Methods and Protocols最新文献

The routine transformation and genetic modification of many plant species other than Arabidopsis thaliana is still an arduous task. In many cases, it is necessary to use special tissues or conditions performed under sterile tissue culture conditions. Nevertheless, this approach is often the most expedient one, and streamlining protocols to maximize efficiency and minimize effort without sacrificing quality is paramount to today's research agendas. The Solanaceae family tends to be amicable to tissue culture and relatively easy to transform. Here, we present our optimized, routine tissue culture protocols for the transformation of Nicotiana benthamiana, Nicotiana tabacum, Solanum tuberosum (potato), and Solanum lycopersicum (tomato). We highlight their commonalities and their differences, thus giving the researcher a framework for optimizing their own protocols in their laboratory if needed. Tissue culture transformation is still an important and dynamic field for the advancement of plant research in molecular genetics, physiology, and plant pathology, and will continue to be a viable and important resource into the future.

Biogenic amines (BAs) are nitrogenous compounds naturally present in protein-rich foods, whose accumulation may indicate spoilage and pose health risks. This study presents the development and validation of a rapid LC-MS/MS method for the simultaneous quantification of six BAs-putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPM)-in meat products, without requiring derivatisation. Sample preparation was optimized to enhance extraction efficiency and reproducibility, using 0.5 M HCl and a double-centrifugation protocol to avoid matrix interference. Chromatographic separation was optimized using a C18 column and acidified ammonium formate/acetonitrile mobile phases. The method showed good linearity (R² > 0.99), trueness between -20% and +20%, and acceptable precision (RSD_r and RSD_R ≤ 25%). Limits of quantification were established at 10 µg/g for all analytes. The method was applied to ten commercial meat samples, where PUT, TYR, and SPD were the most frequently detected amines. Although HIS and TYR levels were below toxicological thresholds for healthy individuals, one sample showed TYR levels potentially concerning for monoamine oxidase inhibitors -treated consumers. The Biogenic Amine Index (BAI) further supported product quality assessment, identifying early spoilage in selected cases. This method offers a rapid, robust and efficient tool for routine monitoring of BAs in meat products, supporting food safety and quality control initiatives.

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. Also, no unified computational framework exists to specifically link miRNA expression with functional gene targets during RV infection. This study aimed to first analyse the impact of RV16 on miRNA expression across the viral life cycle to identify a small panel with altered expression. We then developed a novel bioinformatics pipeline that integrated time-resolved miRNA profiling with multi-database gene-phenotype mapping to identify diagnostic biomarkers and their regulatory networks. Our in-house Python-based tool, combining mirDIP, miRDB and VarElect APIs, predicted seven genes (EZH2, RARG, PTPN13, OLFML3, STAG2, SMARCA2 and CD40LG) implicated in antiviral responses and specifically targeted by RV16 and regulated by our miRNAs. This method therefore offers a scalable approach to interrogate miRNA-gene interactions for viral infections, with potential applications in rapid diagnostics and therapeutic target discovery.

SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus requires enhanced biological containment facilities (i.e., CL3) to reduce the potential of laboratory infection to personnel and the spread of the virus into the community. While the use of an authentic live virus remains the gold standard for biological assays, alternative methods have been developed to effectively evaluate neutralization activity in the absence of a replicating viral agent. Here, we describe a cell-based spike-ACE2 binding assay as a surrogate for neutralization of SARS-CoV-2 spike to identify potential neutralizing antibodies. A main advantage of this approach is the exclusion of infectious viral particles, increasing biosafety for laboratory personnel. The interaction of recombinant SARS-CoV-2 trimeric spike protein with ACE2 is monitored and quantified by flow cytometry. Notably, our previous studies have demonstrated the utility of this assay for other viruses, beyond SARS-CoV-2. The methodology presented here has exhibited a strong correlation to other widely accepted methods, such as pseudotyped lentiviral and live virus neutralization assays, in identifying neutralizing antibodies.

The 90 kDa Heat Shock Protein (Hsp90) is an essential and highly conserved molecular chaperone that supports the folding and maturation of a diverse array of client proteins across prokaryotic and eukaryotic organisms. In bacteria, HtpG, the Hsp90 homolog, plays a central role in stress response and protein homeostasis, particularly under high-temperature and other stress conditions. Despite extensive studies on HtpG from E. coli, the biochemical properties and functional roles of cyanobacterial HtpG remain poorly characterized. Here, we focus on HtpG from the cyanobacterium Synechococcus elongatus PCC 7942 (seHtpG), a model organism for photosynthesis and circadian rhythm research. We developed a method for the overexpression and purification of seHtpG in E. coli, achieving high purity and yield suitable for biochemical and structural studies. Biophysical and biochemical assays show that seHtpG forms dimers and hydrolyzes ATP at a rate of 1.9 ATP/min, 4-fold faster than that of E. coli HtpG. This work establishes seHtpG as a model for studying the roles of HtpG in cyanobacterial protein homeostasis, photosynthesis, and stress response, enabling further exploration of cyanobacterial Hsp90 in ecosystem dynamics and biotechnological applications.

Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or peripheral blood sources, often require extended culture periods and intensive labor, creating bottlenecks for widespread therapeutic application. To address these manufacturing hurdles, we have developed an optimized protocol for ex vivo CAR-NK cell production from human peripheral blood that incorporates lessons learned from previous methodologies while introducing novel efficiency improvements. This protocol offers a practical solution for scalable CAR-NK cell manufacturing that can be readily adapted across different production facilities, potentially accelerating the clinical development of CAR-NK therapies.

