Expression and downstream purification of insulin molecules in Pichia pastoris

In the next decade and beyond global demand for insulin is expected to rise significantly requiring additional manufacturing capacity. The next generation of insulin manufacturing plants will likely be based on new and robust expression and bioprocess platforms that are flexible, safe, simple to implement in a manufacturing setting and capable of step improvements in productivity and cost compared to current manufacturing techniques. Towards this end, Pichia pastoris expression systems has been evaluated for insulin due to its capacity to secrete a variety of heterologous proteins and its ability to grow to high cell densities. Under the well-characterized, tightly regulated AOX1 promoter, yields of 1.5 to nearly 3 g/L of purified insulin have been reported.1,2,3 However, methanol is a very volatile substance requiring specialized facilities, which can hamper large-scale production. Downstream processing of insulin precursors also requires use of organic solvents which can also burden manufacturing. We report development of an insulin process using a constitutive promoter expression system in place of the inducible AOX1 promoter, and a simplified downstream purification process using precipitation. Fermentations were carried out in 2 L scale bioreactors and culture supernatant collected after 65 hours. A design of experiment (DoE) was performed to identify optimal conditions for polyelectrolyte precipitation of the recombinant protein using polyvinyl sulfonic acid (PVS).4 The resulting pellets were then analyzed via SDS-PAGE and HPLC. 1. Gurramkonda, C. et al. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb. Cell Fact. 9, 31 (2010). 2. Polez, S. et al. A Simplified and Efficient Process for Insulin Production in Pichia pastoris. PLoS One 11, e0167207 (2016). 3. Xie, T., Liu, Q., Xie, F., Liu, H. & Zhang, Y. Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin. Prep. Biochem. Biotechnol. 38, 308–17 (2008). 4. Buddha, M. et al. Precipitation as an Alternative to Chromatography in the Insulin Manufacturing Process. BioPharm Int. 30–36 (2016)

Analysis of frontal sinus shape and volume variation between population affinity groups and biological sexes as seen on computed tomography scans

Frontal sinus variation has been used in forensic anthropology to aid in positive identification since the 1920s. As radiographic technology has evolved, so has the quality and quantity of data that radiologists and anthropologists can collect during an individual’s lifetime. This has led to new methodology when comparing antemortem and postmortem radiographic images. The current study aims to look at frontal sinus morphology and dimensional variations on computed tomography (CT) scans, as these are currently the most commonly collected images showing the frontal sinus in its entirety used in clinics in the United States. This study assessed 307 individuals for the morphological analysis and 325 individuals in the dimensional analysis. These individuals represented females and males from Asian, African, European, and Latin American derived groups. It is hypothesized that frontal sinus shape variations will cluster based on assigned sex and population affinities. Similarly, it is hypothesized that dimensional variation, specifically the maximum height, maximum width, and maximum depth, will show statistically significant clustering based on assigned sex and population affinities. The frontal sinus outlines from the CT images were transferred into SHAPE v1.3 in order to run an elliptical Fourier analysis. The dimensional data was measured directly from the images using a MicroDicom viewer. All of the statistical analyses, including Pearson’s Chi-squared and ANOVA tests, were run in R studio. Results indicated that morphologically there is no statistically significant clustering based on assigned sex or population affinity. However, there was statistically significant clustering dimensionally when tested against both assigned sex and population affinity using an ANOVA, indicating that the interactive effects of sexual dimorphism and population affinity influence the dimensions but not the shape of the frontal sinus. These results add to the foundational knowledge that practitioners have surrounding frontal sinus indicating that assigned sex and population affinity have impact on the approximate dimensions of this structure while these variables do not have a statistically significant effect on morphological variation. The results also speak to the idiosyncratic nature of the frontal sinus and bolster confidence using morphological variations as a mean to individuate.

Heat transfer in mixing vessels at low Reynolds numbers. An experimental study of temperature profiles heat transfer rates and power requirements for mechanically agitated vessels operating at low Reynolds numbers.

The present study investigates experimentally the laminar mixing and heat transfer of a range of helical ribbon and anchor impellers for both Newtonian and inelastic non-Newtonian fluids. The work also correlates the experimental data empirically in the form of dimensionless groups. In order to estimate the relative importance and the effect of all the geometrical parameters on the mixing power and heat transfer, data from the published literature sources will be utilized and combined with the results from this study. Thus, reliable empirical correlations will be obtained which are applicable over the widest range of operating conditions. The study also investigates the ablity of the various impellers to level out temerature distributions. The measurement of these temperature gradients and the impeller power requirements gives a measure of the mixing efficiency of the impeller used.Science Research Counci

The Simultaneous Impacts of Seasonal Weather and Solar Conditions on PV Panels Electrical Characteristics

Solar energy usage is thriving day by day. These solar panels are installed to absorb solar energy and produce electrical energy. As a result, the efficiency of solar panels depends on different environmental factors, namely, air temperature, dust (aerosols and accumulated dust), and solar incidence, and photovoltaic panel angles. The effects of real conditions factors on power and efficiency of photovoltaic panels are studied in this paper through testing the panel in real environmental tests. To study the mentioned parameters precisely, two panels with different angles are used. The case study is regarding a region of Tehran, Iran, in summer and winter seasons. The results show that panel efficiency during winter is higher than summer due to air temperature decrement. It is discovered that among air pollutants, Al and Fe have the most share in polluting the air that affect the photovoltaic efficiency. Moreover, measuring the accumulated dust on the panels shows more amount in winter in comparison with summer. The important point in studying the effect of tilt angle is that inconformity between solar incidence and photovoltaic panel angles would result in solar radiation absorption and eventually panel efficiency loss and also, photovoltaic panel installation angle would affect the amount of dust deposited on its surface.© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).fi=vertaisarvioitu|en=peerReviewed

Characterization of a 3D matrix bioreactor for scaled production of human mesenchymal stem cells

Human Mesenchymal Stem Cells (hMSCs) are multipotent, immune-privileged, and possess the capacity to proliferate ex-vivo, making them a good candidate for stem cell therapy. However, a reliable scalable production system for hMSCs is needed to fuel the growing field of regenerative medicine. Current growth of hMSCs is achieved through adherent 2D methods using tissue culture flasks or cell factory systems. These processes are labor intensive and can lead to low purity and poor yield of hMSCs due to the limited control of culture conditions inherent in these systems. In this work, we are investigating a novel 3D honeycomb matrix culture system for controlled high density hMSC production. We have assessed compatibility of the hMSCs on the honeycomb matrix and developed a scale down model bioreactor for development and characterization. Computational Fluid Dynamic (CFD) modeling is used in parallel with the described in-vitro experimentation to characterize shear profiles and oxygen transport for optimization of the conditions to support high cell density hMSC cultures. These techniques will potentially allow for higher yield and purity of hMSCs to meet the large quantities of cells needed for emerging whole cell therapies

Leukemia in Iran: Epidemiology and morphology trends

Background: Leukemia accounts for 8 of total cancer cases and involves all age groups with different prevalence and incidence rates in Iran and the entire world and causes a significant death toll and heavy expenses for diagnosis and treatment processes. This study was done to evaluate epidemiology and morphology of blood cancer during 2003-2008. Materials and Methods: This cross- sectional study was carried out based on re-analysis of the Cancer Registry Center report of the Health Deputy in Iran during a 6-year period (2003 - 2008). Statistical analysis for incidence time trends and morphology change percentage was performed with joinpoint regression analysis using the software Joinpoint Regression Program. Results: During the studied years a total of 18,353 hematopoietic and reticuloendothelial system cancers were recorded. Chi square test showed significant difference between sex and morphological types of blood cancer (P-value < 0.001). Joinpoint analysis showed a significant increasing trend for the adjusted standard incidence rate (ASIR) for both sexes (P-value < 0.05). Annual percent changes (APC) for women and men were 18.7 and 19.9, respectively. The most common morphological blood cancers were ALL, ALM, MM and CLL which accounted for 60 of total hematopoietic system cancers. Joinpoint analyze showed a significant decreasing trend for ALM in both sexes (P-value < 0.05). Conclusions: Hematopoietic system cancers in Iran demonstrate an increasing trend for incidence rate and decreasing trend for ALL, ALM and CLL morphology

A review of the classification of crimes in the light of the new grading of crime

In the present age, with the increasing growth of new science and technologies, fundamental changes in values and norms, and the multiplicity and entanglement of social and political systems; The nature, form, variety and manner of committing crimes have also changed a lot. Given that crime emerges in the context of social conditions, in this study, in an interdisciplinary study between criminology and strategic sciences with a "descriptive-analytical" method and using the criterion of modern social classification, we seek to divide Present a new classification of crimes in accordance with the complex conditions mentioned above. According to the achievements of this study, unlike the major classification system, inspired by the model of "community management system in modern perception" as well as strategic science data, crimes in three new levels: 1- Strategic or imaginary-paradigm crimes ; 2- We have classified tactological or managerial crimes and 3- technological or individual and technological crimes. In this regard, contrary to the common perception in the alloying of crimes into Micro and Macro crimes, this research in the new alloy, crimes in four layers Nano, Micro, Macro And Super is divided according to the two criteria of Sibel or the target environment of the crime and on the other hand the damages. Finally, by combining the above topics, in a matrix structure, a new classification of crimes in the form of a doctrine can be achieved, which in this research is referred to as "crime Rating "

