Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

Non-chemotherapy adjuvant agents in TP53 mutant Ewing sarcoma.

Ewing sarcoma (EWS) is a malignant tumor arising in bone or soft tissue that occurs in adolescent and young adult patients as well as adults later in life. Although non-metastatic EWS is typically responsive to treatment when newly diagnosed, relapsed cases have an unmet need for which no standard treatment approach exists. Recent phase III clinical trials for EWS comparing 7 vs 5 chemotherapy drugs have failed to improve survival. To extend the durability of remission for EWS, we investigated 3 non-chemotherapy adjuvant therapy drug candidates to be combined with chemotherapy. The efficacy of these adjuvant drugs was investigated via anchorage-dependent growth assays, anchorage-independent soft-agar colony formation assays and EWS xenograft mouse models. Enoxacin and entinostat were the most effective adjuvant drug in both long-term in vitro and in vivo adjuvant studies. In the context that enoxacin is an FDA-approved antibiotic, and that entinostat is an investigational agent not yet FDA-approved, we propose enoxacin as an adjuvant drug for further preclinical and clinical investigation in EWS patients.

Skeletal calcification patterns of batoid, teleost, and mammalian models: Calcified cartilage versus bone matrix.

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un-remodeled throughout the life span. Ca2+ and PO4 3- concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2 O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system. HIGHLIGHTS: Comparative physico-chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices. R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values. Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix. These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs. Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix.

Fatal leukodystrophy in Costello syndrome: a case report.

BACKGROUND: Costello syndrome (CS) is a rare genetic condition characterized by dysregulation of the signaling pathway, phenotypic alteration due to fetal macrosomia or growth retardation, facial abnormalities, loose skin, cardiovascular abnormalities, and a variable degree of intellectual disability. CASE PRESENTATION: We describe the case of a 20-month-old male patient with fetal macrosomia and polyhydramnios, presenting psychomotor development delay and growth limitation during the first months of life. CS was diagnosed at four months of age after detecting a variant of the HRAS gene c.35G > C (p.G12A). A clinical description of his condition was recorded throughout his life, including cardiovascular diseases, endocrine disorders, and recurrent infections. At 20 months of age, after presenting events of marked hypotonia and generalized seizures, brain magnetic resonance revealed symmetrical lesions of the infra- and supratentorial white matter in both cerebral hemispheres, which resulted in the diagnosis of cerebral leukodystrophy. The patient had a rapid and progressive deterioration that eventually led to death. CONCLUSIONS: This is the first report of a case of CS in Peru. In addition, this is a case that presented with multisystemic conditions culminating in leukodystrophy, which is a rare event according to the literature.

Fin systems comparative anatomy in model Batoidea Raja asterias and Torpedo marmorata: Insights and relatioships between musculo-skeletal layout, locomotion and morphology.

The macroscopic and microscopic morphology of the appendicular skeleton was studied in the two species Raja asterias (order Rajiformes) and Torpedo marmorata (Order Torpediniformes), comparing the organization and structural layout of pectoral, pelvic, and tail fin systems. The shape, surface area and portance of the T. marmorata pectoral fin system (hydrodynamic lift) were conditioned by the presence of the two electric organs in the disk central part, which reduced the pectoral fin surface area, suggesting a lower efficiency of the "flapping effectors" than those of R. asterias. Otherwise, radials' rays alignment, morphology and calcification pattern showed in both species the same structural layout characterized in the fin medial zone by stiffly paired columns of calcified tiles in the perpendicular plane to the flat batoid body, then revolving and in the horizontal plane to continue as separate mono-columnar rays in the fin lateral zone with a morphology suggesting fin stiffness variance between medial/lateral zone. Pelvic fins morphology was alike in the two species, however with different calcified tiles patterns of the 1st compound radial and pterygia in respect to the fin-rays articulating perpendicularly to the latter, whose tile rows lay-out was also different from that of the pectoral fins radials. The T. marmorata tail-caudal fin showed a muscular and connective scaffold capable of a significant oscillatory forward thrust. On the contrary, the R. asterias dorsal tail fins were stiffened by a scaffold of radials-like calcified segments. Histomorphology, heat-deproteination technique and morphometry provided new data on the wing-fins structural layout which can be correlated to the mechanics of the Batoid swimming behavior and suggested a cartilage-calcification process combining interstitial cartilage growth (as that of all vertebrates anlagen) and a mineral deposition with accretion of individual centers (the tiles). The resulting layout showed scattered zones of un-mineralized matrix within the calcified mass and a less compact texture of the matrix calcified fibers suggesting a possible way of fluid diffusion throughout the mineralized tissue. These observations could explain the survival of the embedded chondrocytes in absence of a canalicular system as that of the cortical bone.

Characterization of Italian Plasmopara viticola populations for resistance to oxathiapiprolin.

BACKGROUND: Oxathiapiprolin is a novel fungicide and the first of the piperidinyl-thiazole-isoxazoline class to be discovered. This fungicide has been reported to have high activity against Plasmopara viticola, the grapevine downy mildew agent, and other plant-pathogenic oomycetes. In this study, the baseline sensitivity of Italian P. viticola populations towards oxathiapiprolin was established on 29 samples collected in 10 different viticultural areas. Two insensitive strains were characterized for their mechanism of resistance. RESULTS: Oxathiapiprolin exhibited substantial inhibitory activity against 27 of the 29 populations tested, with EC50 values ranging from a minimum of under 4 × 10-5  mg L-1 to over 4 × 10-1  mg L-1 , with an average value of 3.2 × 10-2  mg L-1 . Two stable suspected oxathiapiprolin-resistant mutants were isolated from population exhibiting reduced sensitivity, and sequenced for the oxathiapiprolin target gene PvORP1. The comparison with wild-type isolates revealed that the resistant isolates possessed a heterozygous mutation causing the amino acid substitution N837I, recently reported in the literature. CONCLUSION: The results obtained indicate a risk for Italian P. viticola populations to develop resistance to oxathiapiprolin in association with the N837I mutation at PvORP1. Anti-resistance strategies should be carefully implemented and the sensitivity levels to this molecule should be monitored accurately in future to preserve its effectiveness. © 2022 Society of Chemical Industry.

Computer tomography and magnetic resonance for multimodal imaging of fossils and mummies.

The study of fossils and mummies has largely benefited from the use of modern noninvasive and nondestructive imaging technologies and represents a fast developing area. In this review, we describe the emerging role of imaging based on Magnetic Resonance (MR) and Computer Tomography (CT) employed for the study of ancient remains and mummies. For each methodology, the state of the art in paleoradiology applications is described, by emphasizing new technologies developed in the field of both CT, such as micro- and nano-CT, dual-energy and multi-energy CT, and MR, with the description of novel dedicated sequences, radiofrequency coils and gradients. The complementarity of CT and MR in paleoradiology is also discussed, by pointing out what MR provides in addition to CT, with an overview on the state of the art of emerging strategies in the use of CT/MR combination for the study of a sample following a multimodal integrated approach.

Alveolar Bone Remodeling with or without Collagen Filling of the Extraction Socket: A High-Resolution X-ray Tomography Animal Study.

The healing process of the tooth extraction socket often leads to significant resorption of the alveolar bone, eventually causing clinical difficulties for future implant-supported rehabilitations. The aim of the present animal study was to evaluate alveolar bone remodeling after tooth extraction in a rabbit model, either with or without the use of a plain collagen plug inside the socket, by means of micro-computed tomography. The study included the micro-tomography analysis of 36 rabbits' incisor extraction sockets, either left empty or filled with a collagen plug. All animals were euthanized in a staggered manner, in order to address molecular, histologic, and radiographic analyses at different time-points, up to 90 days after surgery. The three-dimensional evaluation was carried out using micro-computed tomography technology on excised bone blocks including the alveolus and the contralateral bone. Both linear and volumetric measures were recorded: the percentage of bone volume change (ΔBV) within the region of interest was considered the primary endpoint of the study. The micro-CT analysis revealed mean volumetric changes of -58.1% ± from baseline to 3 months for the control group, and almost no bone loss for the test group, -4.6%. The sockets treated with the collagen plug showed significantly less dimensional resorption, while the natural-healing group showed an evident collapse of the alveolar bone three months after extraction surgery.

Georgian Grapevine Cultivars: Ancient Biodiversity for Future Viticulture.

Grapevine (Vitis vinifera) is one of the most widely cultivated plant species of agricultural interest, and is extensively appreciated for its fruits and the wines made from its fruits. Considering the high socio-economic impact of the wine sector all over the world, in recent years, there has been an increase in work aiming to investigate the biodiversity of grapevine germplasm available for breeding programs. Various studies have shed light on the genetic diversity characterizing the germplasm from the cradle of V. vinifera domestication in Georgia (South Caucasus). Georgian germplasm is placed in a distinct cluster from the European one and possesses a rich diversity for many different traits, including eno-carpological and phenological traits; resistance to pathogens, such as oomycetes and phytoplasmas; resistance to abiotic stresses, such as sunburn. The aim of this review is to assess the potential of Georgian cultivars as a source of useful traits for breeding programs. The unique genetic and phenotypic aspects of Georgian germplasm were unraveled, to better understand the diversity and quality of the genetic resources available to viticulturists, as valuable resources for the coming climate change scenario.

Condylar Changes in Children with Posterior Crossbite after Maxillary Expansion: Tridimensional Evaluation.

(1) Background: To investigate condylar position in subjects with functional posterior crossbite comparing findings before and after rapid maxillary expansion (RME) treatment through 3D analysis; (2) Methods: Thirty-two Caucasian patients (14 males, mean age 8 y 8 m ± 1 y 2 m; 18 females mean age 8 y 2 m ± 1 y 4 m) with functional posterior crossbite (FPXB) diagnosis underwent rapid palatal expansion with a Haas appliance banded on second deciduous upper molars. Patients' underwent CBCT scans before rapid palatal expansion (T0) and after 12 months (T1). The images were processed through 3D slicer software; (3) Results: The condylar position changes between T1 and T0 among the crossbite and non-crossbite sides were not statistically significant, except for the transversal axis. At T1, the condyles moved forward (y axis) and laterally (x axis), they also moved downward (z axis) but not significantly; (4) Conclusions: Condilar position in growing patients with functional posterior crossbite did not change significantly after rapid maxillary expansion.

Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among "Candidatus Phytoplasma Solani" Strains Associated with Grapevine Bois Noir.

Grapevine Bois noir (BN) is associated with infection by "Candidatus Phytoplasma solani" (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

Anthocyanin Biosynthesis and DNA Methylation Dynamics in Sweet Orange Fruit [Citrus sinensis L. (Osbeck)] under Cold Stress.

The blood red color of pigmented orange fruit varieties [Citrus sinensis L. (Osbeck)] is due to the presence of anthocyanin pigments that largely contribute to determine the high organoleptic qualities and the nutritional properties of the fruits. The content of pigments in sweet orange depends primarily on genetic factors and on environmental conditions. In particular, it has been extensively shown that cold temperature induces an increase of anthocyanin content that is achieved by the induction of the related gene expression. The purpose of our work is to understand the mechanism underlying the color variegation occurring inside the blood oranges during the cold induction of anthocyanin biosynthesis, despite the fact that the entire fruit is genotypically programmed to produce pigments. Therefore, the amount of anthocyanin and the expression of both structural and regulatory genes have been monitored in either high-pigmented (HP) or not/low pigmented (NP) segments of the same fruit during the storage at 4 °C for a total experimental period of 25 days. Our results clearly indicate that the anthocyanin content is directly correlated with the levels of gene transcription, with higher pigmented areas showing higher enhancement of gene expression. Furthermore, we analyzed the reshaping of the DNA methylation status at the promoter regions of genes related to anthocyanin biosynthetic pathway, such as DFR and Ruby. Our results unequivocally demonstrate that in the promoter regions of both DFR and Ruby, the amount of cytosine methylation strongly decreases along the cold storage in the HP areas, whereas it increases in the NP areas of the same fruit, probably causing a partial block of the gene transcription. Finally, by measuring the changes in the expression levels of the Citrus DNA demethylases, we found that DML1 might play a crucial role in determining the observed demethylation of DFR and Ruby promoters, with its expression induced by cold in the HP areas of the fruits. This is the first report in which different levels of gene expression implicated in anthocyanin production in blood orange fruit is correlated with an epigenetic control mechanism such as promoter methylation.

Characterization of fungicide sensitivity profiles of Botrytis cinerea populations sampled in Lombardy (Northern Italy) and implications for resistance management.

