Feasibility of using a combination of staphylococcal superantigen-like proteins 3, 7 and 11 in a fusion vaccine for Staphylococcus aureus.

Staphylococcus aureus is a significant bacterial pathogen in both community and hospital settings, and the escalation of antimicrobial-resistant strains is of immense global concern. Vaccination is an inviting long-term strategy to curb staphylococcal disease, but identification of an effective vaccine has proved to be challenging. Three well-characterized, ubiquitous, secreted immune evasion factors from the staphylococcal superantigen-like (SSL) protein family were selected for the development of a vaccine. Wild-type SSL3, 7 and 11, which inhibit signaling through Toll-like receptor 2, cleavage of complement component 5 and neutrophil function, respectively, were successfully combined into a stable, active fusion protein (PolySSL7311). Vaccination of mice with an attenuated form of the PolySSL7311 protein stimulated significantly elevated specific immunoglobulin G and splenocyte proliferation responses to each component relative to adjuvant-only controls. Vaccination with PolySSL7311, but not a mixture of the individual proteins, led to a > 102 reduction in S. aureus tissue burden compared with controls after peritoneal challenge. Comparable antibody responses were elicited after coadministration of the vaccine in either AddaVax (an analog of MF59) or an Alum-based adjuvant; but only AddaVax conferred a significant reduction in bacterial load, aligning with other studies that suggest both cellular and humoral immune responses are necessary for protective immunity to S. aureus. Anti-sera from mice immunized with PolySSL7311, but not individual proteins, partially neutralized the functional activities of SSL7. This study confirms the importance of these SSLs for the survival of S. aureus in vivo and suggests that PolySSL7311 is a promising vaccine candidate.

Experimental Identification of Potential Martian Biosignatures in Open and Closed Systems.

NASA's Perseverance and ESA's Rosalind Franklin rovers have the scientific goal of searching for evidence of ancient life on Mars. Geochemical biosignatures that form because of microbe-mineral interactions could play a key role in achieving this, as they can be preserved for millions of years on Earth, and the same could be true for Mars. Previous laboratory experiments have explored the formation of biosignatures under closed systems, but these do not represent the open systems that are found in natural martian environments, such as channels and lakes. In this study, we have conducted environmental simulation experiments using a global regolith simulant (OUCM-1), a thermochemically modelled groundwater, and an anaerobic microbial community to explore the formation of geochemical biosignatures within plausible open and closed systems on Mars. This initial investigation showed differences in the diversity of the microbial community developed after 28 days. In an open-system simulation (flow-through experiment), the acetogenic Acetobacterium (49% relative abundance) and the sulfate reducer Desulfosporomusa (43% relative abundance) were the dominant genera. Whereas in the batch experiment, the sulfate reducers Desulfovibrio, Desulfomicrobium, and Desulfuromonas (95% relative abundance in total) were dominant. We also found evidence of enhanced mineral dissolution within the flow-through experiment, but there was little evidence of secondary deposits in the presence of biota. In contrast, SiO2 and Fe deposits formed within the batch experiment with biota but not under abiotic conditions. The results from these initial experiments indicate that different geochemical biosignatures can be generated between open and closed systems, and therefore, biosignature formation in open systems warrants further investigation.

Habitability and Biosignature Formation in Simulated Martian Aqueous Environments.

Water present on early Mars is often assumed to have been habitable. In this study, experiments were performed to investigate the habitability of well-defined putative martian fluids and to identify the accompanying potential formation of biosignatures. Simulated martian environments were developed by combining martian fluid and regolith simulants based on the chemistry of the Rocknest sand shadow at Gale Crater. The simulated chemical environment was inoculated with terrestrial anoxic sediment from the Pyefleet mudflats (United Kingdom). These enrichments were cultured for 28 days and subsequently subcultured seven times to ensure that the microbial community was solely grown on the defined, simulated chemistry. The impact of the simulated chemistries on the microbial community was assessed by cell counts and sequencing of 16S rRNA gene profiles. Associated changes to the fluid and precipitate chemistries were established by using ICP-OES, IC, FTIR, and NIR. The fluids were confirmed as habitable, with the enriched microbial community showing a reduction in abundance and diversity over multiple subcultures relating to the selection of specific metabolic groups. The final community comprised sulfate-reducing, acetogenic, and other anaerobic and fermentative bacteria. Geochemical characterization and modeling of the simulant and fluid chemistries identified clear differences between the biotic and abiotic experiments. These differences included the elimination of sulfur owing to the presence of sulfate-reducing bacteria and more general changes in pH associated with actively respiring cells that impacted the mineral assemblages formed. This study confirmed that a system simulating the fluid chemistry of Gale Crater could support a microbial community and that variation in chemistries under biotic and abiotic conditions can be used to inform future life-detection missions.

Oligotrophic Growth of Nitrate-Dependent Fe2+-Oxidising Microorganisms Under Simulated Early Martian Conditions.

Nitrate-dependent Fe2+ oxidation (NDFO) is a microbially mediated process observed in many anaerobic, low-nutrient (oligotrophic) neutral-alkaline environments on Earth, which describes oxidation of Fe2+ to Fe3+ in tandem with microbial nitrate reduction. Evidence suggests that similar environments existed on Mars during the Noachian epoch (4.1-3.7 Ga) and in periodic, localised environments more recently, indicating that NDFO metabolism could have played a role in a potential early martian biosphere. In this paper, three NDFO microorganisms, Acidovorax sp. strain BoFeN1, Pseudogulbenkiania sp. strain 2002 and Paracoccus sp. strain KS1, were assessed for their ability to grow oligotrophically in simulated martian brines and in a minimal medium with olivine as a solid Fe2+ source. These simulant-derived media were developed from modelled fluids based on the geochemistry of Mars sample locations at Rocknest (contemporary Mars soil), Paso Robles (sulphur-rich soil), Haematite Slope (haematite-rich soil) and a Shergottite meteorite (common basalt). The Shergottite medium was able to support growth of all three organisms, while the contemporary Mars medium supported growth of Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002; however, growth was not accompanied by significant Fe2+ oxidation. Each of the strains was also able to grow in oligotrophic minimal media with olivine as the sole Fe2+ source. Biomineralised cells of Pseudogulbenkiania sp. strain 2002 were identified on the surface of the olivine, representing a potential biosignature for NDFO microorganisms in martian samples. The results suggest that NDFO microorganisms could have thrived in early martian groundwaters under oligotrophic conditions, depending on the local lithology. This can guide missions in identifying palaeoenvironments of interest for biosignature detection. Indeed, biomineralised cells identified on the olivine surface provide a previously unexplored mechanism for the preservation of morphological biosignatures in the martian geological record.

Prevalence of Self-reported Neurologic and Ocular Symptoms in Early Syphilis Cases.

BACKGROUND: Neurosyphilis, a complication of syphilis, can occur at any stage of infection. Measuring the prevalence of neurosyphilis is challenging, and there are limited data on the prevalence of neurologic or ocular symptoms among patients with syphilis. We sought to describe the prevalence of neurologic and/or ocular symptoms among early syphilis (ES) cases and the clinical management of symptomatic cases enrolled in the STD Surveillance Network (SSuN) Neuro/Ocular Syphilis Surveillance project. METHODS: Persons diagnosed with ES were selected for interviews based on current health department protocols in 5 participating SSuN jurisdictions from November 2016 through October 2017. All interviewed ES cases were screened for self-reported neurologic and/or ocular symptoms. Additional clinical information on diagnostic testing and treatment for cases concerning for neurosyphilis/ocular syphilis was obtained from providers. RESULTS: Among 9123 patients with ES who were interviewed, 151 (1.7%; 95% confidence interval [CI], 1.4%-1.9%) reported ≥ 1 neurologic or ocular symptom. Of the 53 (35%) who underwent lumbar puncture, 22 (42%) had documented abnormal cerebrospinal fluid, of which 21 (95%) were treated for neurosyphilis/ocular syphilis. Among the remaining 98 symptomatic patients with no documented lumbar puncture (65%), 12 (12%) were treated for and/or clinically diagnosed with neurosyphilis/ocular syphilis. CONCLUSIONS: We observed a low prevalence of self-reported neurologic and/or ocular symptoms in interviewed ES cases. Approximately one-third of ES cases who self-reported symptoms underwent further recommended diagnostic evaluation. Understanding barriers to appropriate clinical evaluation is important to ensuring appropriate management of patients with possible neurologic and/or ocular manifestations of syphilis.

Estimating Neonatal Herpes Simplex Virus Incidence and Mortality Using Capture-recapture, Florida.

BACKGROUND: Neonatal herpes simplex virus infection (nHSV) leads to severe morbidity and mortality, but national incidence is uncertain. Florida regulations require that healthcare providers report cases, and clinical laboratories report test results when herpes simplex virus (HSV) is detected. We estimated nHSV incidence using laboratory-confirmed provider-reported cases and electronic laboratory reports (ELR) stored separately from provider-reported cases. Mortality was estimated using provider-reported cases, ELR, and vital statistics death records. METHODS: For 2011-2017, we reviewed: provider-reported cases (infants ≤ 60 days of age with HSV infection confirmed by culture or polymerase chain reaction [PCR]), ELR of HSV-positive culture or PCR results in the same age group, and death certificates containing International Classification of Disease, Tenth Revision, codes for herpes infection: P35.2, B00.0-B00.9, and A60.0-A60.9. Provider-reported cases were matched against ELR reports. Death certificates were matched with provider and ELR reports. Chapman's capture-recapture method was used to estimate nHSV incidence and mortality. Mortality from all 3 sources was estimated using log-linear modeling. RESULTS: Providers reported 114 nHSV cases, and ELR identified 197 nHSV cases. Forty-six cases were common to both datasets, leaving 265 unique nHSV reports. Chapman's estimate suggests 483 (95% confidence interval [CI], 383-634) nHSV cases occurred (31.5 infections per 100 000 live births). The nHSV deaths were reported by providers (n = 9), ELR (n = 18), and vital statistics (n = 31), totaling 34 unique reports. Log-linear modeling estimates 35.8 fatal cases occurred (95% CI, 34-40). CONCLUSIONS: Chapman's estimates using data collected over 7 years in Florida conclude nHSV infections occurred at a rate of 1 per 3000 live births.

Synthesis of Organosilicon Ligands for Europium (III) and Gadolinium (III) as Potential Imaging Agents.

The relaxivity of MRI contrast agents can be increased by increasing the size of the contrast agent and by increasing concentration of the bound gadolinium. Large multi-site ligands able to coordinate several metal centres show increased relaxivity as a result. In this paper, an "aza-type Michael" reaction is used to prepare cyclen derivatives that can be attached to organosilicon frameworks via hydrosilylation reactions. A range of organosilicon frameworks were tested including silsesquioxane cages and dimethylsilylbenzene derivatives. Michael donors with strong electron withdrawing groups could be used to alkylate cyclen on three amine centres in a single step. Hydrosilylation successfully attached these to mono-, di-, and tri-dimethylsilyl-substituted benzene derivatives. The europium and gadolinium complexes were formed and studied using luminescence spectroscopy and relaxometry. This showed the complexes to contain two bound water moles per lanthanide centre and T1 relaxation time measurements demonstrated an increase in relaxivity had been achieved, in particular for the trisubstituted scaffold 1,3,5-tris((pentane-sDO3A)dimethylsilyl)benzene-Gd3. This showed a marked increase in the relaxivity (13.1 r1p/mM-1s-1).

PilVax, a novel Lactococcus lactis-based mucosal vaccine platform, stimulates systemic and mucosal immune responses to Staphylococcus aureus.

Most pathogens initiate infection via the mucosa, therefore delivery of vaccines directly to the mucosa is likely to be advantageous for stimulating protective immunity at the site of entry. PilVax is a novel mucosal vaccine platform that harnesses Lactococcus lactis bacteria engineered to stably express multiple copies of vaccine peptide antigens within pili, hair-like structures which extend from the cell wall. This strategy elicited systemic and mucosal antibody responses to a model antigen after intranasal immunization, but has not been tested for its capacity to stimulate protective mucosal immunity. A well-characterized linear B-cell epitope, D3(22-33) , from the fibronectin-binding protein A of Staphylococcus aureus was successfully introduced into PilVax and delivered intranasally to mice. Specific antipeptide immunoglobulin (Ig) G and IgA antibodies were detected in the serum and respiratory mucosa of vaccinated mice. Responses to the major pilus backbone protein Spy0128 were also assessed; robust antibody responses to this antigen were generated both systemically and in the respiratory and intestinal mucosa. Mice were challenged intranasally with the mouse-adapted S. aureus JSNZ strain and the S. aureus load quantified 7 days after challenge. Unexpectedly, exposure to PilVax, irrespective of the presence of the peptide, resulted in a significant reduction in S. aureus load in both the intestine and nasal mucosa (both P < 0.05) when compared with unvaccinated control mice. The mechanism(s) of protection are unclear, but merit further investigation to determine whether PilVax is a suitable platform for delivery of vaccine candidate antigens to the mucosa.

The Microbial Community of a Terrestrial Anoxic Inter-Tidal Zone: A Model for Laboratory-Based Studies of Potentially Habitable Ancient Lacustrine Systems on Mars.

Evidence indicates that Gale crater on Mars harboured a fluvio-lacustrine environment that was subjected to physio-chemical variations such as changes in redox conditions and evaporation with salinity changes, over time. Microbial communities from terrestrial environmental analogues sites are important for studying such potential habitability environments on early Mars, especially in laboratory-based simulation experiments. Traditionally, such studies have predominantly focused on microorganisms from extreme terrestrial environments. These are applicable to a range of Martian environments; however, they lack relevance to the lacustrine systems. In this study, we characterise an anoxic inter-tidal zone as a terrestrial analogue for the Gale crater lake system according to its chemical and physical properties, and its microbiological community. The sub-surface inter-tidal environment of the River Dee estuary, United Kingdom (53°21'15.40″ N, 3°10'24.95″ W) was selected and compared with available data from Early Hesperian-time Gale crater, and temperature, redox, and pH were similar. Compared to subsurface 'groundwater'-type fluids invoked for the Gale subsurface, salinity was higher at the River Dee site, which are more comparable to increases in salinity that likely occurred as the Gale crater lake evolved. Similarities in clay abundance indicated similar access to, specifically, the bio-essential elements Mg, Fe and K. The River Dee microbial community consisted of taxa that were known to have members that could utilise chemolithoautotrophic and chemoorganoheterotrophic metabolism and such a mixed metabolic capability would potentially have been feasible on Mars. Microorganisms isolated from the site were able to grow under environment conditions that, based on mineralogical data, were similar to that of the Gale crater's aqueous environment at Yellowknife Bay. Thus, the results from this study suggest that the microbial community from an anoxic inter-tidal zone is a plausible terrestrial analogue for studying habitability of fluvio-lacustrine systems on early Mars, using laboratory-based simulation experiments.

Nitrate-Dependent Iron Oxidation: A Potential Mars Metabolism.

This work considers the hypothetical viability of microbial nitrate-dependent Fe2+ oxidation (NDFO) for supporting simple life in the context of the early Mars environment. This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1-3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation.

Determination of Geochemical Bio-Signatures in Mars-Like Basaltic Environments.

Bio-signatures play a central role in determining whether life existed on early Mars. Using a terrestrial basalt as a compositional analog for the martian surface, we applied a combination of experimental microbiology and thermochemical modeling techniques to identify potential geochemical bio-signatures for life on early Mars. Laboratory experiments were used to determine the short-term effects of biota on the dissolution of terrestrial basalt, and the formation of secondary alteration minerals. The chemoorganoheterotrophic bacterium, Burkholderia sp. strain B_33, was grown in a minimal growth medium with and without terrestrial basalt as the sole nutrient source. No growth was detected in the absence of the basalt. In the presence of basalt, during exponential growth, the pH decreased rapidly from pH 7.0 to 3.6 and then gradually increased to a steady-state of equilibrium of between 6.8 and 7.1. Microbial growth coincided with an increase in key elements in the growth medium (Si, K, Ca, Mg, and Fe). Experimental results were compared with theoretical thermochemical modeling to predict growth of secondary alteration minerals, which can be used as bio-signatures, over a geological timescale. We thermochemically modeled the dissolution of the basalt (in the absence of biota) in very dilute brine at 25°C, 1 bar; the pH was buffered by the mineral dissolution and precipitation reactions. Preliminary results suggested that at the water to rock ratio of 1 × 107, zeolite, hematite, chlorite, kaolinite, and apatite formed abiotically. The biotic weathering processes were modeled by varying the pH conditions within the model to adjust for biologic influence. The results suggested that, for a basaltic system, the microbially-mediated dissolution of basalt would result in "simpler" secondary alteration, consisting of Fe-hydroxide and kaolinite, under conditions where the abiotic system would also form chlorite. The results from this study demonstrate that, by using laboratory-based experiments and thermochemical modeling, it is possible to identify secondary alteration minerals that could potentially be used to distinguish between abiotic and biotic weathering processes on early Mars. This work will contribute to the interpretation of data from past, present, and future life detection missions to Mars.

Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant.

Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

Engaging blind and partially sighted stakeholders in transformational change.

For non-profit organizations in the disability sector, engaging stakeholders with disabilities on matters of strategic planning is both a responsibility and an expectation. As part of our current strategic plan, which calls for organizational and systemic transformation, the Canadian National Institute for the Blind (CNIB) has engaged blind and partially sighted stakeholders alongside other interest groups to build and advocate for a more holistic model of vision healthcare and rehabilitation. This article describes the CNIB's multi-year process, including early-stage consultations, collaborative strategy development, and political advocacy and shares our organization's key success factors and learnings in creating meaningful, mutually beneficial engagement.

The consistency of beneficial fitness effects of mutations across diverse genetic backgrounds.

Parallel and convergent evolution have been remarkably common observations in molecular adaptation but primarily in the context of the same genotype adapting to the same conditions. These phenomena therefore tell us about the stochasticity and limitations of adaptation. The limited data on convergence and parallelism in the adaptation of different genotypes conflict as to the importance of such events. If the effects of beneficial mutations are highly context dependent (i.e., if they are epistatic), different genotypes should adapt through different mutations. Epistasis for beneficial mutations has been investigated but mainly through measurement of interactions between individually beneficial mutations for the same genotype. We examine epistasis for beneficial mutations at a broader genetic scale by measuring the fitness effects of two mutations beneficial for the ssDNA bacteriophage ID11 in eight different, related genotypes showing 0.3-3.7% nucleotide divergence from ID11. We found no evidence for sign epistasis, but the mutations tended to have much smaller or no effects on fitness in the new genotypes. We found evidence for diminishing-returns epistasis; the effects were more beneficial for lower-fitness genotypes. The patterns of epistasis were not determined by phylogenetic relationships to the original genotype. To improve our understanding of the patterns of epistasis, we fit the data to a model in which each mutation had a constant, nonepistatic phenotypic effect across genotypes and the phenotype-fitness map had a single optimum. This model fit the data well, suggesting that epistasis for these mutations was due to nonlinearity in the phenotype-fitness mapping and that the likelihood of parallel evolution depends more on phenotype than on genotype.

A pilot, open clinical study of depakote in the treatment of cocaine abuse.

The effectiveness of increasing levels of valproic acid on cocaine abstinence was evaluated in an open-label trial of 55 subjects. Significantly lower percentages of individuals with valproate serum levels above 50 mcg/ml reported cocaine use and had less positive urinalyses as compared to those with serum levels less than 50 mcg/ml. The total number of days of cocaine use decreased significantly (p < 0.001) and improved levels of subject functioning were found with increasing serum levels of valproic acid. This pilot study offers confirmation that the divalproex sodium form of valproic acid, when used in sufficient serum levels, may provide an effective pharmacologic adjunct in the management and treatment of cocaine dependency. Copyright 2001 John Wiley & Sons, Ltd.