Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.

A "push and slide" mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.

In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a "push and slide" mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases.

Protein translocation by the SecA ATPase occurs by a power-stroke mechanism.

SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides.

Prolonged antibiotic prophylaxis in tissue reconstruction using autologous fat grafting: Is there a benefit for wound healing?

Fat grafting is a well-established method in plastic surgery. Despite many technical advances, standardised recommendations for the use of prophylactic antibiotics in fat grafting are not available. This retrospective multicentre study aims to analyse the use of prophylactic antibiotics in fat grafting and to compare complication rates for different protocols. A retrospective medical chart review of 340 patients treated with fat grafting of the breast from January 2007 to March 2019 was performed in three plastic surgery centres. Complications, outcomes, and antibiotic regimes were analysed. The Clavien-Dindo classification was applied. All patients received perioperative antibiotic prophylaxis: 33.8% (n = 115) were treated with a single shot (group 1), 66.2% (n = 225) received a prolonged antibiotic scheme (group 2). There was no significant difference in the number of sessions (P = .475). The overall complication rate was 21.6% (n = 75), including graft resorption, fat necrosis, infection, and wound healing problems. Complication rates were not significantly different between groups. Risk factors for elevated complication rates in this specific patient group are smoking, chemotherapy, and irradiation therapy. The complication rate for lipografting of the breast is low, and it is not correlated to the antibiotic protocol. The use of prolonged prophylactic antibiotics does not lower the complication rate.

Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing.

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon.

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

Epitaxial Growth of Room-Temperature Ferromagnetic MnAs Segments on GaAs Nanowires via Sequential Crystallization.

We investigate the incorporation of manganese into self-catalyzed GaAs nanowires grown in molecular beam epitaxy. Our study reveals that Mn accumulates in the liquid Ga droplet and that no significant incorporation into the nanowire is observed. Using a sequential crystallization of the droplet, we then demonstrate a deterministic and epitaxial growth of MnAs segments at the nanowire tip. This technique may allow the seamless integration of multiple room-temperature ferromagnetic segments into GaAs nanowires with high-crystalline quality.

Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation.

While engaged in protein transport, the bacterial translocon SecYEG must maintain the membrane barrier to small ions. The preservation of the proton motif force was attributed to (i) cation exclusion, (ii) engulfment of the nascent chain by the hydrophobic pore ring, and (iii) a half-helix partly plugging the channel. In contrast, we show here that preservation of the proton motif force is due to a voltage-driven conformational change. Preprotein or signal peptide binding to the purified and reconstituted SecYEG results in large cation and anion conductivities only when the membrane potential is small. Physiological values of membrane potential close the activated channel. This voltage-dependent closure is not dependent on the presence of the plug domain and is not affected by mutation of 3 of the 6 constriction residues to glycines. Cellular ion homeostasis is not challenged by the small remaining leak conductance.

Sequential karyotyping in Burkitt lymphoma reveals a linear clonal evolution with increase in karyotype complexity and a high frequency of recurrent secondary aberrations.

Typical Burkitt lymphoma is characterized by an IG-MYC translocation and overall low genomic complexity. Clinically, Burkitt lymphoma has a favourable prognosis with very few relapses. However, the few patients experiencing disease progression and/or relapse have a dismal outcome. Here we report cytogenetic findings of seven cases of Burkitt lymphoma in which sequential karyotyping was performed at time of diagnosis and/or disease progression/relapse(s). After case selection, karyotype re-review and additional molecular analyses were performed in six paediatric cases, treated in Berlin-Frankfurt-Münster-Non-Hodgkin lymphoma study group trials, and one additional adult patient. Moreover, we analysed 18 cases of Burkitt lymphoma from the Mitelman database in which sequential karyotyping was performed. Our findings show secondary karyotypes to have a significant increase in load of cytogenetic aberrations with a mean number of 2, 5 and 8 aberrations for primary, secondary and third investigations. Importantly, this increase in karyotype complexity seemed to result from recurrent secondary chromosomal changes involving mainly trisomy 21, gains of 1q and 7q, losses of 6q, 11q, 13q, and 17p. In addition, our findings indicate a linear clonal evolution to be the predominant manner of cytogenetic evolution. Our data may provide a biological framework for the dismal outcome of progressive and relapsing Burkitt lymphoma.

Mitochondrial DNA and Toll-Like Receptor-9 Are Associated With Mortality in Critically Ill Patients.

OBJECTIVES: Despite underlying pathologies leading to ICU admittance are heterogeneous, many patients develop a systemic inflammatory response syndrome often in the absence of microbial pathogens. Mitochondrial DNA that shows similarities to bacterial DNA may be released after tissue damage and activates the innate immune system by binding to toll-like receptor-9 on immune cells. The aim of this study was to analyze whether levels of mitochondrial DNA are associated with 30-day survival and whether this predictive value is modified by the expression of its receptor toll-like receptor-9. DESIGN: Single-center, prospective, observational study. SETTING: A tertiary ICU in a university hospital. PATIENTS: Two hundred twenty-eight consecutive patients admitted to a medical ICU between August 2012 and August 2013. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was taken within 24 hours after ICU admission, and the levels of circulating mitochondrial DNA were quantified by real-time polymerase chain reaction. Toll-like receptor-9 expression in monocytes was measured by flow cytometry. Median acute physiology and chronic health evaluation II score was 20, and 30-day mortality was 25%. Median mitochondrial DNA levels at admission were significantly higher in nonsurvivors when compared with survivors (26.9, interquartile range = 11.2-60.6 ng/mL vs 19.7, interquartile range = 9.5-34.8 ng/mL; p < 0.05). Patients with plasma levels of mitochondrial DNA in the highest quartile (mitochondrial DNA > 38.2 ng/mL) had a 2.6-fold higher risk (p < 0.001) of dying, independently of age, gender, diagnosis, and acute physiology and chronic health evaluation II score. Mitochondrial DNA improved the c-statistic of acute physiology and chronic health evaluation II score (p < 0.05) and showed enhancement in individual risk prediction indicated by a net reclassification improvement of 32.3% (p < 0.05). Stratification of patients according to toll-like receptor-9 expression above/below median demonstrated that only patients with high expression of toll-like receptor-9 showed an increased risk associated with increased mitochondrial DNA levels (odds ratio, 2.7; p < 0.01), whereas circulating mitochondrial DNA was not associated with mortality in patients with low toll-like receptor-9 expression (odds ratio, 1.1; p = 0.98). CONCLUSIONS: Circulating levels of mitochondrial DNA at ICU admission predict mortality in critically ill patients. This association was in particular present in patients with elevated toll-like receptor-9 expression.

Cancer initiation with epistatic interactions between driver and passenger mutations.

We investigate the dynamics of cancer initiation in a mathematical model with one driver mutation and several passenger mutations. Our analysis is based on a multi-type branching process: we model individual cells which can either divide or undergo apoptosis. In the case of a cell division, the two daughter cells can mutate, which potentially confers a change in fitness to the cell. In contrast to previous models, the change in fitness induced by the driver mutation depends on the genetic context of the cell, in our case on the number of passenger mutations. The passenger mutations themselves have no or only a very small impact on the cell's fitness. While our model is not designed as a specific model for a particular cancer, the underlying idea is motivated by clinical and experimental observations in Burkitt Lymphoma. In this tumor, the hallmark mutation leads to deregulation of the MYC oncogene which increases the rate of apoptosis, but also the proliferation rate of cells. This increase in the rate of apoptosis hence needs to be overcome by mutations affecting apoptotic pathways, naturally leading to an epistatic fitness landscape. This model shows a very interesting dynamical behavior which is distinct from the dynamics of cancer initiation in the absence of epistasis. Since the driver mutation is deleterious to a cell with only a few passenger mutations, there is a period of stasis in the number of cells until a clone of cells with enough passenger mutations emerges. Only when the driver mutation occurs in one of those cells, the cell population starts to grow rapidly.

Mapping polypeptide interactions of the SecA ATPase during translocation.

Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a "clamp" of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel.

[The Emerging Interest in Breast Implant Illness and its impact on Patient Behavior]. / Das wachsende Interesse an Breast Implant Illness und dessen Einfluss auf das Verhalten der Patientinnen.

Breast implant illness (BII) is a loosely defined term for a collection of non-specific systemic symptoms that are hypothesised to be associated with breast implants. BII symptoms include fatigue, hair loss, rashes, chronic pain, and others. However, conclusive evidence for a causal relationship between silicone implants and BII remains lacking. In the light of recent findings that textured implants can, in rare cases, lead to breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), a potential link between breast implants and BII is conceivable and justifies further investigation. We observe a growing number of patients seeking consultation and treatment for systemic symptoms related to breast implants, which is reflected in increasing interest in literature and social media. The aim of this work was to investigate the growing interest in BII. We now describe the clinical features of a patient who suffers from symptoms that are consistent with BII and contextualise clinical presentation in a review of literature and google trend analysis.

Bleeding Risk Assessment in End-Stage Kidney Disease: Validation of Existing Risk Scores and Evaluation of a Machine Learning-Based Approach.

