SEARCH · bioRxiv Science

Results for “Bioengineering”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

bioRxiv2021

Gene signatures and host-parasite interactions revealed by dual single-cell profiling of Plasmodium vivax liver infection

Malaria-causing P. vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell responses to them. Here, we leverage a bioengineered human microliver platform to culture Thai clinical isolates of P. vivax in primary human hepatocytes and conduct transcriptional profiling of infected cultures. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.

microbiology↗

bioRxiv2021

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

cell biology↗

bioRxiv2021

Decoding assembly of alpha-helical transmembrane pores through intermediate states

Membrane-active pore-forming alpha-helical peptides and proteins are well known for their dynamic assembly mechanism and it has been critical to delineate the pore-forming structures in the membrane. Previously, attempts have been made to elucidate their assembly mechanism and there is a large gap due to complex pathways by which these membrane-active pores impart their effect. Here we demonstrate the multi-step structural assembly pathway of alpha-helical peptide pores formed by a 37 amino-acid synthetic peptide, pPorU based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report detectable intermediates states during membrane insertion and pore formation of pPorU. The fully assembled pore is functional and exhibited unusually large stable conductance and voltage-dependent gating, generally applicable to a range of pore-forming proteins. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. Mutant peptides that differ from wild-type peptides produced noisy, unstable intermediate states and low conductance pores, demonstrating sequence specificity in the pore-formation process supported by molecular dynamics simulations. We suggest that our study contributes to understanding the mechanism of action of alpha-helical pores and antimicrobial peptides and should be of broad interest to bioengineers to build peptide-based nanopore sensors.

biophysics↗

bioRxiv2021

Identification of Immunodominant T-lymphocyte Epitope Peptides in HPV 1, 2 and 3 L1 Protein for Novel Cutaneous Wart Vaccine

BackgroundCommon warts and flat warts are caused by the human papillomavirus (HPV). Peak incidence of wart infection occurs in schoolchildren aged 12-16, where prevalence can be as high as 20%. Traditional treatments aimed at destruction of wart tissue have low clearance rates and high recurrence rates. Occasional reports have even shown warts becoming malignant and progressing into verrucous carcinoma. Current licensed HPV vaccines largely target higher-risk oncogenic HPV types, but do not provide coverage of low-risk types associated with warts. To date, little attention has been given to the development of effective, anti-viral wart treatments. ObjectiveThis study aims to identify immunodominant T-lymphocyte epitopes from the L1 major capsid protein of HPV 1, 2 and 3, a foundational step in bioengineering a peptide-based vaccine for warts. MethodsCytotoxic T-cell and helper T-cell epitopes were predicted using an array of immunoinformatic tools against a reference panel of frequently observed MHC-I and MHC-II alleles. Predicted peptides were ranked based on IC50 and IFN-{gamma} Inducer Scores, respectively, and top performing epitopes were synthesized and subjected to in vitro screening by IFN-{gamma} enzyme-linked immunosorbent spot assay (ELISpot). Independent trials were conducted using PBMCs of healthy volunteers. Final chosen peptides were fused with flexible GS linkers in silico to design a novel polypeptide vaccine. ResultsSeven immunodominant peptides screened from 44 predicted peptides were included in the vaccine design, selected to elicit specific immune responses across MHC class I and class II, and across HPV types. Evaluation of the vaccines properties suggest that the vaccine is stable, non-allergenic, and provides near complete global population coverage (>99%). Solubility prediction and rare codon analysis indicate that the DNA sequence encoding the vaccine is suitable for high level expression in Escherichia coli. ConclusionsIn sum, this study demonstrates the potential and lays the framework for the development of a peptide-based vaccine against warts.

immunology↗

bioRxiv2021

TMAO regulates the rigidity of kinesin-propelled microtubules

We demonstrate that the rigidity of the microtubules (MTs), propelled by kinesins in an in vitro gliding assay, can be modulated using the deep-sea osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of the MTs is modulated over a wide range. By employing this approach, we are able to reduce the persistence length of MTs, a measure of MT rigidity, [~]8 fold using TMAO of the concentration of 1.5 M. The rigidity of gliding MTs can be restored by eliminating the TMAO from the gliding assay. This work offers a simple strategy to regulate the rigidity of kinesin-propelled MTs in situ and would widen the applications of biomolecular motors in nanotechnology, materials science, and bioengineering.

biophysics↗

bioRxiv2021

Characterization of pyridomycin B reveals the formation of functional groups in antimycobacterial pyridomycin

Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacteria tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in pyridomycin core system is an important pharmacophoric group and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-F-G). Bioinformatics analysis reveals that SDR family protein Pyr2 functions as a 3-oxoacyl ACP reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module, in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of the pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin and enable creating chemical diversity of pyridomycin derivatives. ImportanceTuberculosis (TB) is one of the worlds leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacteria tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilize a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand the understanding the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.

microbiology↗

bioRxiv2021

Structures of the Cyanobacterial Phycobilisome

The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 [A] (rods) to 2.7 [A] (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

biochemistry↗

bioRxiv2022

basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method

Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These features facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein engineering and multiplexed gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Users can export sequence data and create build instructions for manual or acoustic liquid-handling platforms. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serializable in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection is compatible with BASIC and Golden Gate using BsaI. It encompasses vectors containing six antibiotic resistance markers and five origins of replication from different compatibility groups, including a temperature-sensitive variant. To make the collection accessible we deposited it on Addgene under an OpenMTA agreement. Furthermore, vector sequences are accessible from within the basicsynbio application programming interface along with other collections of parts and linkers, providing an ideal environment to design assemblies for bioengineering applications using BASIC.

synthetic biology↗

bioRxiv2022

A real-time cellular thermal shift assay (RT-CETSA) to monitor target engagement

Determining a molecules mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a proteins unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered that withstood unfolding at temperatures greater than 90 degrees Celsius and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. The RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule. SignificanceValidating target engagement is a critical step when characterizing a small molecule modulator. The cellular thermal shift assay (CETSA) is a common approach to examine target engagement, as alterations in the thermal stability of a protein can be conferred by ligand binding. An advantage of CETSA is that it does not require modification of the protein target or small molecule. Major limitations are the throughput and ease-of-use, as the traditional detection method uses western blots, which limits the number of samples that can be processed. Higher-throughput CETSA methods have been developed but are performed at a single temperature and require target-specific optimization. We developed a high-throughput real-time CETSA to circumvent these challenges, providing a rapid and cost-effective strategy to assess on-target activity of a small molecule in living cells.

pharmacology and toxicology↗

bioRxiv2022

Rubisco forms a lattice inside alpha-carboxysomes

Bacteria employ microcompartments to sequester enzymatic processes, either for purposes of protecting cellular contents from reactive intermediates or as a way of increasing reaction efficiency. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species metabolisms, microcompartments have recently become a target of protein engineering. The shells can be reassembled from purified proteins, and the full operons can be functionally expressed outside their native prokaryotes and can remain functional following purification. Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo electron tomography to image -carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo in situ, and determined a novel arrangement of the enzyme. We find that the H. neapolitanus Rubisco polymerizes in vivo, mediated by the small Rubisco subunit. These fibrils can further pack to form a lattice with six-fold pseudo-symmetry. This arrangement preserves freedom of motion and accessibility around the Rubisco active site and the binding sites for two other carboxysome proteins, CsoSCA (a carbonic anhydrase) and the disordered CsoS2, even at Rubisco concentrations exceeding 800 M. This characterization of Rubisco cargo inside the -carboxysome provides new insight into the balance between order and disorder in microcompartment organization.

biophysics↗

bioRxiv2022

Rhythm of The Night (and Day): Predictive metabolic modeling of circadian growth in Chlamydomonas

Algal cells experience strong circadian rhythms under diurnal light, with regular changes in both biomass composition and transcriptomic environment. However, most metabolic models - critical tools for bioengineering organisms - assume a steady state. The conflict between these assumptions and the reality of the cellular environment make such models inappropriate for algal cells, creating a significant obstacle in engineering cells that are viable under natural light. By transforming a set of discreet transcriptomic measurements from synchronized Chlamydomonas cells grown in a 12/12 diel light regime (1) into continuous curves, we produced a complete representation of the cells transcriptome that can be interrogated at any arbitrary timepoint. We clustered these curves, in order to find genes that were expressed in similar patterns, and then also used it to build a metabolic model that can accumulate and catabolize different biomass components over the course of a day. This model predicts qualitative phenotypical outcomes for the sta6 mutant, including excess lipid accumulation (2) and a failure to thrive when grown diurnally in minimal media (3), representing a qualitative prediction of phenotype from genotype even under dynamic conditions. We also extended this approach to simulate all single-knockout mutants with genes represented in the model and identified potential targets for rational engineering efforts. SIGNIFICANCE STATEMENTWe have developed the first transient metabolic model for diurnal growth of algae based on experimental data and capable of predicting phenotype from genotype. This model enables us to evaluate the impact of genetic and environmental changes on the growth, biomass composition and intracellular fluxes of the model green alga, Chlamydomonas reinhardtii. The availability of this model will enable faster and more efficient design of cells for production of fuels, chemicals and pharmaceuticals.

systems biology↗

bioRxiv2022

Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves

Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 {degrees}C, 500 mol photons m-2 s-1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin-Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin-Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if [≤]21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of [≥]62%. Lastly, we discuss interactions between the OPPP and the cytosolic oxidation-reduction cycle in supplying leaf cytosolic NADPH. Main ConclusionThe oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by {approx}5%.

plant biology↗

bioRxiv2022

Exploring the possible role of hybridization in the evolution of photosynthetic pathways in Flaveria (Asteraceae), the prime model of C4 photosynthesis evolution

Flaveria (Asteraceae) is the prime model for the study of C4 photosynthesis evolution and seems to support a stepwise acquisition of the pathway through C3-C4 intermediate phenotypes, still existing in Flaveria today. Molecular phylogenies of Flaveria based on concatenated data matrices are currently used to reconstruct the complex sequence of trait shifts during C4 evolution. To assess the possible role of hybridization in C4 evolution in Flaveria, we re-analyzed transcriptome data of 17 Flaveria species to infer the extent of gene tree discordance and possible reticulation events. We found massive gene tree discordance as well as reticulation along the backbone and within clades containing C3-C4 intermediate and C4-like species. An early hybridization event between two C3 species might have triggered C4 evolution in the genus. The clade containing all C4 species plus the C4-like species F. vaginata and F. palmeri is robust but of hybrid origin involving F. angustifolia and F. sonorensis (both C3-C4 intermediate) as parental lineages. Hybridization seems to be a driver of C4 evolution in Flaveria and likely promoted the fast acquisition of C4 traits. This new insight can be used in further exploring C4 evolution and can inform C4 bioengineering efforts.

evolutionary biology↗

bioRxiv2022

The optimal docking strength for reversibly tethered kinases

Many kinases use reversible docking interactions to augment the specificity of their catalytic domains. Such docking interactions are often structurally independent of the catalytic domain, which allow for flexible combination of modules in evolution and in bioengineering. The affinity of docking interactions spans several orders of magnitude. This led us to ask how the affinity of the docking interaction affects enzymatic activity, and how to pick the optimal interaction module to complement a given substrate. Here, we develop equations that predict the optimal binding strength of a kinase docking interaction and validate it using numerical simulations and steady-state phosphorylation kinetics for tethered protein kinase A. We show that a kinase-substrate pair has an optimum docking strength that depends on their enzymatic constants, the tether architecture, the substrate concentration and the kinetics of the docking interactions. We show that a reversible tether enhances phosphorylation rates most when: I) The docking strength is intermediate, II) the substrate is non-optimal, III) the substrate concentration is low, IV) the docking interaction has rapid exchange kinetics, and V) the tether optimizes the effective concentration of the intra-molecular reaction. This work serves as a framework for interpreting mutations in kinase docking interactions and as a design guide for engineering enzyme scaffolds.

biophysics↗

bioRxiv2022

Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome

Carboxysomes are proteaceous bacterial microcompartments (BMCs) that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating carbonic anhydrase and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyses the dehydration of bicarbonate into CO2, the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural characterization of native carboxysomes, including the shell and the internal organization, is currently limited to low-resolution tomography studies. Notably, the degree of heterogeneity of the shell, and the internal arrangement of enzymes, remain poorly understood. Here, we report the structural characterization of a native -carboxysome from a marine cyanobacterium by single-particle cryo-EM. We determine the structure of RuBisCO enzyme at 2.9 [A] resolution. In addition, we obtain low-resolution maps of the icosahedral protein shell and the concentric interior organisation. In combination with artificial intelligence (AI)-driven modelling approaches, we exploited these maps to propose a complete atomic model of an intact carboxysome. This study provides insight into carboxysome structure and protein-protein interactions involved in carboxysome assembly. Advanced knowledge about carboxysome architecture and structural plasticity is critical for not only a better understanding of biological carbon fixation mechanism but also repurposing carboxysomes in synthetic biology for biotechnological applications.

biophysics↗

bioRxiv2022

Enhancing the Fitness of Embryoid Bodies and Organoids by Chemical Cytoprotection

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, and kidney) can be optimized by using the CEPT small molecule cocktail, a polypharmacology approach that ensures cytoprotection and cell survival. Application of CEPT (chroman 1, emricasan, polyamines, trans-ISRIB) for just 24 hours during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, and cellular differentiation. Various qualification methods confirmed that CEPT treatment consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

developmental biology↗

bioRxiv2022

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

biophysics↗

bioRxiv2022

Bats as ecosystem engineers in iron ore caves in the Carajas National Forest, Brazilian Amazonia

Ecosystem engineers are organisms able to modify their environment by changing the distribution of materials and energy, with effects on biotic and abiotic ecosystem components. Several ecosystem engineers are known, but for most of them the mechanisms behind their influence are poorly known. We detail the role of bats as ecosystem engineers in iron ore caves in the Carajas National Forest, Brazilian Amazonia, an area with > 1,500 caves, some holding [~]150,000 bats. We analyzed the chemical composition of guano deposits in bat caves, radiocarbon-dated those deposits, and elucidated the chemical mechanisms involved and the role the bat guano has on structuring those caves. The insect-composed guano was rich in organic matter, with high concentrations of carbon, nitrogen, phosphorus pentoxide and ferric oxide, plus potassium oxide, calcium and sulfur trioxide. Radiocarbon dating indicated guano deposits between 22,000 and 1,800 years old. The guano pH was mainly acid (from 2.1 to 5.6). Percolating waters in those bat caves were also acid (pH reaching 1.5), with the presence of phosphate, iron, calcium, nitrate and sulfate. Acid solutions due to guano decomposition and possible microbial activity produced various forms of corrosion on the cave[s] floor and walls, resulting in their enlargement. Caves with active or inactive bat colonies had, on average, horizontal projections 4.5 times larger, areas 4.4 times larger, and volumes 5.0 times bigger than the reginal average, plus more abundant, diversified and bigger speleothems. In an example of bioengineering, the long-term presence of bats (> 22,000 years) and the guano deposits they produce, mediated by biological and chemical interactions along millennia, resulted in very unique ecological, evolutionary and geomorphological processes, whose working are just beginning to be better understood by science. However, the current expansion of mineral activities coupled with the loosening of licensing and cave protection rules is a real conservation threat to the bat caves in Carajas. The destruction of those caves would represent and unacceptable loss of both speleological and biological heritage and we urge that, whenever they occur, bat caves and their colonies must be fully protected and left off-limits of mineral extraction.

ecology↗