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At least 433 records · Page 24

Natural Product Gene Clusters in the Filamentous Nostocales Cyanobacterium HT-58-2

Cyanobacteria are known as rich repositories of natural products. One cyanobacterial-microbial consortium (isolate HT-58-2) is known to produce two fundamentally new classes of natural products: the tetrapyrrole pigments tolyporphins A–R, and the diterpenoid compounds tolypodiol, 6-deoxytolypodiol, and 11-hydroxytolypodiol. The genome (7.85 Mbp) of the Nostocales cyanobacterium HT-58-2 was annotated previously for tetrapyrrole biosynthesis genes, which led to the identification of a putative biosynthetic gene cluster (BGC) for tolyporphins. Here, bioinformatics tools have been employed to annotate the genome more broadly in an effort to identify pathways for the biosynthesis of tolypodiols as well as other natural products. A putative BGC (15 genes) for tolypodiols has been identified. Four BGCs have been identified for the biosynthesis of other natural products. Two BGCs related to nitrogen fixation may be relevant, given the association of nitrogen stress with production of tolyporphins. The results point to the rich biosynthetic capacity of the HT-58-2 cyanobacterium beyond the production of tolyporphins and tolypodiols.

60 APPLIED LIFE SCIENCES↗

A microbiological survey of handwashing sinks in the hospital built environment reveals differences in patient room and healthcare personnel sinks

Handwashing sinks and their associated premise plumbing are an ideal environment for pathogen-harboring biofilms to grow and spread throughout facilities due to the connected system of wastewater plumbing. This study was designed to understand the distribution of pathogens and antibiotic resistant organisms (ARO) within and among handwashing sinks in healthcare settings, using culture-dependent methods to quantify Pseudomonas aeruginosa, opportunistic pathogens capable of growth on a cefotaxime-containing medium (OPP-C), and carbapenem-resistant Enterobacteriaceae (CRE). Isolates from each medium identified as P. aeruginosa or Enterobacteriaceae were tested for susceptibility to aztreonam, ceftazidime, and meropenem; Enterobacteriaceae were also tested against ertapenem and cefotaxime. Isolates exhibiting resistance or intermediate resistance were designated ARO. Pathogens were quantified at different locations within handwashing sinks and compared in quantity and distribution between healthcare personnel (HCP) and patient room (PR) sinks. ARO were compared between samples within a sink (biofilm vs planktonic samples) and between sink types (HCP vs. PR). The drain cover was identified as a reservoir within multiple sinks that was often colonized by pathogens despite daily sink cleaning. P. aeruginosa and OPP-C mean log 10 CFU/cm 2 counts were higher in p-trap and tail pipe biofilm samples from HCP compared to PR sinks (2.77 ± 2.39 vs. 1.23 ± 1.62 and 5.27 ± 1.10 vs. 4.74 ± 1.06) for P. aeruginosa and OPP-C, respectively. P. aeruginosa and OPP-C mean log 10 CFU/ml counts were also higher (p < 0.05) in HCP compared to PR sinks p-trap water (2.21 ± 1.52 vs. 0.89 ± 1.44 and 3.87 ± 0.78 vs. 3.21 ± 1.11) for P. aeruginosa and OPP-C, respectively. However, a greater percentage of ARO were recovered from PR sinks compared to HCP sinks (p < 0.05) for Enterobacteriaceae (76.4 vs. 32.9%) and P. aeruginosa (25.6 vs. 0.3%). This study supports previous work citing that handwashing sinks are reservoirs for pathogens and ARO and identifies differences in pathogen and ARO quantities between HCP and PR sinks, despite the interconnected premise plumbing.

