Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. If the direct suture of a lesion is not possible, i.e., nerve gap > 2 cm, it is necessary to use grafts. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable functional deficit and further morbidity. An alternative to autografting is represented by the acellular nerve allograft (ANA), which avoids disadvantages of autograft harvesting and fresh allograft rejection. In this research, the authors intend to transfer to human nerves a novel technique, previously implemented in animal models, to decellularize nerves. The new method is based on soaking the nerve tissues in decellularizing solutions while associating ultrasounds and freeze–thaw cycles. It is performed without interrupting the sterility chain, so that the new graft may not require post-production γ-ray irradiation, which is suspected to affect the structural and functional quality of tissues. The new method is rapid, safe, and inexpensive if compared with available commercial ANAs. Histology and immunohistochemistry have been adopted to evaluate the new decellularized nerves. The study shows that the new method can be applied to human nerve samples, obtaining similar, and, sometimes better, results compared with the chosen control method, the Hudson technique.     

Complete Characterization of the O-Antigen from the LPS of Aeromonas bivalvium

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 E^T (=CECT 7113^T = LMG 23376^T), a mesophilic bacterium isolated from cockles (Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.     

The Efficacy of Fibrinogen Concentrates in Relation to Cryoprecipitate in Restoring Clot Integrity and Stability against Lysis

Loss of fibrinogen is a feature of trauma-induced coagulopathy (TIC), and restoring this clotting factor is protective against hemorrhages. We compared the efficacy of cryoprecipitate, and of the fibrinogen concentrates RiaSTAP^® and FibCLOT^® in restoring the clot integrity in models of TIC. Cryoprecipitate and FibCLOT^® produced clots with higher maximal absorbance and enhanced resistance to lysis relative to RiaSTAP^®. The fibrin structure of clots, comprising cryoprecipitate and FibCLOT^®, mirrored those of normal plasma, whereas those with RiaSTAP^® showed stunted fibers and reduced porosity. The hemodilution of whole blood reduced the maximum clot firmness (MCF) as assessed by thromboelastography. MCF could be restored with the inclusion of 1 mg/mL of fibrinogen, but only FibCLOT^® was effective at stabilizing against lysis. The overall clot strength, measured using the Quantra^® hemostasis analyzer, was restored with both fibrinogen concentrates but not cryoprecipitate. α₂antiplasmin and plasminogen activator inhibitor-1 (PAI-1) were constituents of cryoprecipitate but were negligible in RiaSTAP^® and FibCLOT^®. Interestingly, cryoprecipitate and FibCLOT^® contained significantly higher factor XIII (FXIII) levels, approximately three-fold higher than RiaSTAP^®. Our data show that 1 mg/mL fibrinogen, a clinically achievable concentration, can restore adequate clot integrity. However, FibCLOT^®, which contained more FXIII, was superior in normalizing the clot structure and in stabilizing hemodiluted clots against mechanical and fibrinolytic degradation.     

Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.     

Ultra-flexible PLA-based blends for the manufacturing of biodegradable tamper-evident screw caps by injection molding

Mono-material in the manufacturing of oil-based plastic bottles has been so far not considered because of the poor flexibility of polyethylene terephthalate (PET). The undercuts of PET caps (i.e., the threads of the screw caps) would be torn-off during the ejection step of the injection/compression molding processes that are commonly used to fabricate them. In this respect, the present work deals with the design and development of innovative biodegradable polyester-based blends with high flexibility, intended for the manufacturing of screw-cap, being this element the most critical in the implementation of the complete bottle. The blend is based on polylactic acid and it is tailored for injection molding of tamper-evident screw plastic caps by the addition of another biodegradable polyester, that is, polybutylene adipate-co-terephthalate. The effect of the addition of a barrier agent, polyvinyl alcohol, on the overall behavior of the blends was also studied in the perspective of the implementation of caps featuring barrier properties against water vapor and oxygen. The same material could be, however, easily tailored for the fabrication of the bottle body and printed label to get to the mono-material bottle. Twin-screw corotating extrusion of the compound, injection molding of the plastic material for the fabrication of the screw caps, and evaluation of the thermophysical and mechanical properties of the screw caps are hereinafter discussed. The biodegradable polyester-based blends are found to be suitable for injection molding of the tamper-evident screw caps, exhibiting adequate flexibility during ejection from the mold. The screw caps can also boast appreciable mechanical strength and impact resistance as well as good thermal stability.     

Glycosyltransferase B4GALNT2 as a Predictor of Good Prognosis in Colon Cancer: Lessons from Databases

Background: glycosyltransferase B4GALNT2 and its cognate carbohydrate antigen Sd^a are highly expressed in normal colon but strongly downregulated in colorectal carcinoma (CRC). We previously showed that CRC patients expressing higher B4GALNT2 mRNA levels displayed longer survival. Forced B4GALNT2 expression reduced the malignancy and stemness of colon cancer cells. Methods: Kaplan–Meier survival curves were determined in “The Cancer Genome Atlas” (TCGA) COAD cohort for several glycosyltransferases, oncogenes, and tumor suppressor genes. Whole expression data of coding genes as well as miRNA and methylation data for B4GALNT2 were downloaded from TCGA. Results: the prognostic potential of B4GALNT2 was the best among the glycosyltransferases tested and better than that of many oncogenes and tumor suppressor genes; high B4GALNT2 expression was associated with a lower malignancy gene expression profile; differential methylation of an intronic B4GALNT2 gene position and miR-204-5p expression play major roles in B4GALNT2 regulation. Conclusions: high B4GALNT2 expression is a strong predictor of good prognosis in CRC as a part of a wider molecular signature that includes ZG16, ITLN1, BEST2, and GUCA2B. Differential DNA methylation and miRNA expression contribute to regulating B4GALNT2 expression during colorectal carcinogenesis.