Compliant DNA Origami Nanoactuators as Size-Selective Nanopores.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Investigating the cause of a 2021 winter wave of COVID-19 in a border region in eastern Germany: a mixed-methods study, August to November 2021.

It is so far unclear how the COVID-19 winter waves started and what should be done to prevent possible future waves. In this study, we deciphered the dynamic course of a winter wave in 2021 in Saxony, a state in Eastern Germany neighbouring the Czech Republic and Poland. The study was carried out through the integration of multiple virus genomic epidemiology approaches to track transmission chains, identify emerging variants and investigate dynamic changes in transmission clusters. For identified local variants of interest, functional evaluations were performed. Multiple long-lasting community transmission clusters have been identified acting as driving force for the winter wave 2021. Analysis of the dynamic courses of two representative clusters indicated a similar transmission pattern. However, the transmission cluster caused by a locally occurring new Delta variant AY.36.1 showed a distinct transmission pattern, and functional analyses revealed a replication advantage of it. This study indicated that long-lasting community transmission clusters starting since early autumn caused by imported or locally occurring variants all contributed to the development of the 2021 winter wave. The information we achieved might help future pandemic prevention.

Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

Compartmentalization and synergy of osteoblasts drive bone formation in the regenerating fin.

Zebrafish regenerate their fins which involves a component of cell plasticity. It is currently unclear how regenerate cells divide labor to allow for appropriate growth and patterning. Here, we studied lineage relationships of fluorescence-activated cell sorting-enriched epidermal, bone-forming (osteoblast), and (non-osteoblast) blastemal fin regenerate cells by single-cell RNA sequencing, lineage tracing, targeted osteoblast ablation, and electron microscopy. Most osteoblasts in the outgrowing regenerate derive from osterix+ osteoblasts, while mmp9+ cells reside at segment joints. Distal blastema cells contribute to distal osteoblast progenitors, suggesting compartmentalization of the regenerating appendage. Ablation of osterix+ osteoblasts impairs segment joint and bone matrix formation and decreases regenerate length which is partially compensated for by distal regenerate cells. Our study characterizes expression patterns and lineage relationships of rare fin regenerate cell populations, indicates inherent detection and compensation of impaired regeneration, suggests variable dependence on growth factor signaling, and demonstrates zonation of the elongating fin regenerate.

Comparing divisome organization between vegetative and sporulating Bacillus subtilis at the nanoscale using DNA-PAINT.

Spore-forming bacteria have two distinct division modes: sporulation and vegetative division. The placement of the foundational division machinery component (Z-ring) within the division plane is contingent on the division mode. However, investigating if and how division is performed differently between sporulating and vegetative cells remains challenging, particularly at the nanoscale. Here, we use DNA-PAINT super-resolution microscopy to compare the 3D assembly and distribution patterns of key division proteins SepF, ZapA, DivIVA, and FtsZ. We determine that ZapA and SepF placement within the division plane mimics that of the Z-ring in vegetative and sporulating cells. We find that DivIVA assemblies differ between vegetative and sporulating cells. Furthermore, we reveal that SepF assembles into ~50-nm arcs independent of division mode. We propose a nanoscale model in which symmetric or asymmetric placement of the Z-ring and early divisome proteins is a defining characteristic of vegetative or sporulating cells, respectively, and regulation of septal thickness differs between division modes.

Balancing the Nanoscale Organization in Multivalent Materials for Functional Inhibition of the Programmed Death-1 Immune Checkpoint.

Dendritic cells (DCs) regulate immune priming by expressing programmed death ligand 1 (PD-L1) and PD-L2, which interact with the inhibitory receptor PD-1 on activated T cells. PD-1 signaling regulates T cell effector functions and limits autoimmunity. Tumor cells can hijack this pathway by overexpressing PD-L1 to suppress antitumor T cell responses. Blocking this inhibitory pathway has been beneficial for the treatment of various cancer types, although only a subset of patients responds. A deepened understanding of the spatial organization and molecular interplay between PD-1 and its ligands may inform the design of more efficacious nanotherapeutics. We visualized the natural molecular PD-L1 organization on DCs by DNA-PAINT microscopy and created a template to engineer DNA-based nanoclusters presenting PD-1 at defined valencies, distances, and patterns. These multivalent nanomaterials were examined for their cellular binding and blocking ability. Our data show that PD-1 nano-organization has profound effects on ligand interaction and that the valency of PD-1 molecules modulates the effectiveness in restoring T cell function. This work highlights the power of spatially controlled functional materials to unravel the importance of multivalent patterns in the PD-1 pathway and presents alternative design strategies for immune-engineering.

IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration.

The influence of aging on intestinal stem cells and their niche can explain underlying causes for perturbation in their function observed during aging. Molecular mechanisms for such a decrease in the functionality of intestinal stem cells during aging remain largely undetermined. Using transcriptome-wide approaches, our study demonstrates that aging intestinal stem cells strongly upregulate antigen presenting pathway genes and over-express secretory lineage marker genes resulting in lineage skewed differentiation into the secretory lineage and strong upregulation of MHC class II antigens in the aged intestinal epithelium. Mechanistically, we identified an increase in proinflammatory cells in the lamina propria as the main source of elevated interferon gamma (IFNγ) in the aged intestine, that leads to the induction of Stat1 activity in intestinal stem cells thus priming the aberrant differentiation and elevated antigen presentation in epithelial cells. Of note, systemic inhibition of IFNγ-signaling completely reverses these aging phenotypes and reinstalls regenerative capacity of the aged intestinal epithelium.

Ångström-resolution fluorescence microscopy.

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1-6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7-14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

Robot-assisted laparoscopic varicocelectomy in a pediatric population.

PURPOSE: To present our experience with robot-assisted laparoscopic varicocelectomy in a pediatric population. METHODS: We reviewed 49 consecutive cases performed by the same experienced surgeon. One-to-four veins were ligated at the internal ring of the inguinal canal, while the testicular artery and lymphatics were spared. Information on patient characteristics, surgical time, complications, and recurrences were collected. RESULTS: Median patient age was 14 (range 10-17) years. Forty-eight had left-sided varicoceles and one had a bilateral varicocele. Forty-five were grade 3. All patients were referred due to discomfort/pain and 20 also had reduced testicular size. The median operating time from skin incision was 48 min (31-89 min) and the median console time was 18 min (7-55 min). Forty-seven patients were discharged the same day. Two patients experienced pain and problems urinating, respectively. These issues had resolved by the first post-operative day. There were no other complications, but at 6 months, eight recurrences were noted (16%). Scrotal complaints had subsided in all patients. Catch-up growth of the affected testicles was seen in 19/20 cases. CONCLUSION: Robot-assisted laparoscopic varicocelectomy is feasible and safe in a pediatric population but with a relatively high recurrence rate.

Corrigendum: A novel type I interferon primed dendritic cell subpopulation in TREX1 mutant chilblain lupus patients.

[This corrects the article DOI: 10.3389/fimmu.2022.897500.].

Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material.

BACKGROUND: Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissues into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs. RESULTS: We show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. At the same time, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq. A direct comparison of scRNAseq and snRNAseq data revealed that both datasets share biologically relevant genes among the most variable genes, and we showed differences in the relative contribution of the two approaches to identified cell types. CONCLUSION: We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue.

Frequency, Risk Factors, and Clinical Outcomes of Late-Onset Atrial Flutter in Patients after Heart Transplantation.

Aims: Atrial flutter (AFL) is a common late-onset complication after heart transplantation (HTX) and is associated with worse clinical outcomes. Methods: This study investigated the frequency, risk factors, and outcomes of late-onset post-transplant AFL. We analyzed 639 adult patients undergoing HTX at the Heidelberg Heart Center between 1989 and 2019. Patients were stratified by diagnosis and type of late-onset post-transplant AFL (>90 days after HTX). Results: A total of 55 patients (8.6%) were diagnosed with late-onset post-transplant AFL, 30 had typical AFL (54.5%) and 25 had atypical AFL (45.5%). Patients with AFL were younger at HTX (p = 0.028), received more biatrial anastomosis (p = 0.001), and presented with moderate or severe tricuspid regurgitation (56.4%). Typical AFL was associated with graft rejection (p = 0.016), whereas atypical AFL was associated with coronary artery disease (p = 0.028) and stent implantation (p = 0.042). Patients with atypical AFL showed a higher all-cause 1-year mortality (p = 0.010) along with a higher rate of graft failure after diagnosis of AFL (p = 0.023). Recurrence of AFL was high (83.6%). Patients with catheter ablation after AFL recurrence had a higher 1-year freedom from AFL (p = 0.003). Conclusions: Patients with late-onset post-transplant AFL were younger at HTX, received more biatrial anastomosis, and showed a higher rate of moderate or severe tricuspid regurgitation. Typical AFL was associated with graft rejection, whereas atypical AFL was associated with myocardial ischemia, graft failure, and mortality. Catheter ablation represents a viable option to avoid further episodes of late-onset AFL after HTX.

