Meriones unguiculatus  and after anti-orthostatic suspension according to E.R. Morey-Holton. A morphometric analysis of α-SMA in the muscularis externa of the stomach and jejunum of mice and Mongolian gerbils from space flight groups revealed a decreased area of the immunopositive regions, a fact indicating a weakening of the SMT functional activity. Gravisensitivity of the contractile structures of the digestive system may be due to changes in the myofilament structural components of the smooth myocytes or myofibroblast actin. A simulated antiorthostatic suspension revealed no significant changes in the content of the α-SMA expression level, a fact supporting an alteration in the functional properties of the muscularis externa of the digestive hollow organs under weightless environment. The data obtained contribute to the novel mechanisms of the SMT contractile apparatus remodeling during orbital flights and can be used to improve preventive measures in space biomedicine.Smooth muscle tissue (SMT) is one of the main structural components of visceral organs, acting as a key factor in the development of adaptive and pathological conditions. Despite the crucial part of SMT in the gastrointestinal tract activity, the mechanisms of its gravisensitivity are still insufficiently studied. The study evaluated the content of smooth muscle actin (α-SMA) in the membranes of the gastric fundus and jejunum in C57BL/6N mice (30-day space flight), in Mongolian gerbils  Developing new space projects for manned flights, scientists are working hard not only to solve technical and engineering problems but also to properly maintain the health of astronauts. Weightlessness, affecting the body physiological functions and being able to cause various diseases, takes one of the leading positions in terms of the major biological effects that accompany the professional activities of the International Space Station (ISS) crews ,4,5,6,7.The gastrointestinal (GI) tract is critical among the vital systems of the human and animal body determining the level of adaptation to remain in extreme conditions . The digSMT is the major structural and functional component of all visceral organs ,20, whicSMT consists of smooth muscle cells (SMCs), which have a high level of alpha-smooth muscle actin, playing an integral role in the regulation of mechanical stress created by cells . The conThe analysis of micropreparations (staining with Gill’s hematoxylin and eosin) involving fragments of the gastric fundus of the animals of the space flight groups helped to detect special morphological changes, which likely resulted from the impact of space flight factors on the tissue structure. The stomach walls of Mongolian gerbils A,B and CWhen analyzing micropreparations of the second block of the study, we detected specific morphological changes in the gastric mucosa of the Mongolian gerbils of the space flight group, probably due to the increased sensitivity of animals to stress factors, including space flight . In partWhen investigating the stomach of mice after antiorthostatic suspension (the third block of the study), no pronounced dystrophic changes were found in the layers of the organ wall: despite the visually detectable flattening of the glandular epithelium, there were no signs of vacuolarization and desquamation.The jejunal wall of Mongolian gerbils F and C57In the analysis of samples of the jejunum in Mongolian gerbils, the second block of the study (Foton-M3 experiment), the space flight group revealed morphological features typical of reactive changes: smoothing of the relief structure of the mucosa due to the appearance of a large number of cells with vacuolized cytoplasm and pyknotic nuclei. The following changes were found in the underlying structures : signs of edema were observed in the space flight group, which can be explained by the pronounced blood content of the vessels, and a proliferation of the fibrous component of the connective tissue both in the submucosa and in the intermuscular layer with an altered architectonics of muscle fiber bundles G.An immunohistochemical technique was used to specify molecular rearrangements in the structure of smooth myocytes.The analysis of micropreparations of the first block of the study involving the material of the Bion-M1 space project detected a decrease in the immunopositive regions of ɑ-SMA expression in the orbital flight group .The morphological evaluation of α-SMA expression in the stomach wall revealed the following differences: In the groups of mice of vivarium stay and control to the readaptation group, uniform α-SMA expression was noted both in the muscularis mucosa and in the muscularis externa A,D. In aImmunopositive α-SMA cells are surrounded by endomysium, a fibrous component of the connective tissue, which accounts for approximately 10% of the total area of the muscularis externa  and doesIn the flight group and readaptation group after the space flight, accumulations of smooth myocytes with weak immunopositivity to α-SMA were observed; their predominant number was identified in the muscularis externa of the gastric fundus of mice after exposure to weightlessness B,E. In mWhen analyzing α-SMA expression in the gastric mucosa, a slight trend was detected towards an increase in the immunopositive region of α-SMA—5.28% of the total area of the mucosa in the space flight group compared to 4.87% in the control group .There were no differences between the groups in the area of α-SMA expression in the samples obtained in a synchronous experiment in the BIOS-MLZH flight equipment model, both in the mucous membrane and in the muscularis externa.A morphological pattern similar to the flight experiment BION-M1 was observed during the analysis of micropreparations of the second block of our study: a-SMA of Muscularis externa in Mongolian gerbils is decreased as in C57BL/N6 mice .Meriones unguiculatus in the space flight group compared to animals from the control group , suspension (85%) and recovery (85.08%). An analysis of the α-SMA expression in the muscularis mucosae of mice demonstrated a slight decrease in parameters in animals from the suspension group, to 4.45% of the total area. In the vivarium control and recovery groups, the rate of immunopositivity to α-SMA was almost the same, amounting to 5.55% and 5.70%, respectively .The analysis of microsections of the jejunum in C57BL/N6 mice and Mongolian gerbils revealed a dynamic of the α-SMA expression similar to that detected in the stomach; it consisted of a decreased α-SMA expression after the space flight. A quantitative calculation of the area of α-SMA expression in the jejunum of C57BL/N6 mice, the first block of the study within the Bion-M1 experiment, is presented in An analysis of α-SMA positive cells in the jejunal mucosa of C57BL/N6 mice demonstrated increased immunopositive structures in animals from the space flight group compared to the animals from the control group . This maMeriones unguiculatus (an experiment Foton-M 3). An Immunomorphometric analysis of muscle fibers revealed numerous clusters of immunonegative cell groups in animals of the space flight group. In animals of the control group, the α-SMA positive regions predominate  of the jejunum of C57BL/N6 mice between the control (4.61% and 86.04%), suspension (3.69% and 89.44%) and recovery groups (4.53% and 86.59%) were identified .Space flight factors resulted in changes in the structure of the smooth muscle tissue of the digestive system with a poorly understood molecular etiology. The results of our study evidence that weightless conditions caused a decrease in the content of α-SMA in the contractile structures of the studied hollow organs of the gastrointestinal tract. This fact was detected in Mongolian gerbils after a 12-day space flight  and in CAccumulated factual material has shown that orbital flights can lead to such changes in the gastrointestinal tract of astronauts, such as the development of hypersecretory syndrome of the stomach, the formation of venous stagnation in the vascular bed, an increase in the size of the liver, modification of the motor-evacuation function, digestive processes and activity of the pancreas and an intensification of the basal secretion of the organs of the gastroduodenal zone of astronauts or test subjects ,28,29,30The balanced coordination of the contractile activity of the structural and functional elements of the gastrointestinal smooth muscles is necessary for adequate digestion; its alteration underlies a number of development disorders of gastric and intestinal motility.Based on the results obtained in the synchronous ground experiment of the BION-M1 project in the BIOS-MLZH flight equipment model, we can conclude that the qualitative nature of the food does not affect the structural rearrangements of the smooth muscle apparatus, namely, the expression area of α-SMA.As stated, unlike other differentiated cells, smooth myocytes are not finally determined, have plasticity and can move from contractile to proliferative, as well as pro-migrating and synthetic phenotypes . Such reOur study indicates the need to further investigate the major molecular mechanisms of switching the SMT phenotype of the digestive system in both post-flight groups and in readaptation groups after space flight. In the experiment involving antiorthostatic suspension, the third block of the study, no significant changes were detected in the area of immunopositive regions of α-SMA expression; this fact may support the multidirectional impact of space flight factors and antiorthostatic suspension.As previously demonstrated, the simulated microgravity with a random positioning apparatus resulted in a decreased content of α-SMA in fibroblast cultures, which worsened their migratory properties ,35. ThisThus, as demonstrated, the gravisensitivity of smooth myocytes of the wall of the hollow gastrointestinal organs is a specific adaptive change to the altered gravity environment; that is similar to other cells of mesodermal origin with the contractile apparatus possessing α-SMA. Further research in this direction will expand the existing understanding of the mechanisms of developing organ-specific features of SMT gravisensitivity, opening up novel options for improving algorithms to prevent adverse biological effects of weightlessness.Meriones unguiculatus; the analysis was conditionally divided into three blocks.The study was performed in the Research Institute of Experimental Biology and Medicine, Burdenko Voronezh State Medical University. We semiquantitatively analyzed the α-SMA content in the membranes of the gastric fundus and in fragments of the jejunum in mammals: C57BL/6N mice and Mongolian gerbils The first block of the study is presented by the findings obtained in a 30-day space flight on board the BION-M1 biosatellite; it involved 26 male C57BL/6N mice  ,39,40. TMeriones unguiculatus , aged 4–4.5 months, with an average weight of all animals of 56.2 g, were on board the spacecraft during a 12-day orbital flight [The second block of our study involved the material obtained during the flight experiment performed within the Foton-M3 research project on the Kontur-L module . Mongolil flight . The firAnimals from three groups were subjected to euthanasia one day after the end of the relevant experiment. The average weight of the animals was about 51.6 g before the flight and about 37.1 g after the flight.Due to a failure in the telemetry system, animals in the synchronous group were fed half the daily ration with a 4-day delay compared to the flight group (from day 9 instead of day 5). The average body weight of the animals was about 63.4 g before the experiment and about 54.2 g after the experiment . The thiAll studies were carried out in compliance with the requirements for the humane treatment of animals in accordance with the decision of the Commission on Biomedical Ethics, the State Scientific Center of the Russian Federation, IMBP RAS (protocol No. 206 dated 7 October 2007).The third block included the experimental material obtained after anti-orthostatic suspension under simulated modified gravity according to the E.R. Morey-Holton method. The simulation lasted 30 days, involved 18 C57BL/6N male mice, aged 19–20 weeks, with an average weight of 27.5 g; the animals were on a standard diet. The animals were divided into 3 experimental groups: group 1—30-day anti-orthostatic suspension (n = 6); group 2—readaptation group, the mice were exposed to 12 h support load after hanging for 30 days (n = 6); and group 3—control group, animals were kept under standard conditions (n = 6) . The expFragments of the gastric fundus and jejunum of the animals were fixed in 10% neutral formalin solution at room temperature, according to the standard sample preparation protocol, immediately after the animal withdrawal from the experiment. For review microscopy to study a microtome, sections were made from paraffin blocks, no more than 4-μm-thick, stained with Gill’s hematoxylin and eosin, using the picro-Mallory staining protocol.Immunohistochemical α-SMA detection was performed using mouse monoclonal antibodies Actin smooth muscle  on 2 µm thick sections according to the standard protocol . HomologFor a representative sample, a minimum of 40 visual fields were evaluated. Photodocumentation and analysis of finished micropreparations were carried out on a ZEISS Axio Imager.A2 research microscope. Quantification of α-SMA expression was determined based on the method of calculating the area of immunopositive regions using the ImageJ 1.51J8 program and expressed in absolute and relative values.t-test was used to assess the significance of differences between the two groups.The data were statistically processed using the Statistica Inc.  12.0 package. The results were presented as mean M ± m (standard error of the mean). A Student’s The decreased intracellular actin expression of smooth muscle cells in the muscular membrane of the stomach and jejunum after both 12-day and 30-day orbital flights of laboratory rodents indicates a high gravisensitivity of the contractile structures of the digestive hollow organs and poses new challenges for space gastroenterology. The data obtained must be considered when developing preventive measures to level the adverse biological effects of long-term space flight factors.


Cryptorchidism, defined as the failure of at least one or both testicles to descend into the scrotal pouches, is the most frequent  congenital anomaly encountered in newborn males, resulting in one of the most frequent causes of non-obstructive azoospermia in men. Similar to other congenital malformations, cryptorchidism is thought to be caused by endocrine and genetic factors, combined with maternal and environmental influences. The etiology of cryptorchidism is unknown, as it involves complex mechanisms aiming to control the testicular development and descent from their initial intra-abdominal location in scrotal pouches. The implication of insulin-like 3 (INSL-3) associated with its receptor (LGR8) is critical. Genetic analysis discloses functionally deleterious mutations in INSL3 and GREAT/LGR8 genes. In this literature review, we discuss and analyze the implication of INSL3 and the INSL3/LGR8 mutation in the occurrence of cryptorchidism in both human and animal models. Cryptorchidism is the clinical condition in which the testes fail to descend  into theWe conducted a Pubmed and Google Academics search from January 2017 to January 2022, using the following search terms (“INSL3”) OR [(“LGR8”) OR (“INSL-3”)] AND [(“cryptorchidism”) OR (“undescended testis”)]. The literature search retrieved 84 references see . All artThe literature search retrieved 84 references . We seleINSLR3 is a small peptide  with the insulin-typical A-B heterodimeric structure, held together by three internal cysteine bonds. During the intrauterine life, the testis migrates from the abdomen into the scrotum by the 15th to the 28th week of gestation . The andINSL3 and androgens represent the primary factors involved in gubernacular development during the testicular descent. INSL3 activation is triggered by Leydig cells throughout embryogenesis. It circulates and connects to receptors located on gubernacular cells, leading to a proliferation of the gubernaculum and modifications in his extracellular matrix. This process will conduct to the retraction of the gubernaculum, followed by the migration of the primitive testis from its retroperitoneal position in the scrotum. The main role of INSL3 is to produce a thickening of the gubernaculum, which keeps the testes in the inguinal region and conducts it in the scrotal pouches. It is thereby considered as the first promoter of the transabdominal phase of testicular descent, being a secretory product of Leydig cells, expressed in a differentiation-dependent manner. Anand-Ivell et al.  have fouAs has been previously mentioned, INSL3 plays a key part in the process of testicular formation and descent, mostly in the bulge reaction of the testis gubernaculum . The exaCurrently, the data on the physiological functions of INSL3 in rodent studies are related to reproduction and internal fertilization and viviparity . INSL3 tMapping the evolution of INSL3 expression (secretion) into peripheral blood in mice and newborn rats, Anand-Ivell et al.  showed tInsl3 resulted in viable organisms, but presented a clinical bilateral cryptorchid phenotype due to gubernaculum deficiencies, leading, thus, to defective spermatogenesis and later infertility [In animals with INSL3 defects, compromised testicular descent is the only manifestation ,25. The ertility ,22,26.In order to verify whether in vivo INSL3-mediated gubernaculum expansion is androgen independent, Adham et al.  designedGreat, revealed that the disarranging of the Great gene leads to failure of the first stage of testicular descent, the transabdominal one [INSL3/Great signaling regulates gubernacular development [Studies carried out by Tomiyama et al.,  with theinal one . This waelopment .Estrogens have been shown to down regulate INSL3 and thereby cause maldescent of the testis. Investigating whether prenatal exposure to diethylstilbestrol, a synthetic estrogen that can conduct to cryptorchidism in humans and rodents, could interfere with testicular INSL3 mRNA expression, Emmen et al.  pointed The occurrence and development of undescended testes in mice is proven to be closely related to exposure to different xenobiotics, which act as endocrine disruptors. The environmental endocrine disruptors are synthetic chemical pollutants, pesticides, and additives that affect the endocrine system. Among them, diethylhexyl phthalate (DEHP)  is an orApart from INSL3, the genitofemoral nerve and hormones (androgens) are involved in the physiopathology of cryptorchidism . Fetal LThe Insulin-like3 gene is composed of two exons incorporating an intron, and encrypted in a single copy in the human genome . An inteMultiple mutations have been researched to explain the implication of INSL3 and its specific receptor, RXFP2, in boys with undescended testis. As stated by Nowacka-Woszuk et al. , the freTestosterone levels and INSL3 vary with age. For example, a small but significant level appears during mini-puberty, and markedly decreased levels are noticed in childhood and then increase during puberty, with the highest levels in adults . de