Monitoring intracellular component pools to identify steady state in mammalian cell perfusion culture

Perfusion cultures of mammalian cells are a well-considered alternative in the process development for new biologic products. Stable operation of the culture should eventually lead to a steady state, positively effecting product quality. Although macroscopic variables such as viable cell density, main metabolite or product concentrations are observed constant, little is known about the extent of steady state on intracellular level. In this study intracellular component pools of CHO cells were monitored at different steady states in perfusion cultures using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). A novel single extraction was used to separate metabolite, lipid as well as protein fractions and quantify their constitution according to associated internal standards. Statistical tools were applied to resolve characteristic steady state attributes This approach allowed a comprehensive insight on metabolic as well as protein expression level. Identified features were used to explain differences between the examined states. Gained knowledge can be applied in further process optimization

Three-point correlations for quantum star graphs

We compute the three point correlation function for the eigenvalues of the Laplacian on quantum star graphs in the limit where the number of edges tends to infinity. This extends a work by Berkolaiko and Keating, where they get the 2-point correlation function and show that it follows neither Poisson, nor random matrix statistics. It makes use of the trace formula and combinatorial analysis.Comment: 10 pages, 2 figure

Effect of process parameters on the energy requirement in ultrasonical treatment of waste sludge

Mechanical treatment methods are used as pre-treatment methods in order to enhance the efficiency of conventional sludge treatment processes and the sludge becomes more suitable for its complete treatment. The ultrasound is an alternative method among other methods, but because of its high energy requirement it should be optimized before utilization. This work gives the optimized parameters such as sonication time, sonication power (these parameters are the two factors which play part for energy calculations), type of sludge, cooling requirements and solid content in the sludge solution. Even if the previous researchers prefer to use the energy (specific energy usually), we have found out that both the sonication time and the sonication power have individual importance. For municipal sludge the main conclusion can be summarized as: “high power-short retention time” is more effective than “low power-long retention time”. As this phenomenon may alter from sludge to sludge, various combinations of power and retention time should be tried while keeping the volume small and the concentration below a certain level. The process should be performed at moderate temperatures and the efficiency increases if the sludge is as homogeneous as possible

Campus Forest Carbon Project Technical Guidance Document

For more information on the Campus Forest Carbon Project visit our webpage: https://geog.umd.edu/project/campus-forest-carbon-project. This project also uses science from NASA's Carbon Monitoring system. Learn more here: https://carbon.nasa.gov/cms/.The technical guidance document was created for the Office of Sustainability to support the inclusion of forest carbon into UMD's Greenhouse Gas Inventory. This document outlines the Campus Forest Carbon's project role within UMD's climate action plan and the approach to calculating forest carbon dynamics on UMD managed and owned properties.Sustainability Fund Gran

DNA-based Diagnosis of Uncharacterized Inherited Macrothrombocytopenias Using Next-generation Sequencing Technology with a Candidate Gene Array

Inherited macrothrombocytopenias comprise a heterogeneous group of inherited platelet disorders that are characterized by large platelets, thrombocytopenia and bleeding tendencies in affected individuals. Diagnostic platforms have traditionally involved a battery of complex phenotypic tests that often fail to reach a diagnosis. Next-generation sequencing lacks the pre-analytical and analytical shortcoming of these tests and provides an attractive alternate diagnostic approach. Our group has developed a candidate gene array targeting genes known to affect platelet function and tested it in a large cohort of Australasian patients with presumed platelet function disorders, particularly macrothrombocytopenia. This array identified causative variants in a significant portion of patients with uncharacterized platelet disorders, including transcription factor mutations that cannot easily be diagnosed with standard platelet phenotyping procedures. We propose that targeted genotypic screening can identify the genetic basis of platelet function defects and has the potential to be developed into a powerful clinical platform to help clinicians diagnose these rare disorders

Anti-glycoprotein VI mediated immune thrombocytopenia: An under-recognized and significant entity?

Idiopathic immune thrombocytopenia (ITP) is an autoimmune disorder characterized by relapsing/remitting thrombocytopenia. Bleeding complications are infrequent with platelet counts above 30x10(9)/L, and this level is commonly used as a threshold for treatment. The question of another/co-existent diagnosis or an alternate mechanism of platelet destruction arises when bleeding is experienced with platelet counts above this threshold. We report a case of anti-GPVI mediated ITP that was diagnosed following investigations performed to address this key clinical question. A patient with ITP experienced exaggerated bruising symptoms despite a platelet count of 91x10(9)/L. Platelet functional testing showed an isolated platelet defect of collagen-induced aggregation. Next generation sequencing excluded a pathogenic variant of GP6, and anti-GPVI antibodies that curtailed GPVI function were confirmed by extended platelet phenotyping. We propose that anti-GPVI mediated ITP may be under-recognized, and that inclusion of GPVI in antibody detection assays may improve their diagnostic utility and in turn, facilitate a better understanding of ITP pathophysiology and aid individualized treatment approaches

Risk Assessment Models for Venous Thromboembolism in Medical Inpatients.

IMPORTANCE Thromboprophylaxis is recommended for medical inpatients at risk of venous thromboembolism (VTE). Risk assessment models (RAMs) have been developed to stratify VTE risk, but a prospective head-to-head comparison of validated RAMs is lacking. OBJECTIVES To prospectively validate an easy-to-use RAM, the simplified Geneva score, and compare its prognostic performance with previously validated RAMs. DESIGN, SETTING, AND PARTICIPANTS This prospective cohort study was conducted from June 18, 2020, to January 4, 2022, with a 90-day follow-up. A total of 4205 consecutive adults admitted to the general internal medicine departments of 3 Swiss university hospitals for hospitalization for more than 24 hours due to acute illness were screened for eligibility; 1352 without therapeutic anticoagulation were included. EXPOSURES At admission, items of 4 RAMs (ie, the simplified and original Geneva score, the Padua score, and the IMPROVE [International Medical Prevention Registry on Venous Thromboembolism] score) were collected. Patients were stratified into high and low VTE risk groups according to each RAM. MAIN OUTCOMES AND MEASURES Symptomatic VTE within 90 days. RESULTS Of 1352 medical inpatients (median age, 67 years [IQR, 54-77 years]; 762 men [55.4%]), 28 (2.1%) experienced VTE. Based on the simplified Geneva score, 854 patients (63.2%) were classified as high risk, with a 90-day VTE risk of 2.6% (n = 22; 95% CI, 1.7%-3.9%), and 498 patients (36.8%) were classified as low risk, with a 90-day VTE risk of 1.2% (n = 6; 95% CI, 0.6%-2.6%). Sensitivity of the simplified Geneva score was 78.6% (95% CI, 60.5%-89.8%) and specificity was 37.2% (95% CI, 34.6%-39.8%); the positive likelihood ratio of the simplified Geneva score was 1.25 (95% CI, 1.03-1.52) and the negative likelihood ratio was 0.58 (95% CI, 0.28-1.18). In head-to-head comparisons, sensitivity was highest for the original Geneva score (82.1%; 95% CI, 64.4%-92.1%), while specificity was highest for the IMPROVE score (70.4%; 95% CI, 67.9%-72.8%). After adjusting the VTE risk for thromboprophylaxis use and site, there was no significant difference between the high-risk and low-risk groups based on the simplified Geneva score (subhazard ratio, 2.04 [95% CI, 0.83-5.05]; P = .12) and other RAMs. Discriminative performance was poor for all RAMs, with an area under the receiver operating characteristic curve ranging from 53.8% (95% CI, 51.1%-56.5%) for the original Geneva score to 58.1% (95% CI, 55.4%-60.7%) for the simplified Geneva score. CONCLUSIONS AND RELEVANCE This head-to-head comparison of validated RAMs found suboptimal accuracy and prognostic performance of the simplified Geneva score and other RAMs to predict hospital-acquired VTE in medical inpatients. Clinical usefulness of existing RAMs is questionable, highlighting the need for more accurate VTE prediction strategies

Microfluidic and nanotechnology based assays for the development of safe biopharmaceuticals

Protein stability towards aggregation represents a potential challenge for the production and administration of pharmaceuticals. In particular, aggregation can compromise the developability and shelf-life of the products, with consequences for yield and safety, respectively. In this work, we discuss two novel approaches for the analysis of the stability of protein formulations: (1) A microfluidic diffusion-sizing platform to analyze protein sizes and interactions at high protein concentration directly in the solution state with minimal perturbation of the sample. The limited dilution of the sample during the analysis and the possibility to characterize properties directly in the solution state make the technique suitable for the analysis of heterogeneous solutions of proteins under dynamic equilibrium. We show how the platform represents an attractive tool for the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation. (2) A highly controlled assay of surface-induced protein aggregation based on nanoparticles. Protein aggregation is often due to heterogeneous nucleation events occurring at interfaces, including air/water interface, impurities and leachable particles. However, the development of screening tools against surface aggregation has been hindered by the difficulty in generating a controlled amount of surface stress in the formulation as well as in decoupling the surface effect from the contribution of hydrodynamic flows. In our assay, we leverage the flexibility of polymer chemistry to finely tune the properties and amount of surfaces provided by the nanoparticles, inducing aggregation of soluble peptides and proteins, including antibodies, in a time scale of a few hours. This platform represents i) an attractive tool for fundamental studies of heterogeneous nucleation events under stagnant and flow conditions, and ii) a high-throughput screening assay of the effect of intrinsic and extrinsic variables on protein stability towards interface-induced aggregation. Please click Additional Files below to see the full abstract