Search Results (1,004)

Conessine is a steroidal alkaloid found in many plants. The pharmacological efficacies of conessine on various ailments, including antiviral effects against Zika, Herpes, and Coronavirus, were reported. However, the effect of conessine on the influenza virus was still unknown. In this study, conessine exhibited a strong inhibitory effect against influenza A virus (IAV) infection. We examined the effect of conessine on IAV using green fluorescent protein (GFP)-expressing Influenza A/PR8/34 and wild-type A/PR8/34. The fluorescence-activated cell sorting, fluorescence microscopy, cytopathic effect analysis, and plaque assay demonstrated that conessine significantly inhibits IAV infection. Consistently, immunofluorescence results showed that conessine strongly reduces the expression of IAV proteins. The time-of-drug-addition assay revealed that conessine could affect the viral attachment and entry into the cells upon IAV infection. Further, conessine eradicated the virus before binding to the cells in the early stage of viral infection. Our results suggest that conessine has strong anti-viral efficacy against IAV infection and could be developed as an anti-influenza viral agent. Full article

Soybean meal (SBM) is extensively used as a predominant protein source in animal feed. However, raw soybean cannot be directly utilized in animal feed, due to the presence of the Kunitz trypsin inhibitor (KTi) and the Bowman–Birk protease inhibitor (BBi). These antinutritional factors inhibit the digestive enzymes in animals, trypsin and chymotrypsin, resulting in poor animal performance. To inactivate the activity of protease inhibitors, SBM is subjected to heat processing, a procedure that can negatively impact the soybean protein quality. Thus, it would be beneficial to develop soybean varieties with little or no trypsin inhibitors. In this study, we report on the creation of experimental soybean lines with significantly reduced levels of Bowman–Birk protease inhibitors. RNA interference (RNAi) technology was employed to generate several transgenic soybean lines. Some of these BBi knockdown soybean lines showed significantly lower amounts of both trypsin and chymotrypsin inhibitor activities. Western blot analysis revealed the complete absence of BBi in selected RNAi-derived lines. RNA sequencing (RNAseq) analysis demonstrated a drastic reduction in the seed-specific expression of BBi genes in the transgenic soybean lines during seed development. Confocal fluorescence immunolabeling studies showed that the accumulation of BBi was drastically diminished in BBi knockdown lines compared to wild-type soybeans. The absence of BBi in the transgenic soybean did not alter the overall protein, oil, and sulfur amino acid content of the seeds compared to wild-type soybeans. The seed protein from the BBi knockdown lines were more rapidly hydrolyzed by trypsin and chymotrypsin compared to the wild type, indicating that the absence of BBi enhances protein digestibility. Our study suggests that these BBi knockdown lines could be a valuable resource in order for plant breeders to incorporate this trait into commercial soybean cultivars, potentially enabling the use of raw soybeans in animal feed. Full article

The misfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non–micellar anionic DMPG (1,2–dimyristoyl–sn–glycero–3–phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates alongside large insulin fibrils was observed in insulin incubated with DMPG via TEM, DLS, and NMR as opposed to insulin aggregates generated without lipids. ¹³C magic angle spinning solid–state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic side chains. Furthermore, the soluble intermediates were found to be more cytotoxic than fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications. Full article

Quantum Dots (QDs) are small semiconductor nanoparticles (<10 nm) with strong, relatively stable, and tunable luminescent properties, which are increasingly applied in the sensing and detection of various analytes, including metal ions, biomarkers, explosives, proteins, RNA/DNA fragments, pesticides, drugs, and pollutants. In this review, we critically assess recent developments and advancements in luminescent QD-based sensors from an analytical perspective. We collected, tabulated, and analyzed relevant data reported in 124 peer-reviewed articles. The key analytical figures of merit, including the limit of detection (LOD), excitation and emission wavelengths, and size of the particles were extracted, tabulated, and analyzed with graphical representations. We calculated the geometric mean and median LODs from those tabulated publications. We found the following geometric mean LODs: 38 nM for QD-fluorescent-based sensors, 26 nM for QD-phosphorescent-based sensors, and an impressively low 0.109 pM for QD-chemiluminescent-based sensors, which demonstrate by far the best sensitivity in QD-based detection. Moreover, AI-based sensing methods, including the ATTBeadNet model, optimized principal component analysis(OPCA) model, and Support Vector Machine (SVM)-based system, were reviewed as they enhance the analytical performance of the detection. Despite these advances, there are still challenges that include improvements in recovery values, biocompatibility, stability, and overall performance. This review highlights trends to guide the future design of robust, high-performance, QD-based luminescent sensors. Full article

Grapevine leafroll disease is one of the most devastating diseases in the global viticulture industry. Grapevine leafroll-associated virus 4 is one of the main pathogens causing this disease. In this study, we obtained the complete genome sequences of two Chinese isolates of GLRaV-4 from ‘Baisainie’ and ‘Fantasy Seedless’ grapevines through high-throughput sequencing and overlapping RT-PCR combined with RACE technology. The sequences contain 13,814 and 13,824 nucleotides and code six open reading frames, respectively. Phylogenetic trees based on the coat protein (CP) and heat shock protein 70 (HSP70) genes show that in addition to other GLRaV-4 strains (strains 5, 6, 9, Pr, and Car), the GLRaV-4 strains were divided into two distinct groups. The two isolates obtained in this study were classified into separate branches within GLRaV-4 Group 1. Additionally, we systematically investigated the interactions between the proteins encoded by GLRaV-4 using the yeast two-hybrid and bimolecular fluorescence complementation techniques. We found significant interactions between the GLRaV-4-encoded p23 and HSP70 and CP. This study first reports the complete genomes of two different GLRaV-4 isolates from China and suggests that p23 protein encoded by GLRaV-4 may play an important role in viral pathogenicity due to its interactions with the other two proteins. Full article

Low-cost mini-bioreactor platforms are becoming increasingly important in synthetic biology and biotechnology for the characterization of genetic constructs and their initial scaling-up to large-scale cultures. Key process variables are the specific growth rate of cells and the synthesis rate of reporter proteins associated with transcriptional units of interest. These variables are of paramount importance for the characterization of gene synthetic circuits. In addition, their on-line estimation can be used for real-time monitoring of cells’ metabolic state and gene circuit dynamic performance, thus allowing for on-line decision taking. In this work, we first describe a procedure for the calibration of absorbance and fluorescence measurements, ensuring standardized and comparable units across different experimental setups and measurement devices. Then, we implement an observer-based software sensor that uses the calibrated on-line measurements to simultaneously estimate the specific growth and protein synthesis rates. We implemented the calibration procedure and software sensor in a Chi.Bio mini-bioreactor platform. The experimental results show very good performance and pave the way for the use of mini-bioreactor platforms in the real-time characterization of gene synthetic circuits under dynamically regulated time-varying complex scenarios. Full article

Orthohantaviruses are tri-segmented negative-sense RNA viruses that can cause severe pathologies in humans. Currently, limited information exists on the molecular interactions driving orthohantavirus assembly in infected cells. Specifically, it is not clear how its glycoproteins (i.e., Gn and Gc) interact with other viral or host molecules. In this study, we use one- and two-color Number and Brightness fluorescence microscopy approaches to quantitatively characterize the interactions between orthohantavirus glycoproteins and the nucleoprotein in transfected cells. Our results indicate that orthohantavirus nucleoprotein homo-interactions are strongly affected by the host environment. Furthermore, we report evidence of Gc–nucleoprotein interactions, based on (i) the high fluorescence cross-correlation between these two proteins and (ii) the increased Gc-Gc interactions observed in the presence of nucleoprotein. Finally, experiments on a Gc deletion mutant suggest that the observed protein–protein interactions are mediated by the cytoplasmic tail of Gc. In conclusion, this study provides new insights into the role of the interactions between orthohantavirus glycoproteins and nucleoprotein in the context of viral assembly. Full article

The APETALA2/ethylene response factor (AP2/ERF) is a class of plant-specific transcription factors, among which the dehydration-responsive element-binding protein (DREB) subfamily has been widely reported to enhance plant resistance to abiotic stresses. A high-temperature-related gene, Apium graveolens DREBA6b (AgDREBA6b; accession number: OR727346), was previously cloned from a heat-tolerant celery variety. In this study, we transformed this gene into Arabidopsis thaliana using an Agrobacterium rhizogenes-mediated method to explore its function. The results showed that overexpressing AgDREBA6b in Arabidopsis thaliana significantly improved plant growth under high-temperature stress (38 °C) compared to the dreb mutant and wild-type (WT) plants. The anatomical structure of the leaves revealed that the number and degree of stomatal openings in the overexpressed plants were significantly higher than those in the WT and dreb plants, suggesting that AgDREBA6b enhances stomatal opening. Additionally, the chlorophyll content, chlorophyll fluorescence properties, proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were higher in the transgenic plants, indicating better stress tolerance. qPCR analysis showed that four heat tolerance-related genes (AtHSP98.7, AtHSP70-1, AtAPX1, and AtGOLS1) were upregulated in the transgenic plants, with higher expression levels than in WT and mutant plants. This study provides valuable genetic resources for understanding the molecular mechanisms of celery’s heat tolerance and offers insights for breeding heat-tolerant celery varieties. Full article

Cardioprotection against ischemia is achieved using openers of mitochondrial ATP-sensitive K⁺ (mitoKATP) channels such as diazoxide (DZX), leading to pharmacological preconditioning (PPC). We previously reported that PPC decreases the abundance of ventricular Cav1.2 channels, but PPC’s effects on other channels remain largely unexplored. In this study, we hypothesized that DZX regulates the expression of hyperpolarization-activated cyclic nucleotide potassium channel 4 (HCN4) channels in sinoatrial node cells (SANCs), the specialized cardiomyocytes that generate the heartbeat. DZX increased the heart rate in intact adult rats. Patch-clamp experiments revealed an increase in the magnitude of ionic currents through HCN4 channels, which was abolished by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the selective mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot assays showed that DZX increased HCN4 channel expression at the mRNA and protein levels. Immunofluorescence analyses revealed that PPC increased HCN4 fluorescence, which was abolished by NAC. DZX increased nuclear translocation of c-Fos and decreased protein abundance of RE1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), suggesting the involvement of these factors. Our results suggest that PPC increases the heart rate by upregulating HCN4 channel expression through a mechanism involving c-Fos, REST, and ROS. Full article

Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell communication. However, the way in which the genome signature regulates EV cargo has not been well studied. This study aimed to understand how genetics impact EV cargo loading. EVs were isolated from vector copy number cells with a fluorescent reporter (GFP) with varying inserted transgene copies and from NIST SRM 2373 cells (MDA-MB-231, MDA-MB-453, SK-BR-3, and BT-474), which contain varying copies of the HER2 gene. Spectradyne nCS1 was utilized to count EVs and measure size distribution. Imaging Flow Cytometry was used to analyze the surface protein content of single EVs and for total EV counts. The RNA content of the EVs was measured using ddPCR. Our results from stable reporter cell lines and breast cancer cell lines suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of a reporter gene (GFP) or a naturally occurring gene (HER2) from breast cancer cells correlated with increasing EV counts positive for the protein cargo compared to total EV counts until a copy threshold was reached. This study has broad implications for understanding EV biology in the context of cancer biology, diagnostics, EV biology/manufacturing, and therapeutic delivery. Full article

