bioRxiv Science⌕ Search

Biology subjects

Wei, J.

Publications and source records attributed to Wei, J..

At least 163 records · Page 9Linked to original sources

A high-accuracy and high-efficiency digital volume correlation method to characterize in-vivo optic nerve head biomechanics from optical coherence tomography

In-vivo optic nerve head (ONH) biomechanics characterization is emerging as a promising way to study eye physiology and pathology. We propose a high-accuracy and high-efficiency digital volume correlation (DVC) method for the purpose of characterizing the in-vivo ONH deformation from volumes acquired by optical coherence tomography (OCT). Using a combination of synthetic tests and analysis of OCTs from monkey ONHs subjected to acute and chronically elevated intraocular pressure, we demonstrate that our proposed methodology overcomes several challenges for conventional DVC methods. First, it accounts for large ONH rigid body motion in the OCT volumes which could otherwise lead to analysis failure; second, sub-voxel-accuracy displacement can be guaranteed despite high noise and low image contrast of some OCT volumes; third, computational efficiency is greatly improved, such that the memory consumption of our method is substantially lower than with conventional methods; fourth, we introduce a parameter measuring displacements confidence. Test of image noise effects showed that the proposed DVC method had displacement errors smaller than 0.028 voxels with speckle noise and smaller than 0.037 voxels with Gaussian noise; The absolute (relative) strain errors in the three directions were lower than 0.0018 (4%) with speckle noise and than 0.0045 (8%) with Gaussian noise. Compared with conventional DVC methods, the proposed DVC method had substantially improved overall displacement and strain errors under large body motions (lower by up to 70%), with 75% lower computation times, while saving about 30% memory. The study thus demonstrates the potential of the proposed technique to investigate ONH biomechanics.

bioengineering↗

Knockout of circRNAs by base editing back-splice sites of circularized exons

A large number of circular RNAs (circRNAs) are produced from back-splicing of exon(s) of precursor mRNAs and generally co-expressed with their cognate linear RNAs from the same gene loci. Methods for circRNA-specific knockout are lacking, largely due to complete sequence-overlaps between circular and cognate linear RNAs. Here, we report to use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress both circular and linear RNAs expression, which confirms the requirement of canonical splice site signals for back-splice. Importantly, by targeting back-splice sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular, but not linear cognate RNAs. As hundreds of exons were found to be predominantly back-spliced to produce circRNAs, this study provides an efficient method to deplete circRNAs for function study.

bioinformatics↗

New insights into the earlier evolutionary history of epiphytic macrolichens

Lichens are well known as pioneer organisms colonizing bare surfaces such as rocks and therefore have been hypothesized to play a role in the early formation of terrestrial ecosystems. Given the rarity of fossil evidence, our understanding of the evolutionary history of lichen-forming fungi is primarily based on molecular dating approaches. These studies suggest extant clades of macrolichens diversified after the K-Pg boundary. Here we corroborate the mid-Mesozoic fossil Daohugouthallus ciliiferus as an epiphytic macrolichen that predates the K-Pg boundary by 100 Mys. Based on new material and geometric morphometric analysis, we demonstrate that the Jurassic fossil is morphologically most similar to Parmeliaceae, but cannot be placed in Parmeliaceae or other similar family-level clades forming macrolichens as these evolved much later. Consequently, a new family, Daohugouthallaceae, is proposed here to accommodate this fossil, which reveals macrolichens may have been diverse long before the Cenozoic diversification of extant lineages.

paleontology↗

Gastric cancer derived exosomes induce peritoneal mesothelial cell EMT through TGF-β1/Smads pathway to promote peritoneal metastasis

Epithelial-mesenchymal transition (EMT) plays an important role in peritoneal metastasis of Gastric cancer (GC). Tumor exosomes can mediate tumor directed metastasis, and TGF-{beta}1 is an important factor in inducing tumor Epithelial mesenchymal transition. However, it is not clear whether GC derived exosomes can induce peritoneal mesothelial cells through the TGF-{beta}1/ Smads pathway and the effect of injured peritoneal mesothelial cells on the biological characteristics of GC cells. In this study, we demonstrated that GC-derived exosomes can activate the TGF-{beta}1/Smads pathway in peritoneal mesothelial cells and induce the corresponding EMT process, and that the injured peritoneal mesothelial cells can improve the migration and adhesion of GC cells. Taken together, these data further support the critical role of exosomes in the remodeling of the pre-metastatic microenvironment.

