A NANOSPHERICAL DENDRIMERIC GALLATE ESTER FOR LONG TERM PRESERVATION OF ESSENTIAL OILS: AN INTEGRATED CHEMOMETRIC ASSISTED FT-IR STUDY

Essential oils (EOs) are hydrophobic concentrated liquids from plants made of volatile chemical compounds. EOs are very popular in the food, cosmetic and pharmaceutical industry as aromas, fragrances and alternative therapeutic devices [1, 2]. EOs are susceptible to degradation reactions, especially of oxidative type, triggered by temperature, light and oxygen availability. A loss of quality and alterations of sensory and pharmacological properties may occur, causing the production of smelly or even harmful compounds, responsible for allergic reactions and skin irritation [3-5]. For preventing and delaying EOs\u2019 spoilage, synthetic preservatives as 2,6-bis(1,1-dimetiletil)-4-metilphenol (BHT) or t-butil-4-hydrohyanisole (BHA) are commonly adopted; but, in addition to a limited efficiency due mainly to poor solubility in oils, they may cause health diseases [6]. Natural polyphenols as gallic acid (GA) are nowadays proposed as safer alternatives, but their efficiency is limited by their low compatibility with hydrophobic material again, or by the occurrence of probable side reactions with oils constituents. Recently, a hydrophobic and biodegradable GA-enriched dendrimer (GAD) (Fig. 1.a) characterised by a nanospherical morphology (Fig. 1.b) and endowed with a remarkable antioxidant activity was synthetized [7]. Further studies currently being completed, have shown that GAD, with respect to free GA, possesses also more efficient antibacterial properties against several antibiotics-resistant G+ strains, inhibits platelet aggregation and ROS accumulation thus representing an excellent alternative to conventional drugs to combat infections and thrombus formation [8]. In this study, based on integrated results obtained from the due investigations, GAD is advised also as an innovative and semi-synthetic preservative additive. a) b) Figure 1. Intuitive representation of GA-enriched dendrimer (GAD) structure (a); SEM images of GAD spherical nanoparticles (b). Scale bars represent 300 nm. In this regard, GAD proved a much more efficient preservative power than free GA and, unlike GA, it never acts as a pro-oxidant. Besides classic oxidation indexes, the desired information was obtained by FT-IR spectroscopy assisted by multivariate analysis (MVA). For further confirmation of the so obtained results, interpretations of FT-IR data by considering the area of some selected informative bands and iodometric titrations to determine the hydro peroxide value (PV) were also performed [9]. References [1] Yamamoto S., SOFW J., 2008, 134, 8. [2] Jiang Y., Wu N., Fu Y.-J., Wang W., Luo M., Zhao C.-J., Zu Y.-G., and Liu Y.-L., Environ. Toxicol. Pharmacol., 2011, 32, 63. [3] Hagvall L., Skold M., Brared-Christensson J., Borje A., and Karlberg A.-T., Contact Dermatitis, 2008, 59, 143. [4] Skold M., Hagvall L., and Karlberg A.-T., Contact Dermatitis, 2008, 58, 9. [5] Brared-Christensson J., Matura M., Gruvberger B., Bruze M., and Karlberg A.-T., Contact Dermatitis, 2010, 62, 32. [6] Hirose M., Takesada Y., Tanaka H., Tamano S., Kato T., and Shirai T., Carcinogenesis, 1998, 19, 207. [7] Alfei S., Catena S., and Turrini F., Drug Deliv. Trans. Res., under review. [8] Alfei S., Signorello M. A., Schito A., Catena S., and Turrini F., results not yet published [9] Alfei S., Oliveri P., and Malegori C., New J. Chem., under review

Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo.

