State Estimation Using a Network of Distributed Observers With Unknown Inputs

State estimation for a class of linear time-invariant systems with distributed output measurements (distributed sensors) and unknown inputs is addressed in this paper. The objective is to design a network of observers such that the state vector of the entire system can be estimated, while each observer has access to only local output measurements that may not be sufficient on their own to reconstruct the entire system’s state. Existing results in the literature on distributed state estimation assume either that the system does not have inputs, or that all the system’s inputs are globally known to all the observers. Accordingly, we address this gap by proposing a distributed observer capable of estimating the overall system’s state in the presence of inputs, while each observer only has limited local information on inputs and outputs. We provide a design method that guarantees convergence of the estimation errors to zero under joint detectability conditions. This design suits undirected communication graphs that may have switching topologies and also applies to strongly connected directed graphs.We also give existence conditions that are consistent with existing results on unknown input observers. Finally, simulation results verify the effectiveness of the proposed estimation scheme for various scenarios

Therapeutic Options in Hereditary Optic Neuropathies

Options for the effective treatment of hereditary optic neuropathies have been a long time coming. The successful launch of the antioxidant idebenone for Leber’s Hereditary Optic Neuropathy (LHON), followed by its introduction into clinical practice across Europe, was an important step forward. Nevertheless, other options, especially for a variety of mitochondrial optic neuropathies such as dominant optic atrophy (DOA), are needed, and a number of pharmaceutical agents, acting on different molecular pathways, are currently under development. These include gene therapy, which has reached Phase III development for LHON, but is expected to be developed also for DOA, whilst most of the other agents (other antioxidants, anti-apoptotic drugs, activators of mitobiogenesis, etc.) are almost all at Phase II or at preclinical stage of research. Here, we review proposed target mechanisms, preclinical evidence, available clinical trials with primary endpoints and results, of a wide range of tested molecules, to give an overview of the field, also providing the landscape of future scenarios, including gene therapy, gene editing, and reproductive options to prevent transmission of mitochondrial DNA mutations

Immunotherapeutic efficacy of retargeted ohsvs designed for propagation in an ad hoc cell line

Our laboratory has pursued the generation of cancer‐specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor‐Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off‐target and off‐tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R‐335 and R‐337 prototypes were armed with murine IL‐12. Intratumorally‐administered R‐337 conferred almost complete protection from LLC‐ 1‐HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long‐term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen‐agnostic vaccines

Amniotic Epithelial Stem Cells Counteract Acidic Degradation By-Products of Electrospun PLGA Scaffold by Improving their Immunomodulatory Profile In Vitro

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H2 O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs’ immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H2 O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days’ culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems

Antimicrobial, antioxidant, anti-inflammatory activities and phytoconstituents of extracts from the roots of Dissotis thollonii Cogn. (Melastomataceae)

