Hepatozoon spp. is the causative agent of a vector-borne parasitic disease in many animal species. In felids, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris have been molecularly isolated. Hepatozoonosis usually causes asymptomatic infections in domestic cats, but clinical cases have recently been reported in Europe. We describe the first Italian case of hepatozoonosis in a cat with an unusual presentation. An 11-year-old neutered European shorthair cat was urgently hospitalized for intestinal intussusception. Hematology, biochemistry, FIV-FeLV tests, blood smears and molecular investigation targeting the 18S rRNA gene of Hepatozoon spp. were performed on blood samples; in addition, histological and molecular investigations were performed to analyze surgical samples to identify Hepatozoon infection. Hepatozoon gamonts were detected in granulocytes in the blood smear, and Hepatozoon spp. DNA was confirmed by PCR on blood. The intussusception was caused by a sessile endoluminal nodule that was surgically removed. Histologically, many elements referring to parasitic tissue forms were identified in the intestinal cells, and then the specimens were molecularly confirmed to harbor H. silvestris. This is the first description of symptomatic hepatozoonosis in a domestic cat in Italy. Hepatozoon silvestris has been described in wild felids, which are usually resilient to the infection, whereas the domestic cat seems to be more susceptible. Indeed, H. silvestris in cats usually presents tropism for skeletal muscle and myocardium with subsequent clinical manifestations. This is the first description of a domestic cat with H. silvestris localized in the intestinal epithelium and associated with intussusception. Hepatozoon life cycle involves an intermediate and a definitive host represented by a vertebrate animal and an arthropod vector, respectively [Babesia spp., Leishmania infantum) transmitted to humans and animals bitten by infected arthropods, in hepatozoonosis, the vertebrate host becomes infected through the ingestion of infected arthropods [Hepatozoon takes place, generating intracellular gamonts that circulate in the bloodstream. The vectors, mostly represented by ticks, ingest Hepatozoon gamonts through blood-feeding from infected animals, and sexual replication takes place within the ticks, ending in the production of mature oocysts that are ready to infect a new vertebrate host and complete the life cycle when the arthropod will be ingested [Hepatozoon canis and Hepatozoon felis life cycles, vertical intrauterine transmission is described, and in the Hepatozoon americanum life cycle, predation (i.e. the ingestion of infected prey) has been proven to be an additional transmission route [Hepatozoonosis is a vector-borne disease affecting many of animals, including reptiles, birds and mammals; it is caused by an apicomplexan parasite, of which almost 340 species are currently described –3. Almosthropods , 2. In ton route , 6.Hepatozoon are recognized in wild and domestic felids in Europe, i.e. Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris [H. silvestris infection [H. felis infection [In felids, hepatozoonosis is still mostly unknown worldwide, but recent epidemiological studies and case reports are raising attention about this parasite and its pathogenicity , 8. Currlvestris , 9–11, blvestris –15. Usualvestris , 16, 17.An 11-year-old neutered European shorthair cat living in a cat household with > 30 individuals in a pre-alpine area at 700 masl  and having free outdoor access was examined for its yearly routine veterinary check-up (day -7). A tick was found attached to the cat’s neck and was removed; then, the cat was treated with fipronil. The blood count presented some alterations in red blood cell, reticulocyte and monocyte total counts, and serum albumin was low ; no other remarkable findings were observed  on a blood sample by PCR targeting the 18S-rRNA gene with a cycle threshold (Ct) of 36.3 .The blood count showed an inflammatory leukogram with left shift and monocytosis, and the biochemical profile presented several alterations  reported in Table Additionally, the cat tested negative for feline immunodeficiency virus (FIV) antibodies and feline leukemia virus (FeLV) antigens . The results were also confirmed by molecular investigations targeting the FeLV proviral DNA . The abdominal ultrasound identified an intestinal intussusception; thus, the cat was urgently admitted to the operating room for a laparotomy.Hepatozoon DNA ; 1 month later (day + 70), it finally became negative. The time line of the events and their brief descriptions are reported in Fig. At surgery, a jejunal endoluminal pedunculated nodule of approximately 1.5 cm, almost occluding the intestinal lumen and causing intussusception, was found. The biochemical parameters improved and normalized within 13 days after surgery . Sequence analysis revealed the presence of Hepatozoon silvestris .The intestinal nodule was submitted to histopathological and molecular investigations (University of Padova). Tissue sections revealed a severe inflammatory reaction characterized by chronic ulcerative enteritis with polypoid proliferation and severe lymphangiectasia. Many protozoal inclusions were revealed within the enterocytes of the intestinal villi and near the lumen Fig. . The proH. silvestris DNA in wildcats from Bosnia and Herzegovina [H. silvestris in a cat was recently reported [Epidemiological studies reported the detection of zegovina , 21 and zegovina , southerzegovina  and centzegovina , 11. Thireported  and wherreported , 23. Eurreported , 21, 24.H. felis DNA in Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks [H. silvestris DNA in Ixodes ricinus ticks [I. ricinus, already known as the forest tick or castor bean tick, is the most widespread tick in European wild areas, it might be considered to have a potential role in the transmission of H. silvestris [Hepatozoon DNA were not possible; in addition, no other ticks were found after the cat was treated with fipronil. The predation of infected prey, e.g. mostly rodents, was considered another possible route of Hepatozoon transmission [Recent studies reported the identification of us ticks , 14, 25 us ticks . This islvestris , 13, 26.Hepatozoon causes a generally subclinical inflammation of skeletal muscles and myocardium [In domestic and wild cats, ocardium , 19, 20 ocardium , 17. Thiocardium , 19. Neuocardium , as obseHepatozoon found a good substrate for replicating. Considering the H. canis life cycle already described by Baneth et al. [H. silvestris, suggesting that the nodule was probably the first site of protozoan replication. In addition, the altered values of CPK and AST at day 0 suggested light skeletal muscle damage , as previously reported in cats with hepatozoonosis [The intestinal intussusception was generated by a sessile endoluminal nodule, which could have been due to (1) the inflammatory local response to the parasite's penetration through the intestinal mucosa and/or (2) an inflammatory process that was already present where the h et al. , the parzoonosis , 17, evezoonosis , 17. ActHepatozoon infection and the scientific publication.Unfortunately, further histological investigations from other muscle sections and organs before and after treatment would have been useful to evaluate the skeletal muscle involvement, infection burden and efficacy of the treatment protocol but would have been unethical. In addition, the cat was treated to ensure its complete recovery without considering the novelty of Hepatozoon infections in felids, particularly in domestic cats, to prevent severe and potentially fatal clinical cases. Increased knowledge regarding the Hepatozoon life cycle in wild and domestic felids as long as arthropod vectors are involved would surely be useful for the adoption of adequate preventative measures in cats.Further investigations are needed to improve the scientific knowledge on H. silvestris caused fatal myocarditis in a domestic cat, in this report, the patient recovered completely after surgical removal of the “parasitic” nodule and monthly doxycycline therapy. The intestinal intussusception caused the sudden worsening of the clinical conditions, and surgical resolution was necessary to save the cat. However, the intestinal nodule was probably the result of a local inflammatory reaction to limit the Hepatozoon penetration, and it became the first site of protozoan replication; its surgical removal helped the cat to rapidly recover. Despite the unusual clinical presentation of this case, surgery should not to be the treatment of choice in every hepatozoonotic infections with intestinal signs and/or ultrasonographic abnormalities. Feline hepatozoonosis is an emerging vector-borne disease, and considering the recent reports of symptomatic cases, monitoring in cat populations is strongly advised.In conclusion, contrary to the other European case  in which


HH gene and the main components in HH signaling are also associated with numerous types of diseases. Before secretion, the HH protein undergoes post-translational cholesterol modification to gain full activity, and cholesterol is believed to be essential for proper HH signaling transduction. In addition, results from recent studies show the reciprocal effect that HH signaling functions in cholesterol metabolism as well as in cholesterol homeostasis, which provides feedback to HH pathway. Here, we hope to provide new insights into HH signaling function by discussing the role of cholesterol in HH protein maturation, secretion and HH signaling transduction, and the potential role of HH in regulation of cholesterol as well.The Hedgehog (HH) signaling is one of the key agents that govern the precisely regulated developmental processes of multicellular organisms in vertebrates and invertebrates. The HH pathway in the receiving cell includes Patched1, a twelve-pass transmembrane receptor, and Smoothened, a seven-transmembrane G-protein coupled receptor (GPCR), and the downstream GLI family of three transcriptional factors (GLI1-GLI3). Mutations of  HH gene mutant Drosophila larvae that is shorter and more spiked , one of the most famous model organisms, has no HH ortholog (Drosophila HH gene can be categorized into three subgroups: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH), among which SHH is the most broadly expressed and is thought to be responsible for the major effects on development of the craniofacial part, spinal cord, axial skeleton and limbs, while IHH is identified to play a major role in cartilage differentiation in the growth of long bones and affects the formation of midface and masticatory system, and DHH is mainly involved in the formation of mouse testis, ovary and development of nerves  family have been recognized as one of the major intercellular signalings involved in controlling cell growth, regulating cell survival, deciding cell fate, and patterning almost all aspects of