JongINSL3 was also measured in human amniotic fluid collected during routine amniocentesis for prenatal genetic tests. Cohen et al.  demonstrHarrison et al.  hypothesThis review described the role of INSL3 in testicular descent in the fetal period. INSL3 gene polymorphism is considered a predisposing factor for cryptorchidism.INSL3 mutations vary from bilateral undescended testes, unilateral undescended testes, and the failure of the testes to descend normally into the scrotum at birth, but with a spontaneous descent during the first year of life.Testicular descent is a complex process, under the influence of many environmental and genetic factors, which involves in the migration of fetal gonads into the scrotum. INSL3 plays a crucial role in the transabdominal migration of the testes. Leydig cells, which are under the control of IGF1, produce both androgens and INSL3. IGFR1 influences the remodeling and development of the cremasteric muscle complex and doubtlessly the testicular descent. Even if the etiology of cryptorchidism in male boys is unknown, it is hypothesized that it is determined by multiple factors, and is most likely due to the altered signaling of INSL3/RXFP2. The phenotypes of males with INSL3 gene polymorphism with cryptorchidism was incriminated in both animal and human studies. The ablation of the gene encoding either INSL3 or its receptor, RXFP2, leads to bilateral undescended testes, which are caused by a failure of the gubernacular ligament to expand. The highest concentration of INSL3 in fetal life is proven between 15–18 weeks, from the moment that corresponds to the outgrowth of the gubernaculum and the initialization of the first phase of the testicular descent. This supports the crucial role of INSL3 in the process of testicular descent. Even higher fetal INSL3 levels than supposed initially in the amniotic fluid, in gestational weeks 18–20, sustain the theory that INSL3 has a base role in all phases of testicular descent.A specific association of -Cryptorchidism is one of the most frequent congenital malformations of the genitourinary tract, with the complex pathogenesis still unclear, and with long term consequences on the further life of the male adult .-INSL3 is one of the main factors involved in the testicular descent, especially in the gubernaculum development, remodeling, and development of the cremasteric muscle complex.-INSL3, a protein hormone produced by the Leydig cells also in the fetal life, has a crucial role in the transabdominal phase of the testicular descent process.-Animal studies were the first to cite INSL3 gene polymorphism as a predisposing factor for cryptorchidism, but the role of INSL3 in human fetal testicular descent and maldescent is less clearly delineated.Our review provides insights from a relatively small number of studies on the role of INSL3 in the process of testicular descent. This is one of the reasons for the inclusion of animal studies among the original articles that were reviewed. Apart from this, the animal studies were the first to emphasize the role of INSL3 in the process of testicular descent and this mutation in cryptorchid rodents. A better picture of the role of INSL3 in cryptorchidism would involve harvesting small biopsies from the undescended testes, which might raise ethical issues due to the difficulties of obtaining informed consent . In conc


This work analyzes on nanoscale spatial domains the mechanical features of electrospun membranes of Polycaprolactone (PCL) loaded with Functionalized Magnetite Nanoparticles (FMNs) produced via an electrospinning process. Thermal and structural analyses demonstrate that FMNs affect the PCL crystallinity and its melting temperature. HarmoniX-Atomic Force Microscopy (H-AFM), a modality suitable to map the elastic modulus on nanometric domains of the sample surface, evidences that the FMNs affect the local mechanical properties of the membranes. The mechanical modulus increases when the tip reveals the magnetite nanoparticles. That allows accurate mapping of the FMNs distribution along the nanofibers mat through the analysis of a mechanical parameter. Local mechanical modulus values are also affected by the crystallinity degree of PCL influenced by the filler content. The crystallinity increases for a low filler percentage (<5 wt.%), while, higher magnetite amounts tend to hinder the crystallization of the polymer, which manifests a lower crystallinity. H-AFM analysis confirms this trend, showing that the distribution of local mechanical values is a function of the filler amount and crystallinity of the fibers hosting the filler. The bulk mechanical properties of the membranes, evaluated through tensile tests, are strictly related to the nanometric features of the complex nanocomposite system. In the last two decades, nanotechnologies have been proposed in many fields. Their potentialities are extremely promising for a wide range of applications ,2,3,4. FTypically, polymeric materials with this exciting peculiarity of being composed of nanostructured fiber mats are those obtained by electrospinning processes. Electrospun membranes are of great interest from an applicative point of view. They can be designed for application as filtering devices ,16,17,18In the production of biomaterials for tissue engineering, for example, it is generally desirable to have materials with the same texture of the primary tissue. The electrospinning process allows the efficient and easy production of nanofibrous membranes, which well-mimic the morphology of human tissue or specific biological tissues. Together with the morphological feature, the possibility of tailoring and controlling the mechanical properties at the macroscopic scale but also at the nanoscopic level in the form of mapping of specific mechanical parameters is of great relevance.The use of nanotechnologies in electrospun membranes is looked at as one of the most promising pathways to tailor mechanical and functional characteristics, for ex. for producing highly flexible, microporous nanomaterials that convincingly mimic the features of the extracellular matrix and human tissue in general. Moreover, the possibility to easily load different types of fillers in the electrospun matrix for all types of applications is attracting great attention because it represents a powerful method to direct the material toward a myriad of diversified performances, including smart functions ,31,32,33Different research papers focused their attention on the control of the smart functionalities in these types of systems. For example, in the case of magnetic nanoparticles, the electrospun mat has demonstrated sensitivity to an external magnetic field, and capability to increase the temperature locally thanks to the application of an alternate magnetic field ,41. HoweIn this paper, membranes loaded with magnetite nanoparticles compatibilized with the human environment were successfully embedded in the mat fibers following the procedure already reported in the literature . These s3) (97%), 1,2-hexadecanediol (90%), oleic acid (OA), citric acid (CA), benzyl ether (90%), ethanol, hexane, and acetone. All of the chemicals used in the testing were of analytical grade. Fe3O4 magnetic nanoparticles (Fe3O4@OA) were made utilizing an experimental method that has previously been documented in the literature [3O4@OA was functionalized by altering the produced nanoparticles via ligand exchange with citric acid (CA) at room temperature, as described by Guadagno et al. [3O4.PCL, with the CAS number 24980-41-4, was supplied by Perstorp  for the membrane preparation. It is a linear polyester with a high molecular weight  that was provided in pellet form (granules with an appr-oximate diameter of 3 mm). Aldrich Chemical Co.  provided the iron (III) acetylacetonate (Fe(acac)terature ,51. Fe3Oo et al. . The fin3O4 concentrations in PCL were calculated at 2, 5, and 10 wt.%, with respect to PCL concentrations. Climate-controlled electrospinning apparatus was used to electrospin the functionalized Fe3O4 nanoparticle-containing solutions . Each combination was put into a stainless-steel syringe (3 mL) with a 0.8 mm needle. The needle had a high-voltage connection to a power source.The procedure followed to obtain the nanofibrous mats of PCL loaded with magnetite nanoparticles had been recently optimized by the research group.  PCL was The counter electrode was a grounded piece of aluminium foil positioned 30 cm away from the spinneret. On the aluminium foil, PCL fibres in the shape of continuous filaments were gathered to form a fibrous membrane. The electrospinning process parameters were suitably set to prevent bead development and, hence, to manufacture mats of fibrous PCL made up of continuous individual fibrils, as homogeneous as possible in diameter along the length.3O4”.During the membrane fabrication process, the parameters were optimized through a process of trial and error. The voltage for electrospinning was set at 25 kV (charge applied to the collector at −4 kV and charge applied to the needle at 21 kV), feeding rate at 4.0 mL/h, the temperature at 25 °C, and relative humidity at 35%. The produced membranes were placed in a vacuum oven for 24 h at room temperature to remove any solvent traces. The membranes made were labelled “PCL_FillerPercentage_F-FeThe thermal stability of the nanocomposite membranes was examined using thermogravimetric investigations (TGA-DTGA) using a Mettler Toledo TGA/STDA851e in flowing air at a 10 K/min heating rate.2 environment at a flow rate of 50 mL/min.Setting a heating rate of 10 °C per minute, differential scanning calorimetry (DSC) was carried out using a Mettler Toledo DSC 822e in an NmH) and comparing it to the enthalpy of fusion of 100% PCL (ΔHm100%), 136.1 J/g, the crystallinity evaluation was evaluated through Equation (1):α is the mass fraction of magnetite in the nanocomposite. By considering the enthalpy of fusion  operating at 35 kV and 40 mA. The analysis was conducted in the 2θ range of 10–30 degrees. The data were analysed with the same methodology reported by Naddeo et al. . The cryGenerally, to study electrospun membranes with functional nanoparticles, the physicochemical properties of the embedded nanoparticles in regulating nano-bio interactions are well-recognized aspects, as well as the effects of the size, shape, and surface charge of nanoparticles on their biological performances. All these aspects were extensively investigated in the literature. The role of nanoparticle mechanical properties in drug delivery has only been recognized recently and remains the least explored. Recently an interesting article review provided an overview of the impacts of nanoparticle mechanical properties on cancer drug delivery . The artThis probe makes mapping nanometric mechanical characteristics possible between 10 MPa and 10 GPa. The elastic modulus was measured with an accuracy of 10 MPa and positioned on the sample surface with a precision of 10 nm. The Derjaguin-Muller-Toporov (DMT) model was used to process the height and local mechanical data using the procedure reported previously in the literature ,60, whicMechanical characterization was carried out using Dual Column Tabletop Testing Systems  in tensile tests. Five samples of each membrane, each measuring 1 cm in width by 5 cm in length and 0.1 mm in thickness, were tested at room temperature. Tensile tests were performed by elevating the beam at 10 mm per minute while collecting information on the force and distance travelled. The data were then detected as stress-strain curves.3O4 nanoparticles are responsible for the random pyrolysis of PCL chains and accelerate the thermal degradation of PCL; a phenomenon already found in the literature for magnetite-based nanoparticles [TGA measurements were carried out on the obtained membranes. The results, reported in articles . As repoarticles ,64. Howearticles ; (ii) tharticles . 3O4 amount. The DSC graph is reported in DSC analyses evidence that these types of systems are thermally stable (no transitions) around human body temperature. In fact, PCL, which is characterized by a low melting point (around 60 °C), tends to increase the melting temperature by increasing the F-Fe3O4) to 64% (for 5 wt.% of F-Fe3O4). In a PCL membrane loaded with 10 wt.% of F-Fe3O4, the large amount of magnetite hinders the crystallization process of the polymer, determining a decrease in the crystallinity degree to 43%.The inclusion of the filler in the polymeric matrix plays a relevant role. It causes an increase in the melting temperature, since it causes an increase in the crystal size in the matrix, as detectable by the narrowing of the melting endotherm profile, which becomes progressively narrower as the amount of magnetite increases with a simultaneous shift at a higher temperature with respect to the PCL alone. The crystallinity of the PCL, evaluated as the difference of melting enthalpy, is compared to that of a theoretical 100% PCL (136.1 g/mol). This analysis (reported in c) and the crystallite coherence lengths perpendicular to reflection planes 110 (D110), 200 (D200), and 111 (D111) for the PCL component of the membranes is described in All these data were confirmed by XRD analysis. The procedure to evaluate the crystallinity degree  investigation. The results shown in The evaluation of F-FeThe local mechanical characteristics of the nanocomposites were evaluated with an innovative approach, using the H-AFM, through which it is possible to obtain an accurate mechanical mapping of the nanofiber surfaces. The height and DMT modulus images are shown in As described before, including magnetite nanoparticles in the matrix affects the crystallinity. This constatation explains the behaviour of the H-AFM results.3O4, with more perfect PCL crystals , the presence of higher peaks is due to the presence of magnetite aggregates in the matrix together with the presence of more perfect crystals. It is also possible to see that the aggregates can reach an extension of the aggregate domains up to 550–750 nm.As reported in 3O4 system presents overall an average mechanical modulus higher than PCL_5%Fe3O4, and a wider distribution, because of a more significant number of magnetite aggregates. This aspect was also considered by evaluating the distribution’s asymmetry. The skewness of the various distributions was evaluated ; in contrast, there is a higher quantity of nanoparticles and clusters, which causes an increase in the local mechanical values. For this reason, the distribution is asymmetrical and shows a strong dependence on the nanoparticle percentage. This parameter gives information about the asymmetry of the distribution of the local mechanical values.  It incre3O4 loading increases the material’s elasticity, so the local mechanical properties well explain the increase in bulk properties. The Young modulus of the PCL (4.58 MPa) slightly increases by increasing the magnetite content from 5 wt.% (6.40 MPa) to 10 wt.% (6.81 MPa), following the trend of the nanometric measurements. The elastic values measured at the nanoscale differ from the bulk values since the H-AFM evaluates the mechanical properties by tapping the sample (similar to the indentation), whereas the bulk tests are in tensile. It is worth noting that the strain at the break of the various membranes is not significantly affected by the inclusion of magnetite nanoparticles, that in fact is around 200% for all the membranes. It is probably due to the fact that the bulk properties of electrospun membranes also depend on other parameters, such as the fibre dimensions formed by the electrospinning process [In synthesis, the mode is a more accurate statistical parameter to monitor the DMT modulus of the matrix, whereas the average is the statistical parameter that describes the properties of the whole nanocomposite. The local mechanical properties of the material were compared to the bulk mechanical properties in tensile mode, and the results are reported in  process  or the fLF is the load force, E* is the reduced Young’s modulus, E* = E/(1 − ν2), ν is the Poisson ratio, R* is the reduced radius 1/R* = 1/Rindenter + 1/Rsurface, i is the indentation depth, and pull-offF is the force at the point of pull-off of the AFM probe, or the pull-off force for short. The pull-off force can easily be found from the force–indentation curves.It is worth noting that by performing a comparison between the elasticity modulus obtained on nanometric domains of the sample surface through H-AFM mode and that obtained from stress-strain curves of the bulk membrane, the values of the elastic modulus detected by H-AFM seem overestimated, even if the trend is the same applying the two different modes. Considering the quantitative difference in the modulus values obtained using the different modes, the following question arises spontaneously: how reliable is the calculation procedure through H-AFM if one wanted to use the data to get a quantitative map of the elastic modulus on the surface? To answer this question, we have to consider two aspects: one concerns the acquisition method through the H-AFM mode, the other concerns the heterogeneity of the material under observation. Although magnetic nanoparticles are functionalized to optimize their dispersion along the fibres, they are still responsible for the heterogeneity in the local mechanical response, as well detectable by DMT Modulus profiles along the fibre lengths in By using the DMT model, the force–indentation is strongly affected by the heterogeneity of the sample. In particular, the indentation force on the magnetic nanoparticles is strongly affected by the rigidity of the magnetite domains. The average DMT modulus of the functionalized magnetite nanoparticle was found to be around 6 GPa. This value agrees with the value found in literature by the other authors for the magnetite hardness alone . In summBoth methods of acquiring the mechanical properties are relevant because they allow the membranes to be designed from two different points of view. The one based on the tensile tests of the material allows considerations on the mechanical consistency of the material from the point of view of the macroscopic application, while the one relating to the local mechanical properties allows information on aspects related to bioreactivity  to be obtained, as described in In this paper, the inclusion of functionalized magnetite nanoparticles in electrospun membranes and their effect on the nanometric properties of the material were explored. The inclusion of nanoparticles affects the thermal and structural properties of the material. Differential scanning calorimetry analyses reveal a strong variation of crystallinity by increasing the magnetite amount from 0 wt.% to 10 wt.%. This dependence is non-monotonous since, for a high amount of nanoparticles, the filler starts to hinder the crystallization of the polymer. The nanometric mechanical properties were explored via an innovative approach through H-AFM, which was used to track the local mechanical modulus along the nanofiber lengths. The results evidence that the nanometric mechanical properties of the samples are a function of the filler percentage and the crystallinity degree. The variation in the bulk mechanical properties is in accordance with the nanometric analysis, reporting an Increase of the Young modulus by increasing the filler percentage.


