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bioRxiv2020

Deciphering the genetic and epidemiological landscape of mitochondrial DNA abundance

Somatically acquired whole chromosome loss in nucleated blood cells is an indicator of immune aging and genomic instability. However, little is known about aging, lifestyle and genetic factors influencing mitochondrial (MT) DNA abundance. In this study, MT DNA abundance was estimated from the weighted intensities of probes mapping to the MT genome in 295,150 participants from the UK Biobank. We found that abundance of MT DNA was significantly elevated in women compared to men, was negatively correlated with advanced age, more packyears, greater body-mass index, higher frailty index as well as elevated red and white blood cell count and, importantly, lower mortality. In addition, several biochemistry markers in blood related to cholesterol metabolism, ion homeostasis and kidney function were found to be significantly associated with MT DNA abundance. By performing a genome-wide association study, we identified 50 independent regions genome-wide significantly associated with MT DNA abundance which harbour multiple genes involved in the immune system, cancer as well as mitochondrial function. Using mixed effects models, we estimated the SNP-heritability of MT DNA abundance to be around 8%. To investigate the consequence of altered MT DNA abundance, we performed a phenome-wide association study and found that MT DNA abundance is involved in risk for leukaemia, hematologic diseases as well as hypertension. Thus, estimating MT DNA abundance from genotyping arrays has the potential to provide novel insights into age- and disease relevant processes, particularly those related to immunity and established mitochondrial functions.

genomics↗

bioRxiv2020

ICP-MS analysis of metal and metalloid concentrations of common microbiological growth media reveals presence of heavy metals

Trace elements such as cobalt and manganese play important roles as cofactors of enzymes, and thus, they collectively impact on biochemistry and cellular metabolism. Hence, it is of importance to gain an understanding of the trace metal and metalloid concentrations of growth medium in order to fully account for the growth performance of the bacterium. But, this aspect of microbial cell cultivation is usually neglected. Advent of instrumented techniques for metals and metalloid analysis such as inductively coupled plasma mass spectrometry (ICP-MS) has significantly facilitated trace element analysis at the parts per billion (ppb) to parts per million (ppm) level in aqueous matrixes free of organic interference. In this work, ICP-MS was utilized as the principal tool for profiling the range of metals and metalloids in different microbiological growth medium ranging from minimal salts medium to complex chemically undefined medium. Growth media examined include: LB Lennox, LB Lennox + 2 g/L glucose, LB Lennox (buffered, 89 mM phosphate), LB Lennox (buffered, 89 mM phosphate) + 6 g/L glucose, formulated medium + 6 g/L glucose, Tryptic Soy Broth, M9 medium, and M9 + 1 g/L yeast extract medium. Results reveal detection of elevated concentrations of chromium, copper and cadmium in different versions of LB Lennox and Tryptic Soy Broth. Concentrations of the above heavy metals in the tens of ppm range meant that the growth media could only support the growth of environmental bacteria with some resistance to heavy metal toxicity. On the other hand, no heavy metals were detected in M9 minimal salts medium or a modified version with supplementation of 1 g/L yeast extract. This indicated that minimal salts media may have less of a problem with heavy metal contamination compared to chemically undefined microbiological growth media. Collectively, the results highlight the essentiality of conducting a comprehensive profiling experiment for detecting different metals and metalloids at trace levels in microbiological growth medium. Such data would in addition to offering a deeper understanding of some peculiar growth behaviour from some microorganisms, may also help identify contamination issue during manufacture that preclude use of the media in cultivating many laboratory domesticated microorganisms. Subject areasbiochemistry, biotechnology, cell biology, microbiology, biochemical engineering,

microbiology↗

bioRxiv2020

DNA Methyltransferase Inhibitors with Novel Chemical Scaffolds

Inhibitors of DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug discovery. They are also chemical tools to understand the biochemistry of epigenetic processes. Herein, we report five distinct inhibitors of DNMT1 characterized in enzymatic inhibition assays that did not show activity with DNMT3B. It was concluded that the dietary component theaflavin is an inhibitor of DNMT1. Two additional novel inhibitors of DNMT1 are the approved drugs glyburide and panobinostat. The DNMT1 enzymatic inhibitory activity of panobinostat, a known pan inhibitor of histone deacetylases, agrees with experimental reports of its ability to reduce DNMT1 activity in liver cancer cell lines. Molecular docking of the active compounds with DNMT1, and re-scoring with the recently developed Extended Connectivity Interaction Features approach, had an excellent agreement between the experimental IC50 values and docking scores.

systems biology↗

bioRxiv2020

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PA01 and define its role in abiotic stress protection. We show that trehalose metabolism in PA01 is integrated with the biosynthesis of branched -glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and -glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is vital to the PA01 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PA01 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived -glucan, with deletion of the glgE synthase gene compromising PA01 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

microbiology↗

bioRxiv2020

NERO: A Biomedical Named-entity (Recognition) Ontology with a Large, Annotated Corpus Reveals Meaningful Associations Through Text Embedding

Machine reading is essential for unlocking valuable knowledge contained in the millions of existing biomedical documents. Over the last two decades 1,2, the most dramatic advances in machine-reading have followed in the wake of critical corpus development3. Large, well-annotated corpora have been associated with punctuated advances in machine reading methodology and automated knowledge extraction systems in the same way that ImageNet 4 was fundamental for developing machine vision techniques. This study contributes six components to an advanced, named-entity analysis tool for biomedicine: (a) a new, Named-Entity Recognition Ontology (NERO) developed specifically for describing entities in biomedical texts, which accounts for diverse levels of ambiguity, bridging the scientific sublanguages of molecular biology, genetics, biochemistry, and medicine; (b) detailed guidelines for human experts annotating hundreds of named-entity classes; (c) pictographs for all named entities, to simplify the burden of annotation for curators; (d) an original, annotated corpus comprising 35,865 sentences, which encapsulate 190,679 named entities and 43,438 events connecting two or more entities; (e) validated, off-the-shelf, named-entity recognition automated extraction, and; (f) embedding models that demonstrate the promise of biomedical associations embedded within this corpus.

systems biology↗

bioRxiv2020

Characterization of the Cannabis sativa glandular trichome proteome

Cannabis sativa has been cultivated since antiquity as a source of fibre, food and medicine. The recent resurgence of Cannabis as a cash crop is mainly driven by the medicinal and therapeutic properties of its resin, which contains compounds that interact with the human endocannabinoid system. Compared to other medicinal crops of similar value, however, little is known about the biology of C. sativa. Glandular trichomes are small hair-like projections made up of stalk and head tissue and are responsible for the production of the resin in C. sativa. Trichome productivity, as determined by Cannabis sativa resin yield and composition, is only beginning to be understood at the molecular level. In this study the proteomes of glandular trichome stalks and heads, were investigated and compared to the proteome of the whole flower tissue, to help elucidate Cannabis sativa glandular trichome biochemistry. The data suggested that the floral tissue acts as a major source of carbon and energy to the glandular trichome head sink tissue, supplying sugars which drive secondary metabolite biosynthesis in the glandular trichome head; the location of the secretory cells. The trichome stalk seems to play only a limited role in secondary metabolism and acts as both source and sink.

plant biology↗

bioRxiv2020

Encoding multiple virtual signals in DNA barcodes with single-molecule FRET

DNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy-to-access method to detect individual DNA barcodes, which can be scaled up to a massively high-throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes-and thus its signal space-that can be detected in a simultaneous manner. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single-molecule FRET occurred. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques.

biophysics↗

bioRxiv2020

Ancestral class-promiscuity as a driver of functional diversity in the BAHD acyltransferase family in plants

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Here, we addressed this question using BAHD acyltransferases as a model, and identified the routes by which duplication, promiscuity and sequence changes influenced BAHD diversification. This fast-evolving family expanded drastically during land plant evolution from 1-5 copies in algae to [~]100 copies in diploid angiosperm genomes. In vitro characterization of fourteen BAHDs against a substrate panel and compilation of >160 published activities revealed the wide prevalence of promiscuity among BAHDs. Using phylogenetic analysis, we predicted the substrate classes that the ancestral enzymes were likely capable of using prior to land plant origins. While the anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms, in vitro testing of BAHDs from non-seed plant lineages suggested that the ability to acylate anthocyanins likely existed promiscuously millions of years prior to its fixation. Motif enrichment analysis in anthocyanin-acylating BAHDs identified two motifs fixed in the largest anthocyanin acylating clade. Molecular dynamic simulations and enzyme kinetics revealed the important role of an active site tryptophan, whose bulkiness, hydrophobicity and aromaticity are critical for anthocyanin acylation. Our results thus describe the molecular processes in robust, evolvable enzymes that drive emergence of functional diversity in enzyme families. One sentence summaryUsing a combination of phylogenetics, biochemistry and protein structure analysis, we investigated how the BAHD acyltransferase family evolved to use a structurally diverse array of substrates.

plant biology↗

bioRxiv2020

Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or monomeric state in solution. However, we have also observed unexpected alternative processing of substrates, such as nicked HJs and a different conformational preference on intact HJs. Moreover, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5-3 exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfills the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

molecular biology↗

bioRxiv2020

Molecular fluctuations as a ruler of force-induced protein conformations

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

biophysics↗

bioRxiv2020

Microparticle-based Biochemical Sensing Using Optical Coherence Tomography and Deep Learning

