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233 records · Page 13

Physiological, genomic, and sulfur isotopic characterization of methanol metabolism by Desulfovibrio carbinolicus

Methanol is often considered as a non-competitive substrate for methanogenic archaea, but an increasing number of sulfate-reducing microorganisms (SRMs) have been reported to be capable of respiring with methanol as an electron donor. A better understanding of the fate of methanol in natural or artificial anaerobic systems thus requires knowledge of the methanol dissimilation by SRMs. In this study, we describe the growth kinetics and sulfur isotope effects of Desulfovibrio carbinolicus, a methanol-oxidizing sulfate-reducing deltaproteobacterium, together with its genome sequence and annotation. D. carbinolicus can grow with a series of alcohols from methanol to butanol. Compared to longer-chain alcohols, however, specific growth and respiration rates decrease by several fold with methanol as an electron donor. Larger sulfur isotope fractionation accompanies slowed growth kinetics, indicating low chemical potential at terminal reductive steps of respiration. In a medium containing both ethanol and methanol, D. carbinolicus does not consume methanol even after the cessation of growth on ethanol. Among the two known methanol dissimilatory systems, the genome of D. carbinolicus contains the genes coding for alcohol dehydrogenase but lacks enzymes analogous to methanol methyltransferase. We analyzed the genomes of 52 additional species of sulfate-reducing bacteria that have been tested for methanol oxidation. There is no apparent relationship between phylogeny and methanol metabolizing capacity, but most gram-negative methanol oxidizers grow poorly, and none carry homologs for methyltransferase (mtaB). Although the amount of available data is limited, it is notable that more than half of the known gram-positive methanol oxidizers have both enzymatic systems, showing enhanced growth relative to the SRMs containing only alcohol dehydrogenase genes. Thus, physiological, genomic, and sulfur isotopic results suggest that D. carbinolicus and close relatives have the ability to metabolize methanol but likely play a limited role in methanol degradation in most natural environments.

54 ENVIRONMENTAL SCIENCES↗

Rapid Computational Identification of Therapeutic Targets for Pathogens

Biological threats continue to persist and evolve as an important challenge to national security. There are multiple ways in which novel viral pathogens could emerge to pose a serious threat to human health. This project developed a pathogen target identification tool that can rapidly respond to a novel or emerging viral biological threat. A set of computational tools were developed that provide detailed information on the newly sequenced genes, their protein products and the drug target sites for the proteins that are best suited for biological countermeasure development. Three key innovations were developed in the project. 1) Development of a new extensive database of protein pocket structures with structure-based search algorithms to rapidly link novel protein targets with the complete collection of previously experimentally solved protein structures. 2) A novel clustering pipeline was introduced to group matching structures and associated small-molecule binding ligands into a consensus protein pocket with the associated small-molecule chemotypes predicted to fit in the pocket site. The matching experimentally solved structures were used to inform the value of different target sites. 3) Where there are viral protein targets with pockets structurally matched to similar human proteins, a biological knowledge graph, which links molecular interactions with human disease, was used to further assess the potential negative impact of a viral protein target with similarities to human proteins that could have important off target side effects. In total, the project produced a new resource for rapid and detailed assessment of promising targets for countermeasures, reflecting the ongoing wet lab, clinical, and computational data being collected. These capabilities will improve the ability to respond to a biological threat in multiple domains.

59 BASIC BIOLOGICAL SCIENCES↗

Adaptation to climate change through strategic integration of long fallow into cropping system in a dryland Mediterranean-type environment

