Glycosylation-on-a-Chip: A Flow-Based Microfluidic System for Cell-Free Glycoprotein Biosynthesis

In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

Glycosylation-on-a-chip: a flow-based microfluidic system for cell-free glycoprotein biosynthesis

In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

Using differential phase for 3D localization of tracer particles in digital inline holographic microscopy PIV/PTV (DIHM-PIV/PTV)

Digital inline holographic microscopy PIV/PTV (DIHM-PIV/PTV) has the ability to provide 4-dimensional (4D), i.e. time-resolved, 3-component 3-dimensional (3C-3D) flow measurement with high spatial and temporal resolution, compact optical setup and minimal calibration Sun et al. (2020) compared to most other volumetric techniques such as tomo-PIV, defocusing PIV, etc. Despite all these advantages DIHMPIV/PTV has not yet developed into a standard laboratory tool due to some major limitations such as the extended depth-of-focus (DOF) problem and the virtual image effect which cause artefacts in the standard reconstruction volume limiting the seeding concentration and thus the achievable velocity spatial resolution. In order to mitigate the above-mentioned limitations we present a novel particle localization and extraction methodology which allows the minimization of these artefacts from the standard reconstruction and perform PIV/PTV analysis on the particle volume fields only. The proposed algorithm is based on the differential phase, which is the axial phase shift of the object wave compared to the reference plane wave propagation.

Serum lipid profile in dengue patients: a simple laboratory tool to predict disease severity

Introduction and Aim: Serum lipids play a pivotal role in the immune response of the host during dengue. The aim of this study was to evaluate the serum lipid abnormalities in dengue patients and to study the relationship between serum lipids with disease severity and platelet count. Materials and Methods: This case control study was carried out in 75 cases of dengue of age group > 18 years divided into three groups namely dengue without warning symptoms (DNWS), dengue with warning symptoms (DWWS) and severe dengue (SD) and 75 age and gender matched healthy controls. Lipid parameters such as total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides were thereafter measured in all the subjects. Results: Among 75 patients with dengue fever, 52 (69.4%) were classified as DNWS, 15 (20%) as DWWS and the remaining 8 (10.6%) as SD. Lower levels of serum total cholesterol (TC) levels (118.8 + 30.71) (p <0.0001) were observed among patients with DNWS when compared to controls. A strong significant positive correlation was seen between the platelets and serum HDL cholesterol levels among the subjects with SD (r = 0.712, p=.047*) and weak negative correlation was observed between the platelets and triglycerides in patients with dengue with warning signs (r = -0.275, p=.048*). Conclusion: We observed a strong association of diminished TC, HDL and LDL cholesterol levels with the severity of dengue. Based on our findings, these three lipid parameters could be utilized as a simple laboratory tool to identify dengue severity in resource limited settings.

The Clinical Utility of Serial Procalcitonin and Procalcitonin Clearance in Predicting the Outcome of COVID-19 Patients

Background: The pandemic of coronavirus disease 2019 (COVID‐19) represents a great threat to global health. Sensitive tests that effectively predict the disease outcome are essentially required to guide proper intervention. Objectives: To evaluate the prognostic ability of serial procalcitonin (PCT) measurement to predict the outcome of COVID-19 patients, using PCT clearance (PCT-c) as a tool to reflect its dynamic changes. Methods: A prospective observational study of inpatients diagnosed with COVID-19 at the Quarantine Hospitals of Ain-Shams University, Cairo, Egypt. During the first five days of hospitalization, serial PCT and PCT-c values were obtained and compared between survivors and non-survivors. Patients were followed up to hospital discharge or in-hospital mortality. Results: Compared to survivors, serial PCT levels of non-survivors were significantly higher (p<0.001) and progressively increased during follow-up, in contrast, PCT-c values were significantly lower (p<0.01) and progressively decreased. Receiver operating characteristic (ROC) curve analysis showed that by using the initial PCT value alone, at a cut off value of 0.80 ng/ml, the area under the curve for predicting in-hospital mortality was 0.81 with 61.1% sensitivity and 87.3% accuracy. Serial measurements showed better predictive performance and the combined prediction value was better than the single prediction by the initial PCT. Conclusions: Serial PCT measurement could be a useful laboratory tool to predict the prognosis and outcome of COVID-19 patients. Moreover, PCT-c could be a reliable tool to assess PCT progressive kinetics.

Analysis of ‘Pre-Fit’ Datasets of gLAB by Robust Statistical Techniques

The GNSS LABoratory tool (gLAB) is an interactive educational suite of applications for processing data from the Global Navigation Satellite System (GNSS). gLAB is composed of several data analysis modules that compute the solution of the problem of determining a position by means of GNSS measurements. The present work aimed to improve the pre-fit outlier detection function of gLAB since outliers, if undetected, deteriorate the obtained position coordinates. The methodology exploits robust statistical tools for regression provided by the Flexible Statistics and Data Analysis (FSDA) toolbox, an extension of MATLAB for the analysis of complex datasets. Our results show how the robust analysis FSDA technique improves the capability of detecting actual outliers in GNSS measurements, with respect to the present gLAB pre-fit outlier detection function. This study concludes that robust statistical analysis techniques, when applied to the pre-fit layer of gLAB, improve the overall reliability and accuracy of the positioning solution.

Diagnostic potential of hypoxia-induced genes in liquid biopsies of breast cancer patients

In tumor cells, higher expression of glucose transporter proteins (GLUT) and carbonic anhydrases (CAIX) genes is influenced by hypoxia-induced factors (HIF).Thus, we aimed to study the expression profile of these markers in sequential peripheral blood collections performed in breast cancer patients in order to verify their predictive potential in liquid biopsies. Gene expressions were analyzed by qPCR in tumor and blood samples from 125 patients and 25 healthy women. Differential expression was determined by the 2(−ΔCq) method. Expression of HIF-1α and GLUT1 in the blood of breast cancer patients is significantly higher (90–91 and 160–161 fold increased expression, respectively; p < 0.0001) than that found in healthy women. Their diagnostic power was confirmed by ROC curve. CAIX is also more expressed in breast cancer women blood, but its expression was detected only in a few samples. But none of these genes could be considered predictive markers. Therefore, evaluation of the expression of HIF-1α and GLUT1 in blood may be a useful laboratory tool to complement the diagnosis of breast cancer, in addition to being useful for follow-up of patients and of women with a family history of breast cancer.

Rock Emissivity Measurement for Infrared Thermography Engineering Geological Applications

Infrared thermography is a growing technology in the engineering geological field both for the remote survey of rock masses and as a laboratory tool for the non-destructive characterization of intact rock. In this latter case, its utility can be found either from a qualitative point of view, highlighting thermal contrasts on the rock surface, or from a quantitative point of view, involving the study of the surface temperature variations. Since the surface temperature of an object is proportional to its emissivity, the knowledge of this last value is crucial for the correct calibration of the instrument and for the achievement of reliable thermal outcomes. Although rock emissivity can be measured according to specific procedures, there is not always the time or possibility to carry out such measurements. Therefore, referring to reliable literature values is useful. In this frame, this paper aims at providing reference emissivity values belonging to 15 rock types among sedimentary, igneous and metamorphic categories, which underwent laboratory emissivity estimation by employing a high-sensitivity thermal camera. The results show that rocks can be defined as “emitters”, with emissivity generally ranging from 0.89 to 0.99. Such variability arises from both their intrinsic properties, such as the presence of pores and the different thermal behavior of minerals, and the surface conditions, such as polishing treatments for ornamental stones. The resulting emissivity values are reported and commented on herein for each different studied lithology, thus providing not only a reference dataset for practical use, but also laying the foundation for further scientific studies, also aimed at widening the rock aspects to investigate through IRT.

Hotplace Magnetic Stirrer Automatic Heat Control and Water Velocity Based on PID (Proportional Integral Derivative)

Hotplate magnetic stirrer is a laboratory tool that is used to heat or warm as well as mix or homogenize chemical solutions. This tool is available in chemical, microbiology and pharmaceutical laboratories. Equipped with a stirrer made of magnets (stir bar) which is used to homogenize chemical solutions. This magnetic stirrer hotplate uses an ATMega 16 microcontroller based system. It uses an infrared temperature sensor MLX90614 to measure and monitor the temperature of the solution, starting from the start of mixing which is assisted by rotating a DC motor as a stirring device until the liquid is mixed or homogeneous. Homogeneous conditions are indicated by the ADC value ( Analog to Digital Convertion) which is obtained constant. If you use the automatic menu, only choose the mixing solution on the automatic menu. There are four choices for the solution speed of 400-1600 rpm with an increase of 400, there are four temperature choices, namely 30-60 degrees Celsius in increments of 10, there is a choice of time used for stirring time at 1-30 minutes. For temperature display, stirring speed and stirring time can be seen on the LCD 20x4 display.