Advancing continuous health monitoring beyond vital signs to biochemistry will revolutionize personalized medicine. Herein, we report a novel platform to achieve remote biochemical monitoring using microparticle-based biosensors and optical coherence tomography (OCT). Stimuli-responsive, polymeric microparticles were designed to serve as freely-dispersible biorecognition units, wherein binding with a target biochemical induces volumetric changes of the microparticle. Analytical approaches to detect these sub-micron changes in 3D using OCT were devised by modeling the microparticle as an optical cavity, enabling estimations far below the resolution of the OCT system. As a proof of concept, we demonstrated the 3D spatiotemporal monitoring of glucose-responsive microparticles distributed throughout a tissue-mimic in response to dynamically-fluctuating levels of glucose. Deep learning was further implemented using 3D convolutional neural networks to automate the vast processing of the continuous stream of three-dimensional time series data, resulting in a robust end-to-end pipeline with immense potential for continuous in vivo biochemical monitoring.

bioengineering↗

bioRxiv2020

Sesbanimide R, a novel cytotoxic polyketide produced by magnetotactic bacteria

Genomic information from various magnetotactic bacteria suggested that besides their common ability to form magnetosomes they potentially also represent a source of bioactive natural products. By using targeted deletion and transcriptional activation, we connected a large biosynthetic gene cluster (BGC) of the trans-AT PKS type to the biosynthesis of a novel polyketide in the alphaproteobacterium Magnetospirillum gryphiswaldense. Structure elucidation by mass spectrometry and NMR revealed that this secondary metabolite resembles sesbanimides which were very recently reported from other taxa. However, sesbanimide R exhibits an additional arginine moiety the presence of which reconciles inconsistencies in the previously proposed sesbanimide biosynthesis pathway when comparing the chemical structure and the potential biochemistry encoded in the BGC. In contrast to sesbanimides D, E and F, we were able to assign the stereocenter of the arginine moiety experimentally and two of the remaining three stereocenters by predictive biosynthetic tools. Sesbanimide R displayed strong cytotoxic activity against several carcinoma cell lines. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY ImportanceThe finding of this study contributes a new secondary metabolite member to the glutarimide-containing polyketides. The determined structure of sesbanimide R correlates with its cytotoxic bioactivity characteristic for members of this family. Sesbanimide R represents the first natural product isolated from magnetotactic bacteria and identifies this highly diverse group as a so far untapped source for the future discovery of novel secondary metabolites.

microbiology↗

bioRxiv2020

Novel insights into the mode of action of 1,4-dioxane using a systems screening approach

1,4-Dioxane (1,4-DX) is an environmental contaminant found in drinking water throughout the United States (US). While it is a suspected liver carcinogen, there is no federal or state maximum contaminant level for 1,4-DX in drinking water. Very little is known about the mechanisms by which this chemical elicits liver carcinogenicity. In the present study, female BDF-1 mice were exposed to 1,4-DX (0, 50, 500 and 5,000 mg/L) in their drinking water for one or four weeks, to explore the toxic effects. Histopathological studies and a multi-omics approach (transcriptomics and metabolomics) were performed to investigate potential mechanisms of toxicity. Immunohistochemical analysis of the liver revealed increased H2AX{gamma}-positive hepatocytes (a marker of DNA double strand breaks), and an expansion of precholangiocytes (reflecting both DNA damage and repair mechanisms) after exposure. Liver transcriptomics revealed 1,4-DX-induced perturbations in signaling pathways predicted to impact the oxidative stress response, detoxification, and DNA damage. Liver, kidney, feces and urine metabolomic profiling revealed no effect of 1,4-DX exposure, and bile acid quantification in liver and feces similarly showed no effect of exposure. We speculate that the results may be reflective of DNA damage being counterbalanced by the repair response, with the net result being a null overall effect on the systemic biochemistry of the exposed mice. Our results show a novel approach for the investigation of environmental chemicals that do not elicit cell death but have activated the repair systems in response to 1,4-DX exposure.

pharmacology and toxicology↗

bioRxiv2021

Dietary electrolyte balance on serum hematological and biochemical parameters of broilers under heat stress

