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Analytical and Bioanalytical Chemistry May 2024Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of...
Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.
Topics: Inclusion Bodies; Protein Refolding; Spectrometry, Fluorescence; Recombinant Proteins; Tryptophan; Escherichia coli; Tyrosine; Fluorescence; Protein Folding
PubMed: 38573344
DOI: 10.1007/s00216-024-05249-1 -
Signal Transduction and Targeted Therapy Apr 2024RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the...
RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC.
Topics: Humans; Autophagy; Colorectal Neoplasms; Family; Phosphoproteins; Proteins; RNA; RNA, Circular; RNA-Binding Proteins
PubMed: 38565536
DOI: 10.1038/s41392-024-01787-3 -
Journal of Dentistry Jun 2024The aim of the present in vitro study was to evaluate the effect of a novel auxiliary geometric device (AGD) on the accuracy of full-arch scans captured with 3 different...
OBJECTIVES
The aim of the present in vitro study was to evaluate the effect of a novel auxiliary geometric device (AGD) on the accuracy of full-arch scans captured with 3 different intraoral scanners (IOS).
METHODS
An edentulous maxillary model with four internal connection implant replicas was scanned using 3 different IOS: iTero Element 5D (ITERO) (Align Technology, Tempe, AZ, USA), Trios 4 (TRIOS) (3Shape A/S, Copenhagen, Denmark), and Carestream 3700 (CS) (Carestream Dental, Atlanta, USA). Thirty-six scans were taken with each IOS, 18 with the AGD in place, and 18 without the AGD. A digital master model was created using an industrial optical scanner (ATOS compact Scan 5M, GOM GmbH, Braunschweig, Germany). The master and IOS models were aligned using the scan bodies as a reference area. A surface comparison was performed, and deviation labels were exported for each scan body to evaluate the linear and angular deviation. Total body, platform and angular deviations were measured.
RESULTS
The use of AGD resulted in a statistically significant increase of angular deviation: 0.87° (SD=0.21) in the AGD group versus 0.64° (SD=0.46) in the no AGD group (p-value=0.005). The difference between the AGD and no AGD groups was not statistically significant for total body and platform deviation values (p-value=0.051 and 0.302 respectively). Using AGD, ITERO showed a statistically significant increase in angular deviation (mean difference=-0.46 µm, p-value=0.002) and a decrease in mean platform deviation (mean difference=63.19 µm, p-value<0.001). No statistically significant differences were found for the other IOS.
CONCLUSIONS
The use of AGD did not add benefit on CS and TRIOS. On ITERO, there was an improvement in platform deviation, that was outweighed by the worsening of the angular deviation.
CLINICAL SIGNIFICANCE
In vitro data suggest that intraoral scans can be successfully used in full-arch cases. The use of AGD has no additional benefit on CS and TRIOS. On ITERO there was an improvement in platform deviation that was outweighed by the worsening of the angular deviation. Translational application to clinical practice deserves further investigation, taking into account patient-related and anatomical variables.
Topics: Humans; Dental Prosthesis, Implant-Supported; Dental Implants; Models, Dental; Maxilla; Computer-Aided Design; Imaging, Three-Dimensional; Jaw, Edentulous; In Vitro Techniques; Dental Arch; Image Processing, Computer-Assisted; Dental Prosthesis Design
PubMed: 38556193
DOI: 10.1016/j.jdent.2024.104979 -
Journal of Alzheimer's Disease Reports 2024The prodromal stage of Lewy body dementia includes a mild cognitive impairment with visual processing and/or attention-executive deficits. A clinical presentation with...
The prodromal stage of Lewy body dementia includes a mild cognitive impairment with visual processing and/or attention-executive deficits. A clinical presentation with progressive visual loss is indeed seldom reported and can be misleading with a posterior cortical atrophy disease. While the neurodegeneration at the occipital cortex can only partially explain the visual disturbances of Lewy body dementia, more recently a retinal dysfunction has been suggested by preliminary optical coherence tomography and autoptic findings. Herein, we present a case of a mild cognitive impairment with Lewy bodies, who presented initially with visual disturbances and signs of both retinal and cortical visual processing dysfunction. A complete neuropsychological, neurophysiological and brain imaging assessment highlighted a prominent ventral visual pathway involvement. This report provides first that the prodromal stage of Lewy body dementia can manifest as a primarily progressive visual loss, second that the involvement of visual pathway, particularly the ventral stream, can be detectable from the retinal to the cortical level.
PubMed: 38549630
DOI: 10.3233/ADR-230176 -
Nature Communications Mar 2024RNA decay is vital for regulating mRNA abundance and gene expression. Existing technologies lack the spatiotemporal precision or transcript specificity to capture the...