cancer biology↗

Amplification-free detection of viral RNA by super resolution imaging-based CRISPR/Cas13a System

RNA detection is crucial for biological research and clinical diagnosis. The current methods include both direct and amplification-based RNA detection. These methods require complicated procedures, suffering from low sensitivity, slow turnaround, and amplification bias. The CRISPR/Cas13a system is a direct RNA detection method via target RNA induced collateral cleavage activity. However, to detect low concentration RNA with CRISPR/Cas13a, target amplification is always required. Herein, we optimize the components of the CRISPR/Cas13a assay to enhance the sensitivity of viral RNA detection which improve the detection limit from 1 pM up to 100 fM. In addition, the integration of CRISPR/Cas13a biosensing and single molecule super resolution imaging is a novel strategy for direct and amplification-free RNA detection. After surface modification, fluorescent RNA reporters are immobilized on the glass coverslip surface and fluorescent signals are captured by total internal reflection fluorescence microscopy (TIRFM), shifting the measurement from spectroscopy to imaging. We quantify the fluorescence signal intensity before and after collateral cleavage of the CRISPR system when viral RNA is present and achieve a detection limit of 10 fM. Therefore, we provide a novel TIRFM-based system to visualize the CRISPR trans-cleavage for direct and robust RNA detection.

bioengineering↗

An oligosaccharide analog exhibited antifungal activity by misleading cell wall organization via targeting PHR transglucosidases

The fungal cell wall is an ideal target for the design of antifungal drugs. In this study we used an analog of cell wall polymer, a highly deacetylated long-chain chitosan oligosaccharide (HCOS), to test its effect against pathogenic Candida strains. Results showed that HCOS was successfully incorporated into the dynamic cell wall organization process and exhibited an apparent antifungal activity against both plankton and mature fungal biofilm, by impairing the cell wall integrity. Unexpectedly, mechanistic studies suggested that HCOS exerts its activity by interfering with family members of PHR {beta}-(1,3)-glucanosyl transferases and affecting the connection and assembly of cell wall polysaccharides. Furthermore, HCOS showed great synergistic activity with different fungicides against Candida cells, especially those in biofilm. These findings indicated HCOS has a great potential as an antifungal drug or drug synergist and proposed a novel antifungal strategy with structure-specific oligosaccharides mimicking cell wall polysaccharide fragments. IMPORTANCEFungal infections have always been a puzzle in clinical medicine. Only a few antifungal drugs are available for medical usage and the widespread use of antifungal drugs increased the incidence of drug resistance. It is an urgent need for the development of novel treatment strategies against fungal infections. In this study, we proposed a novel strategy targeting to fungal cell wall against C. albicans. To our knowledge, it is the first study to show a cell wall polysaccharide fragment analog integrate into and interfere with the fungal cell wall, indicating a novel antifungal strategy using structure-specific oligosaccharides mimicking cell wall polysaccharide fragments.

microbiology↗

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

molecular biology↗

Radiosynthesis and preclinical evaluation of a carbon-11 labeled PDE7 inhibitor for PET neuroimaging

BackgroundDysfunction of cyclic nucleotide phosphodiesterase 7 (PDE7) has been associated with excess intracellular cAMP concentrations, fueling pathogenic processes that are implicated in neurodegenerative disorders. The aim of this study was to develop a suitable PDE7-targeted positron emission tomography (PET) probe that allows non-invasive mapping of PDE7 in the mammalian brain. MethodsBased on a spiro cyclohexane-1,4-quinazolinone scaffold with known inhibitory properties towards PDE7, we designed and synthesized a methoxy analog that was suitable for carbon-11 labeling. Radiosynthesis was conducted with the respective desmethyl precursor using [11C]MeI. The resulting PET probe, codenamed [11C]26, was evaluated by cell uptake studies, ex vivo biodistribution and radiometabolite studies, as well as in vivo PET experiments in rodents and nonhuman primates (NHP). ResultsTarget compound 26 and the corresponding phenolic precursor were synthesized in 2-3 steps with overall yields of 49.5% and 12.4%, respectively. An inhibitory constant (IC50) of 31 nM towards PDE7 was obtained and no significant interaction with other PDE isoforms were observed. [11C]26 was synthesized in high molar activities (170 - 220 GBq/{micro}mol) with radiochemical yields of 34{+/-}7%. In vitro cell uptake of [11C]26 was 6-7 folds higher in PDE7 overexpressing cells, as compared to the controls, whereas an in vitro specificity of up to 90% was measured. Ex vivo metabolite studies revealed a high fraction of intact parent in the rat brain (98% at 5 min and 75% at 30 min post injection). Considerable brain penetration was further corroborated by ex vivo biodistribution and PET imaging studies - the latter showing heterogenic brain uptake. While marginal specific binding was observed by PET studies in rodents, a moderate, but dose-dependent, blockade was observed in the NHP brain following pretreatment with non-radioactive 26. ConclusionIn this work, we report on the preclinical evaluation of [11C]26 (codename [11C]P7-2104), a PDE7-targeted PET ligand that is based on a spiroquinazolinone scaffold. [11C]26 displayed promising in vitro performance characteristics, a moderate degree of specific binding in PET studies with NHP. Accordingly, [11C]26 will serve as a valuable lead compound for the development of a new arsenal of PDE7-targeted probes with potentially improved in vivo specificity.

neuroscience↗

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. One-Sentence SummaryComplementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2.

immunology↗

Dynamic urine proteome changes in a rat model of simvastatin-induced skeletal muscle injury.

BackgroundStatin-associated muscle symptoms (SAMS) are the main side effects of statins. Currently, there are no effective biomarkers for accurate clinical diagnosis. Urine is not subject to homeostatic control and therefore accumulates early changes, making it an ideal biomarker source. We therefore examined urine proteome changes associated with SAMS in an animal model. MethodsHere, we established a SAMS rat model by intragastric intubation with simvastatin (80 mg/kg). Biochemical analyses and hematoxylin and eosin (H&E) staining were used to evaluate the degree of muscle injury. The urine proteome on days 3, 6, 9 and 14 was profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with the data-independent acquisition (DIA) method. ResultsDifferential proteins on day 14 of SAMS were mainly associated with glycolysis/gluconeogenesis, pyruvate metabolism, metabolism of reactive oxygen species and apoptosis, all of which were reported to be associated with the pathological mechanism of SAMS. Among the 14 differentially expressed proteins on day 3, FIBG, OSTP and CRP were associated with muscle damage, while EHD1, CUBN and FINC were associated with the pathogenic mechanisms of SAMS. MYG and PRVA increased dramatically compared with CK elevation in serum on day 14 of SAMS. ConclusionsOur preliminary results indicated that the urine proteome can reflect early changes in the SAMS rat model, providing the potential for monitoring drug side effects in future clinical research.

biochemistry↗

Extraction, purification and anti-TMV effects of α (β)-2,7,11-cembratriene-4,6-diol from tobacco leaves

BackgroundAcetone ethanol extracts from tobacco leaves have antiviral activity against TMV, but the antiviral effects of their specialized metabolites have not been systematically studied yet, especially the underlying mechanism is still unexplored. ResultsThe tobacco cembranoids ({beta})-2,7,11-cembratriene-4,6-diol (({beta})-CBD) were extracted and purified with an effective and green protocol including innovatively added 5% phosphoric acid for elution, one time silica gel chromatographic column separation and impurity removal and further HPLC purification. The results of antiviral activities against tobacco mosaic virus (TMV) with the local lesion counting method showed that ({beta})-CBD have in vivo higher protective effects of 73.2% and 71.6%, at 75.0 M, respectively, than control agent Ningnanmycin (53.1%). Notably, The results of ELISA and and TMV-GFP fluorescent optical imaging assay indicated a obviously reduced viral protein and weaker GFP fluorescence signal and smaller infection area, which confirmed their anti-TMV activities at protein level. Furthermore, the enhanced production of SA and JA and the significantly increased transcription of of JA signaling pathway (COI1 and PDF1.2) and SA signaling pathway genes (PR1, NPR1 and EDS1) in treated plants further conformed that exogenously applied ({beta})-CBD can effectively elicit the tobacco plant immunity against TMV. ConclusionsThe ({beta})-CBD mainly stimulates disease resistance of tobacco plants to resist TMV and it can be used as bioagents to control TMV in the future.

plant biology↗

No observable guide-RNA-independent off-target mutation induced by prime editor

Prime editor (PE) has been recently developed to induce efficient and precise ontarget editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

biochemistry↗

High Gamma and Beta Temporal Interference Stimulation in the Human Motor Cortex Improves Motor Functions

BackgroundTemporal interference (TI) stimulation is a new technique of noninvasive brain stimulation. Envelope-modulated waveforms with two high-frequency carriers can activate neurons in target brain regions without stimulating the overlying cortex, which has been validated in mouse brains. However, whether TI stimulation can work on the human brain has not been elucidate. ObjectiveTo assess the effectiveness and safety aspect of the envelope-modulated waveform of TI stimulation on human primary motor cortex (M1). MethodsParticipants attended three sessions of 30-min TI stimulation at 2 mA during a random reaction time task (RRTT) or a serial reaction time task (SRTT). Motor cortex excitability was measured before and after TI stimulation. ResultsIn the RRTT experiment, only 70 Hz TI stimulation had a promoting effect on the reaction time (RT) performance and excitability of the motor cortex compared to sham stimulation. Meanwhile, compared with the sham condition, only 20 Hz TI stimulation significantly facilitated motor learning in the SRTT experiment, which was significantly positively correlated with the increase in motor evoked potential. ConclusionThese results indicate that the envelope-modulated waveform of TI stimulation has a significant promoting effect on human motor functions, experimentally suggesting the effectiveness of TI stimulation in humans for the first time and pave the way for further explorations.

neuroscience↗

Bacterial Rdl2 dsRNA increased the insecticidal activity of GABAR blockers and allosteric modulators against Plutella xylostella

The utilization of RNA interference (RNAi) for pest management has garnered global interest. The bioassay results suggested the knockout of PxRdl2 significantly increased the insecticidal activities of the{gamma} -aminobutyric acid receptor (GABAR) targeting compounds (Fipronil, two pyrazoloquinazolines and two isoxazolines), thereby presenting a viable target gene for RNAi-mediated pest control. Consequently, we suggest enhancing the insecticidal activities of GABAR-targeting compounds by knockdown the transcript level of PxRdl2. Furthermore, PxRdl2 dsRNA was expressed in HT115 Escherichia coli to reduce costs and protect dsRNA against degradation. In comparison to in vitro synthesized dsRNA, the recombinant bacteria (ds-B) exhibited superior interference efficiencies and greater stability when exposed to UV irradiation. Collectively, our results provide a new strategy of insecticide spray which combined synergistically with insecticidal activities by suppressing PxRdl2 using ds-B, and may be beneficial for reducing the usage of insecticide and slowing pest resistance.

pharmacology and toxicology↗

Combinatorial engineering of bulk-assembled monodisperse coacervate droplets towards logically integrated protocells

Bottom-up synthetic biology seeks to construct artificial cells from simple building blocks for exploring origin and principles of cellular life and material design. Although cellular life may have emerged spontaneously, programmable integration of building blocks into size, membrane property-controlled compartments (liposome or coacervate) towards cellular organization, without using specialized devices, has proven difficult. Here, we report a self-templated route to monodisperse complex droplets in bulk solution from coacervate-liposome synergy, with nanoliposome controlling coacervate size and coacervate templating on-surface nanoliposome fusion. Nanoliposome-coated monodisperse coacervates are self-assembled within 30 seconds, which are sealed by fusing nanoliposomes into size-controlled giant unilamellar vesicles (GUVs), with model building blocks combinatorially integrated into droplets. Defect-free membrane is established on coacervate, which render these complex GUVs surviving at extreme osmotic, salty and pH conditions (4 M NaCl, 100 mM HCl, 1 M NaOH), while providing homeostasis for enzymatic reactions, reminiscent of extremophiles.

synthetic biology↗

Process Development and Scale-up Optimization of the SARS-CoV-2 Receptor Binding Domain-Based Vaccine Candidate, RBD219-N1C1

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel(R) has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMP) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1 and 5 L scale in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.

immunology↗

Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition

SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

microbiology↗

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

immunology↗