Expanding the range of graphene energy transfer with multilayer graphene.

The interaction between single emitters and graphene in the context of energy transfer has attracted significant attention due to its potential applications in fields such as biophysics and super-resolution microscopy. In this study, we investigate the influence of the number of graphene layers on graphene energy transfer (GET) by placing single dye molecules at defined distances from monolayer, bilayer, and trilayer graphene substrates. We employ DNA origami nanostructures as chemical adapters to position the dye molecules precisely. Fluorescence lifetime measurements and analysis reveal an additive effect of graphene layers on the energy transfer rate extending the working range of GET up to distances of approximately 50-60 nm. Moreover, we show that switching a DNA pointer strand between two positions on a DNA origami nanostructure at a height of >28 nm above graphene is substantially better visualized with multilayer graphene substrates suggesting enhanced capabilities for applications such as biosensing and super-resolution microscopy for larger systems and distances. This study provides insights into the influence of graphene layers on energy transfer dynamics and offers new possibilities for exploiting graphene's unique properties in various nanotechnological applications.

Blazed oblique plane microscopy reveals scale-invariant inference of brain-wide population activity.

Due to the size and opacity of vertebrate brains, it has until now been impossible to simultaneously record neuronal activity at cellular resolution across the entire adult brain. As a result, scientists are forced to choose between cellular-resolution microscopy over limited fields-of-view or whole-brain imaging at coarse-grained resolution. Bridging the gap between these spatial scales of understanding remains a major challenge in neuroscience. Here, we introduce blazed oblique plane microscopy to perform brain-wide recording of neuronal activity at cellular resolution in an adult vertebrate. Contrary to common belief, we find that inferences of neuronal population activity are near-independent of spatial scale: a set of randomly sampled neurons has a comparable predictive power as the same number of coarse-grained macrovoxels. Our work thus links cellular resolution with brain-wide scope, challenges the prevailing view that macroscale methods are generally inferior to microscale techniques and underscores the value of multiscale approaches to studying brain-wide activity.

Exploring the Synergies of Single-Molecule Fluorescence and 2D Materials Coupled by DNA.

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided.

Lung Volume Reduction Surgery Reduces Pulmonary Arterial Hypertension Associated With Severe Lung Emphysema and Hypercapnia.

Lung volume reduction surgery (LVRS) represents a standard surgical approach for patients with severe pulmonary emphysema. One of the relevant risk factors for LVRS is the presence of pulmonary arterial hypertension (PAH). The aim of this study is to assess the postoperative changes in pulmonary arterial pressure (PAP) after LVRS for patients with severe pulmonary emphysema compared with preoperative measures. N = 61 consecutive patients with severe pulmonary emphysema and preoperative evidence for PAH (pulmonary arterial systolic pressure [PASP] ≥ 35 mmHg) were prospectively included into this study. In all patients, thoracoscopic LVRS was performed. PASP was assessed by echocardiography before surgery, early postoperatively, and 3 months after surgery. Data were prospectively recorded and analyzed retrospectively. Primary end points were the postoperative changes in PASP as well as the 90 day mortality rate. Secondary endpoints included: pulmonary function test, exercise capacity, quality of life, and dyspnea symptoms (Borg scale). Early after surgery, a significant reduction in PASP was observed at the day of discharge and at 3 month follow-up. In n = 34 patients, no tricuspid valve regurgitation was detectable anymore suggesting normal PAP. In n = 3 patients, venovenous extracorporeal lung support (VV ECLS) was already implemented preoperatively. In the remaining cases, VV ECLS was applied intraoperatively and continued postoperatively. Mean duration of postoperative ECLS support was 2 days. Four patients died due to acute right heart failure, two patients from sepsis with multiorgan failure, and one patient from acute pulmonary embolism. Ninety day mortality was 11.5 %. A significant improvement was postoperatively observed regarding the performance status, dyspnea scale, as well as quality of life. This study suggests a beneficial effect of LVRS on PAP, which may ultimately help to protect and stabilize right ventricular function. Further studies, implementing pre- and postoperative right heart catheterizations including invasive PAP evaluation, are necessary to support the findings in this study in greater detail.

The Complete Phase Diagram of Monolayers of Enantiomeric N-Stearoyl-threonine Mixtures with Preferred Heterochiral Interactions.

