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Microbial Cell Factories Mar 2024Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein...
BACKGROUND
Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPON technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPON tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions.
RESULTS
In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPON-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆G). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆G tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPON tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPON tag increased by 6 kcal/mol. Surprisingly, this new T7AC variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions.
CONCLUSIONS
This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔG), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔG. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPON tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7AC tag in biopharmaceutical industry using the CASPON platform process.
Topics: RNA, Messenger; 5' Untranslated Regions; Escherichia coli; Codon; Recombinant Proteins; Carbon; Recombinant Fusion Proteins
PubMed: 38509572
DOI: 10.1186/s12934-024-02350-z -
Schizophrenia Research. Cognition Jun 2024Deficits in facial identity recognition and its association with poor social functioning are well documented in schizophrenia, but none of these studies have assessed...
Deficits in facial identity recognition and its association with poor social functioning are well documented in schizophrenia, but none of these studies have assessed the role of the body in these processes. Recent research in healthy populations shows that the body is also an important source of information in identity recognition, and the current study aimed to thoroughly examine identity recognition from both faces and bodies in schizophrenia. Sixty-five individuals with schizophrenia and forty-nine healthy controls completed three conditions of an identity matching task in which they attempted to match unidentified persons in unedited photos of faces and bodies, edited photos showing faces only, or edited photos showing bodies only. Results revealed global deficits in identity recognition in individuals with schizophrenia (η = 0.068), but both groups showed better recognition from bodies alone as compared to faces alone (η = 0.573), suggesting that the ability to extract useful information from bodies when identifying persons may remain partially preserved in schizophrenia. Further research is necessary to understand the relationship between face/body processing, identity recognition, and functional outcomes in individuals with schizophrenia-spectrum disorders.
PubMed: 38486791
DOI: 10.1016/j.scog.2024.100307 -
Scientific Reports Mar 2024Axonal terminals of the small ventral lateral neurons (sLNvs), the circadian clock neurons of Drosophila, show daily changes in their arborization complexity, with many...
Axonal terminals of the small ventral lateral neurons (sLNvs), the circadian clock neurons of Drosophila, show daily changes in their arborization complexity, with many branches in the morning and their shrinkage during the night. This complex phenomenon is precisely regulated by several mechanisms. In the present study we describe that one of them is autophagy, a self-degradative process, also involved in changes of cell membrane size and shape. Our results showed that autophagosome formation and processing in PDF-expressing neurons (both sLNv and lLNv) are rhythmic and they have different patterns in the cell bodies and terminals. These rhythmic changes in the autophagy activity seem to be important for neuronal plasticity. We found that autophagosome cargos are different during the day and night, and more proteins involved in membrane remodeling are present in autophagosomes in the morning. In addition, we described for the first time that Atg8-positive vesicles are also present outside the sLNv terminals, which suggests that secretory autophagy might be involved in regulating the clock signaling network. Our data indicate that rhythmic autophagy in clock neurons affect the pacemaker function, through remodeling of terminal membrane and secretion of specific proteins from sLNvs.
Topics: Animals; Drosophila melanogaster; Animals, Genetically Modified; Drosophila Proteins; Circadian Rhythm; Drosophila; Neurons; Circadian Clocks; Autophagy
PubMed: 38480808
DOI: 10.1038/s41598-024-56649-3 -
Updates in Surgery Jun 2024Artificial intelligence is transforming healthcare. Artificial intelligence can improve patient care by analyzing large amounts of data to help make more informed... (Review)
Review
Artificial intelligence is transforming healthcare. Artificial intelligence can improve patient care by analyzing large amounts of data to help make more informed decisions regarding treatments and enhance medical research through analyzing and interpreting data from clinical trials and research projects to identify subtle but meaningful trends beyond ordinary perception. Artificial intelligence refers to the simulation of human intelligence in computers, where systems of artificial intelligence can perform tasks that require human-like intelligence like speech recognition, visual perception, pattern-recognition, decision-making, and language processing. Artificial intelligence has several subdivisions, including machine learning, natural language processing, computer vision, and robotics. By automating specific routine tasks, artificial intelligence can improve healthcare efficiency. By leveraging machine learning algorithms, the systems of artificial intelligence can offer new opportunities for enhancing both the efficiency and effectiveness of surgical procedures, particularly regarding training of minimally invasive surgery. As artificial intelligence continues to advance, it is likely to play an increasingly significant role in the field of surgical learning. Physicians have assisted to a spreading role of artificial intelligence in the last decade. This involved different medical specialties such as ophthalmology, cardiology, urology, but also abdominal surgery. In addition to improvements in diagnosis, ascertainment of efficacy of treatment and autonomous actions, artificial intelligence has the potential to improve surgeons' ability to better decide if acute surgery is indicated or not. The role of artificial intelligence in the emergency departments has also been investigated. We considered one of the most common condition the emergency surgeons have to face, acute appendicitis, to assess the state of the art of artificial intelligence in this frequent acute disease. The role of artificial intelligence in diagnosis and treatment of acute appendicitis will be discussed in this narrative review.
Topics: Humans; Appendicitis; Artificial Intelligence; Acute Disease; Machine Learning; Appendectomy
PubMed: 38472633
DOI: 10.1007/s13304-024-01801-x -
Scientific Reports Mar 2024mGluR2 receptors are widely expressed in limbic brain regions associated with memory, including the hippocampal formation, retrosplenial and frontal cortices, as well as...
mGluR2 receptors are widely expressed in limbic brain regions associated with memory, including the hippocampal formation, retrosplenial and frontal cortices, as well as subcortical regions including the mammillary bodies. mGluR2/3 agonists have been proposed as potential therapeutics for neurological and psychiatric disorders, however, there is still little known about the role of these receptors in cognitive processes, including memory consolidation. To address this, we assessed the effect of the mGluR2/3 agonist, eglumetad, on spatial memory consolidation in both mice and rats. Using the novel place preference paradigm, we found that post-sample injections of eglumetad impaired subsequent spatial discrimination when tested 6 h later. Using the immediate early gene c-fos as a marker of neural activity, we showed that eglumetad injections reduced activity in a network of limbic brain regions including the hippocampus and mammillary bodies. To determine whether the systemic effects could be replicated with more targeted manipulations, we performed post-sample infusions of the mGluR2/3 agonist 2R,4R-APDC into the mammillary bodies. This impaired novelty discrimination on a place preference task and an object-in-place task, again highlighting the role of mGluR2/3 transmission in memory consolidation and demonstrating the crucial involvement of the mammillary bodies in post-encoding processing of spatial information.
Topics: Humans; Rats; Mice; Animals; Spatial Memory; Mammillary Bodies; Bridged Bicyclo Compounds; Brain; Hippocampus
PubMed: 38472268
DOI: 10.1038/s41598-024-56015-3 -
Nanomaterials (Basel, Switzerland) Mar 2024The release of organic contaminants has grown to be a major environmental concern and a threat to the ecology of water bodies. Persulfate-based Advanced Oxidation... (Review)
Review
The release of organic contaminants has grown to be a major environmental concern and a threat to the ecology of water bodies. Persulfate-based Advanced Oxidation Technology (PAOT) is effective at eliminating hazardous pollutants and has an extensive spectrum of applications. Iron-based metal-organic frameworks (Fe-MOFs) and their derivatives have exhibited great advantages in activating persulfate for wastewater treatment. In this article, we provide a comprehensive review of recent research progress on the significant potential of Fe-MOFs for removing antibiotics, organic dyes, phenols, and other contaminants from aqueous environments. Firstly, multiple approaches for preparing Fe-MOFs, including the MIL and ZIF series were introduced. Subsequently, removal performance of pollutants such as antibiotics of sulfonamides and tetracyclines (TC), organic dyes of rhodamine B (RhB) and acid orange 7 (AO7), phenols of phenol and bisphenol A (BPA) by various Fe-MOFs was compared. Finally, different degradation mechanisms, encompassing free radical degradation pathways and non-free radical degradation pathways were elucidated. This review explores the synthesis methods of Fe-MOFs and their application in removing organic pollutants from water bodies, providing insights for further refining the preparation of Fe-MOFs.
PubMed: 38470802
DOI: 10.3390/nano14050473 -
Plant Disease Mar 2024Morel mushrooms (Morchella spp.) are highly regarded globally for their distinctive texture and savory flavor. In 2022, the cultivation area for morel mushrooms in China...
