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Abstract Opioid use disorder (OUD) has emerged as a significant public health crisis affecting individuals across all age groups. However, there remains a critical gap in understanding the specific nuances and challenges associated with OUD in pediatric populations. This article provides a comprehensive review of the epidemiology, definition of OUD, screening recommendations for OUD, and evidence‐based management strategies for OUD in pediatric patients.

Background:. Lymphedema diagnosis relies on effective imaging of the lymphatic system. Indocyanine green (ICG) lymphography has become an essential diagnostic tool, but globally accepted protocols and objective analysis methods are lacking. In this study, we aimed to investigate artificial intelligence (AI), specifically convolutional neural networks, to categorize ICG lymphography images patterns into linear, reticular, splash, stardust, and diffuse. Methods:. A dataset composed of 68 ICG lymphography images was compiled and labeled according to five recognized pattern types: linear, reticular, splash, stardust, and diffuse. A convolutional neural network model, using MobileNetV2 and TensorFlow, was developed and coded in Python for pattern classification. Results:. The AI model achieved 97.78% accuracy and 0.0678 loss in categorizing images into five ICG lymphography patterns, demonstrating high potential for enhancing ICG lymphography interpretation. The high level of accuracy with a low loss achieved by our model demonstrates its effectiveness in pattern recognition with a high degree of precision. Conclusions:. This study demonstrates that AI models can accurately classify ICG lymphography patterns. AI can assist in standardizing and automating the interpretation of ICG lymphographic imaging.

Lung transplantation (LuTx) is an established treatment for patients with end-stage lung diseases, however, outcomes are limited by acute and chronic rejection. One aspect that has received increasing attention is the role of the host’s humoral alloresponse, particularly the formation of de novo donor-specific antibodies (dnDSAs). The aim of this study was to investigate the clinical significance of transient and persistent dnDSAs and to understand their impact on outcomes after LuTx. A retrospective analysis was conducted using DSA screening data from LuTx recipients obtained at the Medical University of Vienna between February 2016 and March 2021. Of the 405 LuTx recipients analyzed, 205 patients developed dnDSA during the follow-up period. Among these, 167 (81%) had transient dnDSA and 38 (19%) persistent dnDSA. Persistent but not transient dnDSAs were associated with chronic lung allograft dysfunction (CLAD) and antibody-mediated rejection (AMR) (p < 0.001 and p = 0.006, respectively). CLAD-free survival rates for persistent dnDSAs at 1-, 3-, and 5-year post-transplantation were significantly lower than for transient dnDSAs (89%, 59%, 56% vs. 91%, 79%, 77%; p = 0.004). Temporal dynamics of dnDSAs after LuTx have a substantial effect on patient outcomes. This study underlines that the persistence of dnDSAs poses a significant risk to graft and patient survival.

Abstract Recent increases in pediatric and adolescent opioid fatalities mandate an urgent need for early consideration of possible opioid exposure and specific diagnostic and management strategies and interventions tailored to these unique populations. In contrast to adults, pediatric methods of exposure include accidental ingestions, prescription misuse, and household exposure. Early recognition, appropriate diagnostic evaluation, along with specialized treatment for opioid toxicity in this demographic are discussed. A key focus is on Naloxone, an essential medication for opioid intoxication, addressing its unique challenges in pediatric use. Unique pediatric considerations include recognition of accidental ingestions in our youngest population, critical social aspects including home safety and intentional exposure, and harm reduction strategies, mainly through Naloxone distribution and education on safe medication practices. It calls for a multifaceted approach, including creating pediatric‐specific guidelines, to combat the opioid crisis among children and to work to lower morbidity and mortality from opioid overdoses.

Cell shape plays a fundamental role in many biological processes, including adhesion, migration, division and development, but it is not clear which shape model best predicts three-dimensional cell shape in structured environments. Here, we compare different modelling approaches with experimental data. The shapes of single mesenchymal cells cultured in custom-made 3D scaffolds were compared by a Fourier method with surfaces that minimize area under the given adhesion and volume constraints. For the minimized surface model, we found marked differences to the experimentally observed cell shapes, which necessitated the use of more advanced shape models. We used different variants of the cellular Potts model, which effectively includes both surface and bulk contributions. The simulations revealed that the Hamiltonian with linear area energy outperformed the elastic area constraint in accurately modelling the 3D shapes of cells in structured environments. Explicit modelling the nucleus did not improve the accuracy of the simulated cell shapes. Overall, our work identifies effective methods for accurately modelling cellular shapes in complex environments.

Abstract Cell crawling on flat substrates stems from intracellular flows of the actin cytoskeleton that are driven by both actin polymerization at the front and myosin contractility at the back. Optogenetics makes it experimentally possible to spatially control contraction and possibly cell migration too. Here we theoretically analyze this situation using a one-dimensional active gel model that reflects the property of myosin II to assemble into minifilaments. Our model predicts bistability between sessile and motile solutions when cell adhesion and contractility are sufficiently large and in balance. We show that one can switch between the different states at realistic parameter values via optogenetic activation or inhibition of contractility, in agreement with recent experiments performed for neutrophils in microchannels. We predict the required activation strengths and initiation times, compare the effects of local and global increases of myosin II levels, and show that actin polymerization alone can affect a switch in direction only at high strength.

Since the early days of clinical lung transplantation the preservation of donor organs has become a fairly standardized procedure and most centers do follow similar processes. This includes the use of low-potassium high dextran flush solutions and static cold storage (SCS) in a cooler filled with ice. Depending on the length of SCS, organs usually arrive at the recipient hospital at a temperature of 0°C–4°C. The question of the optimal storage temperature for donor lung preservation has been revisited as data from large animal experiments demonstrated that organs stored at 10°C experience less mitochondrial damage. Thus, prolonged cold ischemic times can be better tolerated at 10°C—even in pre-damaged organs. The clinical applicability of these findings was demonstrated in an international multi-center observational study including three high-volume lung transplant centers. Total clinical preservation times of up to 24 hrs have been successfully achieved in organs stored at 10°C without hampering primary organ function and short-term outcomes. Currently, a randomized-controlled trial (RCT) is recruiting patients with the aim to compare standard SCS on ice with prolonged SCS protocol at 10°C. If, as anticipated, this RCT confirms data from previous studies, lung transplantation could indeed become a semi-elective procedure.

Accurate representation of aerosol optical properties is essential for the modeling and remote sensing of atmospheric aerosols. Although aerosol optical properties are strongly dependent upon the aerosol size distribution, the use of detailed aerosol microphysics schemes in global atmospheric models is inhibited by associated computational demands. Computationally efficient parameterizations for aerosol size are needed. In this study, airborne measurements over the United States (DISCOVER-AQ) and South Korea (KORUS-AQ) are interpreted with a global chemical transport model (GEOS-Chem) to investigate the variation in aerosol size when organic matter (OM) and sulfate–nitrate–ammonium (SNA) are the dominant aerosol components. The airborne measurements exhibit a strong correlation (r=0.83) between dry aerosol size and the sum of OM and SNA mass concentration (MSNAOM). A global microphysical simulation (GEOS-Chem-TOMAS) indicates that MSNAOM and the ratio between the two components (OM/SNA) are the major indicators for SNA and OM dry aerosol size. A parameterization of the dry effective radius (Reff) for SNA and OM aerosol is designed to represent the airborne measurements (R2=0.74; slope = 1.00) and the GEOS-Chem-TOMAS simulation (R2=0.72; slope = 0.81). When applied in the GEOS-Chem high-performance model, this parameterization improves the agreement between the simulated aerosol optical depth (AOD) and the ground-measured AOD from the Aerosol Robotic Network (AERONET; R2 from 0.68 to 0.73 and slope from 0.75 to 0.96). Thus, this parameterization offers a computationally efficient method to represent aerosol size dynamically.

