SNCA gene, which encodes α-Syn, cause familial forms of PD and are the basis of sporadic PD risk. Given the role of the α-Syn protein in the pathology of PD, animal models that reflect the dopaminergic neuronal loss and the widespread and progressive formation of α-Syn aggregates in different areas of the brain constitute a valuable tool. Indeed, animal models of PD are important for understanding the molecular mechanisms of the disease and might contribute to the development and validation of new therapies. In the absence of animal models that faithfully reproduce human PD, in recent years, numerous animal models of PD based on α-Syn have been generated. In this review, we summarize the main features of the α-Syn pre-formed fibrils (PFFs) model and recombinant adeno-associated virus vector (rAAV) mediated α-Syn overexpression models, providing a detailed comparative analysis of both models. Here, we discuss how each model has contributed to our understanding of PD pathology and the advantages and weakness of each of them.Alpha-synuclein (α-Syn) is a key protein involved in Parkinson's disease (PD) pathology. PD is characterized by the loss of dopaminergic neuronal cells in the substantia nigra pars compacta and the abnormal accumulation and aggregation of α-Syn in the form of Lewy bodies and Lewy neurites. More precisely, the aggregation of α-Syn is associated with the dysfunctionality and degeneration of neurons in PD. Moreover, mutations in the Here, we show that injection of α-Syn PFFs and overexpression of α-Syn mediated by rAAV lead to a different pattern of PD pathology in rodents. First, α-Syn PFFs models trigger the Lewy body-like inclusions formation in brain regions directly interconnected with the injection site, suggesting that there is an inter-neuronal transmission of the α-Syn pathology. In contrast, rAAV-mediated α-Syn overexpression in the brain limits the α-Syn aggregates within the transduced neurons. Second, phosphorylated α-Syn inclusions obtained with rAAV are predominantly nuclear with a punctate appearance that becomes diffuse along the neuronal fibers, whereas α-Syn PFFs models lead to the formation of cytoplasmic aggregates of phosphorylated α-Syn reminiscent of Lewy bodies and Lewy neurites. Parkinson's Disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Currently, PD affects 1%–2% of people over the age of 60 years, rising to 4% at age 80 years . PD is mThe neuropathological mechanisms underlying non-motor symptoms of PD are still poorly understood, but growing evidence suggests that the evolution of these symptoms may arise from the disruption of both dopaminergic and non-dopaminergic systems, and the involvement of diverse structures outside the nigrostriatal system . BesidesThe neuropathological hallmark of PD is the abnormal accumulation and aggregation of alpha synuclein protein (α-Syn) in form of Lewy bodies and Lewy neurites . It is wParkin gene, and some of those with the LRRK2 G2019S mutation, show neuronal degeneration but do not develop Lewy bodies  polyathic PD ; and 3) athic PD .Under normal conditions, native α-Syn exists in a dynamic equilibrium between unfolded monomers and α-helically folded tetramers with a low propensity to aggregation . The decα-Syn undergoes various post-translational modifications, such as phosphorylation, truncation, ubiquitination, and nitration. Phosphorylation of α-Syn at the serine 129 residue is one of the major pathological markers of PD; 90% of α-Syn is phosphorylated in the brain of patients with PD while only 4% of α-Syn is phosphorylated in healthy brains . HoweverIn vivo amperometry recordings in rodents injected with α-Syn PFFs in combination with AAV-mediated overexpression of α-Syn reveal a reduction in dopamine release and reuptake rates in the striatum  the ability of propagation of α-Syn protein. These models can be classified as (a) neural stem cell transplantation into transgenic mice expressing human α-Syn  adman α-Syn  or α-Synan α-Syn , and (c)an α-Syn . Among tAlthough many studies support the prion-like nature of α-Syn and show that its propagation determines the temporal course of the disease, some observations have recently challenged this theory. The Braak staging is neither a proof nor an argument of the spreading hypothesis, it might be rather that certain subsets of neurons are affected by Lewy bodies much earlier than others and that, therefore, the intraneuronal lesions evolve sequentially . AnotherAnother crucial question that is not clear is whether the spreading of α-Syn is a driving factor for neuronal degeneration and progression of PD, or if it is an epiphenomenon that appears as a result of other alterations, such as lysosomal dysfunction . DespiteGBA1 gene are the main genetic risk factor for PD. The GBA1 gene encodes for GCase1, a lysosomal enzyme responsible for degrading the lipid glucosylceramide into ceramide and glucose , whereas others correspond to proteins that are involved in the transport to the lysosome (e.g. VPS35), mitophagy , or other autophagic-related functions , giving special interest to the differences observed between these two models. These observations are based on results obtained by our group and which are in line with the findings of previous studies.in vitro from recombinant α-Syn monomers. Subsequently, the aggregation of monomers into fibrils is induced and the fibrils are sonicated to generate short fibrils which after injection, trigger the aggregation, hyperphosphorylation and ubiquitination of endogenous α-Syn  or brain extracts containing Lewy bodies and α-Syn derived from PD patients or transgenic mice exhibiting α-Syn pathology. PFFs are generated us α-Syn . Intraceus α-Syn  and ratsus α-Syn  and non-us α-Syn . PFFs haus α-Syn , the subus α-Syn  and the us α-Syn . Similarus α-Syn . Most ofus α-Syn . It is wus α-Syn . Some stus α-Syn , and theus α-Syn . Our grous α-Syn Figure 1Also, these models have shown that pathological α-Syn can spread from the injection site to other anatomically interconnected brain regions following a fibrillary retrograde transport Figure 4Snca (i.e. α-Syn KO mice) does not lead to the formation of α-Syn inclusions or degeneration; an observation which supports that endogenous α-Syn is required for the development of the pathology , the sonicated α-Syn PFFs are heterogenous in nature and have different conformational features and biological effects . Rodents injected with different PFFs strains manifest different pathological severity and behavioral phenotypes, reflecting the great variability of the results, especially among different research studies . rAAV are an efficient vehicle for gene delivery in the brain area of interest and offer some characteristics that favor their use in modeling PD. rAAV efficiently transduce various cell types, confer long-lasting transgene expression, and can transduce dividing and non-dividing cells in the absence of an immune reaction . Given tOverexpression of wild type α-Syn or PD-associated mutants (A53T or A30P α-Syn) utilizing rAAV leads to a progressive loss of dopaminergic neurons in the SNc, a loss of dopamine terminals in the striatum , and a rIn the rAAV-α-Syn model, the presence of pα-Syn inclusions in the nigrostriatal system is concomitant with a significant loss of nigral dopaminergic neurons and the reduction in tyrosine hydroxylase immunoreactivity in the striatum. Overexpression of wild type or A53T human α-Syn induces a progressive loss of dopaminergic neurons in the SN over time . At 5 daSome studies show that rAAV-α-Syn expression causes the development of motor alterations, such as an increased apomorphine or amphetamine-induced rotation, defects in the stepping test or increased forepaw asymmetry in the cylinder test . These mIn the rAAV-α-Syn models, the transgene expression is dependent on the serotype, the promoter, the injection site, and the titer of rAAV. The serotype rAAV2 is the most extensively used to date, probably because its production and purification methods are well-established . HoweverThe efficiency of transgene expression is also determined by the design of the vector construct, and the promoter used to control the transgene expression. The most common promoters used are hybrid cytomegalovirus (CMV), chicken β-actin (CBA), phosphoglycerate kinase (PGK), and human synapsin I (Syn-1). These promoters provide high levels of transgene expression. In addition, some studies use post-transcriptional regulatory elements to improve the transgene expression, such as woodchuck hepatitis virus posttranscriptional regulatory element or polyadenylation sequence .SNCA multiplications  exacerbates the neurodegeneration and neuroinflammation induced by overexpression of A53T human α-Syn, reflecting the importance of genetic background , which facilitates the identification of new therapeutic targets . These mMore than two decades ago, α-Syn was identified as the main component of Lewy bodies. Since then, this protein has become established as a possible diagnostic biomarker in PD and therapeutic target. Also, numerous animal models have used this protein in attempts to reproduce PD. Each animal model offers specific aspects of the pathology of human PD, although none of them reproduce all the defining pathological and clinical features of the disease. The lack of comparable phenotypes between rodents overexpressing α-Syn or rodents injected with toxic α-Syn species reflects the difficulty of reproducing PD in animal models. Therefore, a thorough knowledge of the key features of these models is essential to choose the model that best suits the scientific questions that we want to solve. Here, we show that α-Syn PFFs models are suitable for studying the prion-like behavior of α-Syn and its propagation through the brain. While viral α-Syn overexpression models are especially useful to determine the mechanisms of α-Syn-induced toxicity but do not allow the study of their prion-like behavior. Despite these differences, both models are valuable tools for identifying novel therapeutic targets and the design and evaluation of potential therapies aimed at reducing the aggregation of α-Syn to alter disease progression.All experimental procedures were approved by Cajal Institute´s Bioethics Committee in accordance with the guidelines of the European Union Council Directive (DC86/609/CEE).RM conceptualized, arranged the review, and provided funding. MG-B generated the first draft, performed the experiments, and provided the figures. NG performed the experiments and supervised and approved the figures. PG-S contributed with the experiments and provided critical feedback. AM synthetized the α-Syn PFFs and provided mice injected with PFFs, and MD provided antibodies and tools for the experiments.This work was supported by grants from the Spanish Ministries of Science and Innovation (SAF2016-78207-R) and of Health, Consumption and Social Welfare, Instituto de Salud Carlos III (ISCIII), Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas [CIBERNED] (CB06/05/00559 and PNSD2016I033) and from Ramón Areces Foundation (172275) and from Europan Union´s Horizon 2020, research and innovation program, AND-PD project, grant agreement n° 848002 to RM and from the ERA-Net Euronanomed II JTC2015-DiaSyn project through the Spanish Ministries of Innovation, Science and Universities (PCIN-2015-98) to RM and the Walloon Region  to AM and MD (project N° n°1510352). MG-B was supported by a fellowship JAE-Intro 2019 from Consejo Superior de Investigaciones Científicas. The authors acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).AM is working as UCB Biopharma employee.The remaining 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.


The aim of this systematic review was to analyze the types of human chewing simulator described in scientific literature. in vitro”; “dental materials”; “shear strength”; “fatigue fracture”; “bite force”; “prosthetic materials”; “chewing simulator”; “chewing machine”; “simulated mastication”; and “dental wear simulator.” Two researchers worked independently to assess the titles and abstracts of the articles. The quality of the in vitro trials selected was evaluated by means of the Consolidated Standards of Reporting Trials scale. An electronic search was conducted in the databases PubMed, Embase and Scopus. The search strategy included 10 search terms: “The electronic search identified 80 articles related to the topic of interest. After reading the full texts, ten works were selected. The articles focused mainly on the design of chewing simulators. Most of them were considered of moderate quality. Regarding the characteristics that an ideal chewing simulator should encompass, the devices described in articles varied greatly in terms of movement, periodontal ligament simulation, force sensors, and the materials tested. No chewing simulator offers all the characteristics necessary to reproduce human masticatory movements and forces under the humidity and pH conditions of the oral cavity. A simulator that encompasses all these characteristics would make it possible to standardize trials involving simulated mastication. Key words:In vitro, dental materials, dental wear simulator. The mechanical properties of the materials used in dentistry must be thoroughly tested under laboratory conditions before they can enter clinical use. Trials of mechanical resistance  must be complemented by wear and fatigue testing.Wear means “to damage, erode, or destroy by friction or use,” while fatigue means “weakness in metal or other materials caused by repeated variations of stress.” In dentistry the usual terms used to describe these scenarios are aging and fatigue  has established a series of directives for good laboratory practice. But to date, no single standardized system has been determined for measuring wear and aging, as is the case when measuring traction and fracture resistance. In the currently available chewing simulators, variability in terms of the control and regulation of forces impacts negatively on the reproducibility and variability of results, and the difficulty of extrapolating l cavity .The aim of the present systematic review was to analyze the types of chewing simulator described in scientific literature.in vitro studies.This systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . It coulin vitro mastication; C = analysis; and O = human mastication.The review’s PICO  question was: Which chewing simulator most resembles human mastication, in which P= chewing simulators; I = in vitro wear resulting from the action of chewing simulators. Inclusion criteria were: chewing device design was described, and test samples were described (teeth studied/antagonist teeth). These criteria were chosen to center the review on articles involving chewing simulation and oral conditions.The search identified articles that analyzed in vitro”; “dental materials”; “shear strength”; “fatigue fracture”; “bite force”; “prosthetic materials”; “chewing simulator”; “chewing machine”; “simulated mastication”; and “dental wear simulator”. Boolean operators (“OR” and “AND”) were applied to link search terms to the research question  to assess the titles and abstracts of the articles identified in the initial search. The following variables were recorded for each article: author and year of publication, device, study teeth/antagonist teeth, simulated mastication movement, force sensors.in vitro studies included in analysis, their quality was evaluated by means of the Consolidated Standards of Reporting Trials (CONSORT) scale, modified to assess the quality of in vitro trials of dental materials  of load sensors for measuring the forces exerted. Seven articles stated the force applied. Conserva et al.  used a b , made from materials that exhibit resilient rather than rigid behavior. In this context, silicon and acrylic resin have been used to imitate periodontal ligament and alveolar bone, respectively .The simulator must be able to generate movements that imitate human mastication as far as a machine is able to. In mastication, the mandible moves in vertical and horizontal direction, making a bidirectional motion ,17. HoweIf wear testing is to be standardized and obtain comparable results, it is essential for the magnitude of forces generated by chewing simulators to be controlled to create specific test conditions, and for these to be identical for each specimen tested. For this reason, the simulator must be equipped with load sensors to record the loads applied, as in some of the studies reviewed ,8,11-14.In vitro studies may use human saliva or de-ionized water, among other liquids, as a lubricant or even as an abrasive/erosive medium (To simulate conditions in the oral medium, some studies have attended to environmental factors such as humidity/wetness, temperature and pH, which can also influence the mechanical properties and behavior of dental materials in the mouth. The body acts as a heat pump that maintains the temperature in the oral cavity at 37ºC. Teeth and any restoration material are continuously bathed in saliva with a pH of around 7. Humidity in the cavity is 100%. However, the introduction of different foods in the mouth can alter environmental conditions to a large degree, causing fluctuations in pH and temperature. An artificial oral environment should be able to reproduce normal conditions in the oral cavity, and to manage the temperature and pH fluctuations that may occur. e medium ,13,15,18et al. (There are few bibliographic references regarding the design of models for use in chewing simulators for trials involving simulated mastication. Several parameters have not been sufficiently investigated, including the curve of Spee and the curve of Wilson. Alemzadeh et al.  analyzedin vitro study that was the subject of the review. The search did not include the Cochrane database as it does not admit in vitro research. The quality of most of the studies was moderate as it is hard for this type of in vitro research to meet the criteria that would deem it of high quality.The present systematic review suffered certain limitations. Despite conducting a search in the databases, only a small number of relevant articles were identified, which might be explained by the difficulties involved in carrying out the type of The present systematic review did not identify any chewing simulator that includes all the characteristics that would make it possible for complete upper and lower dental arches to reproduce the movements of human mastication under the actual humidity and pH conditions in the oral cavity. There is need for a simulator that meets all these requirements in order to standardize future trials involving simulated mastication.


Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity. Mammals possess two homologs of the leucine-rich repeat protein kinase termed LRRK1 and LRRK2 . Both enDespite overall domain similarity, LRRK1 and LRRK2 appear to possess distinct physiological functions. Genetic evidence in humans, points towards LRRK1 regulating bone biology and LRRK2 contributing to Parkinson's disease. Autosomal recessive variants that induce frameshift or truncating mutations within the C-terminal domain of LRRK1, that are likely loss of function, cause a severe metabolic bone disorder termed osteosclerotic metaphyseal dysplasia (OSMD) . This isLRRK2 phosphorylates a subgroup of Rab proteins  at a conserved Thr/Ser residue (Thr73 for Rab10), located within the effector binding switch-II motif . As mentCo-immunoprecipitation studies revealed that LRRK1 interacts with numerous components of the EGF Receptor signaling pathway . EGF stiOther work has suggested that LRRK1 is recruited to the autolysosome during autophagy, where it activates the Rab7 GTPase-activating protein termed TBC1D2, thereby switching off Rab7A function . LRRK1 h32P]ATP was purchased from PerkinElmer. HG-10-102-01, MLi-2, GSK2578215A, LRRK2-IN1, MRT67307, GDC0941, AG1478, BID1870, H-89, PD0325901, UO1216, Rapamycin and Phos-tag acrylamide were all synthesized by Natalia Shpiro (University of Dundee). Phos-tag acrylamide stocks are stored in aliquots at −80°C and working stocks are stored for a few weeks at 5 mM aqueous solution at 4°C in opaque tubes given the photo-sensitive nature of this reagent. iN04 was purchased from ChemBridge Corporation (#7989904). Full-length recombinant human LRRK2[G2019S] was purchased from Invitrogen (#A15202). GZD-824 was purchased from Cayman Chemical (#21508) and calyculin A (#ab141784) was purchased from Abcam, Inc. Phorbol 12-myristate 13-acetate (PMA) (#P8139), interleukin-1A (#I2778), oligomycin (#75351), antimycin A (#A8674), AICAR (#A9978) and forskolin (#F6886) were all purchased from Sigma–Aldrich. Human EGF (#8916), and human IGF-1 (#3093) were purchased from cell signaling technology (CST). PKC-specific inhibitor GO6983 was purchased from Selleckchem (#S2911). ON-TARGETplus SMARTpool human LRRK1 (#L-005320-00-0005), human LRRK2 (#L-049666-00-0005) and non-targeting control pool (#D-001810-10-20) siRNA were purchased from Horizon Discovery. (DU24813), HA-Rab29 (DU50222), GFP-Empty (DU44062), GFP-Rab7A wt (DU24785), GFP-Rab7A[S72A] (DU24814), GFP-LRRK2 wt (DU48062), GFP-LRRK2[D2017A] (DU48063), GFP-LRRK1 wt (DU30382), GFP-LRRK1[D1409A] (DU67084), GFP-LRRK1 [K746G] (DU67083), GFP-LRRK1[Y971F] (DU67289), GFP-LRRK1[K746G + Y971F] (DU66249), GFP-LRRK1[F1022C] (DU67082), GFP-LRRK1[G1411S] (DU67099), GFP-LRRK1[E929*] (DU66390), GFP-LRRK1[E929K] (DU66384), GFP-LRRK1[E929Q] (DU66391), GFP-LRRK1[Y971C] (DU66385), GFP-LRRK1[I1412F] (DU66395), GFP-LRRK1[E1980A-FS] (DU68320), FLAG-LRRK1[D1409A] (DU17400), FLAG-LRRK1[K746G] (DU67104), GFP-RILP (DU27496), HA-PPM1H wt (DU62789), HA-PPM1H[H153D] (DU62928), HA-PPM1J wt (DU68077), HA-PPM1J[H160D] (DU68170), HA-PPM1M wt (DU68124), and HA-PPM1M[H127D] (DU68165). These are available from MRC PPU Reagents and Services . The rabbits were immunized with peptides C-Ahx-AGQERFQS*LGVAFYR-amide and Ac-AGQERFQS*LGVAFYR-Ahx-C. Three subcutaneous injections were performed using the KLH immunogens, followed by two subcutaneous injections using the ovalbumin immunogens. At the time of each injection, an immunogen aliquot was thawed and combined with Freund's Complete  Adjuvant or Freund's incomplete Adjuvant (for the subsequent injections). Serum bleeds were obtained after the fourth and fifth immunizations. Antibodies were affinity-purified using the phosphorylated epitopes as a positive selection and also passed through a column containing the dephosphorylated peptide as a negative selection step. The affinity-purified antibodies were evaluated in parallel with serum. The subsequent rabbit polyclonal antibodies were diluted in 5% (by mass) BSA  in TBST Buffer  Tween-20) and used for immunoblotting analysis at a final concentration of 1 µg/ml. The antibody was generated by The Michael J. Fox Foundation's research tools program in partnership with Abcam. Development of a monoclonal antibody is underway. Please contact https://mrcppureagents.dundee.ac.uk/). anti-LRRK1 was produced by immunization with C-terminal LRRK1 protein  followed by three further injections 28 days apart, with bleeds performed seven days after each injection. Antibodies were affinity-purified from serum using LRRK1 antigen. For immunoblotting analysis, all sheep polyclonal antibodies were diluted in 5% (by mass) milk in TBST buffer. Rabbit monoclonal phospho-Ser935 LRRK2 (UDD2) was expressed and purified at University of Dundee as described previously  (Novex) and heated at 95°C for 5 min. For normal SDS–PAGE, 10–20 µg samples were loaded on to 4–12% Bis-tris gradient gels and electrophoresed at 180 V for 90 min. Proteins were transferred onto nitrocellulose membranes at 90 V for 90 min on ice in transfer buffer . Transferred membranes were blocked with 5% (by mass) milk in TBST Buffer at room temperature for 60 min. Membranes were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After washing in TBST, membranes were incubated at room temperature for 1 h with near-infrared fluorescent IRDye antibodies (LI-COR) diluted 1 : 10 000 in TBST and developed using the LI-COR Odyssey CLx Western Blot imaging system and signal quantified using the Image Studio software2 before loading. Gels for Phos-tag SDS–PAGE consisted of a stacking gel  and a separating gel . An amount of 10–30 µg of samples were loaded and electrophoresed at 70 V for 30 min and at 120 V for 2 h in running buffer . Gels were washed in transfer buffer containing 10 mM EDTA and 0.05% (by mass) SDS three times for 10 min, followed by one wash in transfer buffer containing 0.05% (by mass) SDS for 10 min. Proteins were transferred onto nitrocellulose membranes  at 100 V for 180 min on ice in the transfer buffer without SDS/EDTA. Subsequent immunoblotting was performed as previously described.Phos-tag SDS–PAGE was carried out as described previously . Samples2HPO4, 1.8 KH2PO4, pH 7.4) for 1 h at room temperature followed with three times for 15 min washes with 0.2% (by mass) BSA in PBS . Then slides were incubated with Alexa Fluor® 405 goat anti-mouse secondary antibody (Invitrogen A-31553) diluted 1 : 500 and HCS CellMask™ Deep Red Stain  at 0.5 µg/ml dilution in 1% (by mass) BSA in PBS for 1 h. Slides were washed three times for 15 min with 0.2% (by mass) BSA in PBS and rinsed with distilled water just before mounting. VECTASHIELD® HardSet™ Antifade Mounting Medium  was used to mount the coverslips on glass slides . Slides were then imaged using Leica TCS SP8 MP Multiphoton Microscope using a 40× oil immersion lens choosing the optimal imaging resolution with 1-pixel size of 63.3 nm × 63.3 nm.For analysis of Rab29 co-localization with LRRK1 and LRRK2, HeLa cells were seeded in six-well plates on glass coverslips . Cells were transfected using Polyethylenimine method  employinE. coli transformed with plasmids encoding for the expression of His-SUMO-Rab7A[wt] (Plasmid DU24781), His-SUMO-Rab7A[S72A] (DU24808) or His-SUMO-Rab10[wt] (Plasmid DU51062). Bacteria were cultured at 37°C to an OD600 of 0.4–0.6, then temperature reduced to 15°C and protein expression was induced by addition of Isopropyl β-d-1-thiogalactopyranoside at 250 µM. Cells were cultured for 16 h before harvesting by centrifugation at 4200 × g for 20 min at 4°C. The pellet was resuspended in 200 ml ice cold E. coli lysis buffer . Cells were lysed using a cell disruptor (passing sample through twice) and extracts clarified by centrifugation at 15 000 rpm for 20 min at 4°C. Subsequent supernatants were run on 15 ml of Cobalt-Agarose resin , previously equilibrated in E. coli lysis buffer, at 4°C for 90 min. The column was then washed with 20 column volumes of High Salt Wash Buffer  until no unbound protein was present in the flow-through. The column was then washed with five column volumes of Low Salt Wash buffer . His-SENP1 (DU39129) (1 : 30 dilution) was then added to the column overnight at 4°C to cleave the His-SUMO tag. Cleaved protein was collected and subjected to gel filtration on a Superdex 75 Hi load column, previously equilibrated in Equilibration buffer . An amount of 0.2 ml fractions were collected at a flow rate of 0.2 ml/min, with protein-containing fractions pooled, subjected to spin concentration using an Amicon Ultra 15 10 KDa and finally snap frozen.4 × 500 ml of Lysogeny broth containing 100 µg/ml ampicillin antibiotic was inoculated with a single colony of BL21-CodonPlus(DE3)-RIPL strain of 06 cells/ml in Lonza Insect-XPRESS medium) were infected with high-titre Baculovirus suspension. After 66 h of incubation (27°C and 90 rpm), cells were harvested by centrifugation. The expressed protein construct contained an N-terminal His6-Z-tag, cleavable with TEV protease. For LRRK1 purification, the pelleted Sf9 cells were washed with PBS, re-suspended in lysis buffer  glycerol) and lysed by sonication. The lysate was cleared by centrifugation and loaded onto a Ni NTA column. After vigorous rinsing with lysis buffer the His6-Z-tagged protein was eluted in lysis buffer containing 300 mM imidazole. Immediately thereafter, the eluate was diluted with buffer containing no NaCl, to reduce NaCl to 250 mM and loaded onto an SP-Sepharose column. His6-Z-TEV-LRRK1 was eluted with a 250 mM to 2.5 M NaCl gradient and treated with TEV protease overnight to cleave the His6-Z-tag. Contaminating proteins, the cleaved tag, uncleaved protein and TEV protease were removed by another combined SP-Sepharose Ni NTA step. Finally, LRRK1 was concentrated and subjected to gel filtration in storage buffer  using an AKTA Xpress system combined with an S200 gel filtration column. The final yield as calculated from UV absorbance was 0.1 mg/L.The DNA coding for the human LRRK1 residues 28 to 2015 (OHu72031 from Genscript) was PCR-amplified using the forward primer TACTTCCAATCCATGGAGACGCTTAACGGTGCCGGGGAC and the reverse primer TATCCACCTTTACTGCTTTACCTTCTCTTGCGAGTGCAAGC. The T4 polymerase-treated amplicon was inserted into the transfer vector pFB-6HZB (SGC) by ligation-independent cloning. The resulting plasmid was utilized for the generation of recombinant Baculoviruses according to the