Methods and Protocols最新文献

The fatty acid composition of some seed oils from plants of emerging interest was studied. The benefits towards human health were evaluated by taking into account current recommendations regarding dietary intake of essential, polyunsaturated, and monounsaturated fatty acids and by discussing the pathologies for which such fatty acids exert protective action. Species studied were Hemp (Cannabis sativa), Flax (Linum usitatissimum), Milk Thistle (Silybum marianum), Perilla (Perilla frutescens), Borage (Borago officinalis), and Black Cumin (Nigella sativa). Seeds were subjected to cold milling in order to maintain their original nutritional characteristics. Chemical analyses were performed via the dual-detector gas chromatography technique by means of Flame Ionization Detection (FID) and mass spectrometry (MS) and by applying a modified version of the AOAC Official Method 991.39, thanks to which it was possible to obtain the fatty acid composition expressed as mg per gram of oil: such information is not always available in the literature for the species studied here. Comparison with the fatty acid international guidelines about the recommended intakes in g/day was made. This allowed us to evaluate whether such oils are suitable to be used as fatty acid food supplements to rebalance the Western diet, which is shown to be inadequate by numerous studies. Results show that seed oils from Cannabis sativa, Linum usitatissimum, and Perilla frutescens are suitable to be used as food supplements while seed oils from Silybum marianum, Borago officinalis, and Nigella sativa are not. It is important to note that any possible benefits from other parts of the plant (leaves, stems, flowers, and roots) are not studied or questioned by the present research, which focuses solely on fatty acids in the oil extracted from the seeds.

Intravenous medications are frequently administered through shared catheter lines in neonatal intensive care units (NICUs) due to the limited venous access in preterm infants, raising concerns about drug incompatibilities that may cause serious complications. Hydrocortisone sodium (HDC), ampicillin (ABPC), and cefotaxime (CTX) are commonly used in NICUs and are often co-administered with unfractionated heparin (UFH), which is routinely infused to prevent catheter occlusion. This study evaluated the physicochemical compatibility of HDC, ABPC, and CTX when mixed with UFH. Each drug was combined with UFH at equal volumes, and the mixtures were assessed immediately and after 3 h of storage by visual inspection, pH measurement, UV absorbance, and HPLC-UV analysis. No precipitation, turbidity, or color changes were observed in any mixture, and UV absorbance showed no relevant deviations compared with controls. Slight pH variations were detected but remained within acceptable limits. In semi-quantitative HPLC analysis, relative peak area changes were all below 10%, indicating no major degradation of the drugs. These findings suggest that HDC, ABPC, and CTX maintain acceptable physicochemical compatibility when co-administered with UFH, supporting their safe concomitant use in NICU practice.

Blood flow restriction (BFR) and body cooling (BC) have been investigated separately during exercise, but little is known about their concurrent use. This study examined acute metabolic responses, respiratory physiology, and rate of perceived exertion (RPE) during interval training (IT) performed with combined BFR and BC (VASPER ON) compared with IT without BFR and BC (VASPER OFF). It was hypothesized that VASPER ON would elicit greater physiological demands. A total of 7 female and 19 male participants (20.2 ± 2.4 years) completed a 21-min IT exercise. In VASPER ON, the participants wore cuffs that simultaneously applied BFR and BC. Total oxygen consumption (TVO₂), total carbon dioxide production (TVCO₂), total breaths (BRTH), and total ventilation (TVE) were measured during exercise (EX) and for 10 min post-exercise (Post-EX). RPE was recorded during EX. During EX, TVE and ventilatory equivalents for both oxygen and carbon were significantly higher in VASPER ON. Post-EX, all variables remained significantly elevated in VASPER ON except for the ventilatory equivalent for carbon dioxide. Sprint interval RPE was significantly lower in VASPER OFF. These findings suggest that concurrent BFR and BC increase post-exercise metabolic and ventilatory demands without attenuating each other's effect.