the body plan during animal embryonic development, depending on the context. Along with establishing the patterns of cellular differentiation to direct complex organ formation, HH signaling also plays an important role in post-embryonic tissue regeneration and repair processes . Dysregue spiked . Furtherortholog . The verf nerves . In the Ski, also known as sightless, rasp or central missing, and HHAT in humans), a palmitic acid moiety is attached to the first amino acid of the N-terminal polypeptide (HH-Np), which is also required for HH-N activity in vivo   . Howevern (HSPG) . In vertach case .Drosophila. This gene, Dispatched (Disp), which encodes the putative 12-pass transmembrane protein Dispatched (DISP) that contains a novel sterol-sensing domain (SSD), is found to be required for secretion of Hh-Np in Drosophila imaginal disc. SSD domain, first identified in proteins implicated in cholesterol homeostasis or trafficking, is required for the cholesterol-dependent regulation . The D95N mutation of SMO is hardly modified by cholesterol and rarely localizes to the primary cilia, resulting in its failure in activation of downstream signaling transduction. Several possible mechanisms by which PTCH1 regulates SMO have been proposed, and these mechanisms are mainly focused on the primary cilium or the ciliary micro-domain: , the seven-transmembrane-span receptor-like protein in HH pathway, and that regulation of SMO by PTCH1 is thought to be dependent on small molecular intermediates, such as cholesterol  summari. The Asp-domain:  PTCH1 ca-domain: . (Kong e-domain:  PTCH1 re-domain: . As a st-domain: . More exXenopus laevis SMO (xSMO) indicated that cholesterol-mediated SMO activation is correlated with the rearrangement of CRD orientation and a cation-lock opening in the transmembrane domain (TMD) of SMO  of SMO . A large) of SMO . For exa) of SMO . Althoug) of SMO .SHH causes axial patterning defects with a phenotype of holoprosencephalic, which is similar to that found in the mice with deficiency of Megalin and in some of the human patients with Smith–Lemli–Opitz syndrome (SLOS) as well. In addition, by using chick embryos some studies also confirmed that treatment of cyclodextrin, a cyclic oligosaccharide that forms non-covalent complexes with sterols and can be thereby used to extract and deplete cholesterol from living cells, causes variable loss of the frontonasal process and other midline structures in chick embryos, and the spectrum of facial defects is similar to that resulted from the exposure to HH-pathway antagonist jervine  in the cytosol. Then, as the rate-limiting enzyme of the pathway, hydroxy-methylglutaryl-CoA reductase (HMG-CoAR) converts HMG-CoA to mevalonate. HMG-CoAR contains a conserved SSD, which is characterized by five membrane-spanning α-helices  and aromatase. As shown in activity , suggestin vivo and decreases expression of proteases in human cartilage in vitro.Given that HH signaling regulates chondrocyte differentiation and is activated in human and murine osteoarthritis, recent study discovers that HH signaling regulates cholesterol homeostasis in osteoarthritis using human osteoarthritis samples by up-regulating genes that govern cholesterol homeostasis, which alters cholesterol accumulation in chondrocytes . MoreoveDrosophila a few decades ago, has been clarified to be involved in the development of both vertebrates and invertebrates and in the progress of cancer. Owing to rapid progress over the last several years, we have a better understanding of the mechanisms by which HH signaling is regulated by cholesterol, and recent work further uncovers the mechanisms by which HH affects cholesterol regulation. Advances in our knowledge of the cross-talk between HH signaling and cholesterol provides a framework which can be used to illustrate unknown mechanisms of clinical diseases, and further fulfill the biological significance of HH signaling to the known mechanisms. Aberrant HH signaling can lead to abnormal lipid homeostasis, which affects atherosclerosis and hepatosteatosis. Further research, especially progress in metabolomics and genetic studies will help reveal novel and intricate details of the biochemical and molecular cellular biological aspects of HH signaling. Currently, there are still some challenges in this area. What is the specific mechanism by which sterol participates in the regulation of SMO by PTCH1? How exactly does cholesterol modification of SMO activate HH signaling and transduce downstream events? How aberrant cholesterol metabolism affects Hh signaling and leads to different clinical manifestations? Deeper investigations in this field will potentially benefit disease therapy, especially in understanding the relation between HH signaling and related sterol metabolisms, which will provide potential targets in developing novel drugs for relevant diseases in the future.The HH signaling that was first identified in


Introducing a Special Issue on mechanism of reproductive disorders in mammals originating from exposure to environmental chemicals during perinatal life. Exposure to chemicals during perinatal life can disrupt reproductive development and ultimately lead to reproductive disorders. This includes disorders manifesting already at birth such as genital malformations, but also late onset disorders such as reproductive cancers and infertility . The undBroadly speaking, male sexual differentiation depends on a surge in androgen signaling during critical stages of development, whereas female sexual differentiation requires androgen levels to be low during the same developmental stages. This is overly simplified, of course, as estrogens and other hormones also play essential roles in reproductive development of both sexes, as do other signaling pathways and biomolecules. Sexual development relies instead on a complex network of signaling pathways that interact to ensure proper spatiotemporal tissue differentiation and many of these signaling pathways may themselves be vulnerable to disruption by exogenous chemical substances.To better account for the many ways by which chemicals can disrupt reproductive development, this special issue invited contributions that could shed new light on causal molecular pathways in addition to classical sex hormone signaling. A more long-term vision is that such new insights can be used in a chemical safety assessment and ultimately regulatory decision-making. Especially in a new era of predictive toxicology where chemical risk assessors are asked to rely much more on non-animal test data than on classical animal toxicity studies when determining if a chemical substance may or may not pose a risk to human health.Disrupted steroid hormone signaling could account for a majority of effect modalities causing reproductive disorders through early-life exposure to chemicals. However, both gonadal differentiation and sexual development rely on additional signaling pathways, for instance the morphoregulatory pathways Wingless-like (WNT), retinoic acid (RA) and Hedgehog (HH). In the first article of this Special Issue, the HH signaling pathway is discussed in the context of reproductive development and disruption by environmental chemicals or drugs . AlthougThe third article uses the Adverse Outcome Pathway (AOP) framework to construct putative causal toxicological pathways linking developmental exposures to EDCs, particularly phthalates, with malformations of the male reproductive tract . The AOPAs a first case for this pragmatic approach to AOP development, the fourth article presents a key event relationship (KER) linking reduced RA signaling in the developing ovary with the failure of oocytes to enter meiosis . The RA Since modern toxicology is moving towards relying more on non-animal test strategies for safety assessments of chemical substances, it is imperative that we understand the underlying mechanisms and modes of action that underpin reproductive toxicity . Only thGiven his role as Guest Editor, Terje Svingen had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Thomas Knudsen.Terje Svingen: Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


TAS1R2 + TAS1R3) genes and another by glucose transporters and the ATP-gated potassium (KATP) channel. Despite these important discoveries, we do not fully understand the mechanisms regulating sweet taste signaling. We will introduce the core components of the above sweet taste signaling pathways and the rationale for having multiple pathways for detecting sweet tastants. We will then highlight the roles of key regulators of the sweet taste signaling pathways, including downstream signal transduction pathway components expressed in sweet taste receptor cells and hormones and other signaling molecules such as leptin and endocannabinoids.Sweet taste, a proxy for sugar-derived calories, is an important driver of food intake, and animals have evolved robust molecular and cellular machinery for sweet taste signaling. The overconsumption of sugar-derived calories is a major driver of obesity and other metabolic diseases. A fine-grained appreciation of the dynamic regulation of sweet taste signaling mechanisms will be required for designing novel noncaloric sweeteners with better hedonic and metabolic profiles and improved consumer acceptance. Sweet taste receptor cells express at least two signaling pathways, one mediated by a heterodimeric G-protein coupled receptor encoded by taste 1 receptor members 2 and 3 ( It is thought that the taste system evolved to rapidly evaluate whether a potential food is suitable for consumption. Generally, five primary taste qualities are thought to exist: sweet, bitter, salty, sour, and umami  and the subsequent demonstration that Gnat3 knockout mice have diminished sweet taste responses strongly suggested that GPCRs mediate sweet taste signaling . L. LATP chgment it .Endocannabinoids such as 2-arachidonoyl glycerol (2AG) and anandamide ((N-arachidonoylethanolamine (AEA)) are orexigenic (appetite promoting) signaling molecules derived from fatty acids. Circulating levels of endocannabinoids are inversely correlated to that of leptin. They primarily act through the cannabinoid receptor 1 (CB1) to promote appetite. Like LEPR, CB1 is coexpressed with the STR in taste cells. Oral application of 2AG and AEA enhances taste nerve responses to both caloric sugars and NCSs in wild type, but not CB1 knockout mice ,120. TheA clearer picture of the mechanisms regulating sweet taste signaling has emerged since the discovery of the sweet taste receptor and that of the caloric sugar-specific pathways, as well as the downstream signaling pathway components. NCSs currently in use have not proved very effective in reducing obesity and diabetes. Development of novel classes of NCSs may benefit from a thorough knowledge of the mechanisms regulating the core signaling pathway downstream of the sweet taste signaling pathways and the hormonal and other signaling pathways that modulate them.


Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn Interestingly, Cu2+ was found to inhibit the UA-mediated proton transport. The results on the transport through the membranes were supplemented by circular dichroism (CD) spectroscopy data, which showed the binding of the metal cations to UA in methanol.Usnic acid (UA) has long been a popular fat-burning pill because of its ability to cause the dissipation of the proton motive force in mitochondria ,3,4,5,6,2 led to a substantial increase in the current, reaching 2.5 nA within 2 min of the stirring of the membrane-bathing solution. Under these conditions, a UA-mediated current, depending on both the pH and Mg2+ concentration, resulted from the electrogenic Mg2+/3H+ exchange [4 caused a substantial decrease in the BLM current, probably due to the strong binding of Cu2+ to UA, interfering with its ability to transport the cations. In the control experiments, the addition of 20 µM (or more) K2SO4 did not affect the UA-mediated BLM current, thereby proving that it was Cu2+ that inhibited the transport  or 1 mM CaCl2 (pink curve) to the buffer solution (pH 6.0) outside the liposomes containing 30 µM UA caused alkalization inside the liposomes, manifesting in an increase in the pyranine fluorescence intensity as a result of the influx of the divalent cations and the efflux of the protons from the liposomes (2 (red track), intermediate in the case of MgCl2 (blue track) and small in the case of CaCl2 (pink track). The black track displayed a weak alkalization of the liposomal lumen induced by UA without the addition of divalent cations. No alkalization took place inside the liposomes in the absence of UA, thereby showing that there was no proton leakage from the liposomes without UA (grey track). In the control experiments without UA, even 1 mM Mn2+ did not elicit a proton leakage . Overall, the spectral data pointed to the chelation of the divalent metal cations by usnic acid.To examine the formation of metal–UA complexes, we measured the CD and UV absorbance spectra of UA in the presence of divalent metal cations in methanol, enabling both lipophilic UA and hydrophilic inorganic salts to be dissolved. The CD spectrum of UA in methanol exhibited two positive bands peaking at about 250 nm and 330 nm and a negative band peaking at about 290 nm -usnic acid (UA) and diphytanoylphosphatidylcholine (DPhPC), were from Sigma-Aldrich, St. Louis, MO, USA.A bilayer lipid membrane (BLM) was formed using the brush technique  from a 2To measure the proton efflux from the liposomes, we prepared large unilamellar vesicles loaded with the pH-sensitive fluorescent dye pyranine and used the method described in  with sli2 or ZnSO4 and the measurements of the Ca2+ (Zn2+) efflux from the liposomes were performed as described in [2+ and Zn2+ were assayed by measuring the fluorescence intensity of the Ca2+ (Zn2+)-sensitive fluorophore Fluo-5N at 520 nm upon excitation at 495 nm with a Solar CM2203 spectrofluorometer . To remove the contribution of 3–5 µM Ca2+, always present in the medium outside the liposomes, to the Fluo-5N fluorescence, we added an aliquot of 1 mM EDTA before the addition of UA.The preparation of liposomes loaded with 100 mM CaClribed in . The outThe electronic circular dichroism (CD) spectra were recorded on a Chirascan CD spectrometer  using a 1 cm quartz cuvette.


Natural Killer (NK) cells provide key resistance against viral infections and tumors. A diverse set of activating and inhibitory NK cell receptors (NKRs) interact with cognate ligands presented by target host cells, where integration of dueling signals initiated by the ligand-NKR interactions determines NK cell activation or tolerance. Imaging experiments over decades have shown micron and sub-micron scale spatial clustering of activating and inhibitory NKRs. The mechanistic roles of these clusters in affecting downstream signaling and activation are often unclear. To this end, we developed a predictive in silico framework by combining spatially resolved mechanistic agent based modeling, published TIRF imaging data, and parameter estimation to determine mechanisms by which formation and spatial movements of activating NKG2D microclusters affect early time NKG2D signaling kinetics in a human cell line NKL. We show co-clustering of NKG2D and the guanosine nucleotide exchange factor Vav1 in NKG2D microclusters plays a dominant role over ligand (ULBP3) rebinding in increasing production of phospho-Vav1(pVav1), an activation marker of early NKG2D signaling. The in silico model successfully predicts several scenarios of inhibition of NKG2D signaling and time course of NKG2D spatial clustering over a short (~3 min) interval. Modeling shows the presence of a spatial positive feedback relating formation and centripetal movements of NKG2D microclusters, and pVav1 production offers flexibility towards suppression of activating signals by inhibitory KIR ligands organized in inhomogeneous spatial patterns . Our in silico framework marks a major improvement in developing spatiotemporal signaling models with quantitatively estimated model parameters using imaging data. Natural Killer cells are lymphocytes of our innate immunity and provide important resistance against viral infections and tumors. NK cells scan the local environment with diverse activating and inhibitory NK cell receptors (NKRs) and remain tolerized or lyse target cells expressing cognate ligands to NKRs. NKRs have been found to form micron sized clusters (or microclusters) as they interact with cognate ligands, and mechanisms regarding how the formation and movements of these microclusters influence NK cell signaling and activation, specifically related to activating NKRs, are often unclear. To this end, we develop a predictive spatially resolved early-time NK cell signaling model to study the interplay between membrane-proximal biochemical signaling events and the kinetics of microclusters of activating NKG2D and inhibitory KIR2DL2 receptors. We used published TIRF imaging data to validate our in silico models and estimate model parameters. Predictions from multiple in silico models are tested against a variety of data obtained from published imaging experiments and immunoassays. Our analysis suggests co-clustering of NKG2D and the guanosine nucleotide exchange factor Vav1 in the microclusters plays a major role in enhancing downstream activating signals. The developed framework can be extended to describe spatiotemporal signaling for other activating NKRs including CD16. Natural Killer (NK) cells are lymphocytes of our innate immune system which provide important immune protection against viral infection and tumors ,2. NK ceSpatial clustering of NKG2D has been well investigated in confocal , total iSpatially resolved computational models have been successfully employed to glean mechanisms that underlie formation, motility, and function of spatial clusters of activating  and inhiMotivated by above reasoning and a potential interplay between signaling kinetics and spatial clustering, we developed a framework combining a spatially resolved mechanistic agent based model and published TIRF imaging data assaying spatiotemporal signaling kinetics in NKL cell lines stimulated via NKG2D receptor or NKG2D and KIR2DL2 receptors. The agent based model developed here represents a major improvement over previous modeling efforts in the following aspects: (1) The model is able to quantitatively describe micron scale details of spatial clustering of activating and inhibitory NKRs. (2) A detailed parameter estimation of model parameters is carried out using spatial data. (3) The model includes interplay between spatial clustering and activating NKR signaling kinetics. We investigated three hypotheses  to show that co-clustering of Vav1 with NKG2D rather than ligand rebinding of ULBP3 is required to produce increased pVav1 production due to the formation of NKG2D microclusters. The presence of the spatial positive feedback allows for an efficient suppression of early time NKG2D signaling by heterogeneously distributed inhibitory HLA-C ligands on target cells.We developed a spatially resolved agent based model involving activating NKG2D receptors, inhibitory KIR2DL2 receptors, cognate NKR ligands (ULBP3 and HLA-C), and signaling proteins: Src family kinases (SFKs), Vav1, and phosphatase SHP1. The NKRs and signaling proteins interact via different rules to describe membrane proximal signaling events in human NK cell line NKL. The model includes rules describing biochemical signaling reactions, spatial movements of signaling complexes, and regulation of the spatial movements by biochemical reactions. The rules in the model are designed to quantitatively capture several distinguishing features of spatiotemporal NK cell signaling kinetics observed in previous imaging experiments, namely, a) formation of mobile and immobile NKG2D microclusters upon NKG2D stimulation, b) centz is discretized into chambers of volume l0× l0×z where, z = l0 or z = 2l0 (l0 = 0.5μm) for molecules residing in the plasma membrane or the cytosol, respectively. The following spatiotemporal processes occur in the models.Our agent based model describes spatiotemporal membrane proximal NK cell signaling in a quasi three-dimensional simulation box representing the interface between NK cell membrane and the plasma membrane of target cell or the lipid bilayer in a TIRF experiment at the IS . The simWe considered key membrane proximal biochemical signaling reactions involved in NKG2D and KIR2DL2 signaling. The specific roles of signaling proteins  considered here in regulating NKG2D signaling have been validated in experiments and modeling over the years. A selection of the pertaining literature is cited below and in NKG2D receptors bind to cognate ligands (ULBP3) to form NKG2D-ULPB3 complexes. NKG2D homodimers are associated with a pair of DAP10 homodimers in human NK cells. The tyrosine residues in two YINM motifs in a DAP10 homodimer are phosphorylated by Src family kinases ,19 upon We modeled movements of NKG2D-ULBP3 complexes for formation of NKG2D microclusters and centripetal movements of NKG2D microclusters towards the center of the IS. These movements are assumed in the model to be dependent on actin remodeling which is regulated by signaling products such as pVav1. The aboThe probabilities of hops generating NKG2D microclusters and centripetal movements of NKG2D microclusters depend on the local and the total number of pVav1 in the simulation box, respectively. The rules capture the interplay between biochemical signaling reactions and regulation of specific spatial movements by signaling reactions which in turn affects signaling reactions. For NKG2D signaling the above interplay represents a positive feedback .We considered three hypotheses encoded in models Model 1, Model 2, and Model 3. The models probe functional outcomes of different types of NKG2D clustering and interplay between NKG2D microclusters and biochemical signaling reactions. In Model 1, mobile NKG2D microclusters and NKG2D bound signaling protein molecules move toward the central region of the IS. In Model 2, NKG2D complexes and membrane proximal Vav1 molecules move simultaneously toward the central region of the IS. This Vav1 species in the model could potentially represent Vav1 molecules that are recruited to the plasma membrane via SOS1. Because2 and a depth of z, and is divided into small cubic chambers of size (l0 × l0 × z), where l0 = 0.5μm and z = l0 (for molecules residing in membrane) or 2l0 (for cytosolic molecules). The molecules are well-mixed in each chamber and molecules in a chamber hop to next nearest neighbor chambers with specific rates to produce diffusive or centripetal movements, or movements leading to microcluster formation. The kinetics of the system is simulated using a kinetic Monte Carlo (kMC) approach via a freely available simulator SPPARKS (https://spparks.sandia.gov/). The kMC simulation includes intrinsic noise fluctuations in biochemical reactions as well as in spatial movements. The list of the processes, and their propensities are listed in The simulations are carried out in a quasi-three-dimensional simulation box representing a thin junction between NK cell and the supported lipid bilayer in TIRF experiments. The simulation box has an area of 15 × 15 μmSpatiotemporal signaling kinetics of NKG2D and associated signaling proteins was simulated in Model 1 and Model 2. NKG2D, ULBP3, SFK, and Vav1 molecules were distributed homogeneously in space in the simulation box at the beginning of the simulation at t = 0. Binding of NKG2D and ULBP3 initiates a series of biochemical signaling reactions  leading The computation of uncertainties in the estimated parameter values showed that about 2/3 of the total number of parameters are estimated well, e.g., (standard deviation)/ < 3. The procedure for estimation of uncertainty is described in detail in the Materials and Methods section, and potential causes behind poor parameter estimations of 1/3 of the parameters are discussed in the Discussion section. Next, we assessed the utility of the parameter estimation scheme for generating predictions at future time points Figs  and provWe investigated the mechanistic role of formation and centripetal movement of NKG2D microclusters in increasing pVav1 production. The average lifetime of a NKG2D-ULBP3 complex within NKG2D microclusters can increase because of the increase in the frequency of ULBP3 rebinding due to higher density of NKG2D molecules in microclusters. The increased ULBP3 rebinding could elevate abundances of NKG2D-ULBP3 complexes leading to higher pVav1 production in Model 1 and Model 2. In addition, in Model 2, the increase in reaction propensities due to co-clustering of NKG2D and Vav1 can enhance pVav1 production. In order to evaluate the roles of ULBP3 rebinding and NKG2D and Vav1 co-clustering in increasing pVav1 production we compared kinetics of pVav1 production in Model 1 and Model 2 under two conditions: Case A: NKG2D is not allowed to form microclusters or perform centripetal movements, and, Vav1 does not co-cluster with NKG2D. This case represents NKG2D stimulation in experiments where NKG2D microcluster formation and migration is blocked by application of drugs inhibiting actin polymerization . Case B:off can diffuse and rebind to the receptors. In the absence of any ligand rebinding, the number of receptor-ligand complex decays exponentially as ∝ exp(-kofft). The presence of rebinding produces a slower and a non-exponential decay of the number of receptor ligand complex with higher numbers of receptor-ligand complex remaining in the system at longer times (t≫ 1/koff) compared to an exponential decay as exp(-kofft). We evaluated the decay kinetics of an initial number of NKG2D-ULBP3 complex where NKG2D-ULBP3 binding-unbinding reactions were the only reactions present in simulations. The NKG2D molecules, held fixed in space, were distributed uniformly and randomly or in a spatially clustered configuration obtained from our simulations for Model 1 or Model 2 at t = 1 min  of ~ 30 seconds and the average number of homogeneously distributed NKG2D molecules in the area occupied by a typical NKG2D microcluster is about two molecules, the higher numbers of NKG2D in microclusters do not result in a large increase in abundances of NKG2D-ULBP3 complexes due to ligand rebinding. Our simulations confirm the above explanation. Co-clustering of signaling proteins could play an important role for increasing downstream signaling for other activating NKRs as well. The density of CD16 on primary NK cells is about 200 times larger than that of NKG2D T) in the simulation box and is given by,Any ULBP3 bound NKG2D receptor complex is assumed to be a part of a microcluster. All molecules of a specific ULBP3-NKG2D complex, e.g., ULBP3-NKG2D-SFK, in a chamber are moved to one of the four nearest neighboring chambers in a single microcluster hopping move. The probabilities of the hops to the neighboring chambers are given by hop is composed of two parts: one generates centripetal movements A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. 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Please don't hesitate to contact us if you have any questions or comments.Sincerely,Mark Alber, Ph.D.Deputy EditorPLOS Computational BiologyMark AlberDeputy EditorPLOS Computational Biology***********************Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This manuscript describes a spatial agent-based model for investigating the interaction between signalling and spatial organisation of receptors and adaptor proteins in the immunological synapses of NK cells. This is an important question for understanding the function of NK cells, as well as T cells that also form immunological synapses and integrate information between activating and inhibitory receptors. The authors show that their model captures key features of previous experimental imaging data of NKG2D clustering and dynamics in NK cell synapses and that co-clustering between Vav1 and NKG2D is more important for robust signalling than rebinding of NKG2D to its ligand. In their modelling Grewal and Das show that NKG2D translocation to the centre of the synapse could initiate a positive feedback of Vav1 phoshorylation, if translocation is movement is dependent on pVav1, and that this allows for efficient inhibition by KIR2DL1.Overall the work presents an interesting hypothesis that is supported by previous microscopy studies. I have some questions surrounding parameter estimation, and suggest some extra discussion of limitations, which are outlined below:Suggest softening the claim in the title from "…Reveals role…" to "…Suggests role…" or "…is consistent with a role…". The authors show their model is consistent with previous work, but to establish that the mechanism they propose exists in the biology, some experimental validation of novel predictions made by the model would be required.doi.org/10.1007/s12195-016-0438-7).Table 1, Rule 2: Are these binding rates the estimate for the catalytic domain of the SFK interacting with the unphosphorylated DAP10 substrate ? If so why are the kinetics of SH2 domain for pY used to estimate this? They are different domains and completely different interactions. Others have estimated these values .doi.org/10.1016/j.bpj.2021.03.019 and others.Table 1, Rule 14: Catalytic rate on pNPP is a very poor indicator of catalytic rates on specific pY targets, which can have large variability depending on flanking sequences. For measurements of SHP1 catalytic rates on phosphotyrosine substrate see doi.org/10.1083/jcb.201009135), and related to Vav1 activity. It would be useful for placing this work in this context. Splitting the limitations section into paragraphs would also make it more readable.Around line 489, a little more discussion about the limitation of not modelling cellular spreading and retraction, both of which are related to activating and inhibitory receptor signalling  I would have liked to see more validation on their model on other signaling pathways, especially their modeling of biochemical signaling reactions. For example, how well would it predict findings from this paper on NKG2D: 2) Along the same lines, would the modeling have to be fitted each time for use in specific signaling systems or would their system be useful to describe microcluster formation in general? For example, the authors cite Steblyanko et. al. as a limitation of their work, however, would the in silico modeling be predictive of these results? If not, I would share Reviewer 1’s concerns about overfitting. Also, it would limit the impact of this type of in silico modeling.3) The other concern related to overfitting, their biochemical signaling processes are limited to a handful of papers. The models would rely heavily on the accuracy of the original paper findings. Can the authors validate their parameters across different publications/published results? Without external validation of the modeling, it’s difficult to evaluate the significance of the findings.doi.org/10.3389/fimmu.2012.00421, doi: 10.1038/nri2381.4) During the discussion of microclusters, please cite these papers: **********Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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Read more information on sharing protocols at  28 Mar 2022Attachmentresponse_to_reviewers.docxSubmitted filename: Click here for additional data file. 18 Apr 2022Dear Prof. Das,We are pleased to inform you that your manuscript 'Spatially Resolved In Silico Modeling of NKG2D Signaling Kinetics Suggests a Key role of NKG2D and Vav1 Co-clustering in Generating Natural Killer cell Activation' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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I have no further concerns.Reviewer #2: All my concerns have been addressed with the new experiments performed to demonstrate the utility of their model in other settings.**********Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: None**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  1223-442824 | ploscompbiol.org | @PLOSCompBiolPLOS Computational Biology | Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom


In the Industry 4.0 era, with the continuous integration of industrial field systems and upper-layer facilities, interconnection between industrial wireless sensor networks (IWSNs) and industrial Internet networks is becoming increasingly pivotal. However, when deployed in real industrial scenarios, IWSNs are often connected to legacy control systems, through some wired industrial network protocols via gateways. Complex protocol translation is required in these gateways, and semantic interoperability is lacking between IWSNs and the industrial Internet. To fill this gap, our study focuses on realizing the interconnection and interoperability between an IWSN and the industrial Internet. The Open Platform Communications Unified Architecture (OPC UA) and joint publish/subscribe (pub/sub) communication between the two networks are used to achieve efficient transmission. Taking the Wireless Networks for Industrial Automation Process Automation (WIA-PA), a typical technology in IWSNs, as an example, we develop a communication architecture that adopts OPC UA as a communication bridge to integrate the WIA-PA network into the industrial Internet. A WIA-PA virtualization method for OPC UA pub/sub data sources is designed to solve the data mapping problem between WIA-PA and OPC UA. Then, the WIA-PA/OPC UA joint pub/sub transmission mechanism and the corresponding configuration mechanism are designed. Finally, a laboratory-level verification system is implemented to validate the proposed architecture, and the experimental results demonstrate its promising feasibility and capability. With the rapidly growing demand for wireless data transmission services in the industrial Internet of things (IIoT) ,2, indusAll three IWSN technologies are based on IEEE 802.15.4 and form wireless personal area networks. The gateway of an IWSN is responsible for data collection from wireless devices and for the downward forwarding of commands to wireless devices. In real industrial scenarios, IWSNs are often connected to legacy control systems  and human–machine interface (HMI)), through industrial wired network protocols  via gateways . HoweverAs an open, platform-independent and service-oriented architecture specification, OPC UA supports the secure and reliable exchange of data, in the industrial automation space and other industries, and ensures the seamless flow of information among devices from multiple vendors . OPC UA Most studies on WIA-PA and OPC UA focus on verifying and implementing their respective protocols. Numerous approaches have been proposed to enable connection between WIA-PA networks and the Internet. For instance, the authors in  proposed(1)Taking WIA-PA, a typical wireless technology in IWSNs, as an example, we propose a communication architecture of WIA-PA/OPC UA joint pub/sub. This architecture can combine WIA-PA pub/sub with MQTT broker-based OPC UA pub/sub to realize the integration of the WIA-PA network into the industrial Internet.(2)A WIA-PA virtualization method for OPC UA pub/sub data sources is designed to solve the data mapping problem between WIA-PA devices and OPC UA publisher/subscriber.(3)To ensure the efficient messages exchange between OPC UA publisher/subscriber and WIA-PA networks, we design a WIA-PA/OPC UA joint pub/sub transmission mechanism and the corresponding configuration mechanism.(4)An experimental system is implemented to evaluate the feasibility and capability of the proposed communication scheme. The results show that the proposed scheme performs well in terms of consumed memory, communication success rate, and publishing delay. Furthermore, in this study, the proposed scheme has strong protocol consistency.Although the IWSNs and OPC UA pub/sub modes have been extensively studied in their respective fields of research ,20, the The rest of this paper is organized as follows. WIA-PA is a wireless network protocol for industrial process measurement, monitoring, and control. It is based on IEEE 802.15.4 and is standardized and approved as IEC 62601 by the IEC . Its topAs shown in The WIA-PA application layer includes the user application process (UAP) and the application sublayer (ASL). A UAP consists of one or more user application objects (UAOs). The properties of a UAO include object name, property name, property identifier, property data type, and supported methods. A UAO supports five operation methods for WIA-PA internal data: read, write, publish, report, and report ACK. To specify the access mode between UAOs, WIA-PA uses the virtual communication relationship (VCR), which distinguishes the paths and communication resources used by different UAOs. VCR_ID uniquely identifies each VCR. According to the supported applications, the VCR is divided into three modes: P/S mode (supports preconfiguration and periodic message transmission), R/S mode (supports aperiodic events and trend reporting), and C/S mode (supports WIA-PA aperiodic and dynamic pairwise unicast information transmission for WIA-PA devices).OPC UA is a platform-independent, service-oriented interoperability technology that is standardized by the IEC and consists of 26 specifications and many companion specifications. It has two communication modes: C/S and pub/sub. In C/S, the client and server are deeply coupled. They must establish a direct connection and send requests and responses to each other. Compared with C/S, pub/sub has a flexible communication structure, easy configuration, and support for many-to-many communication.OPC UA pub/sub has three logical roles: publisher, subscriber, and message middleware. The publisher and subscriber are decoupled through the message middleware. They do not require direct information exchange but need to correctly parse and encapsulate message packets as required. Meanwhile, the OPC UA pub/sub specification defines brokerless and broker-based middlewares. With a brokerless middleware, an OPC UA publisher relies on the network infrastructure to deliver messages to one or more receivers. A broker-based middleware needs to use third-party messaging protocols, such as advanced message queuing protocol (AMQP) and MQTT. Compared with AMQP, MQTT is easier to implement and widely used in industrial automation application scenarios. Therefore, this paper uses MQTT as the WIA-PA/OPC UA joint pub/sub broker-based middleware. Eckhardt et al.  evaluateAccording to the analysis results in A broker in the OPC UA publish/subscribe model can decouple the communication between the entities in space, time, and synchronization. The publisher and subscriber do not have to be directly addressable, which makes the network architecture flexible, while there is no need for resource-constrained WIA-PA nodes to maintain address tables for subscribers. In addition, only the events and data that are of interest need to be transmitted to the broker, and the broker supports a one-to-many communication mode, which can reduce the number of messages to be transmitted and further reduce network conflicts and energy consumption. Hence, broker-based OPC UA pub/sub can meet most requirements of WIA-PA networks, industrial Internet remote applications, and cross-network communication between field networks.In summary, OPC UA pub/sub is deployed in the WIA-PA scheme in this paper as follows. MQTT broker-based OPC UA pub/sub is the broker transport protocol, and JSON and UADP are the message encoding formats. Furthermore, the coupling of OPC UA and the WIA-PA network is realized through the WIA-PA gateway.In an IWSN, the computing resources and energy of sensor devices are relatively limited, whereas those of the gateway are generally greater. Therefore, the use of a WIA-PA gateway as the coupling point between WIA-PA and OPC UA can maximize its abundant computing resources and energy. The system architecture of the WIA-PA/OPC UA joint pub/sub is illustrated in The system architecture includes an OPC UA pub/sub communication unit and a WIA-PA/OPC UA joint pub/sub unit. The former realizes MQTT broker-based OPC UA pub/sub. The latter virtualizes the OPC UA pub/sub data source through a virtualization mechanism. In this way, the device binding and data resource mapping of the OPC UA publisher/subscriber are realized. Meanwhile, through the WIA-PA/OPC UA joint pub/sub transmission configuration mechanism, the WIA-PA network device can communicate with the OPC UA publisher/subscriber. As shown in WIA-PA and OPC UA have different pub/sub mechanisms and cannot be directly combined. Thus, a virtualization mechanism must be designed for the OPC UA pub/sub data source in the WIA-PA application layer. This mechanism is the basis of WIA-PA/OPC UA joint pub/sub communication. The method is to map the OPC UA pub/sub data source to the UAO of the WIA-PA application layer, such that the WIA-PA field devices obtain the data source of the OPC UA publisher/subscriber through the WIA-PA protocol. This method is divided into three steps: device interconnection, resource discovery, and OPC UA pub/sub data source mapping.The device interconnection process is divided into two phases: WIA-PA gateway-based device interconnection and WIA-PA device address binding. In the former phase, the WIA-PA gateway performs resource discovery and communication transmission and realizes the connection between the OPC UA publisher/subscriber and the WIA-PA network. In the latter phase, the OPC UA publisher/subscriber and its corresponding broker middleware are virtualized as a virtual device in the WIA-PA application layer, which has a WIA-PA logical address and names the OPC UA pub/sub virtual device, as shown in The resource discovery process, which is shown in After resource discovery, the discovered OPC UA pub/sub data source should undergo resource mapping. This process entails mapping the OPC UA publisher/subscriber information model to a pub/sub configuration UAO, which is in the WIA-PA gateway. Each OPC UA pub/sub configuration UAO corresponds to a publisher/subscriber. Therefore, through the WIA-PA protocol, the WIA-PA field devices can directly address the desired OPC UA data source.The OPC UA publisher/subscriber information model is completely mapped to the UAO, and the payload becomes overly complex when WIA-PA devices communicate with the OPC UA servers. Therefore, in the design of the resource-mapping mechanism in this paper, the OPC UA pub/sub configuration information model is partially discarded to facilitate the data transmission of the WIA-PA field devices; only the necessary information models of these devices  are retained.Based on the simplified OPC UA pub/sub information model, we construct the communication parameters of the OPC UA pub/sub configuration UAO, which are divided into the public section, publisher section, and subscriber section.The public section contains the name, state, transport protocol, and connection address of the pub/sub connection, which are required by all publishers and subscribers.The publisher section includes the ID information of the publisher, writer group, and dataset writer; the data item and metadata of the published dataset; and the encoding format for message mapping.The subscriber section describes the publisher information subscribed by the OPC UA subscribers. To facilitate WIA-PA device communication, we add SubscriberID to identify the subscriber devices and DataSetReaderID to identify the dataset readers.The parameters of the OPC UA pub/sub configuration UAO are classified as static and dynamic. The static configuration parameters are the various identification information of the OPC UA publishers and subscribers, such as PublisherID and SubscriberID, which are only used within the OPC UA applications and have little relationship with the WIA-PA field devices. We choose static parameters maintained by the WIA-PA gateway. The dynamic configuration parameters mainly are the connection protocol, connection address, topic, pub/sub time interval, and OPC UA message format, which are needed by the field devices to pub/sub data to the OPC UA server and are configured by the field devices before transmission. In this way, the protocol complexity of combining WIA-PA with OPC UA is effectively reduced, and the WIA-PA field devices can execute a pub/sub transmission without fully understanding the parameters of the entire OPC UA pub/sub.OPC UA pub/sub configuration UAO in the gateway. After discovering and mapping the OPC UA publisher/subscriber resources, the WIA-PA gateway establishes the pub/sub configuration mirror within it, namely the OPC UA pub/sub configuration UAO.OPC UA pub/sub message UAO in the gateway. It is responsible for parsing and forwarding the data sent by the WIA-PA field device. Each OPC UA pub/sub message UAO corresponds to an OPC UA pub/sub configuration UAO.Pub/sub configuration UAO in the field device. In the WIA-PA/OPC UA joint pub/sub configuration process, through the pub/sub configuration UAO, the field device registers the pub/sub operation parameters with the OPC UA pub/sub configuration UAO in the gateway and obtains the address of the OPC UA pub/sub message UAO in the gateway. The OPC UA pub/sub configuration UAO in the gateway and the pub/sub configuration UAO in the field device jointly realize the WIA-PA/OPC UA joint pub/sub configuration process.Pub/sub message UAO in the field device. It publishes the data collected by the WIA-PA field device to the OPC UA pub/sub message UAO in the gateway or receives the data from the OPC UA pub/sub message UAO in the gateway. The OPC UA pub/sub message UAO in the gateway and the pub/sub message UAO in the field device jointly realize the WIA-PA/OPC UA joint pub/sub execution process.The functions of the different UAOs in The WIA-PA/OPC UA joint pub/sub configuration process can be divided into two parts: the pub/sub registration phase and the VCR establishment phase. Its time sequence flowchart is shown in In the pub/sub registration phase, the pub/sub configuration UAO in the WIA-PA field device uses the “write” method to write the dynamic configuration parameters to the pub/sub configuration UAO in the gateway, including the broker address and protocol, topic, time interval, and message format (UADP or JSON), and finally enables the publication or subscription.In the VCR establishment phase, the WIA-PA network manager allocates VCRs for the pub/sub message UAO in the WIA-PA field device and pub/sub message UAO in the gateway and configures the parameters, such as the UAO identifier and communication cycle.The pub/sub transmission process between the WIA-PA field device and OPC UA publisher/subscriber entirely relies on the WIA-PA and OPC UA pub/sub modes, as shown in In the publication of the data of the WIA-PA field device to the MQTT broker middleware, the WIA-PA gateway parses the received WIA-PA application layer protocol data and transmits them to the OPC UA publisher through the network cable. Next, the OPC UA publisher parses and enters the received data into the information space of the OPC UA publisher and then sends them to the MQTT broker middleware.In the subscription process of the data from the MQTT broker middleware by the WIA-PA field device, after receiving the data subscribed from the MQTT broker middleware, the OPC UA subscriber enters the data into its information space and forwards them to the WIA-PA gateway through the network cable. The WIA-PA gateway encapsulates the received data into WIA-PA protocol data and sends them to the WIA-PA field device.On the basis of the system architecture  and softIn the verification system, embedded devices with STM32L152 (16KB SRAM and 128KB Flash) microcontroller run the WIA-PA wireless node protocol stack. An embedded device with RT5350 SoC (32MB SDRAM and 8MB NAND Flash) runs the WIA-PA gateway program. The WIA-PA nodes and the WIA-PA gateway form a star topology with the time slotted channel hopping function and the retransmission function (maximum number of retransmissions is three) enabled. The OPC UA publisher, subscriber, and MQTT broker run on a computer running Ubuntu 16.04 (Linux 4.4.0). The WIA-PA gateway connects to the computer through the network cable. The OPC UA publisher and subscriber are implemented using the open62541 SDK . The MosThis section describes system testing, which is divided into functional testing and performance testing. Functional testing includes WIA-PA/OPC UA joint publishing testing and subscription testing. Performance testing includes the memory occupied, the communication success rate, and the publishing delay.In WIA-PA/OPC UA joint publishing, the WIA-PA field device publishes data to the MQTT broker. In WIA-PA/OPC UA joint subscription, the WIA-PA field device subscribes to data from the MQTT broker. The WIA-PA/OPC UA joint publishing process and subscription process are detailed below.WIA-PA/OPC UA joint publishing processThe WIA-PA field device sends data to the OPC UA pub/sub virtual device in the WIA-PA gateway. Then, the OPC UA pub/sub virtual device parses the received data and forwards them to the OPC UA publisher through the network cable. The OPC UA publisher stores the received data, which are the data to be published, in its address space. The dataset writer adds properties to the data to be published and encapsulates them into the publishing dataset. The dataset writer group adds a publisher ID to the publishing dataset and encapsulates it into a message packet. Finally, the writer group encapsulates multiple message packets into a published message packet and then sends this packet to the MQTT broker. The dataset writer, dataset writer group, and writer group are logical concepts in the OPC UA publisher.WIA-PA/OPC UA joint subscription processThe OPC UA subscriber obtains the publisher-related communication parameters from the WIA-PA field device, such as Publisher ID, DataSetWriterID, and DataSetWriterGroupID, and then connects to the MQTT broker, according to the topic of the subscription message. After the OPC UA subscriber receives the subscribed data, it forwards them to the OPC UA pub/sub virtual device in the gateway through the network cable. Finally, the OPC UA pub/sub virtual device encapsulates the data into WIA-PA protocol data and sends them to the WIA-PA field device.We built a laboratory-level experimental system to validate the WIA-PA/OPC UA joint pub/sub process. The experimental result shows that the proposed system can realize the WIA-PA/OPC UA joint pub/sub, supporting both the UADP and JSON message encoding formats and enabling the WIA-PA network to connect to the industrial Internet through OPC UA pub/sub.Memory occupancy testingMemory occupancy testing involves the sizes of the read-only memory (ROM) and random-access memory (RAM) consumed by the OPC UA publisher/subscriber application. The test results are shown in Communication success rate testingThe communication success rate of WIA-PA/OPC UA joint pub/sub in different pub/sub periods  is tested. The number of data packets sent by the WIA-PA field device or the MQTT broker is set to 10,000 in each period. In the joint publishing process, the success rate of publishing communication is the ratio of the number of messages received by the MQTT broker to the number of messages sent by the WIA-PA field device. Similarly, in the joint subscription process, the success rate of subscription communication is the ratio of the number of messages received by the WIA-PA field device to the number of messages sent by the MQTT broker. The test results are shown in Publishing delay testingThe joint publishing delay is defined as the time interval from when a message is received from the gateway’s wireless interface, after the gateway has processed the message and sent it to the OPC UA subscriber, to when the OPC UA subscriber receives this message. In cases with different numbers of field devices, we measure the publishing delay of the experimental system. The number of field devices is set to 1, 5, 10, 50, and 100. In each case, we count 100 sets of data and calculate the average. The results are shown in This paper addresses the problem of achieving interconnection and interoperability between IWSNs and the industrial Internet. Taking WIA-PA, a typical wireless technology in IWSNs, as an example, we propose an architecture of WIA-PA/OPC UA joint pub/sub. On the basis of the WIA-PA standard and OPC UA specification, we designed an OPC UA publisher/subscriber data source virtualization mechanism, the WIA-PA/OPC UA joint pub/sub transmission mechanism, and the corresponding configuration mechanism in the WIA-PA gateway. These mechanisms allow WIA-PA field devices and the OPC UA publisher/subscriber to communicate with each other. The WIA-PA gateway, which plays an important role in linking WIA-PA and OPC UA, was designed to ensure good coupling between WIA-PA and OPC UA and reliable data transmission. After simplifying the OPC UA pub/sub information model, we constructed UAOs for the OPC UA publisher/subscriber, which are used for parameter configuration and data transmission, according to the UAO design method provided by the WIA-PA standard. In addition, a laboratory-level WIA-PA/OPC UA joint pub/sub verification system was assembled to validate the proposed architecture. The experimental results of system execution showed that the proposed system architecture is feasible and performs well in terms of consumed memory, communication success rate, and publishing delay. It can meet most communication requirements of IWSNs.In this study, the system architecture has strong protocol consistency; it does not modify the protocols of WIA-PA and OPC UA. This study serves as a valuable reference for connecting other industrial wireless technologies to the industrial Internet through OPC UA and can inspire more researchers to further advance this field. Future outlooks related to the proposed system architecture include the combination of the aggregation and disaggregation functions of WIA-PA to realize WIA-PA aggregation data transmission. We will add TSN technology to improve the real-time performance and reliability of the system, and we will also test the whole system in a real industrial scenario.


