Recombinant Molecules Research Articles

Molecular characterization of tomato leaf curl Joydebpur virus and tomato leaf curl New Delhi virus associated with severe leaf curl symptoms of papaya in Bangladesh

A survey of viruses in papaya plants exhibiting leaf curl symptoms in Bangladesh showed the occurrence of three begomoviruses: tomato leaf curl Joydebpur virus (ToLCJoV) and its betasatellite (ToLCJoB), tomato leaf curl New Dehli virus (ToLCNDV), and tomato leaf curl Bangladesh virus (ToLCBV). The full-length genome of ToLCJoV (MT127782) was determined and shown to be closely related to ToLCJoV isolates from tomato in Bangladesh or India and chili in India. ToLCJoV from papaya was a recombinant of a ToLCJoV isolate from mungbean in India or an unknown host and another ToLCJoV isolate from tomato in Bangladesh. ToLCJoV betasatellite from papaya (MT161673) showed the highest identity to ToLCJoB from tomato in Bangladesh and chili in India. ToLCJoB from papaya is a recombinant of a ToLCJoB from chili in India and an unknown ToLCJoB. The ToLCNDV DNA-A (MT161674) and DNA-B (MT161675) from papaya are closely related to ToLCNDV isolates from tomato in Bangladesh. The ToLCNDV-papaya DNA-B was a recombinant molecule. Of the 45 papaya leaf samples with leaf curl symptoms, 29 tested positive for ToLCBV, 10 for ToLCJoV and four for ToLCNDV. Our findings documented multiple begomoviruses in papaya exhibiting leaf curl disease in Bangladesh and suggested several recombination events and a host shift from tomato to papaya.

IL-31 induces antitumor immunity in breast carcinoma

BackgroundImmunomodulatory agents that induce antitumor immunity have great potential for treatment of cancer. We have previously shown that interleukin (IL)-31, a proinflammatory cytokine from the IL-6 family, acts as an...

Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells

BackgroundTelomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887–898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2.ResultsOur results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats.ConclusionIdentifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

Safeguards for Using Viral Vector Systems in Human Gene Therapy: A Resource for Biosafety Professionals Mitigating Risks in Health Care Settings.

Health care workers who work daily with human body fluids and hazardous drugs are among those at the highest risk of occupational exposure to these agents. The Occupational Safety and Health Administration's (OSHA) Bloodborne Pathogens Standard (29 CFR 1910.1030) prescribes safeguards to protect workers against health hazards related to bloodborne pathogens. Similarly, the United States Pharmacopeia General Chapter 800 (USP <800>), a standard first published in February 2016 and implementation required by December 2019, addresses the occupational exposure risks of health care workers at organizations working with hazardous drugs. With emerging technologies in the field of gene therapy, these occupational exposure risks to health care workers now extend beyond those associated with bloodborne pathogens and hazardous drugs and now include recombinant DNA. The fifth edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) and the National Institutes of Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) mostly govern work with biohazardous agents and recombinant DNA in a laboratory research setting. When gene therapy products are utilized in a hospital environment, health care workers have very few resources to identify and reduce the risks associated with product use during and after the administration of treatments. At the Abigail Wexner Research Institute at Nationwide Children's Hospital, a comprehensive gap analysis was executed between the research and health care environment to develop a program for risk mitigation. The BMBL, NIH Guidelines, World Health Organization Biosafety Manual, OSHA Bloodborne Pathogens Standard, and USP <800> were used to develop a framework for the gap analysis process. The standards and guidelines for working with viral vector systems in a research laboratory environment were adapted to develop a program that will mitigate the risks to health care workers involved in the preparation, transportation, and administration of gene therapies as well as subsequent patient care activities. The gap analysis identified significant differences in technical language used in daily operations, work environment, training and education, disinfection practices, and policy development between research and health care settings. These differences informed decisions and helped the organization develop a collaborative framework for risk mitigation when a gene therapy product enters the health care setting. With continuing advances in the field of gene therapy, the oversight structure needs to evolve for the health care setting. To deliver the best outcomes to the patients of these therapies, researchers, Institutional Biosafety Committees, and health care workers need to collaborate on training programs to safeguard the public trust in the use of this technology both in clinical trials and as FDA-approved therapeutics.

Rational Vaccine Design in the Time of COVID-19

As scientists consider SARS-CoV-2 vaccine design, we discuss problems that may be encountered and how to tackle them by what we term “rational vaccine design.” We further discuss approaches to pan-coronavirus vaccines. We draw on experiences from recent research on several viruses including HIV and influenza, as well as coronaviruses.

A synthetic heparan sulphate mimetic for enhancing BMP-2-mediated osteogenesis and bone regeneration

<h3>Background & Aim</h3> The bone morphogenetic proteins (BMPs) are a family of morphogens belonging to the TGF-b superfamily. BMP-2, a potent inducer of osteogenesis <i>in vitro</i> and <i>in vivo</i>, has been subject to extensive research for use in bone repair applications. Previously our lab has studied the effects of heparan sulphate (HS), a highly sulphated linear polysaccharides with a complex and heterogeneous structure, on the bioactivity of BMP-2 <i>in vitro</i> and <i>in vivo</i>. Whilst HS displays great potential for therapeutic applications in regenerative medicine and bone repair, animal-derived HS species are less desirable when compared to recombinant or synthetically-derived molecules. In the present study, a panel of 15 fully synthetic HS mimetics were screened against BMP-2 in an effort to identify potential targets for therapeutic applications in bone repair. <h3>Methods, Results & Conclusion</h3> Of the 15 candidates, a four-armed dendrimer functionalized with oversulphated maltose residues and tetrahedral geometry (termed ‘HS20') displayed the highest affinity toward BMP-2 via SPR completion and greatest enhancement BMP-2 bioactivity <i>in vitro</i>. Interestingly, the monomeric equivalent displayed little ability to bind, stabilize and enhance the bioactivity of BMP-2. Further analysis by fluorescence anisotropy and differential scanning fluorimetry confirmed HS20 is capable of avidly binding BMP-2. Moreover, HS20 was able to enhance association between BMP-2 and BMPRII in a dose-dependent manner. <i>In vitro</i> assays indicated that HS20 is capable of enhancing BMP-2-mediated Smad 1/5/9 phosphorylation, osteogenic gene transcription and alkaline phosphatase activity in murine C2C12 cells. HS20 was also able to enhance calcium deposition in Human MSCs during osteogenic differentiation. Additionally, inhibition assays indicated that the pro-osteogenic effects of HS20 can be inhibited through small molecule-mediated BMPR-I antagonism. Finally, HS20 was able to enhance <i>in vivo</i> bone formation in a critical-sized rabbit tibia defect over scaffold alone. These data suggest that a fully synthetic short-armed tetramer bearing oversulphated maltose residues could function as a HS mimetic in bone regeneration applications. The synthesis of this compound is cost-effective and scalable, giving it great potential for translation of this fully synthetic HS mimetic into a clinical setting for bone regeneration applications.

The development of in vitro potency assay for immune checkpoint regulators

Background & Aim Cancer immunotherapy by aiding the immune system in recognition and destruction of tumor cells by enhancing its ability to react to tumor antigens is now been demonstrated to be effective in many instances. Genomic modifications of multiple gene loci to generate potent effector T cells resistant to multiple inhibitory pathways such as PD-1, CTLA-4 is one of major attractive strategies for cell-based immunotherapy. Both PD-1 and CTLA-4 are type I transmembrane T cell inhibitory molecules that express or being recruited on activated T cells. They interact with their ligands, such as PD-L1 and B7-1 respectively to induce co-inhibitory signals on T cells and suppress T-cell mediated immune response against tumors. Thus, the PD-1: PD-L1/PD-L2 and CTL-4: B7-1/B7-2 interactions are key regulators of the threshold of immune response and a consistent, quantitative in vitro potency assay would benefit the development of immune checkpoint-mediated cancer immunotherapy. Methods, Results & Conclusion Recombinant PD-1, CTLA-4 and their specific ligands were expressed in either HEK293 or CHO cells and purified. Purified molecules' biological activities were initially examined and confirmed using T cell-based proliferation assay where applicable. In this study, we would demonstrate how we used Octet instrument to determine the affinities and kinetics of recombinant PD-1 and CTLA-4 Fc chimera molecules’ bindings to their correspondent ligands, and further identified quantitatively different affinities and kinetics of PD-1 and CTLA-4 when bound to their different ligands. Our results indicate the Octet-based assay can serve as a rapid, label-free, quantitatively suitable in vitro potency analysis with very minimal interference for qualifying and studying immune checkpoint regulators.

Mechanistic Insight into Crossing over during Mouse Meiosis

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.

Development of an Enzyme-Linked Immunosorbent Assay Based on CD150/SLAM for the Detection of Peste des Petits Ruminant Virus.

Peste des petits ruminant (PPR) is an economically important severe viral disease of small ruminants that affects primarily the respiratory and digestive tract. Specific detection of the PPR virus (PPRV) antigen plays an important role in the disease control and eradication program. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant goat signaling lymphocyte activation molecule (SLAM) as the capture ligand was successfully developed for the detection of the PPRV antigen (PPRV SLAM-iELISA). The assay was highly specific for PPRV with no cross-reactions among foot and mouth disease virus, Orf virus, sheep pox virus, and goat pox virus and had a sensitivity with a detection limit of 1.56 × 101 TCID50/reaction (50 μl). Assessment of 136 samples showed that the developed PPRV SLAM-iELISA was well correlated with real-time RT-qPCR assays and commercially available sandwich ELISA for detection of PPRV and showed relative sensitivity and specificity of 93.75 and 100.83%, respectively. These results suggest that the developed PPRV SLAM-iELISA is suitable for specific detection of the PPRV antigen. This study demonstrated for the first time that the goat SLAM, the cellular receptor for PPRV, can be used for the development of a diagnostic method for the detection of PPRV.

Baseline levels of dynamic CD4+ T cell adhesion to MAdCAM-1 correlate with clinical response to vedolizumab treatment in ulcerative colitis: a cohort study

BackgroundWhile the number of therapeutic options for treating inflammatory bowel diseases (IBD) is increasing, evidence for rational treatment decisions is scarce in many cases. In particular, appropriate biomarkers to predict the response to the anti-α4β7 integrin antibody vedolizumab are currently lacking.MethodsWe performed a cohort study with 21 patients suffering from ulcerative colitis (UC), in which first-time treatment with vedolizumab was initiated. CD4+ T cells were isolated from the peripheral blood and dynamic adhesion to recombinant mucosal vascular addressin cell adhesion molecule (MAdCAM-)1 in vitro as well as the effect of vedolizumab on such adhesion in vitro was determined. The expression of α4β1 integrin on peripheral blood CD4+ T cells was quantified by flow cytometry. Electronic patient records were reviewed to determine clinical response to vedolizumab.ResultsDynamic adhesion of peripheral blood CD4+ T cells to MAdCAM-1 and the reduction of adhesion following vedolizumab treatment in vitro were higher and the change in α4β1 expression on CD4+ T cells was different in vedolizumab responders and non-responders. Responders could be identified with high specificity and positive-predictive value.ConclusionsDetermining dynamic adhesion of CD4+ T cells to MAdCAM-1 and the in vitro response to vedolizumab before treatment initiation or dynamic integrin regulation in the early course of treatment seem to be promising tools to predict the clinical response to vedolizumab therapy. Larger prospective studies are warranted.

Production of an antibody Fab fragment using 2A peptide in insect cells

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

Viral replicons as valuable tools for drug discovery

RNA viruses can cause severe diseases such as dengue, Lassa, chikungunya and Ebola. Many of these viruses can only be propagated under high containment levels, necessitating the development of low containment surrogate systems such as subgenomic replicons and minigenome systems. Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication but which do not produce infectious virions. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high-throughput testing of drug candidates that act on viral transcription, translation and replication. This review will explore the history and potential for drug discovery of hepatitis C virus, dengue virus, respiratory syncytial virus, Ebola virus and norovirus replicon and minigenome systems.

Bioengineered microRNAs suppress lung cancer cell metabolism and proliferation through targeting of folate cycle

Lung cancer remains as the leading cause of cancer deaths in the world. Ver recently, we have established a novel technology for the production of bioengineered miRNA agents for the study of cancer biology and new therapies. Using recombinant miRNA molecules produced and folded in living cells, we identified a number of the most effective miRNAs for the inhibition of non‐small cell lung cancer (NSCLC) cell proliferation, among which miR‐22‐3p, miR‐9‐5p and miR‐218‐5p are all predicted to interfere with folate cycle and one‐carbon metabolism. The serine hydroxymethyltransferase‐1 (SHMT‐1), a tumor biomarker and important enzyme in folate biotransformation, was verified as a new target for both miR‐9‐5p and miR‐218‐5p. The methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) was proven to be regulated by miR‐9‐5p directly. Furthermore, both methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase‐2 (MTHFD2) were significantly suppressed by miR‐22‐3p in NSCLC cells. As determined by accurate LC‐MS/MS methods, folate metabolites and amino acid metabolome were altered remarkably by individual miRNAs in human NSCLC cells. Consequently, glycolysis rate and mitochondrial function were significantly inhibited in NSCLC cells treated with these miRNAs, accompanied by cell cycle arrest, ROS accumulation, NADP+/NADPH imbalance and other metabolic dysfunctions. In conclusion, our results demonstrated that bioengineered miR‐22‐3p, miR‐9‐5p, and miR‐218‐5p act on folate cycle to suppress NSCLC cell metabolic capacity and exhibit strong antiproliferation activities. These findings provide new insights into mechanistic actions of tumor suppressive miRNAs and development of novel therapeutic strategies.Support or Funding InformationThis study was supported by National Cancer Institute (grant No. R01CA225958) and National Institute of General Medical Sciences (R01GM113888), National Institutes of Health.

Engineering of Fc Multimers as a Protein Therapy for Autoimmune Disease.

The success of Intravenous Immunoglobulin in treating autoimmune and inflammatory processes such as immune thrombocytopenia purpura and Kawasaki disease has led to renewed interest in developing recombinant molecules capable of recapitulating these therapeutic effects. The anti-inflammatory properties of IVIG are, in part, due to the Fc region of the IgG molecule, which interacts with activating or inhibitory Fcγ receptors (FcγRs), the neonatal Fc Receptor, non-canonical FcRs expressed by immune cells and complement proteins. In most cases, Fc interactions with these cognate receptors are dependent upon avidity—avidity which naturally occurs when polyclonal antibodies recognize unique antigens on a given target. The functional consequences of these avid interactions include antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis, degranulation, direct killing, and/or complement activation—all of which are associated with long-term immunomodulatory effects. Many of these immunologic effects can be recapitulated using recombinant or non-recombinant approaches to induce Fc multimerization, affording the potential to develop a new class of therapeutics. In this review, we discuss the history of tolerance induction by immune complexes that has led to the therapeutic development of artificial Fc bearing immune aggregates and recombinant Fc multimers. The contribution of structure, aggregation and N-glycosylation to human IgG: FcγR interactions and the functional effect(s) of these interactions are reviewed. Understanding the mechanisms by which Fc multimers induce tolerance and attempts to engineer Fc multimers to target specific FcγRs and/or specific effector functions in autoimmune disorders is explored in detail.