Abstract Background Dissotis thollonii Cogn. belonging to the Malastomataceae family is used in the West Region of Cameroon for the treatment of inflammation, kidney diseases, pregnancy control and sinusitis. Despite the traditional use of this plant, no scientific report or information was found in the literature regarding neither its biological activity nor its chemical constituents. Aim of the study The present work was designed to determine the antimicrobial, antioxidant and anti-inflammatory activities of different extracts of the roots of D. thollonii Cogn. as well as the isolation and identification of the chemical constituents of this plant. Materials and methods The tests for antimicrobial, antioxidant and anti-inflammatory activities were performed over the MeOH, EtOAc, n-BuOH and aqueous extracts. Compounds were isolated from EtOAc and n-BuOH extracts of the roots of D. thollonii Cogn. through column chromatography and their structures were determined by means of NMR and MS analysis, and published data. Results According to the antimicrobial and antioxidant assays, the EtOAc and n-BuOH extracts were submitted to further separation and purification. This led to the isolation of twelve compounds identified as 3,3′-di- O -methylellagic acid 4′- O-β -D-xylopyranoside 1 , 3- O -methylellagic acid 4′- O-β -D-arabinopyranoside 2 , casuarinin 3 , betulinic acid 4 , β -sitosterol-3- O -D-glucopyranosyl-6′-mirystate 5 , cellobiosylsterol 6 , β -sitosterol 7 , β -sitosterol-3- O-β -D-glucopyranoside 8, arjunolic acid 9 , 3,3′-di- O -methylellagic acid 10 , ellagic acid 11 , and 3,3′-di- O -methylellagic acid 4′- O - β -D-glucopyranoside 12 . The EtOAc extract was the only antimicrobial active sample [diameter of the zone of inhibition (DZI) of 10 mm against Staphyloccocus aureus ] among all the tested extracts. The analysis of fractions of this extract revealed the presence of bioactive compounds with a described antimicrobial activity such as β -sitosterol, β -sitosterol-3- O-β -D-glucopyranoside and arjunolic acid. By using Trolox as the standard drug, all extracts showed antioxidant activity against DPPH in the following order of scavenging ability: Trolox > nBuOH > EtOAc > MeOH > WE (water extract). The ABTS •+ scavenging ability was similar to that found for the DPPH assay, being Trolox > n-BuOH > MeOH > EtOAc > WE. Along with the DPPH and ABTS assays, the FRAP assay showed the scale n-BuOH > MeOH > WE > EtOAc. The phytochemical study of the EtOAc and n-BuOH extracts revealed the presence of important known antioxidant compounds such as ellagic acid derivatives, arjunolic acid, betulinic acid and β -sitosterol. The anti-inflammatory properties of D. thollonii extracts were investigated using RAW 264.7 murine macrophage cells. The MeOH extract reduced the stimulated NO production in a concentration-dependent manner. 86% reduction was observed at the highest tested concentration of 100 μg/ml (IC 50 = 5.9 μg/ml). The n-BuOH extract showed higher dose dependent reduction of NO formation (IC 50 = 6.5 μg/ml) than the EtOAc extract (IC 50 = 18.1 μg/ml), whereas the water extract had no significant influence on the NO production. All the extracts did not have any influence on the macrophage viability. The phytochemical investigation of the EtOAc and n-BuOH extracts revealed that the main compounds identified do have potent anti-inflammatory properties. Conclusion The biological and phytochemical characterization of the root extracts of D. thollonii validates the use of this plant for the treatment of inflammation and sinusitis, thus providing evidence that this plant extracts, as well as some of the isolated compounds, might be potential sources of antioxidant and anti-inflammatory drugs

P-440 Impact of electrospun scaffold topology on the performance of in-vitro Folliculogenesis applied to preantral ovine follicles

