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Abstract Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+’s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p

The chlamydial deubiquitinase Cdu1 of the obligate intracellular human pathogenic bacterium Chlamydia trachomatis plays important roles in the maintenance of chlamydial infection. Despite the structural similarities shared with its homologue Cdu2, both DUBs display remarkable differences in their enzymatic activity towards poly-UB chain substrates. Whereas Cdu1 is highly active towards K48- and K63- poly-UB chains, Cdu2 activity is restricted mostly to mono-UB substrates. Here, we shed light on the molecular mechanisms of the differential activity and the substrate specificity of Cdu1 to better understand the cellular processes it is involved in, including infection-related events. We found that the strikingly elevated activity of Cdu1 relative to its paralogue Cdu2 can be attributed to an N-terminally extended α-helix, which has not been observed in Cdu2. Moreover, by employing isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate the differential recognition of K48- and K63-linked poly-UB substrates by Cdu1. Whereas K63-linked poly-UB substrates appear to be recognized by Cdu1 with only two independent ubiquitin interaction sites, up to four different binding interfaces are present for K48-linked ubiquitin chains. Combined, our data suggest that Cdu1 possesses a poly-UB chain directed activity that may enable its function as a multipurpose DUB with a broad substrate specificity.

Metabolic dysfunction-associated fatty liver disease (MAFLD), the leading cause of end-stage liver disease in developed countries, is expected to increase over the next decade. Characterized by hepatic steatosis, MAFLD is commonly studied in animal models. Here, we generated a human induced pluripotent stem cell (iPSC) line from a patient homozygous of the protective MTARC1 gene variant rs2642438:A. This line displays a normal karyotype and typical pluripotent stem cell morphology and can differentiate into all three germ layers in vitro.

Head and neck squamous-cell carcinoma (HNSCC) is associated with aggressive local invasiveness, being a main reason for its poor prognosis. The exact mechanisms underlying the strong invasive abilities of HNSCC remain to be elucidated. Therefore, there is a need for in vitro models to study the interplay between cancer cells and normal adjacent tissue at the invasive tumor front. To generate oral mucosa tissue models (OMM), primary keratinocytes and fibroblasts from human oral mucosa were isolated and seeded onto a biological scaffold derived from porcine small intestinal submucosa with preserved mucosa. Thereafter, we tested different methods (single tumor cells, tumor cell spots, spheroids) to integrate the human cancer cell line FaDu to generate an invasive three-dimensional model of HNSCC. All models were subjected to morphological analysis by histology and immunohistochemistry. We successfully built OMM tissue models with high in vivo–in vitro correlation. The integration of FaDu cell spots and spheroids into the OMM failed. However, with the integration of single FaDu cells into the OMM, invasive tumor cell clusters developed. Between segments of regular epithelial differentiation of the OMM, these clusters showed a basal membrane penetration and lamina propria infiltration. Primary human fibroblasts and keratinocytes seeded onto a porcine carrier structure are suitable to build an OMM. The HNSCC model with integrated FaDu cells could enable subsequent investigations into cancer cell invasiveness.

3D cell culture models replicate tissue complexity and aim to study cellular interactions and responses in a more physiologically relevant environment compared to traditional 2D cultures. However, the spherical structure of these models makes it difficult to extract meaningful data, necessitating advanced techniques for proper analysis. In silico simulations enhance research by predicting cellular behaviors and therapeutic responses, providing a powerful tool to complement experimental approaches. Despite their potential, these simulations often require advanced computational skills and significant resources, which creates a barrier for many researchers. To address these challenges, we developed an accessible pipeline using open-source software to facilitate virtual tissue simulations. Our approach employs the Cellular Potts Model, a versatile framework for simulating cellular behaviors in tissues. The simulations are constructed from real world 3D image stacks of cancer spheroids, ensuring that the virtual models are rooted in experimental data. By introducing a new metric for parameter optimization, we enable the creation of realistic simulations without requiring extensive computational expertise. This pipeline benefits researchers wanting to incorporate computational biology into their methods, even if they do not possess extensive expertise in this area. By reducing the technical barriers associated with advanced computational modeling, our pipeline enables more researchers to utilize these powerful tools. Our approach aims to foster a broader use of in silico methods in disease research, contributing to a deeper understanding of disease biology and the refinement of therapeutic interventions.