The crop-growing region of Western Australia characterized by a Mediterranean-type climate is projected to become warmer and drier. Appropriate selection of crop sequences will be of importance to cope with these climatic changes for this largest grain-producing region of Australia. Through linking a widely used crop model (APSIM), 26 General Circulation Models (GCMs) with one Shared Socioeconomic Pathway (SSP585) and economic analysis, we explored how the climate change would affect dryland wheat cropping and whether/how long fallow (the practice of leaving a field out of production for an entire growing season) could be integrated into wheat cropping system in Western Australia. The potential adaptation of long fallow into wheat system was assessed with four fixed rotations (fallow-wheat, fallow-wheat-wheat, fallow-wheat-wheat-wheat, and fallow-wheat-wheat-wheat-wheat) and four flexible sowing rule-based rotations (the land was fallowed if sowing rule was not met), compared with continuous wheat. The simulation results at four representing locations show that climate change would have negative impacts on both yield and economic return of continuous wheat cropping in Western Australia. Wheat after fallow out-yielded and out-profited wheat after wheat under future climate. But integrating fallow into wheat cropping systems with the above fixed rotations would lead to yield and economic loss. By contrast, cropping systems in which fallowing took place when sowing condition could not be met at a certain time would achieve comparable yield and economic return to continuous wheat, with wheat yield being only 5% less than continuous wheat and the gross margin being $12ha -1 more than continuous wheat averaged across locations. We highlight strategic integration of long fallow into cropping system in a dryland Mediterranean-type environment would have a great potential to cope with future climate change. These findings can be extended into other Mediterranean-type cropping regions in Australia and beyond.

54 ENVIRONMENTAL SCIENCES↗

Colour gradients of low-redshift galaxies in the DESI Legacy Imaging Survey

ABSTRACT Radial colour gradients within galaxies arise from gradients of stellar age, metallicity, and dust reddening. Large samples of colour gradients from wide-area imaging surveys can complement smaller integral-field spectroscopy data sets and can be used to constrain galaxy formation models. Here, we measure colour gradients for low-redshift galaxies (z < 0.1) using photometry from the DESI Legacy Imaging Survey DR9. Our sample comprises ∼93 000 galaxies with spectroscopic redshifts and ∼574 000 galaxies with photometric redshifts. We focus on gradients across a radial range 0.5Reff to Reff, which corresponds to the inner disc of typical late-type systems at low redshift. This region has been the focus of previous statistical studies of colour gradients and has recently been explored by spectroscopic surveys such as MaNGA. We find that the colour gradients of most galaxies in our sample are negative (redder towards the centre), consistent with the literature. We investigate empirical relationships between colour gradient, average g − r and r − z colour, Mr, M⋆, and sSFR. Trends of gradient strength with Mr (M⋆) show an inflection around Mr ∼ −21 ($\log _{10} \, M_\star /\mathrm{M_\odot }\sim 10.5$). Below this mass, colour gradients become steeper with increasing M⋆, whereas colour gradients in more massive galaxies become shallower. We find that positive gradients (bluer stars at smaller radii) are typical for galaxies of $M_{\star }\sim 10^{8}\, \mathrm{M_\odot }$. We compare our results to age and metallicity gradients in two data sets derived from fits of different stellar population libraries to MaNGA spectra, but find no clear consensus explanation for the trends we observe. Both MaNGA data sets seem to imply a significant contribution from dust reddening, in particular, to explain the flatness of colour gradients along the red sequence.

Astronomy & Astrophysics↗

The direct and indirect drivers shaping RNA viral communities in grassland soils

ABSTRACT Recent studies have revealed diverse RNA viral communities in soils. Yet, how environmental factors influence soil RNA viruses remains largely unknown. Here, we recovered RNA viral communities from bulk metatranscriptomes sequenced from grassland soils managed for 5 years under multiple environmental conditions including water content, plant presence, cultivar type, and soil depth. More than half of the unique RNA viral contigs (64.6%) were assigned with putative hosts. About 74.7% of these classified RNA viral contigs are known as eukaryotic RNA viruses suggesting eukaryotic RNA viruses may outnumber prokaryotic RNA viruses by nearly three times in this grassland. Of the identified eukaryotic RNA viruses and the associated eukaryotic species, the most dominant taxa were Mitoviridae with an average relative abundance of 72.4%, and their natural hosts, Fungi with an average relative abundance of 56.6%. Network analysis and structural equation modeling support that soil water content, plant presence, and type of cultivar individually demonstrate a significant positive impact on eukaryotic RNA viral richness directly as well as indirectly on eukaryotic RNA viral abundance via influencing the co-existing eukaryotic members. A significant negative influence of soil depth on soil eukaryotic richness and abundance indirectly impacts soil eukaryotic RNA viral communities. These results provide new insights into the collective influence of multiple environmental and community factors that shape soil RNA viral communities and offer a structured perspective of how RNA virus diversity and ecology respond to environmental changes. IMPORTANCE Climate change has been reshaping the soil environment as well as the residing microbiome. This study provides field-relevant information on how environmental and community factors collectively shape soil RNA communities and contribute to ecological understanding of RNA viral survival under various environmental conditions and virus-host interactions in soil. This knowledge is critical for predicting the viral responses to climate change and the potential emergence of biothreats.

59 BASIC BIOLOGICAL SCIENCES↗

The influence of spaceflight on the astronaut salivary microbiome and the search for a microbiome biomarker for viral reactivation

Background: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella , Megasphera , and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria , Absconditabacteria , and Abiotrophia and a negative correlation between Oribacterium , Veillonella , and Haemophilus . There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions.

59 BASIC BIOLOGICAL SCIENCES↗

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli

59 BASIC BIOLOGICAL SCIENCES↗

Sulfurospirillum diekertiae sp. nov., a tetrachloroethene-respiring bacterium isolated from contaminated soil

Two anaerobic, tetrachloroethene- (PCE-) respiring bacterial isolates, designated strain ACS DCE T and strain ACS TCE , were characterized using a polyphasic approach. Cells were Gram-stain-negative, motile, non-spore-forming and shared a vibrioid- to spirillum-shaped morphology. Optimum growth occurred at 30°C and 0.1–0.4% salinity. The pH range for growth was pH 5.5–7.5, with an optimum at pH 7.2. Hydrogen, formate, pyruvate and lactate as electron donors supported respiratory reductive dechlorination of PCE to cis-1,2-dichloroethene (cDCE) in strain ACS DCE T and of PCE to trichloroethene (TCE) in strain ACS TCE . Both strains were able to grow with pyruvate under microaerobic conditions. Nitrate, elemental sulphur, and thiosulphate were alternative electron acceptors. Autotrophic growth was not observed and acetate served as carbon source for both strains. The major cellular fatty acids were C 16:1 ω7c, C 16:0 , C 14:0 and C 18:1 ω7c. Both genomes feature a circular plasmid. Strains ACS DCE T and ACS TCE were previously assigned to the candidate species 'Sulfurospirillum acididehalogenans'. Here, based on key genomic features and pairwise comparisons of whole-genome sequences, including average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity, strains ACS DCE T and ACS TCE , 'Ca. Sulfurospirillum diekertiae' strains SL2-1 and SL2-2, and the unclassified Sulfurospirillum sp. strain SPD-1 are grouped into one distinct species separate from previously described Sulfurospirillum species. Compared to Sulfurospirillum multivorans and Sulfurospirillum halorespirans, which dechlorinate PCE to cDCE without substantial TCE accumulation, these five strains produce TCE or cDCE as the end product. In addition, some cellular fatty acids (e.g., C 16:0 3OH, C 17:0 iso 3OH, C 17:0 2OH) were detected in strains ACS DCE T and ACS TCE but not in other Sulfurospirillum species. On the basis of phylogenetic, physiological and phenotypic characteristics, 'Ca. Sulfurospirillum acididehalogenans' and 'Ca. Sulfurospirillum diekertiae' are proposed to be merged into one novel species within the genus Sulfurospirillum, for which the name Sulfurospirillum diekertiae sp. nov. is proposed. Finally, the type strain is ACS DCE T (=JCM 33349 T = KCTC 15819 T =CGMCC 1.5292 T ).