Study question How to improve in-vitro Folliculogenesis (ivF) protocols to address the enlarged demand of fertility preservation? Summary answer Tissue engineering-based approach opens new frontiers for ivF improving 3D-technologies addressed to support immature-ovarian-follicle-growth to obtain an increased number of competent oocytes enrolled in Assisted-Reproductive-Technology. What is known already ivF is a promising Assisted-Reproductive-Technology (ART) for preserving and restoring fertility. This technology potentially reproduces the early stages of folliculogenesis and oogenesis in-vitro allowing to move a large amount of oocyte on individual basis towards the validated protocol of in-vitro maturation/in-vitro fertilization (IVM/IVF). The current availability of biocompatible-supporting materials offers the challenging opportunity to mimic the native organ stroma in order to better reproduce the 3D environmental conditions leading to synergic follicles-oocyte development in-vitro with the aim to improve the performance of ivF in translational large sized mammal models. Study design, size, duration The present research aimed to compare preantral (PA) follicles culture on two different typologies of scaffolds fabricated using PCL(poly(epsilon caprolactone)), respectively made with patterned and randomly aligned fibers (PCL-Patterned/PCL-Randomic) with a standardized-single-follicle scaffold-free-method (3D-oil), widely validated on ovine model (Cecconi et al., 2004). The culture outcomes are compared analyzing follicle/oocyte growth, percentage of antrum differentiation and the incidence of meiotic competence, by exposing ivF growing oocytes to IVM protocol. Participants/materials, setting, methods PA follicles (mean size diameter: 250±4μm), mechanically isolated from slaughterhoused lamb ovaries, were individually cultured on electrospun PCL scaffolds (patterned vs randomic) or using the 3D-oil method. ivF were cultured alphaMEM-Fetal Bovine Serum free medium (5% Knockout Serum Replacement) supplemented with 4 IU/mL of equine Chorionic Gonadotropin (Di Berardino et al., 2021). At the end of ivF (14-days) the fully-grown oocytes isolated from early-antral follicles were tested on IVM. Main results and the role of chance PCL-Patterned electrospun scaffolds were able to strongly support a synergic oocyte and follicular growth. The 3D culture on Patterned electrospun scaffold supported the highest viability of follicles (87.5% vs 63% under 3D-oil conditions). On the contrary, the highest incidence of degenerated follicles was observed in cultures performed using PCL-Randomic materials (55 vs 37% vs 12.5% for PCL-Randomic vs 3D-oil vs PCL-Patterned, respectively; p <0.0004). The greatest follicle diameter increment (74.7±1 vs 70±0.4 vs 60.9±2%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, respectively p <0.0007) and rate of antrum differentiation (87.5% vs 45% and vs 63%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, for both p <0.0001) were observed in PA ovine follicles cultured on PCL-Patterned scaffolds. Furthermore, PCL-Patterned electrospun scaffolds supported a complete functional development of the oocyte compartment. More in detail, the majority of fully grown oocytes isolated from early- antral follicles grown on PCL-Patterned materials reached the metaphase-II stage (MII 80%) at the end of IVM in comparison to the significant lower percentage in 3D-oil (MII 68%, p =0.04) and PCL-Randomic (MII 18%, p <0.0001) protocols, respectively. Limitations, reasons for caution - Wider implications of the findings Tissue engineering scaffold-based approach represents a valid strategy generating a multi-organ in-vitro system, where different compartments may cooperate generating the complexity of paracrine-mechanism controlling early-follicles outcomes. Scaffold topology is essential to control early-follicles development. Indeed, exclusively PCL-Patterned can preserve long-term follicle 3D-microarchitecture supporting in-vitro oogenesis up to a complete meiotic-competence-acquisition. Trial registration number not applicabl

Genotype of Immunologically Hot or Cold Tumors Determines the Antitumor Immune Response and Efficacy by Fully Virulent Retargeted oHSV

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely

Tendon-like Electrospun PLGA Scaffolds with Optimized Physical Cues Induced Tenogenic Differentiation and Boosted Immunomodulatory Properties on Amniotic Epithelial Stem Cells.

Introduction: The advanced strategies in the field of Tissue Engineering might render possible overcoming the unsatisfactory results of conventional treatments to deal with tendinopathies. In this context, the design of tendon biomimetic electrospun scaffolds engineered with Amniotic Epithelial Stem Cells (AECs), which have shown a high teno-regenerative and immunomodulatory potential in tendon-defect models, can represent a promising solution for tendon regeneration. Methods: Poly(lactide-co-glycolic) acid (PLGA) scaffolds were fabricated using the electrospinning technique to mimic the native tendon biomechanics and extracellular matrix by optimizing: fiber alignment and diameter size (1.27 and 2.5 µm), and surface chemistry using the Cold Atmospheric Plasma (CAP) Technique. Moreover, the teno-inductive and immunomodulatory effects of these parameters on AECs have been also assessed. Results: The fabricated PLGA scaffolds with highly aligned fibers and small diameter size (1.27 µm) induced a stepwise tenogenic differentiation on AECs with an early epithelial-mesenchymal transition (EMT), followed by their tenogenic differentiation. Indeed, SCX, an early tendon marker, was significantly more efficiently translated into the downstream effector TNMD, a mature tendon marker. Moreover, 1.27 µm fiber diameter induced on AECs a higher expression of anti-inflammatory interleukin mRNAs (IL-4 and IL-10). The CAP treated PLGA scaffolds showed an improved cell adhesion and infiltration without altering their topological structure and teno-inductive properties. In fact, AECs engineered with CAP treated fibers, expressed in their cytoplasm TNMD. Moreover, CAP treatment did not alter the mechanical properties of PLGA scaffolds. Conclusions: The developed electrospun PLGA scaffolds with the optimized features represent an ideal tendon-like construct that could be applied in in-vivo models to evaluate their biosafety and teno-regenerative potential