A hepatocyte cell line was used to determine the hepatotoxicity of sedatives and opioids, as the hepatotoxicity of these drugs has not yet been well characterized. This might pose a threat, especially to critically ill patients, as they often receive high cumulative doses for daily analgosedation and often already have impaired liver function due to an underlying disease or complications during treatment. A well-established biosensor based on HepG2/C3A cells was used for the determination of the hepatotoxicity of commonly used sedatives and opioids in the intensive care setting (midazolam, propofol, s-ketamin, thiopental, fentanyl, remifentanil, and sufentanil). The incubation time was 2 × 3 days with clinically relevant (Cmax) and higher concentrations (C5× and C10×) of each drug in cell culture medium or human plasma. Afterward, we measured the cell count, vitality, lactate dehydrogenase (LDH), mitochondrial dehydrogenase activity, cytochrome P 450 1A2 (CYP1A2), and albumin synthesis. All tested substances reduced the viability of hepatocyte cells, but sufentanil and remifentanil showed more pronounced effects. The cell count was diminished by sufentanil in both the medium and plasma and by remifentanil only in plasma. Sufentanil and remifentanil also led to higher values of LDH in the cell culture supernatant. A reduction of mitochondrial dehydrogenase activity was seen with the use of midazolam and s-ketamine. Microalbumin synthesis was reduced in plasma after its incubation with higher concentrations of sufentanil and remifentanil. Remifentanil and s-ketamine reduced CYP1A2 activity, while propofol and thiopental increased it. Our findings suggest that none of the tested sedatives and opioids have pronounced hepatotoxicity. Sufentanil, remifentanil, and s-ketamine showed moderate hepatotoxic effects in vitro. These drugs should be given with caution to patients vulnerable to hepatotoxic drugs, e.g., patients with pre-existing liver disease or liver impairment as part of their underlying disease (e.g., hypoxic hepatitis or cholestatic liver dysfunction in sepsis). Further studies are indicated for this topic, which may use more complex cell culture models and global pharmacovigilance reports, addressing the limitation of the used cell model: HepG2/C3A cells have a lower metabolic capacity due to their low levels of CYP enzymes compared to primary hepatocytes. However, while the test model is suitable for parental substances, it is not for toxicity testing of metabolites.

Abstract Background The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. Results In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. Conclusion The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell–matrix interactions and their contribution to a tissue-specific microenvironment.

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

Abstract The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

In plants, an increasing number of traits and new characteristics are being developed using gene editing. Simple traits represented by a single gene can be managed through backcross breeding, but this is typically not the case for more complex traits which may result from the function of a large number of genes. Here, we demonstrate two case studies of improving oleic oil content and developing pod shatter reduction in Brassica napus by using gene editing tools on an industrial scale. There are four BnaFAD2 genes involved in oleic oil content and eight BnaSHP genes involved in pod shatter tolerance. In order to develop these two traits, we delivered nuclease ribonucleoproteins with Gene Repair OligoNucleotides (GRONs) into protoplasts, with subsequent regeneration into plants on an industrial scale, which encompassed robust tissue culture protocols, efficient gene editing, robotics sampling, and molecular screening, vigorous plant regeneration, growth, and phenotyping. We can produce precise loss-of-function-edited plants with two improved agronomically important complex traits, high oleic oil or pod shatter reduction, in elite canola varieties within 1–3 years, depending on the trait complexity. In the edited plants carrying loss of function of four BnaFAD2 genes, the seed fatty acid oleic acid content reached 89% compared to 61% in the non-edited wildtype control. The plants carrying eight edited BnaSHP genes achieved 51% pod shatter reduction in multiple year field testing in the target environment compared to the wildtype control.

The use of inertial measurement units (IMUs) as an alternative to optical marker-based systems has the potential to make gait analysis part of the clinical standard of care. Previously, an IMU-based system leveraging Rauch–Tung–Striebel smoothing to estimate knee angles was assessed using a six-degrees-of-freedom joint simulator. In a clinical setting, however, accurately measuring abduction/adduction and external/internal rotation of the knee joint is particularly challenging, especially in the presence of soft tissue artefacts. In this study, the in vivo IMU-based joint angles of 40 asymptomatic knees were assessed during level walking, under two distinct sensor placement configurations: (1) IMUs fixed to a rigid harness, and (2) IMUs mounted on the skin using elastic hook-and-loop bands (from here on referred to as “skin-mounted IMUs”). Estimates were compared against values obtained from a harness-mounted optical marker-based system. The comparison of these three sets of kinematic signals (IMUs on harness, IMUs on skin, and optical markers on harness) was performed before and after implementation of a REference FRame Alignment MEthod (REFRAME) to account for the effects of differences in coordinate system orientations. Prior to the implementation of REFRAME, in comparison to optical estimates, skin-mounted IMU-based angles displayed mean root-mean-square errors (RMSEs) up to 6.5°, while mean RMSEs for angles based on harness-mounted IMUs peaked at 5.1°. After REFRAME implementation, peak mean RMSEs were reduced to 4.1°, and 1.5°, respectively. The negligible differences between harness-mounted IMUs and the optical system after REFRAME revealed that the IMU-based system was capable of capturing the same underlying motion pattern as the optical reference. In contrast, obvious differences between the skin-mounted IMUs and the optical reference indicated that the use of a harness led to fundamentally different joint motion being measured, even after accounting for reference frame misalignments. Fluctuations in the kinematic signals associated with harness use suggested the rigid device oscillated upon heel strike, likely due to inertial effects from its additional mass. Our study proposes that optical systems can be successfully replaced by more cost-effective IMUs with similar accuracy, but further investigation (especially in vivo and upon heel strike) against moving videofluoroscopy is recommended.

The complexity offered by additive material extrusion (MEX) presents new opportunities for novel design, especially with regard to multi-material components. However, this design freedom has heretofore only been scarcely used thus stifling innovation. One reason for this development is the complicated nature of adhesion at the interface of multi-material parts. Hence, this publication aims to investigate the process-dependent influences in multi-material MEX by conducting tensile tests on ABS-PLA multi-material specimens. By implementing a distance gauge into the tool change procedure, positional fluctuations of the nozzle will be eliminated and the effects of extrusion temperature, line placement, and over- or under-extrusion on composite strength can be determined more precisely. In addition, thermal imaging is conducted to give an informed estimate of the effects of build chamber or build plate temperature on diffusion at the material interface. The results show a clear influence of extrusion temperature and over-extrusion on composite strength, while the effect of line placement is determined to only be minor. The build chamber temperature is predicted to have no meaningful effect on composite strength. Overall, the results suggest that deviations in printer calibration, by as little as 0.04 mm, can have a significant influence on composite strength.

Summary: Experience-driven alterations in neuronal activity are followed by structural-functional modifications allowing cells to adapt to these activity changes. Structural plasticity has been observed for cortical principal cells. However, how GABAergic interneurons respond to experience-dependent network activity changes is not well understood. We show that parvalbumin-expressing interneurons (PVIs) of the dentate gyrus (DG) possess dendritic spines, which undergo behaviorally induced structural dynamics. Glutamatergic inputs at PVI spines evoke signals with high spatial compartmentalization defined by neck length. Mice experiencing novel contexts form more PVI spines with elongated necks and exhibit enhanced network and PVI activity and cFOS expression. Enhanced green fluorescent protein reconstitution across synaptic partner-mediated synapse labeling shows that experience-driven PVI spine growth boosts targeting of PVI spines over shafts by glutamatergic synapses. Our findings propose a role for PVI spine dynamics in regulating PVI excitation by their inputs, which may allow PVIs to dynamically adjust their functional integration in the DG microcircuitry in relation to network computational demands.

Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.

Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.

Intense threat elicits action in the form of active and passive coping. The medial prefrontal cortex (mPFC) executes top-level control over the selection of threat coping strategies, but the dynamics of mPFC activity upon continuing threat encounters remain unexplored. Here, we used 1-photon calcium imaging in mice to probe the activity of prefrontal pyramidal cells during repeated exposure to intense threat in a tail suspension (TS) paradigm. A subset of prefrontal neurons displayed selective activation during TS, which was stably maintained over days. During threat, neurons showed specific tuning to active or passive coping. These responses were unrelated to general motion tuning and persisted over days. Moreover, the neural manifold traversed by low-dimensional population activity remained stable over subsequent days of TS exposure and was preserved across individuals. These data thus reveal a specific, temporally, and interindividually conserved repertoire of prefrontal tuning to behavioral responses under threat.

The bacterial stringent response and its effector alarmone guanosine penta- or tetra – phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (viru-lence). (p)ppGpp does so by binding to its numerous target proteins and repro-graming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identifica-tion of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordi-nates the nucleotide and amino acid metabolic pathways upon stringent re-sponse; however, the mechanistic link between nucleotide and amino acid me-tabolisms remains still incompletely understood. Here we propose the metab-olite ribose 5’-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

Steady states of dynamical systems, whether stable or unstable, are critical for understanding future evolution. Robust steady states, ones that persist under small changes in the model parameters, are desired when modelling ecological systems, where it is common for accurate and detailed information on functional form and parameters to be unavailable. Previous work by Jahedi et al. [Robustness of solutions of almost every system of equations, SIAM J. Appl. Math. 82(5) (2022), pp. 1791–1807; Structured systems of nonlinear equations, SIAM J. Appl. Math. 83(4) (2023), pp. 1696–1716.] has established criteria to imply the prevalence of robust steady states for systems with minimal predetermined structure, including conventional structured systems. We review that work and extend it by allowing symmetries in the system structure, which present added obstructions to robustness.

Functional and transcriptomic comparison of primary neonatal, adult and ESC-derived astrocytes reveals major variations, highlighting the importance for an informed choice of astrocyte origin for research questions.

Abstract Background Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method. Results Here we introduce ReCSAI, a compressed sensing neural network to reconstruct localizations for confocal dSTORM, together with a simulation tool to generate training data. We implemented and compared different artificial network architectures, aiming to combine the advantages of compressed sensing and deep learning. We found that a U-Net with a recursive structure inspired by iterative compressed sensing showed the best results on realistic simulated datasets with noise, as well as on real experimentally measured confocal lifetime scanning data. Adding a trainable wavelet denoising layer as prior step further improved the reconstruction quality. Conclusions Our deep learning approach can reach a similar reconstruction accuracy for confocal dSTORM as frame binning with traditional fitting without requiring the acquisition of multiple frames. In addition, our work offers generic insights on the reconstruction of sparse measurements from noisy experimental data by combining compressed sensing and deep learning. We provide the trained networks, the code for network training and inference as well as the simulation tool as python code and Jupyter notebooks for easy reproducibility.

(1) Antibiotics are an important weapon in the fight against serious bacterial infections and are considered a common cause of drug-induced liver injury (DILI). The hepatotoxicity of many drugs, including antibiotics, is poorly analyzed in human in vitro models. (2) A standardized assay with a human hepatoma cell line was used to test the hepatotoxicity of various concentrations (Cmax, 5× Cmax, and 10× Cmax) of antibiotics. In an ICU, the most frequently prescribed antibiotics, ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, meropenem, rifampicin, tigecycline, and vancomycin, were incubated with HepG2/C3A cells for 6 days. Cell viability (XTT assay, LDH release, and vitality), albumin synthesis, and cytochrome 1A2 activity were determined in cells. (3) In vitro, vancomycin, rifampicin, and tigecycline showed moderate hepatotoxic potential. The antibiotics ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, and meropenem were associated with mild hepatotoxic reactions in test cells incubated with the testes Cmax concentration. Rifampicin and cefuroxime showed significantly negative effects on the viability of test cells. (4) Further in vitro studies and global pharmacovigilance reports should be conducted to reveal underlying mechanism of the hepatotoxic action of vancomycin, rifampicin, tigecycline, and cefuroxime, as well as the clinical relevance of these findings.

Summary: Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled “power stroke.” Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family’s evolutionary success.

Barrier membranes are commonly used as part of the dental surgical technique guided bone regeneration (GBR) and are often made of resorbable collagen or non-resorbable materials such as PTFE. While collagen membranes do not provide sufficient mechanical protection of the covered bone defect, titanium reinforced membranes and non-resorbable membranes need to be removed in a second surgery. Thus, biodegradable GBR membranes made of pure magnesium might be an alternative. In this study a biodegradable pure magnesium (99.95%) membrane has been proven to have all of the necessary requirements for an optimal regenerative outcome from both a mechanical and biological perspective. After implantation, the magnesium membrane separates the regenerating bone from the overlying, faster proliferating soft tissue. During the initial healing period, the membrane maintained a barrier function and space provision, whilst retaining the positioning of the bone graft material within the defect space. As the magnesium metal corroded, it formed a salty corrosion layer and local gas cavities, both of which extended the functional lifespan of the membrane barrier capabilities. During the resorption of the magnesium metal and magnesium salts, it was observed that the membrane became surrounded and then replaced by new bone. After the membrane had completely resorbed, only healthy tissue remained. The in vivo performance study demonstrated that the magnesium membrane has a comparable healing response and tissue regeneration to that of a resorbable collagen membrane. Overall, the magnesium membrane demonstrated all of the ideal qualities for a barrier membrane used in GBR treatment.

An ideal fixation system for guided bone (GBR) regeneration in oral surgery must fulfil several criteria that includes the provision of adequate mechanical fixation, complete resorption when no longer needed, complete replacement by bone, as well as be biocompatible and have a good clinical manageability. For the first time, a biodegradable magnesium fixation screw made of the magnesium alloy WZM211 with a MgF2 coating has been designed and tested to fulfill these criteria. Adequate mechanical fixation was shown for the magnesium fixation screw in several benchtop tests that directly compared the magnesium fixation screw with an equivalent polymeric resorbable device. Results demonstrated slightly superior mechanical properties of the magnesium device in comparison to the polymeric device even after 4 weeks of degradation. Biocompatibility of the magnesium fixation screw was demonstrated in several in vitro and in vivo tests. Degradation of the magnesium screw was investigated in in vitro and in vivo tests, where it was found that the screw is resorbed slowly and completely after 52 weeks, providing adequate fixation in the early critical healing phase. Overall, the magnesium fixation screw demonstrates all of the key properties required for an ideal fixation screw of membranes used in guided bone regeneration (GBR) surgeries.

Abstract Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.

High-grade serous ovarian carcinoma (HGSOC) is the most genomically complex cancer, characterized by ubiquitous TP53 mutation, profound chromosomal instability, and heterogeneity. The mutational processes driving chromosomal instability in HGSOC can be distinguished by specific copy number signatures. To develop clinically relevant models of these mutational processes we derived 15 continuous HGSOC patient-derived organoids (PDOs) and characterized them using bulk transcriptomic, bulk genomic, single-cell genomic, and drug sensitivity assays. We show that HGSOC PDOs comprise communities of different clonal populations and represent models of different causes of chromosomal instability including homologous recombination deficiency, chromothripsis, tandem-duplicator phenotype, and whole genome duplication. We also show that these PDOs can be used as exploratory tools to study transcriptional effects of copy number alterations as well as compound-sensitivity tests. In summary, HGSOC PDO cultures provide validated genomic models for studies of specific mutational processes and precision therapeutics.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7’s ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia. Author summary Human papillomaviruses cause human cancers largely through virally encoded E6 and E7 oncogenes. A murine papillomavirus, MmuPV1, causes lesions in laboratory mice that mimic features of human papillomavirus-induced lesions including cancer. In this study, we investigated how the interaction of MmuPV1 E7 with the cellular tumor suppressor protein, PTPN14, contributes to viral pathogenesis using a MmuPV1 E7 mutant defective for interacting with PTPN14, E7K81S. We examined phenotypic differences between the E7K81S and wild type MmuPV1 in tissue culture and in mice. The E7K81S mutant genome was able to replicate as a nuclear plasmid in tissue culture. In vivo, E7K81S mutant virus caused dysplastic lesions like wild type virus; however, they were smaller in size, arose later, and trended towards being less severe in their grade of neoplastic disease. The E7K81S-induced lesions showed evidence for increased E2F activation and reprogramming of suprabasal cells to support DNA synthesis similar to that seen in lesions induced by wild type virus. One clear molecular difference was that the E7K81S-induced lesions showed an expansion of cells expressing differentiation markers. Our study shows that an intact PTPN14 binding site is important for MmuPV1 E7’s ability to contribute to MmuPV1-induced skin disease.

In recent years, space agencies, such as the National Aeronautics and Space Administration (NASA) and European Space Agency (ESA), have expanded their research activities in the field of manufacturing in space. These measures serve to reduce limitations and costs through fairing size, launch mass capabilities or logistic missions. The objective, in turn, is to develop technologies and processes that enable on-demand manufacturing for long-term space missions and on other celestial bodies. Within these research activities, in-situ resource utilization (ISRU) and recycling are major topics to exploit local resources and save transport capacity and, therefore, costs. On the other hand, it is important to carefully consider which items can be brought and which must be manufactured on the Moon. Consequently, on-demand needs in future space missions are considered regarding frequency, raw material and required manufacturing processes according to investigations by ESA and NASA. In conclusion, manufacturing in space state-of-the-art shows a strong focus on additive processes, primarily considering semicrystalline or amorphous plastics. The subtractive processing of metallic or ceramic materials, in turn, currently represents a research gap. Consequently, an approach for in-situ resource utilization-based subtractive manufacturing in space is presented to supplement the existing processes. The latter uses a high-pressure jet of water, with regolith simulate as abrasive in suspension, being directed at the workpiece, which is moved to separate metal and glass. Proof-of-concept results are presented, including suitable process windows, achieved cutting geometries, as well as the effects of parameter variations on the system technology and consumables used. The focus of the investigations supplements the general requirements for the design of machine tools for space applications with inertial process-specific boundary conditions as a step towards higher technology maturity.

Abstract Chemical probes are important tools for understanding biological systems. However, because of the huge combinatorial space of targets and potential compounds, traditional chemical screens cannot be applied systematically to find probes for all possible druggable targets. Here, we demonstrate a novel concept for overcoming this challenge by leveraging high‐throughput metabolomics and overexpression to predict drug–target interactions. The metabolome profiles of yeast treated with 1,280 compounds from a chemical library were collected and compared with those of inducible yeast membrane protein overexpression strains. By matching metabolome profiles, we predicted which small molecules targeted which signaling systems and recovered known interactions. Drug–target predictions were generated across the 86 genes studied, including for difficult to study membrane proteins. A subset of those predictions were tested and validated, including the novel targeting of GPR1 signaling by ibuprofen. These results demonstrate the feasibility of predicting drug–target relationships for eukaryotic proteins using high‐throughput metabolomics.

The mobility of the β2-adrenergic receptor, from the nanosecond to the second range, is revealed by combining several fluorescent spectroscopy techniques. These data also show a previously hidden mobility of this GPCR.

Abstract Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic reorganization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis, and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. We combined growth dynamics and dynamic changes in intracellular metabolite levels with a coarse‐grained model that integrates fluxes, regulation, protein synthesis, and growth and uncovered fundamental limitations of the regulatory network: After nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade‐offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.

Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn's disease and Parkinson's disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation and cytokine/chemokine secretion with a strong impact for the genotypes found in the twins consistent with a role of the identified mutations in the development of early onset leprosy.

Summary: Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the enzymes catalyzing these modifications remain largely unknown. In addition to enzymatic acetylation, lysine residues can also be chemically acetylated by the metabolite acetyl phosphate. In Escherichia coli, acetylation at over 3,000 sites has been linked to acetyl phosphate, but the functionality of this widespread non-enzymatic acetylation is even less clear than the enzyme-catalyzed one. Here, we investigate the role of acetyl-phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated, only GapA and GpmA are acetylated at high stoichiometry, which inhibits their activity by interfering with substrate binding, effectively reducing glycolysis when flux to or from acetate is high. Extrapolating our results to the whole proteome, maximally 10% of the reported non-enzymatically acetylated proteins are expected to reach a stoichiometry that could inhibit their activity.

Although the genetic code of the yeast Saccharomyces cerevisiae was sequenced 25 years ago, the characterization of the roles of genes within it is far from complete. The lack of a complete mapping of functions to genes hampers systematic understanding of the biology of the cell. The advent of high-throughput metabolomics offers a unique approach to uncovering gene function with an attractive combination of cost, robustness, and breadth of applicability. Here, we used flow-injection time-of-flight mass spectrometry to dynamically profile the metabolome of 164 loss-of-function mutants in TOR and receptor or receptor-like genes under a time course of rapamycin treatment, generating a dataset with >7000 metabolomics measurements. In order to provide a resource to the broader community, those data are made available for browsing through an interactive data visualization app hosted at https://rapamycin-yeast.ethz.ch. We demonstrate that dynamic metabolite responses to rapamycin are more informative than steady-state responses when recovering known regulators of TOR signaling, as well as identifying new ones. Deletion of a subset of the novel genes causes phenotypes and proteome responses to rapamycin that further implicate them in TOR signaling. We found that one of these genes, CFF1, was connected to the regulation of pyrimidine biosynthesis through URA10. These results demonstrate the efficacy of the approach for flagging novel potential TOR signaling-related genes and highlight the utility of dynamic perturbations when using functional metabolomics to deliver biological insight.

Abstract The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Abstract Background Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.

Trinks et al developed a protocol that enables super-resolved visualisation of the interaction between natural killer cells and Aspergillus fumigatus hyphae by expansion microscopy. They describe in detail steps of the formation of the immunological synapse including cytoskeleton re-arrangement, granule convergence and translocation of LAMP1, demonstrating the utility of the method.

We interrogated prefrontal circuit function in mice lacking Disrupted-in-schizophrenia-1 (Disc1-mutant mice), a risk factor for psychiatric disorders. Single-unit recordings in awake mice revealed reduced average firing rates of fast-spiking interneurons (INTs), including optogenetically identified parvalbumin-positive cells, and a lower proportion of INTs phase-coupled to ongoing gamma oscillations. Moreover, we observed decreased spike transmission efficacy at local pyramidal cell (PYR)-INT connections in vivo, suggesting a reduced excitatory effect of local glutamatergic inputs as a potential mechanism of lower INT rates. On the network level, impaired INT function resulted in altered activation of PYR assemblies: While assembly activations defined as coactivations within 25 ms were observed equally often, the expression strength of individual assembly patterns was significantly higher in Disc1-mutant mice. Our data, thus, reveal a role of Disc1 in shaping the properties of prefrontal assembly patterns by setting INT responsiveness to glutamatergic drive.

