pollutant monitoring. It is anticipated that permeability-engineered compartmentalization should pave the way for practical synthetic biology applications such as green bioproduction, environmental sensing, and bacteria-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

Combining computational modeling and experimental library screening to affinity-mature VEEV-neutralizing antibody F5

Engineered monoclonal antibodies have proven to be highly effective therapeutics in recent viral outbreaks. However, despite technical advancements, an ability to rapidly adapt or increase antibody affinity and by extension, therapeutic efficacy, has yet to be fully realized. We endeavored to stand-up such a pipeline using molecular modeling combined with experimental library screening to increase the affinity of F5, a monoclonal antibody with potent neutralizing activity against Venezuelan Equine Encephalitis Virus (VEEV), to recombinant VEEV (IAB) E1E2 antigen. We modeled the F5/E1E2 binding interface and generated predictions for mutations to improve binding using a Rosetta-based approach and dTERMen, an informatics approach. The modeling was complicated by the fact that a high-resolution structure of F5 is not available and the H3 loop of F5 exceeds the length for which current modeling approaches can determine a unique structure. A subset of the predicted mutations from both methods were incorporated into a phage display library of scFvs. This library and a library generated by error-prone PCR were screened for binding affinity to the recombinant antigen. Results from the screens identified favorable mutations which were incorporated into 12 human-IgG1 variants. The best variant, containing eight mutations, improved KD from 0.63 nM (parental) to 0.01 nM. While this did not improve neutralization or therapeutic potency of F5 against IAB, it did increase cross-reactivity to other closely related VEEV epizootic and enzootic strains, demonstrating the potential of this method to rapidly adapt existing therapeutics to emerging viral strains.

affinity-maturation↗

Development of high-affinity nanobodies specific for Na V 1.4 and Na V 1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na V 1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na V isoform has been challenging because they usually show undesired crossreactivity for different Na V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa V 1.4 or CTNa V 1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa V 1.4 and CTNa V 1.5 with high affinity (K D ~ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na V 1.4 and Na V 1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na V 1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na V reagents to capture Na V s from cell lysates and as molecular visualization agents for Na V s.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Phosphate amendment drives bloom of RNA viruses after soil wet-up

Soil rewetting after a dry period results in a surge of activity and succession in both microbial and DNA virus communities. Less is known about the response of RNA viruses to soil rewetting—while they are highly diverse and widely distributed in soil, they remain understudied. We hypothesized that RNA viruses would show temporal succession following rewetting and that phosphate amendment would influence their trajectory, as viral proliferation may cause phosphorus limitation. Using 39 time-resolved metatranscriptomes and amplicon data, 2190 RNA viral populations were identified across five phyla, with 26 % of these predicted to infect bacteria, and 11 % fungi. Only 1.2 % of viral populations had annotated capsid genes, suggesting most persist via intracellular replication without a free virion phase. Phosphate amendment altered RNA viral community composition within the first week and amended vs. unamended communities remained distinguishable for up to three weeks. While the overall host community remained stable, certain bacterial populations showed reduced abundance in phosphate-amended soils, likely due to increased viral lysis, as RNA bacteriophages proliferated significantly. Notably, 60 % of the viruses with increased abundance under phosphate amendment belonged to basal Lenarviricota clades rather than well-known groups like Leviviricetes. We estimate RNA bacteriophage infections may affect 10 7 –10 9 bacteria per gram of soil, aligning with the total bacterial population (10 7 –10 10 g -1 soil), suggesting that RNA phages significantly influence bacterial communities post-wet-up, with phosphorus availability modulating this effect.

59 BASIC BIOLOGICAL SCIENCES↗

Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. SNAbs However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Finally, our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug- and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

60 APPLIED LIFE SCIENCES↗

Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells

We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employs apidaecin-like, anti-microbial peptides observed to temporarily sequester and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent matter, these peptides granted a conditional lambda phage resistance to a recoded Escherichia coli strain with non-essential RF1 activity and promoted multi-site non-standard amino acid (nsAA) incorporation at in-frame amber stop codons in vivo and in vitro. When exogenously added, the peptides stimulated specific nsAA incorporation in a variety of sensitive, wild-type (RF1+) strains including Agrobacterium tumefaciens, a species in which nsAA incorporation has not been previously reported. Improvement in nsAA incorporation was typically 2–15-fold in E. coli BL21, MG1655, DH10B strains and A. tumefaciens with the >20-fold improvement observed in probiotic E. coli Nissle 1917. In-cell expression of these peptides promoted multi-site nsAA incorporation in transcripts with up to 6 amber codons, with a >35-fold increase in BL21 showing moderate toxicity. Leveraging this RF1 sensitivity allowed multiplexed partial recoding of MG1655 and DH10B that rapidly resulted in resistant strains that showed an additional ~2 fold boost to nsAA incorporation independent of the peptide. Lastly, in-cell expression of an apidaecin-like peptide library allowed the discovery of new peptide variants with reduced toxicity that still improved multi-site nsAA incorporation >25-fold. In parallel to genetic reprogramming efforts, these new approaches can facilitate genetic code expansion technologies in a variety of wild-type bacterial strains.

59 BASIC BIOLOGICAL SCIENCES↗

Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants

As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.

54 ENVIRONMENTAL SCIENCES↗