59 BASIC BIOLOGICAL SCIENCES↗

Antimicrobial activity of peptoids against Metallo-β-lactamase-producing Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , and other WHO priority pathogens, including Candida auris

Abstract Aims The World Health Organization has identified ESKAPE bacteria and Candida auris as priority pathogens, emphasizing an urgent need for novel antimicrobials to combat them. This study aimed to explore the therapeutic potential of antimicrobial peptidomimetics, specifically peptoids with sequence-specific N-substituted glycines, against ESKAPEE pathogens, including metallo-β-lactamase (MBL) producers, as well as C. auris strains. Methods and results This study evaluated activity of the peptoids against the multidrug-resistant priority pathogens. The peptoid TM8 (with an N-decyl alkyl chain) demonstrated a geometric mean minimum inhibitory concentration (MIC) of 7.8 μg ml−1 against MBL-producing bacteria, and 5.5 μg ml−1 against C. auris. TM8 showed synergy with ciprofloxacin, enhancing its effectiveness 4-fold against NDM-1-producing Klebsiella pneumoniae. No antagonism was seen when TM8 was used with either conventional antibiotics or antifungals. Peptoids that had therapeutic indices below 3 were generally more hydrophobic, due to either alkyl chains or bromine. Scanning electron microscopy and live-dead staining assay on peptoid-treated C. auris confirmed morphological changes and killing activity, respectively. Furthermore, the peptoid could effectively inhibit biofilm formation by C. auris. Conclusion Peptoids demonstrated antibacterial activity against ESKAPEE, particularly against MBL-producing Gram-negative bacteria. Additionally, they exhibited antifungal and anti-biofilm activities against C. auris strains.

Biotechnology & Applied Microbiology↗

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Finally, sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

59 BASIC BIOLOGICAL SCIENCES↗

Development of a recombinant anti-VEGFR2-EPCAM bispecific antibody to improve antiangiogenic efficiency

Highlights: • Targeted therapy can be a useful therapeutic method along with traditional methods for breast cancer treatment. • Decreasing Akt/Nf-Kβ signaling pathway is crucial target in order to decreasing angiogenesis in TNBC. • The recombinant antibody can have inhibitory effects on production IL6,8, MMP2, and MMP9. Tumor progression and metastasis, especially in invasive cancers (such as triple-negative breast cancer [TNBC]), depend on angiogenesis, in which vascular epithelial growth factor (VEGF)/vascular epithelial growth factor receptor [1] has a decisive role, followed by the metastatic spread of cancer cells. Although some studies have shown that anti-VEGFR2/VEGF monoclonal antibodies demonstrated favorable results in the clinic, this approach is not efficient, and further investigations are needed to improve the quality of cancer treatment. Besides, the increased expression of epithelial cell adhesion molecule (EpCAM) in various cancers, for instance, invasive breast cancer, contributes to angiogenesis, facilitating the migration of tumor cells to other parts of the body. Thus, the main goal of our study was to target either VEGFR2 or EpCAM as pivotal players in the progression of angiogenesis in breast cancer. Regarding cancer therapy, the production of bispecific antibodies is easier and more cost-effective compared to monoclonal antibodies, targeting more than one antigen or receptor; for this reason, we produced a recombinant antibody to target cells expressing EpCAM and VEGFR2 via a bispecific antibody to decrease the proliferation and metastasis of tumor cells. Following the cloning and expression of our desired anti-VEGFR2/EPCAM sequence in E. coli, the accuracy of the expression was confirmed by Western blot analysis, and its binding activities to VEGFR2 and EPCAM on MDA-MB-231 and MCF-7 cell lines were respectively indicated by flow cytometry. Then, its anti-proliferative potential was indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assay to evaluate inhibitory effects of the antibody on tumor cells. Subsequently, the data indicated that migration, invasion, and angiogenesis were inhibited in breast cancer cell lines via the bispecific antibody. Furthermore, cytokine analysis indicated that the bispecific antibody could moderate interleukin 8 (IL-8) and IL-6 as key mediators in angiogenesis progression in breast cancer. Thus, our bispecific antibody could be considered as a promising candidate tool to decrease angiogenesis in TNBC.