The aim of this study was to evaluate the effect of different dietary electrolyte balance (DEB) levels over the hematological parameters and the biochemical profile of broilers under stress caused by cyclic heat. We used 1050 male broilers from Ross lineage, for two experimental phases: phase 1 (broilers from 1 until 21 days old) and phase 2 (broilers from 22 until 42 days old). The broilers were distributed under a completely randomized design, built for five treatments (110; 175; 240; 305; 370 mEq of DEB/kg of ration) and seven repetitions. For the hematological analyses were used two broilers in each repetition, one to blood count evaluation (Hemogram) and another one to the biochemistry evaluation, collected in the end of each experimental phase. During the phase of 1 - 21 days old, the variable hemoglobin number (Heme), mean corpuscular volume (MCV) and Serum Chloride (Cl) presented a linear effect (p<0,05) DEB levels. There was no effects (p>0,05) over the corpuscular volume (CV), total plasma protein (TPP), leukocytes number (Leu), heterophiles (Het), lymphocytes (Lyn), eosinophils (Eos), basophils (Bas), monocytes (Mon), calcium (Ca), phosphor (P), uric acid (UA) e urea (U). During the phase two of 22 - 42 days old there was effect (p<0,05) quadratic to the variables Leu and Het and linear to the variable P and significative difference (p<0,05) between the variable means UA at the levels of DEB tested. There was no effect (p>0,05) over the variable CV, Heme, MCV, TPP, Lyn, Eos, Bas, Mon, Cl, Ca and U on the DEB levels tested. The supplementation of the DEB levels 110, 175 e 240, 305 and 370 to the phase from 1 until 21 days old presented great results in the hematological evaluation and biochemical, because it provided possibly a longer useful life to the Hem without promoting a metabolic imbalance. In the phase from 22 until 42 days old the level of 268 to 280 mEq of DEB/kg of ration presented a better answer from the broilers immune systemic.

zoology↗

bioRxiv2021

Randomized controlled studies comparing traditional lectures versus online modules

We assessed the efficacy of traditional lectures versus online modules with respect to student learning in an undergraduate introductory biochemistry course in two successive years. In the first year, students had the options of attending live lectures by the course instructor and viewing online modules pre-recorded by the instructor, with the lectures and modules covering identical content; in addition, all students had a mandatory weekly application session. Utilizing pre-course and post-course tests as an instrument with which to measure learning during the course, we observed significantly increased learning (0.7 standard deviations) with attendance of traditional lectures and decreased learning with use of online modules, even after adjustment for grade point average. In the second year, the course had the same curriculum, but students were randomized to either live lectures or online modules for the first half of the course, crossing over to the other modality during the second half. With randomization, no difference in learning was observed between the two groups. Furthermore, we found that students self-reported greater engagement when viewing online modules than when attending lectures in person. These findings suggest some aspects of the lecture experience can be shifted to online modules in STEM courses without impacts on student learning so as to use classroom time more fully for application-based active learning interventions.

scientific communication and education↗

bioRxiv2021

Single particle combinatorial multiplexed liposome fusion mediated by DNA

Combinatorial high throughput methodologies are central for both screening and discovery in synthetic biochemistry and biomedical sciences. They are, however, often reliant on large scale analyses and thus limited by long running time and excessive materials cost. We herein present Single PARticle Combinatorial multiplexed Liposome fusion mediated by DNA (SPARCLD), for the parallelized, multi-step and non-deterministic fusion of individual zeptoliter nanocontainers. We observed directly the efficient (>93%), and leakage free stochastic fusion sequences for arrays of surface tethered target liposomes with six freely diffusing populations of cargo liposomes, each functionalized with individual lipidated ssDNA (LiNA) and fluorescent barcoded by distinct ratio of chromophores. The stochastic fusion results in distinct permutation of fusion sequences for each autonomous nanocontainer. Real-time TIRF imaging allowed the direct observation of >16000 fusions and 566 distinct fusion sequences accurately classified using machine learning. The high-density arrays of surface tethered target nanocontainers [~]42,000 containers per mm2 offers entire combinatorial multiplex screens using only picograms of material.

biophysics↗

bioRxiv2021

A Catalog of the Diversity and Ubiquity of Metabolic Organelles in Bacteria

Bacterial microcompartments (BMCs) are organelles that segregate segments of metabolic pathways, which are incompatible with surrounding metabolism. In contrast to their eukaryotic counterparts, the selectively permeable membrane of BMCs, the shell, is composed of protein. While the sequestered enzymes vary among functionally distinct BMCs, the proteins that form diverse BMC shells are structurally homologous; this enables the bioinformatic identification of the organelles by locating genes encoding shell proteins, which are typically proximal to those for the encapsulated enzymes. With recent advances in genome-resolved metagenomics and the emphasis on "microbial dark matter", many new genome sequences from diverse and obscure bacterial clades have become available. We find the number of identifiable BMC loci has increased twenty-fold since the last comprehensive census of 2014. Moreover, the addition of new types we uncovered doubles the number of distinct BMC types known. These expand the range of catalysis encapsulated in BMCs, underscoring that there is dark biochemistry that is compartmentalized in bacterial organelles yet to be discovered through genome sequencing. Our comprehensive catalog of BMCs provides a framework for their identification, correlation with bacterial niche adaptation, and experimental characterization, and broadens the foundation for the development of BMC-based nanoarchitectures for biomedical and bioengineering applications.

microbiology↗

bioRxiv2021

An engineered multifunctional protein tag for advanced fluorescence imaging

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

molecular biology↗