Dem-Aging: autophagy-related pathologies and the "two faces of dementia".

Neuronal ceroid lipofuscinosis (NCL) is an umbrella term referring to the most frequent childhood-onset neurodegenerative diseases, which are also the main cause of childhood dementia. Although the molecular mechanisms underlying the NCLs remain elusive, evidence is increasingly pointing to shared disease pathways and common clinical features across the disease forms. The characterization of pathological mechanisms, disease modifiers, and biomarkers might facilitate the development of treatment strategies.The DEM-AGING project aims to define molecular signatures in NCL and expedite biomarker discovery with a view to identifying novel targets for monitoring disease status and progression and accelerating clinical trial readiness in this field. In this study, we fused multiomic assessments in established NCL models with similar data on the more common late-onset neurodegenerative conditions in order to test the hypothesis of shared molecular fingerprints critical to the underlying pathological mechanisms. Our aim, ultimately, is to combine data analysis, cell models, and omic strategies in an effort to trace new routes to therapies that might readily be applied in the most common forms of dementia.

Study of adhesion and migration dynamics in ubiquitin E3A ligase (UBE3A)-silenced SYSH5Y neuroblastoma cells by micro-structured surfaces.

During neuronal development, neuronal cells read extracellular stimuli from the micro/nano-environment within which they exist, retrieving essential directionality and wiring information. Here, focal adhesions (FAs-protein clusters anchoring integrins to cytoskeleton) act as sensors, by integrating signals from both the extracellular matrix environment and chemotactic factors, contributing to the final neuronal pathfinding and migration. In the processes that orchestrate neuronal development, the important function of ubiquitin E3A ligase (UBE3A) is emerging. UBE3A has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders: the lack of UBE3A leads to Angelman syndrome (AS, OMIN 105830), while its increase causes autisms (Dup15q-autism). By using nano/micro-structured anisotropic substrates we previously showed that UBE3A-deficient neurons have deficits in contact guidance (Tonazzini et al, Mol Autism 2019). Here, we investigate the adhesion and migration dynamics of UBE3A-silenced SH-SY5Y neuroblastoma cells in vitro by exploiting nano/micro-grooved substrates. We analyze the molecular processes regulating the development of FAs by transfection with EGFP-vector encoding for paxillin, a protein of FA clusters, and by live-cell total-internal-reflection-fluorescence microscopy. We show that UBE3A-silenced SH-SY5Y cells have impaired FA morphological development and pathway activation, which lead to a delayed adhesion and also explain the defective contact guidance in response to directional topographical stimuli. However, UBE3A-silenced SH-SY5Y cells show an overall normal migration behavior, in terms of speed and ability to follow the GRs directional stimulus. Only the collective cell migration upon cell gaps was slightly delayed for UBE3Ash SHs. Overall, the deficits of UBE3Ash SHS-SY5Y cells in FA maturation/sensing and in collective migration may have patho-physiological implications, in AS condition, considering the much more complex stimuli that neurons find in vivo during the neurodevelopment.

Assessing the potential outcomes of achieving the World Health Organization global non-communicable diseases targets for risk factors by 2025: is there also an economic dividend?

OBJECTIVES: This study assesses the change in premature mortality and in morbidity under the scenario of meeting the World Health Organization (WHO) global targets for non-communicable disease (NCD) risk factors (RFs) by 2025 in France. It also estimates medical expenditure savings because of the reduction of NCD burden. STUDY DESIGN: A microsimulation model is used to predict the future health and economic outcomes in France. METHODS: A 'RF targets' scenario, assuming the achievement of the six targets on RFs by 2025, is compared to a counterfactual scenario with respect to disability-adjusted life years and healthcare costs differences. RESULTS: The achievement of the RFs targets by 2025 would save about 25,300 (and 75,500) life years in good health in the population aged 25-64 (respectively 65+) years on average every year and would help to reduce healthcare costs by about €660 million on average per year, which represents 0.35% of the current annual healthcare spending in France. Such a reduction in RFs (net of the natural decreasing trend in mortality) would contribute to achieving about half of the 2030 NCD premature mortality target in France. CONCLUSIONS: The achievement of the RF targets would lead France to save life years and life years in good health in both working-age and retired people and would modestly reduce healthcare expenditures. To achieve RFs targets and to curb the growing burden of NCDs, France has to strengthen existing and implement new policy interventions.

Promoting sport and physical activity in Italy: a cost-effectiveness analysis of seven innovative public health policies.

BACKGROUND: Inactive lifestyles are a key risk factor underpinning the development of many chronic diseases, yet more than half of the Italian population does not meet WHO thresholds for at least moderate physical activity. This study aims to make the economic case to upscale investments in policy actions to promote exercise and physical activity. STUDY DESIGN: Modelling-based cost-effectiveness analysis in Italy. METHODS: The study assesses the impact on health and healthcare expenditure of seven public health policies to promote exercise and physical activity against a business as usual scenario. Assessed policies include: promotion of active transport, workplace sedentarily interventions, investments in sports and recreation, mass media campaigns, prescription of physical activity in primary care, school-based interventions and mobile apps. RESULTS: Public policies to promote exercise have the potential to improve population health and produce savings in healthcare expenditure. Assessed policies can avoid hundreds of cases of cardiovascular diseases and diabetes per year and tens of cases of cancer resulting in gains in DALYs in the order of thousands per year. In the medium-term, the vast majority of policies show excellent cost-effectiveness ratio, below internationally recognized thresholds. CONCLUSIONS: Investing in policies to promote active lifestyles is a good investment for Italy.

A prospective evaluation of taste in Parkinson's disease.

It is well known that Parkinson's disease is characterized by a variety of non-motor symptoms. A gustatory deficit is hypothesized to be one of them although few and only cross-sectional studies are available. The aim of our pilot study was to prospectively investigate the taste function in Parkinson's disease patients after some years from the first evaluation (mean follow-up 4.35 ± 0.49 years; time range 3.5-5.6 years). A group of 26 patients was re-examined (16 males and 10 females; mean age 70.9 ± 8.4 years, range 54-88 years). Taste function was assessed in one session, by means of the Whole Mouth Test (WMT) and Taste Strips Test (TST). Olfaction was also evaluated with the Sniffin' Sticks Identification Test (SST). All these tests are commercially available (Burghart Company, Germany). All patients were able to understand and complete the procedure. Although scores decreased over time, no significant difference was found between global taste scores of first and second evaluation, neither comparing every single taste quality (WMT: p = 0.234, Mann-Whitney U test; TST: p = 0.747, Mann-Whitney U test; McNemar chi-square in the range of 0-1.455). These results confirm a persistent but slight and stable taste impairment, in patients with Parkinson's disease. Future studies on a much larger sample of patients are certainly required.

A conformational transition in the myosin VI converter contributes to the variable step size.

Myosin VI (MVI) is a dimeric molecular motor that translocates backwards on actin filaments with a surprisingly large and variable step size, given its short lever arm. A recent x-ray structure of MVI indicates that the large step size can be explained in part by a novel conformation of the converter subdomain in the prepowerstroke state, in which a 53-residue insert, unique to MVI, reorients the lever arm nearly parallel to the actin filament. To determine whether the existence of the novel converter conformation could contribute to the step-size variability, we used a path-based free-energy simulation tool, the string method, to show that there is a small free-energy difference between the novel converter conformation and the conventional conformation found in other myosins. This result suggests that MVI can bind to actin with the converter in either conformation. Models of MVI/MV chimeric dimers show that the variability in the tilting angle of the lever arm that results from the two converter conformations can lead to step-size variations of ∼12 nm. These variations, in combination with other proposed mechanisms, could explain the experimentally determined step-size variability of ∼25 nm for wild-type MVI. Mutations to test the findings by experiment are suggested.

Nanoemulsion of Minthostachys verticillata essential oil. In-vitro evaluation of its antibacterial activity.

Infectious diseases constitute a problem of great importance for animal and human health, as well as the increasing bacterial resistance to antibiotics. In this context, medicinal plants emerge as an effective alternative to replace the use antibiotics. The essential oil (EO) of Minthostachys verticillata (Griseb.) Epling (Lamiaceae) has demonstrated a strong antimicrobial activity. However, its instability and hydrophobicity under normal storage conditions are limitations to its use. Nanoemulsion technology is an excellent way to solubilize, microencapsulate, and protect this compound. This study aimed to obtain a nanoemulsion based on M. verticillata EO and evaluate its antibacterial activity against Staphylococcus aureus. The EO was obtained by steam distillation. Identification and quantification of their components were determined by GC-MS revealing that the dominated chemical group was oxygenated monoterpenes. Nanoemulsions (NE) were characterized by measuring pH, transmittance, separation percentage, release profile, and morphology. The effect of NE on the growth of S. aureus and cyto-compatibility was also evaluated. The results showed that NE containing a higher percentage of tween 20 exhibited higher stability with an approximated droplet size of 10 nm. The effect of encapsulation process was evaluated by GC-MS revealing that the volatile components in EO were no affected. After 24 h, 74.24 ± 0.75% of EO was released from NE and the antibacterial activity of EO was enhanced considerably by its encapsulation. The incubation of S. aureus with the NE and pure EO, show a bacterial growth inhibition of 58.87% ± 0.99 and 46.72% ± 3.32 (p < 0.05), respectively. In addition, nanoemulsión did not cause toxicity to porcine and equine red blood cells. The results obtained showed that NE could be a potential vehicle for M. verticillata EO with promissory properties to emerge as a tool for developing advanced therapies to control and combat infections.

Glial-fibrillary-acidic-protein (GFAP) biomarker detection in serum-matrix: Functionalization strategies and detection by an ultra-high-frequency surface-acoustic-wave (UHF-SAW) lab-on-chip.

Glial-fibrillary-acidic-protein (GFAP) has recently drawn significant attention from the clinical environment as a promising biomarker. The pathologies which can be linked to the presence of GFAP in blood severely affect the human central nervous system. These pathologies are glioblastoma multiforme (GBM), traumatic brain injuries (TBIs), multiple sclerosis (MS), intracerebral hemorrhage (ICH), and neuromyelitis optica (NMO). Here, we develop three different detection strategies for GFAP, among the most popular in the biosensing field and never examined side by side within the experimental frame. We compare their capability of detecting GFAP in a clean-buffer and serum-matrix by using gold-coated quartz-crystal-microbalance (QCM) sensors. All the three detection strategies are based on antibodies, and each of them focuses on a key aspect of the biosensing process. The first is based on a polyethylene glycol (PEG) chain for antifouling, the second on a protein-G linker for controlling antibody-orientation, and the third on antibody-splitting and direct surface immobilization for high-surface coverage. Then, we select the best-performing protocol and validate its detection performance with an ultra-high-frequency (UHF) surface-acoustic-wave (SAW) based lab-on-chip (LoC). GFAP successful detection is demonstrated in a clean-buffer and serum-matrix at a concentration of 35 pM. This GFAP level is compatible with clinical diagnostics. This result suggests the use of our technology for the realization of a point-of-care biosensing platform for the detection of multiple brain-pathology biomarkers.

pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

The oldest old Creutzfeldt-Jakob disease case.

Sporadic Creutzfeldt-Jakob Disease (sCJD) is a rapidly progressive neurodegenerative disorder usually affecting people between 60 and 70 years old, with only anecdotal cases presenting at 90 years or older. The clinical phenotype of sCJD is highly variable. Diagnosis of sCJD should be considered in the differential diagnosis of rapidly evolving ataxic or dementing syndromes with or without epileptic seizures, regardless of the patient age. While the recognition of atypical phenotypes in subject 90 years or older can provide additional diagnostic challenge, it must be underlined that neuropathology is still the "gold standard" for sCJD diagnosis.

Construction of a novel oncogene based on synthetic sequences encoding epidermal growth factor.

The autocrine model postulates that constitutive release of a mitogenic growth factor can lead to uncontrolled proliferation and cell transformation. A synthetic polynucleotide encoding epidermal growth factor conferred a tumorigenic phenotype on cells. These cells were transformed through the action of an autocrine circuit having an extracellular component.

c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling.

Mice lacking the proto-oncogene c-fos develop the bone disease osteopetrosis. Fos mutant mice were found to have a block in the differentiation of bone-resorbing osteoclasts that was intrinsic to hematopoietic cells. Bone marrow transplantation rescued the osteopetrosis, and ectopic c-fos expression overcame this differentiation block. The lack of Fos also caused a lineage shift between osteoclasts and macrophages that resulted in increased numbers of bone marrow macrophages. These results identify Fos as a key regulator of osteoclast-macrophage lineage determination in vivo and provide insights into the molecular mechanisms underlying metabolic bone diseases.

Calculation of free-energy differences by confinement simulations. Application to peptide conformers.

Conformational free-energy differences are key quantities for understanding important phenomena in molecular biology that involve large structural changes of macromolecules. In this paper, an improved version of the confinement approach, which is based on earlier developments, is used to determine the free energy of individual molecular states by progressively restraining the corresponding molecular structures to pure harmonic basins, whose absolute free energy can be computed by normal-mode analysis. The method is used to calculate the free-energy difference between two conformational states of the alanine dipeptide in vacuo, and of the beta-hairpin from protein G with an implicit solvation model. In all cases, the confinement results are in excellent agreement with the ones obtained from converged equilibrium molecular dynamics simulations, which have a much larger computational cost. The systematic and statistical errors of the results are evaluated and the origin of the errors is identified. The sensitivity of the calculated free-energy differences to structure-based definitions of the molecular states is discussed. A variant of the method, which closes the thermodynamic cycle by a quasi-harmonic rather than harmonic analysis, is introduced. The latter is proposed for possible use with explicit solvent simulations.

Allosteric communication in myosin V: from small conformational changes to large directed movements.

The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

Loss of inhibition over master pathways of bone mass regulation results in osteosclerotic bone metastases in prostate cancer.

Prostate cancer is the most common cancer among men in industrialised countries. Most patients with prostate cancer, however, will not die of it. As a result, many of them will experience symptomatic metastasis during the course of the disease. Prostate cancer has a high propensity to metastasize to bone. Unlike many other cancers prostate cancer cells induce a rather osteosclerotic than osteolytic reaction in the bone marrow by interfering with physiological bone remodelling. A proper understanding of the mechanisms of tumour cell-induced bone alterations and exaggerated bone deposition in prostate cancer may open new and urgently needed therapeutic approaches in the field of palliative care for affected patients. In this review we focus on the central role of two major regulators of bone mass, the wingless type integration site family members (WNTs) and the bone morphogenetic proteins (BMPs), in the development of osteosclerotic bone metastases.

CROCC-mutated rhabdoid colorectal carcinoma showing in intercellular spaces lamellipodia and cellular projections revealed by electron microscopy.

BACKGROUND: Rhabdoid colorectal carcinoma (RC) is a rare lesion localized to the proximal colon of patients with a mean age at diagnosis of around 70 years. This tumor shows an aggressive behavior with an overall survival period shorter than 12 months. The diagnostic hallmark is the presence of rhabdoid cells. Alterations in chromatin remodeling (SMARCB1) and in the centrosome structure (CROCC) are reported in RC usually BRAFmut and MSI-H. RKO intestinal neoplastic cells culture (BRAFmut, SMARCB1wt, MSI-H) with CROCC knockdown exhibit rhabdoid features and develop prominent projections from the edge of the cell. METHODS: Here, we investigated two cases of CROCCmutSMARCB1wt RC by scanning and transmission electron microscopy (SEM, TEM). RESULTS: TEM confirmed the diagnostic presence of intermediate cytoplasmic filaments and nucleolar margination. SEM showed cellular protrusions (lamellipodia) in the intercellular spaces not evident at light microscopy. CONCLUSIONS: These protrusions CROCC-related might represent the pathogenetic mechanism underlying the rhabdoid aggressive behavior, independently of tumor staging. To our knowledge, the SEM technique was applied in the study of this neoplasm for the first time.

Impact of primary care-initiated interventions promoting physical activity on body mass index: systematic review and meta-analysis.

INTRODUCTION: Physical inactivity is a risk factor for obesity, overweight and for a number of chronic diseases. Although primary care may be an ideal setting to encourage people to do more exercise, there are still significant gaps in the literature on the effectiveness of primary care-initiated policies to promote physical activity. METHODS: In this paper, we systematically review and meta-analyse the evidence on the impact of primary care-initiated interventions to promote exercise on the body mass index and on physical activity energy expenditure levels among people at an increased risk of having potentially disabling non-communicable diseases (but healthy enough to exercise). RESULTS: We find that such interventions reduce body mass index by about 0.21 kg m-2 (95% confidence interval: -0.41 to -0.01) and increase physical activity-related energy expenditure (based mostly on self-recall) by about 1.77 metabolic equivalent of task-hours a week (95% confidence interval: 0.58 to 2.95). DISCUSSION: This study suggests that primary care-initiated interventions promoting physical activity can be an effective strategy to reduce weight and increase exercise levels in this population group, although the question remains about whether such interventions can be cost-effective.

A molecular dynamics approach to the structural characterization of amyloid aggregation.

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.

Olaratumab for the treatment of soft tissue sarcoma.

Soft tissue sarcoma represents about 1% of all solid malignancies. The standard chemotherapy regimens have included doxorubicin alone or in combination with other agents. Despite recent advances in treatment beyond first line - with the FDA approval of pazopanib, eribulin and trabectidin - overall survival for patients with metastatic disease remains in the region of 12-19 months. Olaratumab is a monoclonal antibody directed against platelet-derived growth factor receptor alpha (PDGFRalpha). It was studied in a phase Ib and randomized phase II study in combination with doxorubicin in patients with soft tissue sarcoma who previously had not received doxorubicin for metastatic disease. The results of the phase II study showed a statistically significant improvement in progression-free survival up to 6 months, and a more dramatic improvement in overall survival to 26.9 months. This is the first randomized trial to show a significant improvement in overall survival compared to doxorubicin alone. An ongoing phase III study has completed accrual and results are being analyzed. Olaratumab has been granted accelerated approval by the United States Food and Drug Administration. Ongoing trials are underway to further demonstrate the mechanism of action. This review will document the studies involved in the development of olaratumab in the treatment of soft tissue sarcomas.