Biology subjects

Schmidt, M.

Publications and source records attributed to Schmidt, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2020

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

Genome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs) however escapes surveillance, which triggers chromosome breakage. Using human cells, we asked here whether such leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition suppresses CFS instability. Repli-Seq and molecular combing analyses consistently showed a burst of replication initiations in mid S phase across large origin-poor domains shaped by transcription, including CFSs. Strikingly, CDC6 or CDT1 depletion or CDC7-DBF4 inhibition during the S phase prevented both extra-initiations and CFS rescue, showing that CDK1 inhibition promotes targeted and mistimed building of functional extra-origins. In addition to delay mitotic onset, checkpoint activation therefore advances replication completion of chromosome domains at risk of under-replication, two complementary roles preserving genome stability.

cell biology↗

bioRxiv2020

Single-femtosecond atomic-resolution observation of a protein traversing a conical intersection

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate energy surfaces. Posited almost a century ago 1, conical intersections remain the subject of intense scientific investigation 2-4. In biology, they play a pivotal role in vision, photosynthesis, and DNA stability 5,6. In ultrafast radiationless de-excitation 1,7, they are vital to ameliorating photon-induced damage. In chemistry, they tightly couple the normally separable nuclear and electronic degrees of freedom, precluding the Born-Oppenheimer approximation 8. In physics, they manifest a Berry phase, giving rise to destructive interference between clockwise and anti-clockwise trajectories around the conical intersection 9. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around 10 atoms, on timescales of about 100 fs or longer 10. Spectroscopy can achieve better time resolution, but provides only indirect structural information. Here, we present single-femtosecond, atomic-resolution movies of a 2,000-atom protein passing through a conical intersection. These movies, extracted from experimental data by geometric machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process, and elucidate the topography of the electronic potential energy surfaces involved.

biophysics↗

bioRxiv2020

Elevation of Gut-Derived p-Cresol Molecular Networks in Astronauts During Spaceflight Informs Drug Metabolism and Performance

Metabolites produced by enteric microbes may have important effects on astronauts on long-duration missions. The NASA Twins Study provided the most comprehensive multi-scale omics data to date from which to extract molecular features of potential clinical significance to spaceflight. From the multivariate data, we identified an elevation of the uremic toxin p-cresol, which is produced by gut microbial fermentation of dietary tyrosine. p-Cresol has adverse metabolic effects via depletion of the hepatic sulfur pool, which impacts metabolism of drugs, endogenous metabolites, and xenobiotics. Moreover, p-cresol reshapes gut microbial community structure by facilitating survival of species such as clostridia and inhibition of butyrate producers. Spaceflight may also impact the genes responsible for the metabolism of p-cresol (e.g. SULT) and for the safe metabolism of common drugs used in space, such as acetaminophen (e.g. SULT, CYP4502E1, GST). Understanding p-cresol production and its related molecular networks in astronauts may lead to precision medicine advances that enhance astronaut safety and performance on long-duration missions.

molecular biology↗

bioRxiv2020

Cryo-EM reveals structural breaks in a patient-derived amyloid fibril from systemic AL amyloidosis

Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present the first ex vivo fibril structures of a {lambda}3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115 residues, mainly from the LC variable domain. The fibril structures imply that a 180{degrees} rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms.

molecular biology↗

bioRxiv2020

High-resolution Crystal Structures of Transient Intermediates in the Phytochrome Photocycle

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting (dark) Pr state and the light activated Pfr state, in which light signals are received and transduced as structural change through the entire protein to modulate the activity of the protein. It is unknown how the Pr-to-Pfr interconversion occurs as the structure of intermediates remain notoriously elusive. Here, we present short-lived crystal structures of the classical phytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray Free Electron Laser 5 ns and 33ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway. The snapshots show with atomic precision how the signal progresses from the chromophore towards the output domains, explaining how plants, bacteria and fungi sense red light.

