Carbohydrate Active enZYmes: functional study and applicative perspectives

Carbohydrates play an important role in a variety of biological and industrial process. The numerous combinatorial possibilities of glycan building blocks explains the variety of competing and sequentially acting enzymatic activies involved in their modification, such as glycosidases, glycosyltransferases and esterases. These enzymes build, modify, and hydrolyze complex carbohydrates and glycoconjugates addressing a large body of biological roles. The discovery of novel thermophilic carbohydrate active enzymes can help us to understand the functional involvement of this class of enzymes in various biological phenomena and how we can exploit their “eco-friendly” activities in numerous industrial processes. This thesis work is dedicated to the study of thermophilic CAZymes and can be subdivided in three principal sections. The first section is focused on the identification of novel thermophilic glycosidases selected in sequence database. Since sustainable biorefineries require numerous biocatalysts, the research topic was addressed at the identification of new thermostable hemicellulases that can be used in the production of second generation bioethanol. The search of novel thermostable enzymes led us to sequence metagenomic DNA isolated from a solfataric field Pisciarelli. In this context, the changes of microbiome colonizing this enviroment in relation to geothermal variations were explored, as described in the second sections. This study allowed to increase our knowledge on habitats with high selective pressure. In the third section, novel cazyme from crenarchaeon S. solfataricus P2 was identified by functional screening. This study led us to achieve additional data concerning the set of cazymes present in S. solfataricus allowing to identification of a novel unclassified enzyme

Effect of argon plasma abutment activation on soft tissue healing: RCT with histological assessment.

OBJECTIVE To assess the peri-implant soft tissue profiles between argon plasma treatment (PT) and non-treated (NPT) healing abutments by comparing clinical and histological parameters 2 months following abutment placement. MATERIALS AND METHODS Thirty participants were randomly assigned to argon-plasma treatment abutments group (PT) or non-treated abutments (NPT) group. Two months after healing abutment placement, soft peri-implant tissues and abutment were harvested, and histological and clinical parameters including plaque index, bleeding on probing, and keratinized mucosa diameter (KM) were assessed. Specialized stainings (hematoxylin-eosin and picrocirious red) coupled with immunohistochemistry (vimentin, collagen, and CK10) were performed to assess soft tissue inflammation and healing, and the collagen content keratinization. In addition to standard statistical methods, machine learning algorithms were applied for advanced soft tissue profiling between the test and control groups. RESULTS PT group showed lower plaque accumulation and inflammation grade (6.71% vs. 13.25%, respectively; p-value 0.02), and more advanced connective tissue healing and integration compared to NPT (31.77% vs. 23.3%, respectively; p = 0.009). In the control group, more expressed keratinization was found compared to the PT group, showing significantly higher CK10 (>47.5%). No differences in KM were found between the groups. SIGNIFICANCE PT seems to be a promising protocol for guided peri-implant soft tissue morphogenesis reducing plaque accumulation and inflammation, and stimulating collagen and soft tissue but without effects on epithelial tissues and keratinization

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

Background: The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini. Results: We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10- XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Conclusion: Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions

Hybrid Funnel Technique: A Novel Approach for Implant Site Preparation: A Pilot Study.

(1) Background: Different techniques and tools have been developed for implant site preparation. In this clinical scenario, Hybrid Funnel Technique (HFT), a novel osteotomy procedure, has been proposed. (2) Aim: The aim of this retrospective observational study was to consider the different responses to compression of the histological bony compartments (cancellus and cortical). HFT involves the use of multiple drills for the cortical layer preparation and of an osteotome for the osteocompaction of the cancellous bone. (3) Materials and Methods: Following computer-supported implant planning and guided surgery, 10 osteotomies with HFT were performed and 10 implants with the same length and diameter were placed in seven healthy and no daily smoking patients. Periapical X-ray and intraoral photographs were performed at baseline and after 12 months of follow-up to evaluate marginal bone level (MBL) changes and aesthetic results obtained from implant prosthetic rehabilitation. (4) Results: At 1 year of follow-up, 100% of the implants were successfully integrated, MBL change mean value was 0.17 mm ± 0.21. No differences in terms of MBL were noted between thin and thick biotypes. Pink esthetic score (PES) and white esthetic score (WES), assessed one year after definitive restoration placement, were 7.5 ± 2.3 and 8.5 ± 1.1, respectively. (5) Conclusions: Based on the findings of this preliminary clinical study, HFT has led to stability of peri-implant tissues and could represent a reliable technique for surgical preparation of the implant site