60 APPLIED LIFE SCIENCES↗

Neutralization profiles of HIV-1 viruses from the VRC01 Antibody Mediated Prevention (AMP) trials

The VRC01 Antibody Mediated Prevention (AMP) efficacy trials conducted between 2016 and 2020 showed for the first time that passively administered broadly neutralizing antibodies (bnAbs) could prevent HIV-1 acquisition against bnAb-sensitive viruses. HIV-1 viruses isolated from AMP participants who acquired infection during the study in the sub-Saharan African (HVTN 703/HPTN 081) and the Americas/European (HVTN 704/HPTN 085) trials represent a panel of currently circulating strains of HIV-1 and offer a unique opportunity to investigate the sensitivity of the virus to broadly neutralizing antibodies (bnAbs) being considered for clinical development. Pseudoviruses were constructed using envelope sequences from 218 individuals. The majority of viruses identified were clade B and C; with clades A, D, F and G and recombinants AC and BF detected at lower frequencies. We tested eight bnAbs in clinical development (VRC01, VRC07-523LS, 3BNC117, CAP256.25, PGDM1400, PGT121, 10–1074 and 10E8v4) for neutralization against all AMP placebo viruses (n = 76). Compared to older clade C viruses (1998–2010), the HVTN703/HPTN081 clade C viruses showed increased resistance to VRC07-523LS and CAP256.25. At a concentration of 1μg/ml (IC80), predictive modeling identified the triple combination of V3/V2-glycan/CD4bs-targeting bnAbs (10-1074/PGDM1400/VRC07-523LS) as the best against clade C viruses and a combination of MPER/V3/CD4bs-targeting bnAbs (10E8v4/10-1074/VRC07-523LS) as the best against clade B viruses, due to low coverage of V2-glycan directed bnAbs against clade B viruses. Overall, the AMP placebo viruses represent a valuable resource for defining the sensitivity of contemporaneous circulating viral strains to bnAbs and highlight the need to update reference panels regularly. Our data also suggests that combining bnAbs in passive immunization trials would improve coverage of global viruses.

60 APPLIED LIFE SCIENCES↗

A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine

African Swine Fever Virus (ASFV) is the causative agent of a highly contagious and lethal vector-borne disease in suids. Recently, a live attenuated virus strain, developed using the currently circulating, virulent Georgia strain (ASFV-G) with a single gene deletion (ASFV-G-ΔI177L), resulted in an effective vaccine. Nevertheless, protective immune response mechanisms induced by this candidate are poorly understood. In this study, Yorkshire crossbred swine intramuscularly vaccinated with 10 6 50% hemadsorption dose (HAD 50 ) of ASFV-G-ΔI177L or a vehicle control were challenged at 28 days post-inoculation (dpi) with 10 2 HAD 50 of ASFV-G. Analysis of purified peripheral blood mononuclear cells following inoculation and challenge revealed that CD4+, CD8+ and CD4+CD8+ central memory T cells (CD44+CD25–CD27–CD62L+CCR7+, T cm ) decreased significantly by 28 dpi in ASFV-G-ΔI177L-vaccinated swine compared to baseline and time-matched controls. Conversely, CD4+, CD8+ and CD4+CD8+ effector memory T cells (CD44+CD25–CD27–CD62–CCR7–, T em ) increased significantly among ASFV-G-ΔI177L-vaccined swine by 28 dpi compared to baseline and time-matched controls. Additionally, the percentage of natural killer (NK), CD4+ and CD4+CD8+ T em and CD8+ T cm and T em positive for IFNγ increased significantly following inoculation, surpassing that of controls by 28 dpi or earlier. These results suggest that NK and memory T cells play a role in prote

60 APPLIED LIFE SCIENCES↗

A simple model of COVID-19 explains disease severity and the effect of treatments

Abstract Considerable effort has been made to better understand why some people suffer from severe COVID-19 while others remain asymptomatic. This has led to important clinical findings; people with severe COVID-19 generally experience persistently high levels of inflammation, slower viral load decay, display a dysregulated type-I interferon response, have less active natural killer cells and increased levels of neutrophil extracellular traps. How these findings are connected to the pathogenesis of COVID-19 remains unclear. We propose a mathematical model that sheds light on this issue by focusing on cells that trigger inflammation through molecular patterns: infected cells carrying pathogen-associated molecular patterns (PAMPs) and damaged cells producing damage-associated molecular patterns (DAMPs). The former signals the presence of pathogens while the latter signals danger such as hypoxia or lack of nutrients. Analyses show that SARS-CoV-2 infections can lead to a self-perpetuating feedback loop between DAMP expressing cells and inflammation, identifying the inability to quickly clear PAMPs and DAMPs as the main contributor to hyperinflammation. The model explains clinical findings and reveal conditions that can increase the likelihood of desired clinical outcome from treatment administration. In particular, the analysis suggest that antivirals need to be administered early during infection to have an impact on disease severity. The simplicity of the model and its high level of consistency with clinical findings motivate its use for the formulation of new treatment strategies.

