Viable Organisms Research Articles

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus.

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yield viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Viability of Phytoplankton, Zooplankton, and Aquatic Macroinvertebrates in Dredged River Sediments of the Upper Mississippi River

ABSTRACTSediment dredging is widely used to manage freshwater ecosystems, including maintaining river navigation corridors. Beneficial uses of the sediments are needed to reduce demands on new and existing stockpile sites. However, using dredged sediments in waterbodies outside the sediment source poses an ecological risk if aquatic organisms remain viable through desiccation‐resistant forms. We assessed viability of aquatic organisms in dredged river sediments from four locations along the Upper Mississippi River. We artificially re‐inundated sediments that had been dry for < 1, 1–5, and 5–10 years and sampled phytoplankton, zooplankton, and macroinvertebrate for 4 weeks. We hypothesized that richness and density of aquatic organisms would be negatively affected by duration of drying. Although individual samples of the most recently dredged material had the greatest richness and density of phytoplankton, zooplankton, and macroinvertebrates, mean richness and density was not different across sediment drying durations. Overall, richness and density of aquatic organisms was low in relation to in situ sampling especially following the first week of sediment re‐inundation. Our results suggest transporting sediments from main channel navigation dredging from the Upper Mississippi River to support beneficial uses in other waterbodies poses some ecological risk given our observations of viable organisms in dredged sediments. We present a multistep risk evaluation procedure that could be applied when considering beneficial uses for dredged sediments.

Characterization of a Mycoplasma hyopneumoniae aerosol infection model in pigs

The purpose of the present study was to develop and characterize an experimental aerosol model for Mycoplasma hyopneumoniae (M. hyopneumoniae) infection and respiratory disease in pigs. The experiment was carried out to determine the pathogenicity, colonization, mucosal immune response, and clinical course of disease of dose-controlled aerosols of M. hyopneumoniae. Four groups of three M. hyopneumoniae-free gilts each were individually exposed to aerosols of diluted lung homogenate containing M. hyopneumoniae strain 232 in a chamber. Each group was exposed to different doses of viable organisms (105 to 106 color changing units/mL during 15-20 or 30-35min in two consecutive days). Nasal, laryngeal, and deep-tracheal secretions were collected from each gilt at 0, 7, 14, 21, and 28 days post-exposure (dpe). Blood samples were collected at 0 and 28 dpe. At necropsy, lung lesions were assessed, and bronchial secretions and bronchoalveolar lavage fluid (BALF) were collected from each lung set. Blood was used to assess seroconversion by means of an indirect ELISA, while BALF, deep-tracheal and nasal secretions were tested by modifying the ELISA to evaluate mucosal IgG and IgA production. Nasal, laryngeal, deep-tracheal, and bronchial secretions were tested by real-time PCR to evaluate bacterial load. Gilts became infected irrespective of the infectious dose, as determined by M. hyopneumoniae detection in deep-tracheal secretions from all gilts at 7 dpe. A specific local humoral immune response starting at 14 dpe was detected in all gilts. While all experimental groups presented gilts with some extent of mycoplasmal pneumonia, only the exposure of gilts to high-dose aerosols consistently reproduced typical clinical signs and severe lung lesions. These results showed that the reproduction of mycoplasmal pneumonia by means of infectious aerosols can be successfully achieved at experimental level, making this model a valuable alternative to evaluate preventive and treatment measures against M. hyopneumoniae.

Essential Genes Discovery in Microorganisms by Transposon-Directed Sequencing (Tn-Seq): Experimental Approaches, Major Goals, and Future Perspectives.

Essential genes are crucial for microbial viability, playing key roles in both the primary and secondary metabolism. Since mutations in these genes can threaten organism viability, identifying them is challenging. Conditionally essential genes are required only under specific conditions and are important for functions such as virulence, immunity, stress survival, and antibiotic resistance. Transposon-directed sequencing (Tn-Seq) has emerged as a powerful method for identifying both essential and conditionally essential genes. In this review, we explored Tn-Seq workflows, focusing on eubacterial species and some yeast species. A comparison of 14 eubacteria species revealed 133 conserved essential genes, including those involved in cell division (e.g., ftsA, ftsZ), DNA replication (e.g., dnaA, dnaE), ribosomal function, cell wall synthesis (e.g., murB, murC), and amino acid synthesis (e.g., alaS, argS). Many other essential genes lack clear orthologues across different microorganisms, making them specific to each organism studied. Conditionally essential genes were identified in 18 bacterial species grown under various conditions, but their conservation was low, reflecting dependence on specific environments and microorganisms. Advances in Tn-Seq are expected to reveal more essential genes in the near future, deepening our understanding of microbial biology and enhancing our ability to manipulate microbial growth, as well as both the primary and secondary metabolism.

A-277 Viability of multi-drug resistant organisms in a fecal collection and transport system stored at ultra-low temperature

Abstract Background Epidemiological studies are constantly investigating new, easier ways to collect and store patient specimens that allow for flexible testing windows and long-term storage as well as accurate quantification of organisms of interest. The FecalSwabTM system (Copan, Brescia, Italy) is intended for the collection and preservation of rectal and fecal specimens. This system includes a preservation medium which serves to maintain the viability of enteric pathogens during transport to a clinical laboratory for up to 72 hours at 4oC. The objective of this study was to assess the continued viability of multi-drug resistant organisms (MDROs) suspended in the FecalSwab sample format when frozen at -80°C and the impact of freeze-thaw cycles. Methods A total of 40 organisms were chosen spanning methicillin-resistant Staphylococcus aureus (MRSA, n=10), carbapenem-resistant Pseudomonas aeruginosa (CRPA, n=5), carbapenem-resistant Enterobacterales (CRE, n=5), vancomycin-resistant Enterobacterales (VRE, n=10) and extended-spectrum beta-lactamase containing Enterobacterales (ESBL, n=10). Each organism was spiked into FecalSwab at standardized concentrations. At each time point, FecalSwab media was plated onto blood agar and colonies were quantified. Each of the 40 organisms were assessed one-week post-freeze and over the course of freeze-thaw cycles at days 3 and 5. The survival of each MDRO was evaluated by calculating a difference between the concentration of organisms recovered (CFU per 10uL media) at each time point and the concentration of organisms confirmed present at the time of inoculation. Percent recovery was calculated from this difference. Results One week after freezing, the percent recovery for MRSA, VRE, ESBL, CRE, and CRPA averaged to 102%, 201%, 17.8%, 7.6%, and 0.8%, respectively (Kruskal Wallis p < 0.001), with significant differences noted between gram positive and gram negative organisms (Kruskal Wallis p <0.001). Conclusions Epidemiological studies interested in MDRO surveillance rely on methodologies that allow for accurate quantification of viable organisms. In this study, we’ve evaluated the viability of several multi-drug resistant organisms when frozen at -80°C in the FecalSwab. The FecalSwab system is intended for the collection of rectal swab and fecal specimens and to preserve the viability of enteric pathogenic bacteria for testing in the clinical laboratory. Although it was not specifically designed to preserve MDROs, it performed well with MRSA and VRE, but less well with CRE, CRPA and ESBL organisms. When using the FecalSwab system, the observed gram-specific differences may inform the suitability of this system depending on the organism(s) of interest.

A new dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms on central venous catheters.

Central venous catheters (CVCs) are widely used for intravenous medication administration. However, biofilm formation along the catheter surface is the main most important cause of catheter-related bloodstream infections. Nowadays, several antimicrobial-coated catheters are available to prevent biofilm development. In this study, we introduced a new dynamic in vitro model to evaluate the antimicrobial activity against bacterial biofilms on CVCs. Rifampicin-minocycline-coated catheters and control catheters without antimicrobial component were assembled into the model to test the antimicrobial activity on external surface and internal surface. After 1 h irrigation of Staphylococcus epidermidis or Staphylococcus aureus preculture and 23 h irrigation of Trypticase Soy Broth, the viable adherent organism was collected and counted. The enumeration results showed that the number of bacteria attached to antibacterial catheter was significantly less than that of the control catheter, both on external surface (P < 0.05) and internal surface (P < 0.05). The results were further confirmed by the scanning electron microscopy. In conclusion, the dynamic in vitro model can be applied to evaluate the antimicrobial activity against bacterial biofilms grown on the external and internal surfaces of CVCs used in clinical practice.IMPORTANCEFor the first time, a new dynamic in vitro model was constructed to evaluate the antimicrobial activity against bacterial biofilms on central venous catheters (CVCs) on both external surface and internal surface. This model could be applied to evaluate the antimicrobial activity against bacterial biofilms not only on CVCs but also other types of catheters.

Hydrochar Utilization for Saline‐Alkali Soil Amelioration and Its Carbon Sequestration Potential Assessment

AbstractFor the current situation of saline‐alkali soil amelioration, it is urgent to explore a multi‐objective amelioration strategy involving crop income increase and environmental benefits. This study used pine‐needle hydrochar to ameliorate saline‐alkali soil and conducted column and pot experiments to investigate its effects on soil properties and crop growth. And the environmental advantages of hydrochar are evaluated through Life Cycle Assessment (LCA). The electrical conductivity, exchangeable sodium percentage, and pH of saline‐alkali soil ameliorated with hydrochar using the column elution method are reduced by 60%, 58%, and 1.2 pH units, respectively, compared to the original soil. Also, the gene copy number of the ameliorated soil has doubled according to qPCR determination. Pot experiment results show that the root length, fresh weight, and germination rate of wheatgrass are increased by 107, 75, and 20%, respectively. These results demonstrated that the exchange of Na+ with H+ released from hydrochar reduced the soil alkalinity and the viability of organisms is enhanced. Moreover, based on this study's data including the hydrochar dosage and wheatgrass yield, the LCA results showed 3.7 × 109 t CO2e carbon sequestration potential and significant environmental benefits.

Colorimetric aptasensor based on self-screened aptamers and cascaded catalytic reaction for the detection of quarantine plant bacteria

Quarantine plant bacteria (QPB) are significant component of invasive alien species that result in substantial economic losses and serious environmental damage. Herein, a colorimetric aptasensor has been proposed based on the sandwich structure and the cascaded catalytic strategy for on-site detecting Xanthomonas hyacinthi, a type of QPB, in natural environments. The self-screened aptamer obtained through SELEX can bind to specific sites on the surface of viable organism with high affinity and specificity, which guarantees the selectivity of aptasensor. As an important part of the aptasensor, MIL-88-NH2(Fe) not only acts as a multifunctional carrier for both aptamers and glucose oxidase, but also catalyzes enzyme-like reaction because of specific surface area, amino and peroxidase-like activity. The present of Xanthomonas hyacinthi can trigger the formation of a sandwich structure and the occurrence of cascade catalytic reaction, enabling the detection with UV–Vis spectra and naked eyes. The proposed aptasensor presents a low detection limit of 2 cfu/mL and a wide linear range of 10 -107 cfu/mL. Compared to traditional detection methods for QPB, the reasonable design, high selectivity and convenience significantly improve the detection efficiency and contribute to environmental protection.

Ecotoxicity Assessment of α-Amino Acid-Derived Polyamidoamines Using Zebrafish as a Vertebrate Model.

The aquatic ecotoxicity of three α-amino acid-derived polyamidoamines (PAAs) was studied using zebrafish embryos as a viable vertebrate model organism. The PAAs examined were water-soluble amphoteric polyelectrolytes with a primarily negative charge, which were efficient flame retardants for cotton. The fish embryo acute toxicity test performed with PAA water solutions using 1.5-500 mg L-1 concentrations showed that toxicity did not statistically differ from the control. The survival rates were indeed >90%, even at the highest concentration; the hatching rates were >80%; and the numbers of morphological defects were comparable to those of the control. Tests using transgenic zebrafish lines indicated that the numbers of microscopic vascular and musculoskeletal defects were comparable to the control, with one random concentration showing doubled alterations. Sensory-motor tests in response to visual and tactile stimuli were also performed. In the presence of PAAs, embryos exposed to alternating light/dark cycles showed an insignificant mobility reduction during the dark phase. Touch-evoked response tests revealed a mild effect of PAAs on the neuromotor system at concentrations > 10 mg L-1. The cystine/glycine copolymer at 100 mg L-1 exhibited the greatest effect. Overall, the studied PAAs showed a minimal impact on aquatic systems and should be further considered as promising ecofriendly materials.

Identifying antibiotic-resistant strains via cell sorting and elastic-light-scatter phenotyping

The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony’s unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern.Key points• Individual bacteria cells are placed on gradient agar plates by a cell sorter• Laser-light scatter patterns are used to recognize antibiotic-resistant organisms• Scatter patterns formed by colonies correspond to AMR-associated phenotypes

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania.

Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

Removal of cresol contaminants from aqueous media using hydrophobic deep eutectic solvents

Cresols have distinct effects on the environment and human health. For example, o-cresol is considered a toxic isomer and can cause serious health problems. Cresol isomers generally have a negative impact on the environment as they can be hazardous to aquatic life and can accumulate in the food chain. The introduction of these chemicals has the potential to disrupt the balance of ecological systems by reducing oxygen concentrations in aqueous environments, thus affecting the viability and reproductive capacity of aquatic organisms. This work involves the use of six hydrophobic deep eutectic solvents (HDESs) for the extraction of m-cresol and o-cresol from wastewater. The HDESs include menthol:thymol, menthol:decanoic acid, thymol:decanoic acid, hydrocinnamic acid:decanoic acid, thymol:coumarin and thymol:camphor. All HDESs were prepared in a molar ratio of 1:1. The effects of various parameters such as contact time, mass ratio of HDESs to water, molar ratio of HDESs and initial concentration were investigated. The experimental results indicate excellent efficiency in the removal of cresols from wastewater using all HDESs. The extraction efficiency of > 94 % was achieved for the removal of cresol isomers from wastewater using all prepared HDESs. In addition, COSMO-RS was used to understand the extraction process.

Fronts affect fish community structure in the Yangtze River estuary and its adjacent area

Dispersal, a critical factor in the viability of marine organisms, is strongly influenced by the physical dynamics of the ocean. Fronts, as a common phenomenon in ocean dynamics, can theoretically impact the dispersal of organisms and ultimately cause spatial homogeneity or heterogeneity of marine communities. However, there is relatively limited understanding concerning whether frontal formation hinders the connectivity between communities and thus results in differences in community structure. To verify whether the formation of fronts caused differences between communities, 60 eDNA samples and their environmental parameters were collected from 32 sites in the Yangtze River estuary and its adjacent area, and four groups, the Yangtze River front-inner group (YRI), the Yangtze River front-outer group (YRO), the Zhejiang costa front-inner group (ZCI), and the Zhejiang coast front-outer group (ZCO), were compared to examine the differences in fish communities between the sides of the front. The results showed that there was a significant difference between YRI and YRO, suggesting that the front affected the community structure. There was no significant difference in fish communities between ZCI and ZCO, indicating that fronts could only hinder dispersal at high intensities. The difference between YRI and YRO possibly resulted from dramatic changes in temperature, salinity, and turbidity related to the front. Fronts also separated water masses with different environmental characteristics and thereby attracted fish to their optimal habitats. Overall, fronts appear to be ecologically significant for fish dispersal and community differentiation.

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Enhancing pathogen detection and characterization of respiratory microbiome signatures of infection through next-generation sequencing

Next-generation sequencing-based (NGS) methods for pathogen detection, which can capture the entire microbial composition in a clinical sample, are used strategically in parallel to culture-based methods. While NGS-based methods cannot confirm organism viability, culture-based methods can but are limited by the number of culture conditions routinely utilized in clinical microbiology laboratories. The relatively wide scope provided by sequencing compensates for the limited scope of culture-based methods but creates obstacles in distinguishing pathogens from an expected body-site-specific microbiome or benign transient colonizers. This study compares NGS-based methods for pathogen detection in respiratory samples to current diagnostics at Texas Children's Hospital in Houston, Texas. Current diagnostics include various culture-based assays, targeted PCR assays, and reference laboratory-generated sequencing. We hypothesize that employing sample metadata (source, type, etc.) and patient data (clinical history, chronic conditions, etc.) during data analysis will help distinguish pathogens from expected flora in respiratory samples. To develop these methods, 27 respiratory samples with suffcient volume for both routine diagnostic testing and this study were collected. Nucleic acid was extracted using the Qiagen DNeasy PowerSoil Pro Kit after collection, and targeted short-read sequencing (16S, 18S/ITS) was performed. These data were then analyzed using a custom bioinformatic pipeline built around Qiime2-2022. In brief, sequences are denoised and quality filtered using DADA2, an algorithm that uses a statistical model for correcting sequencing errors, after which similar amplicon sequence variants (ASVs) are clustered. Next, a feature table of ASVs that are 100% identical is generated, and taxonomy assigned using a naive Bayes taxonomy classifier against the Silva 13.2 database. Finally, unassigned and low abundance ASVs are removed. When species-level identification couldn't be determined through targeted short-read sequencing, untargeted long-read sequencing was used. In one case, we found that a bronchoalveolar lavage sample from the lung of a patient with a history of recurrent pneumonia contained a singular pathogenic bacterium, H. influenzae. This finding agreed with results obtained through reference laboratory-generated sequencing. In another case, we identified &gt;14 unique bacteria at varying concentrations in a wash sample collected from the lung of an asymptomatic patient during a routine bronchoscopy. These findings, however, were determined to be expected respiratory flora. Our NGS-based methods’ enhanced pathogen detection compared to current diagnostics and discriminates between respiratory flora and active infections. Setting thresholds for pathogen detection, evaluating clinical relevance in the context of body site and presentation, and deploying additional molecular techniques when necessary are crucial steps to increase the accuracy of pathogen detection in the clinic. This study was supported by the Texas Children's Hospital Department of Pathology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Population Prediction and Evaluation of Impact Factors of Dandelions

Seeds are the fundamental source of dandelion life on earth, and their germination process is the first step towards plant growth. The seed germination process involves activating a complex series of biochemical reactions that transform the dandelion seed into an active and viable organism. The germination process is crucial for producing plants and flowers, and dandelions require specific environmental conditions for successful growth. In this article, we will explore the growing process of dandelion seeds, the spread of dandelions during their growth, and the impact factors for invasive species, including dandelions. In Problem 1, we answer the question from two perspectives. We first predict the population size of dandelions. We predicted the population of dandelions over the course of one, two, three, six, and twelve months in certain various climatic conditions. We first outlined the life cycle of dandelions in five stages: seed, germination, plant, flower, and seed dispersal. We defined three variables related to the dandelion life cycle as Seed (S), Germination (G), and Flowering (F); with two more additional variables, Resources (R) and Temperature (T) in a given environment, we constructed Dandelion Demography Model using the Ordinary Differential Equations (ODEs). The results showed that the number of dandelion populations gradually increased in summer, and with the advent of autumn and winter, the temperature decreased, and the number of dandelions gradually decreased to 0. After that, we set the average temperature for the whole year higher and carried out the sensitivity analysis of the model. The results showed that dandelion populations could increase more in one year and even in autumn and winter as the temperature increased. We then used Gaussian equations in conjunction with the wind to predict the extent of dandelion coverage. The results show that in the absence of wind, the population will spread outward uniformly. Under windy conditions, the population will concentrate more in the direction of the wind, but the population density decreases as the distance from the mother plant increases. In Problem 2, we continued to study three plant species, including dandelions. We applied the hybrid entropy weight method EWM-TOPSIS model to develop an index-based measurement to determine impact factors for three invasive species: Taraxacum officinale, Amaranthus palmeri, and Alternanthera philoxeroides. EWM is used to calculate the weight of each index and reduce the disadvantages of TOPSIS, which adopts equal weights. The results showed that the influence factors of the three plants were 0.51, 0.69, and 0.71, respectively. After that, we verified the reasonableness of the model by querying the distribution of the plants worldwide.

