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STAR Protocols Jun 2024A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for...
A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for determining the change in interferon response by microinjecting plasmids into one-cell-stage zebrafish embryos. We describe steps for comparing the resistance potential to virus infection in wild-type and knockout zebrafish larvae following plasmid microinjection. We then detail how to link the enhanced interferon immunity to the improved resistance in knockout zebrafish larvae by gene-rescue experiments. For complete details on the use and execution of this protocol, please refer to Qu et al..
PubMed: 38941183
DOI: 10.1016/j.xpro.2024.103156 -
MSphere Jun 2024The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast , because homozygous mutants of...
Probing gene function in wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.
UNLABELLED
The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast , because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, and , which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of gene deletion mutants, which demands a thorough examination of recombination events at the target site.
IMPORTANCE
is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in .
PubMed: 38940507
DOI: 10.1128/msphere.00388-24 -
International Journal of Molecular... Aug 2024Naringenin (NAR) is a prominent flavanone that has been recognized for its capacity to promote the osteogenic differentiation of human periodontal ligament stem cells...
Naringenin (NAR) is a prominent flavanone that has been recognized for its capacity to promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The present study aimed to explore how NAR promotes the osteogenic differentiation of hPDLSCs and to assess its efficacy in repairing alveolar bone defects. For this purpose, a protein‑protein interaction network of NAR action was established by mRNA sequencing and network pharmacological analysis. Gene and protein expression levels were evaluated by reverse transcription‑quantitative and western blotting. Alizarin red and alkaline phosphatase staining were also employed to observe the osteogenic capacity of hPDLSCs, and immunofluorescence was used to examine the co‑localization of NAR molecular probes and AKT in cells. The repair of mandibular defects was assessed by micro‑computed tomography (micro‑CT), Masson staining and immunofluorescence. Additionally, computer simulation docking software was utilized to determine the binding affinity of NAR to the target protein, AKT. The results demonstrated that activation of the nitric oxide (NO)‑cyclic guanosine monophosphate (cGMP)‑protein kinase G (PKG) signaling pathway could promote the osteogenic differentiation of hPDLSCs. Inhibition of AKT, endothelial nitric oxide synthase and soluble guanylate cyclase individually attenuated the ability of NAR to promote the osteogenic differentiation of hPDLSCs. Micro‑CT and Masson staining revealed that the NAR gavage group exhibited more new bone formation at the defect site. Immunofluorescence assays confirmed the upregulated expression of Runt‑related transcription factor 2 and osteopontin in the NAR gavage group. In conclusion, the results of the present study suggested that NAR promotes the osteogenic differentiation of hPDLSCs by activating the NO‑cGMP‑PKG signaling pathway through its binding to AKT.
Topics: Humans; Osteogenesis; Flavanones; Proto-Oncogene Proteins c-akt; Signal Transduction; Cell Differentiation; Nitric Oxide; Cyclic GMP-Dependent Protein Kinases; Stem Cells; Cyclic GMP; Animals; Male; Cells, Cultured
PubMed: 38940332
DOI: 10.3892/ijmm.2024.5391 -
Chemical & Biomedical Imaging Jun 2024Epicuticular wax is the outmost layer of plant leaves that protects them from desiccation and penetration of harmful reagents. There is an intense industrial effort in...
Epicuticular wax is the outmost layer of plant leaves that protects them from desiccation and penetration of harmful reagents. There is an intense industrial effort in the development of softening agents, adjuvants, that can adjust the permeability of the wax toward pesticides and, thus, play an important role in sustainable agriculture. However, mechanistic understanding of the structure and dynamic properties within the plant wax, particularly upon the application of adjuvants, is currently lacking. In this work, we demonstrate that fluorescence lifetime imaging microscopy (FLIM) combined with molecular rotors, fluorescent probes sensitive to viscosity, can directly probe the microviscosity of amorphous and crystalline phases of model plant wax layers. Moreover, this approach is able to quantify the changes in viscosity in both phases upon the addition of water and adjuvant solutions on top of the wax. We show that water permeation mostly perturbs the crystalline phase of the wax, while our chosen adjuvant, Plurafac LF431, mainly softens the amorphous phase of the wax. Our technique provides a facile and quantitative way to monitor dynamic properties within plant waxes with diffraction-limited resolution and reveals the effect of organic substances on wax structure and rigidity, crucial for designing next-generation agents to improve agricultural efficiency.
PubMed: 38939873
DOI: 10.1021/cbmi.4c00005 -
Acta Medica Philippina 2024Retinoblastoma is one of the most common intraocular cancers among children usually caused by the loss of retinoblastoma protein function. Despite being a highly...
BACKGROUND AND OBJECTIVE
Retinoblastoma is one of the most common intraocular cancers among children usually caused by the loss of retinoblastoma protein function. Despite being a highly heritable disease, conventional diagnostic and prognostic methods depend on clinical examination, with limited consideration of cancer genetics in the standard of care. , and are commonly explored genes for their utility in liquid biopsies of cancer including lung adenocarcinoma. To date, there are few extensive molecular studies on retinoblastoma in Filipino patients. To this end, the study aimed to describe the copy number of , and in retinoblastoma samples from a Filipino patient and quantitate the respective expression level of these genes.
METHODS
Hematoxylin & Eosin (H&E) staining was utilized to characterize the retinoblastoma tissue while fluorescence in situ hybridization (FISH) using probes specific to , and was performed to determine the copy number of genes in retinoblastoma samples from a Filipino patient (n = 1). The gene expression of , and was quantitated using RT-qPCR.
RESULTS
The H&E staining in the retinoblastoma tissue shows poorly differentiated cells with prominent basophilic nuclei. was approximately 1.5-fold overexpressed in the retinoblastoma tissue with respect to the normal tissue, while MUC1 and KRT19 are only slightly expressed. Multiple intense signals of each probe were localized in the same nuclear areas throughout the retinoblastoma tissue, with high background noise.
CONCLUSION
These findings suggest that is a potential biomarker for the staging and diagnosis of retinoblastoma in Filipino cancer patients. However, further optimization of the hybridization procedures is recommended.
PubMed: 38939426
DOI: 10.47895/amp.vi0.7666 -
Frontiers in Cellular and Infection... 2024The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence...
BACKGROUND
The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence loop-mediated isothermal amplification assay (cyp141-RealAmp) using six oligonucleotide primers derived from cyp141 to detect MTB. A combined number of 213 sputum samples (169 obtained from clinically diagnosed cases of pulmonary TB and 44 from a control group without tuberculosis) underwent Acid-fast bacillus (AFB) smear, culture, Xpert MTB/RIF assays, and cyp141-RealAmp assay.
RESULTS
By targeting MTB cyp141, this technique could detect as low as 10 copies/reaction within 30 min, and it was successfully rejected by other mycobacteria and other bacterial species tested. Of the 169 patients, there was no statistical difference between the detection rate of cyp141-RealAmp (92.90%, 95% CI: 89.03-96.07) and that of Xpert MTB/RIF (94.67%, 95% CI: 91.28-98.06) ( > 0.05), but both were statistically higher than that of culture (65.68%, 95% CI: 58.52-72.84) (< 0.05) and AFB (57.40%, 95% CI: 49.94-64.86) (< 0.05). Both cyp141-RealAmp and Xpert MTB/RIF had a specificity of 100%. Furthermore, a high concordance between cyp141-RealAmp and Xpert MTB/RIF was found ( = 0.89).
CONCLUSION
The cyp141-RealAmp assay was shown to be effective, responsive, and accurate in this study. This method offers a prospective strategy for the speedy and precise detection of MTB.
Topics: Mycobacterium tuberculosis; Humans; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Molecular Diagnostic Techniques; Sputum; Tuberculosis, Pulmonary; DNA Primers; Female; Fluorescence; Adult; Male; Tuberculosis; Middle Aged
PubMed: 38938885
DOI: 10.3389/fcimb.2024.1349063 -
JACS Au Jun 2024The characterization of intrinsically disordered regions (IDRs) in membrane-associated proteins is of crucial importance to elucidate key biochemical processes,...
The characterization of intrinsically disordered regions (IDRs) in membrane-associated proteins is of crucial importance to elucidate key biochemical processes, including cellular signaling, drug targeting, or the role of post-translational modifications. These protein regions pose significant challenges to powerful analytical techniques of molecular structural investigations. We here applied magic angle spinning solid-state nuclear magnetic resonance to quantitatively probe the structural dynamics of IDRs of membrane-bound α-synuclein (αS), a disordered protein whose aggregation is associated with Parkinson's disease (PD). We focused on the mitochondrial binding of αS, an interaction that has functional and pathological relevance in neuronal cells and that is considered crucial for the underlying mechanisms of PD. Transverse and longitudinal N relaxation revealed that the dynamical properties of IDRs of αS bound to the outer mitochondrial membrane (OMM) are different from those of the cytosolic state, thus indicating that regions generally considered not to interact with the membrane are in fact affected by the spatial proximity with the lipid bilayer. Moreover, changes in the composition of OMM that are associated with lipid dyshomeostasis in PD were found to significantly perturb the topology and dynamics of IDRs in the membrane-bound state of αS. Taken together, our data underline the importance of characterizing IDRs in membrane proteins to achieve an accurate understanding of the role that these elusive protein regions play in numerous biochemical processes occurring on cellular surfaces.
PubMed: 38938811
DOI: 10.1021/jacsau.4c00323 -
ACS Nano Jun 2024Currently, specific cancer-responsive fluorogenic probes with activatable imaging and therapeutic functionalities are in great demand in the accurate diagnostics and...
Currently, specific cancer-responsive fluorogenic probes with activatable imaging and therapeutic functionalities are in great demand in the accurate diagnostics and efficient therapy of malignancies. Herein, an all-in-one strategy is presented to realize fluorescence (FL) imaging-guided and synergetic chemodynamic-photodynamic cancer therapy by using a multifunctional alkaline phosphatase (ALP)-response aggregation-induced emission (AIE) probe, TPE-APP. By responding to the abnormal expression levels of an ALP biomarker in cancer cells, the phosphate groups on the AIE probe are selectively hydrolyzed, accompanied by in situ formation of strong emissive AIE aggregates for discriminative cancer cell imaging over normal cells and highly active quinone methide species with robust chemodynamic-photodynamic activities. Consequently, the activated AIE probes can efficiently destroy cancer cell membranes and lead to the death of cancer cells within 30 min. A superior efficacy in cancer cell ablation is demonstrated in vitro and in vivo. The cancer-associated biomarker response-derived discriminative FL imaging and synergistic chemodynamic-photodynamic therapy are expected to provide a promising avenue for precise image-guided cancer therapy.
PubMed: 38938113
DOI: 10.1021/acsnano.4c03879 -
The Journal of Physical Chemistry. B Jun 2024Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with...
Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe -ethyl -(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.
PubMed: 38937939
DOI: 10.1021/acs.jpcb.4c01915 -
Journal of Medicinal Chemistry Jun 2024Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration....
-Adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as Fluorescent Probes to Detect Microglia Activation through the Imaging of Cannabinoid Receptor Subtype 2 (CB2R).
Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent -adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and microglia cells mimicking inflammation states where CB2R are overexpressed.
PubMed: 38937147
DOI: 10.1021/acs.jmedchem.4c00564