biophysics↗

bioRxiv2020

Ebselen Reacts with SARS Coronavirus-2 Main Protease Crystals

The SARS coronavirus 2 main protease 3CLpro tailor cuts various essential virus proteins out of long poly-protein translated from the virus RNA. If the 3CLpro is inhibited, the functional virus proteins cannot form and the virus cannot replicate and assemble. Any compound that inhibits the 3CLpro is therefore a potential drug to end the pandemic. Here we show that the diffraction power of 3CLpro crystals is effectively destroyed by Ebselen. It appears that Ebselen may be a widely available, relatively cost effective way to eliminate the SARS coronavirus 2.

biophysics↗

bioRxiv2020

Deconvolution of cellular subsets in human tissue based on targeted DNA methylation analysis at individual CpG sites

BackgroundThe complex composition of different cell types within a tissue can be estimated by deconvolution of omics datasets. For example, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. In this study, we investigated if deconvolution is also feasible with individual cell-type-specific CG dinucleotides (CpG sites), which can be addressed by targeted analysis, such as pyrosequencing. ResultsWe compiled and curated a dataset of 579 samples from Illumina 450k BeadChip technology that comprised 14 different purified and characterized human cell types. A training and validation strategy was applied to identify and test cell-type-specific CpGs. Initially, the amount of fibroblasts was estimated using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. This FibroScore correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts and induced pluripotent stem cells. Using previously published BeadChip datasets with cell mixtures the accuracy of this eight CpG signature was comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types. ConclusionThis proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings.

genomics↗

bioRxiv2020

Functional analysis of the fatty acid and alcohol metabolism of Pseudomonas putida using RB-TnSeq

ABSTRACTWith its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, and Apertor Labs. C.B.E has a financial interest in Perlumi Chemicals.View Full Text

microbiology↗

bioRxiv2020

Combining genome-wide studies of breast, prostate, ovarian and endometrial cancers maps cross-cancer susceptibility loci and identifies new genetic associations

We report a meta-analysis of breast, prostate, ovarian, and endometrial cancer genome-wide association data (effective sample size: 237,483 cases/317,006 controls). This identified 465 independent lead variants (P<5x10-8) across 192 genomic regions. Four lead variants were >1Mb from previously identified risk loci for the four cancers and an additional 23 lead variant-cancer associations were novel for one of the cancers. Bayesian models supported pleiotropic effects involving at least two cancers at 222/465 lead variants in 118/192 regions. Gene-level association analysis identified 13 shared susceptibility genes (P<2.6x10-6) in 13 regions not previously implicated in any of the four cancers and not uncovered by our variant-level meta-analysis. Several lead variants had opposite effects across cancers, including a cluster of such variants in the TP53 pathway. Fifty-four lead variants were associated with blood cell traits and suggested genetic overlaps with clonal hematopoiesis. Our study highlights the remarkable pervasiveness of pleiotropy across hormone-related cancers, further illuminating their shared genetic and mechanistic origins at variant- and gene-level resolution.

genetics↗

bioRxiv2020

Spontaneous Axonal ER Ca2+ Waves Mediate A Shift From Action Potential-Dependent to Independent Glutamate Release in the YAC128 HD-Model

Action potential-independent (miniature) neurotransmission occurs at all chemical synapses, but remains poorly understood, particularly in pathological contexts. Spontaneous release of Ca2+ from the axonal endoplasmic reticulum (ER) is thought to facilitated miniature neurotransmission, and aberrant ER Ca2+ handling is notably implicated in the progression of Huntingtons disease (HD) and other neurodegenerative diseases. Here, we report elevated glutamate-mediated miniature synaptic event frequencies in YAC128 (HD-model) cortical neurons, which pharmacological experiments suggest is mediated by enhanced spontaneous ER Ca2+ release. Calcium imaging using an axon-localized sensor revealed slow action potential (AP)-independent axonal Ca2+ waves, which were more common in YAC128 cortical neurons. Conversely, spontaneous axonal ER Ca2+ release was associated with reduced AP-dependent axonal Ca2+ events and consequent glutamate release. Together, our results suggest spontaneous release of axonal ER Ca2+ stores oppositely regulates activity-dependent and -independent neurotransmitter release in HD, with potential implications for the fidelity and plasticity of cortical excitatory signaling.

neuroscience↗

bioRxiv2019

Towards establishing internal validity for correlated gene expression measures in imaging genomics of functional networks: why distance corrections and external face validity alone fall short. Reply to "Distance is not everything in imaging genomics of functional networks: reply to a commentary on Correlated gene expression supports synchronous activity in brain networks"

The primary claim of the Richiardi et. al. 2015 Science article1 is that a measure of correlated gene expression, significant strength fraction (SSF), is related to resting state fMRI (rsfMRI) networks. However, there is still debate about this claim and whether spatial proximity, in the form of contiguous clusters, accounts entirely, or only partially, for SSF2,3. Here, thirteen distributed networks were simulated by combining 34 contiguous clusters randomly placed throughout cortex, with resulting edge distance distributions similar to rsfMRI networks. Cluster size was modulated (6-15mm radius) to test its influence on SSF false positive rate (SSF-FPR) among the simulated noise networks. The contribution of rsfMRI networks on SSF-FPR was examined by comparing simulations using: 1) all cortical samples 2) all samples with non-rsfMRI cluster centers and 3) only non-rsfMRI samples. Results show that SSF-FPR is influenced only by cluster size (r>0.9, p<0.001), not by rsfMRI samples. Simulations using 14mm radius clusters most resembled rsfMRI networks. When thresholding at p<10-4, the SSF-FPR was 0.47. Genes that maximize SF have high global spatial autocorrelation. In conclusion, SSF is unrelated to rsfMRI networks. The main conclusion of Richiardi et. al. 2015 is based on a finding that is [~]50% likely to be a false positive, not less than 0.01% as originally reported in the article1. We discuss why distance corrections alone and external face validity are insufficient to establish a trustworthy relationship between correlated gene expression measures and rsfMRI networks, and propose more rigorous approaches to preclude common pitfalls in related studies.

neuroscience↗

bioRxiv2019

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis

Herpesviruses encode conserved protein kinases to stimulate phosphorylation-sensitive processes during infection. How these kinases bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) kinases. We find that these kinases can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a specific signature of {beta}-herpesvirus kinases. Cyclin A is recruited via RXL-motifs that overlap with nuclear localization signals (NLS) and locate in the non-catalytic N-terminal regions. This architecture is conserved for viral kinases of HHV6, HHV7 and rodent CMVs. Docking to Cyclin A competes with NLS function, enabling dynamic changes in kinase localization and substrate phosphorylation. The viral kinase redirects Cyclin A to the cytosol, which is essential for the inhibition of cellular DNA replication during infection. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

microbiology↗

bioRxiv2019

Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.\n\nAuthor SummaryYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.

genetics↗

bioRxiv2019

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The structures reveal a twist of the D-ring, which lead to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signalling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the water dissociation is key to the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

biophysics↗

bioRxiv2019

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

microbiology↗

bioRxiv2019

TRANSCRIPTION-MEDIATED ORGANIZATION OF THE REPLICATION INITIATION PROGRAM ACROSS LARGE GENES SETS UP COMMON FRAGILE SITES GENOME-WIDE

Common Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

genomics↗

bioRxiv2019

Nicotinamide riboside augments the human skeletal muscle NAD+ metabolome and induces transcriptomic and anti-inflammatory signatures in aged subjects: a placebo-controlled, randomized trial

NAD+ is modulated by conditions of metabolic stress and has been reported to decline with aging, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome, and questioned if tissue NAD+ levels are depressed with aging. We supplemented 12 aged men with NR 1g per day for 21-days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways. NR also depressed levels of circulating inflammatory cytokines. In an additional study, 31P magnetic resonance spectroscopy-based NAD+ measurement in muscle and brain showed no difference between young and aged individuals. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR, while suggesting that NAD+ decline is not associated with chronological aging per se in human muscle or brain.

clinical trials↗