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell-specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8 <sup>+</sup> T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events

Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation

Mitochondrial dynamics and quality control are crucial for neuronal survival and their perturbation is a major cause of neurodegeneration. m-AAA complex is an ATP-dependent metalloprotease located in the inner mitochondrial membrane and involved in protein quality control. Mutations in the m-AAA subunits AFG3L2 and paraplegin are associated with autosomal dominant spinocerebellar ataxia (SCA28) and autosomal recessive hereditary spastic paraplegia (SPG7), respectively. We report a novel m-AAA-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-Dopa-responsive parkinsonism. The proband carried a de-novo AFG3L2 heterozygous mutation (p.R468C) along with a heterozygous maternally-inherited intragenic deletion of SPG7. Functional analysis in yeast demonstrated the pathogenic role of AFG3L2 p.R468C mutation shedding light on its pathogenic mechanism. Analysis of patient's fibroblasts showed an abnormal processing pattern of OPA1, a dynamin-related protein essential for mitochondrial fusion and responsible for most cases of hereditary optic atrophy. Consistently, assessment of mitochondrial morphology revealed a severe fragmentation of the mitochondrial network, not observed in SCA28 and SPG7 patients\u2019 cells. This case suggests that coincidental mutations in both components of the mitochondrial m-AAA protease may result in a complex phenotype and reveals a crucial role for OPA1 processing in the pathogenesis of neurodegenerative disease caused by m-AAA defects

Etiological research in pediatric multiple sclerosis: A tool to assess environmental exposures (PEDiatric Italian Genetic and enviRonment ExposurE Questionnaire)

Background: The etiology of pediatric-onset multiple sclerosis is unknown although putative genetic and environmental factors appear to be involved. Among children multiple sclerosis onset occurs closer to the susceptibility window thank in adults and the exposure to etiological environmental factors is more informative. An Italian multicentre case-control study (the PEDiatric Italian Genetic and enviRonment ExposurE, PEDIGREE study) was designed to investigate environmental exposures in pediatric-onset multiple sclerosis and their interaction with genetics. Objectives: To collect evidence on exposures to environmental risk factors in pediatric-onset multiple sclerosis, a questionnaire was developed for the Italian population (PEDIGREE Questionnaire) and is presented. Methods: PEDIGREE Questionnaire develops from an existing tool used in case-control studies on pediatric-onset multiple sclerosis in US Americans, and was translated, adapted and tested for the contents perceived relevance, acceptability, feasibility and reliability in a population of Italian pediatric subjects and their parents recruited from clinics and general population. Results: PEDIGREE Questionnaire contents were overall deemed relevant by the study population, acceptable for 100% participants and feasible for at least 98%. PEDIGREE Questionnaire degree of reliability ranged 56% to 72%. Conclusion: PEDIGREE Questionnaire proves to be an efficient tool to assess environmental exposures in the Italian pediatric population. We encourage the dissemination of population-specific questionnaires and shared methodology to optimize efforts in MS etiological research

Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation

How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5AH) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer

Consensus protocol for EEG and amplitude-integrated EEG assessment and monitoring in neonates

The aim of this work is to establish inclusive guidelines on electroencephalography (EEG) applicable to all neonatal intensive care units (NICUs). Guidelines on ideal EEG monitoring for neonates are available, but there are significant barriers to their implementation in many centres around the world. These include barriers due to limited resources regarding the availability of equipment and technical and interpretive round-the-clock personnel. On the other hand, despite its limitations, amplitude-integrated EEG (aEEG) (previously called Cerebral Function Monitor [CFM]) is a common alternative used in NICUs. The Italian Neonatal Seizure Collaborative Network (INNESCO), working with all national scientific societies interested in the field of neonatal clinical neurophysiology, performed a systematic literature review and promoted interdisciplinary discussions among experts (neonatologists, paediatric neurologists, neurophysiologists, technicians) between 2017 and 2020 with the aim of elaborating shared recommendations. A consensus statement on videoEEG (vEEG) and aEEG for the principal neonatal indications was established. The authors propose a flexible frame of recommendations based on the complementary use of vEEG and aEEG applicable to the various neonatal units with different levels of complexity according to local resources and specific patient features. Suggestions for promoting cooperation between neonatologists, paediatric neurologists, and neurophysiologists, organisational restructuring, and teleneurophysiology implementation are provided

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype–phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype–phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors

PDCD10 gene mutations in multiple cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype