Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

Multiphoton lithography with protein photoresists.

Recently, 2D/3D direct laser writing has attracted increased attention due to its broad applications ranging from biomedical engineering to aerospace. 3D nanolithography of water-soluble protein-based scaffolds have been envisioned to provide a variety of tunable properties. In this paper, we present a functional protein-based photoresist with tunable mechanical properties that is suitable for multiphoton lithography (MPL). Through the use of methacrylated streptavidin or methacrylated bovine serum albumin in combination with polyethylene glycol diacrylate or methacrylated hyaluronic acid as crosslinkers and a vitamin-based photoinitiator, we were able to write two- and three-dimensional structures as small as 200 nm/600 nm lateral/axial features, respectively. We also demonstrated that Young's modulus can be tuned by the photoresist composition, and we were able to achieve values as low as 40 kPa. Furthermore, we showed that Young's modulus can be recovered after drying and rehydration (i.e. shelf time determination). The retained biological functionality of the streptavidin scaffolds was demonstrated using fluorescently labelled biotins. Using single-molecule fluorescence microscopy, we estimated the density of streptavidin in the written features (1.8 ± 0.2 × 105 streptavidins per 1.00 ± 0.05 µm³ of feature volume). Finally, we showed applicability of our 2D scaffold as a support for a fluorescence absorbance immuno-assay (FLISA), and as a delivery platform of extracellular vesicles to HeLa cells.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Physical Activity, Depression and Quality of Life in COPD - Results from the CLARA II Study.

Background: Symptoms of depression, pain and limitations in physical activity may affect quality of life in COPD patients independent from their respiratory burden. We aimed to analyze the associations of these factors in outpatients with COPD in Austria in a stable phase of disease. Methods: We conducted a national, cross-sectional study among patients with COPD. For depression, the Patient Health Questionnaire-9 (PHQ-9) and for respiratory symptoms the St. George's Respiratory Questionnaire for COPD patients (SGRQ-C) were used along with 10-point scales for physical activity and pain. Results: After exclusion of 211 patients due to non-obstructive spirometry or missing data, 630 patients (62.5% men; mean age 66.8 ± 8.6 (SD) years; mean FEV1%pred. 54.3 ± 16.5 (SD)) were analyzed. Of these, 47% reported one or more exacerbations in the previous year, 10.4% with hospitalization. A negative depression score was found in 54% and a score suggesting severe depression (PHQ-9 score ≥ 15) in 4.7%. In a multivariate linear regression model, self-reported pain, dyspnea, and number of exacerbations were predictors for higher PHQ-9-scores. A negative pain score was found in 43.8%, and a score suggesting severe pain in 2.9% (8-10 points of 10-point scale). Patients reporting severe pain were more often female, had more exacerbations, and reported more respiratory and depressive symptoms, a lower quality of life, and less physical activity. About 46% of patients rated their physical activity as severely impaired. These patients were significantly older, had more exacerbations, concomitant heart disease, a higher pain and depression score, and a lower quality of life (SGRQ-C - total score and all subscores). Conclusions: In Austria, nearly half of stable COPD outpatients reported symptoms of depression, which were associated with lower levels of self-reported physical activity, more pain, and respiratory symptoms. The associations were particularly strong for depression with SGRQ-C.

Entropic barrier of water permeation through single-file channels.

Facilitated water permeation through narrow biological channels is fundamental for all forms of life. Despite its significance in health and disease as well as for biotechnological applications, the energetics of water permeation are still elusive. Gibbs free energy of activation is composed of an enthalpic and an entropic component. Whereas the enthalpic contribution is readily accessible via temperature dependent water permeability measurements, estimation of the entropic contribution requires information on the temperature dependence of the rate of water permeation. Here, we estimate, by means of accurate activation energy measurements of water permeation through Aquaporin-1 and by determining the accurate single channel permeability, the entropic barrier of water permeation through a narrow biological channel. Thereby the calculated value for [Formula: see text] = 2.01 ± 0.82 J/(mol·K) links the activation energy of 3.75 ± 0.16 kcal/mol with its efficient water conduction rate of ~1010 water molecules/second. This is a first step in understanding the energetic contributions in various biological and artificial channels exhibiting vastly different pore geometries.