Standardized GMP-compliant scalable production of human pancreas organoids

BackgroundOrganoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach.MethodsStarting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis.ResultsThis work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%).ConclusionThis work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline.

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one-pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level -1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors.

Создание стабильной высокопродуктивной платформы на основе клеток CHO, адаптированных к химическому и механическому стрессам

Возрастающий спрос на получение рекомбинантных молекул требует создания не только стабильных, но и высокопроизводительных клеточных линий, пригодных для масштабного производства. В настоящей работе предложен дизайн вектора для эффективной и стабильной экспрессии рекомбинатных моноклональных антител в клетках CHO и схема адаптации линии CHO-S (реципиента) к высокой осмоляльности среды в условиях повышенного содержания хлорида натрия (до 100 мМ), лактата натрия (до 170 мМ) и скорости перемешивания в биореакторе до 240 об/мин. Показана эффективность полученной клеточной платформы на примере экспрессии моноклонального антитела IgG2/4k, позволяющей получать до 2,5 г/л продукта без амплификации гена интереса и оптимизации условий культивирования в режиме fed-batch.

On the cause and consequences of IgE to galactose-α-1,3-galactose: A report from the National Institute of Allergy and Infectious Diseases Workshop on Understanding IgE-Mediated Mammalian Meat Allergy

The mammalian meat allergy known as the "α-Gal syndrome" relates to IgE specific for galactose-α-1,3-galactose (α-Gal), an oligosaccharide that is present in cells and tissues of nonprimate mammals. The recognition of delayed reactions to food derived from mammals in patients with IgE to α-Gal and also the association with tick bites have been increasing worldwide. In 2018, the National Institute of Allergy and Infectious Diseases, Division of Allergy, Immunology and Transplantation, sponsored a workshop on this emerging tick-related disease. International experts from the fields of tick biology, allergy, immunology, infectious disease, and dermatology discussed the current state of our understanding of this emerging medical condition. The participants provided suggestions for specific research priorities and for the development of resources to advance our knowledge of the mechanisms, diagnosis, management, and prevention of this allergic disease. This publication is a summary of the workshop and the panel's recommendations are presented herein.

Exploring Serum Proteins to Stabilize the Conformation of the Precursor Protein of ANP

The atrial natriuretic peptide (ANP) is expressed in the form of a precursor, pre-pro-ANP, and is then processed into pro-ANP, which consists of the pro-peptide and the mature peptide. The biological role of ANP has been extensively investigated, but the role of the pro-peptide region continues to remain unclear. Therefore, to study the role of the pro-peptide region of pro-ANP, recombinant pro-ANP molecules were prepared using an E. coli expression system and the resulting proteins were examined in our laboratory. Secondary structure analyses of the pro-ANP by CD in our previous study indicated that pro-ANP is an intrinsically disordered protein and the formation of an α-helical structure rapidly occurs by the addition of organic solvents, such as ethanol and trifluoroethanol. The results suggest that some currently unknown factors contribute to stabilizing the conformation of pro-ANP in vivo. Therefore, to explore these in vivo factors that stabilize the conformation of pro-ANP, the interactions of human serum albumin (HSA) and lipocalin-type prostaglandin D synthase (L-PGDS), major serum proteins, with pro-ANP were examined. The interactions between pro-ANP and candidate interactor proteins were estimated by pull-down assays, DLS, and CD measurements. The binding experiments were carried out in the presence of various concentrations of HSA. The concentrations of HSA bound to pro-ANP in pull-down assays were determined by densitometric analyses of the SDS-PAGE data. The results indicated that HSA specifically binds to pro-ANP in a concentration-dependent manner, suggesting the existence of specific in vivo interactions between the two proteins. In addition, the results of DLS measurements also indicated that the pro-ANP molecule forms a complex with HSA molecule(s). In addition, a pro-ANP and L-PGDS complexes signal was also detected by DLS measurements.