Glyoxalase 2 is a mitochondrial and cytoplasmic protein belonging to the metallo-β-lactamase family encoded by the hydroxyacylglutathione hydrolase (HAGH) gene. This enzyme is the second enzyme of the glyoxalase system that is responsible for detoxification of the α-ketothaldehyde methylglyoxal in cells. The two enzymes glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) form the complete glyoxalase pathway, which utilizes glutathione as cofactor in eukaryotic cells. The importance of Glo2 is highlighted by its ubiquitous distribution in prokaryotic and eukaryotic organisms. Its function in the system has been well defined, but in recent years, additional roles are emerging, especially those related to oxidative stress. This review focuses on Glo2 by considering its genetics, molecular and structural properties, its involvement in post-translational modifications and its interaction with specific metabolic pathways. The purpose of this review is to focus attention on an enzyme that, from the most recent studies, appears to play a role in multiple regulatory pathways that may be important in certain diseases such as cancer or oxidative stress-related diseases. S-D-lactoylglutathione (SLG) from the hemithioacetal (MeCOCH(OH)-SG) formed spontaneously from MGO and GSH. Glo2 catalyzes the hydrolysis of SLG to D-lactic acid, regenerating the GSH consumed in the first reaction. The system, better known as the glyoxalase system, acts in parallel with the glycolytic pathway and serves mainly to eliminate MGO produced spontaneously from α-oxoaldehydes and in particular from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, both intermediates in glycolysis 2 or glutathionylspermidine ,123S-gluproteins . In our onylated . For exaonylated . Indeed,onylated , and ageonylated . Remodelonylated . Dalle Dcal GSSG ,134 and  F-actin . Other s F-actin . The degnditions ,138,139.e enzyme ,141. It ironment . It was ironment . On the residues . Indeed,g lysine . It has rocesses ,144,145.d lysine . James e studied . GSH at proteins . Glo2 isproteins .S-D-lactoylcysteine that rearranges to N-D-lactoylcysteine [S-glutathionylation, N-acetylation and lysine lactoylation) involved in the cell metabolic regulation.Another important post-translational modification in which Glo2 is indirectly involved is the lactoylation of lysines, which consists of a non-enzymatic acyl transfer of the SLG moiety lactate to the protein lysine residues . Gaffneycysteine . Thus, icysteine . Glo2 iscysteine . These acysteine . On the cysteine . On the cysteine . It is pcysteine , which dcysteine . In thisThe study of the signaling pathways regulating Glo2 expression as well as those driven by Glo2 to control specific biological responses dates back to 2006 when Xu and colleagues  demonstr2•−. The depletion of O2•− inhibits the Akt signaling pathway that, in turn, induces the upregulation of mitochondrial Glo2 expression. mGlo2 interacts with the proapoptotic protein Bax, activating apoptosis through the intrinsic pathway (cytochrome c (Cyt c) release from the mitochondrion, activation of Apaf-1, and eventually caspase-3 activation)  control to stimulate cell proliferation and elude apoptosis through a mechanism involving the p53–p21 axis . In the ivation) . Apart from these studies, additional Glo2 regulation by other signaling pathways has not been reported, which encourages research to further explore this interesting field.S-glutathionylated and that functional thiol groups play a critical role in microtubule polymerization. Specifically, a correlation between protein S-glutathionylation and microtubule depolymerization was shown [S-glutathionylation of microtubules (our manuscript in submission), so in their experimental system, the presence of SLG and Glo2 leads to S-glutathionylation of microtubules and inhibition of polymerization. In Norton et al.’s study, inhibitors of Glo2’s thioesterase activity were used, and it has never been investigated whether these substrates can also inhibit Glo2’s S-glutathionylation activity. It is therefore possible to assume two different possibilities: the first is that the same inhibitors were used by Glo2 to glutathionylate the microtubule proteins and thus block polymerization during the first few days of culture in neuronal cells; the second is that these inhibitors induced oxidative stress with GSSG formation, which likewise leads to S-glutathionylation of the proteins and blocking of microtubule polymerization, as shown by Carletti et al. [S-glutathionylation of specific target proteins, its enzymatic activity decreases, so it is possible to suggest that the decrease in Glo2 activity, when occurring at the same time as the increase in Glo1, can lead to a greater substrate for S-glutathionylation by Glo2. On the other hand, this situation is not always the case, because sometimes an increase in both enzymes has been demonstrated, e.g., during secretion processes, so in order to further investigate the regulation of the glyoxalase system, it would be appropriate to measure the ratio between the respective enzyme activities of the two enzymes. In conclusion, it can be claimed that there is probably an interaction between Glo2 and microtubules in terms of PTM, but more extensive studies must be carried out in order to investigate its possible role in microtubule polymerization and other regulative PTMs.In a number of non-recent studies, experiments were conducted showing the involvement of Glo2 in microtubule assembly . In one as shown . Taking i et al. . An impoi et al. . The sami et al. . Since ai et al.  and histi et al. . In lighS-glutathionylation on target proteins, and this aspect indicates a regulatory role for the enzyme that can be more activated for this function under specific pathological conditions. It is interesting to note that in mammalian cells, there is a cytosolic Glo2 and a mitochondrial Glo2 that naturally have different interactions and roles. It has been shown that SLG can enter mitochondria and provide the substrate for mitochondrial Glo2, and from the studies described above, it is clear that mitochondrial Glo2 can play multiple roles in regulating processes such as apoptosis. Indeed, it has been shown that Glo2 can interact with mitochondrial Bax, can limit the S-acetylation of lysine residues and can induce S-glutathionylation. The role of a PTM-specific protein opens up great regulatory possibilities at both the cytosolic and mitochondrial levels that are worth investigating and studying. Many studies on Glo2 have not continued and have never been investigated in depth mainly because the substrate is easily hydrolyzed, the enzyme is sensitive to external redox conditions and the anti-Glo2 antibody has been available for relatively few years. For example, one of our studies showed that there were specific interactions between cytosolic Glo2 and liposomes consisting of negatively charged phospholipids that influenced and inhibited its hydrolase activity, whereas this inhibition on mitochondrial Glo2 was not detected. The data were discussed in relation to a possible regulation of SLG at the cytosolic level such that it was allowed to accumulate and be directed to the mitochondria, which could utilize it via mGlo2, which was not inhibited [Although the glyoxalase system has been studied for more than a hundred years, it still has many unknown aspects. In particular, the enzymatic regulation of the two enzymes that make up the MGO pathway may be of fundamental importance in cell regulation under certain conditions, such as in cancer or in pathological conditions that induce oxidative stress. Glo2 is widely distributed in nature, and by using fluorescent antibodies, it is possible to detect with confocal microscopy a large presence of this enzyme in the cytosol, a presence that overlaps with the cytoskeleton, indicating a possible fundamental role in cells, although Glo2 knockout does not imply any detectable change on cultured cells . This lanhibited . Other s