Transcript Regulation of the Recoded Archaeal α-L-Fucosidase In Vivo

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discusse

Psychological Distress in Patients with Autoimmune Arthritis during the COVID-19 Induced Lockdown in Italy

Lockdowns imposed by governments worldwide as a way to limit the spread of severe atypical respiratory syndrome-coronavirus-2 (SARS-CoV2) have had heavy psychological and economic consequences. Arthritis patients are a vulnerable population at an increased risk of peritraumatic stress. This could be due to several reasons, including the fear of shortage of medicine and difficulty receiving periodical medical checks. In the present case-control study, psychological distress in patients with autoimmune arthritis during the coronavirus disease 2019 (COVID-19) pandemic were investigated. An electronic survey was conducted to gather information on the perceived change in the emotional state, general health (GH), fatigue, joint pain, and disease activity during the lockdown, in 100 patients with autoimmune arthritis and 100 controls. Mental health status was measured using the Depression, Anxiety and Stress Scale (DASS-21). The COVID-19 Peritraumatic Distress Index (CPDI) was used to assess the frequency of peritraumatic stress disorders related to COVID-19. Patients reported a significant worsening of perceived GH (36% vs. 7%; p < 0.001), a significantly higher mean CPDI score (p < 0.001) than controls. Using multivariate analysis, arthritis patients had significantly higher CPDI scores (+3.67 points; p = 0.019), independent of depression, anxiety, and stress symptoms, comorbidities, and sociodemographic and lifestyle characteristics. Logistic regression analysis showed that the risk of reporting worsened GH was 9-fold higher in patients than controls (p < 0.001). Patients with autoimmune arthritis are at higher risk of psychological distress related to COVID-19 pandemic; thus targeted intervention should be designed to strengthen coping capacity in this vulnerable populatio

Destroying the Shield of Cancer Stem Cells: Natural Compounds as Promising Players in Cancer Therapy

In a scenario where eco-sustainability and areduction in chemotherapeutic drug waste are certainly a prerogative to safeguard the biosphere, the use of natural products (NPs) represents an alternative therapeutic approach to counteract cancer diseases. The presence of a heterogeneous cancer stem cell (CSC) population within a tumor bulk is related to disease recurrence and therapy resistance. For this reason, CSC targeting presents a promising strategy for hampering cancer recurrence. Increasing evidence shows that NPs can inhibit crucial signaling pathways involved in the maintenance of CSC stemness and sensitize CSCs to standard chemotherapeutic treatments. Moreover, their limited toxicity and low costs for large-scale production could accelerate the use of NPs in clinical settings. In this review, we will summarize the most relevant studies regarding the effects of NPs derived from major natural sources, e.g., food, botanical, and marine species, on CSCs, elucidating their use in pre-clinical and clinical studies

Seabed mapping in the Pelagie Islands marine protected area (Sicily Channel, southern Mediterranean) using Remote Sensing Object Based Image Analysis (RSOBIA)

In this paper we present the seabed maps of the shallow-water areas of Lampedusa and Linosa, belonging to the Pelagie Islands Marine Protected Area. Two surveys were carried out (“Lampedusa 2015” and “Linosa 2016”) to collect bathymetric and acoustic backscatter data through the use of a Reson SeaBat 7125 high-resolution multibeam system. Ground-truth data, in the form of grab samples and diver video-observations, were also collected during both surveys. Sediment samples were analyzed for grain size, while video images were analyzed and described revealing the acoustic seabed and other bio-physical characteristics. A map of seabed classification, including sediment types and seagrass distribution, was produced using the tool Remote Sensing Object Based Image Analysis (RSOBIA) by integrating information derived from backscatter data and bathy-morphological features, validated by ground-truth data. This allows to create a first seabed maps (i.e. benthoscape classification), of Lampedusa and Linosa, at scale 1:20 000 and 1:32 000, respectively, that will be checked and implemented through further surveys. The results point out a very rich and largely variable marine ecosystem on the seabed surrounding the two islands, with the occurrence of priority habitats, and will be of support for a more comprehensive maritime spatial planning of the Marine Protected Area

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis