Rising sea levels, along with other biological and human factors, have increased erosion rates at a number of important sites located along the Atlantic coastline. Project GaltFish implemented a series of contingency measures to record some of these sites before they degraded further or totally disappeared. This process involved detailed photogrammetric recording of some of the sites under threat over a set period of time. One of the sites selected for this project was Sobreira : a Roman fish-salting factory which was partially destroyed by building activity in the 1980s and the remains of which are under threat from marine erosion and human action. In order to study the site, two photogrammetric models were created to examine the effect of erosive processes across the course of one year. The results illustrate that photogrammetry is an efficient tool for recording and analysing the issue of erosion. The data compiled helped in designing additional action in the factory, which was subject to a rescue excavation to record and help protect the site from further damage. This paper presents the results of this project, as well as the methodology used to produce the models, the data generated and their analysis. It is argued that the methodology can be used to collect and analyse data from other sites, and that this data could inform the political/administrative decision-making processes which concern the future management and preservation of archaeological sites under threat. Gallaecia led, in 2016, to the launching of the research project GaltFish—Salt and Fish Salting in Ancient Gallaecia: looking for the origins of the Galician canned fish industry. The project aimed to collect information about Roman fish-salting factories in the region in order to better understand the operation of these establishments and the commercialisation of their products by analysing commercial routes and links between different regions of the empire . The aim of this was to verify if some of these dubious sites, like Sobreira and O Naso, were, in reality, fish-salting factories, while in other cases, like Adro Vello, the objective was to collect new data about the chronology of the site, as well as to obtain a detailed and complete plan of the factory. Besides, during this intervention, an important ensemble of ichthyofauna was recovered inside one of the vats.http://ramppa.uca.es/), of 19 Roman fish-salting factories, located between the mouth of the Douro River  and the Basque–French border , SCAPE/Sch@arp (http://www.scharp.co.uk/), Rapid Coastal Zone Assessment (https://archaeologydataservice.ac.uk/archives/view/rczas/), Arfordir (http://www.ggat.org.uk/arfordir/) or STORM (http://www.storm-project.eu/), focused on prevention and mitigation as well as the design of specific planning policies. Project GaltFish, on a more local level, aims to measure the effects of these erosive processes—both natural and human—on coastal archaeological sites in order to propose specific measures for the preservation of these sites at the local/regional level.In recent years, a number of projects have been launched to implement mitigating measures to slow or halt the deterioration of archaeological sites affected by climate change and other environmental issues, such as project ALeRT , was being used as a step by beachgoers to travel from one beach to the other Fig. .Fig. 6A In addition to the mortar, which was visible and totally exposed, the ends of at least two walls were visible elsewhere on the site. These walls were also traversed by the pathway that ran across the promontory. In addition to human action, aggressive sea erosion had led to the total loss of the northern face of the site. During the winter months, the surf and high tides reach the wall of the chalet built over the site (sometimes even going far beyond this point), and the effects of the resulting erosion have left the wall foundations exposed. As a result, the northernmost features were particularly exposed. The Roman wall identified to the west, in contrast, was covered by dense vegetation, which protected it from erosion. Moreover, since it was far away from the pathway used by beachgoers, it remained unaffected by human action.In addition to this, the presence of a number of increasingly large sinkholes threatened the integrity of the few layers of soil that still protected the remains, as well as that of the surviving Roman walls. Furthermore, the uncontrolled proliferation of undergrowth, both along the walls of the chalet and the site’s frontal facade, exacerbated the loss of archaeological levels.Under these conditions, the total disappearance of the site seemed imminent. This made the site ideal for monitoring the effects of erosion on coastal sites under severe threat, over the course of a year. The results highlighted the need for certain mitigating measures: the first priority was to record as much archaeological evidence as possible and, as far as practicable, to preserve the original structure of the factory, considering the level of destruction already sustained by the site and the impossibility of removing the pathway that runs across the promontory.Although some of the erosive processes affecting the site were plainly visible Fig. , it was In essence, the research protocol comprised three stages: taking the photographs, generating the 3D models and comparing both models. Efforts were made to ensure that the conditions prevailing when both sets of photographs taken (in May 2016 and May 2017) were as similar as possible in order to generate as closely comparable models as practicable, thus minimising the margin of error. Considering the magnitude of the erosive processes observed in the different visits to the site, the final survey should be accurate enough to quantify centimetre differences between both shots.The initial sequence of photographs was taken in late May 2016 (Sequence 1 – S1), and the second around the same dates in May 2017 (Sequence 2 – S2). We attempted to match as closely as possible the weather and light conditions for both sessions, in order to avoid differences that could have a negative effect on the processing and comparison of both sequences. Some changes, however, had to be made: for instance, the second session (S2) had to be undertaken in the afternoon rather than midday to match the low-tide conditions prevailing during the first session (S1). This leads, however, to a notable light-shadow difference between both models, mainly visible in the final textures.The photographs were taken without the aid of a tripod or any other physical support, owing to the rugged terrain conditions. In addition, the two sequences were taken with different cameras, which slightly affected the quality of the photographs and consequently the final models Table .Table 1Both sequences followed a linear pattern. Photographs were taken along a line with a separation interval of approximately 1 m, and a minimum overlap of 60–70%. Photographs were taken from east to west and from west to east along the pathway that crosses the promontory, resulting in a full photographic sweep of the site. Areas which were deemed to be blind spots, especially several sectors of the rocky outcrop, were photographed separately. In order to ensure that the whole site was covered, additional photographs were taken from farther away, resulting in general overviews that facilitated the assembling of different photographs to generate the final model Fig. .Fig. 8FrA previous evaluation of the terrain revealed that, even using a linear pattern, some of the photographs could not be taken from a regular distance—for example, those taken from the rocky outcrop—as the terrain presented height differences of approximately 1 m and there were few stable standpoints. Besides, some of the blind spots were not fully accessible, changing the distance and the perspective from where they had to be registered Fig. . The firImage processing was carried out with the aid of Agisoft Metashape® software, version 1.6.5. All processes were undertaken at medium resolution Table , some seThe results, as presented in Table In both cases, the final texture applied the highest resolution settings that the equipment in use could handle Fig. , to ensuThe final step was carried out with the aid of CloudCompare® open-access 3D processing software, version 2.11.3. This aimed to obtain, measure and quantify hard evidence for something that was already visible to the naked eye: namely, the progressive destruction of the site. However, before carrying out the comparison, both models, in OBJ format, were cut in Blender® (v. 2.8), to obtain a similar surface and avoid possible errors produced by meshes with different shapes.Imported both meshes in CloudCompare®, the comparison consisted on a process known as “Compute Cloud/Mesh distance”. Firstly, Sequence 1 was used as a reference model, while Sequence 2 was used as a comparison model. This means that data concerning the loss or gain of material on the ground were presented over the model based on Sequence 2.The measure of the variations produced to the terrain by the erosion was carried out stablishing a regulated distance between both meshes of less than 0.5 m, as it was verified that none of the changes exceeded this measure. This way, CloudCompare© could quantify the addition of materials up to 50 cm and the loss of ground till minus 50 cm. The results were expressed by means of a colour scale, that depicts the differences measured in metres between the ground morphology of both models. The chromatic range of this scale was limited to red-yellow-blue, where red represents areas in which there has been an addition of material, and blue those in which there has been a loss of material. The intensity of the colour expresses the intensity of the associated process. This model was designated as C1 Fig. . Using tIt should be noted that to secure a correct measure of the differences between both models, the meshes were imported to CloudCompare© in real-life scale (1:1). Thereof, the comparison displays differences of centimetres on the ground morphology, with an accuracy or error range of ≈3 mm.The results fully confirm the initial on-the-ground observations, while also highlighting other processes that are not visible to the naked eye. Between May 2016 and May 2017, the site was affected by marine erosion as well as by other human and natural factors. A slight loss of volume on the north-eastern face of the rocks is attested (light blue), as well as at other sectors of the site, especially in the western quartz pavement, some areas of the pathway and the layer of soil that still partially cover the rocky outcrop (dark blue). The site was divided into four sectors (1–4) for more in-depth analysis. From left to right (west to east), the following situations are attested Fig. :Fig. 13C3, with the mortar being especially affected: a loss of several centimetres in thickness was noted. The area is exposed to wind erosion and, occasionally, to the direct impact of the sea, which sometimes rises to this level. However, the most important factor for this sector is the transit of humans and animals between the beaches. The steep slope also exacerbates the effects of erosion, as the mortar is used as a step. A marked increased in the volume of vegetation is also attested . The recording of the wall remains allowed us to reconstruct the complex’s northern face and estimate the size of the areas which are now lost, as well as to evaluate changes in coastal physiognomy  dated between the third and the beginning of the fifth century Isings The material collected during fieldwork dates the activity of the site to between the second and early fifth centuries AD. Very scarce materials were recovered during the excavation, adding to their study others from a private collection, recovered from the site along the years, allowing to propose an accurate date of use. As the main dating elements:The chronology of Sobreira matches with the data about other nearby fish-salting factories. For example, the contexts of the factory of Marqués de Valladares state that it was constructed during the first half of the second century and abandoned during the first third of the fifth century . As stated, the human pressure and even the vegetation growth are erosion factors to have in consideration, occurring more likely during the spring and summer months. Hence, the space of a year between the first comparison models seems to be a suitable lapse of time for this type of site, adjusting the successive sequences to the results obtained through this first comparison.The simplicity of the method and the use of common tools allow to easily reproduce the procedure, opening also the possibility to combine it with other monitoring methods or even its application over other non-coastal archaeological sites or even over natural heritage. The time lapses can also be variated, being possible to do as many models as needed to monitor the changes suffered by the terrain or the remains, as well as the accuracy of the surveys, subjected to the needs of each case study. An easy and affordable tool that can provide notable information to the researchers, as it was already demonstrated along this paper with the case of Sobreira.villae a mare, agglomérations secondaires—as the centre of Vigo-Vicus—and hillforts, specialised in fishing activities and in the production of salt and fish-salting products, as well as harbouring and goods redistribution functions.The archaeological excavation allowed definitely to characterise the site as a fish-salting plant, joining the nearby and already known examples of Fiunchal and O Cocho, as well as the ones located in Vigo’s inner city—Marqués de Valladares and Praza de Compostela—and the factory of Igrexiña, in the opposite side of the Ría de Vigo. This fish-salting plant would develop simultaneously to another huge industry: the production of salt though solar evaporation. Arguably, the Ria of Vigo gathers the biggest known group of Roman salt pans. Aside from the large salt complex of O Areal . The seaside and anchorages would progressively be used not only as trade harbours, but also as new living and production areas, with the installation of the fish-salting factories and the largest preserved salt production complex of Roman time, resulting in a vicus with trade and industrial functions  through the generation and comparison of two 3D models separated by a year has proven to be an efficient tool for assessing erosive processes affecting the site. Once these processes had been assessed, we were then able to design measures aimed at palliating the effects of erosion. The methodology followed has great potential for the management of this sort of coastal archaeological site, which is exposed to a wide array of erosive processes. The tool can be used by public agencies for the analysis and protection of coastal heritage.Our results preceded and informed the excavation of the site, which was itself followed by the implementation of conservative preservation strategies, since fully protecting the site against marine erosion was deemed unfeasible . Our work also aimed to raise awareness about the presence of the site and its relevance by ensuring that the local media published stories on the site, the archaeological work being carried out and the measures being adopted to protect it. This contributed to the frequent users of the pathway, who were a factor of erosion, being more aware of the nature of their surroundings, and many decided to start bypassing the site, reducing the degree of human impact.The recovery of the lost archaeological data can be added as a result, as well as to the social awareness and the increase of the scientific knowledge of the Roman past of the Ría de Vigo and this type of industrial facilities. Overall, the proposed tool seems like an effective method to monitor menaced archaeological heritage, a low-cost and easy method, with accurate results and more accessible than other tools.Furthermore, the 3D models allow to preserve in digital format the monitored sites, creating photogrammetric material that can be incorporated into the heritage management lists, so they can be used with scientific, informative and administering purposes. The comparison of the models reveals an image of the past , of the present (monitoring and current state) and also of the future (decision-making processes over the different protection measures) of Sobreira.The visit to the site, a year after the finalisation of the works, demonstrated the positive effects of the measures undertaken Fig. . In addiNon applicable.


Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity.High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17.Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00788-x. Epithelial cells are involved in tissue homeostasis and integrity, and also provide protective barriers for organs , 2. In pcis and trans interactions of its extracellular domain between neighboring cells [cis or trans position allowing adhesion between the cells. The cytoplasmic domains bind to the armadillo proteins plakoglobin (PG) and plakophilins (Pkp 1–3). These are connected to the intermediate filaments of the cytoskeleton of the cell via desmoplakin (DP) [Different types of intercellular adhesions are described to form functional cell-to-cell connections in the epithelium. Tight junctions (TJ) segment epithelial cells into apical and basolateral regions and establish cell polarity by controlling the distribution of membrane proteins and lipids. Further, TJs form a paracellular barrier that controls transport of water, ions, and small molecules , 4. Theyng cells . The cytng cells –8. In cong cells , 10, incng cells . Desmosong cells . The N-tkin (DP) , 14.Helicobacter pylori (H. pylori) in the stomach. H. pylori is a bacterial group-I carcinogen, which persistently colonizes the human mucosa and can induce chronic gastritis, ulceration of the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, or gastric cancer [H. pylori pathogenesis. Upon H. pylori infection, the expression and activity of several hCdh1 cleaving host cell proteases are induced, including members of the A disintegrin and metalloprotease (ADAM) and matrix metalloprotease (MMP) families [H. pylori-infected gastric epithelial cells was originally attributed to the activation of ADAM10 [H. pylori-secreted serine protease high temperature requirement A (HpHtrA) as the main factor responsible for hCdh1 cleavage during H. pylori infection [698QPV↓EAG704 with the valine in position P1 was also found in the linker region of hCdh1 [H. pylori to transmigrate across the epithelial barrier, which is necessary to efficiently translocate the effector protein cytotoxin-associated gene A (CagA) into infected host cells [The intact gastric epithelium is the primary target for c cancer , 16. Abefamilies . In partfamilies , 19. hCdfamilies . Cleavagfamilies , 22. Shef ADAM10 . Howevernfection . Betweennfection . A similof hCdh1 . Functioof hCdh1 . HpHtrA-st cells , 27. In H. pylori HpHtrA deletion mutant and pharmacological inhibitors against matrix metalloproteases (MMPs) or A Disintegrin And Metalloproteinase domain-containing protein 10 and 17 , we showed that HpHtrA is the protease responsible for the cleavage of hDsg2.In this study, we treated intact gastric epithelial cells with recombinant HpHtrA and used LC–MS/MS for identification of membrane proteins released from the cell surface as described previously . We iden221A (SA). Remarkably, hDsg2 exhibited the strongest increase in MS/MS count and signal intensity among all identified membrane protein ectodomains and was also higher than of the well-known substrate hCdh1  was detected by Western blot using an antibody recognizing the extracellular domain of hDsg2. 250 ng active HpHtrA was sufficient to partially cleave rDsg2, which can be observed as a drastic loss of rDsg2FL. This effect was enhanced using increasing amounts of HpHtrA  and increasing amounts of active HpHtrA or its isogenic inactive mutant HpHtrA SA. Full length rDsg2  in the cell culture supernatant was detected  was only decreased in H. pylori P12 wt and N6 wt infected cells, while infection with H. pylori N6 Δhtra did not affect the amount of hDsg2FL compared to the uninfected cells, underlining that hDsg2 shedding on gastric epithelial cells is HpHtrA-dependent. Detection of HpHtrA was performed to verify H. pylori strains and CagA and GAPDH served as infection and loading control  and ADAM10/17, which are activated during inflammatory processes . MMPs anal cells , 31, 32.lleagues , but notH. pylori-infected gastric epithelial cells. Neither in NCI-N87 nor MKN28 cells, H. pylori-induced hDsg2 cleavage was blocked by the MMP and ADAM10/17 inhibitors, while deletion of HtrA in H. pylori clearly diminished hDsg2 shedding. Detection of HpHtrA was performed to verify H. pylori strains and CagA and GAPDH served as infection and loading control  pathovars can be influenced by hDsg2 as well. The type 1 pilus adhesin (FimH) expressed by UPEC binds hDsg2 to establish kidney infection in male C3H/HeN mice [Staphylococcus aureus was shown to cleave hDsg1, which leads to a disruption of hDsg1 function [S. aureus induced kallikreins have been demonstrated to degrade hDsg1 and filaggrin in infected keratinocytes [H. pylori infections.The AJ and TJ proteins hCdh1, occludin and claudin-8 have been previously described as HpHtrA substrates , 27. In myocytes . Generalmyocytes . Moreovemyocytes , 42. BacHeN mice . EPEC diHeN mice . Pathogefunction . Furtherinocytes . Based o2+ binding sites as signature sites for HpHtrA [2+-dependent cadherins and shares structural components with hCdh1, it is tempting to speculate that HpHtrA targets similar signature sites in hDsg2, which are highly conserved between hCdh1 and desmosomal cadherins [H. pylori infection.The identified peptides derived from HpHtrA-mediated hDsg2 cleavage were mainly located in the extracellular region. Previous studies on the classical cadherin hCdh1 identified the Car HpHtrA . Since hadherins . In our adherins . We assuadherins , 39. Thiadherins . FurtherH. pylori-secreted HtrA directly cleaves hDsg2 on the surface of infected gastric epithelial cells. This implies that in addition to TJs and AJs, H. pylori HtrA targets also the integrity of desmosomes. Therefore, we conclude that H. pylori secretes HpHtrA to disrupt all three levels of intercellular adhesions  to obtain access to basolateral regions of the gastric epithelium where it can promote pathogenesis, since loss of function of both, hDsg2 and hCdh1, has been implicated in the development of gastric cancer [Desmosomes are important intercellular adhesion complexes between neighboring cells that link cell–cell contacts to the intermediate filament cytoskeleton in epithelia exposed to high mechanical stress. Downregulation of hDsg expression, mutations in hDsg genes, abnormal ectodomain shedding of hDsg by host proteases or blockage of the extracellular domain interaction through autoantibodies were previously shown to impair desmosome function in different tissues and result in severe disease , 47. Untc cancer , 49.2. For infection experiments, cells were seeded in 6-well plates five days prior to infection. H. pylori strains P12 wild type (wt), N6 wt and N6 ΔhtrA [50 5.3 nM and 541 nM, respectively; Sigma-Aldrich, Vienna Austria) and 20 µM Batimastat . NCI-N87 and MKN28 cells were infected with H. pylori P12 wt, N6 wt and N6 ΔhtrA at a MOI (multiplicity of infection) of 50 for 24 h or left untreated (mock). For activation of MMPs and ADAM proteins, NCI-N87 cells were seeded into 6-well plates and cultivated for five days. Cells were treated with 50 ng/µl TNFα  for 24 h to stimulate activation of MMP-9 and ADAM10 [g for 10 min at 4 °C. Supernatants of infected cells were collected for detection of soluble N-terminal fragments of E-cadherin (hCdh1) and Desmoglein-2 (hDsg2), as well as for zymography. Infection experiments of NCI-N87 cells and MKN28 cells with H. pylori were repeated four times. Experiments for induction of MMP and ADAM10 activation were repeated two times.The gastric epithelial cell lines NCI-N87 and MKN28 were grown in RPMI-1640  supplemented with 10% FCS  and 1% L-glutamine  in a humidified atmosphere at 37 °C and 5% CON6 ΔhtrA   to a final concentration of 100 ng/µl. Recombinant human E-cadherin  was obtained from Sino Biologicals . Lyophilized rCdh1 was reconstituted in sterile water according to the manufacturer’s instructions at a final concentration of 250 ng/µl. Recombinant, active human MMP-7 (EC 3.4.24.23) was purchased from Sigma Aldrich  at concentration of 100 ng/µl. Purification of HpHtrA from H. pylori wild type strain Hp26695  and its isogenic inactive mutant HpHtrA S221A (SA) was performed as previously described [E. coli BL21 transformed with an HpHtrA-GST expression vector were grown in terrific broth (TB) medium to an OD600 ~ 0.7. Expression of GST-HpHtrA was induced by addition of 100 µM IPTG for 3 h at 30 °C. Bacterial cells were harvested by centrifugation and lysed by sonication on ice 6 times 30 s with 50% power  in PBS. GST-tagged HtrA proteins were bound to glutathione sepharose beads  and GST-tag was removed by addition of PreScission protease  for 16 h at 4 °C. Recombinant HpHtrA was eluted and dialyzed against 50 mM HEPES (pH 7.4) and 150 mM NaCl. Purity of recombinant proteins is routinely checked with SDS-PAGE and staining with Coomassie Brilliant Blue G250 .Recombinant human Desmoglein-2  was purchased from R&D Systems . According to the manufacturer´s instructions, lyophilized rDsg2 was reconstituted in sterile Dulbecco’s phosphate buffered saline (PBS) containing CaClescribed . Briefly221A (SA). After the incubation, the supernatant was collected  and prepared for LC–MS/MS analysis as described previously [MKN-28 cells were detached using an enzyme-free cell dissociation solution Hank's based (Millipore). Cells were washed with PBS, resuspended in 50 mM HEPES buffer (pH = 7.4) containing 1 mM EDTA and 4 µM recombinant HpHtrA wt and incubated for 1 h at 37 °C. As a negative control, cells were treated with the isogenic inactive mutant HpHtrA Seviously . Search 221A (SA) or 100 ng recombinant active human MMP-7 in 20 µl 50 mM HEPES (pH 7.4) and 150 mM NaCl for 16 h at 37 °C. 50 ng rCdh1 was incubated with 250 ng recombinant HpHtrA wt, inactive HpHtrA SA or 100 ng recombinant human MMP-7 in 20 µl 50 mM HEPES (pH 7.4) and 150 mM NaCl for 16 h at 37 °C. Where indicated, the broad range MMP inhibitor Batimastat or ADAM10 inhibitor GI254023X was added. For in vitro cleavage experiments using NCI-N87 and MKN28 cell lysates, cells were seeded into 6-well plates and grown for five days. After harvesting in 50 mM HEPES (pH 7.4) and 150 mM NaCl, cells were lysed by pulling 10 times through a 27G needle  and additionally by sonication on ice 6 times 30 s with 50% power . Lysates were cleared by centrifugation at 16,000×g for 10 min at 4 °C. 40 µg NCI-N87 or MKN28 lysate was incubated with 250 ng recombinant HpHtrA wt or inactive HpHtrA SA in 20 µl 50 mM HEPES (pH 7.4) and 150 mM NaCl for 16 h at 37 °C. In vitro cleavage experiments were repeated three times. Protein samples obtained from infection or in vitro cleavage experiments were separated by SDS-PAGE and blotted on a nitrocellulose membrane. For detection of hDsg2, antibodies against the extracellular domain  and the intracellular domain  of hDsg2 were used. hCdh1 was detected using antibodies against the extracellular domain EC5  and the intracellular domain  of hCdh1. Polyclonal sera were used to detect HpHtrA and CagA. GAPDH  was used as a loading control for whole cell lysates. Simultaneous detection of hDsg2 and hCdh1 on the same membrane was done using secondary antibodies coupled to infrared dyes  and analyzed with the Odyssey Fc Imaging System .For in vitro cleavage experiments, 100 ng rDsg2 was incubated with 250 ng, 500 ng or 1 µg recombinant HpHtrA wild type (wt), 1 µg inactive HpHtrA S2, 0.02% Brij-35). After incubation in developing buffer for 16 h at 37 °C gels were stained with 0.5% Coomassie Blue R250 .For detection of proteolytic activity in cell culture supernatants of infected NCI-N87 cells, non-reducing sample buffer  was added to the supernatant and incubated for 10 min at room temperature (RT). Samples were separated with SDS-PAGE containing 0.1% gelatin  or 0.1% casein  as a substrate. Proteins in the gel were renatured by incubation for 2 × 30 min in 2.5% Triton X-100 and equilibrated for 30 min in developing buffer , full length hDsg2 (hDsg2FL) and GAPDH was performed using ImageJ software. Signals were adjusted to average blot intensity and hDsg2FL was normalized to respective GAPDH loading controls. Statistical analysis was performed with GraphPad Prism Software (Ver. 8.0.2) using two-way ANOVA and the Tukey’s multiple comparisons post hoc test to compare each mean to every other mean. Three independent experiments were analyzed for hDsg2NTF and four independent experiments were analyzed for hDsg2FL quantification. Significance is indicated as non-significant (ns) for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001.Densitometric quantification of hDsg2 N-terminal fragment (hDsg2


Neuronal hyperexcitability in the central auditory pathway linked to reduced inhibitory activity is associated with numerous forms of hearing loss, including noise damage, age-dependent hearing loss, and deafness, as well as tinnitus or auditory processing deficits in autism spectrum disorder (ASD). In most cases, the reduced central inhibitory activity and the accompanying hyperexcitability are interpreted as an active compensatory response to the absence of synaptic activity, linked to increased central neural gain control (increased output activity relative to reduced input). We here suggest that hyperexcitability also could be related to an immaturity or impairment of tonic inhibitory strength that typically develops in an activity-dependent process in the ascending auditory pathway with auditory experience. In these cases, high-SR auditory nerve fibers, which are critical for the shortest latencies and lowest sound thresholds, may have either not matured  or are dysfunctional . Fast auditory processing deficits can occur despite maintained basal hearing. In that case, tonic inhibitory strength is reduced in ascending auditory nuclei, and fast inhibitory parvalbumin positive interneuron (PV-IN) dendrites are diminished in auditory and frontal brain regions. This leads to deficits in central neural gain control linked to hippocampal LTP/LTD deficiencies, cognitive deficits, and unbalanced extra-hypothalamic stress control. Under these conditions, a diminished inhibitory strength may weaken local neuronal coupling to homeostatic vascular responses required for the metabolic support of auditory adjustment processes. We emphasize the need to distinguish these two states of excitatory/inhibitory imbalance in hearing disorders: (i) Under conditions of preserved fast auditory processing and sustained tonic inhibitory strength, an excitatory/inhibitory imbalance following auditory deprivation can maintain precise hearing through a memory linked, transient disinhibition that leads to enhanced spiking fidelity  (ii) Under conditions of critically diminished fast auditory processing and reduced tonic inhibitory strength, hyperexcitability can be part of an increased synchronization over a broader frequency range, linked to reduced spiking reliability . This latter stage mutually reinforces diminished metabolic support for auditory adjustment processes, increasing the risks for canonical dementia syndromes. Hearing loss is a very common problem in the aging population of industrial societies. Globally, an estimated 1.57 billion people had hearing loss in 2019, accounting for one in five people (20.3%) . The proHere, we review how impaired auditory input can affect the excitatory/inhibitory balance within the central auditory system and suggest that hearing loss and cognitive decline may be linked through changes in the excitatory/inhibitory balance associated with the functional attenuation of distinct auditory fiber types. We further suggest that these changes in the excitatory/inhibitory balance may, in turn, influence neurovascular coupling, possibly further affecting cognitive function in aging.In the following, we lay out this idea in more detail. First, we provide an overview of the development of inhibitory GABAergic circuits (see Section “Maturation of GABA-Responsive Neurons Prior to Hearing Onset”). Second, we describe the role that different auditory nerve-fiber types might play during development and in regulating the excitatory/inhibitory balance in the auditory system . We then discuss the role of fast