Dynamics in protein translation sustaining T cell preparedness.

In response to pathogenic threats, naive T cells rapidly transition from a quiescent to an activated state, yet the underlying mechanisms are incompletely understood. Using a pulsed SILAC approach, we investigated the dynamics of mRNA translation kinetics and protein turnover in human naive and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion following activation. Furthermore, naive T cells maintained a surprisingly large number of idling ribosomes as well as 242 repressed mRNA species and a reservoir of glycolytic enzymes. These components were rapidly engaged following stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program. Our data elucidate new insights into how T cells maintain a prepared state to mount a rapid immune response, and provide a resource of protein turnover, absolute translation kinetics and protein synthesis rates in T cells ( https://www.immunomics.ch ).

L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity.

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

Social network architecture of human immune cells unveiled by quantitative proteomics.

The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Investigation of the Electrokinetic Properties of HIV-Based Virus-Like Particles.

The antigen density on the surface of HIV-based virus-like particles (VLPs) plays a crucial role in the improvement of HIV vaccine potency. HIV VLPs consist of a dense protein core, which is surrounded by a lipid bilayer and whose surface is usually decorated with antigenic glycoproteins. The successful downstream processing of these particles is challenging, and the high-resolution and cost-efficient purification of HIV-based VLPs has not yet been achieved. Chromatography, one of the major unit operations involved in HIV VLP purification strategies, is usually carried out by means of ion exchangers or ion-exchange membranes. Understanding the electrokinetic behavior of HIV-based VLPs may help to improve the adjustment and efficiency of the corresponding chromatographic processes. In this study, we investigated the electrokinetics and aggregation of both undecorated and decorated VLPs and interpreted the data from the perspective of the soft particle model developed by Ohshima (OSPM), which fails to fully predict the behavior of the studied VLPs. Post-Ohshima literature, and particularly the soft multilayer particle model developed by Langlet et al., provides an alternative theoretical framework to overcome the limits of the OSPM. We finally hypothesized that the electrophoretic mobility of HIV-based VLPs is controlled by an electrohydrodynamic interplay between envelope glycoproteins, lipid bilayer, and Gag envelope.

Functional Renormalization Group Study of Superconductivity in Rhombohedral Trilayer Graphene.

We employ a functional renormalization group approach to ascertain the pairing mechanism and symmetry of the superconducting phase observed in rhombohedral trilayer graphene. Superconductivity in this system occurs in a regime of carrier density and displacement field with a weakly distorted annular Fermi sea. We find that repulsive Coulomb interactions can induce electron pairing on the Fermi surface by taking advantage of momentum-space structure associated with the finite width of the Fermi sea annulus. The degeneracy between spin-singlet and spin-triplet pairing is lifted by valley-exchange interactions that strengthen under the RG flow and develop nontrivial momentum-space structure. We find that the leading pairing instability is d-wave-like and spin singlet, and that the theoretical phase diagram versus carrier density and displacement field agrees qualitatively with experiment.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Spontaneous Valley Spirals in Magnetically Encapsulated Twisted Bilayer Graphene.

Van der Waals heterostructures provide a rich platform for emergent physics due to their tunable hybridization of layers, orbitals, and spin. Here, we find that twisted bilayer graphene stacked between antialigned ferromagnetic insulators can feature flat electronic bands due to the interplay between twist, exchange proximity, and spin-orbit coupling. These flat bands are nearly degenerate in valley only and are effectively described by a triangular superlattice model. At half filling, we find that interactions induce spontaneous valley correlations that favor spiral order and derive a low-energy valley-Heisenberg model with symmetric and antisymmetric exchange couplings. We also show how electric interlayer bias broadens the bands and tunes these couplings. Our results put forward magnetic van der Waals heterostructures as a platform to explore valley-correlated states.

Income support, employment transitions and well-being.

Using specific panel data of German welfare benefit recipients, we investigate the non-pecuniary life satisfaction effects of in-work benefits. Our empirical strategy combines difference-in-difference designs with synthetic control groups to analyse transitions of workers between unemployment, regular employment and employment accompanied by welfare receipt. Working makes people generally better off than being unemployed but employed welfare recipients do not reach the life satisfaction level of regular employees. This implies that welfare receipt entails non-compliance with the norm to make one's own living. Our findings allow us to draw cautious conclusions on employment subsidies paid as welfare benefits.

Tunneling into a Finite Luttinger Liquid Coupled to Noisy Capacitive Leads.

Tunneling spectroscopy of one-dimensional interacting wires can be profoundly sensitive to the boundary conditions of the wire. Here, we analyze the tunneling spectroscopy of a wire coupled to capacitive metallic leads. Strikingly, with increasing many-body interactions in the wire, the impact of the boundary noise becomes more prominent. This interplay allows for a smooth crossover from standard 1D tunneling signatures into a regime where the tunneling is dominated by the fluctuations at the leads. This regime is characterized by an elevated zero-bias tunneling alongside a universal power-law decay at high energies. Furthermore, local tunneling measurements in this regime show a unique spatial dependence that marks the formation of plasmonic standing waves in the wire. Our result offers a tunable method by which to control the boundary effects and measure the interaction strength (Luttinger parameter) within the wire.

The effects of high shear rates on the average hydrodynamic diameter measured in biomimetic HIV Gag virus-like particle dispersions.

HIV Gag virus-like particles (HIV Gag VLPs) are promising HIV vaccine candidates. In the literature, they are often described as shear-sensitive particles, and authors usually recommend the operation of tangential flow filtration (TFF) gently at shear rates below 4,000 s-1 to 6,000 s-1. This in turn poses a severe limitation to the performance of TFF-mediated concentration of VLPs, which would be substantially enhanced by working at higher shear rates. To our knowledge, studies examining the shear sensitivity of HIV Gag VLPs and providing detailed information and evidence for the fragility of these particles have not been conducted yet. Thus, we investigated the effect of high shear rates on the colloidal stability of mosaic VLPs (Mos-VLPs) as relevant examples for HIV Gag VLPs. For this purpose, Mos-VLPs were exposed to different shear rates ranging from 3,395 s-1 to 22, 365 s-1 for 2 h. The average hydrodynamic diameter (AHD) and the polydispersity index (PDI) of the associated particle size distribution were used as stability indicators and measured after the treatment and during storage through dynamic light scattering. At high shear rates, we observed an increase in both AHD and PDI during the storage of HIV Mos1.Gag VLPs (bVLP-without envelope proteins) and Mos1.Gag + Mos2S.Env VLPs (eVLP-with envelope proteins). eVLPs exhibited higher colloidal stability than bVLPs, and we discuss the potential stabilizing role of envelope proteins. We finally demonstrated that the dispersion medium also has a considerable impact on the stability of Mos-VLPs.

The effect of minimum volume standards in hospitals (MIVOS) - protocol of a systematic review.

BACKGROUND: The volume-outcome relationship, i.e., higher hospital volume results in better health outcomes, has been established for different surgical procedures as well as for certain nonsurgical medical interventions. Accordingly, many countries such as Germany, the USA, Canada, the UK, and Switzerland have established minimum volume standards. To date, there is a lack of systematically summarized evidence regarding the effects of such regulations. METHODS: To be included in the review, studies must measure any effects connected to minimum volume standards. Outcomes of interest include the following: (1) patient-related outcomes, (2) process-related outcomes, and (3) health system-related outcomes. We will include (cluster) randomized controlled trials ([C]RCTs), non-randomized controlled trials (nRCTs), controlled before-after studies (CBAs), and interrupted time-series studies (ITSs). We will apply no restrictions regarding language, publication date, and publication status. We will search MEDLINE (via PubMed), Embase (via Embase), CENTRAL (via Cochrane Library), CINHAL (via EBSCO), EconLit (via EBSCO), PDQ evidence for informed health policymaking, health systems evidence, OpenGrey, and also trial registries for relevant studies. We will further search manually for additional studies by cross-checking the reference lists of all included primary studies as well as cross-checking the reference lists of relevant systematic reviews. To evaluate the risk of bias, we will use the ROBINS-I and RoB 2 risk-of-bias tools for the corresponding study designs. For data synthesis and statistical analyses, we will follow the guidance published by the EPOC Cochrane group (Cochrane Effective Practice and Organisation of Care (EPOC), EPOC Resources for review authors, 2019). DISCUSSION: This systematic review focuses on minimum volume standards and the outcomes used to measure their effects. It is designed to provide thorough and encompassing evidence-based information on this topic. Thus, it will inform decision-makers and policymakers with respect to the effects of minimum volume standards and inform further studies in regard to research gaps. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022318883.