60 APPLIED LIFE SCIENCES↗

Unlocking soybean meal pectin recalcitrance using a multi-enzyme cocktail approach

Pectin is a complex plant heteropolysaccharide whose structure and function differ depending on its source. In animal feed, breaking down pectin is essential, as its presence increases feed viscosity and reduces nutrient absorption. Soybean meal, a protein-rich poultry feed ingredient, contains significant amounts of pectin, the structure of which remains unclear. Consequently, the enzyme activities required to degrade soybean meal pectin and how they interact are still open questions. In this study, we produced 15 recombinant fungal carbohydrate-active enzymes (CAZymes) identified from fungal secretomes acting on pectin. After observing that these enzymes were not active on soybean meal pectin when used alone, we developed a semi-miniaturized method to evaluate their effect as multi-activity cocktails. We designed and tested 12 enzyme pools, containing up to 15 different CAZymes, using several hydrolysis markers. Thanks to our multiactivity enzymatic approach combined with a Pearson correlation matrix, we identified 10 fungal CAZymes efficient on soybean meal pectin, 9 of which originate from Talaromyces versatilis. Based on enzyme specificity and linkage analysis, we propose a structural model for soybean meal pectin. Our findings underscore the importance of combining CAZymes to improve the degradation of agricultural co-products.

60 APPLIED LIFE SCIENCES↗

Modeling the emergence of viral resistance for SARS-CoV-2 during treatment with an anti-spike monoclonal antibody

To mitigate the loss of lives during the COVID-19 pandemic, emergency use authorization was given to several anti-SARS-CoV-2 monoclonal antibody (mAb) therapies for the treatment of mild-to-moderate COVID-19 in patients with a high risk of progressing to severe disease. Monoclonal antibodies used to treat SARS-CoV-2 target the spike protein of the virus and block its ability to enter and infect target cells. Monoclonal antibody therapy can thus accelerate the decline in viral load and lower hospitalization rates among high-risk patients with variants susceptible to mAb therapy. However, viral resistance has been observed, in some cases leading to a transient viral rebound that can be as large as 3–4 orders of magnitude. As mAbs represent a proven treatment choice for SARS-CoV-2 and other viral infections, evaluation of treatment-emergent mAb resistance can help uncover underlying pathobiology of SARS-CoV-2 infection and may also help in the development of the next generation of mAb therapies. Although resistance can be expected, the large rebounds observed are much more difficult to explain. We hypothesize replenishment of target cells is necessary to generate the high transient viral rebound. Thus, we formulated two models with different mechanisms for target cell replenishment (homeostatic proliferation and return from an innate immune response antiviral state) and fit them to data from persons with SARS-CoV-2 treated with a mAb. We showed that both models can explain the emergence of resistant virus associated with high transient viral rebounds. We found that variations in the target cell supply rate and adaptive immunity parameters have a strong impact on the magnitude or observability of the viral rebound associated with the emergence of resistant virus. Both variations in target cell supply rate and adaptive immunity parameters may explain why only some individuals develop observable transient resistant viral rebound. Our study highlights the conditions that can lead to resistance and subsequent viral rebound in mAb treatments during acute infection.