auditory processing see  may playWe define fast auditory processing as the increase in auditory acuity that is linked to lowering of hearing thresholds, increased suprathreshold ABR waves I and IV, the shortening of first spike latencies, and widening of response dynamic range with auditory experience, all shown in DCN neurons , IC neurWe define increased central neural gain as the identifiable network homeostasis that increases stimulus-evoked synchronous neural activity at the level of the inferior colliculus (IC) (ABR wave IV) relative to its input at the level of the auditory nerve (ABR wave I). Increased central neural gain can occur following, e.g., auditory deprivation  or sound enrichment. As a multi-level framework, central neural gain includes a positive auditory feedforward and positive fronto-striatal feedback cycle that require co-activation. Mechanistically increased central neural gain likely requires a reinforcement process, as it is also known from auditory perception or improved task performance i) is maintained in most neurons, ensured by the sodium-potassium-chloride co-transporter type 1 (NKCC1)  and at lower auditory brain levels , are spliced to a common protein-encoding exon (IX)    . Both exxon (IX) , cyan anxon (IX) , yellow xon (IX) .Bdnf promoters in SGN to drive the depolarizing-to-hyperpolarizing switch in a bottom-up direction within the ascending auditory circuits both prior to and following hearing onset  . In respng onset , BDNF⇑. ng onset  or CRF lng onset  may be r–]I concentrations.In summary, prior to the first auditory experience and during hearing onset, an initial period of hyperexcitability exists, with excitatory activity dominating over inhibitory activity. Within this transient time period, GABA-responsive neurons have reached their target regions but still react with depolarizing responses, due to the low level of neuronal KCC2 and high [Clvia ribbon synapses  IHC-driven synaptic events that mature during hearing onset, (ii) differences in cochlear IHC output activity through differential maturation of efferent feedback to auditory fibers, as well as (iii) differences in the genetic fate of SGNs. After hearing onset, fast auditory processing  matures The sharpening of cortical receptive fields, i.e., narrower bandwidth responses, occurs for all sensory cortices, including the auditory cortex  as a resImportant to mention here is that the overall process of maturation of fast auditory processing appears to be dispensable for basal hearing function. Thus, when BDNF was deleted in GABAergic precursor neurons in the brainstem of mice under the Pax2 promoter, and despite normal hearing thresholds based on measuring outer hair cell function, supra-threshold auditory nerve (ABR wave I) amplitudes remained low and the late ABR wave IV was delayed, indicating that fast auditory processing may have not matured properly . As a reTo obtain an idea when this critical time period of maturation of inhibitory strength occurs in auditory and associated circuits in humans, we have to consider that the fast inhibitory PV-IN activity regulates not only higher cortical microcircuit functions , but alsWe thus conclude that auditory experience-dependent maturation processes of high-SR auditory nerve fibers in the auditory system are critical for the maturation of fast auditory processing, including the formation of activity-driven, fast inhibitory PV-IN microcircuits. Only then is the neuronal network implemented for a fine-grained resolution of sound discrimination, temporally precise hearing, and fast discrimination of novel auditory information .Numerous studies have indicated that acoustic trauma and age-dependent hearing loss are linked to reduced inhibition and enhanced excitation in ascending auditory circuits . Since lIn addition to low-SR auditory fiber processing sounds , sustainde Kloet et al. (2014)Bdnf transcripts can be seen in nerve endings, glia cells, and capillaries  of default mode network activity, including the prefrontal cortex regions (PFC) . In the 66Met polymorphism  mutant mouse   , elevate(VGlut3)  and from(VGlut3) . Since aexcluded . In geneexcluded  as a resexcluded , or as aexcluded  due to mexcluded . We propexcluded  ; i.e., if not restored early enough, do the relevant auditory brain circuits remain in a stage of insufficient inhibitory strengths that hampers precise sharpening of receptive fields and proper inhibitory strength in the fine-grained microcircuits required for speech discrimination and temporal coding ? The imm1, which is used as an objective measure of developmental hearing experience  showed an autism-like phenotype , linked with enhanced LTP and maintained temporal processing  Figure  Figure , suggestg group, , experimg group, . In thesIn questioning whether reduced tonic inhibitory strength following fast auditory processing may be particularly critical for cognition, as predicted from autism animal models (see Section “Failed Fast Auditory Processing in Autism Spectrum Disorders”), the critical time period of maturation of fast auditory processing and inhibitory strength in auditory and associated limbic circuits - between the 2nd and 3rd postnatal week in rodents  needs toWe questioned whether the maturation of fast (high-SR) auditory processing, of inhibitory PV-IN microcircuits, of hemodynamic BOLD fMRI responses, and of LTP may not only be correlated in time, but also functionally. More precisely, we asked whether the imbalances in excitation and inhibition in hearing disorders that correlated with reduced tonic inhibitory strength, as predicted in the case of impaired fast auditory nerve fiber responses , might aThis hypothesis is based on new insights into the mechanism of hemodynamic responses: Previously, glutamatergic neuronal activity was assumed to mainly trigger hemodynamic responses and vasodilation during a bilateral homeostatic response: Glutamatergic neuronal activity, such as neural feedforward signaling, includes neuronal-derived nitric oxide (NO) release from the glutamatergic synapses that causes a metabolic feedback signal in smooth muscle cells of parenchymal arterioles, finally leading to vasodilation [for a review see The contradicting assumptions, that on the one hand NO-release from GABA-IN may influence endothelia cells of blood vessels, and thereby change their diameter , while oIt is thus challenging to consider that the increase in PV-IN mediated inhibitory strength between the 2nd and 3rd postnatal week  and the During aging or following acoustic trauma, acute or congenital deafness, a critical diminution of fast (high-SR) auditory driving force diminishes activity-dependent BDNF activities and tonic-PV-IN strength, hippocampal LTP, extra-hypothalamic stress control and possibly proper coupling of inhibitory neuronal activity to hemodynamic responses, accelerating a negative feedback cycle (In view of the increasing evidence of a link between hearing loss and dementia, a better understanding of this possible relationship is an important challenge . We suggMK designed, wrote, and revised the manuscript. WS wrote the manuscript and made the figures. KS, GEH, LR, CB, and RL wrote and revised the manuscript. YLH wrote the figure legends and helped writing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Proton pump inhibitors (PPIs) have been associated with an increased risk of fragility fractures in pharmaco-epidemiological studies. The mechanism is unclear, but it has been speculated that by neutralising gastric acid, they may reduce intestinal calcium absorption, causing secondary hyperparathyroidism and bone loss. Here we investigated that hypothesis that the skeletal effects of PPI might be mediated by inhibitory effects on the bone-specific phosphatase PHOSPHO1. We found that the all PPIs tested inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73 µM for esomeprazole to 19.27 µM for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures was inhibited by several PPIs in a concentration dependent manner. In contrast, the histamine-2 receptor antagonists (H2RA) nizatidine, famotidine, cimetidine and ranitidine had no inhibitory effects on PHOSPHO1 activity. Our experiments show for the first time that PPIs inhibit PHOSPHO1 activity and matrix mineralisation in vitro revealing a potential mechanism by which these widely used drugs are associated with the risk of fractures. Proton pump inhibitors (PPIs) are amongst the most commonly prescribed drugs and are used in the treatment of gastroesophageal reflux disease (GORD), peptic ulcer disease and dyspepsia . In the The most commonly accepted explanation is that PPIs predispose to fractures by neutralising gastric acid. This in turn is thought to impair intestinal calcium absorption, secondary hyperparathyroidism and increased osteoclastic bone resorption with bone loss –8. Howevi) through the hydrolysis of phospholipid substrates within the matrix vesicle (MV) membrane  spine  and any-site fractures  in PPI users as compared to controls .Phospho1−/− mice [The PPIs reduce gastric acid secretion through inhibition of H + /K + -ATPases located in stomach parietal cells . In view−/− mice , 35 and −/− mice . It is t−/− mice , 36. Our−/− mice . Therefo−/− mice , 39.Alpl−/− mice, extravesicular crystal propagation is retarded due to an accumulation of inorganic pyrophosphate in the extracellular matrix [Interestingly, the data of this present study indicated no effect of PPIs on TNAP phosphatase activity; a result that is consistent with our previous study that reported lansoprazole and other small molecule inhibitors of PHOSPHO1 had no effect on TNAP activity . The impr matrix . These dr matrix , 43. An We have previously shown TNAP expression and activity to be decreased in PHOSPHO1 knockout osteoblasts, with a concomitant increase in inorganic pyrophosphate levels . However50 data) of PPI inhibition of PHOSPHO1 activity is esomeprazole > omeprazole = lansoprazole > pantoprazole (Fig. The order of potency (based on our ICole Fig. , which pIn summary, we have shown that commonly prescribed PPIs, but not H2RAs, inhibit the activity of the bone-specific phosphatase, PHOSPHO1 in vitro in a dose-dependent manner and at concentrations that are similar to those used clinically. We have also shown that different PPIs differ by more than 25-fold in their ability to inhibit PHOSPHO1 activity when compared with a sevenfold difference in potency for inhibition of acid production . This inConsidering the fact that PHOSPHO1 plays a critical role in bone mineralisation, we hypothesise that the association between PPI use and bone fractures is possibly due to their inhibitory effect on PHOSPHO1 activity. Although this remains to be confirmed by further research it could have clinical implications in allowing clinicians to select PPIs with the least inhibitory effect on PHOSPHO1 activity as the preferred drug in this class in patients at high risk of fragility fractures.