Protocol for the development of a core outcome set for studies on centralisation of healthcare services.

INTRODUCTION: Centralisation defined as the reorganisation of healthcare services into fewer specialised units serving a higher volume of patients is a potential measure for healthcare reforms aiming at reducing costs while improving quality. Research on centralisation of healthcare services is thus essential to inform decision-makers. However, so far studies on centralisation report a variability of outcomes, often neglecting outcomes at the health system level. Therefore, this study aims at developing a core outcome set (COS) for studies on centralisation of hospital procedures, which is intended for use in observational as well as in experimental studies. METHODS AND ANALYSIS: We propose a five-stage study design: (1) systematic review, (2) focus group, (3) interview studies, (4) online survey, (5) Delphi survey. The study will be conducted from March 2022 to November 2023. First, an initial list of outcomes will be identified through a systematic review on reported outcomes in studies on minimum volume regulations. We will search MEDLINE, EMBASE, CENTRAL, CINHAL, EconLIT, PDQ-Evidence for Informed Health Policymaking, Health Systems Evidence, Open Grey and also trial registries. This will be supplemented with relevant outcomes from published studies on centralisation of hospital procedures. Second, we will conduct a focus group with representatives of patient advocacy groups for which minimum volume regulations are currently in effect in Germany or are likely to come into effect to identify outcomes important to patients. Furthermore, two interview studies, one with representatives of the German medical societies and one with representatives of statutory health insurance funds, as well as an online survey with health services researchers will be conducted. In our analyses of the suggested outcomes, we will largely follow the categorisation scheme developed by the Cochrane EPOC group. Finally, a two-round online Delphi survey with all stakeholder groups using predefined score criteria for consensus will be employed to first prioritise outcomes and then agree on the final COS. ETHICS AND DISSEMINATION: This study has been approved by the Research Ethics Committee at the Brandenburg Medical School Theodor Fontane (MHB). The final COS will be disseminated to all stakeholders involved and through peer-reviewed publications and conferences.

Proteomics of immune cells from liver tumors reveals immunotherapy targets.

Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer tissue as well as monocyte/macrophages, CD4+ and CD8+ T cells, and NK cells isolated from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. Our data reveal new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer.

Components of a HIV-1 vaccine mediate virus-like particle (VLP)-formation and display of envelope proteins exposing broadly neutralizing epitopes.

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs.

A Hydrodynamic Approach to the Study of HIV Virus-Like Particle (VLP) Tangential Flow Filtration.

Emerging as a promising pathway to HIV vaccines, Virus-Like Particles (VLPs) have drawn considerable attention in recent years. A challenge of working with HIV VLPs in biopharmaceutical processes is their low rigidity, and factors such as shear stress, osmotic pressure and pH variation have to be reduced during their production. In this context, the purification and concentration of VLPs are often achieved by means of Tangential Flow Filtration (TFF) involving ultrafiltration hollow fiber modules. Despite the urgent need for robust upscaling strategies and further process cost reduction, very little attention has been dedicated to the identification of the mechanisms limiting the performance of HIV VLP TFF processes. In this work, for the first time, a hydrodynamic approach based on particle friction was successfully developed as a methodology for both the optimization and the upscaling of HIV VLP TFF. Friction forces acting on near-membrane HIV VLPs are estimated, and the plausibility of the derived static coefficients of friction is discussed. The particle friction-based model seems to be very suitable for the fitting of experimental data related to HIV VLP TFF as well as for upscaling projections. According to our predictions, there is still considerable room for improvement of HIV VLP TFF, and operating this process at slightly higher flow velocities may dramatically enhance the efficiency of VLP purification and concentration. This work offers substantial guidance to membrane scientists during the design of upscaling strategies for HIV VLP TFF.

The Optimization of Dispersion and Application Techniques for Nanocarbon-Doped Mixed Matrix Gas Separation Membranes.

