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Journal of Visualized Experiments : JoVE Jun 2024Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic...
Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.
Topics: Animals; Neural Stem Cells; Mice; Transfection; Lateral Ventricles; Cytological Techniques
PubMed: 38949388
DOI: 10.3791/66651 -
Small (Weinheim An Der Bergstrasse,... Jul 2024The surface properties of biomaterials interact directly with biological systems, influencing cellular responses, tissue integration, and biocompatibility. Surface...
The surface properties of biomaterials interact directly with biological systems, influencing cellular responses, tissue integration, and biocompatibility. Surface topography plays a critical role in cardiac tissue engineering by affecting electrical conductivity, cardiomyocyte alignment, and contractile function. Current methods for controlling surface properties and topography in cardiac tissue engineering scaffolds are limited, expensive, and lack precision. This study introduces a low-cost, one-step degradation process to create scaffolds with well-defined micro-grooves from multilayered 3D printed poly(lactic acid)/thermoplastic polyurethane composites. The approach provides control over erosion rate and surface morphology, allowing easy tuning of scaffold topographical cues for tissue engineering applications. The findings reported in this study provide a library of easily tuneable scaffold topographical cues. A strong dependence of neonatal rat cardiomyocyte (NRCM) contact guidance with the multilayers' dimension and shape in partially degraded polylactic acid (PLA)/thermoplastic polyurethane (TPU) samples is observed. NRCMs cultured on samples with a layer thickness of 13 ± 2 µm and depth of 4.7 ± 0.2 µm demonstrate the most regular contractions. Hence, the proposed fabrication scheme can be used to produce a new generation of biomaterials with excellent controllability determined by multilayer thickness, printing parameters, and degradation treatment duration.
PubMed: 38949308
DOI: 10.1002/smll.202401902 -
Journal of Visualized Experiments : JoVE Jun 2024The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe...
The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.
Topics: Leptospira; Polymerase Chain Reaction; DNA, Bacterial; Nucleic Acid Hybridization; Water Microbiology
PubMed: 38949304
DOI: 10.3791/65435 -
Journal of Visualized Experiments : JoVE Jun 2024Metal-organic frameworks (MOFs) are materials with potential applications in fields such as gas adsorption and separation, catalysis, and biomedicine. Attempts to...
Metal-organic frameworks (MOFs) are materials with potential applications in fields such as gas adsorption and separation, catalysis, and biomedicine. Attempts to enhance the utility of MOFs have involved the preparation of various composites, including polymer-grafted MOFs. By directly grafting polymers to the external surface of MOFs, issues of incompatibility between polymers and MOFs can be overcome. Polymer brushes grafted from the surface of MOFs can serve to stabilize the MOF while enabling particle assembly into self-assembled metal-organic framework monolayers (SAMMs) via polymer-polymer interactions. Control over the chemical composition and molecular weight of the grafted polymer can allow for tuning of the SAMM characteristics. In this work, instructions are provided on how to immobilize a chain transfer agent (CTA) onto the surface of the MOF UiO-66 (UiO = Universitetet i Oslo). The CTA serves as initiation sites for the growth of polymers. Once polymer chains are grown from the MOF surface, the formation of SAMMs is achieved through self-assembly at an air-water interface. The resulting SAMMs are characterized and shown to be freestanding by scanning electron microscopy imaging. The methods presented in this paper are expected to make the preparation of SAMMs more accessible to the research community and thereby expand their potential use as a MOF-polymer composite.
Topics: Polymers; Organometallic Compounds; Metal-Organic Frameworks; Phthalic Acids
PubMed: 38949297
DOI: 10.3791/66497 -
Inorganic Chemistry Jul 2024The effects of simulated radiolytic degradation of tri-butyl phosphate (TBP) on the chemical speciation of cerium were studied by spectrophotometry and electrochemistry...
The effects of simulated radiolytic degradation of tri-butyl phosphate (TBP) on the chemical speciation of cerium were studied by spectrophotometry and electrochemistry of TBP solutions containing increasing amounts of di--butyl phosphoric acid (HDBP), a common degradation product of TBP. Tetravalent cerium was found to exchange coordinated nitrate for the dibutyl phosphate anion, forming dinuclear complexes of the formula (CeOCe)(NO)(DBP)·3TBP ( = 0-3). Compared to Ce(IV), Ce(III) was complexed less strongly by HDBP in TBP, but HDBP displaced both nitrate and TBP to form the series of mononuclear complexes Ce(NO)(HDBP·DBP)·(3-)TBP ( = 0-3). Dibutyl phosphate coordination caused large negative shifts in the Ce(IV/III) reduction potential in TBP, indicating a strong stabilization of the tetravalent state. Electrochemical investigation of the reduction of Ce(IV) in TBP revealed it to be a two-electron process in accordance with the dinuclear nature of the organic-phase Ce(IV) complexes. The diffusion coefficients of the = 0 dinuclear Ce(IV)-nitrate-TBP complex and mononuclear Ce(III)-nitrate-TBP complex in TBP equilibrated with 7 M HNO were determined to be (1.16 ± 0.06) × 10 cm/s and (1.9 ± 0.4) × 10 cm/s, respectively, which also is consistent with the larger molecular volume of the dinuclear Ce(IV) complexes.
PubMed: 38949275
DOI: 10.1021/acs.inorgchem.4c01309 -
Journal of Agricultural and Food... Jul 2024Aflatoxin B is a notorious mycotoxin with mutagenicity and carcinogenicity, posing a serious hazard to human and animal health. In this study, an AFB-degrading...
Aflatoxin B is a notorious mycotoxin with mutagenicity and carcinogenicity, posing a serious hazard to human and animal health. In this study, an AFB-degrading dipeptidyl-peptidase III mining from HNGD-TM15 (ADPP III) with a molecular weight of 79 kDa was identified. ADPP III exhibited optimal activity toward AFB at 40 °C and pH 7.0, maintaining over 80% relative activity at 80 °C. The key amino acid residues that affected enzyme activity were identified as H450, E451, H455, and E509 via bioinformatic analysis and site-directed mutagenesis. The degradation product of ADPP III toward AFB was verified to be AFD The zebrafish hepatotoxicity assay verified the toxicity of the AFB degradation product was significantly weaker than that of AFB. The result of this study proved that ADPP III presented a promising prospect for industrial application in food and feed detoxification.
PubMed: 38949246
DOI: 10.1021/acs.jafc.4c03531 -
Angewandte Chemie (International Ed. in... Jul 2024This paper describes a concise, asymmetric and stereodivergent total synthesis of tacaman alkaloids. A key step in this synthesis is the biocatalytic Baeyer-Villiger...
This paper describes a concise, asymmetric and stereodivergent total synthesis of tacaman alkaloids. A key step in this synthesis is the biocatalytic Baeyer-Villiger oxidation of cyclohexanone, which was developed to produce seven-membered lactones and establish the required stereochemistry at the C14 position (92% yield, 99% ee, 500 mg scale). Cis- and trans-tetracyclic indoloquinolizidine scaffolds were rapidly synthesized through an acid-triggered, tunable acyl-Pictet-Spengler type cyclization cascade, serving as the pivotal reaction for building the alkaloid skeleton. Computational results revealed that hydrogen bonding was crucial in stabilizing intermediates and inducing different addition reactions during the acyl-Pictet-Spengler cyclization cascade. By strategically using these two reactions and the late-stage diversification of the functionalized indoloquinolizidine core, the asymmetric total syntheses of eight tacaman alkaloids were achieved. This study may potentially advance research related to the medicinal chemistry of tacaman alkaloids.
PubMed: 38949229
DOI: 10.1002/anie.202407149 -
Journal of Medical Virology Jul 2024Hepatitis E virus (HEV) is a prevalent pathogen responsible for acute viral hepatitis, HEV genotypes 3 and 4 infections causing zoonotic infections. Currently, the...
Hepatitis E virus (HEV) is a prevalent pathogen responsible for acute viral hepatitis, HEV genotypes 3 and 4 infections causing zoonotic infections. Currently, the nucleotide similarity analysis between humans and pigs for HEV genotype 4 is limited. In this study, stool samples from an HEV-infected patient who is a pig farmer and from pigs were collected to obtain the near full-length genome of HEV, phylogenetic trees were constructed for genotyping, and similarity of HEV sequences was analyzed. The results showed that HEV-RNA was detected in the stool samples from the patient and six pigs (6/30, 20.0%). Both HEV subtype in the patient and pigs was 4b. Additionally, similarity analysis showed that the range was 99.875%-99.944% between the patient and pigs at the nucleotide level. Four isolates of amino acid sequences (ORFs 1-3) from pigs were 100% identical to the patient. Phylogenetic tree and similarity analysis of an additional nine HEV sequences isolated from other patients in this region showed that the HEV sequence from the pig farmer had the closest relationship with the pigs from his farm rather than other sources of infection in this region. This study provides indirect evidences for HEV subtype 4b can be transmitted from pigs to humans at the nucleotide level. Further research is needed to explore the characteristics of different HEV subtypes.
Topics: Animals; Hepatitis E virus; Swine; Phylogeny; Hepatitis E; China; Genome, Viral; Humans; Feces; Swine Diseases; Genotype; RNA, Viral; Male; Sequence Analysis, DNA
PubMed: 38949212
DOI: 10.1002/jmv.29777 -
Preclinical determination of wound-healing activity of halibut oil cream in rat model of burn wound.Journal of Asian Natural Products... Jul 2024This study investigated the effects of halibut oil cream, containing omega-3 fatty acids, vitamins A and D, and hydroxyproline, on burn wound healing in rats. Acute...
This study investigated the effects of halibut oil cream, containing omega-3 fatty acids, vitamins A and D, and hydroxyproline, on burn wound healing in rats. Acute dermal toxicity tests confirmed its nontoxicity. Wistar rats were divided into five groups: a control, a positive control treated with silver sulfadiazine 1% (SSD), and three groups treated with 3%, 9%, and 27% halibut oil cream Formulation (HBOF). The SSD and HBOF groups showed significant healing improvements compared to the control. Histopathological analysis indicated increased collagen production in the HBOF groups, suggesting halibut oil cream's potential as a topical treatment for burn wounds.
PubMed: 38949206
DOI: 10.1080/10286020.2024.2368835 -
Journal of Asian Natural Products... Jul 2024One new canthinone glycoside (), together with six known compounds (-) including three lignans (-), two coumarins (- and one phenol ( was isolated from the root barks of...
One new canthinone glycoside (), together with six known compounds (-) including three lignans (-), two coumarins (- and one phenol ( was isolated from the root barks of . The structure of new compound was established by the interpretation of UV, IR, MS and NMR data, while its absolute configuration was determined by acid hydrolysis and GIAO NMR calculations with DP4+ probability analysis. The inhibitory effects of all compounds on Nitric oxide (NO) production were investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Results showed that compounds and displayed NO production inhibitory activity with IC values of 30.1 and 15.3 M, respectively.
PubMed: 38949198
DOI: 10.1080/10286020.2024.2360047