Fast and Sustainable Thermo-osmotic DNA Extraction Protocol for Trans-spectrum Contingency and Field Use.

In the field of molecular genetics, DNA extraction protocols and kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Expanding upon previous fast extraction protocols such as alkaline- and detergent-based ones, the use of boiling-hot water to rupture cells, virions, and nuclei, as proposed during the COVID-19 pandemic, might alleviate shortages and costs. Different soft, relatively abundant (highly enriched), and uncomplicated (genomically homogenous and with few inhibitors) biosamples are collected in 1.5 mL tubes, mixed with boiling-hot water, and stirred vigorously, so as to have membranes lysed and proteins deactivated; mechanical disruption may be used as well if necessary. Incubation in boiling water bath for 20-30 min follows. Depending on sample type and quantity, which affects the total extraction volume, 2-5 µL are pipetted off for direct PCR and the same volume for two decimal serial dilutions. The latter are intended to optimize the crude extract to a workable DNA/inhibitor concentration balance for direct PCR. Uncomplicated, highly enriched samples such as mycelial growth in fruits and human swab samples can be processed, contrary to complicated samples such as blood and physically unyielding samples such as plant tissue. The extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing nucleic acid amplification tests (NAAT) being performed in resource-limited settings with low cost and waste footprint or during prolonged crises, where supply chain failures may occur. Key features DNA extraction from different sample types using only boiling water and occasional mechanical assistance. Crude extract serially diluted twice, 10- and 100-fold, to bypass purification and quantification steps. Direct PCR for 2-10 µL of crude lysate and dilutions (conditional to sample type and quantity) to enhance probability of workable DNA-inhibitors' concentrations. Lowers the cost and curtails the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.

Historical microbiology: researching past bioevents by integrating scholarship (re)sources with paleomicrobiology assets.

The analysis of past epidemics and pandemics, either spontaneous or of human origin, may revise the physical history of microbiota and create a temporal context in our understanding regarding pathogen attributes like virulence, evolution, transmission and disease dynamics. The data of high-tech scientific methods seem reliable, but their interpretation may still be biased when tackling events of the distant past. Such endeavors should be adjusted to other cognitive resources including historical accounts reporting the events of interest and references in alien medical cultures and terminologies; the latter may contextualize them differently from current practices. Thus 'historical microbiology' emerges. Validating such resources requires utmost care, as these may be susceptible to different biases regarding the interpretation of facts and phenomena; biases partly due to methodological limitations.

Bacteria and viruses have always impacted humankind. They do this directly by causing illness or indirectly by destroying crops and threatening livestock. We can learn a lot by studying disease events of the past ­ for example, we can see how bacteria and viruses have changed over time and predict how they might change in the future. This knowledge could be important to understanding present disease events and predicting future ones. In this review, we propose the concept of 'historical microbiology', which encourages collaboration between scientists, doctors, historians and linguists to provide historical, linguistic and cultural context to our scientific understanding of diseases of the past.

Classification of olive cultivars by machine learning based on olive oil chemical composition.

Extra virgin olive oil traceability and authenticity are important quality indicators, and are currently the subject of exhaustive research, for developing methods to secure olive oil origin-related issues. The aim of this study was the development of a classification model capable of olive cultivar identification based on olive oil chemical composition. To achieve our aim, 385 samples of two Greek and three Italian olive cultivars were collected during two successive crop years from different locations in the coastline part of western Greece and southern Italy and analyzed for their chemical characteristics. Principal Component Analysis showed trends of differentiation among olive cultivars within or between the crop years. Artificial intelligence model of the XGBoost machine learning algorithm showed high performance in classifying the five olive cultivars from the pooled samples.

Paired associated SARS-CoV-2 spike variable positions: a network analysis approach to emerging variants.

Amino acids in variable positions of proteins may be correlated, with potential structural and functional implications. Here, we apply exact tests of independence in R × C contingency tables to examine noise-free associations between variable positions of the SARS-CoV-2 spike protein, using as a paradigm sequences from Greece deposited in GISAID (N = 6,683/1,078 full length) for the period 29 February 2020 to 26 April 2021 that essentially covers the first three pandemic waves. We examine the fate and complexity of these associations by network analysis, using associated positions (exact P ≤ 0.001 and Average Product Correction ≥ 2) as links and the corresponding positions as nodes. We found a temporal linear increase of positional differences and a gradual expansion of the number of position associations over time, represented by a temporally evolving intricate web, resulting in a non-random complex network of 69 nodes and 252 links. Overconnected nodes corresponded to the most adapted variant positions in the population, suggesting a direct relation between network degree and position functional importance. Modular analysis revealed 25 k-cliques comprising 3 to 11 nodes. At different k-clique resolutions, one to four communities were formed, capturing epistatic associations of circulating variants (Alpha, Beta, B.1.1.318), but also Delta, which dominated the evolutionary landscape later in the pandemic. Cliques of aminoacidic positional associations tended to occur in single sequences, enabling the recognition of epistatic positions in real-world virus populations. Our findings provide a novel way of understanding epistatic relationships in viral proteins with potential applications in the design of virus control procedures. IMPORTANCE Paired positional associations of adapted amino acids in virus proteins may provide new insights for understanding virus evolution and variant formation. We investigated potential intramolecular relationships between variable SARS-CoV-2 spike positions by exact tests of independence in R × C contingency tables, having applied Average Product Correction (APC) to eliminate background noise. Associated positions (exact P ≤ 0.001 and APC ≥ 2) formed a non-random, epistatic network of 25 cliques and 1-4 communities at different clique resolutions, revealing evolutionary ties between variable positions of circulating variants and a predictive potential of previously unknown network positions. Cliques of different sizes represented theoretical combinations of changing residues in sequence space, allowing the identification of significant aminoacidic combinations in single sequences of real-world populations. Our analytic approach that links network structural aspects to mutational aminoacidic combinations in the spike sequence population offers a novel way to understand virus epidemiology and evolution.

Can Water-Only DNA Extraction Reduce the Logistical Footprint of Biosurveillance and Planetary Health Diagnostics? Toward a New Method.

The coronavirus disease-2019 (COVID-19) pandemic has raised the stakes for planetary health diagnostics. Because pandemics pose enormous burdens on biosurveillance and diagnostics, reduction of the logistical burdens of pandemics and ecological crises is essential. Moreover, the disruptive effects of catastrophic bioevents impact the supply chains in both highly populated urban centers and rural communities. One "upstream" focus of methodological innovation in biosurveillance is the footprint of Nucleic Acid Amplification Test (NAAT)-based assays. We report in this study a water-only DNA extraction, as an initial step in developing future protocols that may require few expendables, and with low environmental footprints, in terms of wet and solid laboratory waste. In the present work, boiling-hot distilled water was used as the main cell lysis agent for direct polymerase chain reactions (PCRs) on crude extracts. After evaluation (1) in blood and mouth swabs for human biomarker genotyping, and (2) in mouth swabs and plant tissue for generic bacterial or fungal detection, and using different combinations of extraction volume, mechanical assistance, and extract dilution, we found the method to be applicable in low-complexity samples, but not in high-complexity ones such as blood and plant tissue. In conclusion, this study examined the doability of a lean approach for template extraction in the case of NAAT-based diagnostics. Testing our approach with different biosamples, PCR settings, and instruments, including portable ones for COVID-19 or dispersed applications, warrant further research. Minimal resources analysis is a concept and practice, vital and timely for biosurveillance, integrative biology, and planetary health in the 21st century.

Age and sex associations of SARS-CoV-2 antibody responses post BNT162b2 vaccination in healthcare workers: A mixed effects model across two vaccination periods.

This study aimed to examine the associations with epidemiological, behavioral and clinical parameters of IgG antibody responses against the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after immunization with two doses of the BNT162b2 vaccine in a cohort of healthcare workers (HCWs, n = 439) in Greece. We used a mixed effects model to investigate the potential associations of antibody levels one and three months after vaccination and examined by bootstrapping t-tests the putative effects of gender and age for each period. We also employed exact tests of independence in R × C contingency tables to explore associations between behavioral and gender variables with vaccinations side effects. We found significant differences between males and females as well as between subjects in the youngest (21-30 years) and the older age groups in both study periods. We also detected a decrease in titers with age and time. Males had steeper elimination rates across the age span in both periods, in contrast to females who exhibited a softer elimination titer rate with age in the first period and almost constant titers in the second. Concerning side effects, we found a significant association between pain at the injection site and female sex. Hence, our real-world data analyses revealed potentially important clues into the associations of antibody responses to SARS-CoV-2 spike. We discuss the importance of these findings in view of current mass vaccination perspectives and provide useful clues for the design and optimal timing of booster doses for COVID-19.

Beyond the Microbiome: Germ-ganism? An Integrative Idea for Microbial Existence, Organization, Growth, Pathogenicity, and Therapeutics.

The advances made by microbiome research call for new vocabulary and expansion of our thinking in microbiology. For example, the life-forms presenting in both unicellular and multicellular formats invite us to rethink microbial existence, organization, growth, pathogenicity, and therapeutics in the 21st century. A view of such populations as parts of single organisms with a loose, distributed multicellular organization, introduced here as a germ-ganism, rather than communities, might open up interesting prospects for diagnostics and therapeutics innovation. This study tested and further contextualized the concept of germ-ganism using solid cultures of bacteria and fungi. Based on our findings and the literature reviewed herein, we propose that germ-ganism has synergy with a systems medicine approach by broadening host-environment interactions from cells and microorganisms to a scale of biological ecosystems. Germ-ganism also brings about the possibility of studying the multilevel impacts of novel therapeutic agents within and across networks of microbial ecosystems. The germ-ganism would lend itself, in the long term, to a veritable biocybernetics system, while in the mid-term, we anticipate it will contribute to new diagnostics and therapeutics. Biosecurity applications would be immensely affected by germ-ganism. Industrial applications of germ-ganism are of interest as a more sustainable alternative to costly solutions such as tampered strains/microorganisms. In conclusion, germ-ganism is informed by lessons from microbiome research and invites rethinking microbial existence, organization, and growth as an organism. Germ-ganism has vast ramifications for understanding pathogenicity, and clinical, biosecurity, and biotechnology applications in the current historical moment of the COVID-19 pandemic and beyond.

Integration of biophysical photosynthetic parameters into one photochemical index for early detection of Tobacco Mosaic Virus infection in pepper plants.

Photosynthesis in host plants is significantly reduced by many virus families. The early detection of viral infection before the onset of visual symptoms in both directly and systemically infected leaves is critical in crop protection. Viral pathogens cause a variety of symptoms through modifications of chloroplast structure and function and the response of the photochemistry process is immediate. Therefore, chlorophyll fluorescence monitoring has been extensively investigated the last two decades as a tool for timely assessment of pathogenic threats. Alternatively, the analysis of Chla fluorescence transients offers several interlinked parameters which describe the fate of excitation energy round and through the photosystems. Additionally, OJIP fluorescence transients and leaf reflectance spectra methodologies serve for rapid screening of large number of samples. The objective of the present study was to achieve early detection of viral infection, integrating the multiparametric information of the Chla fluorescence transients and of the leaf reflectance spectra into one photochemical performance index. Infection decreased the maximum quantum yield of PSII (FV/FM), the effective quantum yield of PSII (ΦPSII), the CO2 assimilation rate (A) and the stomatal conductance (gs) in the studied TMV-pepper plant pathosystem, while non-photochemical quenching (NPQ) increased. Some parameters from the OJIP transients and the leaf reflectance spectra were significantly affected 24 h after infection, while others modified three to five days later. Similar results were obtained from systemically infected leaves but with one to three days hysteresis compared to inoculated leaves. Differences between healthy and infected leaves were marginal during the first 24 h post infection. The Integrated Biomarker Response tool was used to create a photochemical infection index (PINFI) which integrates the partial effects of infection on each fluorescence and reflectance index. The PINFI, which to the best of our knowledge is the first photochemical infection index created by the IBR method, discriminated reliably between the infected and healthy leaves of pepper plants from the first 24 h after infection with the TMV.

Fast, Scalable, and Practical: An Alkaline DNA Extraction Pipeline for Emergency Microbiomics Biosurveillance.

Pandemics and environmental crises evident from the first two decades of the 21st century call for methods innovation in biosurveillance and early detection of risk signals in planetary ecosystems. In crises conditions, conventional methods in public health, biosecurity, and environmental surveillance do not work well. In addition, the standard laboratory amenities and procedures may become unavailable, irrelevant, or simply not feasible, for example, owing to disruptions in logistics and process supply chains. The COVID-19 pandemic has been a wakeup call in this sense to reintroduce point-of-need diagnostics with an eye to limited resource settings and biosurveillance solutions. We report here a methodology innovation, a fast, scalable, and alkaline DNA extraction pipeline for emergency microbiomics biosurveillance. We believe that the presented methodology is well poised for effective, resilient, and anticipatory responses to future pandemics and ecological crises while contributing to microbiome science and point-of-need diagnostics in nonelective emergency contexts. The alkaline DNA extraction pipeline can usefully expand the throughput in emergencies by deployment or to allow backup in case of instrumentation failure in vital facilities. The need for distributed public health genomics surveillance is increasingly evident in the 21st century. This study makes a contribution to these ends broadly, and for future pandemic preparedness in particular. We call for innovation in biosurveillance methods that remain important existentially on a planet under pressure from unchecked human growth and breach of the boundaries between human and nonhuman animal habitats.

Glimpses into evolutionary trajectories of SARS-CoV-2: emerging variants and potential immune evasion routes.

Tweetable abstract An opinion on the coronaviruses' evolution paradoxes, the continuing adaptation of the SARS-CoV-2 in humans following the zoonotic transmission, and clues into escape routes from host immune responses.

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples.

Serological methods serve as a direct or indirect means of pathogen infection diagnosis in plant and animal species, including humans. Dot-ELISA (DE) is an inexpensive and sensitive, solid-state version of the microplate enzyme-linked immunosorbent assay, with a broad range of applications in epidemiology. Yet, its applicability is limited by uncertainties in the qualitative output of the assay due to overlapping dot colorations of positive and negative samples, stemming mainly from the inherent color discrimination thresholds of the human eye. Here, we report a novel approach for unambiguous DE output evaluation by applying machine learning-based pattern recognition of image pixels of the blot using an impartial predictive model rather than human judgment. Supervised machine learning was used to train a classifier algorithm through a built multivariate logistic regression model based on the RGB ("Red," "Green," "Blue") pixel attributes of a scanned DE output of samples of known infection status to a model pathogen (Lettuce big-vein associated virus). Based on the trained and cross-validated algorithm, pixel probabilities of unknown samples could be predicted in scanned DE output images, which would then be reconstituted by pixels having probabilities above a cutoff. The cutoff may be selected at will to yield desirable false positive and false negative rates depending on the question at hand, thus allowing for proper dot classification of positive and negative samples and, hence, accurate diagnosis. Potential improvements and diagnostic applications of the proposed versatile method that translates unique pathogen antigens to the universal basic color language are discussed.

Toward High-Throughput Fungal Electroculturomics and New Omics Methodologies in 21st-Century Microbiology and Ecology.

Modern microbiology and drug development are in a watershed moment with the advent of electroceuticals. In addition to genomics, electrical impulses in an organism are believed to contribute to tissue and cellular plasticity. Hence, electroceuticals or bioelectronics offers the promise to identify innovative approaches to treat human diseases. However, applications toward electromicrobiology are still limited and rare, despite the high potential to innovate the fields of both microbiology and therapeutics. For example, electric modalities for manipulating microbial growth are highly sustainable; can be combined with biopharmaceuticals, probiotics, and pharmacobiotics; and, thus, are well poised for use in medicine, public health, and ecology and diverse industries. We report here the introduction of a new research framework and technology platform for electroculturomics, by coupling standard solid-state mycological cultures with conductive treatment using a conformité Européene (CE-)-certified medical ionophoresis device. We share our experience with a diverse range of fungi that have been treated with the electroculturomics approach reported herein. We suggest that this line of inquiry can be extended to electrotranscriptomics and electrometabolomics by deploying electroculturomics in tandem with multi-omics approaches in the future. This article makes a specific contribution to fungal microbiology, and a broader contribution to advance the theory and practice of the field of electroculturomics emerging in 21st-century microbiology and ecology research.

Humanome Versus Microbiome: Games of Dominance and Pan-Biosurveillance in the Omics Universe.

Global governance of pathogens such as Ebola virus and infectious diseases is central to global health, and to innovation in systems medicine. Worrisomely, the gaps in human immunity and healthcare services combined with novel pathogens emerging by travel, biotechnological advances, or the rupture of the host-species barrier challenge infectious diseases' global governance. Such biorisks and biothreats may scale up to global catastrophic biological risks (GCBRs) spatiotemporally, either as individual or as collective risks. The scale and intensity of such threats challenge current thinking on surveillance, and calls for a move toward pan-biosurveillance. New multilayered, cross-sectoral, and adaptable strategies of prevention and intervention on GCBRs should be developed, considering human hosts in entirety, and in close relationship with other hosts (plants and animals). This also calls for the "Humanome," which we introduce in this study as the totality of human subjects plus any directly dependent biological or nonbiological entities (products, constructs, and interventions). Surveillance networks should be implemented by integrating communications, diagnostics, and robotics/aeronautics technologies. Suppression of pathogens must be enforced both before and during an epidemic outbreak, the former allowing more drastic measures before the pathogens harbor the host. We propose in this expert review that microbiome-level intervention might particularly prove as an effective solution in medical and environmental scales against traditional, currently emerging, and future infectious threats. We conclude with a discussion on the ways in which the humanome and microbiome contest and cooperate, and how this knowledge might usefully inform in addressing the GCBRs, bioterrorism, and associated threats in the pursuit of pan-biosurveillance.

Toward Decentralized Agrigenomic Surveillance? A Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Approach for Adaptable and Rapid Detection of User-Defined Fungal Pathogens in Potato Crops.

Agrigenomics is one of the emerging focus areas for omics sciences. Yet, agrigenomics differs from medical omics applications such as pharmacogenomics and precision medicine, by virtue of vastly distributed geography of applications at the intersection of agriculture, nutrition, and genomics research streams. Crucially, agrigenomics can address diagnostics and safety surveillance needs in remote and rural farming communities or decentralized food, crop, and environmental monitoring programs for prompt, selective, and differential identification of pathogens. A case in point is the potato crop that serves as a fundamental nutritional source worldwide. Decentralized potato crop and plant protection facilities are pivotal to minimize unnecessary, preemptive use of broad-spectrum fungicides, thus helping to curtail the costs, environmental burden, and the development of resistance in opportunistic human pathogenic fungi. We report here a polymerase chain reaction-restriction fragment length polymorphism approach that is sensitive and adaptable in detection and broad identification of fungal pathogens in potato crops, with a view to future decentralized agrigenomic surveillance programs. Notably, the fingerprinting patterns obtained by the method fully differentiated 12 fungal species examined in silico, with 10 of them also tested in vitro. The method can be scaled up through improvements in electrophoresis and enzyme panel for adaption to other crops and/or pathogens. We suggest that decentralized and integrated agrosurveillance programs and translational agrigenomic programs can inform future innovations in multidomain biosecurity, particularly across omics applications from agriculture and nutrition to clinical medicine and environmental biosafety.

