Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Diagnostic performance of additional imaging tests for staging purposes in a bicentric German series of low-risk early breast cancer patients.

PURPOSE: Low-risk early breast cancer rarely leads to the development of metastatic disease, and in these patients, additional imaging test is controversial. The aim of our study was to evaluate the conventional staging procedures in a bicentric German series of low-risk breast carcinoma patients. METHODS: Retrospective evaluation of all patients diagnosed with early, low-risk breast cancer at Saarland University Hospital and Freiburg University Hospital in 2017 was performed. Clinical patient characteristics, the number and type of additional imaging examinations, follow-up examinations, and results were evaluated. The detection rate of metastases and the rate of false-positive findings were analyzed. RESULTS: A total of 203 patients were included, with all patients received at least one additional imaging test. Initially, a total of 562 additional imaging examinations were performed: 166 chest X-rays, 169 upper abdominal ultrasounds, 199 bone scans, 27 computer tomographies (CT) chest and abdomen, and 1 CT abdomen. 6.8% of patients had abnormal findings reported, requiring 38 additional imaging examinations. One patient (0.5%) was found to have bone metastases. The rate of false-positive findings in the performed additional imaging procedures was 6.6%. CONCLUSION: Metastatic disease was detected in one of 203 patients with low-risk early breast cancer. A total of 562 examinations and additional 38 follow-up examinations were performed without detection of metastasis (this corresponds to approximately 3 examinations/patient). The rate of false-positive findings was 6.6%. The performance of additional imaging procedures for detection of distant metastases should be critically reconsidered in patients with low-risk early breast cancer.

Sleep consolidates stimulus-response learning.

Performing a motor response to a sensory stimulus creates a memory trace whose behavioral correlates are classically investigated in terms of repetition priming effects. Such stimulus-response learning entails two types of associations that are partly independent: (1) an association between the stimulus and the motor response and (2) an association between the stimulus and the classification task in which it is encountered. Here, we tested whether sleep supports long-lasting stimulus-response learning on a task requiring participants (1) for establishing stimulus-classification associations to classify presented objects along two different dimensions ("size" and "mechanical") and (2) as motor response (action) to respond with either the left or right index finger. Moreover, we examined whether strengthening of stimulus-classification associations is preferentially linked to nonrapid eye movement (non-REM) sleep and strengthening of stimulus-action associations to REM sleep. We tested 48 healthy volunteers in a between-subjects design comparing postlearning retention periods of nighttime sleep versus daytime wakefulness. At postretention testing, we found that sleep supports consolidation of both stimulus-action and stimulus-classification associations, as indicated by increased reaction times in "switch conditions"; that is, when, at test, the acutely instructed classification task and/or correct motor response for a given stimulus differed from that during original learning. Polysomnographic recordings revealed that both kinds of associations were correlated with non-REM spindle activity. Our results do not support the view of differential roles for non-REM and REM sleep in the consolidation of stimulus-classification and stimulus-action associations, respectively.

Explore or exploit? A model-based screening strategy for PETase secretion by Corynebacterium glutamicum.

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide, and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization, and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision-making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.

Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay.

BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh ß-sheet of sfGFP, which can complement a truncated sfGFP that lacks this ß-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress. RESULTS: The biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels. CONCLUSION: Our results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

Data-driven tailoring of molecular dipole polarizability and frontier orbital energies in chemical compound space.

Understanding correlations - or lack thereof - between molecular properties is crucial for enabling fast and accurate molecular design strategies. In this contribution, we explore the relation between two key quantities describing the electronic structure and chemical properties of molecular systems: the energy gap between the frontier orbitals and the dipole polarizability. Based on the recently introduced QM7-X dataset, augmented with accurate molecular polarizability calculations as well as analysis of functional group compositions, we show that polarizability and HOMO-LUMO gap are uncorrelated when considering sufficiently extended subsets of the chemical compound space. The relation between these two properties is further analyzed on specific examples of molecules with similar composition as well as homooligomers. Remarkably, the freedom brought by the lack of correlation between molecular polarizability and HOMO-LUMO gap enables the design of novel materials, as we demonstrate on the example of organic photodetector candidates.

New Twist on the Light-Switch Effect: Controlling the Fate of Excited States with pH in a 4-Hydroxy-thiazol-extended Ruthenium(II) Dppz Complex.

We present a pH-dependent study of the excited state dynamics of a novel Ru complex bearing a 4-hydroxy thiazol-substituted dppz (dipyridophenazine) ligand (RuTzOH) and its deprotonated form (RuTzO-). We combine steady-state and time-resolved absorption and emission spectroscopy with electrochemical investigations to characterize the excited state relaxation, which upon photoexcitation at 400 nm is determined by a multitude of initially populated MLCT states for both complexes. Subsequently, for RuTzOH, two long-lived excited states are populated, leading to dual emission from the complexes, a feature that vanishes upon deprotonation. Upon deprotonation, the electron density on the dppz moiety increases significantly, leading to rapid energy populating ligand-centered states and thus deactivating the initially excited MLCT states.

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin.

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

Tos4 mediates gene expression homeostasis through interaction with HDAC complexes independently of H3K56 acetylation.

Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3's well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.

Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads.

Replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We report a sequencing method for the measurement of replication fork movement on single molecules by detecting nucleotide analog signal currents on extremely long nanopore traces (D-NAscent). Using this method, we detect 5-bromodeoxyuridine (BrdU) incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse-labeling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20-160 kilobases to generate a whole-genome single-molecule map of DNA replication dynamics and discover a class of low-frequency stochastic origins in budding yeast. The D-NAscent software is available at https://github.com/MBoemo/DNAscent.git .

Preoperative Sonographic Prediction of Limited Axillary Disease in Patients with Primary Breast Cancer Meeting the Z0011 Criteria: an Alternative to Sentinel Node Biopsy?

PURPOSE: To assess the accuracy of preoperative sonographic staging for prediction of limited axillary disease (LAD, one or two metastatic lymph nodes) and to identify factors associated with high prediction-pathology concordance in patients with early-stage breast cancer meeting the Z0011 criteria. MATERIALS AND METHODS: Patients treated between January 2015 and January 2020 were included in this retrospective, multicentric analysis of prospectively acquired service databases. The accuracy of LAD prediction was assessed separately for patients with one and two suspicious lymph nodes on preoperative sonography. Test validity outcomes for LAD prediction were calculated for both groups, and a multivariate model was used to identify factors associated with high accuracy of LAD prediction. RESULTS: Of 2059 enrolled patients, 1513 underwent sentinel node biopsy, 436 primary and 110 secondary axillary dissection. For LAD prediction in patients with one suspicious lymph node on preoperative ultrasound, sensitivity was 92% (95% CI 87-95%), negative predictive value (NPV) was 92% (95% CI 87-95%), and the false-negative rate (FNR) was 8% (95% CI 5-13%). For patients with two preoperatively suspicious nodes, the sensitivity, NPV, and FNR were 89% (95% CI 84-93%), 73% (62-83%), and 11% (95% CI 7-16%), respectively. On multivariate analysis, the number of suspicious lymph nodes was associated inversely with correct LAD prediction ([OR 0.01 (95% CI 0.01-0.93), p ≤ 0.01]. CONCLUSIONS: Sonographic axillary staging in patients with one metastatic lymph node predicted by preoperative ultrasound showed high accuracy and a false-negative rate comparable to sentinel node biopsy for prediction of limited axillary disease.

Influence of the Linker Chemistry on the Photoinduced Charge-Transfer Dynamics of Hetero-dinuclear Photocatalysts.