It was shown that, in some cases, the substructures formed during the partial destruction of biopolymer macromolecules also have a significant effect on the classification of lignins of various origins.Lignin is considered a promising renewable source of valuable chemical compounds and a feedstock for the production of various materials. Its suitability for certain directions of processing is determined by the chemical structure of its macromolecules. Its formation depends on botanical origin, isolation procedure and other factors. Due to the complexity of the chemical composition, revealing the structural differences between lignins of various origins is a challenging task and requires the use of the most informative methods for obtaining and processing data. In the present study, a combination of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and multivariate analysis of heteronuclear single quantum coherence (HSQC) spectra is proposed. Principal component analysis and hierarchical cluster analysis techniques demonstrated the possibility to effectively classify lignins at the level of belonging to classes and families of plants, and in some cases individual species, with an error rate for data classification of 2.3%. The reverse transformation of loading plots into the corresponding HSQC loading spectra allowed for structural information to be obtained about the latent components of lignins and their structural fragments (biomarkers) responsible for certain differences. As a result of the analysis of 34 coniferous, deciduous, and herbaceous lignins, 10 groups of key substructures were established. In addition to syringyl, guaiacyl, and  The latter act as precursors for p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) phenylpropane structural units (PPU), respectively. An absence of genetic control of the polymerization process and a great diversity of bonds between the structural units of the macromolecule determine the irregularity of the polymer chains and the dependence of the resulting structure on many factors. Despite the fact that there are a large number of publications devoted to the structure of lignins of various origins [Being the second most abundant biopolymer in nature, lignin has attracted increasing attention from researchers as a renewable source of valuable chemical compounds and a feedstock for the production of various materials. Physical-chemical properties of lignin and thus its suitability for certain directions of processing are determined by the chemical structure of its macromolecules. They are formed as a result of enzymatic dehydrogenative polymerization of  origins ,2,3,4,5, origins ,6,7,8,9. origins . In this regard, a detailed classification of lignins should be based on the most complete consideration of both major and minor structural features of preparations obtained from various plant materials. This is a very difficult and non-trivial task. Its solution is challenging and requires the use of the most sophisticated analytical techniques. Among them, high-resolution mass spectrometry (HRMS) and 2D NMR spectroscopy certainly dominate ,12. The Surprisingly, there are still no data in the literature on the use of multivariate analysis (MVA) of 2D NMR data to study the structural differences of lignins. Nevertheless, this approach has already been successfully used to discriminate wines based on their polyphenolic composition , as wellThe aim of this study is to expand the scope of this approach for the classification of lignins by botanical origin and to determine the key substructures (biomarkers) for their differentiation by PCA and HCA analysis of 2D NMR (HSQC) spectra. As an example, preparations of dioxane lignin isolated from coniferous and deciduous wood, as well as herbaceous plants, were studied.1H-13C HSQC spectra according to the procedure described in Differences in the chemical composition and structure of 34 dioxane lignin preparations , includiy-axis with PC3 made it possible to better differentiate grass lignins highlighting the areas corresponding to monocots and dicotyledons  were converted back to the corresponding HSQC NMR spectra , which aAs expected, the main differences between lignins are in the ratio of syringyl and guaiacyl monomeric units (S/G), namely, in the presence of S-structures in the composition of deciduous lignins, and vice versa, the predominance of G-structures in coniferous lignins. In the case of herbaceous lignins, the important role is played by the presence of H-type structures, which make a significant contribution to PC2. However, the composition of the main dimeric structures also largely affects the differentiation of lignins. In particular, fragments of β-aryl ether and resinol predominate in the composition of deciduous lignins, while the substructures of phenylcoumarane and secoisolariciresinol types are characteristic of coniferous lignin. This is in good agreement with the literature data. It is worth noting that the signals belonging to Hibbert’s ketone and its isomers are observed as negative peaks for PC1, PC2, and PC3 and thus dominate in the composition of coniferous lignins. This may indicate that the destruction of β-O-4 bonds in them during the isolation procedure takes place to a greater extent.Heracleum) is located in the cluster of lignins isolated from hardwood. This may indicate that its chemical composition is distinguished with a significant proportion of S-structures and the absence of cinnamic acids which are characteristic of grass lignins. Another illustrative example is the lignin of saxifrage [The differentiation of grass lignins is not so unambiguous. In particular, the dioxan-lignin of the dicotyledonous plant Sosnowsky’s hogweed  lignin which unexpectedly falls into the zone of dicotyledonous plants can be considered an exception. This is explained by the absence in its structure of flavonoid tricin fragments characteristic of monocots [In the aforementioned differentiation of lignins of monocotyledonous and dicotyledonous herbaceous plants in the PC1-PC3 coordinates b, cattaimonocots . It is aThe combination of PC1 and PC4 for constructing the score plot c proved The results described above show that the coniferous lignins not only differ significantly from the lignins of hardwood and herbaceous plants but are also characterized by a significantly lower structural variability compared to the latter. In this regard, the differences in the composition and structure of dioxane lignins of coniferous trees belonging to different species were analyzed in more detail. The results of the PCA showed that 100% of the variation in the data can be described by 11 principal components, of which PC1 and PC2 account for 29.3 and 21.7%, respectively . CombiniObviously, when using the score plot in PC1-PC2 coordinates a, a cleaBased on the obtained results, two reasons can be suggested to explain the observed picture. First of all, the absence of H-type structures in the composition of spruce lignins may be due to the fact that the age of the plants selected for the isolation of spruce lignin preparations was about 80 years, while other representatives of coniferous trees were aged 25–30 years. Another reason may be associated with inherent limitations of the method caused by the lignin isolation procedure. In the latter, hydrochloric acid solution (0.7%) in dioxane was used as a mild hydrolytic agent facilitating the release of lignin. However, it also contributes to undesirable side processes of acid-catalyzed destruction and transformation of most labile structures in biopolymer macromolecules. They result in the formation of larger amounts of Hibbert’s ketones and other degradation products. Thus, to avoid possible classification errors, it is necessary to know which of the detected structures belong to intact lignin and which were formed during its isolation from a plant material. In the case of differentiation of lignins by classes, this factor may not have a significant effect, while a more detailed analysis, such as classification of coniferous lignins by families, is greatly complicated.Of all the samples, only larch lignin preparations were not grouped together. An analysis of the PC2 loading HSQC spectrum  makes itThe complete separation of the pine, larch, and juniper lignin areas on the score plots was not achieved either in the PC1-PC2 coordinates, or even when using other principal components with the highest contribution (up to PC6). This means that lignins of coniferous tree species have a fairly similar structure; therefore, a distinctive feature for each of the lignins may not be significant for the tested principal components. To this end, other PCs with less contribution to the description of all variations were analyzed revealing more subtle differences in the biopolymer structure. As a result, it was found that the signals described by PC7 b may be The data obtained from the loading spectrum of PC7  showed tSummarizing the results of PCA analysis and structural information from HSQC NMR loading spectra of lignins of various origins, the following classification of the studied biopolymer preparations presented in In general, HCA analysis of the 2D HSQC NMR spectra of the studied lignins showed the same pattern as PCA. However, it allowed for a more detailed and clearer clustering of preparations isolated from the plants of various species and families . Two large clusters are observed on the dendrogram, one of which, in turn, is divided into two separate subclusters of deciduous and herbaceous lignins. Another subcluster involves mainly coniferous lignin preparations. This differentiation is obviously caused by the ratio of the main types of PPU. It should also be noted that 3 of the 34 analyzed samples were differentiated separately. Two of them belong to cereal straw lignin, which is explained by the presence of flavonoid-type substructures and other impurities in them. In addition, it was shown that aspen lignin is not a part of the hardwood lignin subcluster, which is explained by the presence in its structure of fragments that were largely modified during isolation procedure.p-hydroxybenzoates  stems were obtained from Piramida settlement . Other plants were harvested in the Primorskii district of the Arkhangelsk region (Russia). At least three samples of each plant species from different sites were averaged prior to lignin isolation. Dioxane lignins were isolated from plant tissues  by the Pepper’s method [n) and weight-average (Mw) molecular masses were determined by size-exclusion chromatography on an LC-20 chromatographic system  consisted of an LC-20AD pump, a DGU-5A vacuum degasser, an STO-30A column thermostat, an SIL-30AC autosampler, and an SPD-M20A diode array UV-VIS spectrophotometric detector. The separation was carried out on at 40 °C on an MCX column, 300 × 8 mm, pore size 1000 Å . Aqueous solution of sodium hydroxide (0.1 M) was used as a sample solvent and mobile phase. The samples of saxifrage  and transferred to a 5 mm NMR tube. The 1H-13C HSQC spectra were recorded on a Bruker AVANCE III 600 MHz spectrometer  using the Bruker library hsqcedetgpsisp2.3 pulse sequence. The experiments were carried out at 298 K using the following acquisition parameters: size of FID—1024 (F2) and 256 (F1), number of scans—32, relaxation delay—2 s, spectral width—15 ppm (F2) and 239 ppm (F1), transmitter offset—5.719 ppm (F2) and 98.2 ppm (F1), 1JC-H = 145 Hz. After zero filling, the resulting data matrix size was 1024 (F2) × 1024 (F1). The spectra were processed using Bruker’s Topspin software version 3.2. In total, 50–80 mg of the dry lignin powder was dissolved in 0.55 mL of DMSO-dDespite the fact that the assignment of the main cross-peaks is well described in the literature, visual comparison of data and integration of peaks related to the main substructures does not give a complete picture and does not allow us to establish a specific set of fragments characteristic of a particular lignin. The solution to this problem is possible by analyzing the data array of 2D HSQC spectra using multidimensional analysis. In this work, a combination of PCA/HCA methods was used. An overview of the procedure is shown in The initial data was the HSQC spectrum data matrix in the range δC/δH 0-210/0-10 ppm, which includes all cross-peaks characterizing the composition and structure of lignin preparations. Preparation and preprocessing of NMR spectra for multivariate analysis was performed in accordance with protocol described by Hedenström et al. . The obtThe scores used to construct scores plots were used as a distance matrix, allowing us to confirm the clustering of the studied lignin samples using the hierarchical method (HCA).1H-13C HSQC spectra was performed in MATLAB R2021a software , as described in Ref. [POKY software (version 20220114) was used for preliminary modeling of HSQC spectra . For mul in Ref. . PCA ana1H and 13C dimensions, respectively.Qualitative analysis of 2D HSQC NMR spectra of the studied lignins and pseudo-NMR spectra representing loading plots of the main components was carried out by comparison with data from the literature sources ,30,31,32p-hydroxyphenyl PPU, they include various terminal substructures: dihydroconiferyl alcohol, balanopholin, cinnamic acids, and tricin. It was shown that, in some cases, the substructures formed during the partial destruction of biopolymer macromolecules also have a significant effect on the classification of lignins of various origins.The use of 2D NMR spectroscopy in combination with PCA and HCA makes it possible to effectively classify lignins of various botanical origins at the level of belonging to classes and families of plants, and, in some cases, individual species. The reverse transformation of loading plots into the corresponding HSQC NMR loading spectra provides structural information about the latent components of lignin and its structural fragments that cause certain differences. As a result of the study of 34 coniferous, deciduous, and herbaceous lignins, 10 groups of main substructures that determine the differences between lignins were established. In addition to syringyl, guaiacyl, and To the best of our knowledge, this study represents the first attempt to implement 2D NMR spectroscopy in the detailed classification of lignins and identification of related biomarkers in their structure using MVA. Along with the demonstrated advantages of the proposed approach, it is necessary to note its natural limitations revealed in our study. They are primarily related to the difficulty of interpretation and assignment of signals in 2D NMR spectra and the presence of overlapping peaks. In our opinion, overcoming this problem is possible by combining 2D NMR and high-resolution mass-spectrometry data, which may be a part of future research. It should also be focused on the introduction of the PLS-based approaches and expansion of the range of studied lignins, including the technical preparations obtained during the industrial processing of biomass.


Available information in the databases indicated that 7 of these 36 genes are either associated with or potentially related to hearing disorders. In conclusion, the LLN are characterized by specific molecular profiles that reflect their rostrocaudal organization into the three constituent nuclei. This molecular regionalization may be involved in the etiology of some hearing disorders, in accordance with previous functional studies of these genes.The dorsal (DLL), intermediate (ILL), and ventral (VLL) lateral lemniscus nuclei are relay centers in the central auditory pathway of the brainstem, commonly referred to as the lateral lemniscus nuclei (LLN). The LLN are situated in the prepontine and pontine hindbrain, from rhombomeres 1 to 4, extending from the more rostral DLL to the caudal VLL, with the ILL lying in between. These nuclei can be distinguished morphologically and by topological and connectivity criteria, and here, we set out to further characterize the molecular nature of each LLN. We searched  The lateral lemniscus nuclei (LLN) constitute a complex of three independent nuclei in the main auditory pathway of sound perception and spatial sound localization. The LLN transmits signals from lower auditory brainstem nuclei like the cochlear nuclear complex (CN) and the superior olivary complex (SOC), to higher auditory structures like the inferior colliculus (IC) in the midbrain or the medial geniculate nucleus in the thalamus .The LLN is comprised of the dorsal (DLL), intermediate (ILL) and ventral (VLL) nuclei, each of which is embedded within the fibers of the lateral lemniscus tract. These subnuclei are located in the prepontine and pontine hindbrain, with both the DLL and ILL lying within rhombomere (r) 1, while the VLL extends from r2 to r4 .Some studies consider that the LLN in rodents is composed of two nuclei, the DLL and the VLL , which pSlc32a1), a marker of both GABAergic and glycinergic neurons, and very weak expression of the Vesicular Glutamate Transporters  that are markers of glutamatergic neurons  expression has been detected in the DLL  immunoreactivity  has been detected in the DLL and VLL of adult rats  database . AdditioPrior to performing the gene expression analysis, we identified the limits of the LLN in sagittal and coronal sections through the specific labeling of the lateral lemniscus fibers, correlated with Nissl-stained preparations in equivalent planes and following the description of these structures in the literature .1 1  or throu1  into specific brain regions, such that the axonal projections from the infected neurons can be visualized through the expression of EGFP. We studied tracer injection into the ventral cochlear nucleus, allowing visualization of a large part of the lateral lemniscus fibers that reach the IC.in situ hybridization (ISH) data were available from series of sagittal and coronal sections at P56.3 These data consisted of brightfield microphotographs and their counterparts with color-coding of their relative strength of expression. In the brightfield images, the positive cells accumulated a blue precipitate according to standard ISH protocols, while in the color-coded images the intensity of expression ranged from blue (weak expression), through green (medium intensity) to red (strong expression). We analyzed these image series to select genes with regionalized expression within the LLN coherently reproduced in both sagittal and coronal planes. As a result of this screening, we identified 36 genes  for which 36 genes .The images that included the whole LLN were downloaded and cropped to show the region of interest. In the figures, the images from parasagittal sections are orientated with the rostral end to the left, while those from coronal sections show the left side of the original image, with the midline on the right.4 (Images of some selected genes from embryonic (E18.5) and postnatal stages  were also downloaded from the Allen Developing Mouse Brain Atlas (ADMBA)4   can be observed in lateral sagittal sections  as an unIn representative coronal sections, the VLL and ILL can be observed at rostral levels , and theIn a coronal caudal section containing the three nuclei , the DLLIt is noteworthy that while the VLL and DLL are the most caudal and rostral nuclei of the LLN, they nevertheless appear in rostral and caudal coronal sections, respectively .Of the 36 genes identified in the screening, 6 are expressed exclusively in the DLL  and not paranase ; Pax7, Ped box 7 ; Pnoc, Pciceptin ; Cgnl1, Hpse and Pax7 .The expression of and Pax7  in the Dx7  A receptor subunit alpha 5  member 1 (data not shown).There were 8 genes expressed in both the DLL and VLL but not in the ILL : Fstl1, n-like 1 ; Gabra5, alpha 5 ; Gad1, Gxylase 1 ; Inhbb, n beta-B ; Rreb1, rotein 1 ; Gad2, GInhbb and Rreb1 in the VLL is restricted to a caudal portion, that is visible in medial sagittal sections abutting the SOC  receptor 1A .There were 12 genes expressed in both the ILL and VLL : Hcn1, Hhannel 1 ; Igfbp5,rotein 5 ; Tnnt1, ponin T1 ; Cacng5,Tnnt1, Cd24a, Crtac1, and Kcnq4 .Regarding the most caudal nucleus, 7 genes were only expressed in the VLL : Calb1, bindin 1 ; Doc2g, C2 gamma ; Tmem215tein 215 ; Gata3, Gata3, Kcnq4, Gabra5, Hcn1, and Kit. In the Online Mendelian Inheritance in Man (OMIM) database, 3 human orthologous were associated with deafness or hearing loss . From the results of these searches, we discarded other genes whose known hearing phenotypes were related either to ear morphology  or language learning (Foxp2).Five of the genes identified were reported to carry mutations that cause hearing-related mouse phenotypes in the Mouse Genome Informatics (MGI) database: KCNH7 and TMEM215) that belong to the DFNA16 . Hcn1 expression is restricted to the VLL at E18.5  and early postnatal (P4) stages. In the Allen Developing Mouse Brain Atlas there are available ISH experiments for 4 of these genes    labeling the ILL  correspoHcn channel family (Hcn1-Hcn4) participates in the excitability of cells in the auditory system . This latter pattern is on the whole similar to that described in two distant species, the mustached bat , whereas the profile of the ILL is mainly excitatory :  are expressed in the DLL and VLL, while Slc17a6 (glutamatergic marker) is expressed in the ILL. In addition, we found that the GABAA receptor subunit Gabra5 is also expressed in the DLL and VLL, along with other subunits reported previously : . Our reseviously .Inhbb and Rreb1, expressed only in the medial but not in the lateral planes. This may be due to the VLL nuclei extending over several rhombomeres (r2-r4), such that the r2 and r3 parts of the VLL are evident in lateral planes whereas the r4 component of this nucleus is mainly seen in medial planes. The regions of expression in the VLL in which these genes might or might not be expressed could correspond to further subdivisions of the rodent VLL  is not straightforward. In reptiles, a single lemniscal nucleus has been described  in contrGata3, Kcnq4 and Kit) are associated to hearing disorders in both mouse and human. GATA3 is defective in the Barakat syndrome, a disorder that includes sensorineural deafness  Project, NINDS Contracts N01NS02331 and HHSN271200723701C to the Rockefeller University , available from PA conceived the research. IG-G, FM, and PA performed the data mining, analyzed the data, prepared the figures, and wrote the manuscript. All authors contributed to the article and approved the final submitted version.


The relative bandwidth is 2.98%, which satisfies the standard of wide-band patch antennas.This paper proposed a right-handed circularly polarized (RHCP) micro-strip antenna for multi-navigation system applications. The size of the antenna is 70 mm × 70 mm × 2 mm, which is fabricated on an FR4 substrate. A meandering technique on a patch layer and asymmetrical defected ground structures (DGS) are employed to achieve the purpose of miniaturization and increase the bandwidth of the axial ratio. The prototype of this antenna is fabricated according to simulations where the bandwidth of return loss, bandwidth of axial ratio, and radio pattern are further testified. The bandwidth of return loss (S With the development of the civil aviation industry, aircraft are favored for efficiency in medium- and long-distance travel. Air safety is always the critical issue in transportation. With the increasingly dense air routes, which contain both passenger and cargo traffic, high-accuracy tracking and location of aircraft has become the focus of research ,2,3. TheAn antenna is a critical component of airborne equipment. Within the corresponding operating frequency band of targeted navigation systems, an antenna serves as the transceiver of navigation information that would be applied for real-time positioning ,9. SinceUp to now, three polarization modes of antennas have been proposed, namely linear polarization, elliptical polarization, and circular polarization. Recent research has shown that circularly polarized waves will benefit from their strong ability to resist multi-path interference and polarization mismatches affected by various environmental factors. They can also be converted into linear polarized waves as needed ,12,13. IIn the design of navigation antennas at present, in addition to the requirements of wide operating frequency band and gain, it is also necessary to take the installation environment into account. For example, antennas work with other complex avionics, and it is significant to reduce the size to improve the portability ,16. Amon11) and axial ratio (AR) is the limit of the micro-strip antenna [At present, the narrow bandwidth of both return loss . All the design indicators are satisfied after parameter optimizing. In section four, the antenna is tested by a vector network analyzer and microwave anechoic chamber. Comparisons of simulation and experimental results are given, which prove that the antenna meets the design requirements.In this paper, the designed wideband RHCP micro-strip antenna is proposed, which would be available for aircraft tracking in B1I and B1C for BDS-3, L1 of GPS, L1 of SBAS as well as E1 of Galileo. In section two, the principles of the meandering technique and DGS are introduced, which provide the theoretical support for broadening the band width of Sf denotes the resonant frequency, c is the speed of light, L refers to the length of the antenna patch, and rε is the relative dielectric constant of the dielectric substrate.The resonant frequency of the antenna can be retrieved as given by Equation (1),f will be affected by both dielectric constant and patch length. However, this will inevitably increase the risk of occurrence of both wave loss and the shortening of frequency bands. Therefore, our work will focus on structure optimization by applying meandering technology and defect ground structure to broaden the bandwidth of both the return loss and the axial ratio of antennas.From Equation (1), it can be observed that The meandering technique shown in gL and capacitors gC and pC in ω1 and ω2 refer to central frequency and cutoff frequency, and rB and pB are the electromagnetic induction intensity generated by gR and pR, respectively.Recently, more attention has been paid to DGS for performance enhancement of CP antennas. When the electrons move in the periodic potential field formed by the high-impedance surface, an electronic band structure will be generated due to the Bragg scattering , and theStep 1: Determine the basic parameters of the proposed micro-strip antenna patches. The step will be carried out according to the Equations (4)–(8) given in Step 2: According to the meandering technique, T-shaped and F-shaped gaps are added to adjust the current direction to generate the current that can flow counterclockwise and produce right-handed circularly polarized electromagnetic waves. At the same time, a square gap in the center of the patch is employed to increase the forward gain of the antenna. By means of the gap structure, the enhancement of the surface current path will then reduce the patch size of the antenna.Step 3: Design a defective structure by introducing two L-shaped slots on the diagonal of the same side of the coaxial feed, aiming at forming a high-impedance surface and expanding the axial bandwidth.Step 4: Scan the main parameters that would potentially affect the performance of antenna to determine the size of all antenna parameters.The main design steps of the antenna proposed in this article are as follows:In the S-band and below, the FR4 material has the ability to act as an antenna dielectric substrate. To be precise, FR4 material is a glass fiber mixed with epoxy resin. The heat resistance of this material can reach 300 degrees, the operating frequency is about a few GHz, and the nominal value of the permittivity is about 4.4. FR4 material has nearly zero water absorption and can maintain its working performance in both dry and wet conditions, making it suitable for installation on aircraft. At the same time, its price is much lower than the dielectric substrate material of high-frequency boards, which meets the requirements of industry to reduce production costs.Step 1: Complete the preliminary design of the patch antenna using the nominal value of FR4 material;11 and AR distributions, respectively;Step 2: Perform simulations to the designed antenna and extract corresponding S11 and AR of the designed antenna using vector network analyzer;Step 3: Measure the SStep 4: Compare the simulation results of the antenna with the measured results to obtain the deviation distribution. Afterward, the values of FR4 in the simulation are adjusted within a certain range to obtain a series of simulated distributions under different values;rε of FR4 is calculated and set as 4.52;Step 5: Perform least squares analysis on simulated and measured distributions. At this point, the dielectric constant with the smallest least squares estimation value is closest to the true value. In the design of process, permittivity Step 6: Perform antenna design and optimization with the permittivity of FR4 extracted from Step 5.At present, the nominal permittivity value of FR4 material is generally employed for antenna simulations as 4.4. However, due to the deviation of the dielectric substrate material selected by the manufacturer and the inevitable processing errors varying from 4.2 to 4.6, there will be significant differences between the simulated and measurement results of the antenna. This will lead to serious effects on the reliability of the antenna design. To reduce the impact of FR4 parameters on antenna design accuracy, we propose a feedforward optimization method that corrects the estimated value of FR4 through the deviation between sampled simulation and measured results. The critical correction steps are as follows:R denotes the radius of patch, f is the center frequency of working band, h refers to the thickness of antenna, and rε is the permittivity of substrate.In the recent implementations of micro-strip antenna, the features of patch could be generally summarized as circle and square one, respectively. Circular patch antennas possess more characteristics for structure design and more easily generate circularly polarized waves. In addition, circular patch antennas will generate higher radiation and lower losses. The radius of circle patch can be formulated given by Equation (3),f, rε, and h equal 5.8 GHz, 4.4, and 2 mm, respectively. In the same condition, the length of square micro-strip antenna only measures 3.14 mm. This proves that square micro-strip antennas perform better in terms of miniaturization. Compared with circle patch antenna, the square micro-strip antennas now are proven as the reasonable choice in combination with other electronic components, especially in some complex environments such as airborne systems. Moreover, the square patches are easier to produce than the circle ones, saving production costs and resources. Therefore, a square structure will be employed in the design of our antenna, and corresponding simulations as well as analysis will be carried out to improve the effectiveness of our design.Based on this structure, Si et al.  have proWT is printed on the substrate  of the antenna. According to [WT can be calculated according to the equations that follow.According to the analysis above, the circular polarized antenna serving the BDS-3 system is developed and designed with corresponding geometrical configurations as given in rding to , the lenL can be preliminarily defined as Equation (4),c refers to the velocity of light, f is the resonant frequency of antenna, and rε is the permittivity of FR4. Height of the antenna h is set as 2 mm. Then, the effective permittivity of antenna could be defined as Equation (5),refε denotes the effective permittivity. Here, we introduce the length extension of patch given by Equation (6),L refers to length extension. From Equation (4), we can obtain effective length as shown in Equation (7),efL refers to the effective length. The actual length WT could be calculated as Equation (8),The initial length of patch WT is calculated as 45.5 mm according to Equation (8).In this work, p × q is embedded along the x axis. In this patch, a single probe feed is implemented at  and works for exciting right-handed CP radiation. By using the theory of the meandering technique, one T-shaped structure is added to each edge of the square patch, as given by On the patch of the antenna, a narrow slot with dimensions of As shown in As shown in In order to improve antenna performance, especially AR, DGS is designed on the ground of the antenna. 11 and AR can be adjusted by changing parameter values.The geometry of the antenna is further optimized based on the analysis of simulated bandwidth retrieved from software HFSS. As given by 11 and AR shift to a high-frequency band with the increase in parameter dw. 11 and AR move down with the increase in db. In 11 moves up with increase in p, while in pg. In X1g increases, the axis ratio moves downwards to the high frequency, and the effect on S11 is not significant within the operating frequency band.11 and AR as a whole by changing parameters dw and db. Parameter p can be used for changing S11 separately, while pg and X1g are for AR.It is practical to adjust S11 and AR are given in 11 and AR are from 1.540 GHz to 1.612 GHz and 1.554 GHz to 1.601 GHz, which indicate the working band of the design is the second one.The antenna after validation is modeled and processed based on PCB printing. (1)Since the dielectric substrate material is FR4, its dielectric constant is 4.2 to 4.6, the dielectric constant of FR4 material used by the manufacturer cannot be guaranteed to be identical with the set value of 4.52 in thesofware HFSS (version 19.2). And the dielectric constant of the substrate material has a large effect on the resonant frequency of the antenna, which causes the difference between the simulation and the actual measurement.