Langmuir monolayers of chiral amphiphiles are well-controlled model systems for the investigation of phenomena related to stereochemistry. Here, we have investigated mixed monolayers of one pair of enantiomers (l and d) of the amino-acid-based amphiphile N-stearoyl-threonine. The monolayer characteristics were studied by pressure-area isotherm measurements and grazing incidence X-ray diffraction (GIXD) over a wide range of mixing ratios defined by the d-enantiomer mole fraction xD. While the isotherms provide insights into thermodynamical aspects, such as transition pressure, compression/decompression hysteresis, and preferential homo- and heterochiral interactions, GIXD reveals the molecular structural arrangements on the Ångström scale. Dominant heterochiral interactions in the racemic mixture lead to compound formation and the appearance of a nonchiral rectangular lattice, although the pure enantiomers form a chiral oblique lattice. Miscibility was found to be limited to mixtures with 0.27 ≲ xD ≲ 0.73, as well as to both outer edges (xD ≲ 0.08 and xD ≳ 0.92). Beyond this range, coexistence of oblique and rectangular lattices occurs, as is clearly seen in the GIXD patterns. Based on the results, a complete phase diagram with two eutectic points at xD ≈ 0.25 and xD ≈ 0.75 is proposed. Moreover, N-stearoyl-threonine was found to have a strong tendency to form a hydrogen-bonding network between the headgroups, which promotes superlattice formation.

Structure-based peptide ligand design for improved epidermal growth factor receptor targeted gene delivery.

The epidermal growth factor receptor EGFR allows targeted delivery of macromolecular drugs to tumors. Its ligand, epidermal growth factor, binds EGFR with high affinity but acts mitogenic. Non-mitogenic peptides are utilized as targeting ligands, like the dodecapeptide GE11, although its low binding affinity warrants improvement. We applied a two-step computational approach with database search and molecular docking to design GE11 variants with improved binding. Synthesized peptides underwent binding studies on immobilized EGFR using surface plasmon resonance. Conjugates of peptides coupled via heterobifunctional PEG linker to linear polyethylenimine (LPEI) were used for transfection studies on EGFR-overexpressing cells using reporter gene encoding plasmid DNA. Docking studies unraveled similarities between GE11 and the EGFR dimerization arm. By skipping non-overlapping amino acids, a less hydrophobic segment (YTPQNVI) was identified to be directly involved in EGFR binding. By replacing valine by tyrosine, a full-length version with proposed enhanced binding (GE11m3) was developed. While hydrophobic or hydrophilic segments and variations thereof exhibited low binding, GE11m3 exhibited 3-fold increase in binding compared to GE11, validating in silico predictions. In transfection studies, polyplexes with GE11m3 induced a significantly higher reporter gene expression when compared to GE11 polyplexes both on murine and human cancer cells overexpressing EGFR.

Multidisciplinary management of pleural infection after ventricular assist device implantation.

BACKGROUND: Postsurgical pleural infection is a life-threatening complication after implantation of artificial devices such as ventricular assist devices (VADs). The treatment can be challenging and the evidence in the literature is very limited. Here we report our multidisciplinary approach of the management of pleural infection after VAD implantation. METHODS: Between March 2014 and December 2019, 33 patients developed postoperative pleural infection after VAD implantation and underwent thoracic surgical intervention at our institution. All patients were prospectively enrolled in this analysis. Data were retrospectively analyzed. Primary outcome was the 90-day mortality rate. Length of ICU stay related to pleural infection, chest tube duration, re-thoracotomy rate and length of ventilatory support represented secondary outcomes. RESULTS: The 90-day mortality rate was 6% (2 patients). The mean ICU stay related to the pleural infection was 6 days (2-24 days). Video-assisted thoracoscopic surgery (VATS) was performed in all patients. Conversion to thoracotomy was necessary in 12 cases. Decortication and parietal pleurectomy in addition to hematoma and empyema removal was performed in all patients. Due to diffuse bleeding, packing of the thoracic cavity with temporary thoracic closure was necessary in 10 patients. Depacking was performed after a mean of 3 days (3-7 days). Recurrent empyema or bleeding after definitive chest closure was not observed. Lung resection was performed in 3 patients. CONCLUSIONS: Thoracic surgical management of pleural infection in patients after VAD implantation is challenging and complicated due to the inevitable anticoagulative therapy. A perioperative multidisciplinary management which includes the early involvement of thoracic surgical expertise helps to improve survival in this very complex patient cohort.

Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope.

The advent of highly sensitive photodetectors and the development of photostabilization strategies made detecting the fluorescence of single molecules a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g., in point-of-care diagnostic settings, where costly equipment would be prohibitive. Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in NACHOS emit up to 461-fold (average of 89 ± 7-fold) brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia on the road.

Pulsed Interleaved MINFLUX.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.

Moving targets in drug discovery.

Drug Discovery is a lengthy and costly process and has faced a period of declining productivity within the last two decades resulting in increasing importance of integrative data-driven approaches. In this paper, data mining and integration is leveraged to inspect target innovation trends in drug discovery. The study highlights protein families and classes that have received more attention and those that have just emerged in the scientific literature, thus highlighting novel opportunities for drug intervention. In order to delineate the evolution of target-driven research interest from a biological perspective, trends in biological process annotations from Gene Ontology and disease annotations from DisGeNET are captured. The analysis reveals an increasing interest in targets related to immune system processes, and a recurrent trend for targets involved in circulatory system processes. At the level of diseases, targets associated with cancer-related pathologies, intellectual disability, and schizophrenia are increasingly investigated in recent years. The methodology enables researchers to capture trends in research attention in target space at an early stage during the drug discovery process. Workflows, scripts, and data used in this study are publicly available from https://github.com/BZdrazil/Moving_Targets . An interactive web application allows the customized exploration of target, biological process, and disease trends (available at https://rguha.shinyapps.io/MovingTargets/ ).

Veno-Venous Extracorporeal Lung Support as a Bridge to or Through Lung Volume Reduction Surgery in Patients with Severe Hypercapnia.

Extracorporeal lung support (ECLS) represents an essential support tool especially for critically ill patients undergoing thoracic surgical procedures. Lung volume reduction surgery (LVRS) is an important treatment option for end-stage lung emphysema in carefully selected patients. Here, we report the efficacy of veno-venous ECLS (VV ECLS) as a bridge to or through LVRS in patients with end-stage lung emphysema and severe hypercapnia. Between January 2016 and May 2017, 125 patients with end-stage lung emphysema undergoing LVRS were prospectively enrolled into this study. Patients with severe hypercapnia caused by chronic respiratory failure were bridged to or through LVRS with low-flow VV ECLS (65 patients, group 1). Patients with preoperative normocapnia served as a control group (60 patients, group 2). In group 1, VV ECLS was implemented preoperatively in five patients and in 60 patients intraoperatively. Extracorporeal lung support was continued postoperatively in all 65 patients. Mean length of postoperative VV ECLS support was 3 ± 1 day. The 90 day mortality rate was 7.8% in group 1 compared with 5% in group 2 (p = 0.5). Postoperatively, a significant improvement was observed in quality of life, exercise capacity, and dyspnea symptoms in both groups. VV ECLS in patients with severe hypercapnia undergoing LVRS is an effective and well-tolerated treatment option. In particular, it increases the intraoperative safety, supports de-escalation of ventilatory strategies, and reduces the rate of postoperative complications in a cohort of patients considered "high risk" for LVRS in the current literature.

A structure-kinetic relationship study using matched molecular pair analysis.

The lifetime of a binary drug-target complex is increasingly acknowledged as an important parameter for drug efficacy and safety. With a better understanding of binding kinetics and better knowledge about kinetic parameter optimization, intentionally induced prolongation of the drug-target residence time through structural changes of the ligand could become feasible. In this study we assembled datasets from 21 publications and the K4DD (Kinetic for Drug Discovery) database to conduct large scale data analysis. This resulted in 3812 small molecules annotated to 78 different targets from five protein classes (GPCRs: 273, kinases: 3238, other enzymes: 240, HSPs: 160, ion channels: 45). Performing matched molecular pair (MMP) analysis to further investigate the structure-kinetic relationship (SKR) in this data collection allowed us to identify a fundamental contribution of a ligand's polarity to its association rate, and in selected cases, also to its dissociation rate. However, we furthermore observed that the destabilization of the transition state introduced by increased polarity is often accompanied by simultaneous destabilization of the ground state resulting in an unaffected or even worsened residence time. Supported by a set of case studies, we provide concepts on how to alter ligands in ways to trigger on-rates, off-rates, or both.

Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity.

Smartphone-based fluorescence microscopy has been rapidly developing over the last few years, enabling point-of-need detection of cells, bacteria, viruses, and biomarkers. These mobile microscopy devices are cost-effective, field-portable, and easy to use, and benefit from economies of scale. Recent developments in smartphone camera technology have improved their performance, getting closer to that of lab microscopes. Here, we report the use of DNA origami nanobeads with predefined numbers of fluorophores to quantify the sensitivity of a smartphone-based fluorescence microscope in terms of the minimum number of detectable molecules per diffraction-limited spot. With the brightness of a single dye molecule as a reference, we compare the performance of color and monochrome sensors embedded in state-of-the-art smartphones. Our results show that the monochrome sensor of a smartphone can achieve better sensitivity, with a detection limit of ∼10 fluorophores per spot. The use of DNA origami nanobeads to quantify the minimum number of detectable molecules of a sensor is broadly applicable to evaluate the sensitivity of various optical instruments.