BACKGROUND: Resistance to fungicides is one of the aspects that must be considered when planning treatments to achieve an optimal control of grey mold, caused by Botrytis cinerea, in vineyards. In this study, extensive fungicide resistance monitoring was carried out in Northern Italy (Lombardy region) to evaluate several aspects of fungicide resistance (frequency of resistance, effect of field treatments, mechanism of resistance and fitness) on 720 B. cinerea strains isolated from 36 vineyards. RESULTS: Of the characterized strains, 12% were resistant to a single fungicide class (3% to the succinate dehydrogenase inhibitor boscalid, 4% to the anilinopirimidine cyprodinil; 5% to the phenylpirrole fludioxonil; 0.1% to the ketoreductase inhibitor fenhexamid) and 0.8% to two fungicide classes contemporaneously. Resistance was associated with mutations reported in the literature for boscalid (H272Y/R) and fenhexamid (P238S or I232M). Two new mutations in sdhC (A187F) and in sdhD (I189L) could be related to boscalid resistance. Strains resistant to fludioxonil did not show any known mutations. No significant differences were found in the fitness of sensitive and resistant strains. CONCLUSION: Overall, field populations of B. cinerea showed a relatively low frequency of resistance, but the geographical distribution of resistance, genetic mechanisms of resistance and fitness of resistant strains suggest that management of resistance should be implemented, at local and regional levels. Particular attention should be given to the fungicide sprays planned before veraison, since they seem to be associated with a higher frequency of resistant strains in vineyards. © 2020 Society of Chemical Industry.

Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy.

A topic that has attracted considerable interest in recent years is the possibility to perform thermodynamic studies of proteins directly in-cell or in complex environments which mimic the cellular interior. Nuclear magnetic resonance (NMR) could be an attractive technique for these studies but its applicability has so far been limited by technical issues. Here, we demonstrate that 2D NMR methods can be successfully applied to measure thermodynamic parameters provided that a suitable choice of the residues used for the calculation is made. We propose a new parameter, named RAD, which reflects the level of protection of a specific amide proton in the protein core and can guide through the selection of the resonances. We also suggest a way to calibrate the volumes to become independent of technical limitations. The methodology we propose leads to stability curves comparable to that calculated from CD data and provides a new tool for thermodynamic measurements in complex environments.

Numerical simulation of novel concept 4D cardiac microtomography for small rodents based on all-optical Thomson scattering X-ray sources.

Accurate dynamic three-dimensional (4D) imaging of the heart of small rodents is required for the preclinical study of cardiac biomechanics and their modification under pathological conditions, but technological challenges are met in laboratory practice due to the very small size and high pulse rate of the heart of mice and rats as compared to humans. In 4D X-ray microtomography (4D µCT), the achievable spatio-temporal resolution is hampered by limitations in conventional X-ray sources and detectors. Here, we propose a proof-of-principle 4D µCT platform, exploiting the unique spatial and temporal features of novel concept, all-optical X-ray sources based on Thomson scattering (TS). The main spatial and spectral properties of the photon source are investigated using a TS simulation code. The entire data acquisition workflow has been also simulated, using a novel 4D numerical phantom of a mouse chest with realistic intra- and inter-cycle motion. The image quality of a typical single 3D time frame has been studied using Monte Carlo simulations, taking into account the effects of the typical structure of the TS X-ray beam. Finally, we discuss the perspectives and shortcomings of the proposed platform.

Combined Effect of Fatty Diet and Cognitive Decline on Brain Metabolism, Food Intake, Body Weight, and Counteraction by Intranasal Insulin Therapy in 3×Tg Mice.

Obesity and cognitive decline can occur in association. Brain dysmetabolism and insulin resistance might be common underlying traits. We aimed to examine the effect of high-fat diet (HFD) on cognitive decline, and of cognitive impairment on food intake and body-weight, and explore efficacy of chronic intranasal insulin (INI) therapy. We used control (C) and triple transgenic mice (3×Tg, a model of Alzheimer's pathology) to measure cerebral mass, glucose metabolism, and the metabolic response to acute INI administration (cerebral insulin sensitivity). Y-Maze, positron emission-computed tomography, and histology were employed in 8 and 14-month-old mice, receiving normal diet (ND) or HFD. Chronic INI therapy was tested in an additional 3×Tg-HFD group. The 3×Tg groups overate, and had lower body-weight, but similar BMI, than diet-matched controls. Cognitive decline was progressive from HFD to 3×Tg-ND to 3×Tg-HFD. At 8 months, brain fasting glucose uptake (GU) was increased by C-HFD, and this effect was blunted in 3×Tg-HFD mice, also showing brain insulin resistance. Brain mass was reduced in 3×Tg mice at 14 months. Dentate gyrus dimensions paralleled cognitive findings. Chronic INI preserved cognition, dentate gyrus and metabolism, reducing food intake, and body weight in 3×Tg-HFD mice. Peripherally, leptin was suppressed and PAI-1 elevated in 3×Tg mice, correlating inversely with cerebral GU. In conclusion, 3×Tg background and HFD exert additive (genes*lifestyle) detriment to the brain, and cognitive dysfunction is accompanied by increased food intake in 3×Tg mice. PAI-1 levels and leptin deficiency were identified as potential peripheral contributors. Chronic INI improved peripheral and central outcomes.

Barcoding the Neotropical freshwater fish fauna using a new pair of universal COI primers with a discussion of primer dimers and M13 primer tails.

Designing primers for DNA barcoding is a significant challenge for the rich Neotropical fish fauna, which is comprised of ∼6000 species. Previously, researchers required multiple pairs of PCR primers or primer cocktails to obtain standard COI (i.e., mitochondrial cytochrome c oxidase subunit I) barcode sequences from assemblages of freshwater fish in this region. To simplify DNA barcoding and metabarcoding studies of Neotropical freshwater fish, we present a new pair of COI primers, which have yielded high quality barcodes across six teleost orders-Characiformes, Cichliformes, Cyprinodontiformes, Gymnotiformes, Siluriformes, and Synbranchiformes-native to South America. Following previous fish barcoding studies, we also tailed our primers with M13 forward and reverse primers to facilitate the DNA sequencing process. Although this practice generates primer dimers, we obtained complete and high quality COI barcode sequences for all samples. We discuss the problem of primer dimers and suggest strategies for neutralizing their influence on data quality.

Metabolic effects of treatment in patients with obstructive sleep apnea: a systematic review.

INTRODUCTION: Growth retardation was frequently observed in obstructive sleep apnea (OSA) patients but the complex mechanisms causing this condition are still unclear. Several findings suggested growth catch-up after surgical treatment but other studies did not confirm the results, showing no improvement in OSA patients after tonsillectomy and adenoidectomy (T&A). The aim of the present study was to systematically review scientific literature of the relationships between OSA and metabolic changes involving growth hormone (GH) axis before and after treatment in patients. EVIDENCE ACQUISITION: Different databases were searched without limitations up to August 2017. Additionally, the bibliographies of the finally selected articles were hand searched to identify any relevant publications that were not previously identified. EVIDENCE SYNTHESIS: From the 37 studies collected from all the databases based on their title and abstract, only 12 studies actually fulfilled the selection criteria. From the twelve finally selected articles, eight focused on growth mediators, two evaluated endothelial mediators, one focused on neurocognitive function and mediators and one focused on local inflammation. CONCLUSIONS: OSA children present lower levels of growth mediators (IGF-I and IGFBP-3) thus indicating growth retardation, significantly higher cardiovascular disease risk and decreased cognitive functions when compared to controls. T&A seems to improve all the above mentioned functions with great impact on general health.

Studies of Microbiota Dynamics Reveals Association of "Candidatus Liberibacter Asiaticus" Infection with Citrus (Citrus sinensis) Decline in South of Iran.

Citrus Decline Disease was recently reported to affect several citrus species in Iran when grafted on a local rootstock variety, Bakraee. Preliminary studies found "Candidatus Phytoplasma aurantifoliae" and "Candidatus Liberibacter asiaticus" as putative etiological agents, but were not ultimately able to determine which one, or if an association of both, were causing the disease. The current study has the aim of characterizing the microbiota of citrus plants that are either asymptomatic, showing early symptoms, or showing late symptoms through amplification of the V1­V3 region of 16S rRNA gene using an Illumina sequencer in order to (i) clarify the etiology of the disease, and (ii) describe the microbiota associated to different symptom stages. Our results suggest that liberibacter may be the main pathogen causing Citrus Decline Disease, but cannot rule out the possibility of phytoplasma being involved as well. The characterization of microbiota shows that the leaves show only two kinds of communities, either symptomatic or asymptomatic, while roots show clear distinction between early and late symptoms. These results could lead to the identification of bacteria that are related to successful plant defense response and, therefore, to immunity to the Citrus Decline Disease.

3D fiber deposited polymeric scaffolds for external auditory canal wall.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.