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.     

On the limit theory of the Gaussian SQMLE in the EGARCH(1,1) model

We derive the limit theory of the Gaussian stable quasi maximum likelihood estimator for the stationary EGARCH(1,1) model when the squared innovation process has marginals with regularly varying tails. We derive regularly varying rates and limiting stable distributions. We perform Monte Carlo experiments to assess the extent of the parameter space corresponding to the invertibility condition, and the quality of the asymptotic approximation.     

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (K_m ~1 µM), while inosine and guanosine displayed K_i values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.     

Increased 24-hour endothelin-1 urinary excretion in patients with chronic obstructive pulmonary disease

Abnormalities in endothelin-1 (ET-1) pulmonary metabolism have been reported in patients with pulmonary hypertension, asthma and chronic obstructive pulmonary disease (COPD). In this study we have evaluated the 24-hour urinary excretion of ET-1 in COPD patients both during acute exacerbation and stable phase of the disease. ET-1 plasma and urinary levels were measured in 13 COPD patients on admission to the hospital for an acute exacerbation and at the recovery period. Ten healthy volunteers were also studied. Determination of plasma and 24-Hour urinary ET-1 levels were carried out with a radioimmunoassay test. Plasma ET-1 levels in COPD patients were similar during exacerbation and recovery and were not significantly different from those in the healthy subjects. 24-hour urinary excretion of ET-1 was increased in COPD patients during acute exacerbation; it decreased during recovery, but remained elevated when compared to normal subjects. A negative correlation was found between arterial oxygen pressure and ET-1 excretion; no correlation was found between plasma and urinary ET-1 values. In conclusion, COPD patients excrete higher amounts of ET-1 compared to healthy subjects. Urinary ET-1 values are further increased during acute exacerbation of the disease.     

Senescence-Independent Anti-Inflammatory Activity of the Senolytic Drugs Dasatinib,Navitoclax, and Venetoclax in Zebrafish Models of Chronic Inflammation

Telomere shortening is the main molecular mechanism of aging, but not the only one. The adaptive immune system also ages, and older organisms tend to develop a chronic pro-inflammatory status with low-grade inflammation characterized by chronic activation of the innate immune system, called inflammaging. One of the main stimuli that fuels inflammaging is a high nutrient intake, triggering a metabolic inflammation process called metainflammation. In this study, we report the anti-inflammatory activity of several senolytic drugs in the context of chronic inflammation, by using two different zebrafish models: (i) a chronic skin inflammation model with a hypomorphic mutation in spint1a, the gene encoding the serine protease inhibitor, kunitz-type, 1a (also known as hai1a) and (ii) a non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) model with inflammation induced by a high-fat diet. Our results show that, although these models do not manifest premature aging, the senolytic drugs dasatinib, navitoclax, and venetoclax have an anti-inflammatory effect that results in the amelioration of chronic inflammation.     

Accuracy of FIB-4 to Detect Elevated Liver Stiffness Measurements in Patients with Non-Alcoholic Fatty Liver Disease: A Cross-Sectional Study in Referral Centers

The identification of advanced fibrosis by applying noninvasive tests is still a key component of the diagnostic algorithm of NAFLD. The aim of this study is to assess the concordance between the FIB-4 and liver stiffness measurement (LSM) in patients referred to two liver centers for the ultrasound-based diagnosis of NAFLD. Fibrosis 4 Index for Liver Fibrosis (FIB-4) and LSM were assessed in 1338 patients. A total of 428 (32%) had an LSM ≥ 8 kPa, whereas 699 (52%) and 113 (9%) patients had an FIB-4 < 1.3 and >3.25, respectively. Among 699 patients with an FIB-4 < 1.3, 118 (17%) had an LSM ≥ 8 kPa (false-negative FIB-4). This proportion was higher in patients ≥60 years, with diabetes mellitus (DM), arterial hypertension or a body mass index (BMI) ≥ 27 kg/m². In multiple adjusted models, age ≥ 60 years (odds ratio (OR) = 1.96, 95% confidence interval (CI) 1.19–3.23)), DM (OR = 2.59, 95% CI 1.63–4.13), body mass index (BMI) ≥ 27 kg/m² (OR = 2.17, 95% CI 1.33–3.56) and gamma-glutamyltransferase ≥ 25 UI/L (OR = 2.68, 95% CI 1.49–4.84) were associated with false-negative FIB-4. The proportion of false-negative FIB-4 was 6% in patients with none or one of these risk factors and increased to 16, 31 and 46% among those with two, three and four concomitant risk factors, respectively. FIB-4 is suboptimal to identify patients to refer to liver centers, because about one-fifth may be false negative at FIB-4, having instead an LSM ≥ 8 KPa.     