A Reflection on the Theoretical and Practical Aspects of the Cooperation of Third States with the International Criminal Court

The idea of establishing an International Criminal Court can be considered as a transformational and developmental idea in the field of international law. Following the Nuremberg trials, the drafting and adoption of the Rome Statute is considered an important document in the realm of international criminal law. This document, unlike the founding documents of the Ad Hoc tribunals, is an international treaty. Therefore, it is subject to the provisions of customary international law governing international treaties. This statute is a development and codifying treaty. In addition, not all state are parties to this treaty and therefore some states are considered third parties to it. In the present article, the concept of a third state in the framework of the Rome Statute has been studied descriptively-analytically. The question then arises as to whether the principle of relativity of treaties applicable to the Rome Statute also is subject to exceptions. According to the findings of this study, the Rome Statute is subject to some exceptions, such as: the existence of a customary rule, the existence of a jus cogense rule, the decision of the Security Council and the consent of the concerned state to the application of the treaty. It is important to note, however, that the extension of the Court's jurisdiction to a state not party to the Statute, outside of what is provided for in the Statute, cannot be consistent with the spirit of criminal law as set forth in the Statute

Computational Fluid Dynamics (CFD) modelling and experimental confirmation of hollow fiber tangential flow filtration (HFTFF) and alternating tangential flow filtration (ATF) In a perfusion bioreactor

Hollow fiber tangential flow filtration (HFTFF) and Alternating tangential flow filtration (HFATF) are technologies of choice in continuous (perfusion) bioreactor operations. A major drawback of these technologies is membrane fouling and associated reduction in membrane permeability. Membrane fouling leads to a gradual decline in trans-membrane flux and the sieving of the protein product. Additionally, experimental data suggests that under otherwise similar conditions protein sieving may be different in TFF vs. ATF, indicating that flow behavior patterns in the two technologies may be different. Many models of fouling and protein sieving have been reported in the literature for HFTFF. In comparison, however, there is limited research work on HFATF, making it hard to compare mechanisms of fouling and product sieving between HFTFF and HFATF. Additionally, almost all mechanisms of fouling and predictive models make sweeping assumptions with regards to the complex flow patterns prevailing in HFTFF and HFATF. In this study, we provide experimental data and computational fluid dynamics (CFD) information to gain insight into factors that impact fouling and product sieving. Specifically, first we present the confirmation of CFD model outputs by comparing experimentally measured trans-membrane flux and pressure with model predictions. Next, we compare the CFD model predictions of pressure drop, shear rate profile and axial and radial fluid velocity distributions between HFTFF and HFATF. Subsequently, we investigate the shear effect on cell damage, using the concept of constant Camp number, defined as Gt = constant, where G is the prevailing shear rate and t is the exposure time. Our CFD model predicts that shear rate (G) and hence the resulting stress experienced by cells in HFATF has a distribution that is determined by the operation of the diaphragm pump. Finally, we use CFD to compare Gt profile generated by imposing different pump condition

Development and qualification of a scale-down model of a commercial mammalian cell culture bioreactor using Computational Fluid Dynamics

The use of computational fluid dynamics (CFD) techniques can be used to develop and/or optimize a scale-down model to investigate mixing, oxygen mass transfer characteristics and turbulence, strain rate, and bubble size distribution in laboratory-scale stirred-tank bioreactors. In this work, CFD was used to test and modify a laboratory-scale bioreactor model of a manufacturing-scale bioreactor. The laboratory-scale model was originally established based on power per volume (P/V) and volume of gas per bioreactor volume per minute (vvm). CFD simulations of mixing time, power input, and gas volume hold-up were performed to demonstrate comparability between the laboratory-scale model and the manufacturing-scale bioreactor. These simulations were verified with experimental measurement of mixing time and gas hold-up. The results were used to propose sparge rate and impeller agitation as factors in a Design of Experiments (DoE) study in laboratory-scale bioreactors. The impact of sparge rate and impeller agitation on cell growth, productivity, and product quality attributes were evaluated in the DOE study. The laboratory-scale production bioreactor model was compared to the manufacturing-scale production bioreactor. The results confirmed that CFD techniques could be used to establish sparge rate and impeller agitation to improve a scale-down model