A Novel Type I Interferon Primed Dendritic Cell Subpopulation in TREX1 Mutant Chilblain Lupus Patients.

Heterozygous TREX1 mutations are associated with monogenic familial chilblain lupus and represent a risk factor for developing systemic lupus erythematosus. These interferonopathies originate from chronic type I interferon stimulation due to sensing of inadequately accumulating nucleic acids. We here analysed the composition of dendritic cell (DC) subsets, central stimulators of immune responses, in patients with TREX1 deficiency. We performed single-cell RNA-sequencing of peripheral blood DCs and monocytes from two patients with familial chilblain lupus and heterozygous mutations in TREX1 and from controls. Type I interferon pathway genes were strongly upregulated in patients. Cell frequencies of the myeloid and plasmacytoid DC and of monocyte populations in patients and controls were similar, but we describe a novel DC subpopulation highly enriched in patients: a myeloid DC CD1C+ subpopulation characterized by the expression of LMNA, EMP1 and a type I interferon- stimulated gene profile. The presence of this defined subpopulation was confirmed in a second cohort of patients and controls by flow cytometry, also revealing that an increased percentage of patient's cells in the subcluster express costimulatory molecules. We identified a novel type I interferon responsive myeloid DC subpopulation, that might be important for the perpetuation of TREX1-induced chilblain lupus and other type I interferonopathies.

Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis.

Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48-/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.

Robot-Assisted vs. Open Appendicovesicostomy in Pediatric Urology: A Systematic Review and Single-Center Case Series.

Introduction: Appendicovesicostomy (APV) is the preferred choice of continent catheterizable channels in pediatric urology. The introduction of robot-assisted laparoscopic techniques has been correlated to superior cosmesis and convalescence and is now increasingly implemented for APV procedures. We aimed to perform a systematic review of the literature comparing open vs. robotic APV regarding possible differences in postoperative outcomes and to evaluate these findings with our own initial experiences with robotic APV compared to our previous open procedures. Methods: We evaluated the first five patients undergoing robotic APV at our institution and compared 1-year outcomes with a consecutive series of 12 patients undergoing open APV. In a systematic literature review, we screened studies from PubMed, EMBASE, and CENTRAL comparing open and robotic APV in pediatric urology (current to December 2021) and performed meta-analyses on postoperative outcomes comparing the two groups and evaluated the grade of evidence. Results: We found significantly shortened postoperative length of stay in the robotic group (p = 0.001) and comparable 1-year complication rates in robotic vs. open APV patients. We systematically screened 3,204 studies and ultimately included three non-randomized studies comparing postoperative outcomes of robotic and open APV for quantitative analysis. The open and robotic approaches performed equally well regarding overall postoperative complications, surgical reintervention, and stomal stenosis. Two of the included studies reported comparable stomal continence rates and shortened postoperative length of stay in the robotic group, in agreement with the findings in our own series. Conclusion: Robotic APV is equally safe to the conventional open approach with additional advantages in postoperative hospitalization length.

Single-Cell Analysis and Tracking of Antigen-Specific T Cells: Integrating Paired Chain AIRR-Seq and Transcriptome Sequencing: A Method by the AIRR Community.

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) offers the possibility to access the nucleotide sequences of paired receptor chains from T-cell receptors (TCR) or B-cell receptors (BCR ). Here we describe two protocols and the downstream bioinformatic approaches that facilitate the integrated analysis of paired T-cell receptor (TR ) alpha/beta (TRA /TRB ) AIRR-seq, RNA sequencing (RNAseq), immunophenotyping, and antigen-binding information. To illustrate the methodologies with a use case, we describe how to identify, characterize, and track SARS-CoV-2-specific T cells over multiple time points following infection with the virus. The first method allows the analysis of pools of memory CD8+ cells, identifying expansions and contractions of clones of interest. The second method allows the study of rare or antigen-specific cells and allows studying their changes over time.