Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy

BACKGROUND: Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. METHODS: We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. RESULTS: The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. CONCLUSION: The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis

Low-Cost and Eco-Friendly Hydroxyapatite Nanoparticles Derived from Eggshell Waste for Cephalexin Removal

This work describes the hydroxyapatite nanoparticle (HAP) preparation from eggshell waste and their application as an adsorbent for Cephalexin (Ceph) antibiotic removal from aqueous solutions. Chemical precipitation with phosphoric acid was used to evaluate the feasibility of calcium oxide for HAP preparation. The structural properties of HAP were characterized by X-ray diffraction, which revealed the formation of the hydroxyapatite crystalline phase formation. In addition, transmitting electron spectroscopy showed an irregular shape with a variation in size. The impact of various experimental conditions on the removal efficiency such as the solution’s pH, contact time, HAP mass, solution temperature, and Ceph concentration were studied. Experimental data showed that HAP could remove most Ceph species from aqueous solutions within 1 h at pH = 7 with 70.70% adsorption efficiency utilizing 50 mg of the HAP. The removal process of Ceph species by HAP was kinetically investigated using various kinetic models, and the results showed the suitability of the pseudo-second-order kinetic model for the adsorption process description. Moreover, the removal process was thermodynamically investigated; the results showed that the removal was spontaneous endothermic and related to the randomness increase. The data confirmed that HAP had high efficiency in removing Ceph antibiotics from an aqueous solution

Predictors and Direct Cost Estimation of Long Stays in Pediatric Intensive Care Units in Saudi Arabia: A Mixed Methods Study

Mohamad-Hani Temsah,1,2,* Noura Abouammoh,2,3,* Ayman Al-Eyadhy,1,2 Yazed AlRuthia,4,5 Marwah Hassounah,3 Fahad Alsohime,1,2 Ali Alhaboob,1,2 Mohammed Almazyad,1,2 Majed Alabdulhafid,1,2 Fadi Aljamaan,2,6 Khalid Alhasan,1,2 Amr Jamal2,3,7 1Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 2King Saud University Medical City, King Saud University, Riyadh, Saudi Arabia; 3Family and Community Medicine Department, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 4Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 5Pharmacoeconomics Research Unit, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 6Department of Critical Care, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 7Evidence-Based Health Care & Knowledge Translation Research Chair, King Saud University, Riyadh, Saudi Arabia*These authors contributed equally to this workCorrespondence: Amr Jamal Email [email protected]: Several factors influence patients’ length of stay (LOS) in pediatric intensive care units (PICUs). This study explored the factors and cost associated with long LOS among pediatric patient population across various PICUs and pediatric surgical cardiac intensive care units (SCICUs).Methods: This is a multi-center concurrent mixed-methods study that was conducted in different PICUs and SCICUs in Riyadh, Saudi Arabia. The units’ leaders were qualitatively interviewed to explore the potential factors contributing to long LOSs in PICUs and SCICUs, and deductive content analysis was used. Microcosting was utilized to estimate the inpatient costs for a sample of long-stay patients (LSPs) and the potential cost savings if those patients were transferred to step-down care units (SDUs) for those who stayed ≥ 60 days.Results: Ten (90%) leaders of PICUs and SCICUs who were invited had participated. Changing the admission criteria, patient transfer to SDUs, family engagement in the decision-making process, and adopting a national do-not-resuscitate (DNR) policy were factors contributing to short LOSs. On the other hand, administrative constraints, staff avoidance of code status decisions, lack of palliative care resources, and complex patient characteristics were factors associated with long LOSs. The mean inpatient cost per patient was SAR 3.63 million (USD 0.97 million), and the total cost for the 48 patients was SAR 172.95 million (USD 46.12 million).Conclusion: If the recommendation to transfer LSPs after a 60-day PICU stay to SDUs is taken, the estimated cost savings among this sample of patients alone can be as high as SAR 109.47 million (USD 29.19 million). Therefore, future studies should examine the factors contributing to long LOSs in PICUs and SCICUs as well as the effectiveness of different policies aimed at improving the resource allocation and management using more robust study designs and diverse patient population.Keywords: pediatric critical care, long-stay patients, cost of long-stay patients, content analysi

Absolute and Direct MicroRNA Quantification Using DNA–Gold Nanoparticle Probes

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis