In silico analysis of design of experiment methods for metabolic pathway optimization.

Microbial cell factories allow the production of chemicals presenting an alternative to traditional fossil fuel-dependent production. However, finding the optimal expression of production pathway genes is crucial for the development of efficient production strains. Unlike sequential experimentation, combinatorial optimization captures the relationships between pathway genes and production, albeit at the cost of conducting multiple experiments. Fractional factorial designs followed by linear modeling and statistical analysis reduce the experimental workload while maximizing the information gained during experimentation. Although tools to perform and analyze these designs are available, guidelines for selecting appropriate factorial designs for pathway optimization are missing. In this study, we leverage a kinetic model of a seven-genes pathway to simulate the performance of a full factorial strain library. We compare this approach to resolution V, IV, III, and Plackett Burman (PB) designs. Additionally, we evaluate the performance of these designs as training sets for a random forest algorithm aimed at identifying best-producing strains. Evaluating the robustness of these designs to noise and missing data, traits inherent to biological datasets, we find that while resolution V designs capture most information present in full factorial data, they necessitate the construction of a large number of strains. On the other hand, resolution III and PB designs fall short in identifying optimal strains and miss relevant information. Besides, given the small number of experiments required for the optimization of a pathway with seven genes, linear models outperform random forest. Consequently, we propose the use of resolution IV designs followed by linear modeling in Design-Build-Test-Learn (DBTL) cycles targeting the screening of multiple factors. These designs enable the identification of optimal strains and provide valuable guidance for subsequent optimization cycles.

Combinatorial optimization of pathway, process and media for the production of p-coumaric acid by Saccharomyces cerevisiae.

Microbial cell factories are instrumental in transitioning towards a sustainable bio-based economy, offering alternatives to conventional chemical processes. However, fulfilling their potential requires simultaneous screening for optimal media composition, process and genetic factors, acknowledging the complex interplay between the organism's genotype and its environment. This study employs statistical design of experiments to systematically explore these relationships and optimize the production of p-coumaric acid (pCA) in Saccharomyces cerevisiae. Two rounds of fractional factorial designs were used to identify factors with a significant effect on pCA production, which resulted in a 168-fold variation in pCA titre. Moreover, a significant interaction between the culture temperature and expression of ARO4 highlighted the importance of simultaneous process and strain optimization. The presented approach leverages the strengths of experimental design and statistical analysis and could be systematically applied during strain and bioprocess design efforts to unlock the full potential of microbial cell factories.

Machine Learning-Guided Optimization of p-Coumaric Acid Production in Yeast.

Industrial biotechnology uses Design-Build-Test-Learn (DBTL) cycles to accelerate the development of microbial cell factories, required for the transition to a biobased economy. To use them effectively, appropriate connections between the phases of the cycle are crucial. Using p-coumaric acid (pCA) production in Saccharomyces cerevisiae as a case study, we propose the use of one-pot library generation, random screening, targeted sequencing, and machine learning (ML) as links during DBTL cycles. We showed that the robustness and flexibility of the ML models strongly enable pathway optimization and propose feature importance and Shapley additive explanation values as a guide to expand the design space of original libraries. This approach allowed a 68% increased production of pCA within two DBTL cycles, leading to a 0.52 g/L titer and a 0.03 g/g yield on glucose.

Biosignature Detection and MinION Sequencing of Antarctic Cryptoendoliths After Exposure to Mars Simulation Conditions.

In the search for life in our Solar System, Mars remains a promising target based on its proximity and similarity to Earth. When Mars transitioned from a warmer, wetter climate to its current dry and freezing conditions, any putative extant life probably retreated into habitable refugia such as the subsurface or the interior of rocks. Terrestrial cryptoendolithic microorganisms (i.e., those inhabiting rock interiors) thus represent possible modern-day Mars analogs, particularly those from the hyperarid McMurdo Dry Valleys in Antarctica. As DNA is a strong definitive biosignature, given that there is no known abiotic chemistry that can polymerize nucleobases, we investigated DNA detection with MinION sequencing in Antarctic cryptoendoliths after an ∼58-sol exposure in MARTE, a Mars environmental chamber capable of simulating martian temperature, pressure, humidity, ultraviolet (UV) radiation, and atmospheric composition, in conjunction with protein and lipid detection. The MARTE conditions resulted in changes in community composition and DNA, proteins, and cell membrane-derived lipids remained detectable postexposure. Of the multitude of extreme environmental conditions on Mars, UV radiation (specifically UVC) is the most destructive to both cells and DNA. As such, we further investigated if a UVC exposure corresponding to ∼278 martian years would impede DNA detection via MinION sequencing. The MinION was able to successfully detect and sequence DNA after this UVC radiation exposure, suggesting its utility for life detection in future astrobiology missions focused on finding relatively recently exposed biomarkers inside possible martian refugia.

The Atacama Rover Astrobiology Drilling Studies (ARADS) Project.

With advances in commercial space launch capabilities and reduced costs to orbit, humans may arrive on Mars within a decade. Both to preserve any signs of past (and extant) martian life and to protect the health of human crews (and Earth's biosphere), it will be necessary to assess the risk of cross-contamination on the surface, in blown dust, and into the near-subsurface (where exploration and resource-harvesting can be reasonably anticipated). Thus, evaluating for the presence of life and biosignatures may become a critical-path Mars exploration precursor in the not-so-far future, circa 2030. This Special Collection of papers from the Atacama Rover Astrobiology Drilling Studies (ARADS) project describes many of the scientific, technological, and operational issues associated with searching for and identifying biosignatures in an extreme hyperarid region in Chile's Atacama Desert, a well-studied terrestrial Mars analog environment. This paper provides an overview of the ARADS project and discusses in context the five other papers in the ARADS Special Collection, as well as prior ARADS project results.

In Situ Real-Time Monitoring for Aseptic Drilling: Lessons Learned from the Atacama Rover Astrobiology Drilling Studies Contamination Control Strategy and Implementation and Application to the Icebreaker Mars Life Detection Mission.

In 2019, the Atacama Rover Astrobiology Drilling Studies (ARADS) project field-tested an autonomous rover-mounted robotic drill prototype for a 6-Sol life detection mission to Mars (Icebreaker). ARADS drilled Mars-like materials in the Atacama Desert (Chile), one of the most life-diminished regions on Earth, where mitigating contamination transfer into life-detection instruments becomes critical. Our Contamination Control Strategy and Implementation (CCSI) for the Sample Handling and Transfer System (SHTS) hardware (drill, scoop and funnels) included out-of-simulation protocol testing (out-of-sim) for hardware decontamination and verification during the 6-Sol simulation (in-sim). The most effective five-step decontamination combined safer-to-use sterilants (3%_hydrogen-peroxide-activated 5%_sodium-hypochlorite), and in situ real-time verification by adenosine triphosphate (ATP) and Signs of Life Detector (SOLID) Fluorescence Immunoassay for characterization hardware bioburden and airborne contaminants. The 20- to 40-min protocol enabled a 4-log bioburden reduction down to <0.1 fmoles ATP detection limit (funnels and drill) to 0.2-0.7 fmoles (scoop) of total ATP. The (post-cleaning) hardware background was 0.3 to 1-2 attomoles ATP/cm2 (cleanliness benchmark background values) equivalent to ca. 1-10 colony forming unit (CFU)/cm2. Further, 60-100% of the in-sim hardware background was ≤3-4 bacterial cells/cm2, the threshold limit for Class <7 aseptic operations. Across the six Sols, the flux of airborne contaminants to the drill sites was ∼5 and ∼22 amoles ATP/(cm2·day), accounting for an unexpectedly high Fluorescence Intensity (FI) signal (FI: ∼6000) against aquatic cyanobacteria, but negligible anthropogenic contribution. The SOLID immunoassay also detected microorganisms from multiple habitats across the Atacama Desert (anoxic, alkaline/acidic microenvironments in halite fields, playas, and alluvial fans) in both airborne and post-cleaning hardware background. Finally, the hardware ATP background was 40-250 times lower than the ATP in cores. Similarly, the FI peaks (FImax) against the microbial taxa and molecular biomarkers detected in the post-cleaned hardware (FI: ∼1500-1600) were 5-10 times lower than biomarkers in drilled sediments, excluding significant interference with putative biomarker found in cores. Similar protocols enable the acquisition of contamination-free materials for ultra-sensitive instruments analysis and the integrity of scientific results. Their application can augment our scientific knowledge of the distribution of cryptic life on Mars-like grounds and support life-detection robotic and human-operated missions to Mars.

Life Detection and Microbial Biomarker Profiling with Signs of Life Detector-Life Detector Chip During a Mars Drilling Simulation Campaign in the Hyperarid Core of the Atacama Desert.

The low organic matter content in the hyperarid core of the Atacama Desert, together with abrupt temperature shifts and high ultraviolet radiation at its surface, makes this region one of the best terrestrial analogs of Mars and one of the best scenarios for testing instrumentation devoted to in situ planetary exploration. We have operated remotely and autonomously the SOLID-LDChip (Signs of Life Detector-Life Detector Chip), an antibody microarray-based sensor instrument, as part of a rover payload during the 2019 NASA Atacama Rover Astrobiology Drilling Studies (ARADS) Mars drilling simulation campaign. A robotic arm collected drilled cuttings down to 80 cm depth and loaded SOLID to process and assay them with LDChip for searching for molecular biomarkers. A remote science team received and analyzed telemetry data and LDChip results. The data revealed the presence of microbial markers from Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Cyanobacteria to be relatively more abundant in the middle layer (40-50 cm). In addition, the detection of several proteins from nitrogen metabolism indicates a pivotal role in the system. These findings were corroborated and complemented on "returned samples" to the lab by a comprehensive analysis that included DNA sequencing, metaproteomics, and a metabolic reconstruction of the sampled area. Altogether, the results describe a relatively complex microbial community with members capable of nitrogen fixation and denitrification, sulfur oxidation and reduction, or triggering oxidative stress responses, among other traits. This remote operation demonstrated the high maturity of SOLID-LDChip as a powerful tool for remote in situ life detection for future missions in the Solar System.

A Mission Simulating the Search for Life on Mars with Automated Drilling, Sample Handling, and Life Detection Instruments Performed in the Hyperarid Core of the Atacama Desert, Chile.

We report on a field demonstration of a rover-based drilling mission to search for biomolecular evidence of life in the arid core of the Atacama Desert, Chile. The KREX2 rover carried the Honeybee Robotics 1 m depth The Regolith and Ice Drill for Exploration of New Terrains (TRIDENT) drill and a robotic arm with scoop that delivered subsurface fines to three flight prototype instruments: (1) The Signs of Life Detector (SOLID), a protein and biomolecule analyzer based on fluorescence sandwich microarray immunoassay; (2) the Planetary In Situ Capillary Electrophoresis System (PISCES), an amino acid analyzer based on subcritical water extraction coupled to microchip electrophoresis analysis; and (3) a Wet Chemistry Laboratory cell to measure soluble ions using ion selective electrodes and chronopotentiometry. A California-based science team selected and directed drilling and sampling of three sites separated by hundreds of meters that included a light-toned basin area showing evidence of aqueous activity surrounded by a rocky desert pavement. Biosignatures were detected in basin samples collected at depths ranging from 20 to 80 cm but were not detected in the surrounding area. Subsurface stratigraphy of the units drilled was interpreted from drill sensor data as fine-scale layers of sand/clay sediments interspersed with layers of harder material in the basins and a uniform subsurface composed of course-to-fine sand in the surroundings. The mission timeline and number of commands sent to accomplish each activity were tracked. The deepest sample collected (80 cm) required 55 commands, including drilling and delivery to three instruments. Elapsed time required for drilling and sample handling was less than 3 hours to collect sample from 72 cm depth, including time devoted to recovery from a jammed drill. The experiment demonstrated drilling, sample transfer technologies, and instruments that accomplished successful detection of biomolecular evidence of life in one of the most biologically sparse environments on Earth.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three ß-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest ß-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt.

Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB.

Mars-like UV Flux and Ionizing Radiation Differently Affect Biomarker Detectability in the Desert Cyanobacterium Chroococcidiopsis as Revealed by the Life Detector Chip Antibody Microarray.

The effect of a Mars-like UV flux and γ-radiation on the detectability of biomarkers in dried cells of Chroococcidiopsis sp. CCMEE 029 was investigated using a fluorescence sandwich microarray immunoassay. The production of anti-Chroococcidiopsis antibodies allowed the immunoidentification of a reduced, though still detectable, signal in dried cells mixed with phyllosilicatic and sulfatic Mars regolith simulants after exposure to 6.8 × 105 kJ/m2 of a Mars-like UV flux. No signal was detected in dried cells that were not mixed with minerals after 1.4 × 105 kJ/m2. For γ-radiation (60Co), no detectable variations of the fluorescence signal occurred in dried cells exposed to 113 kGy compared to non-irradiated dried cells. Our results suggest that immunoassay-based techniques could be used to detect life tracers eventually present in the martian subsurface in freshly excavated materials only if shielded from solar UV. The high structural integrity of biomarkers irradiated with γ-radiation that mimics a dose accumulated in 13 Myr at 2 m depth from the martian surface has implications for the potential detectability of similar organic molecules/compounds by future life-detection missions such as the ExoMars Rosalind Franklin rover.

Lipid Profiles From Fresh Biofilms Along a Temperature Gradient on a Hydrothermal Stream at El Tatio (Chilean Andes), as a Proxy for the Interpretation of Past and Present Biomarkers Beyond Earth.

Hydrothermal systems and their deposits are primary targets in the search for fossil evidence of life beyond Earth. However, to learn how to decode fossil biomarker records in ancient hydrothermal deposits, we must first be able to interpret unambiguously modern biosignatures, their distribution patterns, and their association with physicochemical factors. Here, we investigated the molecular and isotopic profile of microbial biomarkers along a thermal gradient (from 29 to 72°C) in a hot spring (labeled Cacao) from El Tatio, a geyser field in the Chilean Andes with abundant opaline silica deposits resembling the nodular and digitate structures discovered on Mars. As a molecular forensic approach, we focused on the analysis of lipid compounds bearing recognized resistance to degradation and the potential to reconstruct the paleobiology of an environment on a broader temporal scale than other, more labile, biomolecules. By exploiting the lipid biomarkers' potential to diagnose biological sources and carbon fixation pathways, we reconstructed the microbial community structure and its ecology along the Cacao hydrothermal transect. The taxonomic adscription of the lipid biomarkers was qualitatively corroborated with DNA sequencing analysis. The forensic capacity of the lipid biomarkers to identify biosources in fresh biofilms was validated down to the genus level for Roseiflexus, Chloroflexus, and Fischerella. We identified lipid biomarkers and DNA of several new cyanobacterial species in El Tatio and reported the first detection of Fischerella biomarkers at a temperature as high as 72°C. This, together with ecological peculiarities and the proportion of clades being characterized as unclassified, illustrates the ecological singularity of El Tatio and strengthens its astrobiological relevance. The Cacao hydrothermal ecosystem was defined by a succession of microbial communities and metabolic traits associated with a high- (72°C) to low-(29°C) temperature gradient that resembled the inferred metabolic sequence events from the 16S rRNA gene universal phylogenetic tree from thermophilic to anoxygenic photosynthetic species and oxygenic phototrophs. The locally calibrated DNA-validated lipidic profile in the Cacao biofilms provided a modern (molecular and isotopic) end member to facilitate the recognition of past biosources and metabolisms from altered biomarkers records in ancient silica deposits at El Tatio analogous to Martian opaline silica structures.

Enzyme-constrained models predict the dynamics of Saccharomyces cerevisiae growth in continuous, batch and fed-batch bioreactors.

Genome-scale, constraint-based models (GEM) and their derivatives are commonly used to model and gain insights into microbial metabolism. Often, however, their accuracy and predictive power are limited and enable only approximate designs. To improve their usefulness for strain and bioprocess design, we studied here their capacity to accurately predict metabolic changes in response to operational conditions in a bioreactor, as well as intracellular, active reactions. We used flux balance analysis (FBA) and dynamic FBA (dFBA) to predict growth dynamics of the model organism Saccharomyces cerevisiae under different industrially relevant conditions. We compared simulations with the latest developed GEM for this organism (Yeast8) and its enzyme-constrained version (ecYeast8) herein described with experimental data and found that ecYeast8 outperforms Yeast8 in all the simulations. EcYeast8 was able to predict well-known traits of yeast metabolism including the onset of the Crabtree effect, the order of substrate consumption during mixed carbon cultivation and production of a target metabolite. We showed how the combination of ecGEM and dFBA links reactor operation and genetic modifications to flux predictions, enabling the prediction of yields and productivities of different strains and (dynamic) production processes. Additionally, we present flux sampling as a tool to analyse flux predictions of ecGEM, of major importance for strain design applications. We showed that constraining protein availability substantially improves accuracy of the description of the metabolic state of the cell under dynamic conditions. This therefore enables more realistic and faithful designs of industrially relevant cell-based processes and, thus, the usefulness of such models.

Time-Integrative Multibiomarker Detection in Triassic-Jurassic Rocks from the Atacama Desert: Relevance to the Search for Basic Life Beyond Earth.

Detecting evidence of life on other planetary bodies requires a certain understanding of known biomarkers and their chemical nature, preservation potential, or biological specificity. In a planetary search for life, carbonates are of special interest due to their known association with life as we know it. On Earth, carbonates serve as an invaluable paleogeochemical archive of fossils of up to billions of years old. Here, we investigated biomarker profiles on three Chilean Triassic-Jurassic sedimentary records regarding our search for signs of past and present life over ∼200 Ma. A multianalytical platform that combines lipid-derived biomarkers, metaproteomics, and a life detector chip (LDChip) is considered in the detection of biomolecules with different perdurability and source-diagnosis potential. The combined identification of proteins with positive LDChip inmunodetections provides metabolic information and taxonomic affiliation of modern/subrecent biosignatures. Molecular and isotopic analysis of more perdurable hydrocarbon cores allows for the identification of general biosources and dominant autotrophic pathways over time, as well as recreation of prevailing redox conditions over ∼200 Ma. We demonstrate how extraterrestrial life detection can benefit from the use of different biomarkers to overcome diagnosis limitations due to a lack of specificity and/or alteration over time. Our findings have implications for future astrobiological missions to Mars.

Geomicrobiological Heterogeneity of Lithic Habitats in the Extreme Environment of Antarctic Nunataks: A Potential Early Mars Analog.

Nunataks are permanent ice-free rocky peaks that project above ice caps in polar regions, thus being exposed to extreme climatic conditions throughout the year. They undergo extremely low temperatures and scarcity of liquid water in winter, while receiving high incident and reflected (albedo) UVA-B radiation in summer. Here, we investigate the geomicrobiology of the permanently exposed lithic substrates of nunataks from Livingston Island (South Shetlands, Antarctic Peninsula), with focus on prokaryotic community structure and their main metabolic traits. Contrarily to first hypothesis, an extensive sampling based on different gradients and multianalytical approaches demonstrated significant differences for most geomicrobiological parameters between the bedrock, soil, and loose rock substrates, which overlapped any other regional variation. Brevibacillus genus dominated on bedrock and soil substrates, while loose rocks contained a diverse microbial community, including Actinobacteria, Alphaproteobacteria and abundant Cyanobacteria inhabiting the milder and diverse microhabitats within. Archaea, a domain never described before in similar Antarctic environments, were also consistently found in the three substrates, but being more abundant and potentially more active in soils. Stable isotopic ratios of total carbon (δ 13C) and nitrogen (δ 15N), soluble anions concentrations, and the detection of proteins involved in key metabolisms via the Life Detector Chip (LDChip), suggest that microbial primary production has a pivotal role in nutrient cycling at these exposed areas with limited deposition of nutrients. Detection of stress-resistance proteins, such as molecular chaperons, suggests microbial molecular adaptation mechanisms to cope with these harsh conditions. Since early Mars may have encompassed analogous environmental conditions as the ones found in these Antarctic nunataks, our study also contributes to the understanding of the metabolic features and biomarker profiles of a potential Martian microbiota, as well as the use of LDChip in future life detection missions.

A multiplex antigen microarray for simultaneous IgG and IgM detection against SARS-CoV-2 reveals higher seroprevalence than reported.

The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 µg ml-1 ) and IgG (~0.017 µg ml-1 ) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.

Similar incidence of coronavirus disease 2019 (COVID-19) in patients with rheumatic diseases with and without hydroxychloroquine therapy.

BACKGROUND: Hydroxychloroquine is not efficacious as post-exposure prophylaxis against coronavirus disease 2019 (COVID-19). It is not known whether as pre-exposure prophylaxis it may prevent COVID-19. OBJECTIVE: To compare the incidence of COVID-19 in Spanish patients with autoimmune rheumatic diseases treated with and without hydroxychloroquine. PATIENTS AND METHODS: Retrospective electronic record review, from February 27th to June 21st, 2020, of patients with autoimmune inflammatory diseases followed at two academic tertiary care hospitals in Seville, Spain. The cumulative incidence of confirmed COVID-19, by PCR or serology, was compared between patients with and without hydroxychloroquine as part of their treatment of autoimmune inflammatory diseases. RESULTS: Among 722 included patients, 290 (40%) were receiving hydroxychloroquine. During the seventeen-week study period, 10 (3.4% [95% CI: 1.7%-6.7%] cases of COVID-19 were registered among patients with hydroxychloroquine and 13 (3.0% [1.6%-5.1%]) (p = 0.565) in those without hydroxychloroquine. COVID-19 was diagnosed by PCR in four (1.4%, 95% CI 0.38%-3.5%) subject with hydroxychloroquine and six (1.4%, 95% CI 0.5%-3.0%) without hydroxychloroquine (p = 0.697). Three patients on hydroxychloroquine and four patients without hydroxychloroquine were admitted to the hospital, none of them required to be transferred to the intensive care unit and no patient died during the episode. CONCLUSIONS: The incidence and severity of COVID-19 among patients with autoimmune rheumatic diseases with and without hydroxychloroquine was not significantly different.

Oncolytic adenovirus with hyaluronidase activity that evades neutralizing antibodies: VCN-11.

Tumor targeting and intratumoral virus spreading are key features for successful oncolytic virotherapy. VCN-11 is a novel oncolytic adenovirus, genetically modified to express hyaluronidase (PH20) and display an albumin-binding domain (ABD) on the hexon. ABD allows the virus to self-coat with albumin when entering the bloodstream and evade neutralizing antibodies (NAbs). Here, we validate VCN-11 mechanism of action and characterize its toxicity. VCN-11 replication, hyaluronidase activity and binding to human albumin to evade NAbs was evaluated. Toxicity and efficacy of VCN-11 were assessed in mice and hamsters. Tumor targeting, and antitumor activity was analyzed in the presence of NAbs in several tumor models. VCN-11 induced 450 times more cytotoxicity in tumor cells than in normal cells. VCN-11 hyaluronidase production was confirmed by measuring PH20 activity in vitro and in virus-infected tumor areas in vivo. VCN-11 evaded NAbs from different sources and tumor targeting was demonstrated in the presence of high levels of NAbs in vivo, whereas the control virus without ABD was neutralized. VCN-11 showed a low toxicity profile in athymic nude mice and Syrian hamsters, allowing treatments with high doses and fractionated administrations without major toxicities (up to 1.2x1011vp/mouse and 7.5x1011vp/hamster). Fractionated intravenous administrations improved circulation kinetics and tumor targeting. VCN-11 antitumor efficacy was demonstrated in the presence of NAbs against Ad5 and itself. Oncolytic adenovirus VCN-11 disrupts tumor matrix and displays antitumor effects even in the presence of NAbs. These features make VCN-11 a safe promising candidate to test re-administration in clinical trials.

The Complex Molecules Detector (CMOLD): A Fluidic-Based Instrument Suite to Search for (Bio)chemical Complexity on Mars and Icy Moons.

Organic chemistry is ubiquitous in the Solar System, and both Mars and a number of icy satellites of the outer Solar System show substantial promise for having hosted or hosting life. Here, we propose a novel astrobiologically focused instrument suite that could be included as scientific payload in future missions to Mars or the icy moons: the Complex Molecules Detector, or CMOLD. CMOLD is devoted to determining different levels of prebiotic/biotic chemical and structural targets following a chemically general approach (i.e., valid for both terrestrial and nonterrestrial life), as well as their compatibility with terrestrial life. CMOLD is based on a microfluidic block that distributes a liquid suspension sample to three instruments by using complementary technologies: (1) novel microscopic techniques for identifying ultrastructures and cell-like morphologies, (2) Raman spectroscopy for detecting universal intramolecular complexity that leads to biochemical functionality, and (3) bioaffinity-based systems (including antibodies and aptamers as capture probes) for finding life-related and nonlife-related molecular structures. We highlight our current developments to make this type of instruments flight-ready for upcoming Mars missions: the Raman spectrometer included in the science payload of the ESAs Rosalind Franklin rover (Raman Laser Spectrometer instrument) to be launched in 2022, and the biomarker detector that was included as payload in the NASA Icebreaker lander mission proposal (SOLID instrument). CMOLD is a robust solution that builds on the combination of three complementary, existing techniques to cover a wide spectrum of targets in the search for (bio)chemical complexity in the Solar System.