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell–cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell–cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern (‘cell doublet’). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell–matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Migration of animal cells is based on the interplay between actin polymerization at the front, adhesion along the cell-substrate interface, and actomyosin contractility at the back. Active gel theory has been used before to demonstrate that actomyosin contractility is sufficient for polarization and self-sustained cell migration in the absence of external cues, but did not consider the dynamics of adhesion. Likewise, migration models based on the mechanosensitive dynamics of adhesion receptors usually do not include the global dynamics of intracellular flow. Here we show that both aspects can be combined in a minimal active gel model for one-dimensional cell migration with dynamic adhesion. This model demonstrates that load sharing between the adhesion receptors leads to symmetry breaking, with stronger adhesion at the front, and that bistability of migration arises for intermediate adhesiveness. Local variations in adhesiveness are sufficient to switch between sessile and motile states, in qualitative agreement with experiments.

Abstract Alemtuzumab is a monoclonal antibody targeting CD52, used as induction therapy after lung transplantation (LTx). Its engagement produces a long-lasting immunodepletion; however, the mechanisms driving cell reconstitution are poorly defined. We hypothesized that miRNAs are involved in this process. The expression of a set of miRNAs, cytokines and co-signaling molecules was measured with RT-qPCR and flow cytometry in prospectively collected serum samples of LTx recipients, after alemtuzumab or no induction therapy. Twenty-six LTx recipients who received alemtuzumab and twenty-seven matched LTx recipients without induction therapy were included in the analysis. One year after transplantation four miRNAs were differentially regulated: miR-23b (p = 0.05) miR-146 (p = 0.04), miR-155 (p

Sylvatic plague, a primarily flea-borne zoonosis, is a significant threat to prairie dogs (Cynomys spp., PDs) and their specialized predators, endangered black-footed ferrets (Mustela nigripes, BFFs). Host-fed fipronil baits have proven effective in controlling fleas on PDs for the purposes of plague mitigation and BFF conservation. Currently, annual treatments are the norm. We tested the long-term efficacy of fipronil bait treatments with black-tailed PDs (C. ludovicianus, BTPDs) and BFFs in South Dakota, USA. During 2018–2020, we provided BTPDs on 21 sites with grain bait formula, laced with 0.005% fipronil (50 ​mg/kg); 18 non-treated sites functioned as baselines. In 2020–2022, we live-trapped, anesthetized, and combed BTPDs for fleas. Flea control was significant for at least 639–885 days. Flea abundance on the treated sites was < 0.5 fleas/BTPD for ∼750 days. During 2020–2022, we sampled BFFs for fleas on 4 BTPD colonies treated with fipronil grain bait and 8 non-treated colonies. Flea control was significant with BFFs, but flea abundance began to rebound within ∼240 days post-treatment. When feasible, the combination of insecticide treatments, such as fipronil baits, and BFF vaccination against plague provide a “two-pronged” protection approach for these endangered carnivores. If fipronil bait treatments are less effective with predatory BFFs than PDs, as found herein, the “two-pronged” approach might be used to protect BFFs and biennial fipronil bait treatments might be used to protect PDs. If BFF vaccination is not possible, or few BFFs can be vaccinated, annual fipronil bait treatments might be used as a precaution to protect BFFs. Flea densities might be surveyed to determine when/where more frequent treatments seem useful.

AbstractObjectives To define the care cascade for patients with serious injection drug use related infections (SIRI) in a tertiary hospital system and compare outcomes of those who did and did not participate in an opioid use disorder (OUD) treatment referral program.Methods The medical records of patients admitted with both OUD and SIRI including endocarditis, osteomyelitis, septic arthritis, epidural abscess, thrombophlebitis, myositis, bacteremia, and fungemia from 2016-2019 were retrospectively reviewed. Patient demographics, clinical covariates, 90-day readmission rates, and outcomes data were collected. We compared data from those who were successfully referred to outpatient care through Engaging Patients in Care Coordination (EPICC), a peer recovery specialist-run OUD treatment referral program, to those who did not receive outpatient referral.Results During the study period 334 persons who inject opioids were admitted with SIRI. Fourteen admitted patients died and were excluded from the analysis. The all-cause readmission rate was lower among patients referred to the EPICC program (18/76 [23.7%]) compared to those not referred to EPICC (100/244 [41.0%]) (OR 0.44; 95% CI 0.25–0.80).Conclusion An OUD care cascade evaluation for patients with SIRI demonstrated that referral to peer recovery services with outpatient OUD treatment was associated with reduced 90-day readmission rate.