In this work, supported cellulose acetate (CA) mixed matrix membranes (MMMs) were prepared and studied concerning their gas separation behaviors. The dispersion of carbon nanotube fillers were studied as a factor of polymer and filler concentrations using the mixing methods of the rotor-stator system (RS) and the three-roll-mill system (TRM). Compared to the dispersion quality achieved by RS, samples prepared using the TRM seem to have slightly bigger, but fewer and more homogenously distributed, agglomerates. The green γ-butyrolactone (GBL) was chosen as a polyimide (PI) polymer-solvent, whereas diacetone alcohol (DAA) was used for preparing the CA solutions. The coating of the thin CA separation layer was applied using a spin coater. For coating on the PP carriers, a short parameter study was conducted regarding the plasma treatment to affect the wettability, the coating speed, and the volume of dispersion that was applied to the carrier. As predicted by the parameter study, the amount of dispersion that remained on the carriers decreased with an increasing rotational speed during the spin coating process. The dry separation layer thickness was varied between about 1.4 and 4.7 µm. Electrically conductive additives in a non-conductive matrix showed a steeply increasing electrical conductivity after passing the so-called percolation threshold. This was used to evaluate the agglomeration behavior in suspension and in the applied layer. Gas permeation tests were performed using a constant volume apparatus at feed pressures of 5, 10, and 15 bar. The highest calculated CO2/N2 selectivity (ideal), 21, was achieved for the CA membrane and corresponded to a CO2 permeability of 49.6 Barrer.

CO2/CH4 and He/N2 Separation Properties and Water Permeability Valuation of Mixed Matrix MWCNTs-Based Cellulose Acetate Flat Sheet Membranes: A Study of the Optimization of the Filler Material Dispersion Method.

The main scope of this work is to develop nano-carbon-based mixed matrix cellulose acetate membranes (MMMs) for the potential use in both gas and liquid separation processes. For this purpose, a variety of mixed matrix membranes, consisting of cellulose acetate (CA) polymer and carbon nanotubes as additive material were prepared, characterized, and tested. Multi-walled carbon nanotubes (MWCNTs) were used as filler material and diacetone alcohol (DAA) as solvent. The first main objective towards highly efficient composite membranes was the proper preparation of agglomerate-free MWCNTs dispersions. Rotor-stator system (RS) and ultrasonic sonotrode (USS) were used to achieve the nanofillers' dispersion. In addition, the first results of the application of the three-roll mill (TRM) technology in the filler dispersion achieved were promising. The filler material, MWCNTs, was characterized by scanning electron microscopy (SEM) and liquid nitrogen (LN2) adsorption-desorption isotherms at 77 K. The derivatives CA-based mixed matrix membranes were characterized by tensile strength and water contact angle measurements, impedance spectroscopy, gas permeability/selectivity measurements, and water permeability tests. The studied membranes provide remarkable water permeation properties, 12-109 L/m2/h/bar, and also good separation factors of carbon dioxide and helium separations. Specifically, a separation factor of 87 for 10% He/N2 feed concentration and a selectivity value of 55.4 for 10% CO2/CH4 feed concentration were achieved.

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling.

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

Transradial versus transfemoral approach for coronary angiography and intervention in patients above 75 years of age.

AIMS: The transradial (TR) approach has potentially lower complication rates than transfemoral (TF) approach coronary angiography. However, it may be technically more challenging, especially in elderly patients with alterations in vascular anatomy. We thus determined success rates, procedural data, and complication rates of TR angiography in comparison to the TF approach in elderly patients in a randomized, prospective trial. METHODS AND RESULTS: Four hundred consecutive patients >or=75 years with known or suspected coronary artery disease were included in the study. After exclusion of 93 patients with contraindications to the radial approach, 152 patients were randomized to the TR and 155 to TF coronary angiography and intervention. In 13 patients randomized to TR, cross-over to TF was necessary (9%). Total examination time was significantly longer for the TR approach (18.1 vs. 15.0 min, P = 0.009), but no difference was found for fluoroscopy time, number of catheters used, or amount of contrast agent. The rate of major complications (bleeding requiring surgery or transfusion, stroke) was 0% for TR and 3.2% for TF approach (P < 0.001). Minor complications occurred in 1.3% versus 5.8% of patients (P < 0.001). CONCLUSION: In elderly patients, TR coronary angiography and intervention has a high technical success rate and lower complication rates than the TF approach.