Neutrophils and galectin-3 defend mice from lethal bacterial infection and humans from acute respiratory failure.

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.

Sexual dysfunctions in psoriatic patients.

Introduction: Psoriasis is one the most common skin diseases associated with a great decrease in the quality of patients' lives. Methods: We aimed to study sexual dysfunctions in psoriatic patients using the Female Sexual Function Index (FSFI) for women and the International Index of Erectile Function (IIEF) for men via an anonymous online survey. The study included 80 psoriatic patients and 75 controls without dermatoses. Results: There was a downward trend in the total IIEF score in psoriatic men compared to controls. 58% of male patients and 76% of controls had a normal IIEF score. There was no significant difference in IIEF between patients treated and not with systemic agents. 62% of female patients had a decreased FSFI score, whereas in the control group, the majority of subjects (54%) had a normal FSFI score. There was no significant difference in FSFI score between patients and controls. Female patients treated with systemic antipsoriatic agents had significantly worse lubrication, satisfaction with sexual life, and pain. Discussion: Our study has shown that the majority of questioned female psoriatic patients had sexual dysfunction according to FSFI, particularly they had worse satisfaction with sexual life and less sexual desire compared to women without psoriasis. The majority of male patients did not have sexual dysfunction according to IIEF, however, they had significantly worse overall satisfaction with sexual life and confidence to keep an erection. Systemic antipsoriatic treatment does not probably influence sexual dysfunctions in men but it does in women although we were not able to assess the severity or resolution of lesions after those treatments. However embarrassing, psoriatic patients should be questioned about their sexual lives by dermatologists, and more studies are needed to explore this matter.

Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.

Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane bound CD163. Hemolysis and increase in hepatic heme, hemoglobin and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates membrane bound CD163 expression independent of NRF2 signaling in SCD liver. We further demonstrate that of the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in membrane bound CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants.

Traditional methods for the enrichment of microorganisms rely on growth in a selective liquid medium or on an agar plate, followed by tedious characterization. Droplet microfluidic techniques have been recently used to cultivate microorganisms and preserve enriched bacterial taxonomic diversity. However, new methods are needed to select droplets comprising not only growing microorganisms but also those exhibiting specific properties, such as the production of value-added compounds. We describe here a droplet microfluidic screening technique for the functional selection of biosurfactant-producing microorganisms, which are of great interest in the bioremediation and biotechnology industries. Single bacterial cells are first encapsulated into picoliter droplets for clonal cultivation and then passively sorted at high throughput based on changes in interfacial tension in individual droplets. Our method expands droplet-based microbial enrichment with a novel approach that reduces the time and resources needed for the selection of surfactant-producing bacteria.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Lung microvascular occlusion by platelet-rich neutrophil-platelet aggregates promotes cigarette smoke-induced severe flu.

Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.

A lone spike in blood glucose can enhance the thrombo-inflammatory response in cortical venules.

How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.

Dry eye syndrome symptoms in patients with psoriasis.

BACKGROUND: Psoriasis is one of the most common dermatoses associated with a variety of comorbidities. There have been some reports on its possible association with ocular disorders however dry eye syndrome (DES) in such patients has been poorly investigated. OBJECTIVES: To investigate the frequency of DES symptoms in psoriatic patients, also regarding psoriasis severity in PASI, manifestation and therapy. METHODS: 40 patients with psoriasis and 40 volunteers without dermatoses were enrolled in the study. They completed Ocular Surface Disease Index (OSDI) questionnaire and were objectively examined by IDRA® device to perform automatic interferometry, automatic meibography of lower eyelid glands, non-invasive break-up time (NIBUT), blink quality and tear meniscus height. RESULTS: Patients with psoriasis had statistically significantly thicker lipid layer (p = 0.0042 left eye, p = 0.0313 right eye) and greater loss of Meibomian glands compared to controls (p = 0.0128 left eye, p = 0.048 right eye). The patients had lower, although insignificantly, eye blink quality and tear meniscus height than the control group, as well as shorter NIBUT and higher score in OSDI. After the division of patients into two groups-with or without nails involvement/psoriatic arthritis/systemic treatment- we did not observe any significant differences between the groups. PASI did not correlate with any DES parameter. CONCLUSIONS: This is the first study of DES symptoms with an objective IDRA® analyzer. We managed to observe that patients with psoriasis have thicker lipid layer and higher Meibomian glands' loss in lower eyelids. Based on all assessed objective and subjective parameters psoriatics do not seem to have an increased risk of DES. The presence of psoriatic arthritis or nail involvement does not seem to be a predisposing factor for DES development. PASI probably cannot be a prognostic factor for any of the DES-associated parameters. Nevertheless, DES in psoriasis requires further research on bigger samples to establish reliable recommendations.

Impaired hemoglobin clearance by sinusoidal endothelium promotes vaso-occlusion and liver injury in sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in the mice liver as well as flow cytometric analysis and confocal imaging of primary human LSECs, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSECs, leading to LSEC senescence. Hb uptake by LSECs was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSECsenescence by accelerating HbS clearance in the liver of SCD mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and HbS induced LSEC-senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.

Long-Term L-Glutamine Treatment Reduces Hemolysis without Ameliorating Hepatic Vaso-Occlusion and Liver Fibrosis in a Mouse Model of Sickle Cell Disease.