The reduced presence of ERp57 within the ER of CD biopsies suggests a regulatory function of this protein within CD pathogenesis. PX12 and L-cystine oxidatively inhibit TG2 and might serve as treatment options in CD. mTG is poorly regulated and could contribute to the accumulation of immunogenic peptides within the gut with potential pathogenic effects.Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a central step in celiac disease (CD) pathogenesis. Microbial transglutaminase (mTG) mimics the enzymatic function of TG2 and might play a role in CD. TG2 is inhibited by endogenous oxidative endoplasmic reticulum-resident protein 57 (ERp57), but data about mTG are lacking. We investigated the localization of ERp57 in duodenal biopsies and examined inhibition of TG2, and mTG by competitive, and oxidative molecules. Localization of ERp57 was investigated in duodenal biopsies from CD, and control patients by electron microcopy. Inhibition of TG2 and mTG was analyzed on an in vitro level using a photometric assay. ERp57 was observed within the lamina propria and its abundance within the endoplasmic reticulum (ER) was reduced in CD patients. TG2 was oxidatively inhibited by up to 95% by PX12 ( The absolute amount of ERp57 within the ER of RACE was significantly higher compared to enterocytes , an effect that could be reduced by trend using the TG2-specific competitive inhibitor ERW1041  by treatment with TRX  and 5 µM , whereas ERp57 treatment trended to reduce crosslinking at a dose of 0.5 µM   B. Next,  = 0.06) C.p = 0.08) excess and decreasing crosslinking of 5BP by about 40% at a 1,000-fold excess . mTG activity was not influenced by ERW1041 underlining the specificity of this competitive inhibitor. mTG-blocker showed strong inhibitory effect at a 10 to 1000-fold excess, reducing mTG activity by 28% , 37%  and 61% . This effect was confirmed using concentration series of PX12, where higher doses of PX12 markedly reduced TG2 activity below basic enzyme activity  and 20 µM  was calculated using a variable slope model. First, we could show that a fourfold excess of PX12 (20 µM) abolishes TRX (5 µM)-mediated activation of TG2 , 1 mM , and 10 mM . To investigate whether those effects were TG2-specific, we tested the influence of PX12 and L-cystine on mTG activity  might provide a multitude of neo-epitopes and increase the load of immunogenic peptides within the intestinal epithelium and the lamina propria ,35,36. FIn conclusion, we showed that the presence of the endogenous TG2 inhibitor ERp57 differs within the ER of duodenal biopsies of CD and control patients. This indicates a potential role of this protein in CD, potentially by inhibiting TG2 activity within the ER and preventing cross presentation of immunogenic gliadin peptides in healthy individuals. Our functional transglutaminase assay revealed PX12 and L-cystine as potent TG2 inhibitors using an oxidative mechanism. Both substances nearly abolished TG2 activity at micromolar doses and appeared to be much more potent than the competitive inhibitor ERW1041 in this setting. However, further research is clearly needed to investigate the effect of those molecules in cell culture and in vivo. Lastly, we could demonstrate that mTG activity is less regulated than TG2 and that common competitive TG2 inhibitors do not affect mTG. Since unregulated enzyme activity might increase the intestinal load of immunogenic peptides, additional effort is needed to clarify its role within food products and the intestinal microbiota.This study was approved by the local ethics committee (reference number 119/16) and written informed consent was given from every patient. Excess duodenal biopsies were obtained during clinically indicated upper gastrointestinal endoscopy. At the time of endoscopy, all patients were on gluten-containing diet. Tissue samples from 5 CD (14 (9–15) years, 4 females) and 5 non-celiac disease patients  years, 3 females) were obtained. Diagnosis of CD was made according to current guidelines of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) ,42. All Duodenal biopsies were sectioned and labelled using the postembedding technique according to Tokuyasu  and GrifSubcellular quantification was performed using a transmission electron microscope  by meander-shaped counting of 100 gold particles (12 nm) within the epithelium. Specificity of ERp57-antibody was assessed by evaluating the labelling of goblet cell granules as negative control. For quantifying the intracellular distribution of ERp57 within the epithelium, enterocytes and RACE were screened in a meander-shaped manner and 100 gold particles (12 nm) were counted. To investigate the concentration of ERp57 in the ER, 10 standardized images  of enterocytes and RACE of each patient were taken. To evaluate the ER volume and the concentration of ERp57 within this compartment, a standardized grid (square side length: 4000 µm) and the following formulas were used to calculate organelle circumference (OC) and labelling density (LD) as adapted from Griffiths .OC (µm)2 (pH 7.5) for 3 h. After fixation with PFA 4% and blocking overnight with BSA 2%, streptavidin-HRP  was applied for 1 h. Finally, 100 µL per well of the substrate TMB  was added and the reaction was monitored continuously for 15 min using a microplate reader at 640 nm. The redox reagent dithiothreitol (DTT), which is commonly used to stabilize disulfide bonds within enzymes, was used as positive control. The molecular formula of mTG-blocker  was not disclosed by the manufacturer. All tested substances are listed in Evaluation of in vitro transamidation activity of both transglutaminases was performed according to Yi et al. . In briet test (with Welch’s correction where appropriate), or Mann-Whitney U test were used where appropriate. Data are given as mean ± standard deviation , or as median with interquartile range (IQR) .Statistical analysis was performed using SPSS 21  and GraphPad Prism 9 . Students unpaired two-tailed


Gracilaria lemaneiformis polysaccharide (GLP) exhibits good physiological activities, and it is more beneficial as it is degraded. After its degradation by hydrogen peroxide combined with vitamin C (H2O2-Vc) and optimized by Box–Behnken Design (BBD), a new product of GLP-HV will be generated. While using GLP as control, two products of GLP-H (H2O2-treated) and GLP-V (Vc-treated) were also produced. These products chemical characteristics  were assessed. The results showed that the optimal conditions for H2O2-Vc degradation were as follows: H2O2-Vc concentration was 18.7 mM, reaction time was 0.5 h, and reaction temperature was 56 °C. The total sugar content of GLP and its degradation products  were more than 97%, and their monosaccharides are mainly glucose and galactose. The SEM analysis demonstrated that H2O2-Vc made the structure loose and broken. Moreover, GLP, GLP-HV, GLP-H, and GLP-V had significantly inhibition effect on α-glucosidase, and their IC50 value were 3.957, 0.265, 1.651, and 1.923 mg/mL, respectively. GLP-HV had the best inhibition effect on α-glucosidase in a dose-dependent manner, which was the mixed type of competitive and non-competitive. It had a certain quenching effect on fluorescence of α-glucosidase, which may be dynamic quenching. Gracilaria lemaneiformis belongs to Rhodophyta Phylum, Gigartinales Order, Gracilariaceae Family, and Gracilaria Genus, which is a kind of economic red algae, used as agar, feed, food, and drug resources  curve. Such data are summarized in Ka was 9.5082 L/mol, and the number of binding sites (n) was 0.8137 (close to 1). We noticed that the value of Ka was relatively low, and the value of n was only 1, which indicated that the binding effect of the GLP-HV to the α-glucosidase was weak and there was only one binding site.F . It demoeratures . Kq was  L/mol/s , the max50, inhibition kinetics and fluorescence spectrum analysis, GLP-HV had a good inhibition effect on α-glucosidase in a dose-dependent manner, which was the mixed type of competitive and non-competitive. It had a certain quenching effect on fluorescence, which may be dynamic quenching. GLP-HV may have the potential to be developed as new hypoglycemic agents. According to the results of inhibition rate, IC2O2-Vc concentration, degradation time and temperature according to single factor and response surface experiments. H2O2-Vc concentration in the system was very small, and the degradation time was short, which had the advantages of rapid reaction, high efficiency, and low cost. The Mw of H2O2 and Vc were both lower than 300 g/mol, respectively, which can be removed by dialysis without introducing impurities and interfering with the activity of polysaccharide. This method is safe and reliable that can be used in industrial production. The total sugar and protein content were not changed after degradation, indicating that H2O2-Vc did not introduce other impurities. In addition, the reducing content of polysaccharide was higher after degradation, and its lower Mw, exhibiting the effective degradation of H2O2-Vc. And the degradation effect was better than fermentation, and similar to HPT treatment (high temperature and pressure combined with Vc) from the comparison of molecular weight [The degradation degree of polysaccharide was related to Hr weight ,15.2O2 or Vc treatment may break the similar position or numbers of glyosidic bond. They may cut the glyosidic bonds on both sides of galactose, promoting the produce of galactose, compared with H2O2-Vc treatment. The degradation of polysaccharide led to the change of monosaccharide composition, which may be the main reason for the change of glucosidase inhibition effect by polysaccharide and its production products. Polysaccharide degradation is attributed to the breaking of glyosidic bonds, resulting in changes in monosaccharide composition. Various degradation methods act on different glyosidic bonds, so the breakage of glyosidic bonds’ location and number may be diverse, resulting in differences in the amount and content of monosaccharides exposed ,20,21. X2O2-Vc obviously degraded the Gracilaria lemaneiformis polysaccharide effectively, thus could break its structure, and changed its surface morphology. Furthermore, H2O2-Vc treatment exhibited the better degradation effect than alone H2O2 or Vc treatment, might be due to the differences in the distance and degree of crosslinking between molecules. Gong et al. [2O2. However, the polysaccharide structure was more fragmented and looser degraded by H2O2-Vc. Perhaps Vc reacted with H2O2 to produce more free radicals, which had a stronger effect on the polysaccharide molecules. The Mw of polysaccharides dreaded by H2O2 or Vc were only slightly less than GLP, and H2O2-Vc exhibited the excellent degradation effect with the minimum Mw. This finding validated the SEM result.The SEM results were consistent with the change of reducing sugar and molecular weight, which exhibited that Hg et al.  also fou2O2-Vc enhanced the hypoglycemic activity in vitro, compared with H2O2 or Vc treatment alone. GLP-HV presented higher galactose, ribose, and xylose than GLP, and lower glucose and rhamnose. Both GLP-H and GLP-V expressed similar changes with GLP-HV, but the effect were not as significant as GLP-HV. The degraded polysaccharides showed better inhibition, especially GLP-HV. It possibly was related to these changes in monosaccharide compositions. Cao et al. [Lentinus edodes mycelia polysaccharide containing mannose, arabinose, galactose, xylose, and rhamnose. Combined with the results of molecular weight, GLP-HV had the lower Mw than GLP, GLP-H, and GLP-V. For another, the Mw of GLP-V was smaller than GLP and GLP-H, but its inhibition rate on α-glucosidase was worse than GLP-H and higher than GLP. Therefore, the good inhibition effect of GLP-HV may not only be due to the low molecular weight and monosaccharide composition, but also to the connection mode of glycoside chains, which needs to be verified by subsequent studies. Lv et al. [α-glucosidase is one of the hydrolases in the digestive tract, which is closely related to hypoglycemic activity . The inho et al.  exerted v et al.  research2O2-Vc is also an excellent degradation method, which has the advantages of high efficiency, low cost, and no by-products. Low molecular weight polysaccharide with high hypoglycemic activity could be obtained by H2O2-Vc degradation. Polysaccharide degradation products showed the inhibition on α-glucosidase, and GLP-HV presented the best effect in a dose-dependent manner, which was the mixed type of competitive and non-competitive. It had a certain quenching effect on fluorescence of α-glucosidase, which may be dynamic quenching. The best inhibition effect on α-glucosidase may be related to its molecular weight, monosaccharide composition and other factors.The method of hot water extraction and alcohol precipitation is a suitable extraction method for polysaccharide, with high total sugar content and without protein. HGracilaria lemaneiformis was purchased from Nan’ao Island , the Coomassie Bright Blue kit was purchased from Shanghai Biyuntian Biotechnology Co., LTD ; vitamin C (Vc) and α-glucosidase were purchased from Shanghai Yuanye Biotechnology Co., LTD ; P-nitrobenzene-α-d-glucoside (PNPG) was purchased from Shanghai Maclin Biochemical Technology Co., LTD .Gracilaria lemaneiformis was cleaned many times to remove impurities, dried in an oven at 50 °C, crushed and sifted through 40 mesh to obtain uniform powder. The powder was soaked on a shaking table (50 °C) with an ethanol volume of nine times to remove pigment, fat, and alcohol-soluble impurities. After 24 h, the filter residue was extracted and placed in a drying oven at 50 °C for drying. The GLP was extracted by hot water and precipitated by ethanol according to the previous reported methods with some modifications [Gracilaria lemaneiformis was extracted with water at a ratio of 1:45 (w/v) at 90 °C for 4 h under oscillation, and then cooled to room temperature to add 1% papain and 0.5% cellulase (w/v). The mixture was incubated at 60 °C for 2 h under oscillation, and then rapidly heated in boiling water for 10 min to denature the papain and cellulase. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 15 min, and the supernatant was collected and condensed to one third of the original volume by rotary evaporation (60 °C). The concentrated solution was precipitated at 4 °C for 12 h, centrifuged for precipitation, filtered with 200 mesh gauze, and then dialyzed (7 kDa) at 4 °C for 72 h, and pure water was changed once at 4 h. After freeze-drying, GLP values were obtained. ications . After 32O2-Vc (mole ratio of H2O2 and Vc was 1:1). In brief, GLP (5 mg/mL) and H2O2-Vc at different concentrations  were added directly into the solution. Degradation times were 0.1, 0.3, 0.5, 0.7, and 0.9 h, and the temperature were maintained at 30, 40, 50, 60, and 70 °C. Reducing sugar content and α-glucosidase inhibition rate were used as screening indexes.The degradation was adopted based on the reported method . GLP wasAccording to the single-factor experimental results, the response surface method was designed and implemented using the Box–Behnken Design (BBD) principle of Design-Expert 8.0.6 software, and the response surface results were fitted. v/v). After centrifuging , the precipitation was dissolved with pure water in the magnetic blender, and then dialyzed (300 Da) at 4 °C for 48 h, and pure water was changed once at 4 h. After freeze-drying, the degradation product was obtained, named GLP-HV (Gracilaria lemaneiformis polysaccharide degraded by H2O2-Vc). Only H2O2 in the same concentration was used for polysaccharide, the other conditions were the same, the sample was obtained, named GLP-H (Gracilaria lemaneiformis polysaccharide degraded by H2O2). Only Vc in the same concentration was used for polysaccharide, the other conditions were the same, the sample was obtained, named GLP-V (Gracilaria lemaneiformis polysaccharide degraded by Vc). GLP-H and GLP-V were the controls of GLO-HV.The polysaccharide was degraded by the optimal process obtained by the results of single-factor experiment and response surface analysis. The degradation product was neutralized (pH 7.0) with 1 M NaOH, concentrated to 1/3 volume at 60 °C using a rotary evaporator, and precipitated at 4 °C for 12 h with absolute ethanol  method with D-glucose as the standard compound, and Coomassie Bright Blue kit.Monosaccharide composition was determined using high performance liquid chromatography (LC-20AD) according to the method reported by Kang et al. , with a Derivatives of standard products: After the monosaccharide standards were dissolved in water, 250 uL 0.6 mol/L NaOH and 500 uL 0.4 mol/L PMP-methanol were added and reacted at 70 °C for 1 h. Subsequently, the solution was cooled in cold water for 10 min, 500 uL 0.3 mol/L HCl was added for neutralization, and 1 mL chloroform was added and mixed. Centrifugation was performed at 3000 r/min for 10 min. The supernatant was carefully taken and extracted three times. Hydrolysis and derivatization of samples: appropriate amounts of samples were accurately weighed, 2 mL 2 mol/L trifluoroacetylacetone (TFA) was added and acidolized at 120 °C for 4 h. TFA was blow-dried with nitrogen and redissolved with 2 mL water. The derivatization procedure of the hydrolyzed sample solution was consistent with that of the standard. 3) and 0.06% Sodium azide (NaN3) at a flow rate of 0.6 mL/min.Molecular weight was determined by Gel Permeation Chromatography , referred to the report , with a The method was referred to the previous research . The abs2-KI reagent  for 10 min. The absorbance was detected by a UV-vis spectrophotometer  with the range of 300–700 nm [A polysaccharide and degradation products solution  was mixed with I0–700 nm .Polysaccharide and degradation products solution  were prepared, and 2 mL of 80 uM Congo red was added, and then 1 M NaOH was dropped to make the concentration of NaOH in different solutions vary from 0 to 0.5 mol/L. UV-vis spectrophotometer scanning (400–700 nm) was conducted to determine the maximum absorption wavelength under various concentration gradients of NaOH .The morphology of GLP, GLP-HV, GLP-H, and GLP-V were analyzed by SEM  . These sThe inhibition effect on α-glucosidase was detected according to the previous report , with soThe inhibition rate: 30 μL polysaccharide and degradation products solution , mixed with 100 μL phosphate buffer solution  and 30 μL α-glucoside enzyme solution  in 96-well plates, which incubated at 37 °C for 15 min. 30 μL PNPG solution  was mixed into the reaction mixture and incubated at 37 °C for 20 min. Then measured the absorbance value at 405 nm, and calculated the inhibitory activity according to the following formula:1: Samples and enzyme; A2: The enzyme was replaced by PBS; A3: Samples are replaced with pure water; A4: Samples and enzyme were replaced with pure water and PBS, respectively.AKm), the maximum reaction rate (Vmax), according to the following formula:Inhibition kinetics: The sample concentration was 0, 1, and 5 mg/mL, and the concentration of PNPG was 1, 2.5, 5, 7.5, and 10 mM, respectively. The reaction time was 20 min, and the determination was performed every 2 min. The Lineweaver–Burk curve was drawn by double reciprocal plotting method with reciprocal of substrate concentration as abscissa and reciprocal of reaction rate as ordinate, which calculated the Michaelis constant . Duncan’s multiple range test was applied to identify differences between the mean values for each group by IBM SPSS software (version 22). 