(2)In the manufacture of the antenna, high manufacturing accuracy is required due to the use of a loaded seam and defective ground structure. Manufacturing errors within the process allowable errors can also cause errors in the test results of the antenna’s performance.(3)When welding the coaxial feed probe, the corner of the L-shaped slot of the defective ground structure is inevitably blocked by the SM joint, resulting in a deviation in the measured results.(4)The unavoidable presence of non-insulating materials in the measurement environment would impact the measurement reliability.11 and AR retrieved from experiments, the operating band is 0.047 GHz. The relative band width (RBW) is defined by Equation (9),Hf and Lf are the are the highest and lowest values of the operating band.According to the bandwidths of SRBW of this design is calculated as 2.98%, which is greater than 1% and satisfies the definition of a wideband patch antenna. The following table shows a comparison between the proposed antenna and other designs.Although there exist some deviations between the measurement results and the theoretical values, our proposed concept for micro-strip antenna design still has significant advantages compared to the existing methods in refs ,33,34,3511 and radiation patterns. The performance of the antenna and quality of our work are validated according to the comparisons between simulated and measured results. The relative bandwidth of the antenna is determined as 2.98%. The suggested antenna also possesses high application potential compatible with L1 of GPS, L1 of SBAS, and E1 of Galileo.In this work, a circularly polarized antenna is proposed for BDS-3 applications. By means of the meandering technique, the geometry of the antenna is obtained as 7 mm × 7 mm × 2 mm. Afterward, rectangular-shaped and L-shaped DGS are introduced to broaden the bandwidth of the axial ratio of the antenna. After a series of optimizations, the bandwidth of return loss and axial ratio of antenna can be retrieved as 1.540 GHz to 1.612 GHz and 1.554 GHz to 1.601 GHz. The suggested antenna is fabricated and experiments are carried out on analysis of S


BackgroundThe coronary sinus (CS) and its tributaries have been used to perform various electrophysiological and cardiac interventional procedures which require cannulation. The Thebesian valve (TV) guarding the coronary sinus orifice (CSO) exhibits morphological variations which might make cannulation unsuccessful leading to the failure of invasive cardiac procedures. This study aimed to analyze in detail the morphological features of the TV in fresh autopsied human hearts which were representative of the adult population of this region owing to its practical implications in invasive cardiac procedures.MethodologyThis was a cross-sectional, descriptive study conducted in the Department of Anatomy in collaboration with the Department of Forensic Medicine and Toxicology at Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. A total of 104 fresh adult heart specimens were collected during the autopsy. The CSO was located, and the characteristic shape, composition, position, and extent of coverage of the CSO by the TV were observed and analyzed.ResultsThe TV was present in 65% of heart specimens. The most common shape was remnant (33%), and the most common site of origin was inferior (63%). The valve composition was thin and membranous in 63% of heart specimens. In 7% of heart specimens, the TV covered more than 75% of the CSO diameter, of which in 4% of heart specimens, the CSO was completely closed and found to be obstructive.ConclusionsThis study highlights the variability in the morphological structure of the TV in adult human hearts and its potential implications in unsuccessful CS cannulation and failure of invasive cardiac procedures. Thus, prior imaging of the TV should be an integral part of CS cannulation procedures to avoid unsuccessful cannulation and complications related to repeated forceful cannulation. The coronary sinus (CS) is the major venous trunk draining 60% of venous blood from the heart to the right atrium. Its orifice is situated in the right atrium posteromedially between the right atrioventricular orifice and the inferior vena cava. The coronary sinus orifice (CSO) is guarded by the Thebesian valve (TV), which is embryologically the caudal remnant of the right sinoatrial valve .In this era of percutaneous transcatheter therapies for cardiac diseases, the cannulation of CS has become inevitable. The CSO, the primary point of entry into the coronary venous network, is guarded by the TV which shows a prevalence of 65-95%, along with a large variability in its morphology . The CSOData of morphological variations of the TV described in earlier studies were from formalin-fixed cadaveric hearts. The goal of this study was to highlight in detail the morphological features of the TV in fresh autopsied human hearts, which are lacking in the adult south Indian population, owing to its practical implications in invasive cardiac procedures.This cross-sectional, descriptive study was conducted in the Research Laboratory of the Department of Anatomy in collaboration with the Department of Forensic Medicine and Toxicology at Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry from June 2015 to June 2017. This study was approved by Postgraduate Research Monitoring Committee and Institute Ethics Sub-committee (Human Studies) vide approval number JIP/IEC/SC/2015/19/781 dated July 17, 2015.After obtaining written informed consent from the close relatives of the deceased, 104 fresh adult heart specimens of both sexes aged 21-86 years were obtained during the autopsy by convenient sampling technique method after being subjected to inclusion and exclusion criteria. Exclusion criteria included trauma, previous history of heart surgery, myocardial infarction, cardiomyopathy, and pericarditis. Baseline parameters of the deceased such as name, age, gender, residential address, hospital number, postmortem number with date, the weight of the heart, and the cause of death were noted in the data collection proforma.Heart specimens were washed thoroughly to remove blood stains, and the right atrium was opened by making an incision close to the sulcus terminalis along the right border of the heart from the superior vena cava to the inferior vena cava. The interior of the right atrium was exposed, the CSO was identified, and the presence of the TV and its morphological features were noted, documented, photographed in situ Figures , 2, and A total of 104 fresh autopsied adult hearts were observed and interpreted. Out of the 104 heart specimens, 71 (68%) were males and 33 (32%) were females. The age ranged from 21 to 86 years, with a mean (SD) of 45 (16.2) years.The CSO was present in all heart specimens in normal locations between the inferior vena cava and the tricuspid orifice. Then heart specimens were examined for the occurrence of the TV. The TV was present in 65% 68/104) and absent in 35% (36/104) of heart specimens. Thereafter, the shape of the valve was observed. Six types of valves, namely, remnant, semilunar, fold, cord, mesh , and fenestrated , were observed  and 22% (15/68), respectively. Two cases in fold type and one case in fenestrated type were completely closing the CSO which constituted 4% (3/68).The inferior position of the TV was dominant with a frequency of 63% (43/68). Subsequent morphologies included the valve along the posterior position in 30% 20/68) and the anterior position in 7% (5/68), while the superior margin of the CSO was free from valve attachment (Table 0/68 and The composition (nature) of the TV was assessed subjectively and observed to be thin and membranous in 63% (43/68), thick and fibrous in 24% (16/68), and mixed in 13% (9/68) of heart specimens (Table The extent of coverage of the CSO by the TV was assessed subjectively. In 35% 36/104) of heart specimens, the TV was absent and the CSO was fully open. The remaining 65% (68/104) of heart specimens showed valves covering the CSO to various extents (Table  of heartIn the majority of hearts (63/68), the extent of coverage of the CSO by the TV was <75%. In five out of 68 hearts studied, the valve covered >75% of the CSO, of which three valves were almost covering the CSO completely Figure .This study aimed to describe the morphological features of the TV in fresh autopsied human hearts which are representative of the adult population. The parameters studied included the morphology and position of the TV, composition of the valve, and extent of coverage of the CSO.The TV exhibits variations in its occurrence, size, shape, and extent of coverage . AccordiThe shape of the TV is highly variable. Several authors have tried to classify the TV based on shape ,12,13-17The site of attachment of the TV was from the inferior margin 63%) or posterior margin (30%) of the CSO Table . In five3% or posThe composition of TV was noted to be membranous in 63% (43/68), fibrous in 24% (16/48), and mixed type in 13% (9/68) of heart specimens in this study. The composition of TV studied by authors based either on observation or on histological grounds was membranous ,7,17.The assessment of the extent of coverage of the CSO by the TV revealed that in 63 hearts, the occlusion was <75%. In five hearts (7%), the ostial coverage was found to be >75%. Out of these five hearts, two cases in fold type and one case in fenestrated type were completely closing the CSO, which constituted 4% (3/68) and were designated as obstructive TV, potentially complicating CS cannulation. The definition of obstructive TV shown in Table Several studies demonstrated the usefulness of both non-invasive and invasive visualization methods which can identify the morphology of the valve before invasive procedures. Scholten et al. in a case report demonstrated the usefulness of intracardiac echocardiography to identify the anatomical variations of TV which helped in the successful catheterization of CS . ShinbanThe cannulation of CS is required to perform various diagnostic and therapeutic cardiac procedures which can become difficult or unsuccessful due to the presence of large, fibrous, dynamic, and anomalously originating TV. Thus, pre-procedural imaging of CS and its valve should be integral while planning for CS cannulation. This study highlights the variability in the morphological structure of the TV and its potential implications in unsuccessful CS cannulation and failure of invasive cardiac procedures. Thus, detailed anatomical knowledge about the morphology of the TV is pivotal to planning and adapting procedural strategies during various invasive cardiac procedures.