Bac-to-Bac expression system protocol (Invitrogen). Exponentially growing Sf9 cells (2 × 102 and 1 mM ATP. The kinase reactions were carried out at 30°C for the indicated times and reactions terminated by the addition of 4× SDS–PAGE sample buffer containing 1% (by vol) 2-mercaptoethanol. Twenty percent of total reaction volume was used for Phos-tag Coomassie staining analysis; <10% was used for conventional immunoblot analysis.Kinase assays were set in a 20 µl final reaction mixture containing purified Rab proteins , recombinant LRRK1  or full-length LRRK2[G2019S]  and 50 mM Tris–HCl pH 7.5, 0.1 mM EGTA, 10 mM MgCl32P]ATP (specific activity: ∼3000 Ci/pmol) for 1 h at 30°C. The reactions were stopped by the addition of 4× SDS–PAGE sample buffer containing 1% (by vol) 2-mercaptoethanol, and samples were subjected to electrophoresis on a 4–12% SDS–PAGE gels. The gels were was stained with Coomassie blue and the band corresponding to Rab7A was excised and washed with water (10 min) and subsequently subjected to two rounds of washes with 100% acetonitrile and 50 mM Tris–HCl pH 8.0 (5 min per wash). Gel pieces were then reduced by incubation in 5 mM DTT in 50 mM Tris–HCl pH 8.0 at 65°C for 20 min. This was followed by alkylation of the samples by addition of 20 mM iodoacetamide in 50 mM Tris–HCl pH 8.0 for 20 min. The gel pieces were then washed with 100% acetonitrile, 50 mM Triethylammonium bicarbonate and again 100% acetonitrile (5 min each). Any remaining acetonitrile was removed with a pipette before adding trypsin (5 mg/ml in 50 mM Triethylammonium bicarbonate in a volume sufficient to cover the gel pieces) for overnight digestion at 30°C. The resulting tryptic peptides were extracted from the gel pieces by addition of 100% acetonitrile. These were subsequently resuspended in 0.1% (by vol) trifluoroacetate (TFA) in 5% (by vol) acetonitrile before being separated on a reverse-phase HPLC Vydac C18 column (Separations Group) with an on-line radioactivity detector. The column was equilibrated in 0.1% (v/v) trifluoroacetic acid and developed with a linear acetonitrile gradient at a flow rate of 0.2 ml/min. Fractions (0.1 ml each) were collected and analyzed for 32P radioactivity by Cerenkov counting with a tricarb scintillation counter. All the identified phosphopeptide fractions were analyzed by liquid chromatography (LC)–MS/MS using a Thermo U3000 RSLC nano liquid chromatography system (Thermo Fisher Scientific) coupled to a Thermo LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) to determine the primary sequence of the phosphopeptides. Data files were searched using Mascot (www.matrixscience.com) run on an in-house system against a database containing the appropriate Rab sequences, with a 10 ppm mass accuracy for precursor ions, a 0.6 Da tolerance for fragment ions, and allowing for Phospho (ST), Phospho (Y), Oxidation (M), and Dioxidation (M) as variable modifications. Individual MS/MS spectra were inspected using Xcalibur 2.2 (Thermo Fisher Scientific), and Proteome Discoverer with phosphoRS 3.1 (Thermo Fisher Scientific) was used to assist with phosphosite assignment. The site of phosphorylation of 32P-labeled peptides was determined by solid-phase Edman degradation on a Shimadzu PPSQ33A Sequencer of the peptide coupled to Sequelon-AA membrane (Applied Biosystems) as described previously  mutant with Rab7A in HEK293 cells. Immunoblot analysis of cell extracts revealed that wild type LRRK1 but not kinase inactive LRRK1[D1409A] induced significant phosphorylation of Rab7A . In para32P-ATP  in Sf9 insect cells . We also32P-ATP  and subjly Ser72 . We alsote Rab7A . These rAs previous work had demonstrated that LRRK1 co-immunoprecipitates with components of the EGF receptor signaling pathway , we testin vivo, especially after short time points following mitochondrial depolarization  , LRRK1[F1022C] , LRRK1[G1411S]  and LRRK1[I1412T]  . In addihttps://www.ncbi.nlm.nih.gov/snp/;   and LRRK1[I1412T]  promoted Rab7A phosphorylation also indicates that LRRK1 could be regulated similarly to LRRK2. The LRRK2[R1441G] mutation in the ROC domain increases LRRK2 mediated phosphorylation of Rab10, 3 to 4-fold. The LRRK2[R1441G] mutation also enhances affinity of LRRK2 towards its upstream activator, Rab29 . The LRRMutations that delete the C-terminal eight residues of LRRK2 ablate kinase activity . Recent Previous work indicated that Rab7A phosphorylation is mediated by the PINK1/TBK1 signaling pathways in experiments undertaken in HeLa cells overexpressing Parkin . These sOur data show that the PPM1H protein phosphatase previously shown to dephosphorylate Rab proteins phosphorylated by LRRK2 , was alsIn future work it would be also be important to probe the functional consequences of Rab7A Ser72 phosphorylation. In a previous study where stoichiometry of Rab7A was not verified, it was reported that overexpression of LRRK1[Y971F] mutant enhanced binding of Rab7A to one of its well characterized receptors termed RILP . In our d.r.alessi@dundee.ac.uk). All reagents (and associated datasheets) generated at the MRC Protein Phosphorylation and Ubiquitylation Unit at the University of Dundee can be requested through our reagent's website https://mrcppureagents.dundee.ac.uk/.All of the primary data that is presented in this study can be requested in electronic form by contacting Dario Alessi (


In the paper, we provide a detailed description of the enzyme catalytic cycle, model calibration based on a series of in vitro kinetic data, and model validation using experimental data on the regulatory properties of PGHS-1. The validated model of PGHS-1 with a unified set of kinetic parameters is applicable for in silico screening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in different in vitro and in vivo conditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate  and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold; and synthesis of intermediate prostaglandin G The enzyme state containing heme with two oxidizing equivalents—one on the iron and another one on the porphyrin radical cation—is referred to as Intermediate 1  state. The E2 state can be converted back to the resting state E1 by 2 consecutive reactions with RC by followed release of oxidized cosubstrate, OC (reactions 12 and 10). Alternatively, the E2 state can undergo one-electron internal reduction via intramolecular electron transfer from Tyr385 residue in the COX site to protoporphyrin (reaction 13) resulting in the formation of the E5 state  containing Tyr385 radical (Tyr385*) and a ferryl heme. This state is referred to as Intermediate II  referred to as Intermediate II  (E9), containing AA in the COX site. Reaction 2 corresponds to the abstraction of a hydrogen atom from AA by Tyr385 radical and formation of the E13 state containing AA radical in the COX site, . Further, AA* reacts with two oxygen molecules, and the intermediate product PGG2 radical (PGG2*) is produced (reaction 3). We assumed that binding of the first and second oxygen molecules is a rapid process, during which the redox state of the heme remains unchanged  with one oxidising equivalent on iron, and therefore, each of the enzyme states E5, E8, E9, and E13 can potentially participate in redox reactions with RC , producing catalytic states E12, E15, E16, and E20. The later states can be considered as intermediates of an additional COX2 cycle. Note that similar COX cycle was included in the model developed by Kulmacz et al.  (state E11). Note, that similar COX cycle was considered in the extended BC mechanism model , which further converts into intermediate,  (E13), containing the arachidonyl radical AA* (reaction 56). Consideration of two alternative mechanisms of AA radical formation in our model allowed us to analyse their possible contributions to overall COX activity.In our model, we also included an alternative route for initiation of COX reactions, which was proposed in an early version of the TC mechanism of PGHS-1 function —a directPGHS-1 activity undergoes irreversible self-inactivation (SI) during catalysis . The exaBased on the experimental data, we considered several possible mechanisms for enzyme SI in our model. These mechanisms included separate inactivation of the COX and POX activities, originating from enzyme intermediates with different redox state of heme group and containing one or two radicals in the catalytic domains . All theFirst, inactivation of POX activity resulting in fully inactivation enzyme  Reactions 38–42, 53, and 67 originate from Intermediate I and lead to POX and further COX inactivation due to radical state of heme prosthetic group () ,22. In o5, E9, E15, and E20) and lead to POX and further COX inactivation due to the presence of oxidising equivalent Fe(IV) in heme.(b) Reactions 43–46 originate from Intermediate II (states E5 cycle (reactions 25–27). A similar POX cycle was considered in the model developed by Kulmacz et al.  (E17) and  (E4). It should be noted that the remaining POX activity can also further be inactivated due to damage caused by the presence of the radical state in heme (reaction 42).Second, we considered the direct inactivation of COX activity with remaining POX activity which can be caused by the presence of radical states in the COX site in the model . In our z et al. . AccordiAs a result of the joint fitting of three series of the experimental data (see Methods), a unified set of kinetic parameters was determined. The results of fitting are shown in 2 and PGH2 production by PGHS-1 treated by AA and phenol as RC , E2) by RC was obtained to be about zero 8k (0.001 μM−1 s−1); that means that the Intermediate I is rather reduced by intramolecular process of electron abstraction from Tyr385 with radical formation than by exogenous RC.A small value of reaction rate 12k of AA binding to the states in the POX2 cycle , while it is sensitive to the dissociation constant d,12K of the same reactions. The model also showed weak sensitivity to parameter in2k, which corresponds to one of the SI reactions of the enzyme, but it is highly sensitive to parameters in1k and ink characterising the other SI reactions considered in the model.To estimate uncertainty of the parameters obtained in the fitting procedure of the model against experimental data, we carried out global sensitivity analysis (GSA) of the model (see Methods). GSA revealed that the calibrated model showed uniform sensitivity to the most kinetic parameters . The mod2O2 and adrenalin as RC  (Ection 18 .5) state much more effectively than to the resting state (E1), Intermediate I (E2), and E3 state. Maximum flux 1(t)V exceeds maximum values of 30(t)V, 31(t)V, and 32(t)V by an order of magnitude (see comparison of 1(t)V and 31(t)V in 31V) has a high peak in an initial stage of the COX reaction, but its contribution to overall catalysis is negligible because of the short duration of the peak (1 ms).The performed flux analysis showed that AA binds to the Intermediate II . Thus, reduction of the heme group due to Tyr385 radical formation in the presence of AA in the COX site is weaker than that in the case of the empty COX site. Therefore, substrate–enzyme complex  (E9) is formed mainly from Intermediate II according to the experimental data , as opposed to the kinetic models of PGHS-1 , being common for the COX2 and POX4 cycles, participates simultaneously in the production of the intermediate product PGG2 (reaction 23) and the final product PGH2 (reaction 8). As discussed above, these reactions make maximum contributions to the synthesis of PGG2 and PGH2 . For this, we first calculated the kinetics of the oxygen consumption rate O2V(t) and compared the results with the experimental data  (E15). This conclusion contradicts the models where only the ferril state  (E5) was considered in the COX reactions  E1. This coeactions . To remo14 state in POX4 cycle) occurs, when AA is in the COX site. The flux analysis allowed us to compare this mechanism of AA radical formation with the alternative mechanism through Tyr385 radical, proposed in framework of the BC mechanism   and ferryl states  and  . Alternatively, AA can bind to the enzyme state containing Tyr385 radical . Therefore, it was assumed that the reactions of a similar type would have similar rate constants.In general, all the reactions in the developed catalytic cycle shown in However, in general case, kinetic constants of the elementary reactions taking place in one of the catalytic sites can depend on the state of all other catalytic components, located in the nearest proximity and involved in the further catalytic steps. For example, there is experimental evidence that AA binding to the COX site may depend on the presence/absence of the radical on Tyr385 . PGHS-1 We tried different approaches in grouping the model parameters and analysed the results of fitting of the corresponding models to the same set of experimental data. Analysis of the fitting results allowed us to hypothesise which reaction rates do not depend much on the processes taking place in the neighbour site and therefore which kinetic constants can be assumed to be equal to each other. This analysis also allowed us to make conclusions on the cooperativity between COX and POX activities. The approach we used allowed us to significantly reduce the number of parameters subject to fitting. The best fitting of the model to experimental data was achieved with the set of 18 parameters given in When identifying the model parameters, we were trying to find a unified set of parameters which would allow us to consistently describe a majority of the in vitro kinetic data known for PGHS-1 catalysis. Identification of the model parameters and their further validation was performed as follows. We chose several experimental data sets obtained in different research laboratories to fit the model parameters against the data and then validated the calibrated model by checking its applicability to the description of experimental data which were not taken in the fitting procedure.2 and PGH2 production .To avoid ambiguity, for the initial stage of the fitting, among all kinetic information available for PGHS-1, we chose three homogeneous datasets which were obtained for the same object—purified PGHS-1 from sheep seminal vesicles—under approximately the same experimental conditions (temperature and pH). The first set included kinetic data on PGGd AA see . Note thμ and σ are two sensitivity measures in the Morris GSA method, i.e., μ assesses the impact of a parameter on the model output, and σ describes the non-linear effects and interactions of the parameters. As the model output in the calculation, we took an area under the curve (AUC) of PGH2 time course.Global sensitivity analysis (GSA) of the model to the kinetic parameters obtained in the fitting procedure was based on the Morris GSA method for the calculation of elementary effects ,33. The The key aim of this work was the development of a reliable and validated kinetic model of PGHS-1 which reproduces the main features of its complex kinetics and can be used as a computational tool for accurate prediction of inhibition effect of NSAIDs and their combinations in a range of cellular conditions. The cycle-based network model integrates both the current models of the molecular mechanism of PGHS-1 catalysis and a large dataset of experimental data on the kinetics and regulatory properties of PGHS-1. This integrative approach helped us to overcome the limitation of the previously developed PGHS-1 models mainly applicable to the description of individual in vitro data in a specific range of substrate and cosubstrate concentrations.The developed model of PGHS-1 catalysis is a platform for the further development of a kinetic model of inducible isoform PGHS-2 (COX-2) which is expressed together with PGHS-1 in vascular endothelia cells and other organs and is a target of COX-2 selective NSAIDs. Development of the kinetics models of two isoforms within a unified integrative approach would allow for the investigation of the different physiological roles of these isoforms in the same cells and analysis of the effect of NSAID inhibition on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandins in platelets and endothelial cells.


Immobilization of photocatalysts on supports is an important method of adding highly active photocatalysts to a continuous flowing system without the need for photocatalyst recovery. However, direct immobilization prevents exposure to all photocatalytically active surfaces. Therefore, to immobilize particulate photocatalysts, while exposing the photocatalytic surface to organic pollutant water in a continuous flowing system, in this study, we employed double-inverse-opal (DIO) with periodically arranged, interconnected macropores, each containing a single photocatalytic particle. Increasing the macropore size successfully enhanced the decomposition rate of organic dye due to the high diffusion rate of dye molecules in the macropores of thin DIOs. However, an excessive increase in macropore size lowered the decomposition rate of dye molecules because an increase in DIO thickness caused the attenuation of light used to excite the photocatalytic particles. This study presents novel, immobilized photocatalytic DIO-structured particles that can be employed in continuous flowing reaction systems by tuning the photocatalytic particle size, macropore size, and DIO thickness. In theO2 cores d. To pret 500 °C .ϕ = 28 mm and height = 2 mm , 162–1100). As shown in 2 particles, we conducted the circulation of the MB solution for 1.5 h in the dark (without UV light irradiation). It was confirmed that the adsorption equilibrium of MB was attained within 1.5 h in this preliminary experiment (see C0) was examined on the basis of the absorption peak intensity at 664 nm, which was measured using a UV–VIS spectrophotometer . The photocatalytic reaction was initiated by the irradiation of the assembly with UV light . After initiating UV irradiation, we withdrew 1.5 mL of the solution from the reactor at various times throughout the reaction (0.5–5 h), and we determined the residual MB concentration (C) using the 664 nm peak. A xenon lamp  with a band-pass filter , HQBP-UV ϕ25) that shields visible light was used as the UV light source. During the reaction, the MB solution was continuously circulated at a flow rate of 20 mL/min, a figure chosen due to the results of a preliminary photocatalytic experiment  was determined using the following equation:dV was determined by measuring the diameters of more than 200 particles using TEM images. The size of the macropores in the DIO assemblies (dp) was estimated by direct measurement of macropores in SEM images. The average thickness of the assemblies (T) was determined by measuring more than 50 spots in the cross-sectional SEM images of each assembly.The obtained particles and particle assemblies were observed via field-emission scanning transmission electron microscopy  and field-emission scanning electron microscopy , respectively. The transmittance of the particle assemblies was measured using the UV–VIS spectrophotometer mentioned previously. The volume-averaged diameter  is shown in 2 inverse opal frame because of scattering by the TiO2 particles within the DIO. In the UV region, the transmittance of the DIO assembly was lower than it was in the visible light region. This suggests that the TiO2 particles inside the DIO assembly can be excited through UV light absorption.An SEM image of the DIO photocatalytic assembly is shown in EM image , the voi2 core and the SiO2 frame) on the photocatalytic activity of the DIO assembly, we fabricated a TiO2 particle assembly (TiO2 in 2 particle assembly immobilized by a SiO2 frame without voids (TiO2/SiO2 in (C/C0) plotted against irradiation time for each assembly. The TiO2 particle assembly was highly fragile; under the flow of MB aqueous solution, the assembly collapsed within 2 h of initiating UV irradiation, and almost all the assembled particles flowed out of the substrate. In contrast, the TiO2/SiO2 assembly was sufficiently strong, withstanding the flow of the MB solution for 5 h, owing to immobilization with the SiO2 frame. The MB molecules in the solution were initially decomposed, similar to those in the solution employed for the TiO2 assembly, indicating almost no contact between the MB molecules and the TiO2 interface in the assemblies. The DIO assembly developed for the continuous photocatalytic system possessed a mechanical strength similar to that of the TiO2/SiO2 assembly and exhibited a higher activity than those of both the TiO2 and TiO2/SiO2 assemblies.The photocatalytic activity of the DIO assembly was examined on the basis of the decomposition of MB under UV irradiation. To investigate the effect of voids  of macropores were prepared by tuning the PSt shell thickness on a TiO2 core. The variation in the thickness of the PSt shell increased the macropore diameter from 400 to 730 nm. In 2 core and the SiO2 frame, which simultaneously affects the diffusion of the MB molecules and the propagation of UV light in a macropore, should be considered.DIO photocatalytic assemblies with four different diameters  incorporated in them; the thickness of the DIO assemblies increased from 7.2 µm for V400 to 29 µm for V730. The increase in thickness of the DIO assemblies attenuates the irradiation because of the increased optical length. To examine the two factors separately, we fabricated DIO assemblies with varying thicknesses using core-shell particles with similar PSt shells.The larger spaces in V450, when compared with V400, for the diffusion of MB could prolong the presence of MB in the former, thus offering a high frequency of contact between the MB and the TiO2 precursor. A macropore diameter of 450 nm was confirmed by direct measurement of their sizes in SEM images. The photocatalytic activities of the DIO assemblies are shown on the left side of 2 components in the assembly increased with an increase in the thickness of the DIO. The amount of MB molecules that decomposed for each particle are presented in 2 particle in T10, T19, and T29 were 9.5 × 10−19, 7.3 × 10−19, and 5.6 × 10−19 mol/particle, respectively. This suggests the inhomogeneous excitation of the TiO2 particles due to the attenuation of light propagation in the thick DIO assemblies. A similar trend of non-proportionality in the decomposition rate for photocatalyst-supported films has been reported [The SEM images on the right side of reported ,43. The 2 particle number, the decomposition amount of MB in V730 with a DIO thickness of 29 μm . This indicates that the thickness of the DIO assemblies is an important factor in determining the decomposition rate of the MB molecules, and the time spent inside the macropores is less important in DIOs with sufficient space for MB diffusion.When the photocatalytic activity was normalized by TiOof 29 μm  was 5.3 2 cores in the DIO. A precise design—for the efficient use of light energy—is required for the development of a continuous photocatalytic system that is practical for industry use . Another possible option is the employment of the “slow photon effect”. This is a phenomenon that slows down the propagation of light with wavelengths near the photonic band gap in certain photonic crystals [On the basis of the results, an increase in the macropore size in this novel DIO assembly could promote the diffusion of reactants; however, it simultaneously attenuated the incident light meant to excite the TiOcrystals ,44. Ther2 particles encapsulated within macropores of a SiO2 frame was developed as a new type of immobilized photocatalyst. The fabricated DIO assembly exhibited higher photocatalytic activity than that of TiO2 particles immobilized within the SiO2 frame without voids. The SiO2 frame played an important role in preventing TiO2 particles from flowing out of the assembly. The interconnecting pores between the macropores provided a channel for the reactant solution to flow from one macropore to another with a single TiO2 particle. DIO assemblies with large macropores can increase the contact frequency of the reactant molecules with the photocatalytic surface exposed in the assembly. However, the increase in macropore size causes thickening of the DIO assembly, resulting in the inhomogeneous excitation of TiO2 particles in the assembly caused by attenuation of light propagation. This novel DIO offers a new type of immobilized photocatalytic structure to be applied to continuous photocatalytic reaction systems. To optimize the performance of DIO assembly system, the following factors should be considered: the photocatalytic particle sizes, macropore sizes, and film thickness.In this study, a DIO photocatalytic assembly of crystallized TiO


The presence of residual cardiovascular disease (CVD) risk is a current dilemma in clinical practice; indeed, despite optimal management and treatment, a considerable proportion of patients still undergo major CV events. Novel lipoprotein biomarkers are suggested as possible targets for improving the outcomes of patients at higher risk for CVD, and their impact on major CV events and mortality have previously been investigated. Innovative antidiabetic therapies have recently shown a significant reduction in atherogenic lipoproteins, beyond their effects on glucose parameters; it has also been suggested that such anti-atherogenic effect may represent a valuable mechanistic explanation for the cardiovascular benefit of, at least, some of the novel antidiabetic agents, such as glucagon-like peptide-1 receptor agonists. This emphasizes the need for further research in the field in order to clearly assess the effects of innovative treatments on different novel biomarkers, including atherogenic lipoproteins, such as small dense low-density lipoprotein (LDL), lipoprotein(a) (Lp(a)) and dysfunctional high-density lipoprotein (HDL). The current article discusses the clinical importance of novel lipid biomarkers for better management of patients in order to overcome residual cardiovascular risk. A body of evidence from epidemiological, genetic and interventional studies strongly supports causality between low-density lipoprotein cholesterol (LDL-C) and CVD risk, emphasizing LDL-C level as a major risk factor and principal therapeutic target . We are In contrast to elevated LDL-C concentrations, there is no definitive evidence of whether low high-density lipoprotein cholesterol (HDL-C) levels should be treated , althougThe main characteristics of an ideal lipid biomarker include its ability to predict clinically significant outcomes and the patients’ response to therapy in a cost-effective manner . HoweverCirculating LDL particles consist of several discrete subclasses, differing in size, density, composition and metabolic origin, and sdLDL is the most atherogenic fraction as a consequence of their prolonged residence in plasma, easier penetration in subendothelial space and increased susceptibility to oxidative and glycation modifications . The chaStatins are able to favourably modulate LDL subclass distribution  and, morAs already mentioned, the atherogenic lipoprotein phenotype is a cluster of lipid abnormalities, including the elevation of triglyceride-rich lipoproteins . As a coThe results from the TNT trial clearly showed the greatest benefit of intensive statin treatment on major adverse cardiovascular events (MACE) in CVD patients with the highest remnant cholesterol levels . Data frAnother important aspect of residual risk is elevated Lp(a). The structure of this lipoprotein particle resembles LDL, with the addition of apo(a), a polymorphic apolipoprotein with several isoforms of different sizes ,41. FurtAs reported by the investigators of the ODYSSEY Outcomes trial, the greatest benefit of PCSK9i on MACE risk among patients with recent acute coronary syndrome were those with optimal LDL-C levels and mildly elevated Lp(a) . Taking Overall, the data discussed here suggest a clear benefit of targeting atherogenic lipoproteins for further reduction of residual risk , which iRecent findings of the CANHEART Study  have demThe term “dysfunctional HDL” has been forged with an aim to illustrate the incapability of HDL particles to perform protective, anti-atherosclerotic activities . It has Previous studies demonstrated that S1P exhibits atheroprotective effects by regulation of survival, proliferation, differentiation, and mobility of different types of cells, which participate in maintaining vascular homeostasis ,51. S1P Besides lipid species, numerous protein constituents of HDL are responsible for its antioxidative, anti-inflammatory, anti-apoptotic and endothelium-protective effects . In addiOne interesting constraint should be noted with respect to the proposed antiatherogenic characteristics of HDL and its capacity as a biomarker. As recently summarized by Beazer et al. , given tFinally, it should be noted that, in parallel with futile attempts to decisively confirm the independent contribution of low HDL-C to CVD development, a plethora of evidence suggest modulatory roles of HDL in non-cardiovascular diseases, such as diabetes, disorders of the central nervous system, infections, kidney diseases or cancer ,61,62,63There is increasing interest in the effects of novel antidiabetic treatments on atherogenic lipoproteins since such therapies significantly impact the cardiovascular outcome in patients with type-2 diabetes; sodium-glucose cotransporter-2 inhibitors (SGLT-2i) and glucagon-like peptide-1 receptor agonists (GLP-1RAs) have been shown over the last few years to reduce cardiovascular events and mortality, while dipeptidyl peptidase-4 inhibitors (DPP-4i) have a neutral effect with cardiovascular safety, although with no benefit ,65. The Agents in both classes of SGLT-2i and GLP1-RAs, including dapagliflozin, exenatide and liraglutide, have shown the ability to significantly reduce levels of small, dense LDL in patients with type-2 diabetes ,69,70,71All the above suggest a divergent effect of novel antidiabetic therapies on atherogenic small, dense LDL: SGLT-2i and GLP1-RAs reduce their levels, while DPP-4i do not. This is fully in line with the divergent effect of such therapies on the cardiovascular outcome, as previously discussed. Of interest, some of the agents able to reduce small dense LDL, such as exenatide and liraglutide, are also concomitantly reducing the concentrations of the pro-atherogenic adipokine resistin ,78; sincHowever, it is somewhat puzzling that findings on Lp(a) are totally oppositive: agents in both classes of SGLT-2i and GLP1-RAs, such as empagliflozin and liraglutide, have shown no significant effect ,81 whileThe presence of residual cardiovascular risk is a current dilemma in clinical practice; indeed, despite optimal management and treatment, a considerable proportion of patients still undergo major cardiovascular events. This emphasizes the need for further research in the field in order to clearly assess the effects of innovative treatments on different novel biomarkers, including atherogenic lipoproteins, such as small dense LDL, Lp(a) and dysfunctional HDL. This is particularly important during the current coronavirus COVID-19 pandemic since a higher number of cardiovascular diseases, such as acute myocardial infarction, have been reported . Insight


Nitrene transfer reactions represent one of the key reactions to rapidly construct new carbon-nitrogen bonds and typically require transition metal catalysts to control the reactivity of the pivotal nitrene intermediate. Herein, we report on the application of iminoiodinanes in amination reactions under visible light photochemical conditions. While a triplet nitrene can be accessed under catalyst-free conditions, the use of a suitable photosensitizer allows the access of a nitrene radical anion. Computational and mechanistic studies rationalize the access and reactivity of triplet nitrene and nitrene radical anion and allow the direct comparison of both amination reagents. We conclude with applications of both reagents in organic synthesis and showcase their reactivity in the reaction with olefins, which underline their markedly distinct reactivity. Both reagents can be accessed under mild reaction conditions at room temperature without the necessity to exclude moisture or air, which renders these metal-free, photochemical amination reactions highly practical. Gaining in-depth understanding of photochemical processes is key for developing more sustainable and efficient chemical transformations. Here the authors show that under visible light photochemical conditions, iminoiodinanes undergo formation of triplet nitrenes or nitrene radical anions, depending on the use of a photosensitizer; These reagents are studied in amination reactions with olefins. It features light as the main source of energy to conduct chemical transformations and can thus be regarded as a key pillar in the development of sustainable approaches and reduced ecological footprint. Today, photochemistry is one of the most rapidly developing fields in chemistry and the past decades witnessed the development of major milestones and concepts, such as dye-sensitized solar cells2, polymerization chemistry3, protein labeling4, photoredox catalysis8, and classic photochemical applications11 that leverage photochemistry as one of the key technologies in the advancement of all chemical disciplines.Photochemistry is a classic discipline in chemistry and early reports date back even to the beginning of the 208 or reagents14. Significant advances have been made in the context of organic synthesis methodology, for example in the utilization of radicals in the presence of photoredox catalysts8 or carbenes via the photolysis reaction of appropriate reagents14. The access and application of nitrenes22 or analogs therof23 under visible light photochemical conditions however remains largely underestimated although it would allow for direct amination reactions with vast potential in modern drug synthesis22. Such photochemical nitrene transfer reactions would significantly expand currently available concepts in classic metal-catalyzed nitrene transfer reactions , relying on strong UV-light for their photolysis29. UV-light can however significantly reduce reaction efficiency and increase by-product formation. Yet, UV light still remains a prerequisite to access pivotal nitrene intermediate under photochemical conditions . Other light sources proved by far less efficient, which might be related to weaker absorption or side-reactions due to the high-energy UV light. A surprising observation was made when switching to photocatalytic reaction conditions. Using simple [Ru(bpy)3]Cl2 as photocatalyst, a complete reversal of reactivity was observed and selective aziridination reaction to yield 11a occurred. Other photocatalysts, such as [Ru(bpz)3](PF6)2, iridium-based photocatalysts, or organic dyes did not alter the reactivity and the C-H functionalization product 10a was formed selectively. Only in the case of Eosin Y as photocatalyst, a mixture of C-H amination (10a) and aziridination (11a) was observed. Further optimization steps included investigations on the solvent, concentration, reaction stoichiometry, yet no further improvements were observed  relaxation on the singlet spin surface leads to the direct formation of a singlet nitrene that features a very short N-O distance, which can be interpreted as a stabilization of the low-valent nitrene with the lone pair of an oxygen atom of the pendant sulfonyl group. B) relaxation involving intersystem crossing (ISC) results in the formation of a triplet intermediate 1a-T, which features a very long N-I bond, close to a non-bonding situation. Scanning of different N-I bond lengths indicated that further elongation of the N-I bond proceeds in a barrier-free fashion to directly lead to a triplet nitrene intermediate. This triplet nitrene intermediate is the energetically favored intermediate and can alternatively be accessed from the high-lying singlet nitrene via intersystem crossing. This theoretical analysis of the photochemistry of iminioiodinane 1a now rationalizes for the formation of a triplet nitrene intermediate under photochemical conditions and shows a marked difference to the ground state reactivity31. Under photocatalytic conditions, calculations are in line with our previous report30 and a very facile reduction of the iodinane, leading to iodinane radical anion INT1. This anion features a very long N-I bond that is close to non-existent and therefore a rapid cleavage of the N-I bond can occur in a barrierless process to give a nitrene radical anion.Further studies on the reaction mechanism involved theoretical calculations to better rationalize this photochemical nitrene transfer reaction Fig. . We therFor further analysis and understanding of the reactivity of triplet nitrene and nitrene radical anion, we examined their structural and electronic properties. The calculations show that the nitrene radical anion possesses a higher electron density at the nitrogen atom compared to the triplet nitrene, which in turn leads to higher nucleophilicty of the nitrene radical anion . A second pathway involves the addition of the triplet nitrene to α-methyl styrene via a TS3 with an activation free energy of only 6.1 kcal mol−1, which is significantly favored over the hydrogen transfer pathway. This addition product can undergo intersystem crossing to give an open shell singlet species that can either cyclize to give the aziridine 11a, or undergo hydrogen atom transfer to give the C-H functionalization product 10a. Analysis of the respective transition states reveals significant steric hindrance of rotation around the central C-C bond, which renders cyclization energetically unfavorable over hydrogen atom transfer. The latter is favored by 2.5 kcal mol−1 and now reasons the reactivity of triplet nitrene intermediates with α-methyl styrene to yield the C-H functionalization product 10a. Further studies concerned the reaction of the nitrene radical anion  that can arise from an addition – hydrogen atom transfer mechanism were observed under photochemical conditions. The theoretical analysis of reaction pathways for the hydrogen atom transfer revealed that pathways involving different hydrogen atom transfer can occur due to the presence of the ethyl group. These are very similar in energy and thus reason the unselective reaction and represent a limitation of the present C-H amination method in the case different hydrogen atom transfer reactions are possible , we could observe a selective reaction towards the trans-aziridine 20, which is indicative of a stepwise mechanism under both photochemical and photocatalytic reaction conditions. Further control experiments involved the use of spin trapping reagents, such as 2,2,6,6-Tetramethylpiperidinyloxyl (TEMPO), 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), or DNP, and in all cases a complete suppression of the photochemical and photocatalytic reactions were observed, which is indicative of radical intermediates or participating in the reaction mechanism . Importantly, ortho-substitution  had a slightly detrimental effect on the product yield. Similarly, different sulfonyl groups were tolerated under the present photochemical reaction conditions and the allyl amines products were obtained in high isolated yield . When applying this photochemical protocol to cyclic, trisubstitued, cyclic olefins, 1-alkynyl-1-methyl or aliphatic 1,2-disubstituted olefins, the C-H amination products 10r–v were selectively obtained in good isolated yield. Then, the photocatalytic protocol was employed in the aziridination of α-methyl styrenes . Most notably, in this case also different α-alkyl-substituted styrene derivatives, such as a very bulky tert.-butyl group (11s), and nucleophilic N-heterocycles  were well tolerated to yield the corresponding aziridines in high yield, which are commonly very challenging substrates in aziridination reactions. Furthermore, a range of trisubstitued, cyclic olefins, and further examples of 1,1-disusbtituted olefins were studied under the present photocatalytic conditions to yield the aziridine products 11t–11ad.We next turned our attention towards applications of the above protocols in amination reactions Fig. . The phoion Fig. . Halogennes Fig. . We exam22a–t). Most notably, even in the presence of two sterically demanding ortho-chloro substituents (22t) the desired aziridine product was obtained in good isolated yield. Further studies involved the reaction of cyclic olefins, such as indene, which smoothly reacted to the aziridine product (22u) in high isolated yield. An important difference in the reactivity of nitrene and nitrene radical anion was however observed in the reaction with aliphatic olefins . Further examples under investigation concerned the utilization of 1,2-disubstituted olefins. In this case, different aryl-alkyl and alkyl-alkyl disubstituted olefins smoothly underwent selective aziridination reaction to afford the trans-azridines 20a–i in high yield.Finally, we embarked on the reaction of simple styrene derivatives under both photochemical and photocatalytic conditions Fig. . In bothins Fig. . While oINT3S , equipped with a magnetic stirring bar, iminoiodinane  and alkene (5.0 equiv) are dissolved in 2 mL DCM under air atmosphere. The reaction is stirred and irradiated with a 3 W LED lamp (5 cm distance) for 4 h. A cooling fan is used to maintain room temperature (25–28 °C). The product was obtained after column chromatography using n-hexane/EtOAc as eluent.In an oven-dried tube (10 mL), equipped with a magnetic stirring bar, iminoiodinane , alkene (5.0 equiv), and catalyst (1 mol%) are dissolved in 2 mL DCM under air atmosphere. The reaction is stirred and irradiated with a 3 W LED lamp (5 cm distance) for 4 h. A cooling fan is used to maintain room temperature (25–28 °C). The product was obtained after column chromatography using 32. All structures were optimized at the (U)M06-2X level33 of theory in combination with D3 dispersion corrections34, in which all atoms were described with the def2-SVP basis set35. Analytical frequency calculations were carried out at the same level of theory in order to confirm each stationary point as either an intermediate (no imaginary frequencies) or a transition state (only one imaginary frequency). Key transition-state structures were confirmed to connect corresponding reactants and products by intrinsic reaction coordinate (IRC) calculations37. The electronic energy was then refined using def2-TZVP basis set35 at the (U)M06-2X level on the optimized geometries in combination with D3 dispersion corrections. Solvation energies in dichloromethane  were evaluated by IEFPCM calculations with radii and non-electrostatic terms for SMD solvation model38 based on the optimized structures. Time-dependent (TD)-DFT calculations were carried out on the optimized structures of the PhINTs to obtain the absorption wavelength.All calculations were performed using the Gaussian 16 series of programsSupplementary Information


A new data transformation method for micro-manufacturing using a topological model for a micro-/nano-texture was proposed for a surface-decorated product. Femtosecond laser printing was utilized to form the micro-/nano-textures into the hardened thick layer of dies by plasma nitriding. At first, the plasma-nitrided AISI316L flat substrate was laser-printed as a punch to imprint the tailored nano-textures onto the AA1060 aluminum plate for its surface decoration with topological emblems. Second, the plasma-nitrided SKD11 cylindrical punch was laser-trimmed to form the nanostructures on its side surface. This nano-texture was imprinted onto the hole surface concurrently with piercing a circular hole into electrical steel sheet. The fully burnished surface had a shiny, metallic quality due to the nano-texturing. The plasma nitriding, the laser printing and the CNC  imprinting provided a way of transforming the tailored textures on the metallic product. Digital manufacturing requires innovative changes to the tools used on the production-line . The taiIn the latter, a mother punch works as a tailored tool to transform a perfect shearing process design onto highly qualified product surfaces with use of the nano-texturing method, as shown in Femtosecond laser processing is commonly employed as tooling to perform the laser printing of micro-/nano-textures onto mother dies ,5. AmongIn the present paper, a three-step procedure is proposed to design micro-/nano-textures for the surface decoration of products, to apply tailored textures onto the surface of nitrided punch for micro-embossing and for micro-piercing, respectively, and to imprint them onto product surfaces. These formed nano-textures on the surface of punch are imprinted onto an AA1060 plate surface as well as the pierced hole surface of an electrical steel sheet by using the CNC  stamper. The product quality is improved by these imprinted micro-/nano-textures.The three-step procedure is depicted by a scheme in In the third stage, this laser-printed die is fixed into a die set and loaded to imprint the designed textures onto the product surface. The flow stress of the product materials and the loading sequence in the stamping have a significant influence on the dimensional accuracy of micro-/nano-textures in duplication by stamping. Various product materials are also selected for this imprinting procedure.Nano-textures induced by LIPSS are controllable through optical path control. The six orientations above, in This qualitative correlation between the nano-textures by LIPSS and the surface plasmonic colors is employed to identify the presence of nano-textures on the laser-processed surfaces on the nitrided punch as well as the replica surfaces on the products. Through this nano-texturing together with color-grating though micro-texturing, the laser-printed die surfaces and the imprinted product surfaces develop surface plasmonic brilliance.The insertion of a thick, hardened surface layer into the punch and die is necessary to make laser printing with a sufficient depth. The low-temperature plasma nitriding system  in The femtosecond laser machining system  in The CNC stamping system  in Two types of nitrided punches are prepared for the CNC imprinting of seven micro-textured emblems to the metallic plate and for nano-texturing the metallic sheets with piercing. SEM (scanning electron microscopy)–EDX (electron dispersive X-ray spectroscopy) was utilized to perform the microstructure analysis and nitrogen mapping, respectively.High-density RF (radio frequency)–DC (direct current) plasma nitriding was employed to harden and strengthen the AISI316L flat and SKD11 cylindrical punches. Two types of stamping punch were fabricated, as depicted in The average nitrogen content in the nitrided layer reaches 3.4 mass%. Its surface roughness remains as it was before nitriding. The nitrided layer has a fine microstructure, in which the average grain size is reduced to 0.1 μm.These two punches were processed by femtosecond laser printing to form the tailored micro-/nano-textures onto them. The nitrided flat AISI316L punch in This M6 emblem is composed of 5 × 6 = 30 triangular segments by repeating the micro-texturing five times process to form six different segments, as depicted in Both the head and the side surfaces of the plasma-nitrided cylindrical SKD11 punch with a diameter of 2 mm were trimmed by the femtosecond laser processing to sharpen their edges and to reduce the surface roughness. An AA1060 aluminum plate with a thickness of 1mm with a normal flow stress of 200 MPa was employed. The CNC stamping system was utilized to imprint seven micro-textures with the tailored nano-textures onto this substrate. A single-mode loading sequence was used to compress the upper punch with a constant velocity. We will now describe the CNC-imprinting behavior of each emblem on the laser-printed die onto the aluminum plate. An M6 emblem was also employed for this comparison. As shown in An SEM analysis with high magnification was performed to describe the nano-texture imprinting onto the aluminium plate together with the transcription of the micro-texture M6 in The laser-trimmed SKD11 punch was fixed into the upper die set for piercing the electrical steel sheet with a thickness of 0.15 mm. The core die was also cemented to the lower die set. The CNC stamping system was also utilized for the fine piercing experiment. We experimentally evaluated the laser-trimmed SKD11 punch life and, here, we describe the debris particle deposition’s behavior. With respect to the former, the recent demand for highly cost-competitive motor cores has resulted in the prolongation of punch life together with the high qualification of the pierced electrical steel sheets. In the latter, chipping and micro-cracking was expected to occur through the severe deposition of debris particles from the work. The original nano-texture was designed to have a straight orientation along the piercing direction in order to visualize the deposition process.Plasma nitriding, femtosecond laser printing, and the CNC imprinting were employed as a three-step procedure to improve the design flexibility of the micro-/nano-textures of products and to prolong the tool life, in order to ensure high cost-competitiveness. The plasma nitriding process is the first step in the formation of thick, hardened layers into stainless steel and tool steel die, roll and tool substrates. Owing to the low-temperature plasma nitriding, this layer is uniformly nitrogen-supersaturated without nitride precipitates ; the dieThe data transformation from the tailored model in CAD (computer-aided design) to the surface-decorated product by using the three-step procedure becomes a key process to improve the quality of metal and polymer products. The original color grating by micro-texturing and the surface plasmonic brilliance by nano-texturing are duplicated onto the product surface. The colored emblems, symbols, codes, and fonts are embedded into the product surface, as tailored in CAD. In particular, their topology and geometry work as functional convex and concave textures to control the surface properties and to stimulate the engineering performance across the textured interface.In addition to aluminum and aluminum alloy works, low-flow-stress metals and alloys, such as tin and copper, as well as polymers, such as PET  and PMMA (polymethyl methacrylate) can be employed as work materials for cold and warm imprinting. When using the aluminum alloy, the micro-textures are nearly fully imprinted by embossing the tool surface; the nano-textures are often partially transcribed. These metals with low flow stress and polymers are suitable for the full transcription of micro- and nano-textures onto their surfaces.The fine piercing and punching of metals and alloys requires their sharp separation without the formation of fractured surfaces . As repoThe present three-step procedure provides a hardened SKD11 punch and die with a sharpened edge and micro-/nano-textured side surfaces. As discovered in , the useThe laser nano-texturing process works directly as a tool to build up a master-piece for the duplication of tailored nano-textures onto metallic and polymer products. Owing to the nitrogen supersaturation of the die substrate at 673 K, the laser-nano-textured die is responsible for long-term usage in this duplication processes. In particular, this nitrided die has sufficient chemical stability and heat resistance to perform high-temperature injection molding and mold stamping for repetitive duplication operations. Femtosecond laser nano-texturing also works to form the unidirectional nano-textures onto the trimmed punch side surface. The laser trimming and nano-texturing processes work simultaneously to adjust the surface roughness and geometric irregularities by trimming and to superpose the nano-textures onto the trimmed surface.