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International Journal of Molecular... May 2024Vedolizumab (VDZ) is used for treating inflammatory bowel disease (IBD) patients. A study investigating colonic epithelial barrier function ex vivo following VDZ is...
Vedolizumab (VDZ) is used for treating inflammatory bowel disease (IBD) patients. A study investigating colonic epithelial barrier function ex vivo following VDZ is lacking. This work aims to evaluate ex vivo the colonic epithelial barrier function in IBD patients at baseline and during VDZ treatment, and to investigate the relationships between barrier function and clinical parameters. Colonic specimens were obtained from 23 IBD patients before, and at 24 and 52 weeks after VDZ treatment, and from 26 healthy volunteers (HV). Transepithelial electrical resistance (TEER, permeability to ions) and paracellular permeability were measured in Ussing chambers. IBD patients showed increased epithelial permeability to ions (TEER, 13.80 ± 1.04 Ω × cm vs. HV 20.70 ± 1.52 Ω × cm, < 0.001) without changes in paracellular permeability of a 4 kDa probe. VDZ increased TEER (18.09 ± 1.44 Ω × cm, < 0.001) after 52 weeks. A clinical response was observed in 58% and 25% of patients at week 24, and in 62% and 50% at week 52, in ulcerative colitis and Crohn's disease, respectively. Clinical and endoscopic scores were strongly associated with TEER. TEER < 14.65 Ω × cm predicted response to VDZ (OR 11; CI 2-59). VDZ reduces the increased permeability to ions observed in the colonic epithelium of IBD patients before treatment, in parallel to a clinical, histological (inflammatory infiltrate), and endoscopic improvement. A low TEER predicts clinical response to VDZ therapy.
Topics: Humans; Antibodies, Monoclonal, Humanized; Male; Female; Adult; Middle Aged; Permeability; Intestinal Mucosa; Inflammatory Bowel Diseases; Colon; Ions; Gastrointestinal Agents; Electric Impedance; Colitis, Ulcerative; Crohn Disease; Aged
PubMed: 38892004
DOI: 10.3390/ijms25115817 -
International Journal of Molecular... May 2024The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric...
The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric homo-analogues, styryl moieties arranged around a central aromatic core. The interactions with the most common biorelevant targets, ds-DNA and ds-RNA, were studied by a set of spectrophotometric methods (UV-VIS, fluorescence, circular dichroism, thermal denaturation). All studied dyes showed strong light absorption in the 350-420 nm range and strongly Stokes-shifted (+100-160 nm) emission with quantum yields () up to 0.57, whereby the mentioned properties were finely tuned by the type of the terminal cationic substituent and number of styryl components (tetramers being red-shifted in respect to trimers). All studied dyes strongly interacted with ds-DNA and ds-RNA with 1-10 nM affinity, with dye emission being strongly quenched. The tetrameric analogues did not show any particular selectivity between ds-DNA or ds-RNA due to large size and consequent partial, non-selective insertion into DNA/RNA grooves. However, smaller trimeric styryl series showed size-dependent selective stabilization of ds-DNA vs. ds-RNA against thermal denaturation and highly selective or even specific recognition of several particular ds-DNA or ds-RNA structures by induced circular dichroism (ICD) bands. The chiral (ICD) selectivity was controlled by the size of a terminal cationic substituent. All dyes entered efficiently live human cells with negligible cytotoxic activity. Further prospects in the transfer of ICD-based selectivity into fluorescence-chiral methods (FDCD and CPL) is proposed, along with the development of new analogues with red-shifted absorbance properties.
Topics: Circular Dichroism; Humans; DNA; Fluorescent Dyes; RNA, Double-Stranded; Cations; Spectrometry, Fluorescence; Styrenes; Nucleic Acid Denaturation
PubMed: 38891911
DOI: 10.3390/ijms25115724 -
International Journal of Molecular... May 2024The chiral H-BINOL derivatives R- and R- were efficiently synthesized via a Suzuki coupling reaction, and they can be used as novel dialdehyde fluorescent probes for the...
The chiral H-BINOL derivatives R- and R- were efficiently synthesized via a Suzuki coupling reaction, and they can be used as novel dialdehyde fluorescent probes for the enantioselective recognition of R/S-2-amino-1-phenylethanol. In addition, R- is much more effective than R-. Scanning electron microscope images and X-ray analyses show that R- can form supramolecular vesicles through the self-assembly effect of the π-π force and strong hydrogen bonding. As determined via analysis, the fluorescence of the probe was significantly enhanced by mixing a small amount of S-2-amino-1-phenylethanol into R-, with a redshift of 38 nm, whereas no significant fluorescence response was observed in R-2-amino-1-phenylethanol. The enantioselective identification of S-2-amino-1-phenylethanol by the probe R- was further investigated through nuclear magnetic titration and fluorescence kinetic experiments and DFT calculations. The results showed that this mechanism was not only a simple reactive probe but also realized object recognition through an ICT mechanism. As the intramolecular hydrogen bond activated the carbonyl group on the probe R-, the carbonyl carbon atom became positively charged. As a strong nucleophile, the amino group of S-2-amino-1-phenylethanol first transferred the amino electrons to a carbonyl carbocation, resulting in a significantly enhanced fluorescence of the probe R- and a 38 nm redshift. Similarly, S-2-amino-1-phenylethanol alone caused severe damage to the self-assembled vesicle structure of the probe molecule itself due to its spatial structure, which made R- highly enantioselective towards it.
Topics: Stereoisomerism; Amino Alcohols; Hydrogen Bonding; Fluorescent Dyes; Kinetics; Molecular Structure; Models, Molecular; Naphthols
PubMed: 38891794
DOI: 10.3390/ijms25115606 -
International Journal of Molecular... May 2024Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene () can... (Review)
Review
Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene () can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene , and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.
Topics: Humans; Muscular Dystrophies, Limb-Girdle; Dysferlin; Genetic Therapy; Mutation; Oligonucleotides, Antisense; Animals
PubMed: 38891760
DOI: 10.3390/ijms25115572 -
Foods (Basel, Switzerland) Jun 2024The fermentation process of Chinese Baijiu's fermented grains involves the intricate succession and metabolism of microbial communities, collectively shaping the...
Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing.
The fermentation process of Chinese Baijiu's fermented grains involves the intricate succession and metabolism of microbial communities, collectively shaping the Baijiu's quality. Understanding the composition and succession of these living microbial communities within fermented grains is crucial for comprehending fermentation and flavor formation mechanisms. However, conducting high-throughput analysis of living microbial communities within the complex microbial system of fermented grains poses significant challenges. Thus, this study addressed this challenge by devising a high-throughput analysis framework using light-flavor Baijiu as a model. This framework combined propidium monoazide (PMA) pretreatment technology with amplicon sequencing techniques. Optimal PMA treatment parameters, including a concentration of 50 μM and incubation in darkness for 5 min followed by an exposure incubation period of 5 min, were identified. Utilizing this protocol, viable microorganism biomass ranging from 8.71 × 10 to 1.47 × 10 copies/μL was successfully detected in fermented grain samples. Subsequent amplicon sequencing analysis revealed distinct microbial community structures between untreated and PMA-treated groups, with notable differences in relative abundance compositions, particularly in dominant species such as , , , , and , as identified by LEfSe analysis. The results of this study confirmed the efficacy of PMA-amplicon sequencing technology for analyzing living microbial communities in fermented grains and furnished a methodological framework for investigating living microbial communities in diverse traditional fermented foods. This technical framework holds considerable significance for advancing our understanding of the fermentation mechanisms intrinsic to traditional fermented foods.
PubMed: 38891011
DOI: 10.3390/foods13111782 -
Human Genomics Jun 2024In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly...
BACKGROUND
In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted.
RESULTS
We ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure.
CONCLUSION
This study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.
Topics: Humans; Female; Germ-Line Mutation; Breast Neoplasms; Genetic Predisposition to Disease; Colombia; Middle Aged; Adult; BRCA2 Protein; BRCA1 Protein; Exome Sequencing; Aged; Genetic Testing; Ataxia Telangiectasia Mutated Proteins
PubMed: 38890714
DOI: 10.1186/s40246-024-00623-7 -
BMC Cancer Jun 2024Tumor hypoxia is associated with prostate cancer (PCa) treatment resistance and poor prognosis. Pimonidazole (PIMO) is an investigational hypoxia probe used in clinical...
BACKGROUND
Tumor hypoxia is associated with prostate cancer (PCa) treatment resistance and poor prognosis. Pimonidazole (PIMO) is an investigational hypoxia probe used in clinical trials. A better understanding of the clinical significance and molecular alterations underpinning PIMO-labeled tumor hypoxia is needed for future clinical application. Here, we investigated the clinical significance and molecular alterations underpinning PIMO-labeled tumor hypoxia in patients with localized PCa, in order to apply PIMO as a prognostic tool and to identify potential biomarkers for future clinical translation.
METHODS
A total of 39 patients with localized PCa were recruited and administered oral PIMO before undergoing radical prostatectomy (RadP). Immunohistochemical staining for PIMO was performed on 37 prostatectomy specimens with staining patterns evaluated and clinical association analyzed. Whole genome bisulfite sequencing was performed using laser-capture of microdissected specimen sections comparing PIMO positive and negative tumor areas. A hypoxia related methylation molecular signature was generated by integrating the differentially methylated regions with previously established RNA-seq datasets.
RESULTS
Three PIMO staining patterns were distinguished: diffuse, focal, and comedo-like. The comedo-like staining pattern was more commonly associated with adverse pathology. PIMO-defined hypoxia intensity was positively correlated with advanced pathologic stage, tumor invasion, and cribriform and intraductal carcinoma morphology. The generated DNA methylation signature was found to be a robust hypoxia biomarker, which could risk-stratify PCa patients across multiple clinical datasets, as well as be applicable in other cancer types.
CONCLUSIONS
Oral PIMO unveiled clinicopathologic features of disease aggressiveness in localized PCa. The generated DNA methylation signature is a novel and robust hypoxia biomarker that has the potential for future clinical translation.
Topics: Humans; Male; Prostatic Neoplasms; Aged; Middle Aged; Epigenesis, Genetic; Prostatectomy; DNA Methylation; Nitroimidazoles; Tumor Hypoxia; Biomarkers, Tumor; Prognosis; Administration, Oral
PubMed: 38890593
DOI: 10.1186/s12885-024-12505-1 -
Scientific Reports Jun 2024This study aimed to explore naringin's potential to promote the osteogenic differentiation of MC3T3-E1 under oxidative stress. It delved into Nar's connection with the...
This study aimed to explore naringin's potential to promote the osteogenic differentiation of MC3T3-E1 under oxidative stress. It delved into Nar's connection with the Wnt/β-catenin and PI3K/Akt signaling pathways. Initially, 2911 OP-related genes were analyzed, revealing close ties with the PI3K/Akt and Wnt pathways alongside oxidative stress. Nar's potential targets-ESR1, HSP90AA1, and ESR2-were identified through various databases and molecular docking studies confirmed Nar's affinity with ESR1 and HSP90AA1. Experiments established optimal concentrations for Nar and HO. HO at 0.3 mmol/L damaged MC3T3-E1 cells, alleviated by 0.1 µmol/L Nar. Successful establishment of oxidative stress models was confirmed by DCFH-DA probe and NO detection. Nar exhibited the ability to enhance osteogenic differentiation, counteracting oxidative damage. It notably increased osteoblast-related protein expression in MC3T3-E1 cells under oxidative stress. The study found Nar's positive influence on GSK-3β phosphorylation, β-catenin accumulation, and pathway-related protein expression, all critical in promoting osteogenic differentiation. The research concluded that Nar effectively promotes osteogenic differentiation in MC3T3-E1 cells under oxidative stress. It achieved this by activating the Wnt/β-catenin and PI3K/Akt pathways, facilitating GSK-3β phosphorylation, and enhancing β-catenin accumulation, pivotal in osteogenesis.
Topics: Flavanones; Oxidative Stress; Osteogenesis; Animals; Mice; Cell Differentiation; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Wnt Signaling Pathway; beta Catenin; Osteoblasts; Hydrogen Peroxide; Cell Line; Molecular Docking Simulation; Signal Transduction
PubMed: 38890371
DOI: 10.1038/s41598-024-64952-2 -
Protein Science : a Publication of the... Jul 2024Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of...
Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.
Topics: Dependovirus; Capsid Proteins; Genetic Vectors; Genetic Therapy; Capsid; Hydrogen Deuterium Exchange-Mass Spectrometry; Protein Stability; Humans; Protein Conformation; Models, Molecular
PubMed: 38888268
DOI: 10.1002/pro.5074 -
RSC Advances Jun 2024Bovine serum albumin-stabilized Au nanoclusters (BSA-Au NCs) have emerged as promising contenders for imaging agents and highly sensitive fluorescence sensors due to...
Bovine serum albumin-stabilized Au nanoclusters (BSA-Au NCs) have emerged as promising contenders for imaging agents and highly sensitive fluorescence sensors due to their biocompatibility and strong photoluminescence. Optimizing the synthesis conditions of BSA-Au NCs is crucial for enhancing fluorescence imaging and other nanocluster applications. In this study, for the first time, we systematically investigated the effects of BSA concentration and Au on both particle size and optical characteristics of BSA-Au NCs. When the two components achieved a suitable concentration ratio, it was beneficial to form BSA-Au NCs with a high quantum yield (QY = 74.30%) and good fluorescence stability. In contrast, an inappropriate concentration ratio would lead to the formation of gold nanoparticles (Au NPs), and their internal filtration effect (IFE) would attenuate the fluorescence emission of BSA-Au NCs. The BSA-Au NCs were then employed as efficient fluorescence sensors for detecting Hg. Furthermore, the growth mechanism of BSA-Au NCs was elucidated by monitoring fluorescence changes during different incubation times. The BSA-Au NCs with a high quantum yield introduce a novel synthetic concept for sensitive fluorescent probes and expanding versatile applications of BSA-Au NCs in catalysis, chemical sensing and biomedicine.
PubMed: 38887651
DOI: 10.1039/d4ra01140f