2O2-Vc degradation were as follows: H2O2-Vc concentration was 18.7 mM, reaction time was 0.5 h, and reaction temperature was 56 °C. The total sugar content of GLP, GLP-HV, GLP-H and GLP-V were 98.77%, 98.43%, 97.28%, and 97.5%, respectively, and their reducing sugar were 2.47%, 46.92%, 1.95%, and 50.2%, respectively. Moreover, the Mw was reduced after degradation, and the monosaccharides were mainly glucose and galactose before and after degradation. In addition, GLP and its degradation products did not have protein, starch, and triple helix structure. The degradation method of H2O2-Vc is feasible, and no by-products can be produced. The SEM analysis demonstrated that H2O2-Vc made the structure loose and broken. All samples showed the inhibition on α-glucosidase, and GLP-HV presented the best effect in a dose-dependent manner, which was the mixed type of competitive and non-competitive. It had a certain quenching effect on fluorescence of α-glucosidase, which may be dynamic quenching. The polysaccharide degraded by H2O2-Vc, with low Mw, exerted the good inhibition effect on glucosidase activity.The optimal conditions for H


Persicaria hydropiper L. (P. hydropiper), perform its phytochemical analysis, and explore its in vitro antidiabetic effects.  Medicinal plants and essentials oils are well known for diverse biological activities including antidiabetic potential. This study was designed to isolate essential oils from the leaves of P. hydropiper leaves essential oils (Ph.Los) were extracted using a hydrodistillation apparatus and were subjected to phytochemical analysis using the gas chromatography mass spectrometry (GC-MS) technique. Ph.Lo was tested against two vital enzymes including α-glucosidase and α-amylase which are important targets in type-2 diabetes. The identified compounds were tested using in silico approaches for their binding affinities against the enzyme targets using MOE-Dock software. α-bulnesene, nerolidol, β-caryophyllene epoxide, and decahydronaphthalene were the most abundant compounds. Ph.Lo exhibited considerable inhibitory potential against α-glucosidase enzyme with 70% inhibition at 1000 μg mL−1 which was the highest tested concentration. The inhibitory activity of positive control acarbose was 77.30 ± 0.61% at the same tested concentration. Ph.Lo and acarbose exhibited IC50 of 170 and 18 µg mL−1 correspondingly. Furthermore, dose-dependent inhibitions were observed for Ph.Lo against α-amylase enzyme with an IC50 of 890 μg mL−1. The top-ranked docking conformation was observed for β-caryophyllene epoxide with a docking score of -8.3182 against α-glucosidase, and it has established seven hydrogen bonds and one H-pi interaction at the active site residues . Majority of the identified compounds fit well in the binding pocket of Tyr 62, Asp 197, Glu 233, Asp 300, His 305, and Ala 307 active residues of α-amylase. β-Caryophyllene epoxide was found to be the most active inhibitor with a docking score of -8.3050 and formed five hydrogen bonds at the active site residues of α-amylase. Asp 197, Glu 233, and Asp 300 active residues were observed to be making polar interactions with the ligand.  GC-MS analysis revealed the presence of 141 compounds among which dihydro-alpha-ionone, cis-geranylacetone,  The current study revealed that Ph.Lo is rich in bioactive metabolites which might contribute to its enzyme inhibitory potential. Inhibition of these enzymes is the key target in reducing postprandial hyperglycemia. However, further detailed in vivo studies are required for their biological and therapeutic activities. Diabetes mellitus (DM) is a metabolic syndrome associated with hyperglycemia due to the body's inability to produce sufficient amount of insulin or abnormalities in its secretion or tissue resistance to its action , 2. Hypeα-amylase and α-glucosidase enzymes are of high pharmacological interest and are used to control elevated glucose level in T2DM. These enzymes cause metabolic breakdown of complex dietary carbohydrates to simple sugars which are subsequently absorbed heptane,-3-cyclopropyl,-7-hydroxymethyl, trans , nerolidol , bicyclo[2.2.2]oct-2-ene, 1,2,3,6-tetramethyl , -4,4,8-trimethyltricyclo [6.3.1.0] dodeca-2-en-9-ol , β-caryophyllene epoxide , and decahydronaphthalene   .α-glucosidase enzyme as shown in μg mL−1 correspondingly. The standard drug acarbose inhibitory activity showed 77.30 ± 0.61, 73.00 ± 0.00, 69.00 ± 0.00, 55.50 ± 1.04, 49.83 ± 0.44, and 41.00 ± 0.00% using the abovementioned doses, respectively. For test (Ph.Lo) and control (acarbose), IC50 of 170 and 18 μg mL−1 was calculated.In the present study, Ph.Lo was found to be highly active against −1, 51.91% at 500 μg mL−1, 42.66% at 250 μg mL−1, 32.00% at 125 μg mL−1, 24.00% at 62.50 μg mL−1, and 14.50% at 31.25 μg mL−1. Positive control showed inhibition rates of 77.3% at 1000 μg mL−1, 73.00% at 500 μg mL−1, 69.00% at 250 μg mL−1, 55.50% at 125 μg mL−1, 49.83% at 62.50 μg mL−1, and 41.00% at 31.25 μg mL−1. Overall, concentration-dependent amylase inhibitory activities were observed for Ph.Lo as shown in 50 of 890 μg mL−1.Results of alpha-amylase inhibitory potential of Ph.Lo are summarized in β-caryophyllene epoxide, and decahydronaphthalene were the most abundant compounds. It has been suggested by Jabeen et al. that, in a molecule, the presence of lipophilic side chain is responsible for the inhibition of alpha-glucosidase enzymes [Eruca vesicaria subsp. longirostris. Here, erucin was suggested to inhibit alpha-glucosidase. Apart from erucin, it was also reported that β-elemene may inhibit alpha-glucosidase activity [Hertia cheirifolia essential oils obtained from its leaves and flowers and were suggested to contribute α-amylase inhibitory activity [H. cheirifolia volatile oils possess inhibitory potential against α-glucosidase enzyme [Nepeta curviflora volatile oils [β-caryophyllene which are suggested for their antiamylase and antiglucosidase activities. These compounds in addition to other bioactive metabolites were identified in Ph.Lo analysis and might contribute to the overall enzyme inhibitory potential.In GC-MS characterization, 141 phytochemicals were identified, among which dihydro-alpha-ionone, cis-geranylacetone, alpha-bulnesene, nerolidol,  enzymes . Inhibitactivity . In esseactivity . Recentle enzyme . Apart file oils . In thesα-amylase enzyme. Docking studies revealed that the β-caryophyllene epoxide is the most active inhibitor with a docking score of -8.3050 and formed five hydrogen bonds with the active site residues of α-amylase. Asp 197, Glu 233, and Asp 300 active residues were observed to be making polar interactions with the ligand  for the e ligand .in silico performance of the compound. Interaction reports of the remaining inhibitors are given in Enzyme inhibition properties of the phytochemicals might be attributed to electron-donating group (-CH3) on the identified compound. The oxygen atom of the ligand might be implicated in the considerable α-glucosidase receptor active sites. Considerable docking conformations were observed for ß-caryophyllene epoxide with a docking score of −8.3182 which indicates that the compound established seven hydrogen bonds and one H-pi interaction with the residues of active sites   .A considerably high inhibitory potential of the identified metabolite might be attributed to the existence of the two methyl moieties and oxygen atom attached to the (S)-2-methyloxirane moiety of the ligand .In summary, findings of this study showed that Ph.Lo is rich in bioactive phytochemicals which might contribute to the antidiabetic and health-promoting potentials of the oils. The test samples exhibited concentration-dependent inhibition of the vital enzymes implicated in the gastrointestinal absorption of postprandial glucose and thus might help in reducing the hyperglycemia in type-2 diabetes. The binding mode and energies of the identified phytochemicals against the target enzymes using the molecular docking approach further supported our claim regarding the antidiabetic potential of our test samples. Nevertheless, we suggest that, in future, in vivo studies be performed for the therapeutic and beneficial effects of these compounds in metabolism-associated disorders.