The centriole exhibits an evolutionarily conserved 9-fold radial symmetry that stems from a cartwheel containing vertically stacked ring polymers that harbor 9 homodimers of the protein SAS-6. Here the authors show how dual properties inherent to surface-guided SAS-6 self-assembly possess spatial information that dictates correct scaffolding of centriole architecture.

Our goal is to describe superconformal structures on super Riemann surfaces (SRSs) based on data assigned to a fatgraph. We start from the complex structures on punctured (1|1)-supermanifolds, characterizing the corresponding moduli and the deformations using Strebel differentials and certain Čech cocycles for a specific covering, which we reproduce from fatgraph data, consisting of U(1)-graph connection and odd parameters at the vertices. Then, we consider dual (1|1)-supermanifolds and related superconformal structures for N=2 super Riemann surfaces. The superconformal structures, N=1 SRS, are computed as the fixed points of involution on the supermoduli space of N=2 SRS.

Introduction:. Vascularized lymph node transfer (VLNT) restores physiological lymphatic function. Although effective, postoperative impairment of donor-site lymphatic function and iatrogenic lymphedema following lymph node transfer remains a pressing concern. Methods:. Prospective analysis of VLNT patients undergoing dual fluorescent tracers-assisted harvest was performed at our institution from September 2013 to April 2022. Reverse lymphatic mapping of the lower extremity was performed with indocyanine green (ICG). Blue dye was utilized in both white light and near-infrared spectra for visualization of donor-site lymphatic structures. Demographics, intraoperative details, and surgical outcomes were recorded. Results:. Twenty-five patients were included. Median age was 52.9 years with a body mass index of 29.1 kg/m2 and mean follow-up of 44 months (range 24 to 90 months). Lymphedema stage ranged from Campisi 2 to 4. Inguinal VLNT was performed in 13 patients, and 12 patients received combined VLNT and free flap breast reconstruction. No patients required change in lymph node donor site intraoperatively. All ICG stained nodes were preserved in situ. No cases of iatrogenic lower extremity lymphedema were observed. Postoperative bioimpedance spectroscopy, circumferential, and volumetric measurements of the donor-site limb did not show evidence of subclinical or clinical lymphedema. The donor site healed appropriately in 92% of patients; one patient developed methylene blue-induced skin necrosis. Conclusion:. Reverse lymphatic mapping and surgical guidance with dual ICG and blue dye fluorescent tracers provides surgeons with real-time surgical guidance without radioisotope, improves surgical visualization in both white light and near-infrared spectra, and avoids iatrogenic lymphatic dysfunction in the donor limb.

BackgroundInsect-vectored Leishmania are responsible for loss of more disability-adjusted life years than any parasite besides malaria. Elucidation of the environmental factors that affect parasite transmission by vectors is essential to develop sustainable methods of parasite control that do not have off-target effects on beneficial insects or environmental health. Many phytochemicals that inhibit growth of sand fly-vectored Leishmania-which have been exhaustively studied in the search for phytochemical-based drugs-are abundant in nectars, which provide sugar-based meals to infected sand flies.Principle findingsIn a quantitative meta-analysis, we compare inhibitory phytochemical concentrations for Leishmania to concentrations present in floral nectar and pollen. We show that nectar concentrations of several flowering plant species exceed those that inhibit growth of Leishmania cell cultures, suggesting an unexplored, landscape ecology-based approach to reduce Leishmania transmission.SignificanceIf nectar compounds are as effective against parasites in the sand fly gut as predicted from experiments in vitro, strategic planting of antiparasitic phytochemical-rich floral resources or phytochemically enriched baits could reduce Leishmania loads in vectors. Such interventions could provide an environmentally friendly complement to existing means of disease control.

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II paralogs A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modeling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between paralogs A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-localizes with pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two paralogs form a balanced system for regulated force generation.

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.

Many intracellular pathogens mimic extracellular matrix motifs to specifically interact with the host membrane which may influences virus particle uptake. Here authors use single molecule tension sensors to reveal the minimal forces exerted on single virus particles and demonstrate that the uptake forces scale with the adhesion energy.

Essential cellular processes such as cell adhesion, migration and division strongly depend on mechanical forces. The standard method to measure cell forces is traction force microscopy (TFM) on soft elastic substrates with embedded marker beads. While in 2D TFM one only reconstructs tangential forces, in 2.5D TFM one also considers normal forces. Here we present a systematic comparison between two fundamentally different approaches to 2.5D TFM, which in particular require different methods to deal with noise in the displacement data. In the direct method, one calculates strain and stress tensors directly from the displacement data, which in principle requires a divergence correction. In the inverse method, one minimizes the difference between estimated and measured displacements, which requires some kind of regularization. By calculating the required Green's functions in Fourier space from Boussinesq-Cerruti potential functions, we first derive a new variant of 2.5D Fourier Transform Traction Cytometry (FTTC). To simulate realistic traction patterns, we make use of an analytical solution for Hertz-like adhesion patches. We find that FTTC works best if only tangential forces are reconstructed, that 2.5D FTTC is more precise for small noise, but that the performance of the direct method approaches the one of 2.5D FTTC for larger noise, before both fail for very large noise. Moreover we find that a divergence correction is not really needed for the direct method and that it profits more from increased resolution than the inverse method.

ABSTRACT Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPR interference (CRISPRi)-mediated repression of the cdrS gene caused slow growth and cell division defects and changed the expression of multiple genes and their products associated with cell division, protein degradation, and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) identified 18 DNA-binding sites of the CdrS protein, including one upstream of the promoter for a cell division gene, ftsZ1, and another upstream of the essential gene dacZ, encoding diadenylate cyclase involved in c-di-AMP signaling, which is implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. IMPORTANCE Cell division is a central mechanism of life and is essential for growth and development. Members of the Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in members of the Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in members of the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here, we identify the small protein CdrS as essential for survival and a central regulator of cell division in the euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways, including synthesis of signaling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.

Here, using an integrative experimental and computational approach, Imle et al. show how cell motility and density affect HIV cell-associated transmission in a three-dimensional tissue-like culture system of CD4+ T cells and collagen, and how different collagen matrices restrict infection by cell-free virions.

Purpose: Immediate lymphatic reconstruction (ILR), performed at the time of axillary lymph node dissection (ALND), has demonstrated promising reductions in the development of breast-cancer associated lymphedema. However, questions remain regarding the effects of adjuvant therapies on the continued patency of the lymphaticovenous anastomosis. The aim of our study is to assess lymphographic outcomes, including ICG pattern and LVB patency, following axillary ILR in patients at high risk for breast cancer associated lymphedema.. Methods: Baseline ICG studies of 15 patients who underwent ILR were compared to studies obtained during secondary stage breast reconstructive procedures to assess for any changes in lymphatic morphology and transit in the at-risk arm. Results: All 15 patients in this study demonstrated linear lymphatic flow in intra-operative lymphography studies performed during the initial lymphatic reconstruction. An average of 2.4 (range 1-4) LVBs were performed per patient. Only 1 patient in this study group had preservation of in-continuity lymphatics at time of ALND. Followup lymphographic studies showed clear, linear lymphatic transit in 12/15 patients. Of these 12 patients, an average of 2.5 LVBs were performed, 10 received chemotherapy (7 neoadjuvant, 3 adjuvant), and all 12 received post mastectomy radiation (PMRT). Dermal backflow patterns of varying severity were recorded in 3/15 patients, two of whom showed signs of lymphedema prior to their followup study and the last went on to develop clinically detectable lymphedema. Of these 3 patients, an average of 2 LVBs were performed, all received chemotherapy (2 neoadjuvant, 1 adjuvant) and 2/3 underwent PMRT. Of the 12 patients that remain lymphedema-free, 7 post-operative studies demonstrated clear visualization of linear ICG flow from the lymphatics of the arm into the axilla without evidence of lymphatic collateralization. An average of 3 LVBs were performed in this group and 100% of these patients received adjuvant radiation. Conclusion: We have demonstrated that ICG lymphography can be implemented as a post-operative tool to assess lymphatic function in patients who have undergone ILR in the axilla. Post-operative imaging studies in the majority of patients demonstrated linear ICG flow with evidence of lymphatic contractility and velocity similar to baseline studies obtained at the time of lymphadenectomy and ILR. Additionally, ICG flow patterns through the axilla in post-operative imaging studies provided visual evidence supporting sustained LVB patency despite inflammation and tissue fibrosis associated with axillary irradiation.

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5′ UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5′ UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.

Recent experiments with super-resolution live cell microscopy revealed that nonmuscle myosin II minifilaments are much more dynamic than formerly appreciated, often showing plastic processes such as splitting, concatenation and stacking. Here we combine sequence information, electrostatics and elasticity theory to demonstrate that the parallel staggers at 14.3, 43.2 and 72 nm have a strong tendency to splay their heads away from the minifilament, thus potentially initiating the diverse processes seen in live cells. In contrast, the straight antiparallel stagger with an overlap of 43 nm is very stable and likely initiates minifilament nucleation. Using stochastic dynamics in a newly defined energy landscape, we predict that the optimal parallel staggers between the myosin rods are obtained by a trial-and-error process in which two rods attach and re-attach at different staggers by rolling and zipping motion. The experimentally observed staggers emerge as the configurations with the largest contact times. We find that contact times increase from isoforms C to B to A, that A-B-heterodimers are surprisingly stable and that myosin 18A should incorporate into mixed filaments with a small stagger. Our findings suggest that nonmuscle myosin II minifilaments in the cell are first formed by isoform A and then convert to mixed A-B-filaments, as observed experimentally.

Complex energy landscapes often arise in biological systems, e.g. for protein folding, biochemical reactions, or intracellular transport processes. Their physical effects are frequently reflected in the first-passage times (FPTs) arising from these energy landscapes. However, their calculation is notoriously challenging and it is often difficult to identify the most relevant features of a given energy landscape. Here we show how this can be achieved by coarse-graining the Fokker–Planck equation to a master equation and decomposing its FPTs in an iterative process. We apply this method to the electrostatic interaction between two rods of nonmuscle myosin II (NM2), which is the main molecular motor for force generation in nonmuscle cells. Energy landscapes are computed directly from the amino acid sequences of the three different isoforms. Our approach allows us to identify the most relevant energy barriers for their self-assembly into NM2 minifilaments and how they change under force. In particular, we find that antiparallel configurations are more stable than parallel ones, but also show more changes under mechanical loading. Our work demonstrates the rich dynamics that can be expected for NM2-assemblies under mechanical load and in general shows how one can identify the most relevant energy barriers in complex energy landscapes.

During collective cell migration, how leader cells emerge is poorly understood. Here, the authors find that small groups of mechanically-interacting follower cells pull on the future leaders to stochastically elect them to their fate.

The sequence of structural and molecular events during clathrin-mediated endocytosis is unclear. Here the authors combine correlative microscopy and simple mathematical growth laws to demonstrate that the flat patch starts to curve when around 70% of the final clathrin content is reached.

Surface contraction waves (SCWs) are prominent shape changes coupled to cell cycle transitions in oocytes. Here the authors show that SCWs are patterned by the spatiotemporal integration of two conserved modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.

Cellular contractility is regulated by the GTPase RhoA, but how local signals are translated to a cell-level response is not known. Here the authors show that targeted RhoA activation results in propagation of force along stress fibres and actin flow, and identify zyxin as a regulator of stress fibre mechanics and homeostasis.