Morel mushrooms (Morchella spp.) are highly regarded globally for their distinctive texture and savory flavor. In 2022, the cultivation area for morel mushrooms in China reached nearly 20,000 hectares, with predominant cultivars including M. sextelata, M. importuna and M. exima (Bian et al., 2024). In March 2022, however, deformities of friting bodies were observed in M. importna at morel mushroom farms in Huaihua city (28.43°N, 110.47°), China, with an incidence rate ranging from 5% to 10%. The disease symptoms begin with the invasion of the hymenium of morel mushroom by white cotton-like mycelia, ultimately resulting in halted fruiting body growth and the manifestation of anomalous fruiting body morphology. Infected samples were collected from the morel growers. Following sterilization with 75% ethanol of the surrounding tissue of infected samples, the white hyphae from the morel lesions were picked out using a dissecting needle, and incubated onto potato saccharose agar medium supplemented with 60 mg/L streptomycin at 25°C. Studies showed that seven out of nine fungal isolates exhibiting identical morphological features rapidly grew on the same culture medium described above, reaching a length of 75 mm in 4 to 5 days at 25°C. The white and thick hyphal colonies of these isolates gradually filled with brown spore powder. Generally, the conidia of the hyphal colonies were polyblastic with protrusions at the tips, measuring 75 to 165 × 36 to 50 μm (n = 30) in width and length, displaying colors varying from light reddish brown to grayish brown, and possessing one or five septa. To confirm the identity of the pathogen, the region of the internal transcribed spacer region (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II second largest subunit (rpb2) genes of the representative isolate H2 were amplified by PCR (Taguiam, et al. 2021). The generated ITS (OR338304), rpb2 (OR452112) and LSU (OR338334) from the isolate H2 had 98-100% similarity to the Alternaria alternata strains ATCC 6663 and CBS 880.95 in BLASTn analysis. ITS, rpb2 and LSU sequences were assembled using Sequence Matrix, and their homogeneity was assessed with PAUP (Vaidya et al., 2011). Bayesian (MrBayes-3.2.7a) and maximum-likelihood (RAxML1.3.1) methods, utilizing the best fit GTR+G+I model obtained from MrModeltest 2.3, were employed for phylogenetic analysis (Aveskamp et al. 2010). Based on morphological characteristics and phylogenetic analysis, the isolate H2 was identified as A. alternata. In the second year post-disease, disease-free morels, with a height of 3 cm, were cultivated in field greenhouses and used for test. A 15 ml suspension (1 × 106 conidia/ml) was applied to 15 young fruiting bodies and their corresponding substrate soil. The results showed that the reappearance of white cotton-like mycelia and deformed M. importuna fruiting bodies within 7 days post-inoculation with the spore suspension, as opposed to the controls. The isolates (H2-1, H2-2 and H2-3) were reisolated from the infected tissues and identified as A. alternata based on its morphological features and phylogenetic analyses. In this study, a similar investigation was previously conducted on cultivated quinoa (Chenopodium quinoa) in Eastern Denmark (Colque-Little et al., 2023). This study marks the first documentation of A. alternata causing deformities in M. importuna fruiting bodies. These deformities occur under conditions of high-temperature (>22°C) and high humidity (>88%). Our findings provide crucial insights for managing A. alternata in M. importuna cultivation in China.
PubMed: 38468140
DOI: 10.1094/PDIS-10-23-2122-PDN -
BioRxiv : the Preprint Server For... Feb 2024Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component...
Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events. We used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, . We found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies , the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes. The increase in alternative splicing of the gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.
PubMed: 38464190
DOI: 10.1101/2024.02.26.582162 -
Cell Reports Mar 2024Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62...
Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity.
Topics: Humans; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Sequestosome-1 Protein; Processing Bodies; Inflammation; Autophagy
PubMed: 38460129
DOI: 10.1016/j.celrep.2024.113935 -
Frontiers in Artificial Intelligence 2024Mesoscale eddies, which are fast-moving rotating water bodies in the ocean with horizontal scales ranging from 10 km to 100 km and above, are considered to be the...
Mesoscale eddies, which are fast-moving rotating water bodies in the ocean with horizontal scales ranging from 10 km to 100 km and above, are considered to be the weather of the oceans. They are of interest to marine biologists, oceanographers, and geodesists for their impact on water mass, heat, and nutrient transport. Typically, gridded sea level anomaly maps processed from multiple radar altimetry missions are used to detect eddies. However, multi-mission sea level anomaly maps obtained by the operational processors have a lower effective spatiotemporal resolution than their grid spacing and temporal resolution, leading to inaccurate eddy detection. In this study, we investigate the use of higher-resolution along-track sea level anomaly data to infer daily two-dimensional segmentation maps of cyclonic, anticyclonic, or non-eddy areas with greater accuracy than using processed sea level anomaly grid map products. To tackle this challenge, we propose a deep neural network that uses spatiotemporal contextual information within the modality of along-track data. This network is capable of producing a two-dimensional segmentation map from data with varying sparsity. We have developed an architecture called Teddy, which uses a Transformer module to encode and process spatiotemporal information, and a sparsity invariant CNN to infer a two-dimensional segmentation map of classified eddies from the ground tracks of varying sparsity on the considered region. Our results show that Teddy creates two-dimensional maps of classified eddies from along-track data with higher accuracy and timeliness when compared to commonly used methods that work with less accurate preprocessed sea level anomaly grid maps. We train and test our method with a carefully curated and independent dataset, which can be made available upon request.
PubMed: 38455471
DOI: 10.3389/frai.2024.1298283