Here, we identified casein kinase I alpha (CK1α) as a regulator of WAVE, thereby controlling cell shape and cell motility in Drosophila macrophages. CK1α binds and phosphorylates WAVE in vitro. Phosphorylation of WAVE by CK1α appears not to be required for activation but, rather, regulates its stability. Pharmacologic inhibition of CK1α promotes ubiquitin-dependent degradation of WAVE. Consistently, loss of Ck1α but not ck2 function phenocopies the depletion of WAVE. Phosphorylation-deficient mutations in the CK1α consensus sequences within the VCA domain of WAVE can neither rescue mutant lethality nor lamellipodium defects. By contrast, phosphomimetic mutations rescue all cellular and developmental defects. Finally, RNAi-mediated suppression of 26S proteasome or E3 ligase complexes substantially rescues lamellipodia defects in CK1α-depleted macrophages. Therefore, we conclude that basal phosphorylation of WAVE by CK1α protects it from premature ubiquitin-dependent degradation, thus promoting WAVE function in vivo.The WAVE regulatory complex (WRC) is the main activator of the Arp2/3 complex, promoting lamellipodial protrusions in migrating cells. The WRC is basally inactive but can be activated by Rac1 and phospholipids, and through phosphorylation. However, the This article has an associated First Person interview with the first author of the paper. Summary: We identified CK1α as a novel regulator of WAVE controlling cell shape and motility in immune response. Basal phosphorylation of WAVE by CK1α protects it from premature proteasomal degradation. Together with Abi, NCKAP1/Nap1, CYFIP/Sra-1 and BRK1/HSPC300, WAVE forms a conserved hetero-pentameric complex, the WAVE regulatory complex (WRC). Within the WRC, activity of WAVE towards the Arp2/3 complex is inhibited by intracomplex sequestration of its Arp2/3 activating domain, i.e. the verprolin homology, cofilin homology, acidic region (VCA) domain. One of the central WRC activators is the small RhoGTPase Rac1, which directly binds to the WRC subunit Sra-1 and activates the WRC by allosterically releasing the bound Arp2/3-activating VCA domain of WAVE. Several studies have shown that phosphorylation plays an important role in regulating WRC-Arp2/3-mediated actin filament branching and lamellipodia formation . WAVE practivity . Howeveractivity . A more dynamics .Drosophila in vivo. By using RNA interference (RNAi), we identified casein kinase 1α (CK1α) but not CK2 as an important regulator of lamellipodia formation and immune cell migration. Ck1α-mutant macrophages exhibit a stellate morphology and an altered migratory behavior that phenocopies wave-deficient cells. We also found that CK1α can interact physically with WAVE. Drosophila WAVE contains two conserved CK1α consensus sequences that are located in the N-terminal WHD and the C-terminal acidic domain, overlapping with two conserved CK2 phosphorylation sites within mammalian WAVE2. Phosphorylation-deficient mutations in the N-terminal but not C-terminal domain of WAVE can fully rescue the lethality of the wave mutant and the lamellipodium defects of macrophages deficient for wave. Loss-of and gain-of-function analysis, and pharmacological inhibition of CK1α further suggest that basal phosphorylation of VCA domain by CK1α is crucial for WAVE stability rather than its activity in vivo.In this work, we analyze the role of phosphorylation of WAVE  in arp2- or wave-depleted macrophages results in complete loss of lamellipodial protrusions . Transgene RNAis were specifically coexpressed with GFP in the macrophage lineage using the hmlΔ-Gal4 driver . We identified the casein kinase 1α (Ck1α) gene as a candidate that most strongly affected lamellipodia formation and phenocopied wave-depleted cells, characterized by a prominently reduced circularity index (Table S1).Wild type macrophages acquire a round, pancake-like shape with a broad lamellipodial actin filament network A. Expresty index B–D,I. WeCk1α mutants, bearing distinct missense mutations . Of those, Ck1α8B12 (Ck1αG43D) is the only functionally characterized Ck1α allele carrying a mutation that transforms the conserved glycine residue at position 43 into an aspartic acid (G43D). It has been first described as a strong hypomorph or amorphic allele (Ck1αA (Ck1αL141M) and Ck1αB (Ck1αG148S) have been previously isolated in a large EMS screen, but neither allele has so far been functionally characterized (Ck1αA carries a mutation that leads to replacement of a conserved lysine residue with methionine at position 141 (L141M), constituting the only mutation that yields removal of an H-bond – which might affect the active site of the CK1 . By contrast, Ck1αB replaces glycine with a serine at position 148 (G148S) without any obvious structural changes.Next, we analyzed c allele . Ck1αA . Whereas CK1α is a monomeric serine kinase, CK2 is composed of two catalytic CK2α and two regulatory CK2β subunits that form a hetero-tetrameric (α2β2) holoenzyme -mutant larvae . Ck1α-deficient macrophages still respond to the cell damage, however, due to reduced lamellipodia formation, Ck1αL141M mutant cells were impaired in their ability to migrate towards the ablated cell. To better characterize defects in the migratory behavior we first counted the number of cells that reached the wound within the first 30 min after wounding (Ck1αL141M as well as in myospheroid (mys1) and waveΔ37 mutant cells (Movie 3). To further describe the impaired migratory behavior of Ck1αL141M mutant cells we also measured the bias angle, i.e. the angle between the motion vector (a step of the cell) and the direction vector pointing towards the wound  mutant macrophages  – which has 65% identity to Drosophila CK1α – in the presence of purified glutathione S-transferase (GST)-tagged Drosophila WAVE protein (GST-WAVE). In this assay, ATPγS served as the phosphate donor from which mono-thiophosphate instead of phosphate is transferred to the substrate , which function as scaffolds to assemble distinct E3 ligase complexes . However, induced expression of full-length CK1α did neither induce a phosphorylation-dependent mobility shift of endogenous WAVE nor did it yield increased levels of WAVE protein , suggesting that prominent basal phosphorylation by CK1α already stabilizes endogenous WAVE levels.We also tested whether forced overexpression of CK1α might increase WAVE protein levels. For this, we generated a stably transfected S2 cell line expressing CK1α under the control of a Cu3x and C-terminal SA5x mutant variants)  /T(p)-X-X-S/T] A FRT19A/FM7c, Kr-GAL4, UAS-GFP (BL#57084), y1w* Ck1α8B12 FRT19A/FM7i, twi-GAL4, UASt-GFP; sna[Sco]/CyO (BL# 63802); y1w* Ck1α[A] FRT19A/FM7c, Kr-GAL4, UAS-GFP (BL# 64459); hsFLP, w*, y; tubP-GAL80, FRT40A; hmlΔ-GAL4, UAS-eGFP/TM6B; Δ37, FRT40A/CyohsFLP; wave; y* w*;; ck2α[TikR]/TM3 (BL# 24511); y* w*;; ck2αTIK/TM3 (BL# 24512); y* w*;; ck2αP1/TM6B, Tb1 (Kyoto 141869). MARCM   males weDrosophila S2R+ cells were propagated in 1× Schneider's Drosophila Medium, as described previously . S2R+ cells were transfected as described previously . Cells were harvested at indicated time points.ged CK1α . For selThe CK1α inhibitor D4476 (Sigma) was resuspended in DMSO to 5000 µM stock dilution. D4476 was then diluted in cell culture medium and added to cells at final concentrations of 100 µM in 24-well tissue culture plates. Cells were treated for 4 h. The proteasome inhibitor MG132 (Sigma) was resuspended in DMSO to 1 mM stock dilution. Cells were treated with 10 µM MG132 for 4 h. Control-treated cells were treated with equal volumes of DMSO under conditions identical to those of drug treatment.Drosophila WAVE protein (GST-WAVE) as substrate (0.2 µg/µl in 50 mM Tris buffer pH 8.0). First, GST-WAVE was incubated with ATPγS. Which serves as the phosphate donor from which mono-thiophosphate instead of phosphate is transferred to the substrate. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues is allowed by addition of para-nitrobenzylmesylate (PNBM). Alkylated thiophosphate creates an epitope for thiophosphate ester-specific antibodies, which allows to detection phosphorylation (Phosphorylation assays were conducted using recombinant glutathione S-transferase (GST)-tagged rylation . Kinasesrylation  and Abl rylation ], both pCo-immunoprecipitation experiments were performed as described previously . SamplesProtein extracts were separated by SDS-PAGE and analyzed by western blotting. The following antibodies were used: anti-WAVE 1:1000; , anti-TuPupal macrophages were isolated as described previously . Wing imStructure illumination microscopy images were taken with an ELYRA S.1 microscope . Confocal fluorescence images were taken using a Leica TCS SP8 with an HC PL APO CS2 63×/1.4 oil objective. Live imaging of macrophage cultures was performed using a Zeiss CellObserver Z.1 with a Yokogawa CSU-X1 spinning disc scanning unit and an Axiocam MRm CCD camera (6.45 µm×6.45 µm). Ablation experiments were done using a 355 nm pulsed UV laser , as reported previously .UCSF Chimera package  and 100 (perfect circle), 4π×area÷perimeter2 .Tracking of migrating macrophages was performed using the spots module of Imaris 9.3  software. The reference frame module was set at the ablation site. After automatic tracking, all time-lapse movies were checked and were manually corrected if neccessary. The mean track speed was measured by using the Imaris software and values were analyzed with Graph Pad Prism. The bias angle between the vector towards the ablated cell and the direction vector of the cell was calculated in R software (R Studios Version 1.4). The angle between the vector directly towards the ablation cell and the direction vector of the cell at each time point was calculated using a and b divided by the multiplication product of the length of each vector i.e. the scalar product package ggplot2. For directed migration only cells within a 10–80 µm radius of the wounding site were analyzed. Results were statistically analyzed with GraphPad Prism 8.Polar histograms were generated using the FIJI software. F-actin and WAVE intensities were quantified within the same plane. The intensities of three different regions (10×10 µm size) within the posterior and anterior compartment were quantified for each experiment. The integrated density of each square was measured using FIJI software. The mean value of each side was taken to calculate the quotient of posterior over anterior intensity.Confocal microscopy images were processed and quantified with Results were statistically analyzed with GraphPad Prism 8.