The Transdermal Drug Delivery System (TDDS) offers several benefits, such as enhanced patient adherence, controlled release, reduced gastric irritation, and the bypassing of the first-pass metabolism. However, not all drugs can be delivered through this route in effective doses. Biodegradable microneedles (BMn) are designed to improve TDDS. This review outlines various types of BMn and their fabrication methods. BMn are produced in different forms, including hollow, solid, dissolve, and hydrogel-forming versions, which have garnered significant attention. These innovative BMn do not contain drugs themselves but instead absorb interstitial fluid to create continuous channels between the dermal microcirculation and a drug-containing patch. Several types of BMn have been tested and approved by regulatory bodies. The use of BMn technology is rapidly growing in point-of-care applications, attracting significant interest from both researchers and healthcare providers. BMn-based Point-of-care (POC) devices have high efficacy for finding various analytes of clinical interests and transdermal drug administration in a minimally invasive manner owing to BMn' micro-size sharp tips and ease of use. Porous BMn technology may have a very rising future in the case of a vaccine delivery system.

Background: Shoulder soft tissue disorders, such as rotator cuff tears and subacromial impingement, are among the most common causes of musculoskeletal disability. Both physical examination tests and imaging techniques are routinely used in clinical settings; however, their respective contributions to patient outcomes and their potential complementarity remain underexplored.

Methods: A systematic review and meta-analysis were conducted following PRISMA 2020 guidelines. Controlled clinical studies comparing pre- and post-intervention outcomes in adults with suspected or confirmed shoulder soft tissue pathology were included. Two groups were analyzed: studies using musculoskeletal imaging (ultrasound or MRI) and studies applying orthopedic physical examination tests (e.g., Neer, Hawkins, and Jobe). Functional outcomes were converted into standardized mean differences (SMDs) and synthesized using a random-effects model. Heterogeneity was quantified using the I² statistic.

Results: In total, 11 studies met the inclusion criteria (n = 6 imaging, n = 5 orthopedic tests). Imaging-based studies showed a pooled SMD of 4.85 (95% CI: 2.77-6.93), indicating substantial clinical improvement. Orthopedic test-based studies yielded a pooled SMD of 2.34 (95% CI: 1.27-3.41). Heterogeneity was high across both groups (I² > 90%).

Conclusions: Imaging was associated with a larger overall clinical effect, while orthopedic tests provided functional insight valuable for screening and monitoring. These findings support the complementary use of both strategies to enhance diagnostic accuracy and treatment planning in shoulder care.

Background: Developing clinically relevant experimental models of the human airway can significantly advance our understanding of the mechanisms underlying airway diseases and aid in translating potential therapies to clinical settings. The aim of this study is to establish an ex vivo human airway tissue culture model.

Methods: Human donor airway tissues were obtained from clinical cases of lung transplantation. Our established method is based on the concept of scavenging metabolic activity and controlling bacterial growth and includes increased media volume, frequent media exchange, and antifungal additives to efficiently maintain the homeostatic culture environment. After a 3-day culture period, the airway was investigated, and its viability and function were compared with a standard cell culture method.

Results: Control tissue exhibited significant acidosis after 3 days, suggesting high metabolic activity of airway tissue and bacterial contamination. The airway epithelial viability-after culturing in our established method for 3 days-was better than that of the controls. We only performed an acute but early investigation of the cultures as airway complications have been known to start early at the proximal bronchus after transplantation. H&E and alcian blue staining showed intact morphology of the epithelium of airway tissue and mucus layers after 3 days in our model, while controls showed remarkable damage to the epithelial layer. Newly synthesized glycoproteins were detected in the epithelial layer using metabolic labeling and the click chemistry technique, suggesting cellular protein synthesis of the airway tissue in our established ex vivo model.

Conclusions: We successfully established a reproducible model of human ex vivo airway tissue culture (n = 3 independent biological samples) that may be useful for investigating airway complications and developing their therapies.

In the nuclear industry, the decontamination of nuclear metallic structures is an essential process to reduce radiation exposure during maintenance or dismantling. The oxide layer, such as chromium (III) oxide (Cr₂O₃), formed on stainless steel and nickel-based alloys, contributes significantly to surface radioactivity by trapping radioactive contaminants. To address this, permanganic acid (HMnO₄) has proven to be a promising oxidizing agent for dissolving these oxide layers-particularly chromium oxide-on stainless steel and nickel-based alloys. In this study, HMnO₄ was synthesized via ion exchange using AmberLite IRN97 H resin and potassium permanganate (KMnO₄). The optimized process yielded a highly acidic solution (pH~1.6) with potassium concentrations below 0.1 ppm, indicating near-complete exchange efficiency. Dissolution kinetics were investigated at HMnO₄ concentrations ranging from 240 to 1920 ppm and temperatures from 30 °C to 80 °C. At a constant temperature, increasing HMnO₄ concentration significantly improved Cr dissolution, with up to 31% of total chromium solubilized after 33 h. Lower temperatures favored higher dissolution efficiency, likely due to improved thermal stability of HMnO₄. For durations shorter than 4 h, the influence of temperature was limited compared to the effect of acid concentration. To assess post-treatment options, HMnO₄ decomposition was studied using oxalic acid (H₂C₂O₄) at 80 °C. Results showed that a minimum H₂C₂O₄/HMnO₄ molar ratio above 2.75 was necessary to achieve effective reduction while preventing MnO₂ precipitation. However, even under strongly acidic conditions and with a large excess of reductant, Mn²⁺ yields remained below 55%, suggesting that thermal degradation of oxalic acid and possible formation of undetected manganese species limited the reduction process.

Cancer development in BRCA1 carriers is a multi-step process, which is triggered by several factors and mechanisms that are not clearly understood. Most BRCA1 carriers develop triple-negative breast cancer (TNBC)-estrogen receptor (ER)-, progesterone receptor (PR)-, and HER2 -negative cancers-which originates from ER/PR/HER2-negative breast progenitor cells. Due to a lack of ER/PR/HER2-negative cell models with BRCA mutations, the processes inducing cancer development in BRCA carriers have not been comprehensively studied. Thus, studies characterizing ER/PR/HER2-negative cells carrying a BRCA1 germline mutation are needed to gain more in-depth knowledge about the steps leading to cancer initiation in BRCA1 carriers. To study the cancer development in these patients, we established a protocol for the generation of human ER/PR/HER2-negative breast organoids carrying a BRCA1 germline mutation. We confirmed that these organoids are unresponsive to estrogen, can self-renew, and express the stem/progenitor marker CD44. In addition, we observed that these organoids contain outgrowths that resemble the mature ductal and lobular units of the mammary gland, thus making it a suitable model system to study how cancer develops in ER/PR/HER2-negative mammary cells that carry a BRCA1 germline mutation.

This study discusses the challenges encountered in implementing a detailed protocol for upper abdominal imaging using magnetic resonance imaging (MRI), ranging from patient preparation and sequence selection to clinical applications. MRI is a valuable non-invasive imaging modality employed both in the early detection of diseases and as a complementary tool for the detailed characterization of various pathologies. Nevertheless, performing an abdominal MRI examination can be challenging; therefore, the understanding of sequences is particularly important, as changing the parameters can not only influence the quality of the images but also optimize scanning time improve patient experience during the examination. The methodology illustrates the purpose of each sequence and the critical role of appropriate patient preparation. Results highlighted the significance of these factors in the evaluation of hepatic lesions, showing that the proper choice of sequences and parameters is essential for distinguishing benign from malignant findings and for achieving an accurate diagnosis. It was also shown that MRI plays an important role as a complementary technique in investigation of upper abdominal pathologies in order to avoid overexposure to radiation.