Melanoma is the most common type of skin cancer. Melanomas are well known for their ability to metastasize to other organs, including the lungs, liver, brain, and bones. The ability of melanoma cells to switch among different phenotypes is a key mechanism that underscores their metastatic potential. The objective of this work is to report here on the effect of calcium sulfide (CaS) dispersions in melanoma cells. Melanomas with the epithelial- and mesenchymal-like phenotypes were observed during cell culture preparation. The dose-dependent viability was explored up to slightly less than 3% per volume of cell culture. The dispersion reduced the relative percentage of melanomas with the epithelial- and mesenchymal-like phenotypes to (57 ± 5) and (55 ± 5)%, respectively, at 24 h post treatment. In contrast, the viability of normal fibroblasts treated with the dispersion or melanoma cells treated with the reactants used to prepare the dispersion remained nearly constant, with a value range of (100.0 ± 0.2)% for the control and (97 ± 4)% and (93 ± 2)% for doses as high as 2 and 3% per volume of cell culture, respectively. Fluorescence imaging measurements were consistent with the release of cytochrome c from the mitochondria and its translocation to the cell nuclei. The average expression of caspases 3 and 9 was found to be 3 and 1.4 times higher than in the corresponding melanoma control, respectively, which was consistent with intrinsic apoptosis. The response of vinculin expression was slightly different in both cell phenotypes. Vinculin was found to delocalize in the cytoplasm of treated mesenchymal melanoma cells, with a slightly higher concentration at the end of the actin fibers. A statistically significant increase (p < 0.0001) in the number of focal adhesion points (FAP) at the edge of the cell membrane–external cellular matrix (ECM) interphase was observed in post-treated melanoma that exhibited the epithelial-like phenotype. The changes in vinculin expression and FAP and the reduced viability of the melanomas were consistent with regulation of proteins associated with programmed cell death. It is thus proposed that the sulfides produced from the reactions of the nanoclusters in the acidic environment facilitate the regulation of proteins required to initiate apoptosis, although other processes may also be involved. We conclude that CaS may be an adequate chemical to selectively reduce melanoma viability with little effect on benign fibroblasts. Full article

Human respiratory syncytial virus (RSV) causes acute respiratory illness, attributing to deaths among young children and older adults worldwide. RSV neutralization assay is an important tool to measure RSV neutralization antibody that can prevent infection and severe complication of RSV. Conventional RSV neutralization assays have some limitations of speed and cost, especially for expensive kits, reagents or instruments required for detection. To solve this problem, this paper describes an improved simple and economical RSV neutralization assay protocol using recombinant RSV (rRSV) expressing reporter fluorescent protein to measure RSV growth as reporter activity with plate reader. The condition of 3 days culture demonstrated sufficient fluorescent activity even when small amounts of rRSV were used to inoculate Hep-2 cells. In addition, white 96-well cell culture plate showed better stable reporter activities than black plate. Furthermore, RSV neutralization assay protocol using rRSV-reporter fluorescent protein demonstrated similar signal detection capacity for RSV antibody titer detection compared to other protocols, such as rRSV-Luciferase and ELISA assay. The new RSV neutralization assay protocol can be applied to RSV antibody titration of numerous samples necessary for RSV surveillance or antiviral testing. Full article

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G₁-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers. Full article

Enteroviruses are a group of highly prevalent human pathogens responsible for a wide range of illnesses, ranging from common cold symptoms to life-threatening diseases. A deep understanding of enterovirus biology, evolution, and host interaction is required for the development of effective vaccines and antivirals. Recombinant reporter viruses encoding luminescent or fluorescent proteins have been developed to facilitate such investigation. In this work, using coxsackievirus B3 as our model, we analyze how the insertion of fluorescent reporter genes at three distinct sites within the viral polyprotein affects viral fitness, identifying the most tolerant site for reporter insertion. We then describe a set of experimental workflows for measuring viral fitness, sera neutralization, antiviral efficacy, and recombination using fluorescent reporter viruses. The high homology between different enteroviruses suggests these assays can be readily adapted to study additional members of this medically and economically relevant group of viruses. Full article

CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls. Full article