Characterization of the mass transfer of lyophilized products based on X-ray micro-computed tomography images

ABSTRACT

This paper proposes a systematic work process for the rapid determination of resistance to vapor flow given by the products being freeze-dried. This approach couples mathematical modeling and 3D nondestructive X-ray imaging to reconstruct the internal structure of lyophilized samples. Knowledge of the internal structure is fundamental to better understand the relationship between freezing and within-product heterogeneity as well as between freezing and the drying behavior of the product being lyophilized. The method was validated upon mannitol-based formulations using various freezing protocols. This stepwise approach was demonstrated to be helpful to lyophilization professionals for the control of within-product heterogeneity and for the optimization of the cycle.     

Network Analysis of Gut Microbiome and Metabolome to Discover Microbiota-Linked Biomarkers in Patients Affected by Non-Small Cell Lung Cancer

Several studies in recent times have linked gut microbiome (GM) diversity to the pathogenesis of cancer and its role in disease progression through immune response, inflammation and metabolism modulation. This study focused on the use of network analysis and weighted gene co-expression network analysis (WGCNA) to identify the biological interaction between the gut ecosystem and its metabolites that could impact the immunotherapy response in non-small cell lung cancer (NSCLC) patients undergoing second-line treatment with anti-PD1. Metabolomic data were merged with operational taxonomic units (OTUs) from 16S RNA-targeted metagenomics and classified by chemometric models. The traits considered for the analyses were: (i) condition: disease or control (CTRLs), and (ii) treatment: responder (R) or non-responder (NR). Network analysis indicated that indole and its derivatives, aldehydes and alcohols could play a signaling role in GM functionality. WGCNA generated, instead, strong correlations between short-chain fatty acids (SCFAs) and a healthy GM. Furthermore, commensal bacteria such as Akkermansia muciniphila, Rikenellaceae, Bacteroides, Peptostreptococcaceae, Mogibacteriaceae and Clostridiaceae were found to be more abundant in CTRLs than in NSCLC patients. Our preliminary study demonstrates that the discovery of microbiota-linked biomarkers could provide an indication on the road towards personalized management of NSCLC patients.     

Copy Number Analysis of 24 Oncogenes: MDM4 Identified as a Putative Marker for Low Recurrence Risk in Non Muscle Invasive Bladder Cancer

Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher’s test p = 0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p = 0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.     

The mineral profile affects the coagulation pattern and cheese-making efficiency of bovine milk

Natural variations in milk minerals, their relationships, and their associations with the coagulation process and cheese-making traits present an opportunity for the differentiation of milk destined for high-quality natural products, such as traditional specialties or Protected Designation of Origin (PDO) cheeses. The aim of this study was to quantify the effects of the native contents of Ca, P, Na, K, and Mg on 18 traits describing traditional milk coagulation properties (MCP), curd firming over time (CF_t) equation parameters, cheese yield (CY) measures, and nutrient recoveries in the curd (REC) using models that either included or omitted the simultaneous effects of milk fat and casein contents. The results showed that, by including milk fat and casein and the minerals in the statistical model, we were able to determine the specific effects of each mineral on coagulation and cheese-making efficiency. In general, about two-thirds of the apparent effects of the minerals on MCP and the CF_t equation parameters are actually mediated by their association with milk composition, especially casein content, whereas only one-third of the effects are direct and independent of milk composition. In the case of cheese-making traits, the effects of the minerals were mediated only negligibly by their association with milk composition. High Ca content had a positive effect on the coagulation pattern and cheese-making traits, favoring water retention in the curd in particular. Phosphorus positively affected the cheese-making traits in that it was associated with an increase in CY in terms of curd solids, and in all the nutrient recovery traits. However, a very high P content in milk was associated with lower fat recovery in the curd. The variation in the Na content in milk only mildly affected coagulation, whereas with regard to cheese-making, protein recovery was negatively associated with high concentrations of this mineral. Potassium seemed not to be actively involved in coagulation and the cheese-making process. Magnesium content tended to slow coagulation and reduce CY measures. Further studies on the relationships of minerals with casein and protein fractions could deepen our knowledge of the role of all minerals in coagulation and the cheese-making process.     

Increasing Cold Tolerance of Cactus Pear Fruit by High-Temperature Conditioning and Film Wrapping

Pre-storage high-temperature conditioning (HTC, 38 °C, and 95% RH for 24 h) and individual film wrapping (IFW) with a perforated polyolefinic heat-shrinkable film were used as individual treatments or in combination to mitigate chilling injury of first crop cactus pear cv ‘Gialla’. The fruit was stored for 21 days at either 2 or 8 °C (CS) plus 1 week of simulated marketing conditions (SMC) at 20 °C. The reduction in peel disorders and decay in HTC-treated fruit stored at 2 °C was comparable to that detected in control fruit stored at 8 °C. IFW was more efficient than HTC in reducing peel disorders, almost completely inhibited weight loss, and preserved freshness in fruit stored at 8 °C as well as in those stored at 8 °C. The internal quality of the fruit (pH, total soluble solids, titratable acidity, vitamin C, polyphenols, betaxanthins, antioxidant capacity, acetaldehyde, and ethanol) was slightly affected by treatments. Combining HTC with IFW did not improve fruit tolerance to low temperature but reduced decay with respect to individual treatments. IFW with a perforated film in combination with HTC is a good means of overcoming the stringent conditions of cold quarantine treatments, maintaining fruit freshness and reducing decay in cold-stored cactus pears.     

Preclinical Development of FA5, a Novel AMP-Activated Protein Kinase (AMPK) Activator as an Innovative Drug for the Management of Bowel Inflammation

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.     

Condensation Heat Transfer and Pressure Losses of High- and Low-Pressure Refrigerants Flowing in a Single Circular Minichannel

A 0.96 mm circular minichannel is used to measure both heat transfer coefficients during condensation and two-phase pressure losses of the refrigerants R32 and R245fa. Test runs have been performed at around 40°C saturation temperature, corresponding to 24.8 bar saturation pressure for R32 and 2.5 bar saturation pressure for R245fa. The pressure drop tests have been performed in adiabatic flow conditions, to measure only the pressure losses due to friction. The heat transfer experimental data are compared against predicting models to provide a guideline for the design of minichannel condensers.     

Elements essential for accumulation and function of small nucleolar RNAs directing site-specific pseudouridylation of ribosomal RNAs

During site-specific pseudouridylation of eukaryotic rRNAs, selection of correct substrate uridines for isomerization into pseudouridine is directed by small nucleolar RNAs (snoRNAs). The pseudouridylation guide snoRNAs share a common 'hairpin-hinge- hairpin-tail' secondary structure and two conserved sequence motifs, the H and ACA boxes, located in the single-stranded hinge and tail regions, respectively. In the 5'- and/or 3'-terminal hairpin, an internal loop structure, the pseudouridylation pocket, selects the target uridine through formation of base-pairing interactions with rRNAs. Here, essential elements for accumulation and function of rRNA pseudouridylation guide snoRNAs have been analysed by expressing various mutant yeast snR5, snR36 and human U65 snoRNAs in yeast cells. We demonstrate that the H and ACA boxes that are required for formation of the correct 5' and 3' ends of the snoRNA, respectively, are also essential for the pseudouridylation reaction directed by both the 5'- and 3'-terminal pseudouridylation pockets. Similarly, RNA helices flanking the two pseudouridylation pockets are equally essential for pseudouridylation reactions mediated by either the 5' or 3' hairpin structure, indicating that the two hairpin domains function in a highly co-operative manner. Finally, we demonstrate that by manipulating the rRNA recognition motifs of pseudouridylation guide snoRNAs, novel pseudouridylation sites can be generated in yeast rRNAs.