Lipoprotein Particles as Shuttles for Hydrophilic Cargo.

Lipoprotein particles (LPs) are excellent transporters and have been intensively studied in cardiovascular diseases, especially regarding parameters such as their class distribution and accumulation, site-specific delivery, cellular internalization, and escape from endo/lysosomal compartments. The aim of the present work is the hydrophilic cargo loading of LPs. As an exemplary proof-of-principle showcase, the glucose metabolism-regulating hormone, insulin, was successfully incorporated into high-density lipoprotein (HDL) particles. The incorporation was studied and verified to be successful using Atomic Force Microscopy (AFM) and Fluorescence Microscopy (FM). Single-molecule-sensitive FM together with confocal imaging visualized the membrane interaction of single, insulin-loaded HDL particles and the subsequent cellular translocation of glucose transporter type 4 (Glut4).

Structural Role of Plasma Membrane Sterols in Osmotic Stress Tolerance of Yeast Saccharomyces cerevisiae.

Yeast S. cerevisiae has been shown to suppress a sterol biosynthesis as a response to hyperosmotic stress. In the case of sodium stress, the failure to suppress biosynthesis leads to an increase in cytosolic sodium. The major yeast sterol, ergosterol, is known to regulate functioning of plasma membrane proteins. Therefore, it has been suggested that the suppression of its biosynthesis is needed to adjust the activity of the plasma membrane sodium pumps and channels. However, as the sterol concentration is in the range of thirty to forty percent of total plasma membrane lipids, it is believed that its primary biological role is not regulatory but structural. Here we studied how lowering the sterol content affects the response of a lipid bilayer to an osmotic stress. In accordance with previous observations, we found that a decrease of the sterol fraction increases a water permeability of the liposomal membranes. Yet, we also found that sterol-free giant unilamellar vesicles reduced their volume during transient application of the hyperosmotic stress to a greater extent than the sterol-rich ones. Furthermore, our data suggest that lowering the sterol content in yeast cells allows the shrinkage to prevent the osmotic pressure-induced plasma membrane rupture. We also found that mutant yeast cells with the elevated level of sterol accumulated propidium iodide when exposed to mild hyperosmotic conditions followed by hypoosmotic stress. It is likely that the decrease in a plasma membrane sterol content stimulates a drop in cell volume under hyperosmotic stress, which is beneficial in the case of a subsequent hypo-osmotic one.

Clinical, imaging, and blood biomarkers to assess 1-year progression risk in fibrotic interstitial lung diseases-Development and validation of the honeycombing, traction bronchiectasis, and monocyte (HTM)-score.

Introduction: Progression of fibrotic interstitial lung disease (ILD) leads to irreversible loss of lung function and increased mortality. Based on an institutional ILD registry, we aimed to evaluate biomarkers derived from baseline patient characteristics, computed tomography (CT), and peripheral blood for prognosis of disease progression in fibrotic ILD patients. Methods: Of 209 subsequent ILD-board patients enregistered, 142 had complete follow-up information and were classified fibrotic ILD as defined by presence of reticulation or honeycombing using a standardized semi-quantitative CT evaluation, adding up typical ILD findings in 0-6 defined lung fields. Progression at 1 year was defined as relative loss of ≥10% in forced vital capacity, of ≥15% in diffusion capacity for carbon monoxide, death, or lung transplant. Two-thirds of the patients were randomly assigned to a derivation cohort evaluated for the impact of age, sex, baseline lung function, CT finding scores, and blood biomarkers on disease progression. Significant variables were included into a regression model, its results were used to derive a progression-risk score which was then applied to the validation cohort. Results: In the derivation cohort, age, monocyte count ≥0.65 G/L, honeycombing and traction bronchiectasis extent had significant impact. Multivariate analyses revealed the variables monocyte count ≥0.65 G/L (1 point) and combined honeycombing or traction bronchiectasis score [0 vs. 1-4 (1 point) vs. 5-6 lung fields (2 points)] as significant, so these were used for score development. In the derivation cohort, resulting scores of 0, 1, 2, and 3 accounted for 1-year progression rates of 20, 25, 46.9, and 88.9%, respectively. Similarly, in the validation cohort, progression at 1 year occurred in 0, 23.8, 53.9, and 62.5%, respectively. A score ≥2 showed 70.6% sensitivity and 67.9% specificity, receiver operating characteristic analysis for the scoring model had an area under the curve of 71.7%. Conclusion: The extent of honeycombing and traction bronchiectasis, as well as elevated blood monocyte count predicted progression within 1 year in fibrotic ILD patients.

Multiple pore lining residues modulate water permeability of GlpF.

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin "open state" is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States.

The reconstitution of secondary active transporters into liposomes shed light on their molecular transport mechanism. The latter are either symporters, antiporters or exchangers, which use the energy contained in the electrochemical gradient of ions to fuel concentrative uptake of their cognate substrate. In liposomal preparations, these gradients can be set by the experimenter. However, due to passive diffusion of the ions and solutes through the membrane, the gradients are not stable and little is known on the time course by which they dissipate and how the presence of a transporter affects this process. Gradient dissipation can also generate a transmembrane potential (VM). Because it is the effective ion gradient, which together with VM fuels concentrative uptake, knowledge on how these parameters change within the time frame of the conducted experiment is key to understanding experimental outcomes. Here, we addressed this problem by resorting to a modelling approach. To this end, we mathematically modeled the liposome in the assumed presence and absence of the sodium glucose transporter 1 (SGLT1). We show that 1) the model can prevent us from reaching erroneous conclusions on the driving forces of substrate uptake and we 2) demonstrate utility of the model in the assignment of the states of SGLT1, which harbor a water channel.

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold.

Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of four representatives, key aspects of AQP stability, gating, selectivity, pore geometry, and oligomerization, with a potential impact on channel functionality, are identified. The general view of AQPs possessing a continuous open water pore is challenged and it is depicted that AQPs' selectivity is not exclusively shaped by pore-lining residues but also by the relative arrangement of transmembrane helices. Moreover, this analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, a numbering scheme of the conserved AQP scaffold is established, facilitating direct comparison of, for example, disease-causing mutations and prediction of potential structural consequences. Additionally, the results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.

Determinants of Lipid Domain Size.

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.

GaN Heterostructures as Innovative X-ray Imaging Sensors-Change of Paradigm.

Direct conversion of X-ray irradiation using a semiconductor material is an emerging technology in medical and material sciences. Existing technologies face problems, such as sensitivity or resilience. Here, we describe a novel class of X-ray sensors based on GaN thin film and GaN/AlGaN high-electron-mobility transistors (HEMTs), a promising enabling technology in the modern world of GaN devices for high power, high temperature, high frequency, optoelectronic, and military/space applications. The GaN/AlGaN HEMT-based X-ray sensors offer superior performance, as evidenced by higher sensitivity due to intensification of electrons in the two-dimensional electron gas (2DEG), by ionizing radiation. This increase in detector sensitivity, by a factor of 104 compared to GaN thin film, now offers the opportunity to reduce health risks associated with the steady increase in CT scans in today's medicine, and the associated increase in exposure to harmful ionizing radiation, by introducing GaN/AlGaN sensors into X-ray imaging devices, for the benefit of the patient.

First-Line Pembrolizumab Mono- or Combination Therapy of Non-Small Cell Lung Cancer: Baseline Metabolic Biomarkers Predict Outcomes.

