PyKleeBarcode: Enabling representation of the whole animal kingdom in information space.

As biological sequence databases continue growing, so do the insight that they promise to shed on the shape of the genetic diversity of life. However, to fulfil this promise the software must remain usable, be able to accommodate a large amount of data and allow use of modern high performance computing infrastructure. In this study we present a reimplementation as well as an extension of a technique using indicator vectors to compute and visualize similarities between sets of nucleotide sequences. We have a flexible and easy to use python program relying on standard and open-source libraries. Our tool allows analysis of very large complement of sequences using code parallelization, as well as by providing routines to split a computational task in smaller and manageable subtasks whose results are then merged. This implementation also facilitates adding new sequences into an indicator vector-based representation without re-computing the whole set. The efficient synthesis of data into knowledge is no trivial matter given the size and rapid growth of biological sequence databases. Based on previous results regarding the properties of indicator vectors, the open-source approach proposed here efficiently and flexibly supports comparative analysis of genetic diversity at a large scale. Our software is freely available at: https://github.com/WandrilleD/pyKleeBarcode.

The Coronavirus Calendar (CoronaCal): a simplified SARS-CoV-2 test system for sampling and retrospective analysis.

Objectives: To develop a biological diary (CoronaCal) that allows anyone in the community to collect and store serial saliva samples and chart symptoms on ordinary printer paper. Methods: Diaries were analyzed for the presence of SARS-CoV-2 RNA using established polymerase chain reaction (PCR) procedures. CoronaCal diaries were distributed to volunteer subjects in the community during the peak of the COVID-19 outbreak in New York. Volunteers collected their own daily saliva samples and self-reported symptoms. Results: SARS-CoV-2 RNA extracted from CoronaCals was measured using qPCR and RNA levels were correlated with reported symptoms. SARS-CoV-2 RNA was detected in CoronaCals from nine of nine people with COVID-19 symptoms or exposure to someone with COVID-19, and not in one asymptomatic person. CoronaCals were stored for up to 70 days at room temperature during collection and then frozen for up to four months before analysis, suggesting that SARS-CoV-2 RNA is stable once dried onto paper. Conclusions: Sampling saliva on simple paper provides a useful method to study the natural history and epidemiology of COVID-19. The CoronaCal collection and testing method is easy to implement, inexpensive, non-invasive and scalable. The approach can inform the historical and epidemiological understanding of infections in individuals and populations.

Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection.

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

Archiving time series sewage samples as biological records of built environments.

This commentary encourages the regular archiving of nucleic-acid-stabilized serial samples of wastewaters and/or sewage. Stabilized samples would facilitate retrospective reconstitution of built environments' biological fluids. Biological time capsules would allow retrospective searches for nucleic acids from viruses such as SARS-CoV-2. Current resources for testing need not be diverted if samples are saved in case they become important in the future. Systematic storage would facilitate investigation into the origin and prevalence of viruses and other agents. Comparison of prevalence data from individual and clinical samplings with community wastewater would allow valuable comparison, contrast and correlation among different testing modalities. Current interest is focused on SARS-CoV-2, but archived samples could become valuable in many contexts including surveys for other infectious and chemical agents whose identity is not currently known. Archived time series of wastewater will take their place alongside other biological repositories and records including those from medical facilities, museums, eDNA, living cell and tissue collections. Together these will prove invaluable records of the evolving Anthropocene.

Precision public health to inhibit the contagion of disease and move toward a future in which microbes spread health.

Antimicrobial resistance continues to outpace the development of new chemotherapeutics. Novel pathogens continue to evolve and emerge. Public health innovation has the potential to open a new front in the war of "our wits against their genes" (Joshua Lederberg). Dense sampling coupled to next generation sequencing can increase the spatial and temporal resolution of microbial characterization while sensor technologies precisely map physical parameters relevant to microbial survival and spread. Microbial, physical, and epidemiological big data could be combined to improve prospective risk identification. However, applied in the wrong way, these approaches may not realize their maximum potential benefits and could even do harm. Minimizing microbial-human interactions would be a mistake. There is evidence that microbes previously thought of at best "benign" may actually enhance human health. Benign and health-promoting microbiomes may, or may not, spread via mechanisms similar to pathogens. Infectious vaccines are approaching readiness to make enhanced contributions to herd immunity. The rigorously defined nature of infectious vaccines contrasts with indigenous "benign or health-promoting microbiomes" but they may converge. A "microbial Neolithic revolution" is a possible future in which human microbial-associations are understood and managed analogously to the macro-agriculture of plants and animals. Tradeoffs need to be framed in order to understand health-promoting potentials of benign, and/or health-promoting microbiomes and infectious vaccines while also discouraging pathogens. Super-spreaders are currently defined as individuals who play an outsized role in the contagion of infectious disease. A key unanswered question is whether the super-spreader concept may apply similarly to health-promoting microbes. The complex interactions of individual rights, community health, pathogen contagion, the spread of benign, and of health-promoting microbiomes including infectious vaccines require study. Advancing the detailed understanding of heterogeneity in microbial spread is very likely to yield important insights relevant to public health.

Microwave detection and quantification of water hidden in and on building materials: implications for healthy buildings and microbiome studies.

BACKGROUND: Excess water in all its forms (moisture, dampness, hidden water) in buildings negatively impacts occupant health but is hard to reliably detect and quantify. Recent advances in through-wall imaging recommend microwaves as a tool with a high potential to noninvasively detect and quantify water throughout buildings. METHODS: Microwaves in both transmission and reflection (radar) modes were used to perform a simple demonstration of the detection of water both on and hidden within building materials. RESULTS: We used both transmission and reflection modes to detect as little as 1 mL of water between two 7 cm thicknesses of concrete. The reflection mode was also used to detect 1 mL of water on a metal surface. We observed oscillations in transmitted and reflected microwave amplitude as a function of microwave wavelength and water layer thickness, which we attribute to thin-film interference effects. CONCLUSIONS: Improving the detection of water in buildings could help design, maintenance, and remediation become more efficient and effective and perhaps increase the value of microbiome sequence data. Microwave characterization of all forms of water throughout buildings is possible; its practical development would require new collaborations among microwave physicists or engineers, architects, building engineers, remediation practitioners, epidemiologists, and microbiologists.

Toward a microbial Neolithic revolution in buildings.

The Neolithic revolution--the transition of our species from hunter and gatherer to cultivator--began approximately 14,000 years ago and is essentially complete for macroscopic food. Humans remain largely pre-Neolithic in our relationship with microbes but starting with the gut we continue our hundred-year project of approaching the ability to assess and cultivate benign microbiomes in our bodies. Buildings are analogous to the body and it is time to ask what it means to cultivate benign microbiomes in our built environment. A critical distinction is that we have not found, or invented, niches in buildings where healthful microbial metabolism occurs and/or could be cultivated. Key events affecting the health and healthfulness of buildings such as a hurricane leading to a flood or a burst pipe occur only rarely and unpredictably. The cause may be transient but the effects can be long lasting and, e.g., for moisture damage, cumulative. Non-invasive "building tomography" could find moisture and "sentinel microbes" could record the integral of transient growth. "Seed" microbes are metabolically inert cells able to grow when conditions allow. All microbes and their residue present actinic molecules including immunological epitopes (molecular shapes). The fascinating hygiene and microbial biodiversity hypotheses propose that a healthy immune system requires exposure to a set of microbial epitopes that is rich in diversity. A particular conjecture is that measures of the richness of diversity derived from microbiome next-generation sequencing (NGS) can be mechanistically coupled to--rather than merely correlated with some measures of--human health. These hypotheses and conjectures inspire workers and funders but an alternative is also consequent to the first Neolithic revolution: That the genetic uniformity of contemporary foods may also decrease human exposure to molecular biodiversity in a heath-relevant manner. Understanding the consequences--including the unintended consequences of the first Neolithic revolution--will inform and help us benignly implement the second--the microbial--Neolithic revolution.

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies.

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648-bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein-encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most - possibly all - synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well-curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.

DNA barcoding works in practice but not in (neutral) theory.

BACKGROUND: DNA barcode differences within animal species are usually much less than differences among species, making it generally straightforward to match unknowns to a reference library. Here we aim to better understand the evolutionary mechanisms underlying this usual "barcode gap" pattern. We employ avian barcode libraries to test a central prediction of neutral theory, namely, intraspecific variation equals 2 Nµ, where N is population size and µ is mutations per site per generation. Birds are uniquely suited for this task: they have the best-known species limits, are well represented in barcode libraries, and, most critically, are the only large group with documented census population sizes. In addition, we ask if mitochondrial molecular clock measurements conform to neutral theory prediction of clock rate equals µ. RESULTS: Intraspecific COI barcode variation was uniformly low regardless of census population size (n = 142 species in 15 families). Apparent outliers reflected lumping of reproductively isolated populations or hybrid lineages. Re-analysis of a published survey of cytochrome b variation in diverse birds (n = 93 species in 39 families) further confirmed uniformly low intraspecific variation. Hybridization/gene flow among species/populations was the main limitation to DNA barcode identification. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first large study of animal mitochondrial diversity using actual census population sizes and the first to test outliers for population structure. Our finding of universally low intraspecific variation contradicts a central prediction of neutral theory and is not readily accounted for by commonly proposed ad hoc modifications. We argue that the weight of evidence-low intraspecific variation and the molecular clock-indicates neutral evolution plays a minor role in mitochondrial sequence evolution. As an alternate paradigm consistent with empirical data, we propose extreme purifying selection, including at synonymous sites, limits variation within species and continuous adaptive selection drives the molecular clock.

Specialized transduction designed for precise high-throughput unmarked deletions in Mycobacterium tuberculosis.

UNLABELLED: Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. IMPORTANCE: This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.

φ(2)GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples.

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

Reporter phage and breath tests: emerging phenotypic assays for diagnosing active tuberculosis, antibiotic resistance, and treatment efficacy.

The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or ß-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics.

Einstein Contained Aerosol Pulmonizer (ECAP): Improved Biosafety for Multi-Drug Resistant (MDR) and Extensively Drug Resistant (XDR) Mycobacterium tuberculosis Aerosol Infection Studies.

A new apparatus enhances the biosafety of containment (biosafety level 3 [BSL-3]) and provides experimental reproducibility for aerosol infection experiments with MDR and XDR Mycobacterium tuberculosis. The methods are generally applicable to the study of airborne pathogens.

Life-threatening infectious diseases of childhood: single-gene inborn errors of immunity?

The hypothesis that inborn errors of immunity underlie infectious diseases is gaining experimental support. However, the apparent modes of inheritance of predisposition or resistance differ considerably among diseases and among studies. A coherent genetic architecture of infectious diseases is lacking. We suggest here that life-threatening infectious diseases in childhood, occurring in the course of primary infection, result mostly from individually rare but collectively diverse single-gene variations of variable clinical penetrance, whereas the genetic component of predisposition to secondary or reactivation infections in adults is more complex. This model is consistent with (i) the high incidence of most infectious diseases in early childhood, followed by a steady decline; (ii) theoretical modeling of the impact of monogenic or polygenic predisposition on the incidence distribution of infectious diseases before reproductive age; (iii) available molecular evidence from both monogenic and complex genetics of infectious diseases in children and adults; (iv) current knowledge of immunity to primary and secondary or latent infections; (v) the state of the art in the clinical genetics of noninfectious pediatric and adult diseases; and (vi) evolutionary data for the genes underlying single-gene and complex disease risk. With the recent advent of new-generation deep resequencing, this model of single-gene variations underlying severe pediatric infectious diseases is experimentally testable.

Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria.

BACKGROUND: PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H(2)O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. CONCLUSIONS/SIGNIFICANCE: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved "treatment-free" attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.

Tests of the single-hit DNA damage model.

The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA (S) surviving doses (D) of three damaging agents: gamma irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (lnS=kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For gamma irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.

Improving introspection to inform free will regarding the choice by healthy individuals to use or not use cognitive enhancing drugs.

A commentary in Nature entitled "Towards responsible use of cognitive-enhancing drugs by the healthy" (Greely et al 2008 Nature 456: 702-705) offers an opportunity to move toward a humane societal appreciation of mind-altering drugs. Using cognitive enhancing drugs as an exemplar, this article presents a series of hypotheses concerning how an individual might learn optimal use. The essence of the proposal is that individuals can cultivate sensitivity to the effects of ever-smaller amounts of psychoactive drugs thereby making harm less likely and benign effects more probable. Four interrelated hypotheses are presented and briefly discussed. 1. Humans can learn to discriminate ever-smaller doses of at least some mind-altering drugs; a learning program can be designed or discovered that will have this outcome. 2. The skill to discriminate drugs and dose can be generalized, i.e. if learned with one drug a second one is easier and so on. 3. Cultivating this skill/knack would be beneficial in leading to choices informed by a more accurate sense of mind-body interactions. 4. From a philosophical point of view learning the effects of ever-smaller doses of psychoactive agents offers a novel path into and to transcend the objective/subjective barrier and the mind/body problem.Whatever the fate of these specific hypotheses, discussion of cognitive enhancing drugs for healthy individuals has the potential to inspire innovative educational and public policy initiatives toward all types of mind-altering drugs and the people who use them.

The cytoplasmic structure hypothesis for ribosome assembly, vertical inheritance, and phylogeny.

Fundamental questions in evolution concern deep divisions in the living world and vertical versus horizontal information transfer. Two contrasting views are: (i) three superkingdoms Archaea, Eubacteria, and Eukarya based on vertical inheritance of genes encoding ribosomes; versus (ii) a prokaryotic/eukaryotic dichotomy with unconstrained horizontal gene transfer (HGT) among prokaryotes. Vertical inheritance implies continuity of cytoplasmic and structural information whereas HGT transfers only DNA. By hypothesis, HGT of the translation machinery is constrained by interaction between new ribosomal gene products and vertically inherited cytoplasmic structure made largely of preexisting ribosomes. Ribosomes differentially enhance the assembly of new ribosomes made from closely related genes and inhibit the assembly of products from more distal genes. This hypothesis suggests experiments for synthetic biology: the ability of synthetic genomes to "boot," i.e., establish hereditary continuity, will be constrained by the phylogenetic closeness of the cell "body" into which genomes are placed.