Physical and chemical quality characteristics and antioxidant properties of strawberry cultivars (Fragaria × ananassa Duch.) in Greece: assessment of their sensory impact.

BACKGROUND: There are many factors determining the strawberry organoleptic profile and they are difficult to define. In this study, the sensory, physical, and chemical quality characteristics, the antioxidant properties as examined using ferric reducing antioxidant power (FRAP) and 1-diphenyl-2-picrylhydrazyl (DPPH) assays, the lactone concentration, and the FaFAD1 expression of ripe strawberries (cv. Camarosa, Florida Fortuna, and Sabrina) from Greece were evaluated and their interrelationships were investigated. RESULTS: 'Camarosa' had the highest antioxidant capacity and polyphenol content, although significant intra-cultivar variations of sugars, solid soluble content/titratable acidity (SSC/TA), red color intensity, sweetness, and hardness were recorded. In 'Sabrina' there was a constant lactone presence and FaFAD1 expression; it also had the lowest ascorbic acid content, the highest pH, SSC/TA index, firmness, and sweetness. 'Fortuna' showed the lowest sweetness and aroma indices, whereas 'Camarosa' had intermediate ones. Overall, firmness was correlated with hardness, while pH and SSC/TA index correlated with juiciness and sweetness. Both γ-decalactone and γ-dodecalactone concentrations were correlated with FaFAD1 expression and pH, but they did not solely determine the aroma sensory perception. In total, FRAP values were positively correlated with ascorbic acid and polyphenol content, and negatively with pH. CONCLUSIONS: Significant inter- and intra-cultivar variation was recorded, revealing the impact of the genotype and underlining the effect of microenvironmental and cultivation conditions on quality and sensory perception. © 2018 Society of Chemical Industry.

Rebooting Bioresilience: A Multi-OMICS Approach to Tackle Global Catastrophic Biological Risks and Next-Generation Biothreats.

Global Catastrophic Biological Risks (GCBRs) refer to biological events-natural, deliberate, and accidental-of a global and lasting impact. This challenges the life scientists to raise their game on two hitherto neglected innovation frontiers: a veritable "futures" thinking to "think the unthinkable," and "systems thinking" so as to see both the trees and the forest when it comes to GCBRs. This innovation analysis article outlines the promise of Omics systems science biotechnologies, for example, to deploy rapid fire diagnostics for health security crises at GCBR level, possibly involving neopathogens and/or incurring epidemics (e.g., severe acute respiratory syndrome [SARS] and Ebola) that collectively threaten the lives of global society and interdependent biological ecosystems. Moreover, Omics encourages thinking beyond immediacy and in long-term strategies for biopreparedness and response innovation when the timelines are aggressive and compressed in response to crises such as GCBRs, but also to non-global but surging, multiple threats occurring as successive, overlapping, or distinct events, rather than as distinct entities-a prospect enforcing a reboot in Bioresilience. We define Next-Generation Bioresilience as "a systems approach against natural, accidental and perpetrated GCBRs using Omics technologies, and a shift in mentality, whereby the systems approach is expanded to include multiple plausible futures and expose unchecked assumptions attendant to risks, beyond technological determinism." In sum, it is time to think about the realistic potential of Omics biotechnologies beyond clinical practice and precision medicine so as to harness the opportunities and address the uncertainties associated not only with GCBRs but also with other emerging Omics applications in health and society.

Wireless electrostimulation: a new approach in combating infection?

Electrostimulation (ES), hitherto successfully employed in wound treatment, has shown potential in antimicrobial applications, suggesting its use as synergistic to or replacement of antibiotics. The differential susceptibility of pathogens and host tissue and organs to various ES modalities might allow selective use against specific infections. The use of ES is cheaper in terms of development/testing, routine application and environmental footprint. If extensive substitution of chemical compounds is achieved, the development of resistance might be reversed through negative selection. A promising setup of ES seems to be the noncontact current transfer, due to low amperage similar to innate bioelectricity, painlessness, simple logistics and low risk for treatment-caused infection.