Direkli Cave, located in the Taurus Mountains of southern Turkey, was occupied by Late Epipaleolithic hunters-gatherers for the seasonal hunting and processing of game including large numbers of wild goats. We report genomic data from new and published Capra specimens from Direkli Cave and, supplemented with historic genomes from multiple Capra species, find a novel lineage best represented by a ~14,000 year old 2.59 X genome sequenced from specimen Direkli4. This newly discovered Capra lineage is a sister clade to the Caucasian tur species (Capra cylindricornis and Capra caucasica), both now limited to the Caucasus region. We identify genomic regions introgressed in domestic goats with high affinity to Direkli4, and find that West Eurasian domestic goats in the past, but not those today, appear enriched for Direkli4-specific alleles at a genome-wide level. This forgotten ‘Taurasian tur’ likely survived Late Pleistocene climatic change in a Taurus Mountain refuge and its genomic fate is unknown.

Antimicrobials (e.g., antibiotics and biocides) are invaluable chemicals used to control microbes in numerous contexts. Because of the simultaneous use of antibiotics and biocides, questions have arisen as to whether environments commonly treated with biocides (e.g., hospitals, food processing, wastewater, agriculture, etc.) could act as a reservoir for the development of antibiotic cross-resistance. Theoretically, cross-resistance could occur if the mechanism of bacterial tolerance to biocides also resulted in antibiotic resistance. On the other hand, biocides would likely present a higher evolutionary barrier to the development of resistance given the different modes of action between biocides and antibiotics and the broad-based physicochemical effects associated with most biocides. Published studies have shown that the induction of biocide tolerance in a laboratory can result in cross-resistance to some antibiotics, most commonly hypothesized to be due to efflux pump upregulation. However, testing of environmental isolates for biocide tolerance and antibiotic cross-resistance has yielded conflicting results, potentially due to the lack of standardized testing. In this review, we aim to describe the state of the science on the potential linkage between biocide tolerance and antibiotic cross-resistance. Questions still remain about whether the directed evolution of biocide tolerance and the associated antibiotic cross-resistance in a laboratory are or are not representative of real-world settings. Thus, research should continue to generate informative data to guide policies and preserve these tools’ utility and availability.

Garitano-Trojaola et al. used a combination of human acute myeloid leukemia (AML) cell lines and primary samples to show that RAC1-dependent actin cytoskeleton remodeling through BCL2 family plays a key role in resistance to the FLT3 inhibitor, Midostaurin in AML. They showed that by targeting RAC1 and BCL2, Midostaurin resistance was diminished, which potentially paves the way for an innovate treatment approach for FLT3 mutant AML.

Abstract Background Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. Results Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. Conclusion Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (

Abstract Microorganisms adjust metabolic activity to cope with diverse environments. While many studies have provided insights into how individual pathways are regulated, the mechanisms that give rise to coordinated metabolic responses are poorly understood. Here, we identify the regulatory mechanisms that coordinate catabolism and anabolism in Escherichia coli. Integrating protein, metabolite, and flux changes in genetically implemented catabolic or anabolic limitations, we show that combined global and local mechanisms coordinate the response to metabolic limitations. To allocate proteomic resources between catabolism and anabolism, E. coli uses a simple global gene regulatory program. Surprisingly, this program is largely implemented by a single transcription factor, Crp, which directly activates the expression of catabolic enzymes and indirectly reduces the expression of anabolic enzymes by passively sequestering cellular resources needed for their synthesis. However, metabolic fluxes are not controlled by this regulatory program alone; instead, fluxes are adjusted mostly through passive changes in the local metabolite concentrations. These mechanisms constitute a simple but effective global regulatory program that coarsely partitions resources between different parts of metabolism while ensuring robust coordination of individual metabolic reactions.

Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.