Assessing the gene silencing potential of AuNP-based approaches on conventional 2D cell culture versus 3D tumor spheroid.

Three-dimensional (3D) cell culture using tumor spheroids provides a crucial platform for replicating tissue microenvironments. However, effective gene modulation via nanoparticle-based transfection remains a challenge, often facing delivery hurdles. Gold nanoparticles (AuNPs) with their tailored synthesis and biocompatibility, have shown promising results in two-dimensional (2D) cultures, nevertheless, they still require a comprehensive evaluation before they can reach its full potential on 3D models. While 2D cultures offer simplicity and affordability, they lack physiological fidelity. In contrast, 3D spheroids better capture in vivo conditions, enabling the study of cell interactions and nutrient distribution. These models are essential for investigating cancer behavior, drug responses, and developmental processes. Nevertheless, transitioning from 2D to 3D models demands an understanding of altered internalization mechanisms and microenvironmental influences. This study assessed ASO-AuNP conjugates for silencing the c-MYC oncogene in 2D cultures and 3D tumor spheroids, revealing distinctions in gene silencing efficiency and highlighting the microenvironment's impact on AuNP-mediated gene modulation. Herein, we demonstrate that increasing the number of AuNPs per cell by 2.6 times, when transitioning from a 2D cell model to a 3D spheroid, allows to attain similar silencing efficiencies. Such insights advance the development of targeted gene therapies within intricate tissue-like contexts.

Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples.

The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.

Dexmedetomidine Alone or Combined With Morphine for Epidural Anesthesia in Bitches Undergoing Elective Ovariohysterectomy.

The purpose of this study was to assess perioperative analgesia provided by the combination of epidural dexmedetomidine and morphine in bitches undergoing elective ovariohysterectomy. Twenty-four bitches were included in the study and allocated into 3 groups: GM, morphine 0.1 mg/kg; GD, dexmedetomidine 2 µg/kg; and GDM, dexmedetomidine and morphine at the same doses. All solutions were diluted in saline to a total of 0.36 mL/kg. Heart rate (HR), respiratory rate (FR) and systolic blood pressure (SAP) were recorded prior to epidural analgesia (TB), immediately following epidural analgesia (TEA), at surgical incision (TSI), at the first ovarian pedicle clamping (TOP1), at the second pedicle clamping (TOP2), at uterine stump clamping (TUC), at the start of abdominal cavity closure (TSC) and at the end of skin closure (TEC). Rescue analgesia with fentanyl was administered at 2 µg/kg IV if nociception corresponding to a 20% increase of any cardiorespiratory variables was noted. Postoperative pain assessment was performed using a modified composite Glasgow pain scale along the first 6 hours following the end of surgery. Numeric data were compared using ANOVA for repeated measures followed by Tukey test and ovarian ligament relaxation was analyzed using chi-square test under 5% significance. No differences were found on FR among times or groups, although HR showed significant differences between GM and GD at TSI, TOP1, TOP2, TSC and TEC and between GM and GDM at TEA and TSI (significantly lower HR values recorded in dexmedetomidine groups). Differences among time points were found on HR between TB and TEA in GD and on PAS between TOP1 and TSC in GM and between TOP1 and TUC in GDM (P < .05). Ovarian ligament relaxation was significantly more present in groups using dexmedetomidine, although the number of rescue analgesia administrations did not differ among groups. Kaplan-Meyer analysis failed to show significant differences on time of rescue analgesia administration among groups (P > .05). In conclusion, the combination of epidural dexmedetomidine and morphine is a more interesting choice for elective ovariohysterectomy in bitches for producing analgesia comparable to that of each drug alone, with noticeable relaxation of ovarian ligaments and lesser cardiovascular consequences.

Engineering gold nanoparticles for molecular diagnostics and biosensing.

Advances in nanotechnology and medical science have spurred the development of engineered nanomaterials and nanoparticles with particular focus on their applications in biomedicine. In particular, gold nanoparticles (AuNPs) have been the focus of great interest, due to their exquisite intrinsic properties, such as ease of synthesis and surface functionalization, tunable size and shape, lack of acute toxicity and favorable optical, electronic, and physicochemical features, which possess great value for application in biodetection and diagnostics purposes, including molecular sensing, photoimaging, and application under the form of portable and simple biosensors (e.g., lateral flow immunoassays that have been extensively exploited during the current COVID-19 pandemic). We shall discuss the main properties of AuNPs, their synthesis and conjugation to biorecognition moieties, and the current trends in sensing and detection in biomedicine and diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Water safety screening via multiplex LAMP-Au-nanoprobe integrated approach.

Contaminated water resources remain a major global concern regarding public health. The majority of water safety protocols include indicators of microbial contamination to evaluate the potential risk to public health and are key elements of quality guidelines. Among these, markers for total coliforms and fecal coliforms are strong indicators of co-contamination with other pathogens. Traditional methods, recurring to slow and cumbersome culture-based approaches, have been gradually replaced by molecular methods, capable of faster and more specific screening. These are usually PCR-based methods that may allow for multiple pathogen detection but require dedicated laboratory equipment, hindering the rapid on-site assessment. Here, we used a multiplex Loop-Mediated Isothermal Amplification (mLAMP) strategy for the amplification of two markers associated with the contamination by total and fecal coliforms (e.g. Escherichia coli) - lacZ and uidA genes, respectively - thus allowing for single tube multiplex detection. The mLAMP products were then subject to an Au-nanoprobe colorimetric detection assay for precise discrimination of targets. This approach was validated in 22 water samples that were also screened for the presence of lacZ and uidA using standard and quantitative PCR, with the capability for discriminating the contamination level, e.g. a semi-quantitative evaluation of water quality.