60 APPLIED LIFE SCIENCES↗

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress–budding at the inner nuclear membrane–is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

60 APPLIED LIFE SCIENCES↗

A novel candidate hepatitis C virus genotype 4 subtype identified by next generation sequencing full-genome characterization in a patient from Saudi Arabia

Background and aim: Hepatitis C virus (HCV) infection is a major global public health concern, being a leading cause of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The virus is classified into 8 genotypes and 93 subtypes, each displaying distinct geographic distributions. Genotype 4 is the most predominant in the Middle East and Eastern Mediterranean and is associated with high rates of hepatitis C infection worldwide. This study used next-generation sequencing to fully characterize the HCV genome and identify a novel subtype within genotype 4 isolated from a 64-year-old Saudi man diagnosed with hepatitis C. Methods: We analyzed the complete genome of the 141-HCV isolate using whole-genome sequencing. Results: Our phylogenetic reconstructions, based on the entire genome of HCV-4 strains, revealed that the 141-HCV isolate formed a distinct group within the genotype 4 classification, providing valuable new insights into the variability of HCV. Conclusion: This discovery of a previously unclassified HCV subtype within genotype 4 sheds light on the ongoing evolution and diversity of the virus. Such knowledge has significant implications for diagnostic and therapeutic approaches, as different subtypes may exhibit varying drug sensitivities and resistance profiles.

60 APPLIED LIFE SCIENCES↗

Torsional twist of the SARS ‐ CoV and SARS ‐ CoV ‐2 SUD ‐N and SUD ‐M domains

Abstract Coronavirus non‐structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication–transcription complex. Nsp3 in SARS‐like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD‐N (Macrodomain 2) and SUD‐M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x‐ray structure of SARS‐CoV SUD‐N/M as well as a mutant variant of SARS‐CoV‐2 SUD‐N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD‐N and SUD‐M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.

Rosas‐Lemus, Monica [Department of Microbiology‐Im↗

Microbial Influence on the Mobility of +3 Actinides from a Salt-Based Nuclear Waste Repository

Biologically enhanced transport of radionuclides is one of several processes that can affect the performance of a nuclear waste repository. In this work, several microbial isolates from the Waste Isolation Pilot Plant (WIPP) were tested for their influence on the concentration of neodymium, as an analog for +3 actinides, in simple sodium chloride solutions and in anoxic WIPP brines. Batch sorption experiments were carried out over a period of 4–5 weeks. In many cases, the effect on neodymium in solution was immediate and extensive and assumed to be due to surface complexation. However, over time, the continued loss of Nd from the solution was more likely due to biologically induced precipitation and/or mineralization and possible entrapment in extracellular polymeric substances. The results showed no correlation between organism type and the extent of its influence on neodymium in solution. However, a correlation was observed between different test matrices (simple NaCl versus high-magnesium brine versus high-NaCl brine). Further experiments were conducted to test these matrix effects, and the results showed a significant effect of magnesium concentration on the ability of microorganisms to remove Nd from solution. Possible mechanisms include cation competition and the alteration of cell surface structures. This suggests that the aqueous chemistry of the WIPP environs could play a larger role in the final disposition of +3 actinides than the microbiology.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Machine learning-led semi-automated medium optimization reveals salt as key for flaviolin production in Pseudomonas putida

Although synthetic biology can produce valuable chemicals in a renewable manner, its progress is still hindered by a lack of predictive capabilities. Media optimization is a critical, and often overlooked, process which is essential to obtain the titers, rates and yields needed for commercial viability. Here, we present a molecule- and host-agnostic active learning process for media optimization that is enabled by a fast and highly repeatable semi-automated pipeline. Its application yielded 60% and 70% increases in titer, and 350% increase in process yield in three different campaigns for flaviolin production in Pseudomonas putida KT2440. Explainable Artificial Intelligence techniques pinpointed that, surprisingly, common salt (NaCl) is the most important component influencing production. The optimal salt concentration is very high, comparable to seawater and close to the limits that P. putida can tolerate. The availability of fast Design-Build-Test-Learn (DBTL) cycles allowed us to show that performance improvements for active learning are rarely monotonous. This work illustrates how machine learning and automation can change the paradigm of current synthetic biology research to make it more effective and informative, and suggests a cost-effective and underexploited strategy to facilitate the high titers, rates and yields essential for commercial viability.

59 BASIC BIOLOGICAL SCIENCES↗

Protocol to detect dilution cycles in chemostat experiments and estimate growth rate slopes with linear modeling with R software chemostat_regression

Chemostat growth chambers measure optical density over time and require manual calculation of growth rates. Here, we present chemostat_regression, R software that enables users to automatically identify chemostat cycles and estimate growth rate using a linear regression approach. We describe steps for creating requisite software environment(s), formatting input data, executing the software via command line/RStudio/R-Shiny, interpreting results, assessing the validity of results, and modifying input parameters.

59 BASIC BIOLOGICAL SCIENCES↗

High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope

A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages.

60 APPLIED LIFE SCIENCES↗

Structure and antigenicity of the divergent human astrovirus VA1 capsid spike

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

60 APPLIED LIFE SCIENCES↗

Understanding early HIV-1 rebound dynamics following antiretroviral therapy interruption: The importance of effector cell expansion

Most people living with HIV-1 experience rapid viral rebound once antiretroviral therapy is interrupted; however, a small fraction remain in viral remission for an extended duration. Understanding the factors that determine whether viral rebound is likely after treatment interruption can enable the development of optimal treatment regimens and therapeutic interventions to potentially achieve a functional cure for HIV-1. We built upon the theoretical framework proposed by Conway and Perelson to construct dynamic models of virus-immune interactions to study factors that influence viral rebound dynamics. We evaluated these models using viral load data from 24 individuals following antiretroviral therapy interruption. The best-performing model accurately captures the heterogeneity of viral dynamics and highlights the importance of the effector cell expansion rate. Our results show that post-treatment controllers and non-controllers can be distinguished based on the effector cell expansion rate in our models. Furthermore, these results demonstrate the potential of using dynamic models incorporating an effector cell response to understand early viral rebound dynamics post-antiretroviral therapy interruption.

60 APPLIED LIFE SCIENCES↗

Evaluation of Inactivation Methods for Rift Valley Fever Virus in Mouse Microglia

Rift Valley fever phlebovirus (RVFV) is a highly pathogenic mosquito-borne virus with bioweapon potential due to its ability to be spread by aerosol transmission. Neurological symptoms are among the worst outcomes of infection, and understanding of pathogenesis mechanisms within the brain is limited. RVFV is classified as an overlap select agent by the CDC and USDA; therefore, experiments involving fully virulent strains of virus are tightly regulated. Here, we present two methods for inactivation of live virus within samples derived from mouse microglia cells using commercially available kits for the preparation of cells for flow cytometry and RNA extraction. Using the flow cytometry protocol, we demonstrate key differences in the response of primary murine microglia to infection with fully virulent versus attenuated RVFV.

60 APPLIED LIFE SCIENCES↗

Growth and inactivation of Listeria monocytogenes in sterile extracts of fruits and vegetables: Impact of the intrinsic factors pH, sugar and organic acid content

Intrinsic characteristics of fresh produce, such as pH, water activity, acid content and nutrient availability are critical factors in determining the survival and growth of Listeria monocytogenes (Lm). In this study, sterile fresh produce juice was used to analyze Lm growth potential among 14 different commodities and to identify physicochemical characteristics in those juices that affect Lm growth. Significant growth of Lm was observed in juices with pH ≥5.6 and low acidity (0.04–0.07 % titratable acidity (TA)) (cantaloupe, carrot, celery, green pepper, parsley, and romaine lettuce), slight reduction of Lm was observed in juices with pH 4.1 (tomato) and pH 3.9 (mango), and no Lm counts were recovered from juices with pH ≤3.8 and high acidity (0.28–1.17 % TA) (apple, blueberry, grape, peach, and pineapple). Although these acidic fruit juices possessed a high sugar content, the pH and acidity of produce juice seemed to be the primary determinants for Lm growth. The neutralization of acidic juices (i.e., Fuji and Gala apple, blueberry, grape, mango, pineapple, peach, and tomato) enabled Lm growth at 37 °C in all juices except for Gala apple and peach. Strong decline in Lm populations in Gala apple, grape and peach juices might be linked to sensitivity to organic acids, such as malic acid. As a result, Lm populations significantly decreased in pH-neutral (7.6) cauliflower juice, suggesting that potential antilisterial substances may play a role in Lm decline in cauliflower juice.

60 APPLIED LIFE SCIENCES↗