Metabolic and DNA Repair Variations in Susceptibility to Genotoxins

An overview on the influence of metabolic and DNA repair polymorphisms on biological indicators of genotoxic risk in occupational, environmental, or lifestyle exposure is presented in this article. Indicators of genotoxic risk include biomarkers of internal dose (urinary concentration of the substance or its metabolites and urinary mutagenicity), biological effective dose (protein and DNA adducts), and indicators of early biological effects (chromosome aberrations, sister chromatid exchanges, micronuclei, COMET assay, HPRT mutants). Genetic polymorphisms include those involved in the activation and detoxification (i.e., P-450 cytochrome, acetyltransferase, glutathione transferase) of various classes of carcinogens and the newly discovered polymorphisms affecting DNA repair enzymes. Genetic polymorphisms are assessed for their importance in detecting the impact of genotype on biological indicators of genotoxic risk.     

Effect of mechanical and air-particle cleansing protocols of provisional cement on immediate dentin sealing layer and subsequent adhesion of resin composite cement

Immediate dentin sealing (IDS) could avoid contamination of dentin from impression material and provisional cement but prior to final cementation of indirect restorations, removal of the provisional cement may damage the IDS. The objectives of this study were to investigate the effect of mechanical and air-particle cleansing protocols of provisional cement on IDS layer and subsequent adhesion of resin composite cement. The cuspal dentin surfaces of human third molars (N = 21, n_quadrant = 84) were exposed by a low-speed diamond saw under water cooling and conditioned with an adhesive system based on the three-step etch and rinse technique (OptiBond FL). Provisional cement (Freegenol) was applied on each specimen. They were then randomly divided into six subgroups where the provisional cement was removed either by (1) air-borne particle abrasion with 50-μm Al₂O₃ particles at 2 bar (AL2), (2) air-borne particle abrasion with 50-μm Al₂O₃ particles at 3.5 bar (AL3.5), (3) air-borne particle abrasion with 30-μm SiO₂ particles at 2 bar (SL2), (4) air-borne particle abrasion with 30-μm SiO₂ particles at 3.5 bar (SL3.5), (5) prophylaxy paste (Cleanic) (PP) or (6) pumice-water slurry (PW) at 1500 rpm for 15 s. The dentin surface on each tooth was assigned to four quadrants and each quadrant received the cleansing methods in a clockwise sequence. The non-contaminated and non-cleansed teeth acted as the control (C). Two separate teeth, contaminated and cleansed according to six cleansing protocols, were allocated for scanning electron microscopy (SEM) analysis (×2000). The dentin surfaces in each quadrant received resin composite luting cement (Variolink II, Ivoclar Vivadent) incrementally in a polyethylene mould (diameter: 1 mm²; height: 4 mm) and photopolymerized. The specimens were stored in distilled water for 24 h at 37 °C until the testing procedures and then shear force was applied to the adhesive interface until failure occurred in a universal testing machine (0.5 mm/min). Microshear bond (μSBS) was calculated by dividing the maximum load (N) by the bonding surface area of the resin cement. Failure types were analysed using optical microscope and SEM. Data (MPa) were analysed using one-way ANOVA (α = 0.05). Two-parameter Weibull distribution values including the Weibull modulus, scale (m) and shape (₀), values were calculated. Mean μSBS results (MPa) showed a significant difference between the experimental groups (p = 0.011) and were in a descending order as follows: C (8 ± 2.3)^a < AL2 (6.7 ± 2.4)^b < PP (6.9 ± 2)^b < PW (6.5 ± 2.1)^b < AL3.5 (5.8 ± 1.1)^b < SL2 (5.3 ± 1)^b < SL3.5 (5.2 ± 1)^b. Failure types were predominantly mixed failure type between the dentin and the adhesive resin which is a combination of adhesive and cohesive failures in the adhesive resin. Cohesive failure in the dentin was not observed in any of the groups. Weibull distribution presented lower shape (₀) for C (3.9), AL2 (3.2), PP (3.5) and PW (3.6). SEM analysis showed rough surfaces especially in the air-abraded groups whereas mechanical cleansing methods presented smoother surfaces and partially covered by particle remnants all of which occluded the dentin tubuli.     

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.     

Structure Formation of Integral Asymmetric Composite Membranes of Polystyrene‐block‐Poly(2‐vinylpyridine) on a Nonwoven

The formation of an integral asymmetric membrane composed of a cylinder‐forming polystyrene‐block‐poly(2‐vinylpyridine) on a nonwoven by using solvent casting followed by solvent/nonsolvent exchange (phase inversion) is reported for the first time. The influence of parameters such as solvent composition, evaporation time of the solution‐cast block copolymer film before phase inversion, and immersion bath temperature is demonstrated. The optimized membranes are characterized in terms of stimuli‐responsive water flux properties. The morphologies of the membranes as well as of the bulk of the block copolymer are imaged by scanning force microscopy, scanning electron microscopy, and transmission electron microscopy.