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At least 1,423 records · Page 79

Identifying Genomic Islands with Deep Neural Networks

Background Horizontal gene transfer is the main source of adaptability for bacteria, through which genes are obtained from different sources including bacteria, archaea, viruses, and eukaryotes. This process promotes the rapid spread of genetic information across lineages, typically in the form of clusters of genes referred to as genomic islands (GIs). Different types of GIs exist, and are often classified by the content of their cargo genes or their means of integration and mobility. While various computational methods have been devised to detect different types of GIs, no single method is capable of detecting all types. Results We propose a method, which we call Shutter Island, that uses a deep learning model (Inception V3, widely used in computer vision) to detect genomic islands. The intrinsic value of deep learning methods lies in their ability to generalize. Via a technique called transfer learning, the model is pre-trained on a large generic dataset and then re-trained on images that we generate to represent genomic fragments. We demonstrate that this image-based approach generalizes better than the existing tools. Conclusions We used a deep neural network and an image-based approach to detect the most out of the correct GI predictions made by other tools, in addition to making novel GI predictions. The fact that the deep neural network was re-trained on only a limited number of GI datasets and then successfully generalized indicates that this approach could be applied to other problems in the field where data is still lacking or hard to curate.

Computer Vision↗

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015

Background: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. Methods: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual’s RDT results. Dose–response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. Results: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). Conclusions: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

60 APPLIED LIFE SCIENCES↗

BMC Caller: a webtool to identify and analyze bacterial microcompartment types in sequence data

Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a “do-it yourself” analysis for the public we have devised a webserver, BMC Caller (https://bmc-caller.prl.msu.edu), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism’s metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

59 BASIC BIOLOGICAL SCIENCES↗

A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

Abstract Background Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO 2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions.

09 BIOMASS FUELS↗

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13 C), even though the soils’ overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera . Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

Background - Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. Results - We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. Conclusions - Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.

59 BASIC BIOLOGICAL SCIENCES↗

Identifying a forward-scattering superconductor through pump-probe spectroscopy

Electron-boson scattering that is peaked in the forward direction has been suggested as an essential ingredient for enhanced superconductivity observed in FeSe monolayers. Here, we study the superconducting state of a system dominated by forward scattering in the time domain and contrast its behavior against the standard isotropic BCS case for both s- and d-wave symmetries. An analysis of the electron's dynamics in the pump-driven non-equilibrium state reveals that the superconducting order in the forward-focused case is robust and persistent against the pump-induced perturbations. The superconducting order parameter also exhibits a non-uniform melting in momentum space. We show that this behavior is in sharp contrast to the isotropic interaction case and propose that time-resolved approaches are a potentially powerful tool to differentiate the nature of the dominant coupling in correlated materials.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

The Value of {sup 99m}Tc-PSMA SPECT/CT-Guided Surgery for Identifying and Locating Lymph Node Metastasis in Prostate Cancer Patients

Background: This study evaluated the effect of technetium-99m ({sup 99m}Tc)-labeled prostate-specific membrane antigen (PSMA)-based image-guided surgery on the oncologic outcomes for patients with primary or recurrent prostate cancer (PCa). Methods: This study retrospectively analyzed 54 consecutive patients with PCa who underwent {sup 99m}Tc-labeled PSMA-based image-guided surgery between January 2016 and September 2017. These patients received a radical prostatectomy (RP) with pelvic lymph node dissection (PLND) or salvage lymph node dissection (sLND). The resected specimens were compared with findings of postoperative histologic analysis. The responses to the treatment were recorded during the follow-up period. Results: In 31 patients, PSMA single-photon emission computerized tomography (SPECT) and computed tomography (CT) could find 52 suspicious lymph node metastases (LNMs). With the help of PSMA SPECT/CT, 12 patients with recurrence received sLND, 19 primary PCa patients received RP with extended PLND, and 23 primary PCa patients received RP with standard PLND. The findings showed that PSMA SPECT/CT could detect LNMs with high sensitivity and specificity. In six patients, PSMA SPECT/CT could find more LNMs that were not found by MRI and help to modify the extent of lymphadenectomy. At the latest follow-up evaluation, 39 patients showed a biochemical response (BR), 9 patients showed a biochemical recurrence (BCR) after BR, and 6 patients never exhibited BR. The patients who received RP with standard PLND or extended PLND had a better prostate-specific antigen (PSA) response than the patients who received sLND. The patients with pelvic LNMs also had a better PSA response than the patients with retroperitoneal LNMs. Conclusions: This study showed that {sup 99m}Tc-PSMA SPECT/CT-guided surgery can remove more LNMs than conventional imaging with high sensitivity and specificity and delay disease progression in PCa patients.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Data for Identifying the best high-biomass sorghum hybrids based on biomass yield potential and feedstock quality affected by nitrogen fertility management under various environments

Data were collected from agronomy fields in Urbana and Ewing, IL, during the 2022 and 2023 growing seasons. The dataset includes dry biomass yield, nitrogen, phosphorus, and potassium concentrations and removals, and chemical composition elements (cellulose, hemicellulose, lignin, and soluble fractions) for 13 high-biomass sorghum hybrids. data_sharing.xlsx contains 20 columns and 104 rows. Below is the explanation of all variables in the file: Year: 2022; 2023 Location: Urbana, IL; Ewing, IL N rate (kg-N/ha): 0; 112 Hybrid #: H1-H13 Pedigree: Pedigree for 13 hybrids Dry biomass yield (Mg/ha): Aboveground dry biomass yield N (g/kg): Nitrogen concentration in plant tissue P (g/kg): Phosphorus concentration in plant tissue K (g/kg): Potassium concentration in plant tissue N (kg/ha): Nitrogen removal by aboveground biomass P (kg/ha): Phosphorus removal by aboveground biomass K (kg/ha): Potassium removal by aboveground biomass Cellulose (g/kg): Cellulose concentration in plant tissue Hemicellulose (g/kg): Hemicellulose concentration in plant tissue Lignin (g/kg): Lignin concentration in plant tissue Soluble (g/kg): Soluble concentration in plant tissue Cellulose (Mg/ha): Cellulose content in aboveground biomass Hemicellulose (Mg/ha): Hemicellulose content in aboveground biomass Lignin (Mg/ha): Lignin content in aboveground biomass Soluble (Mg/ha): Soluble content in aboveground biomass

environmental adaptability↗

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called “replication factories”), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.

59 BASIC BIOLOGICAL SCIENCES↗

Machine learning identifies novel signatures of antifungal drug resistance in Saccharomycotina yeasts

Antifungal drug resistance is a major challenge in fungal infection management. Numerous genomic changes are known to contribute to acquired drug resistance in clinical isolates of specific pathogens, but whether they broadly explain natural resistance across entire lineages is unknown. We leveraged genomic, ecological, and phenotypic trait data from naturally sampled strains from nearly all known species in subphylum Saccharomycotina to examine the evolution of resistance to eight antifungal drugs. The phylogenetic distribution of drug resistance varied by drug; fluconazole resistance was widespread, while 5-fluorocytosine resistance was rare, except in Lipomycetales. A random forest algorithm trained on genomic data predicted drug-resistant yeasts with 54–75% accuracy. Fluconazole resistance was consistently predicted with the highest accuracy (75.2%). Furthermore, fluconazole resistance prediction accuracy was similar between models trained on genome-wide variation in the presence and number of InterPro protein annotations across Saccharomycotina (75.2%) and those trained on amino acid sequence alignment data of Erg11, a protein known to be involved in fluconazole resistance (74.3-74.9%). Interestingly, the top Erg11 residues for predicting fluconazole resistance across Saccharomycotina do not overlap with, are not spatially close to, and are less conserved than those previously linked to resistance in clinical isolates of Candida albicans. In silico deep mutational scanning of the C. albicans Erg11 protein reveals that amino acid variants implicated in clinical cases of resistance are almost universally destabilizing while variants in our most informative residues are energetically more neutral, explaining why the latter are much more common than the former in natural populations. Importantly, previous experimental analyses of C. albicans Erg11 have shown that amino acid variation in our most informative residues, despite having never been directly implicated in clinical cases, can directly contribute to resistance. Our results suggest that studies of natural resistance in yeast species never encountered in the clinic will yield a fuller understanding of antifungal drug resistance.

Harrison, Marie-Claire [Vanderbilt Univ., Nashvill↗

Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples.

59 BASIC BIOLOGICAL SCIENCES↗

Gene network analysis of poplar root transcriptome in response to drought stress identifies a PtaJAZ3PtaRAP2.6-centered hierarchical network

Using time-series transcriptomic data from poplar roots undergoing polyethylene glycol (PEG)-induced drought stress, we built a genetic network model of the involved putative molecular responses. We found that the network resembled a hierarchical structure. The highest hierarchical level in this structure is occupied by 9 genes, which we called superhubs because they were primarily connected to 18 hub genes, which are then connected to 2,934 terminal genes. We were only able to regenerate transgenic plants overexpressing two of the superhubs, suggesting that the majority of the superhubs might interfere with the regeneration process and did not allow recovery of transgenic plants. The two superhubs encode proteins with closest homology to JAZ3 and RAP2.6 genes of Arabidopsis and were consequently named PtaJAZ3 and PtaRAP2.6. PtaJAZ3 and PtaRAP2.6 overexpressing transgenic lines showed a significant increase in both root elongation and lateral root proliferation and these responses were specific for the drought stress conditions and were highly correlated with the levels of overexpression of the transgenes. Several lines of evidence suggest of regulatory interactions between the two superhubs. Both superhubs were significantly induced by methyl jasmonate (MeJA). Because jasmonate signaling involves ubiquitin-mediated proteasome degradation, treatment with proteasome inhibitor abolished the MeJA induction for both genes. PtaRAP2.6 was upregulated in PtaJAZ3 transgenics but PtaJAZ3 expression was not affected in the PtaRAP2.6 overexpressors. The discovery of the two genes and further future insights into the associated mechanisms can lead to improved understanding and novel approaches to regulate root architecture in relation to drought stress.

59 BASIC BIOLOGICAL SCIENCES↗