BACKGROUND: Patients with end-stage kidney disease (ESKD) on hemodialysis (HD) are at increased risk for bleeding. However, despite relevant clinical implications regarding dialysis modalities or anticoagulation, no bleeding risk assessment strategy has been established in this challenging population. METHODS: Analyses on bleeding risk assessment models were performed in the population-based Vienna InVestigation of Atrial fibrillation and thromboemboLism in patients on hemoDialysIs (VIVALDI) study including 625 patients. In this cohort study, patients were prospectively followed for a median observation period of 3.5 years for the occurrence of major bleeding. First, performances of existing bleeding risk scores (i.e., HAS-BLED, HEMORR2HAGES, ATRIA, and four others) were evaluated in terms of discrimination and calibration. Second, four machine learning-based prediction models that included clinical, dialysis-specific, and laboratory parameters were developed and tested using Monte Carlo cross-validation. RESULTS: Of 625 patients (median age: 66 years, 37% women), 89 (14.2%) developed major bleeding, with a 1-year, 2-year, and 3-year cumulative incidence of 6.1% (95% confidence interval [CI]: 4.2-8.0), 10.3% (95% CI: 8.0-12.8), and 13.5% (95% CI: 10.8-16.2), respectively. C-statistics of the seven contemporary bleeding risk scores ranged between 0.54 and 0.59 indicating poor discriminatory performance. The HAS-BLED score showed the highest C-statistic of 0.59 (95% CI: 0.53-0.66). Similarly, all four machine learning-based predictions models performed poorly in internal validation (C-statistics ranging from 0.49 to 0.55). CONCLUSION: Existing bleeding risk scores and a machine learning approach including common clinical parameters fail to assist in bleeding risk prediction of patients on HD. Therefore, new approaches, including novel biomarkers, to improve bleeding risk prediction in patients on HD are needed.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Keystone Flap Type IIIB: A New Variation for Coverage of Defects at Joint Regions.

Soft tissue defect reconstruction at joint regions is a challenging problem due to the sparse excessive tissue and late complication of constrigent scar formation. Priorly irradiated tissue, often the case in sarcoma patients, is especially problematic. The keystone design perforator island flap is safe and reliable. We now present a new keystone flap design, which is particularly suitable for the reconstruction of large soft tissue defects at joint regions. It provides a cutaneous component without the need for a skin graft and therefore minimizes the risk of contracture. Donor site morbidity is negligible. Furthermore, it offers a favorable aesthetic result compared to other flaps, eg, a muscular flap. We propose a new keystone flap design as an extension of Behan's classification, the Keystone flap type IIIb.

Exploration of zebrafish larvae as an alternative whole-animal model for nephrotoxicity testing.

Due to an increasing demand for testing of new and existing chemicals and legal restrictions for the use of animals, there is a strong need for alternative approaches to assess systemic toxicity. Embryonic and larval zebrafish (Danio rerio) are increasingly recognized as a promising alternative whole-animal model that may be able to overcome limitations of cell-based in vitro assays and bridge the gap between high-throughput in vitro screening and low-throughput in vivo tests in animals. Despite the relatively simple anatomical structure of the zebrafish larval kidney (pronephros) - composed of only two nephrons - the pronephros shares major functions and cell types with mammalian nephrons. Glomerular filtration begins at 48 h post fertilization. The aim of the present study was to investigate if early zebrafish larvae might be a suitable model for nephrotoxicity testing. On day 3 post fertilization, larval zebrafish were treated with selected nephrotoxins (aristolochic acid, cadmium chloride, potassium bromate, ochratoxin A, gentamicin) for 48 h. Histological evaluation of zebrafish larvae exposed to model nephrotoxins revealed tubule injury as evidenced by dilated tubules with loss of the brush border, tubule cell necrosis and disorganization of the tubular epithelium. These changes were most severe after treatment with gentamicin, which also impaired pronephros function as evidenced by reduced clearance of FITC-dextran. Whole-mount in situ hybridization showing loss of cdh17 expression revealed site-specific injury to the proximal tubule segment. Analysis of genes previously identified as novel biomarkers of kidney injury in mammals showed upregulation of the kidney injury marker genes heme oxygenase 1 (hmox1), clusterin (clu), secreted phosphoprotein/osteopontin (spp1), connective tissue growth factor (ctgf) and kim-1 (havcr-1) in response to nephrotoxin treatment, although the response of individual genes varied across compounds. Consistent with the severity of lesions and impaired kidney function, the most prominent gene expression changes occurred in larvae exposed to gentamicin. Overall, our results suggest that larval zebrafish may be a suitable alternative model organism for nephrotoxicity screening, yet further improvements and integration with quantitative in vitro to in vivo extrapolation will be needed to predict human toxicity.

Position controlled self-catalyzed growth of GaAs nanowires by molecular beam epitaxy.

GaAs nanowires are grown by molecular beam epitaxy using a self-catalyzed, Ga-assisted growth technique. Position control is achieved by nano-patterning a SiO(2) layer with arrays of holes with a hole diameter of 85 nm and a hole pitch varying between 200 nm and 2 µm. Gallium droplets form preferentially at the etched holes acting as catalyst for the nanowire growth. The nanowires have hexagonal cross-sections with {110} side facets and crystallize predominantly in zincblende. The interdistance dependence of the nanowire growth rate indicates a change of the III/V ratio towards As-rich conditions for large hole distances inhibiting NW growth.

Quantifying the heterogeneity of macromolecular machines by mass photometry.

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.