RNA decay is vital for regulating mRNA abundance and gene expression. Existing technologies lack the spatiotemporal precision or transcript specificity to capture the stochastic and transient decay process. We devise a general strategy to inducibly recruit protein factors to modulate target RNA metabolism. Specifically, we introduce a Rapid Inducible Decay of RNA (RIDR) technology to degrade target mRNAs within minutes. The fast and synchronous induction enables direct visualization of mRNA decay dynamics in cells. Applying RIDR to endogenous ACTB mRNA reveals rapid formation and dissolution of RNA granules in pre-existing P-bodies. Time-resolved RNA distribution measurements demonstrate rapid RNA decay inside P-bodies, which is further supported by knocking down P-body constituent proteins. Light and oxidative stress modulate P-body behavior, potentially reconciling the contradictory literature about P-body function. This study reveals compartmentalized RNA decay kinetics, establishing RIDR as a pivotal tool for exploring the spatiotemporal RNA metabolism in cells.
Topics: Processing Bodies; RNA, Messenger; Proteins; RNA Stability
PubMed: 38548718
DOI: 10.1038/s41467-024-46943-z -
Behavioral Sciences (Basel, Switzerland) Mar 2024Attentional bias towards threatening information is a crucial factor contributing to the development and persistence of social anxiety. However, the attentional bias...
Attentional bias towards threatening information is a crucial factor contributing to the development and persistence of social anxiety. However, the attentional bias towards threat information and the preferential processing pattern of emotional cues in individuals with social anxiety disorder during integrated facial and physical stimuli processing remain unclear. In this study, we employed a dot-probe paradigm to investigate the attentional bias towards integrated emotions (facial-body) among students with high and low levels of social anxiety (Experiment 1). Experiments 2 and 3 examined the attentional bias of socially anxious individuals when faced with conflicting emotional cues from faces or bodies in relation to integrated emotions. The data revealed that participants both high and low levels of social anxiety participants exhibited accelerated orienting and biased attention towards facial-body emotional processing. When there was inconsistency between emotional cues from faces or bodies and integrated emotions, higher levels of social anxiety were associated with increased vigilance towards threatening faces or bodies. These findings underscore that individuals with social anxiety possess an ability to rapidly capture threatening cues during the processing of facial-body emotional stimuli while also demonstrating a tendency to avoid relying solely on facial cues by compensating through bodily cues for emotion perception.
PubMed: 38540547
DOI: 10.3390/bs14030244 -
Frontiers in Bioscience (Elite Edition) Jan 2024Xrn1 exoribonuclease is the major mRNA degradation enzyme in In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA...
BACKGROUND
Xrn1 exoribonuclease is the major mRNA degradation enzyme in In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown.
METHODS
We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δ cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy.
RESULTS
We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy.
CONCLUSIONS
The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.
Topics: Saccharomyces cerevisiae; Exoribonucleases; Population Growth; RNA, Messenger
PubMed: 38538525
DOI: 10.31083/j.fbe1601001 -
The Journal of Cell Biology Jun 2024Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress...
Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA-RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.
Topics: Adenosine Triphosphatases; Processing Bodies; RNA; Stress Granules; Humans; Cell Line, Tumor; DEAD-box RNA Helicases
PubMed: 38536035
DOI: 10.1083/jcb.202306022 -
Frontiers in Bioengineering and... 2024[This corrects the article DOI: 10.3389/fbioe.2023.1249196.].
[This corrects the article DOI: 10.3389/fbioe.2023.1249196.].
PubMed: 38532877
DOI: 10.3389/fbioe.2024.1392514 -
The Journal of Neuroscience : the... May 2024The visual perception of individuals is thought to be mediated by a network of regions in the occipitotemporal cortex that supports specialized processing of faces,...
The visual perception of individuals is thought to be mediated by a network of regions in the occipitotemporal cortex that supports specialized processing of faces, bodies, and actions. In comparison, we know relatively little about the neural mechanisms that support the perception of multiple individuals and the interactions between them. The present study sought to elucidate the visual processing of social interactions by identifying which regions of the social perception network represent interpersonal synchrony. In an fMRI study with 32 human participants (26 female, 6 male), we used multivoxel pattern analysis to investigate whether activity in face-selective, body-selective, and interaction-sensitive regions across the social perception network supports the decoding of synchronous versus asynchronous head-nodding and head-shaking. Several regions were found to support significant decoding of synchrony/asynchrony, including extrastriate body area (EBA), face-selective and interaction-sensitive mid/posterior right superior temporal sulcus, and occipital face area. We also saw robust cross-classification across actions in the EBA, suggestive of movement-invariant representations of synchrony/asynchrony. Exploratory whole-brain analyses also identified a region of the right fusiform cortex that responded more strongly to synchronous than to asynchronous motion. Critically, perceiving interpersonal synchrony/asynchrony requires the simultaneous extraction and integration of dynamic information from more than one person. Hence, the representation of synchrony/asynchrony cannot be attributed to augmented or additive processing of individual actors. Our findings therefore provide important new evidence that social interactions recruit dedicated visual processing within the social perception network that extends beyond that engaged by the faces and bodies of the constituent individuals.
Topics: Humans; Female; Male; Social Perception; Adult; Magnetic Resonance Imaging; Young Adult; Brain Mapping; Nerve Net; Photic Stimulation; Interpersonal Relations; Facial Recognition; Visual Perception; Brain
PubMed: 38527811
DOI: 10.1523/JNEUROSCI.2009-22.2024