60 APPLIED LIFE SCIENCES↗

The direct and indirect drivers shaping RNA viral communities in grassland soil

Recent studies have revealed diverse RNA viral communities in soils. Yet, how environmental factors influence soil RNA viruses remains largely unknown. Here, we recovered RNA viral communities from 24 metatranscriptomes sequenced from grassland soils managed under a range of environmental conditions including 1) water content: 100% and 25% water holding capacity, 2) plant presence: planted with tall wheatgrass (Thinopyrum ponticum) and bare soil, 3) cultivar type: Alkar and Jose, and 4) soil depth: 0-5 cm and 15-25 cm. The recovered RNA viral communities were novel with nearly one-third of the RNA viral contigs uniquely detected in the studied grassland. The classified RNA viral contigs are mostly known as eukaryotic RNA viruses (74.7%) belonging to Phyla Duplornaviricota, Kitrinoviricota, Lenarviricota, and Pisuviricota. Eukaryotic RNA viruses of Family Mitoviridae as well as their natural hosts, Fungi, are one of the most dominant taxa. Consistent with the results of nonmetric multidimensional scaling analysis, the four environmental conditions (water content, plant presence, cultivar, and soil depth) significantly influence the assemblages of soil RNA viral communities as suggested by the correlation analysis and the random forest algorithm. The modularity analysis of the factor network and the structural equation modeling further support the hierarchical associations among the four environmental factors and the community factors representing the co-existing eukaryotic, prokaryotic, and RNA viral communities. The soil water content, plant presence, and type of cultivar demonstrate a significant positive impact on eukaryotic RNA viral richness directly as well as indirectly on eukaryotic RNA viral abundance via influencing the co-existing eukaryotic members in this soil. Our data also provide statistical support for the negative influence of soil depth on soil eukaryotic richness and abundances resulting in its indirect impact on soil eukaryotic RNA viral communities. This study provides field-relevant information on how environmental and community factors collectively shape soil RNA communities and contribute to ecological understanding of RNA viral survival under various environmental conditions and virus-host interactions in soil.

Wu, Ruonan↗

Depth-dependent links between microbial taxa and nitrous oxide emissions in a long-term cotton cropping system employing soil health practices

Long-term management practices can shape soil microbial communities in ways that influence nitrogen (N) dynamics and nitrous oxide (N 2 O) emissions. We leverage a 41-year continuous cotton cropping experiment with contrasting tillage, cover cropping, and N fertilization regimes to investigate how these long-term strategies influence soil microbial communities and their associations with N 2 O fluxes during the cotton growing season. Using 16S rRNA gene metabarcoding, we assessed microbial composition in surface and subsurface soils and evaluated its relationship with temporal N 2 O emissions. Among the management practices, N fertilization – a known driver of N 2 O emissions – had the strongest effect on microbial community composition and was linked to a greater number of taxa correlated to N 2 O emissions, particularly in surface soils. Soil pH emerged as a key variable influencing microbial structure across depth and was negatively associated with both N 2 O emissions and microbial composition in the surface layers of fertilized soils. In total, 57 archaeal/bacterial taxa were correlated with N 2 O fluxes, but only seven were shared across depths, suggesting distinct microbial contributors in surface and subsurface soils. Several of these taxa have been previously reported to be associated with N and C cycling processes such as nitrate respiration or carbon turnover, indicating functional context to their correlation with N 2 O fluxes. Temporal shifts in the abundance of key taxa aligned with seasonal peaks in N 2 O emissions, notably in early and late August, and were most pronounced under conventional tillage, hairy vetch cover cropping, and N fertilization. While 16S-based associations cannot confirm functional gene presence or activity, these findings demonstrate that long-term fertilization and associated soil acidification are dominant drivers of microbial shifts linked to N 2 O emissions and highlight the importance of accounting for depth-specific and seasonal microbial dynamics when evaluating management impacts on greenhouse gas emissions.

16S rRNA gene sequencing↗

Rapid Patterning and Advanced Device Structures for Low Cost Manufacturable Crystalline Si IBC Cells

Recent record efficiency Si cells have had an interdigitated back contact (IBC) design where both positive and negative contacts are fabricated on the back side of the Si wafer. Many industry groups have investigated IBC cells but used photolithography to pattern the rear contacts although it is considered impractical for low cost, high volume manufacturing. Alternative patterning methods using lasers and mechanical masking have been utilized in fabrication of patterned regions in IBC Si solar cells to replace photolithography. Another exciting advance in Si solar cell technology has been replacing the high temperature diffusion of doped regions inside the crystalline Si wafer with low temperature deposition of very thin layers of doped amorphous hydrogenated Si (a-Si:H) on the Si wafer. This is called a Si heterojunction (HJ) device. The a-Si:H provides excellent passivation of surface defects and has produced the highest open circuit voltages (VOC) of any Si solar cell device structure. The Institute of Energy Conversion (IEC) demonstrated the first IBC-HJ solar cell combining these two strategies in 2007 and has fabricated IBC-HJ cells with 20% efficiency using three photolithography steps. Despite the demonstrated efficiency potential of this device by industry groups (>26%) there is no commercial production because of the challenges of patterning and processing the structure in an industrial environment. Our work sought to address that challenge. The objective of this 3 year project was to develop the processing for the IBC-HJ Si solar cell using lasers for patterning and contact formation instead of photolithography. Laser patterning enables rapid, contactless manufacturing of patterned regions. We intensively studied laser fired contacts, laser patterning of the a-Si multi-layer stacks and metal layers and the use of plasma shadow masks. After an exhaustive focus in the first year on the laser fired emitter (LFE) and contact (LFC), we were unable to achieve VOC greater than 660 mV, compared to the 720 mV required to meet our milestones. We discovered that an additional issue with our original IBC structure was the presence of an inversion layer at the back surface connecting the p and n regions which reduced the VOC and fill factor (FF). We developed innovative methods for characterizing the inversion layer. These two limitations in the original design lead to development of a new IBC-HJ process sequence and device structure which we called Plasma Masked Laser Processed (PMLP). It retained the original high efficiency features including manufacturability. The LFC was replaced with a standard n-type a-Si HJ contact. The inversion layer was eliminated by replacing the previous p-type stack in the gap with an n-type or SiN stack. The PMLP used laser ablation of a multi-layer a-Si stack followed by chemical etching to open the n-contact. It required deposition of a patterned stack through a mask in the plasma deposition chamber. This turned out to be a source of significant problems due to inevitable unwanted deposition ‘leakage’ under the mask. This formed a blocking contact on the emitter which significantly reduced Voc and FF. Several iterations in PMLP device structure and chemical etching steps resulted in a large improvements, e.g. the efficiency increasing from 3 to 15% and Voc from 450 to 660 mV but they were unable to completely eliminate it. The best IBC-HJ device we fabricated had only 15% efficiency. For perspective, our standard FHJ devices had 20% efficiency.

14 SOLAR ENERGY↗

STITCHES: creating new scenarios of climate model output by stitching together pieces of existing simulations

Abstract. Climate model output emulation has long been attempted to support impact research, mainly to fill in gaps in the scenario space. Given the computational cost of running coupled earth system models (ESMs), which are usually the domain of supercomputers and require on the order of days to weeks to complete a century-long simulation, only a handful of different scenarios are usually chosen to externally force ESM simulations. An effective emulator, able to run on standard computers in times of the order of minutes rather than days could therefore be used to derive climate information under scenarios that were not run by ESMs. Lately, the necessity of accounting for internal variability has also made the availability of initial-condition ensembles, under a specific scenario, important, further increasing the computational demand. At least so far, emulators have been limited to simplified ESM-like output, either seasonal, annual, or decadal averages of basic quantities, like temperature and precipitation, often emulated independently of one another. With this work, we propose a more comprehensive solution to ESM output emulation. Our emulator, STITCHES, uses existing archives of earth system models' (ESMs) scenario experiments to construct ESM-like output under new scenarios or enrich existing initial-condition ensembles, which is what other emulators also aim to do. Importantly, however, STITCHES' output has the same characteristics of the ESM output it sets out to emulate: multivariate, spatially resolved, and high frequency, representing both the forced component and the internal variability around it. STITCHES extends the idea of time sampling – according to which climate outcomes are stratified by the global warming level at which they manifest themselves, irrespective of the scenario and time at which they occur – to the construction of a continuous history of ESM-like output over the whole 21st century, consistent with a 21st-century trajectory of global surface air temperature (GSAT) derived from the scenario that has been chosen as the target of the emulation. STITCHES does so by first splitting the target GSAT trajectory into decade-long windows, then matching each window in turn to a decade-long window within an existing model simulation from the available scenario runs according to its proximity to the target in absolute size of the temperature anomaly and its rate of change. A look-up table is therefore created of a sequence of existing experiment–time-window combinations that, when stitched together, create a GSAT trajectory “similar” to the target. Importantly, we can then stitch together much more than GSAT from these windows, i.e., any output that the ESM has saved for these existing experiment–time-window combinations, at any frequency and spatial scale available in its archive. We show that the stitching does not introduce artifacts in the great majority of cases (we look at temperature and precipitation at monthly frequency and on the native grid of the ESM and at an index of ENSO activity, the Southern Oscillation Index). This is true even if the criteria for the identification of the decades to be stitched together are chosen to work for a smoothed time series of annual GSAT, a result we expect given the larger amount of noise affecting most other variables at finer spatial scales and higher frequencies, which therefore are more “forgiving” of the stitching. We successfully test the method's performance over many ESMs and scenarios. Only a few exceptions surface, but these less-than-optimal outcomes are always associated with a scarcity of the archived simulations from which we can gather the decade-long windows that form the building blocks of the emulated time series. In the great majority of cases, STITCHES' performance is satisfactory according to metrics that reward consistency in trends, interannual and inter-ensemble variance, and autocorrelation structure of the time series stitched together. The method therefore can be used to create ESM-like output according to new scenarios, on the basis of a trajectory of GSAT produced according to that scenario, which could be easily obtained by a simple climate model. It can also be used to increase the size of existing initial-condition ensembles. There are aspects of our emulator that will immediately disqualify it for specific applications, like when climate information is needed whose characteristics result from accumulated quantities over windows of times longer than those used as pieces by STITCHES, droughts longer than a decade for example. But for many applications, we argue that a stitched product can satisfy the climate information needs of impact researchers. STITCHES cannot emulate ESM output from scenarios that result in GSAT trajectories outside of the envelope available in the archive, nor can it emulate trajectories with shapes different from existing ones (overshoots with negative derivative, for example). Therefore, the size and characteristics of the available archives of ESM output are the principal limitations for STITCHES' deployment. Thus, we argue for the possibility of designing scenario experiments within, for example, the next phase of the Coupled Model Intercomparison Project according to new principles, relieved of the need to produce a number of similar trajectories that vary only in radiative forcing strength but more strategically covering the space of temperature anomalies and rates of change.

54 ENVIRONMENTAL SCIENCES↗

Identification and characterization of mono- and bifunctional galactan synthases in the pediatric pathogen Kingella kingae

The emerging pediatric pathogen Kingella kingae elaborates a lipopolysaccharide (LPS) that is extended with a galactofuranose homopolymer called galactan, which is a key virulence determinant that contributes to resistance to complement-mediated and neutrophil-mediated killing. Previous work has demonstrated that the pamABCDE locus is required for galactan synthesis. In this study, mutational studies suggested that the pamC gene product is a UDP-galactofuranose (Galf) transferase and is the galactan synthase. Analysis of genome sequence data revealed two distinct pamC alleles designated pamC1 and pamC2, which correlate with the two galactan structures in K. kingae. Examination of isogenic mutants expressing either pamC1 or pamC2 demonstrated that the pamC alleles are the determinants of galactan structure. Experiments with recombinant PamC1 and PamC2 in vitro established that these proteins are galactan synthases capable of extending synthetic Galf disaccharide acceptors in the presence of UDP-Galf. Homology analysis identified critical amino acids that are essential for PamC1 and PamC2 enzymatic activity both in vitro and in K. kingae. Structural analysis of the in vitro-modified synthetic acceptors implicated PamC1 as a monofunctional enzyme capable of generating a β-(1 → 5) Galf linkage and PamC2 as a bifunctional enzyme capable of generating β-(1 → 3) and β-(1 → 6) Galf linkages. This study advances our understanding of the GT2 family of UDP-galactofuranosyltransferases.

60 APPLIED LIFE SCIENCES↗