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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 -
Reviews in Medical Virology Jul 2024Human T-lymphotropic virus type-1 (HTLV-1) was the first discovered human oncogenic retrovirus, the etiological agent of two serious diseases have been identified as... (Review)
Review
Novel insights into human T-lymphotropic virus type-1 (HTLV-1) pathogenesis-host interactions in the manifestation of HTLV-1-associated myelopathy/tropical spastic paraparesis.
Human T-lymphotropic virus type-1 (HTLV-1) was the first discovered human oncogenic retrovirus, the etiological agent of two serious diseases have been identified as adult T-cell leukaemia/lymphoma malignancy and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a debilitating chronic neuro-myelopathy. Despite more than 40 years of molecular, histopathological and immunological studies on HTLV-1-associated diseases, the virulence and pathogenicity of this virus are yet to be clarified. The reason why the majority of HTLV-1-infected individuals (∼95%) remain asymptomatic carriers is still unclear. The deterioration of the immune system towards oncogenicity and autoimmunity makes HTLV-1 a natural probe for the study of malignancy and neuro-inflammatory diseases. Additionally, its slow worldwide spreading has prompted public health authorities and researchers, as urged by the WHO, to focus on eradicating HTLV-1. In contrast, neither an effective therapy nor a protective vaccine has been introduced. This comprehensive review focused on the most relevant studies of the neuro-inflammatory propensity of HTLV-1-induced HAM/TSP. Such an emphasis on the virus-host interactions in the HAM/TSP pathogenesis will be critically discussed epigenetically. The findings may shed light on future research venues in designing and developing proper HTLV-1 therapeutics.
Topics: Humans; Human T-lymphotropic virus 1; Paraparesis, Tropical Spastic; HTLV-I Infections; Host-Pathogen Interactions; Animals; Host Microbial Interactions
PubMed: 38937135
DOI: 10.1002/rmv.2567 -
In Vivo (Athens, Greece) 2024Gliomas are highly heterogeneous malignancies originating from diverse cell types within the brain. Although their precise etiology is frequently unknown, risk factors,...
BACKGROUND/AIM
Gliomas are highly heterogeneous malignancies originating from diverse cell types within the brain. Although their precise etiology is frequently unknown, risk factors, such as chemical exposure, radiation, and specific uncommon genetic disorders have been identified. Diagnosis typically entails imaging tests, such as magnetic resonance imaging and computed tomography, complemented by a biopsy for confirmation, which may be further validated through genetic testing.
CASE REPORT
Next-generation sequencing technology revealed germline co-deletion deletion of cyclin-dependent kinase inhibitor 2 A and B genes (CDKN2A and CDKN2B) in a patient diagnosed with pleomorphic xanthoastrocytoma based on the tumor's molecular characteristics. Following this result, we performed focused genetic analysis with use of multiplex ligation-dependent probe amplification technology for the mother that revealed the same co-deletion. Moreover, due to the father's neuroendocrine pancreatic cancer, application of the NGS technology detected a pathogenic variant in the BRCA1-interacting helicase 1 (BRIP1) gene. Comprehensive multi-gene testing conducted within the familial context, marked by a varied spectrum of cancer type, revealed a constellation of genetic predispositions.
CONCLUSION
This case study underscores the critical importance of molecular testing for tumor characterization and highlights the pivotal role of genetic testing in facilitating early intervention and screening for at-risk family members. Furthermore, the identification of germline co-deletions in cancer lays the foundation for the development of targeted therapeutic strategies aimed at restoring normal cellular regulation and improving patient management.
Topics: Humans; Cyclin-Dependent Kinase Inhibitor p16; Astrocytoma; Cyclin-Dependent Kinase Inhibitor p15; Germ-Line Mutation; High-Throughput Nucleotide Sequencing; Genetic Predisposition to Disease; Male; Female; Adult; Brain Neoplasms; Pedigree; Magnetic Resonance Imaging; Gene Deletion
PubMed: 38936911
DOI: 10.21873/invivo.13617 -
International Journal of Biological... Jun 2024Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address...
Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address this need, a novel fluorescent aptasensor, assisted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), have been developed for food allergens. In this study, aptamer offers distinctive recognition capabilities in binding specific targets, while CRISPR-associated-12a protein (Cas12a) holds precise cis-cleavage for cutting fluorescent signal probes. Notably, the utilization of Cas12a cis-cleavage activity, rather than trans-cleavage, eliminates the necessity for additional fluorescent probes, thus reducing interference between substances and enhancing sensitivity. Throughout the process, complementary DNA (cDNA) plays a crucial dual role in target recognition conversion and signal presentation, representing a key challenge and innovative aspect of this study. To evaluate the performance of the aptasensor, lysozyme (LYS) is employed as a representative model target of food allergens. Under optimal conditions, the developed aptasensor could achieve an exceptional low limit of detection (LOD) of 6.10 pM with a dynamic detection range of 10 pM-320 pM. The aptasensor demonstrates high selectivity and great recovery rates. This strategy yields promising outcomes, holding the potential to serve as a valuable reference for various food allergens detection.
PubMed: 38936584
DOI: 10.1016/j.ijbiomac.2024.133444 -
Tissue & Cell Jun 2024Lung ischemia/reperfusion injury (LIRI) is a pathological process caused by the deficiency and subsequent reperfusion of oxygen and blood to the lung. Literature reports...
Downregulation of HDAC6 mitigates lung ischemia/reperfusion injury depending on activation of Nrf2/HO-1 signaling pathway and inactivation of ERK/NF-κB signaling pathway.
INTRODUCTION
Lung ischemia/reperfusion injury (LIRI) is a pathological process caused by the deficiency and subsequent reperfusion of oxygen and blood to the lung. Literature reports that the catalytic activity and expression of HDAC6 can be induced in response to IRI. HDAC6 inhibition confers protective effects against a series of IRI and also exerts pulmonary protection against various lung damage. The present study was formulated to investigate the functional role of HDAC6 inhibitor in LIRI and to probe into the intrinsic mechanisms underlying the protective role of HDAC6 inhibitor against LIRI.
METHODS
Lung epithelial cell line MLE-12 cells were subjected to H/R injury to construct in vitro cell culture model of LIRI. For functional experiments, MLE-12 cells were pre-treated with various concentrations of selective HDAC6 inhibitor ACY-1215 (1, 5, 10 μM) to evaluate the biological role of HDAC6 in LIRI. For rescue experiments, MLE-12 cells were pre-treated with Nrf2 inhibitor ML385 (10 μM) or ERK activator LM22B-10 (50 μM) to discuss the molecular mechanisms.
RESULTS
It was verified that HDAC6 inhibition repressed H/R-induced apoptosis, oxidative stress, inflammation and mitochondrial dysfunction of MLE-12 cells. HDAC6 inhibition activated Nrf2/HO-1 signaling pathway and inactivated ERK/NF-κB signaling pathway in MLE-12 cells. The repressing effects of HDAC6 inhibition on H/R-induced apoptosis, oxidative stress, inflammation and mitochondrial dysfunction of MLE-12 cells were partially abolished upon pre-treatment with Nrf2 inhibitor ML385 or ERK activator LM22B-10.
CONCLUSION
HDAC6 inhibition may mitigate H/R-induced lung epithelial cell injury depending on activation of Nrf2/HO-1 signaling pathway and inactivation of ERK/NF-κB signaling pathway.
PubMed: 38936199
DOI: 10.1016/j.tice.2024.102446 -
European Journal of Medicinal Chemistry Jun 2024The serotonin type 6 receptor (5-HTR) displays a strong constitutive activity, suggesting it participates largely in the physiological and pathological processes...
The serotonin type 6 receptor (5-HTR) displays a strong constitutive activity, suggesting it participates largely in the physiological and pathological processes controlled by the receptor. The active states of 5-HTR engage particular signal transduction pathways that lead to different biological responses. In this study, we present the development of 5-HTR neutral antagonists at Gs signaling built upon the 2-phenylpyrrole scaffold. Using molecular dynamics simulations, we outline the relationship between the exposure of the basic center of the molecules and their ability to target the agonist-activated state of the receptor. Our study identifies compound 30 as a potent and selective neutral antagonist at 5-HTR-operated Gs signaling. Furthermore, we demonstrate the cytoprotective effects of 30 and structurally diverse 5-HTR neutral antagonists at Gs signaling in C8-D1A cells and human astrocytes exposed to rotenone. This effect is not observed for 5-HTR agonists or inverse agonists. In light of these findings, we propose compound 30 as a valuable molecular probe to study the biological effects associated with the agonist-activated state of 5-HTR and provide insight into the glioprotective properties of 5-HTR neutral antagonists at Gs signaling.
PubMed: 38936149
DOI: 10.1016/j.ejmech.2024.116615