Cannabis sativa (cannabis). The regulation of glandular trichome development has not been characterized in cannabis. We recently identified an R2R3-MYB transcription factor, CsMIXTA, which could be involved in trichome morphogenesis in cannabis. Some homologous genes of CsMIXTA are known to function in glandular trichome initiation in other plant species. CsMIXTA is highly expressed in flower tissue compared to vegetative tissues. Interestingly, CsMIXTA is also highly expressed in trichomes isolated from female flower tissue. In addition, CsMIXTA is upregulated during the peak stages of female flower maturation in correlation with some cannabinoid biosynthetic genes. Transient expression in Nicotiana benthamiana showed that CsMIXTA is localized in the nucleus. Furthermore, yeast transcriptional activation assay demonstrated that CsMIXTA has transactivation activity. Overexpression of CsMIXTA in Nicotiana tabacum resulted in higher trichome density, larger trichome size, and more branching on stalked glandular trichomes. The results indicate that CsMIXTA not only promotes glandular trichome initiation in epidermal cells, but also regulates trichome development in tobacco leaves. In this report, we characterized the novel function of the first cannabis transcription factor that may be critical for glandular trichome morphogenesis.Cannabinoids are synthesized in glandular stalked trichomes on the female flowers of  Glandular trichomes are epidermal outgrowths characterized by the presence of a secretory cavity or “gland”, which houses secretory gland cells capable of synthesizing secondary metabolites and exporting them into a secretory reservoir ,2. GlandCurrent models indicate that the regulation of secondary metabolite production is associated with the regulation of flowering and trichome development ,10. AfteA complex of transcription factors (TFs) in the R2R3-MYB and HD-ZIP IV families has been shown to regulate the development of glandular trichomes in other plant species ,12,13. TCsMIXTA, which could positively regulate cannabis CSG development. We showed that CsMIXTA was highly expressed in flowers; CsMIXTA also showed higher expression in trichome cells isolated from female flower tissue. Ectopic expression of CsMIXTA in tobacco not only significantly increased glandular trichome size and density but also promoted trichome branching on tobacco leaves. Our study suggests that CsMIXTA is a TF that could potentially control glandular trichome initiation and development in female cannabis flowers.In cannabis, the genetic regulation of glandular trichome initiation and development is still unknown. In this study, we identified a novel R2R3-MYB TF in cannabis, Cannabis sativa. We named the cannabis gene CsMIXTA. We first determined CsMIXTA expression in different tissues and during female flower development in a hemp variety, Cherry Wine (CW). CsMIXTA is highly expressed in flower tissues compared to the vegetative tissues  and Olivetolic Acid Cyclase (OAC)) required to generate the substrates  necessary for the synthesis of the CBG, the first cannabinoid in the biosynthetic pathway. This analysis indicated that during flower development, CsMIXTA expression was also positively correlated with cannabinoid levels and expression of Cannabichromenic Acid Synthase (CBCAS) which generates the end product cannabinoid CBCa , like cannabis, has glandular stalked trichomes but in the case of tobacco, GSTs are abundant on the leaves [CsMIXTA (controlled by a constitutive (35S CaMV) promoter) in tobacco plants to evaluate if this cannabis TF could regulate glandular trichome initiation/development on tobacco leaves.Our work monitoring recently ,17,18, be leaves . We ectoCsMIXTA overexpressing tobacco line (cco line .CsMIXTA expression in L1 (We further examined trichome morphology on tobacco leaves using SEM. Two transgenic lines (L1 and L3) were evaluated in this experiment . Both lion in L1 . Close-uon in L1 E,F and ton in L1 D. Floralon in L1 . Anecdoton in L1 .CsMIXTA plays an essential role in glandular trichome initiation and development. Our findings provide evidence that CsMIXTA also functions in CSG cells to modulate trichome growth and development, consistent with the higher CsMIXTA expression detected in isolated female flower glandular trichomes  [GPPS, monoterpenes. Sessile  glandular trichomes produce minimal monoterpenes or cannabinoids before maturation, possessing a sesquiterpene-dominant chemotype. However, after the sessile trichomes develop into CSGs, cannabinoid and monoterpene biosynthesis takes place. Previous studies have shown that GPPS was more highly expressed in stalked trichomes, but OAC expression was similar among different glandular trichome types [There is a strong positive correlation between ctively)  for bothme types .CsMIXTA in tobacco has shown a significant impact on trichome development with increased density, enlarged size and more branching . Cuttings were allowed to root for 3 weeks before transplant. All plants were grown in #600 standard nursery pots filled with Promix-BX25  soilless medium and Osmocote 15-9-12 . Plants were grown under 16 h light/8 h dark conditions with supplemental lighting from high-pressure sodium lighting. The light intensity from the high-pressure sodium lights is 568 µmol mDifferent tissue samples were collected in the eighth week of flowering. Amounts totaling 100 mg of plant tissues were collected and immediately frozen in liquid nitrogen. The NucleoSpin Plant and Fungi RNA Isolation Kit (Macherey-Nagel) was used for RNA isolation according to manufacturer’s manual. cDNA was synthesized from 1 µg RNA using the iScript Reverse Transcriptase Master Mix (BioRad).Trichome isolation was performed following the protocol developed by Livingston et al. (2020)  with sliCsUbiquitin was used as the internal reference [CsMIXTA Primers were forward 5′-TCCATGCTTTACTAGGCAACAG-3′, reverse 5′-CCACCGTCTTGTTGAGAGAG-3′. Experiments were performed with four biological replicates.qPCR analysis was performed using Bio-Rad CFX. iTaq Universal Sybr Green Master Mix was used (Bio-Rad). For all qPCR reactions, eference . The CsMCsMIXTA was cloned out of cDNA using iProof HF Master Mix (Bio-Rad). The primers for cloning were forward 5′-CAGTCGACTGGATCCGGTACCATGGGTCGGTCACCATGCTG-3′, reverse 5′-GAAAGCTGGGTCTAGATATCTCGAGAACATAGGAGAATCTG-3′. NEBuilder HiFi DNA Assembly kit (New England Biolabs) was used for the cloning of CsMIXTA into KpnI and XhoI digested pENTR-3C vector . LR reaction was then performed to clone CsMIXTA into the pB7YWG2 binary vector with a 35S CaMV promoter using LR Clonase II (Invitrogen), as well as the pAS2 and pUBC vectors.N. tabacum (tobacco) seeds were acquired from Dr. Yi Li at the University of Connecticut. Seeds were sterilized using 3% bleach and 70% ethanol and planted in sterile magenta boxes containing 50 mL full strength Murashige and Skoog (MS) salts , MES (adjusted to pH 5.7 with Tris), 1% sucrose, and 1% agar. Seedlings were grown under fluorescent lighting in a growth chamber at 28 °C with a 16-h photoperiod. Leaves from 3-week-old sterile tobacco plants were used for transformation by Agrobacterium tumefacience GV3101 harboring pB7YWG2-CsMIXTA. Tobacco leaf disc transformation was performed as described previously . Rooted transformants were transferred to Promix-BX amended with 12 g/gal Osmocote 15-9-12 and maintained in the greenhouse with 16 h photoperiod.CsMIXTA was fused with the GAL4-BD domain in the pAS2 vector using LR Clonase II (Invitrogen). The resulting plasmid was transformed into yeast strain AH109  using the Frozen-EZ Yeast Transformation II Kit . A known Arabidopsis TF, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1) (provided by Dr. Huanzhong Wang), was used as a positive control and empty vector as a negative control. The transformed cells were plated on synthetic defined (SD) media to select positive transformants. A 4 μL amount of yeast culture harboring corresponding constructs was dropped onto a solid SD medium with or without histidine to check for activation of reporter genes. Yeast growth was observed and documented after cultivation at 28 °C for 2 days.CsMIXTA was fused to YFP using a ubiquitin promoter-containing plasmid (pUBC-YFP) and LR Clonase II (Invitrogen), and the resulting construct was transformed into Agrobacterium tumefacience GV3101. Transient transfection protocol was performed as described by Espinoza-Patharkar et al., modified from [ied from .2O). These samples were then transferred to a 70% ethanol solution twice, and then dehydrated through a graded ethanol series. Samples were collected and placed into stainless steel containers for critical point drying while completely submerged in 100% ethanol. Dehydrated and dried samples were mounted onto SEM stubs using double-sided carbon tape, and sputter coated with gold nanoparticles. The samples were mounted into a Nova Nanosem 450 and imaged.Squares of tobacco leaf tissue 3 mm in size were excised with midribs and the veins were removed. These squares were fixed for 24 h in formaldehyde-acetic acid-ethanol  as well as a red filter (700 nm). Z-stacks were collected at a step size of 27 microns. Composite channel/stack images/scale bars were produced in ImageJ. Trichomes were counted in photographs using “cell counter” in ImageJ.CsMIXTA in tobacco resulted in an increase in trichome density, size, and branching on the leaves of multiple transgenic lines, demonstrating that CsMIXTA is involved in trichome initiation and development. In cannabis, CsMIXTA is strongly expressed in inflorescence tissue and has an expression pattern which coordinates with key cannabinoid biosynthesis genes during female flower development. Through analyzing cannabis gene expression data, we established an association between the expression of CsMIXTA and cannabinoid biosynthetic genes as well as flower/trichome development. CsMIXTA is also highly expressed in the isolated trichome, suggesting a function within the trichome. CsMIXTA expression has a significantly positive correlation with the expression of GPPS and OAC: two key enzymes in the cannabinoid biosynthesis pathway. These data suggest that CsMIXTA not only regulates glandular trichome morphogenesis, but also cannabinoid and/or cutin biosynthesis. Together, these data create a strong case to support the hypothesis that CsMIXTA is a TF involved in the coordination of CSG development in female cannabis flowers; increasing CsMIXTA expression in cannabis could potentially enhance cannabinoid production in female flowers.Overexpression of the cannabis TF A provisional patent application has been submitted based on the findings in this report.