To optimize light-driven catalytic processes, light-mediated multi-electron transfer dynamics in molecular dyads need to be studied and correlated with structural changes focusing on the catalytically active metastable intermediates. Here, spectro-electrochemistry has been employed to investigate the structure-dependent photoelectron transfer kinetics in catalytically active intermediates of two Ru-Rh catalysts for light-driven NAD+ reduction. The excited-state reactivity of short-lived intermediates was studied along different photoreaction pathways by resonance Raman and time-resolved transient absorption spectro-electrochemistry with sub-picosecond time resolution under operando conditions. The results demonstrate, for the first time, how the bridging ligand serves as a (multi-)electron storage structure, mediates the strength of the electronic coupling of catalytic and photocenter and impacts the targeted electron transfer as well as parasitic electron-transfer kinetics.

A Combined Spectroscopic and Theoretical Study on a Ruthenium Complex Featuring a π-Extended dppz Ligand for Light-Driven Accumulation of Multiple Reducing Equivalents.

The design of photoactive systems capable of storing and relaying multiple electrons is highly demanded in the field of artificial photosynthesis, where transformations of interest rely on multielectronic redox processes. The photophysical properties of the ruthenium photosensitizer [(bpy)2 Ru(oxim-dppqp)]2+ (Ru), storing two electrons coupled to two protons on the π-extended oxim-dppqp ligand under light-driven conditions, are investigated by means of excitation wavelength-dependent resonance Raman and transient absorption spectroscopies, in combination with time-dependent density functional theory; the results are discussed in comparison to the parent [(bpy)2 Ru(dppz)]2+ and [(bpy)2 Ru(oxo-dppqp)]2+ complexes. In addition, this study provides in-depth insights on the impact of protonation or of accumulation of multiple reducing equivalents on the reactive excited states.

Photocatalytic Reduction of Nicotinamide Co-factor by Perylene Sensitized RhIII Complexes.

The ambitious goal of artificial photosynthesis is to develop active systems that mimic nature and use light to split water into hydrogen and oxygen. Intramolecular design concepts are particularly promising. Herein, we firstly present an intramolecular photocatalyst integrating a perylene-based light-harvesting moiety and a catalytic rhodium center (RhIII phenPer). The excited-state dynamics were investigated by means of steady-state and time-resolved absorption and emission spectroscopy. The studies reveal that photoexcitation of RhIII phenPer yields the formation of a charge-separated intermediate, namely RhII phenPer⋅+ , that results in a catalytically active species in the presence of protons.

Pyrimidoquinazolinophenanthroline Opens Next Chapter in Design of Bridging Ligands for Artificial Photosynthesis.

The synthesis and detailed characterization of a new Ru polypyridine complex containing a heteroditopic bridging ligand with previously unexplored metal-metal distances is presented. Due to the twisted geometry of the novel ligand, the resultant division of the ligand in two distinct subunits leads to steady state as well as excited state properties of the corresponding mononuclear Ru(II) polypyridine complex resembling those of prototype [Ru(bpy)3 ]2+ (bpy=2,2'-bipyridine). The localization of the initially optically excited and the nature of the long-lived excited states on the Ru-facing ligand spheres is evaluated by resonance Raman and fs-TA spectroscopy, respectively, and supported by DFT and TDDFT calculations. Coordination of a second metal (Zn or Rh) to the available bis-pyrimidyl-like coordination sphere strongly influences the frontier orbitals, apparent by, for example, luminescence quenching. Thus, the new bridging ligand motif offers electronic properties, which can be adjusted by the nature of the second metal center. Using the heterodinuclear Ru-Rh complex, visible light-driven reduction of NAD+ to NADH was achieved, highlighting the potential of this system for photocatalytic applications.

KiMoPack: A python Package for Kinetic Modeling of the Chemical Mechanism.

Herein, we present KiMoPack, an analysis tool for the kinetic modeling of transient spectroscopic data. KiMoPack enables a state-of-the-art analysis routine including data preprocessing and standard fitting (global analysis), as well as fitting of complex (target) kinetic models, interactive viewing of (fit) results, and multiexperiment analysis via user accessible functions and a graphical user interface (GUI) enhanced interface. To facilitate its use, this paper guides the user through typical operations covering a wide range of analysis tasks, establishes a typical workflow and is bridging the gap between ease of use for less experienced users and introducing the advanced interfaces for experienced users. KiMoPack is open source and provides a comprehensive front-end for preprocessing, fitting and plotting of 2-dimensional data that simplifies the access to a powerful python-based data-processing system and forms the foundation for a well documented, reliable, and reproducible data analysis.

Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: • Automated workflows for strain construction and screening of protein secretion • Comparison of spacer, signal peptide, and host combinations for cutinase secretion • Signal peptide screening for secretion by C. glutamicum using the split GFP assay.

Environment-dependent fitness gains can be driven by horizontal gene transfer of transporter-encoding genes.

Many microbes acquire metabolites in a "feeding" process where complex polymers are broken down in the environment to their subunits. The subsequent uptake of soluble metabolites by a cell, sometimes called osmotrophy, is facilitated by transporter proteins. As such, the diversification of osmotrophic microorganisms is closely tied to the diversification of transporter functions. Horizontal gene transfer (HGT) has been suggested to produce genetic variation that can lead to adaptation, allowing lineages to acquire traits and expand niche ranges. Transporter genes often encode single-gene phenotypes and tend to have low protein-protein interaction complexity and, as such, are potential candidates for HGT. Here we test the idea that HGT has underpinned the expansion of metabolic potential and substrate utilization via transfer of transporter-encoding genes. Using phylogenomics, we identify seven cases of transporter-gene HGT between fungal phyla, and investigate compatibility, localization, function, and fitness consequences when these genes are expressed in Saccharomyces cerevisiae Using this approach, we demonstrate that the transporters identified can alter how fungi utilize a range of metabolites, including peptides, polyols, and sugars. We then show, for one model gene, that transporter gene acquisition by HGT can significantly alter the fitness landscape of S. cerevisiae We therefore provide evidence that transporter HGT occurs between fungi, alters how fungi can acquire metabolites, and can drive gain in fitness. We propose a "transporter-gene acquisition ratchet," where transporter repertoires are continually augmented by duplication, HGT, and differential loss, collectively acting to overwrite, fine-tune, and diversify the complement of transporters present in a genome.

Imaging the Renner-Teller effect using laser-induced electron diffraction.

Structural information on electronically excited neutral molecules can be indirectly retrieved, largely through pump-probe and rotational spectroscopy measurements with the aid of calculations. Here, we demonstrate the direct structural retrieval of neutral carbonyl disulfide (CS2) in the [Formula: see text] excited electronic state using laser-induced electron diffraction (LIED). We unambiguously identify the ultrafast symmetric stretching and bending of the field-dressed neutral CS2 molecule with combined picometer and attosecond resolution using intrapulse pump-probe excitation and measurement. We invoke the Renner-Teller effect to populate the [Formula: see text] excited state in neutral CS2, leading to bending and stretching of the molecule. Our results demonstrate the sensitivity of LIED in retrieving the geometric structure of CS2, which is known to appear as a two-center scatterer.

Multifunctional Polyoxometalate Platforms for Supramolecular Light-Driven Hydrogen Evolution*.

Multifunctional supramolecular systems are a central research topic in light-driven solar energy conversion. Here, we report a polyoxometalate (POM)-based supramolecular dyad, where two platinum-complex hydrogen evolution catalysts are covalently anchored to an Anderson polyoxomolybdate anion. Supramolecular electrostatic coupling of the system to an iridium photosensitizer enables visible light-driven hydrogen evolution. Combined theory and experiment demonstrate the multifunctionality of the POM, which acts as photosensitizer/catalyst-binding-site[1] and facilitates light-induced charge-transfer and catalytic turnover. Chemical modification of the Pt-catalyst site leads to increased hydrogen evolution reactivity. Mechanistic studies shed light on the role of the individual components and provide a molecular understanding of the interactions which govern stability and reactivity. The system could serve as a blueprint for multifunctional polyoxometalates in energy conversion and storage.