Impact of diabetes type on treatment and outcome of patients with peripheral artery disease.

BACKGROUND: The prevalence of diabetes mellitus and its associated complications such as peripheral artery disease is increasing worldwide. We aimed to explore the distinct impact of type 1 diabetes mellitus and type 2 diabetes mellitus on treatment and on short- and long-term outcome in patients with peripheral artery disease. METHODS: Retrospective analysis of anonymized data of hospitalized patients covered by a large German health insurance. Assessment of patient's characteristics (comorbidities, complications, etc.) and outcome using multivariable Cox regression and Kaplan-Meier curves. RESULTS: Among 41,702 patients with peripheral artery disease, 339 (0.8%) had type 1 diabetes mellitus and 13,151 (31.5%) had type 2 diabetes mellitus. Patients with diabetes mellitus had more comorbidities and complications than patients without diabetes mellitus ( p < 0.001). Type 1 diabetes mellitus patients exhibited the highest risk for limb amputation at 4-year follow-up (44.6% vs 35.1%, p < 0.001), while type 2 diabetes mellitus patients had higher mortality than type 1 diabetes mellitus (43.6% vs 31.0%, p < 0.001). CONCLUSION: Although the fraction of type 1 diabetes mellitus among patients with peripheral artery disease and diabetes mellitus is low, it represents a subset of patients being at particular high risk for limb amputation. Research focused on elaborating the determinants of limb amputation and mortality in peripheral artery disease patients with diabetes mellitus is warranted to improve the poor prognosis of these patients.

SAR-Guided Scoring Function and Mutational Validation Reveal the Binding Mode of CGS-8216 at the α1+/γ2- Benzodiazepine Site.

The structural resolution of a bound ligand-receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a challenging endeavor. In the absence of structural knowledge, scientists resort to structure-activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.

Ligand Desolvation Steers On-Rate and Impacts Drug Residence Time of Heat Shock Protein 90 (Hsp90) Inhibitors.

Residence time and more recently the association rate constant kon are increasingly acknowledged as important parameters for in vivo efficacy and safety of drugs. However, their broader consideration in drug development is limited by a lack of knowledge of how to optimize these parameters. In this study on a set of 176 heat shock protein 90 inhibitors, structure-kinetic relationships, X-ray crystallography, and molecular dynamics simulations were combined to retrieve a concrete scheme of how to rationally slow down on-rates. We discovered that an increased ligand desolvation barrier by introducing polar substituents resulted in a significant kon decrease. The slowdown was accomplished by introducing polar moieties to those parts of the ligand that point toward a hydrophobic cavity. We validated this scheme by increasing polarity of three Hsp90 inhibitors and observed a 9-, 13-, and 45-fold slowdown of on-rates and a 9-fold prolongation in residence time. This prolongation was driven by transition state destabilization rather than ground state stabilization.

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p<0.05), indicating that non-cycling (G0) cells entered the cell cycle following the doxorubicin exposure. Support for this notion was obtained also in vitro. A pharmacologically relevant dose (1µM) resulted in a marked accumulation of Ki67 positive BE(2)-C cells (p<0.0001), as well as a >3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation.

Topliss Batchwise Schemes Reviewed in the Era of Open Data Reveal Significant Differences between Enzymes and Membrane Receptors.

In 1977, John G. Topliss introduced the Topliss Batchwise Scheme, a straightforward nonmathematical procedure to assist medicinal chemists in optimizing the substitution pattern of a phenyl ring. Despite its long period of application, a thorough validation of this method has been missing so far. Here, we address this issue by gathering 129 congeneric series from the ChEMBL database, suitable to retrospectively assess the approach. Frequency analysis of Topliss' schemes showed that the π, Es, σ, and -σ scheme occurred in 17, 20, 6, and 4 congeneric series, respectively. We observed a significant difference of π scheme frequency in enzymes versus membrane receptors, with 12 versus only 2 occurrences. Validation of Topliss schemes in potency optimization showed a remarkable performance increase after restricting the data set to analogue series tested solely against enzymes. In this setting, the Es and the π scheme were successful in 50% and 56% of the analogue series, respectively.

Loss of miR-514a-3p regulation of PEG3 activates the NF-kappa B pathway in human testicular germ cell tumors.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.