Quantitative Analysis of Genomic DNA Degradation of E. coli Using Automated Gel Electrophoresis under Various Levels of Microwave Exposure.

The problem that this study addresses is to understand how microwave radiation is able to degrade genomic DNA of E. coli. In addition, a comparative study was made to evaluate the suitability of a high-throughput automated electrophoresis platform for quantifying the DNA degradation under microwave radiation. Overall, this study investigated the genomic DNA degradation of E. coli under microwave radiation using automated gel electrophoresis. To examine the viable organisms and degradation of genomic DNA under microwave exposure, we used three methods: (1) post-microwave exposure, where E. coli was enumerated using modified mTEC agar method using membrane filtration technique; (2) extracted genomic DNA of microwaved sample was quantified using the Qubit method; and (3) automated gel electrophoresis, the TapeStation 4200, was used to examine the bands of extracted DNA of microwaved samples. In addition, to examine the impacts of microwaves, E. coli colonies were isolated from a fecal sample (dairy cow manure), these colonies were grown overnight to prepare fresh E. coli culture, and this culture was exposed to microwave radiation for three durations: (1) 2 min; (2) 5 min; and (3) 8 min. In general, Qubit values (ng/µL) were proportional to the results of automated gel electrophoresis, TapeStation 4200, DNA integrity numbers (DINs). Samples from exposure studies (2 min, 5 min, and 8 min) showed no viable E. coli. Initial E. coli levels (at 0 min microwave exposure) were 5 × 108 CFU/mL, and the E. coli level was reduced to a non-detectable level within 2 min of microwave exposure. The relationships between Qubit and TapeStation measurements was linear, except for when the DNA level was lower than 2 ng/µL. In 8 min of microwave exposure, E. coli DNA integrity was reduced by 61.7%, and DNA concentration was reduced by 81.6%. The overall conclusion of this study is that microwave radiation had a significant impact on the genomic DNA of E. coli, and prolonged exposure of E. coli to microwaves can thus lead to a loss of genomic DNA integrity and DNA concentrations.

Optimized Bags of Artificial Neural Networks Can Predict the Viability of Organisms Exposed to Nanoparticles.

Prediction of organismal viability upon exposure to a nanoparticle in varying environments─as fully specified at the molecular scale─has emerged as a useful figure of merit in the design of engineered nanoparticles. We build on our earlier finding that a bag of artificial neural networks (ANNs) can provide such a prediction when such machines are trained with a relatively small data set (with ca. 200 examples). Therein, viabilities were predicted by consensus using the weighted means of the predictions from the bags. Here, we confirm the accuracy and precision of the prediction of nanoparticle viabilities using an optimized bag of ANNs over sets of data examples that had not previously been used in the training and validation process. We also introduce the viability strip, rather than a single value, as the prediction and construct it from the viability probability distribution of an ensemble of ANNs compatible with the data set. Specifically, the ensemble consists of the ANNs arising from subsets of the data set corresponding to different splittings between training and validation, and the different bags (k-folds). A machine uses a single partition (or bag) of k - 1 ANNs each trained on 1/k of the data to obtain a consensus prediction, and a k-bag machine quorum samples the k possible machines available for a given partition. We find that with increasing k in the k-bag or machines, the viability strips become more normally distributed and their predictions become more precise. Benchmark comparisons between ensembles of 4-bag machines and fraction machines suggest that the fraction machine has similar accuracy while overcoming some of the challenges arising from divergent ANNs in the 4-bag machines.