Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system

Objectives

The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic.

Results

Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme.

Conclusions

Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.

     

How the chemical features of molecules may have addressed the settlement of metabolic steps

Introduction

While the evolutionary adaptation of enzymes to their own substrates is a well assessed and rationalized field, how molecules have been originally selected in order to initiate and assemble convenient metabolic pathways is a fascinating, but still debated argument.

Objectives

Aim of the present study is to give a rationale for the preferential selection of specific molecules to generate metabolic pathways.

Methods

The comparison of structural features of molecules, through an inductive methodological approach, offer a reading key to cautiously propose a determining factor for their metabolic recruitment.

Results

Starting with some commonplaces occurring in the structural representation of relevant carbohydrates, such as glucose, fructose and ribose, arguments are presented in associating stable structural determinants of these molecules and their peculiar occurrence in metabolic pathways.

Conclusions

Among other possible factors, the reliability of the structural asset of a molecule may be relevant or its selection among structurally and, a priori, functionally similar molecules.

     

Evidence-informed policy formulation and implementation: a comparative case study of two national policies for improving health and social care in Sweden

Background

Evidence has come to play a central role in health policymaking. However, policymakers tend to use other types of information besides research evidence. Most prior studies on evidence-informed policy have focused on the policy formulation phase without a systematic analysis of its implementation. It has been suggested that in order to fully understand the policy process, the analysis should include both policy formulation and implementation. The purpose of the study was to explore and compare two policies aiming to improve health and social care in Sweden and to empirically test a new conceptual model for evidence-informed policy formulation and implementation.

Methods

Two concurrent national policies were studied during the entire policy process using a longitudinal, comparative case study approach. Data was collected through interviews, observations, and documents. A Conceptual Model for Evidence-Informed Policy Formulation and Implementation was developed based on prior frameworks for evidence-informed policymaking and policy dissemination and implementation. The conceptual model was used to organize and analyze the data.

Results

The policies differed regarding the use of evidence in the policy formulation and the extent to which the policy formulation and implementation phases overlapped. Similarities between the cases were an emphasis on capacity assessment, modified activities based on the assessment, and a highly active implementation approach relying on networks of stakeholders. The Conceptual Model for Evidence-Informed Policy Formulation and Implementation was empirically useful to organize the data.

Conclusions

The policy actors’ roles and functions were found to have a great influence on the choices of strategies and collaborators in all policy phases. The Conceptual Model for Evidence-Informed Policy Formulation and Implementation was found to be useful. However, it provided insufficient guidance for analyzing actors involved in the policy process, capacity-building strategies, and overlapping policy phases. A revised version of the model that includes these aspects is suggested.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

The effect of colloid formulation on colloid osmotic pressure in horses with naturally occurring gastrointestinal disease

Background

Naturally occurring gastrointestinal disease is an important cause of acute hypoproteinemia in adult horses and hydroxyethyl starch colloid fluid treatment is a component of supportive care in these cases to improve plasma volume and maintain colloid osmotic pressure (COP). The objectives of the present study were to compare 2 formulations of high molecular weight hydroxyethyl starch and their relative effect on COP, acid-base status, and survival of horses with acute hypoproteinemia secondary to gastrointestinal disease.

Methods

Twenty adult horses, ≥ 1 year of age, were prospectively enrolled, with informed client consent, if they developed acute hypoproteinemia, defined as a plasma total protein <5.0 g/dL or albumin <2.2 g/dL during hospitalization while undergoing treatment for gastrointestinal disease. Horses were randomly assigned to receive a rapid infusion of either 6% hydroxyethyl starch in 0.9% saline or 6% hydroxyethyl starch in lactated ringers solution at a dose of 10ml/kg. Venous blood gas analysis, COP, and PCV were evaluated before and after colloid administration.

Results

For both groups, average COP prior to treatment was 11.0 mmHg (9.7 – 12.2 mmHg) and post colloid treatment was 13.2 mmHg (12.0 -14.7 mmHg) [Normal range 18 – 22 mmHg]. COP was significantly increased with colloid treatment (p<0.001) but this increase was not significantly different between treatment groups. Venous pH did not change significantly with treatment. Twelve horses survived to hospital discharge and survival did not differ significantly between treatment groups.

Conclusions

Post-treatment COP improved approximately 20% regardless of the formulation used, however, values did not reach the normal range of COP observed in healthy horses. Acid-base parameters were not significantly impacted by either treatment. Further study is needed to determine how these two products compare with regards to other outcome measures. Evaluation of the relative effects of colloid formulation in horses with clinical disease is a future area of interest.

     

Characterization of trotter horses urine metabolome by means of proton nuclear magnetic resonance spectroscopy

Background

Metabolomics has been recognized as a powerful approach for disease screening. In order to highlight potential health issues in subjects, a key factor is the possibility to compare quantitatively the metabolome of their biofluids with reference values from healthy individuals. Such efforts towards the systematic characterization of the metabolome of biofluids in perfect health conditions, far from concluded for humans, have barely begun on horses.

Objectives

The present work attempts, for the first time, to give reference quantitative values for the molecules mostly represented in the urine metabolome of horses at rest and under light training, as observable by ¹H-NMR.

Methods

The metabolome of ten trotter horses, four male and six female, ranging from 3 to 8 years of age, has been observed by ¹H-NMR spectroscopy before and after three training sessions.

Results

We could characterize and quantify 54 molecules in trotter horse urine, originated from diet, protein digestion, energy generation or gut-microbial co-metabolism.

Conclusion

We were able to describe how gender, age and exercise affected their concentration, by means of a two steps protocol based on univariate and robust principal component analysis.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A novel bispecific antibody fusion protein co-targeting EGFR and CD47 with enhanced therapeutic index

Objective

To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the “off-target” effects caused by CD47 expression on red blood cells.

Results

The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy.

Conclusions

Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.

     

NMR-based metabolic characterization of chicken tissues and biofluids: a model for avian research

Introduction

Poultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments.

Objectives

As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus).

Methods

Metabolic extractions were performed prior to ¹H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized.

Results

Nearly 80 metabolites were identified. A cross-comparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice.

Conclusion

This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.

     

A metabolomics guided exploration of marine natural product chemical space

Introduction

Natural products from culture collections have enormous impact in advancing discovery programs for metabolites of biotechnological importance. These discovery efforts rely on the metabolomic characterization of strain collections.

Objective

Many emerging approaches compare metabolomic profiles of such collections, but few enable the analysis and prioritization of thousands of samples from diverse organisms while delivering chemistry specific read outs.

Method

In this work we utilize untargeted LC–MS/MS based metabolomics together with molecular networking to inventory the chemistries associated with 1000 marine microorganisms.

Result

This approach annotated 76 molecular families (a spectral match rate of 28 %), including clinically and biotechnologically important molecules such as valinomycin, actinomycin D, and desferrioxamine E. Targeting a molecular family produced primarily by one microorganism led to the isolation and structure elucidation of two new molecules designated maridric acids A and B.

Conclusion

Molecular networking guided exploration of large culture collections allows for rapid dereplication of know molecules and can highlight producers of uniques metabolites. These methods, together with large culture collections and growing databases, allow for data driven strain prioritization with a focus on novel chemistries.

     

High salt and fat intake,inflammation, and risk of cancer

Background

Inflammatory conditions are involved in the pathophysiology of cancer. Recent findings have revealed that excessive salt and fat intake is involved in the development of severe inflammatory reactions.

Methods

literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of high salt and fat intake in the induction of inflammatory reactions and their roles in the etiopathogenesis of cancer.

Results

The results indicate that high salt and fat intake can induce severe inflammatory conditions. However, various inflammatory conditions have been strongly linked to the development of cancer. Hence, high salt and fat intake might be involved in the pathogenesis of cancer progression via putative mechanisms related to inflammatory reactions.

Conclusion

Reducing salt and fat intake may decrease the risk of cancer.

     

Synthesis and physicochemical characterization of the one-carbon carrier 10-formyltetrahydrofolate; a reference standard for metabolomics

Introduction

Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism, where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis.

Objectives

The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling.

Methods

Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of antioxidant was utilized to prepare 10-formyltetrahydrofolate.

Results

Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate have been determined.

Conclusion

This study describes the facile preparation of pure (>90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.

     

Treatment of the Midwestern Endemic Mycoses,Blastomycosis and Histoplasmosis

Purpose of Review

The purpose of this review is to assess the recommended treatment regimens for the major endemic mycoses, histoplasmosis and blastomycosis, which occur in the Midwestern USA and to provide information about the use of newer antifungal agents for these diseases.

Recent Findings

The basic approach to treatment of histoplasmosis and blastomycosis outlined in the IDSA Guidelines is helpful in managing these diseases. However, changes since these guidelines were published provide safer and better tolerated treatment regimens. Prolonged treatment with amphotericin B is rarely required, and lipid formulations of this drug have largely replaced the amphotericin B deoxycholate formulation. Although no clinical trials have been performed and the data are anecdotal, voriconazole and posaconazole are increasingly used in patients who cannot tolerate itraconazole. Voriconazole is especially useful when central nervous system infection is present. Posaconazole tablets provide consistently appropriate serum levels and the drug is well tolerated.

Summary

New azole agents provide alternative therapeutic options for histoplasmosis and blastomycosis.

     

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector

Objective

To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.

Results

The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.

Conclusions

The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

     

Production,purification, characterization,immobilization, and application of Serrapeptase: a review

Background

Serrapeptase is a proteolytic enzyme with many favorable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases. Although Serrapeptase is widely used, there are very few published papers and the information available about the enzyme is very meagre. Hence this review article compiles all the information about this important enzyme Serrapeptase.

Methods

A literature search against various databases and search engines like PubMed, SpringerLink, Scopus etc. was performed.

Results

We gathered and highlight all the published information regarding the molecular aspects, properties, sources, production, purification, detection, optimizing yield, immobilization, clinical studies, pharmacology, interaction studies, formulation, dosage and safety of the enzyme Serrapeptase.

Conclusion

Serrapeptase is used in many clinical studies against various diseases for its anti-inflammatory, fibrinolytic and analgesic effects. There is insufficient data regarding the safety of the enzyme as a health supplement. Data about the antiatherosclerotic activity, safety, tolerability, efficacy and mechanism of action of the Serrapeptase are still required.

     

Autodisplay of an avidin with biotin-binding activity on the surface of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli.

Results

Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA).

Conclusions

Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

     

Generalized logical model based on network topology to capture the dynamical trends of cellular signaling pathways

Background

Cellular responses to extracellular perturbations require signaling pathways to capture and transmit the signals. However, the underlying molecular mechanisms of signal transduction are not yet fully understood, thus detailed and comprehensive models may not be available for all the signaling pathways. In particular, insufficient knowledge of parameters, which is a long-standing hindrance for quantitative kinetic modeling necessitates the use of parameter-free methods for modeling and simulation to capture dynamic properties of signaling pathways.

Results

We present a computational model that is able to simulate the graded responses to degradations, the sigmoidal biological relationships between signaling molecules and the effects of scheduled perturbations to the cells. The simulation results are validated using experimental data of protein phosphorylation, demonstrating that the proposed model is capable of capturing the main trend of protein activities during the process of signal transduction. Compared with existing simulators, our model has better performance on predicting the state transitions of signaling networks.

Conclusion

The proposed simulation tool provides a valuable resource for modeling cellular signaling pathways using a knowledge-based method.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.