ARM-Cortex M3-Based Two-Wheel Robot for Assessing Grid Cell Model of Medial Entorhinal Cortex: Progress towards Building Robots with Biologically Inspired Navigation-Cognitive Maps

This article presents the implementation and use of a two-wheel autonomous robot and its effectiveness as a tool for studying the recently discovered use of grid cells as part of mammalian’s brains space-mapping circuitry (specifically the medial entorhinal cortex). A proposed discrete-time algorithm that emulates the medial entorhinal cortex is programed into the robot. The robot freely explores a limited laboratory area in the manner of a rat or mouse and reports information to a PC, thus enabling research without the use of live individuals. Position coordinate neural maps are achieved as mathematically predicted although for a reduced number of implemented neurons (i.e., 200 neurons). However, this type of computational embedded system (robot’s microcontroller) is found to be insufficient for simulating huge numbers of neurons in real time (as in the medial entorhinal cortex). It is considered that the results of this work provide an insight into achieving an enhanced embedded systems design for emulating and understanding mathematical neural network models to be used as biologically inspired navigation system for robots

application of advanced process control techniques to a pusher type reheating furnace

In this paper an Advanced Process Control system aimed at controlling and optimizing a pusher type reheating furnace located in an Italian steel plant is proposed. The designed controller replaced the previous control system, based on PID controllers manually conducted by process operators. A two-layer Model Predictive Control architecture has been adopted that, exploiting a chemical, physical and economic modelling of the process, overcomes the limitations of plant operators' mental model and knowledge. In addition, an ad hoc decoupling strategy has been implemented, allowing the selection of the manipulated variables to be used for the control of each single process variable. Finally, in order to improve the system flexibility and resilience, the controller has been equipped with a supervision module. A profitable trade-off between conflicting specifications, e.g. safety, quality and production constraints, energy saving and pollution impact, has been guaranteed. Simulation tests and real plant results demonstrated the soundness and the reliability of the proposed system

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

ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction

P-030 ACE2 receptor and its isoform short-ACE2 are expressed on human spermatozoa

STUDY QUESTION: Do human spermatozoa express angiotensin-converting enzyme 2 (ACE2) receptor? What would be its localization? SUMMARY ANSWER: Human spermatozoa express uniformly ACE2 on the sperm head and the flagellum. Moreover, the short-ACE2 isoform is concentrated on the post-acrosomal region and midpiece. WHAT IS KNOWN ALREADY: The Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection is generating important concerns regarding not only the possible consequences on the respiratory system, but also on other organs, including the reproductive system. ACE2 is considered the main point of entry for the SARS-CoV-2 within the cells through the binding with the spike protein on the virus surface. Furthermore, ACE2 is expressed in human testes cells including Leydig cells, Sertoli cells and spermatogonia. However, to date, the expression and location of ACE2 in mature human spermatozoa has not been investigated yet. STUDY DESIGN, SIZE, DURATION: This was an in vitro study for the evaluation of the expression and immune-localization of full-length ACE2 and its isoform, short-ACE2, in human spermatozoa. Thirthyfour non-immunized healthy normozoospermic volunteers were enrolled in the study. The study was conducted from May to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were collected by masturbation from non-immunized healthy normozoospermic voluntaries. Motile sperm suspensions were obtained by swim-up procedure. The expression of ACE2 was assessed by Western-blot analysis, while the immune-localization of ACE2 was evaluated by immune-cytochemical analysis under confocal microscopy. Flow-cytometry experiments were also performed to assess the surface protein expression on a large number of cells. MAIN RESULTS AND THE ROLE OF CHANCE: The Western-blot analysis of sperm extracts demonstrated two specific bands, one of approximately 120 KDa, corresponding to the glycosylated full-length ACE2, and a second one of approximately 52 KDa, the molecular weight of the protein recently termed short-ACE2. The immune-cytochemical analysis showed a uniformly localization of full-length ACE2 along both the sperm head and the flagellum, whereas the short isoform was preferentially located in the post-acrosomal region of the sperm head and the midpiece. At the flow cytometer, semen samples displayed a wide between-subject variability both in the percentage of ACE2-positive spermatozoa and the density of protein surface expression. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to determine whether short-ACE2 is a cleavage product from the full-length protein or if it is originated during spermatogenesis. Moreover, the role and the interaction of ACE2 with SARS-CoV-2 in human spermatozoa should be clarified to evaluate the possible impact of the virus on sperm biology. WIDER IMPLICATIONS OF THE FINDINGS: Since mature spermatozoa are transcriptionally silent and SARS-CoV-2 is an RNA virus, it is unlikely that the virus could affect sperm biology by replicating itself. Nevertheless, the potential effects related to modifications of the sperm membrane or interaction with other receptors or specific proteins cannot be ruled out. TRIAL REGISTRATION NUMBER: not applicabl

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

26S PROTEASOME AND PKA MODULATE MAMMALIAN SPERM CAPACITATION BY CREATING AN INTEGRATED DIALOGUE: A COMPUTATIONAL ANALYSIS

Recent experimental evidence suggests the involvement of the 26S proteasome, the main protease active in eukaryotic cells, in the process that leads mammalian sperm to become fully fertile, so-called capacitation. Unfortunately, its role in male gametes signaling is still far from being completely understood. For this reason, here, we realized a computational model as an attempt to rebuild and explore 26S proteasome signaling cascade, aggregating all the molecular data available to date and realizing the Proteasome Interactome Network (PIN). Once obtained the network (i.e., a graph to represent the molecules as nodes and the interactions among them as links), we assessed its topology to infer important biological information. PIN is composed of 157 nodes, 248 links and it is characterized by a scale-free topology, following the Barabasi Albert model. In other words, it possesses a large amount of scarcely linked nodes and a small set of highly linked nodes, the hubs, which act as system controllers. This peculiar topology confers to the network relevant biological features: it is robust against random attacks, easily navigable and controllable and it is possible to infer new information from it. Indeed, the analysis of PIN showed that PKA and 26S proteasome were strongly interconnected and both were active in sperm signaling by influencing the protein phosphorylation pattern and then controlling several key events in sperm capacitation, such as membrane and cytoskeleton remodeling. In conclusion, the network model could explain many biological aspects of sperm physiology that are out of focus looking at the single molecular determinant, overcoming the reductionist approach which did not consider the complexity of molecules and their interactions. This could be helpful to identify potential diagnostic markers and therapeutic strategies concurring in explaining and approaching male infertility

Diffusion tensor imaging mapping of brain white matter pathology in mitochondrial optic neuropathies

BACKGROUND AND PURPOSE: Brain white matter is frequently affected in mitochondrial diseases; optic atrophy gene 1-autosomal dominant optic atrophy and Leber hereditary optic neuropathy are the most frequent mitochondrial monosymptomatic optic neuropathies. In this observational study, brain white matter microstructure was characterized by DTI in patients with optic atrophy gene 1-autosomal dominant optic atrophy and Leber hereditary optic neuropathy, in relation to clinical and genetic features. MATERIALS AND METHODS: Nineteen patients with optic atrophy gene 1-autosomal dominant optic atrophy and 17 with Leber hereditary optic neuropathy older than 18 years of age, all genetically diagnosed, and 19 healthy volunteers underwent DTI by using a 1.5T MR imaging scanner and neurologic and ophthalmologic assessments. Brain white matter DTI metrics were calculated for all participants, and, in patients, their correlations with genetics and clinical findings were calculated. RESULTS: Compared with controls, patients with optic atrophy gene 1-autosomal dominant optic atrophy had an increased mean diffusivity in 29.2% of voxels analyzed within major white matter tracts distributed throughout the brain, while fractional anisotropy was reduced in 30.3% of voxels. For patients with Leber hereditary optic neuropathy, the proportion of altered voxels was only 0.5% and 5.5%, respectively, of which half was found within the optic radiation and 3.5%, in the smaller acoustic radiation. In almost all regions, fractional anisotropy diminished with age in patients with optic atrophy gene 1-autosomal dominant optic atrophy and correlated with average retinal nerve fiber layer thickness in several areas. Mean diffusivity increased in those with a missense mutation. Patients with Leber hereditary optic neuropathy taking idebenone had slightly milder changes. CONCLUSIONS: Patients with Leber hereditary optic neuropathy had preferential involvement of the optic and acoustic radiations, consistent with trans-synaptic degeneration, whereas patients with optic atrophy gene 1-autosomal dominant optic atrophy presented with widespread involvement suggestive of a multisystemic, possibly a congenital/developmental, disorder. White matter changes in Leber hereditary optic neuropathy and optic atrophy gene 1-autosomal dominant optic atrophy may be exploitable as biomarkers. ABBREVIATIONS: DOA autosomal dominant optic atrophy; FA fractional anisotropy; LHON Leber hereditary optic neuropathy; MD mean diffusivity; OPA1 optic atrophy gene 1 ;O R optic radiation; RNFL retinal nerve fiber layer; TBSS tract-based spatial statistic

Longitudinal Study of Optic Disk Perfusion and Retinal Structure in Leber's Hereditary Optic Neuropathy

PURPOSE. The purpose of this study was to evaluate optic disk perfusion and neural retinal structure in patients with subacute Leber's hereditary optic neuropathy (LHON) and LHON carriers, as compared with healthy controls. METHODS. This study included 8 patients with LHON in the subacute stage, 10 asymptomatic carriers of a LHON-associated mitochondrial DNA mutation, and 40 controls. All subjects underwent measurement of the retinal nerve fiber layer (RNFL) thickness, the ganglion cell-inner plexiform layer (GCIPL) thickness using optical coherence tomography and optic disk microvascular perfusion (Mean Tissue [MT]) using laser speckle flowgraphy (LSFG). Patients were re-examined after a median interval of 3 months from the baseline visit. RESULTS. LHON carriers had higher values of RNFL thickness, GCIPL thickness, and disk area than controls (P < 0.05), whereas MT was not different between the two groups (P = 0.936). Median MT and RNFL thickness were 32% and 15% higher in the early subacute stage of the disease than in controls (P < 0.001 and P = 0.001). MT declined below the values of controls during the late subacute stage (P = 0.024), whereas RNFL thickness declined later during the dynamic stage (P < 0.001). GCIPL thickness was lower in patients with LHON than in controls independently of the stage of the disease (P < 0.001). CONCLUSIONS. The high blood flow at the optic disk during the early subacute stage may be the consequence of vasodilation due to nitric oxide release as compensation to mitochondrial impairment. Optic disk perfusion as measured by LSFG is a promising biomarker for LHON diagnosis and monitoring as well as an objective outcome measure for assessing response to therapies