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Biotechnology Advances Jun 2024Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently... (Review)
Review
Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.
PubMed: 38950872
DOI: 10.1016/j.biotechadv.2024.108402 -
Molecular and Biochemical Parasitology Jun 2024The study aimed to conduct in vitro biological assessments of hydantoin and thiohydantoin compounds against mature Schistosoma mansoni worms, evaluate their cytotoxic...
The study aimed to conduct in vitro biological assessments of hydantoin and thiohydantoin compounds against mature Schistosoma mansoni worms, evaluate their cytotoxic effects and predict their pharmacokinetic parameters using computational methods. The compounds showed low in vitro cytotoxicity and were not considered hemolytic. Antiparasitic activity against adult S. mansoni worms was tested with all compounds at concentrations ranging from 200 to 6.25μM. Compounds SC01, SC02, and SC03 exhibited low activity. Compounds SC04, SC05, SC06 and SC07 caused 100% mortality within 24h of incubation at a concentration of 100 and 200μM. Thiohydantoin SC04 exhibited the highest activity, resulting in 100% mortality after 24h of incubation at a concentration of 50μM and IC of 28µM. In the ultrastructural analysis (SEM), the compound SC04 (200µM) induced integumentary changes, formation of integumentary blisters, and destruction of tubercles and spicules. Therefore, the SC04 compound shows promise as an antiparasitic against S. mansoni.
PubMed: 38950658
DOI: 10.1016/j.molbiopara.2024.111646 -
Vector Borne and Zoonotic Diseases... Jul 2024Chagas disease or American trypanosomiasis, caused by and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico,...
Chagas disease or American trypanosomiasis, caused by and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Our results re-emphasize dogs' utility as sentinels for in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.
PubMed: 38949980
DOI: 10.1089/vbz.2023.0110 -
Mikrochimica Acta Jun 2024A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for...
A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/μL in a model plasmid containing the malB gene and 3 CFU/μL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.
Topics: Nucleic Acid Amplification Techniques; Escherichia coli; Limit of Detection; Milk; Animals; Molecular Diagnostic Techniques; Microfluidic Analytical Techniques; DNA, Bacterial
PubMed: 38949666
DOI: 10.1007/s00604-024-06509-8 -
Chemistry, An Asian Journal Jul 2024Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered,...
Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.
PubMed: 38949517
DOI: 10.1002/asia.202400608 -
Journal of Visualized Experiments : JoVE Jun 2024Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere....
Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.
Topics: Mycorrhizae; Polymers; Autotrophic Processes; Plant Roots
PubMed: 38949309
DOI: 10.3791/66848 -
Analytical Chemistry Jul 2024Plastic pollution represents a critical threat to soil ecosystems and even humans, as plastics can serve as a habitat for breeding and refuging pathogenic microorganisms...
Plastic pollution represents a critical threat to soil ecosystems and even humans, as plastics can serve as a habitat for breeding and refuging pathogenic microorganisms against stresses. However, evaluating the health risk of plastispheres is difficult due to the lack of risk factors and quantification model. Here, DNA sequencing, single-cell Raman-DO labeling, and transformation assay were used to quantify key risk factors of plastisphere, including pathogen abundance, phenotypic resistance to various stresses (antibiotic and pesticide), and ability to acquire antibiotic resistance genes. A Bayesian network model was newly introduced to integrate these three factors and infer their causal relationships. Using this model, the risk of pathogen in the plastisphere is found to be nearly 3 magnitudes higher than that in free-living state. Furthermore, this model exhibits robustness for risk prediction, even in the absence of one factor. Our framework offers a novel and practical approach to assessing the health risk of plastispheres, contributing to the management of plastic-related threats to human health.
PubMed: 38949233
DOI: 10.1021/acs.analchem.4c01433 -
BioRxiv : the Preprint Server For... Jun 2024The multidrug-resistant, nosocomial pathogen is a major threat to human health. A sensor kinase-response regulator system, BfmS-BfmR, is a potential antimicrobial...
The multidrug-resistant, nosocomial pathogen is a major threat to human health. A sensor kinase-response regulator system, BfmS-BfmR, is a potential antimicrobial target in the bacterium due to its essential role in promoting drug resistance and virulence. Important questions remain, however, about how the system controls resistance and pathogenesis. Although knockout of BfmR is known to alter expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls BfmR is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and reactions using a small phosphodonor to study how phosphorylation affects BfmR function. We show that phosphorylation requires the BfmR active site Asp58 and is essential to gene regulation, antibiotic resistance, and virulence in a sepsis model. Consistent with activation of the protein, phosphorylation induces dimerization and increases its affinity for target DNA. Integrated analysis of the genome-wide binding and transcriptional profiles of BfmR led to several key findings: (1) Phosphorylation dramatically expands the number of genomic sites bound by BfmR, from 4 to >250; (2) BfmR recognizes a direct repeat motif widespread across promoters; (3) BfmR directly regulates >300 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (eg, type IV pilus machinery); (4) The regulator also directly controls a set of non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving resistance and pathogenicity and unravel the extensive gene regulatory network under its control.
PubMed: 38948834
DOI: 10.1101/2024.06.16.599214 -
BioRxiv : the Preprint Server For... Jun 2024Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a...
Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of 'Cascade-TniQ.' Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 'bundle.' Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.
PubMed: 38948825
DOI: 10.1101/2024.06.21.600075 -
BioRxiv : the Preprint Server For... Jun 2024Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These...
UNLABELLED
Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA ) and CypA-depleted (CypA ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA cells but not in CypA cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA cells but not in CypA cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA cells had lower activity compared to those from CypA cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA and CypA cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor.
IMPORTANCE
HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.
PubMed: 38948800
DOI: 10.1101/2024.06.15.599180