CRISPR interference screens reveal growth-robustness tradeoffs in Synechocystis sp. PCC 6803 across growth conditions.

Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and noncoding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in 1 condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive data set assigns condition-specific importance to many previously unannotated genes and suggests additional functions for central metabolic enzymes. Phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions are compiled in an interactive web application.

Comparison of Mortality Prediction Scores in Intermediate-Care Patients with Liver Cirrhosis at a German University Transplant Centre: A Prospective Study.

BACKGROUND AND AIMS: Mortality prediction models help to extract and relate patient data upon admission to intensive or intermediate care units (ImCUs). Considering technical and economic healthcare developments, re-evaluations of score performances are required to warrant their validity. This study validates and compares established scoring systems in cirrhotic ImCU patients. METHODS: Acute Physiology and Chronic Health Evaluation (APACHE) II, Simplified Acute Physiology Score (SAPS) 2 and 3, Sepsis Organ Failure Assessment (SOFA), Mortality Probability Model at ICU admission (MPMo) II and III, Model for End stage Liver Disease (MELD), CLIF-Consortium Acute-on-Chronic Liver Failure (CLIF-C ACLF), CLIF-Consortium Acute Decompensation (CLIF-C AD), and Intermediate Care Unit Severity Score (ImCUSS) were calculated in patients with cirrhosis (n = 98) at ImCU admission. Discrimination performances were evaluated by area under the receiver operating characteristic curves (AUROCs), calibration performances with calibration belt plots, and their corresponding p values. RESULTS: Overall, SAPS 3 and CLIF-C ACLF have shown the best 90-day mortality prediction outcomes with AUROCs of 0.825 and 0.783 along with calibration belt p values of 0.128 and 0.061, respectively. In a subgroup analysis of patients with acute-on-chronic liver failure (ACLF), expanded SAPS 2, SOFA, and SAPS 3 reached the best AUROCs, i.e., 0.760, 0.750, and 0.714, but none of the tested scores reached an acceptable calibration. CONCLUSION: Ninety-day mortality risk prediction of the SAPS 3 and CLIF-C ACLF was accurate in our cohort of patients with liver cirrhosis admitted to ImCUs. A particular challenge remains that is the mortality prediction in patients with ACLF requiring ImCU-level care; here, further developments are needed to generate scores with acceptable predictive performances.

Blood-saving dissection with monopolar tungsten needle electrodes and Teflon-coated spatula electrodes in tumor orthopedics.

INTRODUCTION: Resection of musculoskeletal tumors and reconstruction with tumor endoprostheses often results in blood loss requiring transfusion of blood products. We assessed the blood-saving potential of using monopolar tungsten needle electrodes and polytetrafluoroethylene (PTFE)-coated spatula electrodes (intervention) compared with conventional dissection with sharp instruments and coagulation with uncoated steel electrodes (control). METHODS: We retrospectively analyzed data of 132 patients (79 interventions, 53 controls) undergoing surgery by one single experienced surgeon in our tertiary referral center between 2012 and 2021. RESULTS: Intraoperative blood loss in the intervention group was reduced by 29% [median (IQR): 700 (400-1200) vs 500 (200-700) ml; p = 0.0043]. Postoperative wound drainage decreased by 41% [median (IQR): 1230 (668-2041) vs 730 (450-1354) ml; p = 0.0080]. Additionally, patients in need of PRBCs during surgery declined from 43% to 15% (23/53 vs 12/79; p = 0.0005), while the transfusion rate after surgery did not change notably. The number of patients in need of revision surgery due to wound healing disorders was low in both groups (control group: 4/53 vs intervention group: 4/79). Only one patient in the control group and two patients in the intervention group underwent revision surgery due to hemorrhage. Baseline characteristics were similar between groups (sex, Charlson Comorbidity score, tumor entity). CONCLUSION: Dissection with tungsten needle electrodes and PTFE-coated spatula electrodes appears an effective surgical blood-saving measure without increased risk of wound healing disorders. LEVEL OF EVIDENCE: III, retrospective comparative study. CLINICAL TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov. Identifier: NCT05164809.

Thermodynamic limitations of PHB production from formate and fructose in Cupriavidus necator.

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.

A novel protocol for in-depth analysis of recombinant adeno-associated virus capsid proteins using UHPLC-MS/MS.

RATIONALE: In-depth characterization of the three capsid viral proteins (VPs 1, 2, and 3) of adeno-associated viruses (AAVs) is immediately needed to ensure the consistency in gene therapy products and processes. These proteins are typically present at very low concentrations in matrices containing high concentrations of excipients and salts. Thus, there is a need for convenient methods for sample preparation before proteomic analysis. The aim of this study was to meet this need by developing a fast, reliable approach for isolating VPs in a manner enabling their efficient digestion and in-depth characterization using liquid chromatography-mass spectrometry (LC-MS). METHODS: VPs from Anc80 were precipitated with different organic solvents, and the resulting precipitates were dissolved in either sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) or guanidine hydrochloride (Gu-HCl). The peptides obtained by the following enzymatic digestion by either trypsin or Asp-N were analyzed using LC-MS/MS. RESULTS: We found that precipitation with chloroform/methanol/water results in fast, efficient preparation of VP samples, allowing 100% and 99.2% amino acid sequence coverage of VP1 for trypsin and Asp-N digestion, respectively. This also allowed complete sequence confirmation of VP1, VP2, and VP3 of Anc80, as well as characterization of the amino acid sequences of the N- and C-terminal regions of each VP, together with their post-translational modifications (PTMs). CONCLUSIONS: The presented method enables fast, reliable, and relatively cheap sample preparation for identifying AAV serotypes and characterizing the heterogeneity of capsid viral proteins, including their PTMs.

Analysis of Ascites-Challenged Blood in Patients with Liver Cirrhosis Using Rotational Thromboelastometry: How Robust Is the Evidence on Ascites-Attributed Fibrinolysis?

INTRODUCTION: For over 30 years, ascites has been postulated to facilitate fibrinolysis in patients with liver cirrhosis. In contrast to previous research employing conventional coagulation tests, this study aimed to characterize hemostatic interactions between blood and ascites using the rotational thromboelastometry (ROTEM). METHODS: Blood samples - pure or mixed with ascites in a ratio of 1:1 - from cirrhotic patients (n = 10) were subjected to ROTEM analysis. In addition, a negative control group was built with cirrhotic patients (n = 10) whose blood was mixed with physiologic sodium chloride (0.9% NaCl) solution in a ratio of 1:1. Subsequently, ROTEM measurements were subjected to statistical analysis. RESULTS: During ascites challenge, clotting time (CT, measured in seconds) was significantly prolonged in EXTEM (blood: 70.40 ± 20.40 vs. ascites/blood: 109.8 ± 47.7) and APTEM (blood: 66.50 ± 14.55 vs. ascites/blood: 138.7 ± 105.8), likely reflecting a dilution effect. However, CT in INTEM remained unchanged, suggesting a sustained intrinsic pathway function. Maximal clot firmness (measured in millimeters) in FIBTEM decreased significantly (blood: 14.70 ± 9.55 vs. ascites/blood: 6.00 ± 5.66), thus indicating depletion of fibrinogen in ascites. Strikingly, maximum lysis (measured in %) significantly decreased in EXTEM (blood: 9.30 ± 2.79 vs. ascites/blood: 5.50 ± 2.84), APTEM (blood: 8.50 ± 3.10 vs. ascites/blood: 5.60 ± 2.88), and INTEM (blood: 7.50 ± 2.27 vs. ascites/blood: 5.10 ± 3.48). CONCLUSIONS: ROTEM provided new evidence that ascites may not primarily induce fibrinolysis in cirrhotic patients. This finding seems to be of significant importance for the clinical management of cirrhotic patients experiencing complications, for example, abdominal hemorrhage after liver biopsy or paracentesis; here, replacement of prothrombin complex concentrates and/or fibrinogen concentrates may prove more beneficial than the use of fresh frozen plasma or antifibrinolytic drugs.

Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation.

PURPOSE: Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations. METHODS: The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone. RESULTS: First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations. CONCLUSION: The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.

Effect of Regional Citrate Anticoagulation vs Systemic Heparin Anticoagulation During Continuous Kidney Replacement Therapy on Dialysis Filter Life Span and Mortality Among Critically Ill Patients With Acute Kidney Injury: A Randomized Clinical Trial.

Importance: Although current guidelines suggest the use of regional citrate anticoagulation (which involves the addition of a citrate solution to the blood before the filter of the extracorporeal dialysis circuit) as first-line treatment for continuous kidney replacement therapy in critically ill patients, the evidence for this recommendation is based on few clinical trials and meta-analyses. Objective: To determine the effect of regional citrate anticoagulation, compared with systemic heparin anticoagulation, on filter life span and mortality. Design, Setting, and Participants: A parallel-group, randomized multicenter clinical trial in 26 centers across Germany was conducted between March 2016 and December 2018 (final date of follow-up, January 21, 2020). The trial was terminated early after 596 critically ill patients with severe acute kidney injury or clinical indications for initiation of kidney replacement therapy had been enrolled. Interventions: Patients were randomized to receive either regional citrate anticoagulation (n = 300), which consisted of a target ionized calcium level of 1.0 to 1.40 mg/dL, or systemic heparin anticoagulation (n = 296), which consisted of a target activated partial thromboplastin time of 45 to 60 seconds, for continuous kidney replacement therapy. Main Outcomes and Measures: Coprimary outcomes were filter life span and 90-day mortality. Secondary end points included bleeding complications and new infections. Results: Among 638 patients randomized, 596 (93.4%) (mean age, 67.5 years; 183 [30.7%] women) completed the trial. In the regional citrate group vs systemic heparin group, median filter life span was 47 hours (interquartile range [IQR], 19-70 hours) vs 26 hours (IQR, 12-51 hours) (difference, 15 hours [95% CI, 11 to 20 hours]; P < .001). Ninety-day all-cause mortality occurred in 150 of 300 patients vs 156 of 296 patients (Kaplan-Meier estimator percentages, 51.2% vs 53.6%; unadjusted difference, -2.4% [95% CI, -10.5% to 5.8%]; unadjusted hazard ratio, 0.91 [95% CI, 0.72 to 1.13]; unadjusted P = .38; adjusted difference, -6.1% [95% CI, -12.6% to 0.4%]; primary adjusted hazard ratio, 0.79 [95% CI, 0.63 to 1.004]; primary adjusted P = .054). Of 38 prespecified secondary end points, 34 showed no significant difference. Compared with the systemic heparin group, the regional citrate group had significantly fewer bleeding complications (15/300 [5.1%] vs 49/296 [16.9%]; difference, -11.8% [95% CI, -16.8% to -6.8%]; P < .001) and significantly more new infections (204/300 [68.0%] vs 164/296 [55.4%]; difference, 12.6% [95% CI, 4.9% to 20.3%]; P = .002). Conclusions and Relevance: Among critically ill patients with acute kidney injury receiving continuous kidney replacement therapy, anticoagulation with regional citrate, compared with systemic heparin anticoagulation, resulted in significantly longer filter life span. The trial was terminated early and was therefore underpowered to reach conclusions about the effect of anticoagulation strategy on mortality. Trial Registration: ClinicalTrials.gov Identifier: NCT02669589.

Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.

BACKGROUND: Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA). RESULTS: In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively. CONCLUSIONS: The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.

Music devices for the fetus? An evaluation of pregnancy music belts.

BACKGROUND: The implementation of music during pregnancy is a topic of interest for parents-to-be accompanied by a growing commercial interest. We evaluated acoustic properties of commercially available music devices. MATERIALS AND METHODS: Sound characteristics of three different music devices designed for fetal acoustical stimulation were analyzed. A white noise sample was presented at a high volume to produce a standardized acoustic stimulus. Sound emissions were registered for each loudspeaker with a sound level meter in order to document the sound pressure levels (SPLs) and to analyze the long-term averaged spectra (LTAS) with the help of PRAAT-sound-analyzing software. Measurements were conducted in open air and under attenuated conditions with interposition of a pork uterus of 5 mm thickness covered by porcine tissue from the abdominal wall of either 3 or 5 cm thickness. RESULTS: Under attenuated conditions, SPLs of all three devices were hardly detectable and interfered with the basal noise of around 50-55 dB (SPL), particularly low and high frequencies ranges were attenuated. CONCLUSION: Pregnancy music belts seem to be a useless tool to support fetal development. The poor sound characteristics of the loudspeakers and the concept of an isolated stimulation appear not promising to effectively support the complex multimodal maturation of the sensory system. Traditional implementation of music appears maternal singing appears more reasonable.

Controlled Extraction Studies Applied to Polyvinyl Chloride and Polyethylene Materials: Conclusions from the ELSIE Controlled Extraction Pilot Study.

The effective management of leachables in pharmaceutical products is a critical aspect of their development. This can be facilitated if extractables information on the materials used in a packaging or delivery system is available to assist companies in selecting materials that will be compatible with the drug product formulation and suitable for the intended use. The Extractables and Leachables Safety Information Exchange (ELSIE) materials working group developed and executed a comprehensive extraction study protocol that included a number of extraction solvents, extraction techniques, and a variety of analytical techniques. This was performed on two test materials, polyethylene (PE) and polyvinyl chloride (PVC), that were selected due to their common use in pharmaceutical packaging. The purpose of the study was to investigate if the protocol could be simplified such that (i) a reduced number or even a single extraction technique could be used and (ii) a reduced number of solvents could be used to obtain information that is useful for material selection regardless of product type. Results indicate that, at least for the PVC, such reductions are feasible. Additionally, the studies indicate that levels of extractable elemental impurities in the two test materials were low and further confirm the importance of using orthogonal analytical detection techniques to gain adequate understanding of extraction profiles.

Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

Integrated strategy for deep profiling of host cell proteins in downstream processing of therapeutic monoclonal antibodies: Novel approach to isolate and digest host cell proteins.

Host cell proteins (HCPs) are process-related impurities generated during the production of biopharmaceuticals, which may contaminate the final product unless they are efficiently removed. Due to their potential impact on product safety, quality and efficacy, regulatory authorities require removal of HCPs during processing down to trace amounts in final manufactured biopharmaceuticals. The current standard method for detecting HCPs is enzyme-linked immunosorbent assay (ELISA), which should reveal the total amount of HCPs. A necessary orthogonal technique to get more granular information on HCPs is obtained by application of liquid chromatography-mass spectrometry (LC-MS) techniques that permit identification and quantification of individual HCPs. However, differences in sample preparation methods and MS acquisition techniques have led to discrepancies in detected HCPs between studies, which may compromise product safety, quality and efficacy. To address this issue, we have developed a novel and reproducible workflow for isolation, digestion, and mass spectrometry detection of HCPs that is applicable to downstream process characterization of therapeutic monoclonal antibodies (mAbs). This article describes a rapid and efficient workflow for the isolation, digestion and identification of HCPs. For the first time, Fc-receptor (FcγRIIIa) affinity chromatography is employed to isolate the HCP fraction from the mAb. Next, the HCPs are precipitated with acetone and digested using a newly developed "single-pot" method that improves digestion performance and prevents sample loss of problematic low-abundant HCPs. The new HCP isolation method outperforms protein A affinity chromatography for monitoring problematic HCPs.

Protein-polyanion interactions for the controlled release of monoclonal antibodies.

The objective of the present study was to investigate ionic interactions between alginate and a monoclonal antibody (mAb1) and to utilize those interactions for the sustained release of mAb1. The existence of ionic interactions between alginate and mAb1 was strongly reflected by their rheological behavior. A 3-4 times increase in storage modulus (G') was observed by addition of 30 mg/mL mAb1 to a 20 mg/mL alginate solution. This increase was strongly dependent on pH and ionic strength. In vitro release studies revealed a marked pH-dependence of release rates and the reversibility of alginate-mAb1 complexation under physiological conditions. Two alginate-mAb1 sustained release formulations were developed by an internal gelation technique using CaCO(3) and CaHPO(4) as calcium sources for physical cross-linking. The CaCO(3) formulation provided a stable pH-environment, optimally suited for pH-sensitive proteins. CaHPO(4) led to a lower pH and stronger alginate-mAb1 interactions. The CaHPO(4) cross-linked alginate released mAb1 over a period of 10-15 days. The long release period and changes in viscoelastic properties of alginate, when being mixed with mAb1, indicate the incorporation of mAb1 molecules into a mixed network with alginate. The results of this study demonstrate that ionic interactions between polyanions and mAb1 are present and that they can be exploited for sustained release delivery of mAb1.

Temporal and spatial properties of a yeast multi-cellular amplification system based on signal molecule diffusion.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α-factor as key signaling molecule between two cell types. One cell type represents the α-factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 x 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α-factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

No Differences in Rotational Thromboelastometry Measurements between Portal and Peripheral Circulation in Cirrhotic Patients Undergoing TIPS.

BACKGROUND: In patients with liver cirrhosis, transjugular intrahepatic portosystemic shunt (TIPS) is considered a standardized treatment of refractory ascites or variceal bleeding. TIPS thrombosis (TT) and/or portal vein thrombosis (PVT) are possible complications during/after TIPS placement. Previous studies suggested increased clotting activity in portal circulation (PORC). This pilot study aimed to evaluate alterations and differences of coagulation function in PORC and in peripheral circulation (PERC) via rotational thromboelastometry during TIPS. METHODS: Blood samples were collected from cirrhotic patients (n = 13; median Model of End Stage Liver Disease, MELD Score: 12; median age: 60 years) undergoing TIPS (10/13 TIPSs were elective procedures due to refractory ascites) as follows: median cubital vein (MCV; PERC)-confluence of the three hepatic veins to the inferior cava vein (HV/ICV; PORC)-portal vein (PV; PORC)-TIPS (PORC). This research utilized four variables of the extrinsic test EXTEM, i.e., clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), and maximum lysis (ML). RESULTS: EXTEM results [mean, M (range) ± standard deviation, SD (range)] showed no significant differences for CT [M (70-73) ± SD (9-13); p = 0.93] or CFT [M (137-155) ± SD (75-112); p = 0.97] or MCF [M (51-54) ± SD (9-10); p = 0.90] or ML [M (9-10) ± SD (4-5); p = 0.89] between the compartments, i.e., MCV vs. HV/ICV vs. PV vs. TIPS. Overall, we detected no differences in coagulation function between PERC and PORC. CONCLUSION: These results are in contrast to previous reports suggesting increased clotting activity in PORC vs. PERC in association with liver cirrhosis. Rotational thromboelastometry-based evaluation of coagulation function in PERC appears to reliably reflect coagulation function in PORC with respect to risk estimation for TT and/or PVT in cirrhotic patients undergoing TIPS.

Expression of Interferon Regulatory Factor 8 (IRF8) and Its Association with Infections in Dialysis Patients.

Patients on dialysis have dysfunctions of innate and adaptive immune system responses. The transcriptional factor IRF8 (interferon regulatory factor 8) is primarily expressed in plasmacytoid cells (pDCs) and myeloid dendritic cells (mDCs), playing a crucial role in the maturation of dendritic cells, monocytes, and macrophages, and contributing to protection against bacterial infections. The current study analyzed the expression patterns of IRF8 and assessed its association with the risk of infections in 79 dialysis patients compared to 44 healthy controls. Different subsets of leukocytes and the intracellular expression of IRF8 were measured using flow cytometry. Compared to the healthy controls, the dialysis patients showed significantly reduced numbers of pDCs and significantly increased numbers of natural killer cells and classical and intermediate monocytes. The dialysis patients exhibited decreased numbers of IRF8-positive dendritic cells (pDC p < 0.001, mDC1 p < 0.001, mDC2 p = 0.005) and increased numbers of IRF8-positive monocytes (p < 0.001). IRF8 expression in pDC, mDC, and classical monocytes was lower in the dialysis patients than in the controls. Dialysis patients who required hospitalization due to infections within one year of follow-up displayed significantly reduced IRF8 expression levels in pDCs compared to patients without such infections (p = 0.04). Our results suggest that reduced IRF8 expression in pDCs is a potential risk factor predisposing dialysis patients to serious infections.

Therapeutic Plasma Exchange in Early-Onset Preeclampsia: A 7-Year Monocentric Experience.

Different therapeutic apheresis techniques have been clinically tested to delay preterm delivery in the case of eoPE (early-onset preeclampsia). Our study evaluated the feasibility of TPE (therapeutic plasma exchange) compared to standard-of-care treatment. Twenty patients treated with 95 TPE sessions were included in the final analysis and retrospectively matched with 21 patients with comparable placental dysfunction. Gestational age at admission was 23.75 ± 2.26 versus 27.57 ± 2.68 weeks of gestation (WoG) in the control group (p = < 0.001), mean sFlt-1/PlGF ratio was 1946.26 ± 2301.63 versus 2146.70 ± 3273.63 (p = 0.821) and mean sEng was 87.63 ± 108.2 ng/mL versus 114.48 ± 88.78 ng/mL (p = 0.445). Pregnancy was prolonged for 8.25 ± 5.97 days when TPE was started, compared to 3.14 ± 4.57 days (p = 0.004). The median sFlt-1/PlGF Ratio was 1430 before and 1153 after TPE (-18.02%). Median sEng fell from 55.96 ng/mL to 47.62 mg/mL (-27.73%). The fetal survival rate was higher in TPE-treated cases. NICU (Neonatal Intensive Center Unit) stay was in the median of 63 days in the TPE group versus 48 days in the standard-of-care group (p = 0.248). To date, this monocentric retrospective study, reports the largest experience with extracorporeal treatments in eoPE worldwide. TPE could improve pregnancy duration and reduce sFlt-1 and sEng in maternal serum without impairing neonatal outcomes.

Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive. Here we employ a structure-driven mutagenesis approach in combination with an extensive panel of functional signaling readouts to investigate the importance of conserved state-stabilizing residues in FZD5 for signal specification. Similar data were obtained for FZD4 and FZD10 suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD5 mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD5 and FZDs in general prefer coupling to DVL rather than heterotrimeric G proteins and that distinct active state micro-switches in the receptor are essential for pathway selection arguing for conformational changes in the receptor protein defining transducer selectivity.