Quantitative biomarkers derived from positron-emission tomography/computed tomography (PET/CT) have been suggested as prognostic variables in immune-checkpoint inhibitor (ICI) treated non-small cell lung cancer (NSCLC). As such, data for first-line ICI therapy and especially for chemotherapy-ICI combinations are still scarce, we retrospectively evaluated baseline 18F-FDG-PET/CT of 85 consecutive patients receiving first-line pembrolizumab with chemotherapy (n = 70) or as monotherapy (n = 15). Maximum and mean standardized uptake value, total metabolic tumor volume (MTV), total lesion glycolysis, bone marrow-/and spleen to liver ratio (BLR/SLR) were calculated. Kaplan-Meier analyses and Cox regression models were used to assess progression-free/overall survival (PFS/OS) and their determinant variables. Median follow-up was 12 months (M; 95% confidence interval 10-14). Multivariate selection for PFS/OS revealed MTV as most relevant PET/CT biomarker (p < 0.001). Median PFS/OS were significantly longer in patients with MTV ≤ 70 mL vs. >70 mL (PFS: 10 M (4-16) vs. 4 M (3-5), p = 0.001; OS: not reached vs. 10 M (5-15), p = 0.004). Disease control rate was 81% vs. 53% for MTV ≤/> 70 mL (p = 0.007). BLR ≤ 1.06 vs. >1.06 was associated with better outcomes (PFS: 8 M (4-13) vs. 4 M (3-6), p = 0.034; OS: 19 M (12-/) vs. 6 M (4-12), p = 0.005). In patients with MTV > 70 mL, concomitant BLR ≤ 1.06 indicated a better prognosis. Higher MTV is associated with inferior PFS/OS in first-line ICI-treated NSCLC, with BLR allowing additional risk stratification.

Descriptors of Secondary Active Transporter Function and How They Relate to Partial Reactions in the Transport Cycle.

Plasmalemmal solute carriers (SLCs) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino acid sequence that often leads to impaired protein function and onset of monogenic disorders. To understand how these altered proteins cause disease, it is necessary to undertake relevant functional assays. These experiments reveal descriptors of SLC function such as the maximal transport velocity (Vmax), the Michaelis constant for solute uptake (KM), potencies for inhibition of transporter function (IC50/EC50), and many more. In several instances, the mutated versions of different SLC transporters differ from their wild-type counterparts in the value of these descriptors. While determination of these experimental parameters can provide conjecture as to how the mutation gives rise to disease, they seldom provide any definitive insights on how a variant differ from the wild-type transporter in its operation. This is because the experimental determination of association between values of the descriptors and several partial reactions a transporter undergoes is casual, but not causal, at best. In the present study, we employ kinetic models that allow us to derive explicit mathematical terms and provide experimental descriptors as a function of the rate constants used to parameterize the kinetic model of the transport cycle. We show that it is possible to utilize these mathematical expressions to deduce, from experimental outcomes, how the mutation has impinged on partial reactions in the transport cycle.

Computed Tomography Findings as Determinants of Local and Systemic Inflammation Biomarkers in Interstitial Lung Diseases: A Retrospective Registry-Based Descriptive Study.

PURPOSE: To evaluate the association of peripheral blood (PBL) and broncho-alveolar lavage (BAL) biomarkers with inflammatory versus fibrotic high-resolution computed tomography (HRCT) findings in interstitial lung disease (ILD) patients. METHODS: HRCT findings of 127 consecutive ILD-board patients were semi-quantitatively evaluated: reticulation/honeycombing (RET), traction bronchiectasis (TBR) and emphysema (EMP) were classified as non-inflammatory/fibrotic; consolidations (CON), ground glass opacities (GGO), parenchymal nodules (NDL) and mosaic attenuation (MOS) as active inflammatory. Each HRCT finding was assessed in six distinct lung regions, resulting scores were graded as minimal (0-1 regions involved), medium (2-4) or extensive (5-6). Associations of routinely assessed PBL/BAL biomarkers with these HRCT scores were evaluated using Spearman correlation coefficients and graphical presentation; significance was tested by applying Kruskal-Wallis tests. RESULTS: Blood neutrophil, lymphocyte and eosinophil fraction, neutrophil to lymphocyte ratio (NLR) and BAL lymphocyte fraction consistently showed opposite correlations with inflammatory versus non-inflammatory/fibrotic HRCT finding scores. Blood lymphocyte fraction significantly differed by graded GGO (p = 0.032) and CON (p = 0.027) extent, eosinophil fraction by TBR (p = 0.006) and NLR by CON (p = 0.009). C-reactive protein was significantly related to GGO (p = 0.023) and CON (p = 0.004), BAL lymphocyte fraction to GGO (p = 0.017) extent. CONCLUSION: Blood lymphocyte and eosinophil fraction, NLR, CRP and BAL lymphocyte fraction may aid to differentiate inflammatory from non-inflammatory/fibrotic ILD patterns. TRIAL REGISTRATION: This evaluation was based on data from the ILD registry of Kepler University Hospital Linz, as approved by the ethics committee of the Federal State of Upper-Austria (EK Number. I-26-17).

Native-like membrane models of E. coli polar lipid extract shed light on the importance of lipid composition complexity.

BACKGROUND: Lipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments. RESULTS: To enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37∘C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly. CONCLUSIONS: The here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.

Scattering versus fluorescence self-quenching: more than a question of faith for the quantification of water flux in large unilamellar vesicles?

The endeavors to understand the determinants of water permeation through membrane channels, the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications all rely on accurate ways to quantify water permeabilities (P f). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate P f values. By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate for the first time the influence of the LUVs size distribution, channel distribution between vesicles and remaining detergent after protein reconstitution on P f values. We point out that results such as the single channel water permeability (p f) depend on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure.

Sex-Based Clinical Outcome in Advanced NSCLC Patients Undergoing PD-1/PD-L1 Inhibitor Therapy-A Retrospective Bi-Centric Cohort Study.

Men with non-small cell lung cancer (NSCLC) have a more favorable response to immune-checkpoint inhibitor (ICI) monotherapy, while women especially benefit from ICI-chemotherapy (CHT) combinations. To elucidate such sex differences in clinical practice, we retrospectively analyzed two cohorts treated with either ICI monotherapy (n = 228) or ICI-CHT combination treatment (n = 80) for advanced NSCLC. Kaplan-Meier analyses were used to calculate progression-free (PFS) and overall survival (OS), influencing variables were evaluated using Cox-regression analyses. No significant sex differences for PFS/OS could be detected in either cohort. Men receiving ICI monotherapy had a statistically significant independent impact on PFS by Eastern Cooperative Oncology Group performance status (ECOG) ≥2 (hazard ratio (HR) 1.90, 95% confidence interval (CI): 1.10-3.29, p = 0.021), higher C-reactive protein (CRP; HR 1.06, 95%CI: 1.00-1.11, p = 0.037) and negative programmed death-ligand 1 (PD-L1) status (HR 2.04, 95%CI: 1.32-3.15, p = 0.001), and on OS by CRP (HR 1.09, 95%CI: 1.03-1.14, p = 0.002). In men on ICI-CHT combinations, multivariate analyses (MVA) revealed squamous histology (HR 4.00, 95%CI: 1.41-11.2, p = 0.009) significant for PFS; and ECOG ≥ 2 (HR 5.58, 95%CI: 1.88-16.5, p = 0.002) and CRP (HR 1.19, 95%CI: 1.06-1.32, p = 0.002) for OS. Among women undergoing ICI monotherapy, no variable proved significant for PFS, while ECOG ≥ 2 had a significant interaction with OS (HR 1.90, 95%CI 1.04-3.46, p = 0.037). Women treated with ICI-CHT had significant MVA findings for CRP with both PFS (HR 1.09, 95%CI: 1.02-1.16, p = 0.007) and OS (HR 1.11, 95%CI: 1.03-1.19, p = 0.004). Although men and women responded similarly to both ICI mono- and ICI-CHT treatment, predictors of response differed by sex.