Sickle cell disease (SCD) is an autosomal recessive monogenic disorder caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, hemolysis, vaso-occlusion, and sterile inflammation. The administration of oral L-glutamine has been shown to reduce the frequency of pain in SCD patients; however, the long-term effect of L-glutamine in SCD remains to be determined. To understand the long-term effect of L-glutamine administration in the liver we used quantitative liver intravital microscopy and biochemical analysis in humanized SCD mice. We here show that chronic L-glutamine administration reduces hepatic hemoglobin-heme-iron levels but fails to ameliorate ischemic liver injury. Remarkably, we found that this failure in the resolution of hepatobiliary injury and persistent liver fibrosis is associated with the reduced expression of hepatic Kupffer cells post-L-glutamine treatment. These findings establish the importance of investigating the long-term effects of L-glutamine therapy on liver pathophysiology in SCD patients.

Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.

Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in vitro compartmentalization in water-in-oil emulsion of picoliter droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for carbohydrate-active enzymes (CAZymes) catalyzing biotechnologically valuable reactions in this format presents an additional challenge because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by the incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make the observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabeled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid, and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route toward faster and easier development of specific and efficient biocatalysts for biovalorization, directing enzyme discovery by challenging catalysts for reaction with natural rather than model substrates.

spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content.

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Circulating ANGPTL8 as a Potential Protector of Metabolic Complications in Patients with Psoriasis.

Angiopoietin-like protein 8 (ANGPTL8) exerts pleiotropic effects, taking part in lipid and carbohydrate metabolism, inflammation, hematopoiesis and oncogenesis. So far, the exact molecular targets of ANGPTL8 remain poorly defined. We aimed to evaluate the serum concentration of ANGPTL8 in individuals with psoriasis and examine how systemic therapy affects the concentration of ANGPTL8. The study enrolled 35 patients with plaque-type psoriasis that were followed for 3 months of treatment with methotrexate or acitretin, and 18 healthy volunteers without psoriasis as controls. Serum ANGPTL8 concentrations were analyzed by ELISA and differences between groups were determined using Student's t-test or the Mann-Whitney test, while correlations were assessed using Spearman's rank test. The average concentration of ANGPTL8 differed significantly between the psoriasis group (before and after therapy) and the control group (p < 0.05). Significant negative correlations between ANGPTL8 and total cholesterol and LDL levels were noted (both p < 0.05). A significant increase in ANGPTL8 concentration was observed after acitretin (p < 0.05), whereas in patients treated with methotrexate the ANGPTL8 did not change significantly (p > 0.05). Additionally, a negative, statistically significant correlation with PASI was found after treatment (p < 0.05). Based on our study, it appears that elevated levels of ANGPTL8 may reduce the likelihood of atherogenic dyslipidemia in individuals with psoriasis, and treatment for psoriasis may impact the protective effects of ANGPTL8.

Ultra-High-Throughput Absorbance-Activated Droplet Sorting for Enzyme Screening at Kilohertz Frequencies.

Droplet microfluidics is a valuable method to "beat the odds" in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance-activated droplet sorting (AADS) is currently ∼10-fold slower than typical fluorescence-activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of sorting at this increased frequency with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of "bias oil" providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultra-high-throughput absorbance-activated droplet sorter increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.

Osteopontin and Regulatory T Cells in Effector Phase of Allergic Contact Dermatitis.

Studies have shown that osteopontin (OPN) and regulatory T cells play a role in allergic contact dermatitis (ACD), but the mechanisms responsible for their function are poorly understood. The study aimed to determine CD4 T lymphocytes producing intracellular osteopontin (iOPN T cells) and assess the selected T lymphocyte subsets including regulatory T cells in the blood of patients with ACD. Twenty-six patients with a disseminated form of allergic contact dermatitis and 21 healthy controls were enrolled in the study. Blood samples were taken twice: in the acute phase of the disease and during remission. The samples were analyzed by the flow cytometry method. Patients with acute ACD showed significantly higher percentage of iOPN T cells compared with healthy controls which persisted during remission. An increase in the percentage of CD4CD25 and a reduced percentage of regulatory T lymphocytes (CD4CD25highCD127low) were also found in the patients with acute stage of ACD. The percentage of CD4CD25 T lymphocytes showed a positive correlation with the EASI index. The increase in the iOPN T cells can indicate their participation in acute ACD. The decreased percentage of regulatory T lymphocytes in the acute stage of ACD may be related to the transformation of Tregs into CD4CD25 T cells. It may also indicate their increased recruitment to the skin. The positive correlation between the percentage of CD4CD25 lymphocytes and the EASI index may be indirect evidence for the importance of activated lymphocytes-CD4CD25 in addition to CD8 lymphocytes as effector cells in ACD.

Ultrahigh-Throughput Directed Evolution of a Metal-Free α/ß-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase.

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

Vector redesign and in-droplet cell-growth improves enrichment and recovery in live Escherichia coli.

Directed evolution (DE) is a widely used method for improving the function of biomolecules via multiple rounds of mutation and selection. Microfluidic droplets have emerged as an important means to screen the large libraries needed for DE, but this approach was so far partially limited by the need to lyse cells, recover DNA, and retransform into cells for the next round, necessitating the use of a high-copy number plasmid or oversampling. The recently developed live cell recovery avoids some of these limitations by directly regrowing selected cells after sorting. However, repeated sorting cycles used to further enrich the most active variants ultimately resulted in unfavourable recovery of empty plasmid vector-containing cells over those expressing the protein of interest. In this study, we found that engineering of the original expression vector solved the problem of false positives (i.e. plasmids lacking an insert) cells containing empty vectors. Five approaches to measure activity of cell-displayed enzymes in microdroplets were compared. By comparing various cell treatment methods prior to droplet sorting two things were found. Substrate encapsulation from the start, that is prior to expression of enzyme, showed no disadvantage to post-induction substrate addition by pico-injection with respect to recovery of true positive variants. Furthermore in-droplet cell growth prior to induction of enzyme production improves the total amount of cells retrieved (recovery) and proportion of true positive variants (enrichment) after droplet sorting.