Objectives: The present study mainly aimed to identify whether the envelope and triangular flaps affected wound healing and patient quality of life differently. Secondarily, the study aimed to investigate whether some anatomical and operational variables may also affect healing.

Study design: A prospective randomized study was conducted with 56 fully impacted lower third molars, randomly divided into two groups, one treated with the envelope flap and the other with the bayonet flap. Qualitative variables were transformed into quantitative ones and then analyzed using independent samples t-tests or analysis of variance. An analysis of bivariate correlations with Pearson's coefficient was also used. The chi-square test was used to verify the association between each flap and the categorical variables considered.

Results: No statistically significant associations were found between flap types and dehiscence, although the mean dehiscence diameter was consistently greater in the envelope flap group. The maximum diameter of the dehiscence at 14 days was found to be significantly and negatively related to the 14-day wound healing indices. Analyses relating to the quality of life did not show significant associations.

Conclusions: Despite some significant healing differences between the two considered flaps exist, they do not have relevant effects on the patient's post-operative quality of life.

Cannabis sativa (C. sativa) is a psychoactive plant that has been used for millennia for medicinal, recreational, and industrial purposes. The main constituents of cannabis are the cannabinoids, with other constituents including terpenes and flavonoids that contribute to its bioactivity. Among the flavonoid class, there is a subclass, specific to cannabis, namely the cannflavins (A, B, and C), which are biologically active. This study is directed to the analysis of these constituents in various cannabis chemovars. In this study, an HPLC-PDA method was validated and applied to determine the content of cannflavins, namely, cannflavin A (CF-A), cannflavin B (CF-B), and cannflavin C (CF-C), in six different cannabis chemovars. The HPLC separation was achieved using a Luna^® C18 (150 × 4.6 mm × 3 μm) with isocratic elution using a mobile phase consisting of acetonitrile and water (65:35, v/v), both containing 0.1% formic acid at a flow rate of 1 mL/min, with the detector set at 342.4 nm. The method was validated according to the ICH guidelines and exhibited a linear relationship in the 5-500 ppm range with R² > 0.99. The method showed good recovery, ranging from 82% to 98%. The intra-day and inter-day relative standard deviations (% RSDs) were ≤5.29%. Consequently, the method was applied for the determination of all these cannflavins in the different cannabis chemovars. CF-A was the most abundant cannflavin in the examined samples (15.2-478.38 ppm). The method was shown to be simple, accurate, and selective.

Yeasts of the Candida genus are part of the normal human microbiota but can cause infections (candidiasis) under certain conditions. While Candida albicans remains the most common etiological agent, the prevalence of non-albicans Candida species-such as C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, C. lusitaniae, and the emerging multidrug-resistant C. auris-has been increasing. Effective treatment of candidiasis requires rapid and accurate identification of the causative species, particularly due to species-specific antifungal agent resistance patterns. The aim of this study was to evaluate the usefulness of five chromogenic media for the differentiation of Candida species: BD CHROMagar Candida (Becton Dickinson), CHROM ID Candida (bioMérieux), CHROMAgar Candida Plus (CHROMAgar France, Biomaxima), CHROMAgar Candida Plus (GRASO Biotech), and Brilliance Candida Agar (OXOID). A total of 175 strains from the following species were tested: C. albicans, C. parapsilosis, C. dubliniensis, C. lusitaniae, C. tropicalis, C. glabrata, C. kefyr, C. krusei, and C. auris. Species identification was confirmed by MALDI-TOF mass spectrometry using the MALDI Biotyper system (Bruker). Colony morphology, especially color characteristics, was assessed on each medium. The morphological features of most Candida species were consistent with the manufacturer's descriptions and allowed for presumptive species-level identification. However, some species showed reproducible but previously undescribed morphological traits, including variations in colony shade. Notably, C. auris could not be reliably identified using BD, bioMérieux, or OXOID media. In conclusion, while chromogenic media are a helpful preliminary diagnostic tool, subtle differences in colony coloration can complicate interpretation. Diagnostic caution is recommended, and confirmatory methods such as MALDI-TOF remain essential for reliable identification, especially for emerging or less common Candida species.

Pharmaceutical formulations, in addition to the medicinal substance(s), contain added excipients that make it possible to create a pharmaceutical product that exhibits required properties in terms of mechanical, physical, chemical, and microbiological stability. Additionally, these substances can act as release modifiers or improve bioavailability parameters. Literature data indicate that excipients, especially polymeric ones, can also affect the polymorphism of the active substance, resulting in drug bioavailability enhancement or reduction. This influence can be evaluated using thermal and spectroscopic methods. In the study, differential scanning calorimetry (DSC), vibrational spectroscopic studies (Fourier transform infrared spectroscopy, FTIR), Raman spectroscopy, and X-ray diffraction (XRD) assay of ibuprofen, naproxen, and naproxen sodium standards and pharmaceutical preparations containing these medicinal substances in their compositions were carried out. DSC results indicated that a sharp melting peak was observed on the DSC curves of the standards, confirming their crystalline form. DSC results obtained for pharmaceutical formulations also indicated that the enthalpy of melting is sometimes lower than calculated from the percentage of active ingredients in the formulations. In addition, the melting peak is often broadened and shifted toward lower temperatures, suggesting the influence of excipients on the polymorphism of drug substances. The FTIR and Raman spectra of pharmaceutical formulations contained all characteristics of the active substances. XRD analysis was also performed. Therefore, possible chemical interactions between the components of the preparations have been excluded. At the same time, FTIR and Raman spectroscopy results as well as XRD assay showed a reduction in the height of signals corresponding to the crystalline API form, confirming the possibility of reducing API crystallinity in pharmaceutical formulations.