Such a robust adhesion mainly stems from a high yield of DHI moieties through a delicate synergy of leading oxidation and subsidiary cyclization within self-polymerization, allowing for governing mussel-inspired adhesion by the substituent chemistry and self-polymerization manner. The adhesion mechanisms revealed in this work offer a useful paradigm for the exploitation of functional mussel-inspired materials.Mussel-inspired chemistry has become an ideal platform to engineer a myriad of functional materials, but fully understanding the underlying adhesion mechanism is still missing. Particularly, one of the most pivotal questions is whether catechol still plays a dominant role in molecular-scale adhesion like that in mussel adhesive proteins. Herein, for the first time, we reveal an unexplored adhesion mechanism of mussel-inspired chemistry that is strongly dictated by 5,6-dihydroxyindole (DHI) moieties, amending the conventional viewpoint of catechol-dominated adhesion. We demonstrate that polydopamine (PDA) delivers an unprecedented adhesion of 71.62 mN m in situ force measurements with molecular-scale simulations.A 5,6-dihydroxyindole (DHI)-dominated mechanism in the interfacial adhesion of mussel-inspired chemistry is first proposed and demonstrated by the fusion of  Other moieties such as amine and phosphate ester can also work with catechol groups to synergistically enhance wet adhesion through some unique mechanisms such as cation–π interactions,25,28 surface salt displacement,29,30 and anion–π interactions.20 However, in spite of extensive achievements in molecular-level measurements, most cases revolve around some specific-design research objects such as single catechol molecule and mussel adhesive proteins. The catechol-dominated role in these previous research objects still remains challenging to elucidate the adhesion mechanism of mussel-inspired chemistry from an integrated perspective, because most mussel-inspired derivatives encounter a sophisticated oxidative-polymerization process to produce complex, dispersed chemical compositions.31–33 Taking dopamine as a typical example, its polymerization process normally involves oxidation, intramolecular cyclization, intramolecular rearrangement, and covalent coupling.14 As a result, the as-polymerized product, termed polydopamine (PDA), features extremely complex compositions containing catechol, leucodopaminechrome, dopaminechrome, 5,6-dihydroxyindole (DHI), 5,6-indolequinone, amine, and imine groups. Moreover, many other common mussel-inspired derivatives also bear some distinct substitutional groups such as hydroxyl, methyl, and carboxyl moieties. All of these tiny groups actually have a substantial impact on mussel-inspired chemistry from self-polymerization to eventual adhesion.34–36 However, to date, very few reports focus on this molecular-level impact and current views still follow the previous catechol-dominated adhesion mechanism. To this end, it is imperative to revisit mussel-inspired chemistry and uncover the underneath adhesion mechanism thoroughly from the perspectives of molecular architecture.Several advanced force measurement techniques have been recently deployed to investigate the adhesion mechanism of mussel-inspired chemistry, which include those involving single-molecule atomic force microscopy (SM-AFM),In situ SFA measurements show that PDA exhibits an unparalleled adhesion as high as ∼71.62 mN m−1, which is approximately 121, 68, 18, and 5-fold higher than that of polycatechol, poly(l-DOPA), polyadrenaline, and polynoradrenaline, respectively. By combining the results of force measurements with molecular-scale simulations, we attribute the strong adhesion of PDA to its high yield of DHI moieties by virtue of the synergy effect of oxidation and intramolecular cyclization for forming sufficient DHI-enabled interactions . We also demonstrate that the substituent chemistry and polymerization manner can govern the adhesion of mussel-inspired chemistry by modulating the yield and properties of DHI moieties. Our findings offer new insights into the fundamental understanding of mussel-inspired adhesion as well as the rational design of mussel-inspired materials.Herein, in striking contrast to the conventional catechol-based adhesion, we report a DHI-dominated mechanism in the interfacial adhesion of mussel-inspired chemistry from the perspectives of molecular architecture. 1, R2, R3 = H), l-DOPA , adrenaline , and noradrenaline   after 1 h (−1 (Wad = 11.21 mJ m−2), with extending the polymerization time to 2 h. The deteriorated adhesion results from the enhanced steric repulsion between the two interacting surfaces, because of the generation of large PDA aggregates that lead to relatively higher surface roughness   , which pA Fig. S1. The adhfter 1 h  and S2†.e.g., catechol) as controls. We first selected catechol, the simplest mussel-inspired derivative that is able to self-polymerize into polycatechol, to evaluate its contribution in PDA-based adhesion. −1 (Wad = 0.13 mJ m−2) is measured between two polycatechol layers, which is 121-fold lower than that of PDA. Considering that the molecular structure of polycatechol is short of amine groups compared with PDA, we further measured the adhesion of catechol and amine  with various molar ratios, in which catechol and amine can form poly(catechol-amine) by the Michael reaction  to 6.4 mN m−1 (Wad = 1.36 mJ m−2), yet it is still ten times lower than that of PDA. Even though catechol is replaced by polyphenols like tannic acid containing numerous catechol groups, the maximized adhesion of poly(tannic acid–amine) is only 20.86 mN m−1 (Wad = 4.43 mJ m−2),40 which is still far away from that of PDA. In addition to the adhesion between coatings, we further studied the adhesion between various coatings , and PDA) and mica in an asymmetric measurement configuration by using an SFA. As shown in Fig. S7,−1) is 3-fold and 15-fold higher than that of poly(catechol-amine) (4.2 mN m−1) and polycatechol (0.83 mN m−1), respectively. These adhesion results in both symmetric and asymmetric measurement configurations manifest that PDA has stronger adhesion capability than polycatechol and poly(catechol-amine). Therefore, we speculate that the strong adhesion of PDA originates from an unexplored interaction mechanism, which is different from the conventional PDA-based adhesion and mussel adhesive proteins that rely on the major contribution of catechol groups and their synergy with amine groups.28,41Elucidating which groups are mainly responsible for the strong adhesion of PDA can be implemented by splitting dopamine into single functional moieties , adrenaline , and noradrenaline , all of which possess dopamine-like capacities to generate DHI moieties and form nanoscale-roughness coatings , polyadrenaline, and polynoradrenaline is 1.06 mN m−1 (Wad = 0.23 mJ m−2), 3.89 mN m−1 (Wad = 0.83 mJ m−2), and 15.28 mN m−1 (Wad = 3.24 mJ m−2), respectively  and hydroxyl group (R1 = OH) as the electron-withdrawing and electron-donating substituent, respectively, whereas adrenaline has a methyl substituent (R3 = CH3) to become a secondary amine compared with dopamine. These substituents are capable of governing the chemical micro-environment of oxidation and cyclization sites, determining the formation and properties of DHI moieties   > noradrenaline (0.851 eV) > adrenaline (0.862 eV) (−1) > noradrenaline (49.48 kJ mol−1) > dopamine (50.75 kJ mol−1) > l-DOPA (56.53 kJ mol−1). Remarkably, adrenaline possesses the highest cyclization activity, whereas l-DOPA exhibits the poorest cyclization activity, which are distinct from their oxidation abilities. Such a huge difference results from the substituent impact on the electron densities of the amine group. For instance, the carboxylate group of l-DOPA can pair with its cationic amine so as to impact the activity of Michael addition based cyclization.To study the substituent chemistry effect on DHI formation, the UV-vis spectra of renaline . The subrenaline . By calc.862 eV) . In contl-DOPA and noradrenaline showed that although noradrenaline shows a slower oxidation activity, it has better cyclization ability than l-DOPA  and their concentrations. As shown in −1 (Wad = 15.21 mJ m−2) to 19.38 mN m−1 (Wad = 4.11 mJ m−2) with the increase of KCl concentration to 10 mM. This Li+/Na+-insensitive yet K+-sensitive adhesion behavior coincides well with the cation–π interaction, in which K+ can form stronger cation–π interactions with DHI moieties compared with Li+ and Na+.25,49 As a result, PDA adhesion mainly originates from DHI-enabled cation–π interactions over the other interactions such as π–π interactions, hydrogen bonds, and hydrophobic interactions because they are normally insensitive to salt species and concentrations. We further examined the contribution of these DHI-enabled interactions by measuring the adhesion of PDA to Phe-, OH-, and N(CH3)3+-terminated surfaces in an asymmetric configuration using the SFA. 3)3+-terminated surface is as high as 42.9 mN m−1 (cation–π interaction), which far surpasses that of Phe-terminated surface  and OH-terminated surface   . Togethe−1, 19.21 kJ mol−1, 27.36 kJ mol−1, and 25.25 kJ mol−1 in the case of l-DOPA, adrenaline, noradrenaline, and dopamine, respectively. In striking contrast to the others, l-DOPA exhibits the lowest cation–π interaction strength because of its electron-withdrawing substituent (R2 = COOH) which significantly decreases the electron densities of DHI (l-DOPA displays the poorest adhesion ability (1 = OH)  and hence the strength of DHI-enabled cation–π interactions.Owing to different π-conjugated electron densities within DHI caused by the substitutes , dopamins of DHI . Such lo ability . Notably(1 = OH) . Thus, iNext, we investigated the number of DHI-enabled cation–π interaction sites in the coatings of PDA and derivatives using XPS spectra. Interestingly, the N element content in PDA is around 7.40%, which is 2.2- and 5.6-fold higher than that of polynoradrenaline (3.42%) and polyadrenaline (1.27%) . By furtin situ polymerized dopamine at a wide range of pH values from acidity to alkalinity. The measured adhesion at pH 3.5 and pH 7.0 is 2.98 mN m−1 (Wad = 0.63 mJ m−2) and 6.18 mN m−1 (Wad = 1.31 mJ m−2), respectively, which are extremely weaker than that at pH 8.5  , which highly coincides with very limited deposition capability of dopamine under strong alkaline conditions .53,54 In strong alkaline media, despite possessing high yield of DHI moieties, the resultant DHI moieties are fast, substantially oxidized into 5,6-indolequinone because of the large dissociations of phenolic hydroxyl groups, which destroys the π-conjugated electron densities within DHI and thus weakens DHI-enabled interactions including cation–π interactions, π–π interactions and hydrogen bonding , the measured adhesion has an obvious decrease from 71.62 mN m−1 (Wad = 15.21 mJ m−2) to 27.66 mN m−1 (Wad = 5.87 mJ m−2). This is because Na2S2O8, a stronger oxidant compared with oxygen, can induce more oxidation of DHI into 5,6-indolequinone, which undermines DHI-enabled interactions and eventually results in a reduced adhesion.To demonstrate the validity and generality of DHI-dominated adhesion, we also examined the effect of many synthesis conditions that may vary the yield and properties of DHI and affect DHI-enabled interactions . First,  mN m−1)  and S13† bonding . Second,−1 (Wad = 3.85 mJ m−2), which is 4-fold smaller than that of then pure PDA case. It suggests that the introduction of cationic amine significantly deteriorates PDA adhesion. Such a dramatically decreased adhesion results from the reduction of DHI yield by a competition mechanism, in which DETA can react with the oxidized dopamine by Michael addition for undermining the cyclization of dopamine , indicating that DETA is more prone to react with the oxidized dopamine. As a result, the yield of DHI obtained by cyclization suffers from an obvious decrease, eventually leading to an obvious reduction of DHI-enabled interactions and adhesion. Together, these clear facts powerfully underpin the reliability of DHI-dominated adhesion over both catechol and amines in mussel-inspired chemistry, as well as offer a general, viable strategy to dynamically regulate mussel-inspired adhesion by the meticulous control of oxidation and cyclization.The DHI-dominated adhesion mechanism can be further demonstrated by inspecting the adhesion variation of PDA after incorporating amine-terminated molecules. We selected diethylenetriamine (DETA) as a typical amine molecule to carry out a similar SFA experiment . Fig. 5gdopamine . To demo−1, which is 121, 68, 18, and 5-fold higher than that of polycatechol, poly(l-DOPA), polyadrenaline and polynoradrenaline, respectively. Such robust adhesion probably stems from the highly efficient yield of DHI by the synergy of oxidation playing a leading role and cyclization an assistant role. Moreover, the DHI-dominated adhesion can be elegantly governed by manipulating the substituent chemistry and polymerization manner to tailor the formation and properties of DHI. From a broader perspective, our work provides a new paradigm for re-understanding fundamental adhesion science of mussel-inspired chemistry, as well as re-thinking the design principle of developing advanced mussel-inspired materials.In summary, we discover and demonstrate the dominant role of DHI moieties in the interfacial adhesion of mussel-inspired chemistry over the conventional catechol group from the perspectives of molecular architecture and polymerization mechanisms. The SFA measurement results show that the adhesion of PDA is as high as 71.62 mN mAll data needed to evaluate the conclusions in the paper are present in the paper and/or ESI.†Z.-K. X., H. Z., and C. Z. conceived the research. C. Z. and L. X. designed the experiment and prepared the samples. J. Z. and C. L. conducted the molecular simulations. Z.-K. X., H. Z., Z. W., and C. Z. wrote the paper, and all the authors discussed the manuscript.There are no conflicts to declare.SC-013-D1SC05512G-s001


N-ethylmaleimide sensitive factor) initially purified from an in vitro reconstitution of Golgi membranes  needs to be transported around the cell by the help of motor molecules such as kinesins and dyneins. These molecular machines use the energy of ATP hydrolysis to generate processive and efficient distribution of cargo and their carriers along the microtubule network of the cell. A set of comprehensive reviews from Schiavon et al. discuss previous work on mitochondria intracellular transport and provide an example where impaired mobility of mitochondria may be playing a central role in the pathogenesis of Charcot-Marie-Tooth (CMT) disease, a neurodegenerative disorder. Additionally, Sneeggen et al. describe how the role of intracellular trafficking in cell proliferation, epithelial to mesenchymal transition, and invasion contributes to the regulation of energy consumption and metabolism during cancer progression. Lastly, Tavares et al. delineate the capacity of the HIV-1 virus to hijack key machinery of the intracellular pathway in order to ensure efficient viral replication and survival in the host.Finally, this Research Topic includes three insightful articles that put the role of intracellular trafficking in the context of disease. Altogether, these contributions should give the reader an updated view of different aspects of the intracellular traffic field and a glimpse on the future directions of research in each sub-field. We hope the reader can also find the gaps and the many still unanswered questions in the field while navigating through this Research Topic: how is the motor molecules' activity regulated during development and disease progression? What is the next technology that will thrust this field into the next century of discovery? At the molecular level, how do we modulate cargo localization through targeting strategies, and in doing so, influence the adaptive requirements of a cell? How can we prevent future public health crises by manipulating the host-microorganism energy supply and demand flux? How can we generate a more comprehensive understanding of the molecular machinery of protein trafficking? The dysregulation of the machinery involved in controlling intracellular traffic is frequently associated with diseases that include cancer, developmental and degenerative diseases, and multiple immunity disorders, highlighting the urgency of unraveling the mechanisms that regulate the movement of cargo inside the cell.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.AH was funded by the Spanish Ministry of Economy and Competitiveness (BFU2017-88766-R and PID2020-119132GB-I00). DCG was funded by a Sir Henry Dale Fellowship awarded from the Wellcome Trust/Royal Society (Grant No. 210481).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Over the past two decades, organic molecules adsorbed on atomically defined metal surfaces have been intensively studied to obtain an in-depth understanding of their self-assembly behavior, on-surface reactivity, as well as their structural and electronic properties –6. An imThe molecule–substrate decoupling can either be achieved by suitable molecular design or manipulation of the molecular building blocks, or passivation of the substrate. Molecular engineering approaches include rigid spacers added to the molecular core –10 or sp2O3 [hBN) [2 [Ultrathin semiconducting or insulating decoupling layers can be epitaxially grown as mono- and multilayers on many metallic substrates by either physical or chemical vapor deposition. Among others, ultrathin dielectric layers of either alkali halides  or meta2O3 , and CuO2O3 ), or nit2O3 ) have beO3 [hBN) –23, grapO3 [hBN) –27, and [hBN) [2 , have em[hBN) [2 –32. Addi[hBN) [2 .For semiconductors, for example, bare silicon or germanium, electronic decoupling of molecules can be achieved by either the growth of ultrathin dielectric layers on top of the surface –35 or a Most of these ultrathin interfacial layers significantly reduce both the molecular adsorption energy and the hybridization of molecular states with the electronic bands in metals and semiconductors. However, charge transport is not completely inhibited, and electrons can still tunnel from the metal through these decoupling layers to the organic molecules and vice versa. This has the great advantage that these systems can still be examined by scanning tunneling microscopy (STM) and spectroscopy (STS), which gives insight into structural and electronic properties of individual molecules. For applications such vertical tunneling through the interfacial layers can be undesirable since it prevents the current from flowing across electrode–molecule–electrode junctions  or leads2, and calcite) have mostly served as model systems. To a lesser extent, metal oxides have also been used, for which defects and charging often pose additional challenges [For studying molecules on bulk insulators, ionic crystals  of molecular resonances observed in tunneling spectroscopy since the hybridization of molecular states with the ones of the metallic/semiconducting support is prevented. Thereby, it became feasible to investigate molecular orbitals , observe10This Thematic Issue highlights recent experimental and theoretical developments in realizing and understanding physically and electronically decoupled single molecules and molecular assemblies on surfaces. Several decoupling strategies at the solid–vacuum and solid–liquid interface were explored to elucidate structural, electronic, vibronic, and chemical properties of decoupled molecular structures.S)-camphor on Cu(111) [Physical decoupling by molecular design often relies on non-planar adsorbates with bulky spacer groups, which can adopt various conformations. From a theoretical point of view, finding the energetically most stable conformational structure can be challenging and costly because conventional atomistic simulations are often limited to the partial exploration of the potential energy landscape due to the complexity of the system. Recently developed structure search methods that combine machine learning with density functional theory provide the possibility of reliable structure identification of non-planar molecules, as demonstrated for the example of (1 Cu(111) . Such con-tetradecane) and tip material , which indicates that the ligands have to be decoupled from the substrates [With an appropriate molecular design, the built-in functionality of the active part of the molecule can be preserved upon adsorption on a surface. An example of the preservation of catalytic properties is demonstrated for the redox behavior of manganese porphyrins at the solid–liquid interface. Redox reactions at the axial ligands attached to the metal center of the porphyrin were observed regardless of the type of surface (highly oriented pyrolytic graphite (HOPG) and Au surfaces were used), solvent  as substrate that the HOMO of tetracyanoquinodimethane (TNCQ) is not decoupled because it is located in the MoS2 valence band, while the lowest unoccupied molecular orbital narrows but still suffers from lifetime broadening because it is situated at the conduction band onset of MoS2. Despite this, the vibronic states of the transiently negatively charged TCNQ could be resolved by STS.The semiconducting 2D material MoSd et al.  found ushBN is an insulator and has therefore been widely used to decouple organic molecules from metal substrates. Three articles within this Thematic Issue successfully employed hBN to investigate the pristine properties of particular molecules. Schaal et al. [hBN on Ni(111) electronically decoupled tetraphenyldibenzoperiflanthene such that the molecular vibronic progression was observable by in situ differential reflectance spectroscopy, which is otherwise only achieved for multilayers on the bare Ni. On hBN/Cu(111), Zimmermann et al. [hBN, ordered molecular films were observed. The decoupling even allowed for the formation of complex self-assemblies such as kagome lattices by tuning the number and position of the substituents of the pyrenes derivatives [l et al.  showed tn et al.  could vin et al.  measuredivatives .Metal oxide thin films on top of metal substrates are another interesting class of ultrathin interfacial layers to decouple organic molecules and to enable the study of their electronic properties without the contribution of the underlying metal substrate. Hurdax et al. reported that both charged and neutral species of sexiphenyl can co-exist on thin dielectric MgO films on Ag(100) . Due to 2 thin films on Si(111) [2, and on a thicker CaF2 layer. While isolated PTCDA molecules were pinned to defects on Si and also on the thin CaF2 layer, PTCDA was physically decoupled via the thicker CaF2 films and self-assembled into small islands. For FePc on H-passivated Ge(001), efficient physical decoupling facilitated the growth of large islands with upright oriented molecules, similar to their arrangement in molecular crystals.Physical decoupling of molecules from a semiconducting substrate is discussed for the example of both insulating CaF Si(111)  and hydr Si(111) . In the In summary, the articles collected in this Thematic Issue highlight recent experimental and theoretical developments in the atomic- and molecular-scale understanding of physically and electronically decoupled single molecules and molecular assemblies on surfaces. Over the last decade, significant progress in this field led to a manifold of decoupling strategies for single molecules and surface-supported molecular architectures. Decoupling strategies are highly relevant to preserve the intrinsic structural, electronic, and optical properties of the molecules for performing insightful fundamental surface-science-based studies on them and, thus, play a crucial role in designing new molecule-based electronic and optoelectronic devices.Sabine Maier and Meike StöhrErlangen and Groningen, July 2021


Urinary tract infection (UTI) is one of the most common bacterial infections responsible for increased annual incidence of antimicrobial resistance (AMR) cases. Clinical diagnosis of UTI AMR relies heavily on conventional urine culture and antibiotic susceptibility testing (AST) which has a turnaround time of ∼3 days. Often, irrespective of the infection status, antibiotics are prescribed to patients even before the test results are available, leading to non-judicious use of antibiotics. Over the years, several technologies have been developed for the rapid detection and diagnosis of UTI AMR, however, most of them are limited to traditional microbiological techniques and large laboratory equipment that are not readily available in low-to-middle income countries (LMICs). To address these diagnostic limitations, we are developing a rapid and affordable UTI-AMR diagnostic microfluidic device that is clinical friendly aimed at improving UTI management and AMR stewardship.Escherichia coli expressing kanamycin resistance gene on chemically surface modified polysaccharide-based polymer containing con A and the antibiotic susceptibility of captured bacteria against different antibiotics with and without the porous membrane. We have quantitatively determined the limit of detection of E. coli on multifunctional polysaccharide-based polymer material.Our device enables the flow of a large volume of urine specimens for the capture/enrichment of uropathogenic bacteria and determination of AST via a porous membrane that is augmented with a multifunctional polymer-based material. Important objectives for the development of UTI AMR diagnostic microfluidic device are: (i) development of a multifunctional polymer-based material; and (ii) validation of UTI AMR diagnostic device. We have successfully developed a polysaccharide-based platform to (i) selectively capture uropathogenic bacteria from urine specimen by immobilizing concanavalin A (con A) lectin as bacterial capture agent on the polymer surface via chemical modification; (ii) encapsulate and release bacterial nutrient media and antibiotics for AST; and (iii) detect AST via encapsulation of bacterial growth indicator. In addition, we have also determined the development of methacrylate-based and acrylamide-based synthetic polymer-based material for our application. Further, we have demonstrated the uniform augmentation of the polysaccharide-based polymer onto porous membrane via dip-coating technique for on-chip bacterial capture/enrichment and AST in fluid (urine) flow conditions. The porous membrane is a conducting material which enables us to perform electrochemical measurements such as impedance spectroscopy that accelerates the detection process of antibiotic susceptibility. As a proof-of-concept, we have determined the capture of biosafety level I The utility of the UTI AMR microfluidic device in clinical settings enables clinicians to make informed decisions on the most appropriate antibiotic for treatment in less than a day. Integration of impedance spectroscopy will further accelerate the detection by significantly reducing the time of detection. Further, the device allows for off-chip analysis by retrieving the captured uropathogenic bacteria to perform high throughput sequencing for identifying AMR genetic determinants. Therefore, with the ability to selectively capture uropathogenic bacteria and determine AST in a short time, our technology has the potential to overcome some of the current limitations in UTI AMR diagnostics.


Additionally, VP was significantly lower in animals administered rotigotine compared to those injected with vehicle (p < 0.05). Compared to vehicle-injected animals, subcutaneous administration of rotigotine  led to a significantly higher ICI at 2 h after injection (p < 0.05); however, there was no change in ICI after injection with (+)-SCH23390 hydrochloride. Dermal administration of rotigotine in a rat model of PD could suppress an overactive bladder.Parkinson’s disease (PD) is a neurodegenerative condition caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. As activation of dopaminergic receptors is fundamentally involved in the micturition reflex in PD, the objective of this study was to determine the effect of a single dose of rotigotine ([−]2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin) on intercontraction interval (ICI) and voiding pressure (VP) in a rat model of PD. We used 27 female rats, PD was induced by injecting 6-hydroxydopamine , and rotigotine was administrated at doses of 0.125, 0.25, or 0.5 mg/kg, either intravenous or subcutaneous injection. In rats with 6-OHDA-induced PD, intravenous injection of 0.25 or 0.5 mg/kg rotigotine led to a significantly lower ICI than after vehicle injection ( PD is characterized by both motor and non-motor symptoms, and a recent review has demonstrated that the prevalence of lower urinary tract symptoms (LUTS) ranges from 27 to 63.9% in patients with PD2. Among non-motor symptoms, such as constipation, REM sleep behavior disorder, hyposmia, fatigue, and urinary symptoms, autonomic neuropathy has been reported to greatly impair patients’ quality of life. Specifically, an overactive bladder is seen even in the early stages of PD3 and one study has reported that istradefylline, an adenosine A2A receptor antagonist, can effectively improve not only motor symptoms but also LUTS in patients with PD in clinical settings4.Parkinson’s disease (PD) is a chronic neurodegenerative condition caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. The number of PD patients who are older than 50 years is expected to double by 2030, compared to 2005, due to an aging population and greater life expectancy5.Normal micturition cycles, which consist of filling and emptying, are maintained through coordination between the bladder and the urethra. Cystometry has demonstrated that the detrusor muscle, supplied by the inferior hypogastric plexus, maintains low pressure within the bladder, and that the urethral sphincter, supplied by the inferior hypogastric plexus and the pudendal nerve, closes the bladder outlet during the filling phase. This coordinated activity in the lower urinary tract is mediated by the central nervous system, including the brain and the spinal cord, and the supraspinal site, which projects into the pontine micturition center (PMC), plays an important role in controlling bladder activity7 and that various anti-parkinsonian drugs affect the micturition reflex in this model; specifically, while D1 and D5 receptor agonists can antagonize the micturition reflex (bladder hyperactivity), D2 and D4 agonists have a role in stimulating the reflex8.A previous study has reported that the micturition reflex is accelerated in a 6-hydroxydopamine (6-OHDA)-induced PD model10. The effects of rotigotine have been confirmed in clinical settings, which translate to an improvement in detrusor-related overactive bladder in patients with PD11. Therefore, we investigated the direct effect of rotigotine on the micturition reflex in a rat model of PD.Rotigotine ([-]2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetraline) is a dopamine agonist with an affinity for all dopamine receptors (D1 to D5), which when delivered daily as a single dose through a transdermal patch, provides consistent and stable plasma concentrations over the next 24 hICI and VP values after administration of vehicle or rotigotine  are shown in Table p < 0.05); however, 0.125 mg/kg i.v rotigotine did not significantly affect ICI. Intravenous administration of rotigotine led to significantly lower VP values than after vehicle injection (p < 0.05).In 6-OHDA-treated rats, intravenous injection of 0.25 or 0.5 mg/kg rotigotine led to a significantly lower ICI than after vehicle injection (p = 0.004); 0.25 mg/kg: 9 min 41 s ± 1 min 20 s vs. 10 min 15 s ± 2 min 35 s (p = 0.042); 0.5 mg/kg: 9 min 30 s ± 2 min 3 s vs. 12 min 15 s ± 2 min 19 s (p = 0.002)]  and (+)-SCH23390 hydrochloride (1.0 mg/kg), a D1/D5 receptor antagonist, in PD rats led to a significantly higher ICI at 2 h than after vehicle injection [0.125 mg/kg: 9 min 58 s ± 1 min 51 s vs. 12 min 7 s ± 2 min 0 s  dopamine receptors, and that ICI varies based on route of rotigotine administration, i.e., it significantly decreased following intravenous administration but increased following subcutaneous injection. These contrasting effects of rotigotine may be attributed to complete agonism of the dopamine receptor family  in the micturition reflex and the affinity of dopamine receptors.8, indicating that degeneration of dopaminergic neurons in the substantia nigra pars compacta can cause an overactive bladder. In fact, compared to healthy animals, bladder contraction was elicited at a significantly smaller volume threshold in a primate model of PD induced by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine12. Further, 6-OHDA-treated rats exhibited significantly smaller bladder capacity during micturition, as measured by cystometry8, which is consistent with our data on ICI in PD rats.Previous studies in 6-OHDA-treated rats have demonstrated detrusor hyperactivitymax at 4–8 h following a single subcutaneous injection of 3, 6, or 12 mg/kg in the plasma of rats. But, given the opposing changes in micturition reflex upon two different routes of rotigotine administration described here, we speculate that, following subcutaneous administration, rotigotine may stimulate more D1-like dopamine receptors as if it were a D1/D5 dopamine receptor agonist. In contrast, when administered intravenously, rotigotine may have enhanced affinity for D2/D3/D4 (D2-like) dopamine receptors, mimicking a D2/D3/D4 dopamine receptor agonist. This divergence can be accounted for by the high affinity of rotigotine for D1, D2, and D3 receptors  and a relatively lower affinity for D4 and D5 receptors 13. An additional mechanism may involve D2-like dopamine receptors in a high affinity state14, which would be affected by low concentrations of dopamine.The pharmacokinetic characteristics of rotigotine include a median T8. These previous findings suggest robust D2-like receptor activation immediately after injection and gradual D1-like receptor activation, probably due to differences in the level of affinity, blood concentration, and/or the effect of the dopamine family on the micturition reflex. Similarly, another report has stated that a D2-like agonist seemed to reduce bladder capacity due to worsening of detrusor overactivity in patients with PD15. Therefore, rotigotine may predominantly act as a D1-like dopamine receptor agonist rather than as a D2-like receptor agonist, thereby affecting the micturition reflex. Moreover, apomorphine, an anti-parkinsonian drug, has a higher affinity for dopamine D2 receptors and displays a dose- and time-dependent biphasic effect on bladder filling16. Additionally, low-dose apomorphine induced an initial increase in micturition volume, while medium- and high-dose apomorphine first decreased micturition volume and subsequently increased it. As an improvement in clinical motility and bladder function due to rotigotine has been reported in PD17, we speculated that transdermal delivery of rotigotine can improve bladder function in clinical use.Previous reports of dopamine agonists on micturition in 6-OHDA-treated rats show that, compared to ICI of 6.2 ± 0.4 min in controls, a dose of 0.5 mg/kg significantly increased ICI to 7.9 ± 0.5 min and to 9.3 ± 0.7 min at 1.0 mg/kg; in contrast, a D2-like receptor agonist significantly reduced ICI to 4.1 ± 0.5 min at 0.2 mg/kg and 2.3 ± 0.3 min at 0.4 mg/kg compared to 6.3 ± 0.6 min in control animalsWe demonstrate significantly greater ICI values after subcutaneous administration of rotigotine compared to vehicle injection in PD rats; however, no change was seen after injection with (+)-SCH23390 hydrochloride, which is a D1 antagonist. Thus, we hypothesized that ICI increases after subcutaneous rotigotine injection but that it decreases after (+)-SCH23390 hydrochloride, and hence, administered the antagonist only after confirming changes in ICI at 2 h after subcutaneous rotigotine injection.8. It has been reported that D1 agonists can inhibit the micturition reflex in a rat model of PD, suggesting that D1-like dopamine receptors may inhibit the micturition reflex8. (+)-SCH23390 hydrochloride facilitates the micturition reflex in normal conscious rats18, and congruently, inhibition of bladder hyperactivity was reversed by intracerebroventricular administration of SCH23390 hydrochloride after the micturition reflex was inhibited by stimulation of the substantia nigra pars compacta in cats19. Thus, the increase in ICI following rotigotine administration may be due to its effect on D1-like dopamine receptors. In contrast to a previous report where (+)-SCH23390 hydrochloride facilitated micturition reflex18, no changes were observed upon the use of a D1-like dopamine receptor antagonist in our experiment even though both dose and time were optimized based on pharmacokinetics20. SKF 38,393, a D1 agonist, has been shown to be effective in inhibiting bladder hyperactivity8 and other neurotransmitters might be involved in regulating bladder function in PD. Notably, clinical and animal studies have not been able to completely elucidate lower urinary tract dysfunction in PD, and the results of the current study can be attributed to the complexity of dopamine receptor activation.Dopamine released from the substantia nigra and the striatum can inhibit bladder contraction by stimulating D1 receptors on striatal neurons related to bladder storage; however, dopamine depletion due to degeneration of the neurons of the substantia nigra and the striatum can reduce such stimulation of D1 receptors, resulting in impaired bladder storage. In PD, bladder hyperactivity is suppressed by D1 agonists, and compared to sham rats, bladder activity was facilitated in a PD model, indicating that dopamine depletion can cause bladder hyperactivity8. Rotigotine is a unique dopamine agonist with an affinity for all dopamine receptor subtypes, which may be because while other neurotransmitters bind to the site projecting into the PMC, including γ-aminobutyric acid (GABA) and/or adenosine. Further, an in vivo microdialysis study by Kitta et al. demonstrated that a GABAergic mechanism is involved in the midbrain periaqueductal gray projection to the PMC21. The micturition reflex is under dopaminergic regulation through D1 receptors with potential GABAergic involvement. Micturition-induced inhibition of GABA was not observed in rat model of PD, and 6-OHDA-treated rats exhibit bladder hyperactivity, which mimics facilitation of the micturition reflex induced by D1 receptor blockade.It is known that stimulation of dopamine D2 and D4 receptors, but not the D3 receptor, can facilitate the micturition reflex, whereas stimulation of D1 and D5 receptors can inhibit the micturition reflex22. A prolonged micturition reflex after subcutaneous rotigotine administration in this study prompted us to hypothesize that rotigotine-induced inhibition of micturition reflux was due, at least in part, to D1-like dopamine receptor agonism. Hence, we examined the potential involvement of D1-like dopamine receptors in rotigotine-induced inhibition of micturition reflux. To prove this hypothesis, we deem it important to investigate the effects of GABA in future studies. Similarly, adenosine A2A receptor antagonism significantly increases ICI compared to pre-administration in PD rats, indicating that the adenosine A2A receptor may be involved in bladder overactivity6. Also, it has been reported that rotigotine may have higher binding affinity for 5-HT receptors24.In the -supraspinal pathway under normal conditions, tonic activation of dopaminergic neurons in the substantia nigra pars compacta activates D1-like dopamine receptors expressed on GABAergic inhibitory neurons in the striatum to inhibit the micturition reflex. In contrast, dopaminergic neurons in the substantia nigra pars compacta are lost in PD, leading to the loss of D1-like dopamine receptor activation and reduced activation of inhibitory GABAergic neurons in the striatum11. Our basic findings show that subcutaneous administration of rotigotine significantly improved urinary symptoms in a rat model of PD, which supports results from previous clinical studies. The rotigotine transdermal patch (Neupro®) is routinely used in the clinic after being approved by the European Medicines Agency in 2006 and the Food and Drug Administration in 2007. It is well-known that PD is characterized by a profound disruption of motor function, including bradykinesia, tremor, rigidity, perturbed gait, and postural instability3. A meta-analysis found that rotigotine, launched as a dopamine receptor agonist, can reduce motor dysfunction and improve activities of daily living when used in the treatment of idiopathic PD25. As PD symptoms are caused by a massive loss of dopaminergic neurons from the substantia nigra pars compacta, rotigotine would improve not only motor symptoms but also micturition-related symptoms after transdermal administration, apart from its overall effects to ameliorate pollakiuria in PD patients.A clinical study investigating the effect of rotigotine on bladder function in PD patients reported that bladder overactivity was suppressed, probably due to stimulation of D1 receptorsWe provide corroborating evidence that an overactive bladder is suppressed by the administration of rotigotine in rat model of PD and that transdermal administration, rather than an intravenous route, is critical for the control of the micturition reflex. Importantly, as this mechanism could have a role in activating D1-like dopamine receptors, transdermal administration of rotigotine could be effective in improving not only motor symptoms but also an overactive bladder in patients with PD.Female Sprague–Dawley rats (n = 27), aged 12–14 weeks and weighing 250–375 g, were used in this study. All experiments conformed to National Institutes of Health animal care guidelines. This study was approved by the Hokkaido university ethics committee  and conformed to ARRIVE guidelines.6. Additionally, 6-OHDA (10 μg) was injected into the lateral striatum of rats before intravesical pressure was measured to obtain a rat model of unilateral PD. Motor dysfunction was confirmed in these animals to establish PD.Rats were intraperitoneally injected with 60 mg/kg of pentobarbital  before surgery for producing unilateral 6-OHDA-induced lesions. 6-OHDA  was microinjected unilaterally into the left substantia nigra pars compacta of the brain at a point 5.3 mm posterior to the bregma, 8.0 mm ventral to the dura, and 2.2 mm laterallyRats were anesthetized with isoflurane  and urethane . The lower abdomen was dissected, polyethylene tubing  was inserted into the bladder dome to record bladder filling and pressure, and then ligated using thread. After confirming that no liquid was leaking from the bladder dome, the abdominal wound was sutured. Rats were allowed to stabilize for approximately 2 h before the experimental protocol was initiated.26.ICI and VP for micturition were measured using cystometry Fig. , as prevICI was defined as time-to-contraction under constant filling conditions and VP was defined as maximum intravesical pressure obtained upon detrusor contractility for micturition. In addition, PRV was measured by withdrawing intravesical fluid through the catheter using gravity before drug injection and at the end of the micturition reflex after drug administration. PRV was measured to confirm that changes in ICI caused by the drugs were not due to increased PRV. The bladder was emptied using a syringe after micturition to measure residual volume. For cystometry, a catheter from the bladder was connected via a three-way stopcock to a pressure transducer for monitoring bladder pressure. Saline at room temperature was continuously infused into the bladder using a syringe pump  at a constant rate (3 mL/h). These parameters were monitored by a pressure amplifier, incorporated into a personal computer via a PowerLab system analog-to-digital converter , and recorded on chart data acquisition software at a sampling rate of 400 Hz. Cystometry to assess the effects of drug administration was initiated after stable bladder pressure baseline data were obtained for 60 min.8. Intravenous rotigotine was administered at least 3 micturition reflexes after vehicle injection. Drugs for intravenous administration were dissolved in saline   was administrated cumulatively, either intravenously or subcutaneously, to a rat model of PD. Cystometry was conducted 1–2 h after inserting the bladder catheter and was based on previous experiments of bladder function in parkinsonian ratsCystometry was performed after at least three micturition reflexes post (+)-SCH23390 injection, which was dissolved in cone oil  for subcutaneous administration -SCH23390 hydrochloride  was intravenously administrated after at least 3 micturition reflexes and at 2 h after rotigotine injection (0.25 mg/kg s.c.)  was administered as the intravenous vehicle while cone oil (0.1 mL/kg s.c) was administered as a subcutaneous vehicle. (+)-SCH23390 hydrochloride was administered intravenously at 1.0 mg/kg. All doses were determined based on previous reportsp < 0.05 was considered significant.The results of urinary variables, such as ICI and VP, are expressed as the mean ± standard error, and data in the PD model were statistically analyzed using repeated analysis of variance and Tukey’s test;


Ceramic fiber photocatalysts fabricated by electrospinning hold great potential in alleviating global environmental and energy issues. However, many challenges remain in improving their photocatalytic efficiencies, such as the limited carrier lifetime and solar energy utilization. To overcome these predicaments, various smart strategies have been invented and realized in ceramic fiber photocatalysts. This review firstly attempts to summarize the fundamental principles and bottlenecks of photocatalytic processes. Subsequently, the approaches of doping, surface plasmon resonance, and up-conversion fluorescent to enlarge the light absorption range realized by precursor composition design, electrospinning parameter control, and proper post heat-treatment process are systematically introduced. Furthermore, methods and achievements of prolonging the lifetime of photogenerated carriers in electrospun ceramic fiber photocatalysts by means of introducing heterostructure and defective composition are reviewed in this article. This review ends with a summary and some perspectives on the future directions of ceramic fiber photocatalysts. Electrospinning is a fascinating technique that allows the fabrication of continuous fibers with diameters down to a few nanometers ,2,3. Sin2 electrode in 1972 [Energy shortages and environmental pollution are urgent issues facing humanity, despite the fact that the solar energy reaching the Earth far exceeds the global energy consumption ,19. Semi in 1972 , photoca in 1972 ,25,26. N in 1972 ,28,29. A2 nanofibers [The further application of photocatalytic technology is mainly restricted by two aspects: (1) the narrow light response range; and (2) the limited photogenerated carrier utilization. During the past decades, a variety of strategies have been employed to improve the photocatalytic efficiencies of electrospun ceramic fibers. To expand the range of light response and develop visible-light-response photocatalysts, doping , solid snofibers . We beliEg) can absorb photons whose energy is equal to or greater than their band gap energy. Then, the electrons (e−) at the valance band (VB) will be excited to the conduct band (CB), leaving an equal number of holes (h+) at the VB. The photogenerated e− and h+ at CB and VB hold different chemical potentials according to the band position and will participate in the subsequent reduction and oxidation reaction, respectively [−/·OH reaction, the holes at VB can react with absorbed OH− and produce hydroxyl radicals with high oxidation activity for pollutant decomposition [+/H2, hydrogen can be generated with the assistance of photogenerated electrons [2 (Eg = 3.2 eV) can only be excited by ultraviolet (UV) light, which occupies 4% of the solar spectrum. Second, the photogenerated e- and h+ pairs recombine easily and rapidly, accompanied by heat or light emission. Therefore, developing visible-light-response semiconductor photocatalysts and improving the utilization of photogenerated carriers still pose great challenges.From the perspective of photochemistry, semiconductor photocatalysts can achieve the aims of degradation, water splitting, and hydrogen production by initiating a serial of redox reactions under irradiation ,18. Howeectively . For exaposition . Similarlectrons . During Taking advantage of the high aspect ratio, good flexibility, proper surface area, and ease of recycling, 1D nanofibers are attractive for practical applications as photocatalysts ,47. For The setup of an electrospinning device is not complicated; typically, a high-voltage resource, syringe, metallic needle, and a grounded collector are enough , as showAlthough the setup for electrospinning is simple, its mechanism is intricate and involves several physical instability processes . Before 2O, and acetic acid as starting materials, and calcinated the precursor fibers at 400 °C for 2 h in air to obtain anatase TiO2 nanofibers [2 nanofibers. With a decrease in the diameter from 245 to 205 nm, the photocatalytic activity of the TiO2 nanofiber is improved. However, further reduction in the diameter below 205 nm results in a deterioration in the photocatalytic activity, as shown in 2 nanofibers with a diameter around 200 nm show a better performance, which is comparable with that of the commercial TiO2 photocatalyst nanopowders . In addition to TiO2, ZnO is also a representative photocatalytic material with a direct band gap (Eg = 3.4 eV), strong oxidation ability, and a large excitation binding energy, and can also be electrospun into nanofibers. Lin et al. prepared ZnO nanofibers by electrospinning and demonstrated its good photocatalytic property under UV light irradiation [2 and ZnO ceramic fibers need to absorb UV light to initiate the photocatalytic process. The search for novel visible-light-response ceramic fiber photocatalysts has been the main research trend in recent years.For example, titanium dioxide is a pioneer of electrospun ceramic fiber photocatalysts, and is also a popular, excellent, and commercialized photocatalyst ,81,82. Lnofibers . They foadiation . However5 TW), in which visible light is abundant, exceeds the global human energy consumption (16 TW) by nearly four orders of magnitude [Efficient utilization of solar energy is essential for photocatalysts. The solar energy reaching the surface of the Earth  and monoclinic sheelite (s-m) phase BiVO4 nanofibers for the first time by electrospinning [4 nanofibers was around 2.55–2.58 eV, which is suitable for visible light irradiation. The results of RhB degradation demonstrated that more than 70% RhB was degraded by both single s-t and s-m phase BiVO4 nanofibers after 40 min visible light irradiation. A mat comprising electrospun Bi2WO6 nanofibers with a band gap of 2.68 eV exhibited enhanced photocatalytic activity in the decomposition of CH3CHO for CO2 evaluation. Shang et al. proposed that the hybridization of the Bi 6s and O 2p largely dispersed the VB, which facilitated the transfer of the generated charge carriers, thereby enhancing the photocatalytic oxidation of organic pollutants [In recent years, a large amount of research has been devoted to finding visible-light-response photocatalysts, such as BiVO4 , Bi2WO6 4 [2WO6 , AgNbO3 [2WO6 [3 , AgVO3 [O6 [3 [3 , Ag3VO4 [3 [3VO4 , Ag3PO4 O4 [3PO4 , CdS [67PO4 [2O5 , and InV [2O5 [4 . Due to spinning . The banllutants . AlthougThe light-absorbing range, limited by the intrinsic band gap, determines the theoretical solar energy conversion efficiency of semiconductors. To date, significant efforts have been made to expand the light-absorbing range of semiconductor photocatalysts from UV light to visible light. Doping, surface plasmon resonance, and up-conversion fluorescence have been widely proven to be efficient approaches and can be realized in electrospun ceramic fiber photocatalysts when controlling the precursor composition, electrospinning parameters, and the thermal treatment process.4) in the precursor solution [2/polyacrylonitrile (PAN) composite nanofibers were prepared using a mixture of dimethylformamde (DMF) and N-methyl-2-pyrrolidone (NMP) as solvents [2/PAN nanofibers demonstrated a relatively strong absorption in the visible region, whereas the pure PAN, TiO2 and TiO2/PAN nanofibers has no significant absorbing peak. Thus, up to 90% degradation of MO was achieved by the PANI modified TiO2/PAN nanofibers in less than 60 min in comparison with the neat nanofibers (about 10%). Moreover, the choice of salt is also important. Instead of the normally used nitrate, Lv et al. utilized the organic salt (Vanadium(IV)oxy Acetylacetonate) to induce the electrospinning gradient effect [4 could be fabricated by single-spinneret electrospinning without a template. Benefiting from the unique tubular structure, BiVO4 nanotubes possess a hollow interior, leading to strong light harvesting ability and better photocatalytic performance than solid BiVO4 nanofibers. The authors proposed that this gradient effect is not subject to the usage amount, ratio, or kind of as-employed salt, which can also be applied for the fabrication of other tubular ceramic photocatalysts.The precursor solution for electrospinning is composed of the soluble salt of target components, the polymer skeleton, and solvents. The choice of polymer, solvent system, and concentration have a great influence on the crystal structure, facet, and morphology of the electrospun ceramic fibers, thereby changing their photocatalytic performance. For example, Li et al. found that the diameter of titanium oxide fibers increases linearly with the increase in the concentration of tetrabutyl titanate (Ti(oBu)solution . Becausesolution ,66,67, csolution , metal msolution , and polsolution , that hasolvents . The autt effect . Then, hd orbital to build a t2g impurity level and let the absorption edge of semiconductor red shift [3+-doped TiO2/SnO2 necklace-structured nanofiber by electrospinning [3+ and hybrid component SnO2 were introduced into the precursor solution by adding Fe(NO3)3·9H2O and H2SnO3 nanoparticles. The XRD results shown in 2 lattice with no third phase other than anatase and rutile appearing. They found that TiO2/SnO2 nanofibers with 0.5 at.% Fe doping possess the best photocatalytic performance, which exceeded that of non-doped nanofibers by a factor of more than 3  ,103,104.adiation . Gao et nofibers . They pr2 fiber photocatalyst based on the up-conversion luminescent effect [2 nanofibers were prepared by electrospinning and a subsequent calcination process. Then, glucose was converted to CQDs under the assistance of alkali and ultrasound. After immersing TiO2 nanofibers into the CQD solution, ultrasonic treatment began and the time was controlled to obtain the different thicknesses of carbon shells in which CQDs were dispersed  and pure CQDs. The C2 samples had the best performance with complete RhB degradation achieved in 20 min of visible light irradiation  with an up-conversion luminescence effect, can absorb visible or even near-infrared light and emit ultraviolet or visible light ,108,109.t effect . This naispersed a,b. Finaadiation d. Moreov2 nanotubes were fabricated by coaxial electrospinning using the PVP solution as the core fluids and TiO2 sol as the shell [2 nanotubes and solid TiO2 nanofibers by MB degradation and confirmed the superior performance of hollow TiO2 nanotubes [After completing the blend of the precursor solution, the variety of resulting fiber architectures becomes even greater with spinning parameter design. The main parameters are spinneret structure, number of nozzles, applied voltage, and tip to collector distance. By controlling the voltage and tip to collector distance, the intensity and distribution of the electrostatic field can be changed. Then, various microstructure containing soft surface, rough surface and beaded structure can be obtained in electrospun ceramic fibers. Lin et al. found that the jet diameter was a strong function of the applied voltage and the nozzle-to-ground distance and the formation of beads structure appeared with the increase in applied voltage . Coaxialhe shell . Chang eanotubes . The mor2 nanofibers with different phase ratios by controlling the calcination temperature [3 to prevent the transfer of the doping element Mn2+ to Mn4+ and synthesized a visible-light-response Mn-ZnO ceramic fiber photocatalyst [3N5 [2ZnSnS4 [2S4 [2O2S [p and S 3p orbitals can help to form a valence band at potentials more negative than those of O 2p orbitals, resulting in a narrow band gap. Electrospinning can also be used to fabricate oxynitride nanofibers by controlling the calcination atmospheres. Li et al. annealed the electrospun TiO2 nanofibers under NH3 atmosphere and obtained surface nitride N-TiO2 nanofibers  materials has led to new research directions [It can be seen that the electrospun ceramic fibers possess excellent photocatalytic activity and are suitable for the water treatment process. Their performance usually exceeds the particle catalysts with the same composition and the commercial TiOrections . HE materections . However−) at the valance band (VB) will be excited to the conduction band (CB), leaving behind an equal number of holes (h+) at the VB, as illustrated in When the light shines on electrospun ceramic fiber photocatalysts, electrons (em > φs), electrons will pass from the semiconductor into the metal to equalize the Fermi energy levels and bend the energy band upward [4 [3 [2 [4 [The introduction of well-dispersed metal particles into the electrospun ceramic fibers forms a metal–ceramic heterojunction. The rectifying characteristics of a metal–semiconductor system can provide an efficient and feasible pathway for carrier separation . For exad upward , as illud upward , Ag-BiVOpward [4 , Ag-BaTird [4 [3 , Au-TiO2[4 [3 [2 , and Pt/[3 [2 [4 .Lin et al. added silver nitrate into the precursor solution and fabricated Ag-ZnO nanofibers by electrospinning . During 4 with a narrow band gap to construct a novel heterojunction with Ag [4 nanofibers with different Ag contents were fabricated by electrospinning. Then, their photocatalytic activity was determined by dye degradation under visible light. As shown in 4 nanofibers by introducing Ag particles. The photocatalytic activity of Ag-BiVO4 nanofibers increased with the increase in Ag content, and the composite fibers containing 10 at.% Ag showed the best performance, with almost 100% RhB degradation achieved in 20 min. By band structure analysis, they proposed that the photogenerated electrons migrated from CB of BiVO4 to Ag, impeding the recombination of photogenerated carriers  heterojunction, as illustrated in ase TiO2 . The anaficiency b.4 nanofiber [2-SnO2 nanofibers [3PO4-TiO2-CNFs [2 nanofibers [−1, which is 10 times higher than that of pure ZnO, as shown in Various ceramic fiber photocatalysts with a type 2 heterojunction have been developed in recent years, including ZnO-ZnS core-shell nanofibers , tetragoanofiber , MnO-ZnOanofiber , TiO2-Snnofibers , Ag3PO4-iO2-CNFs , and Carnofibers . In a tynofibers . The thi4 nanofiber with tetragonal-monoclinic phase junction by electrospinning [4 nanofibers calcinated at 450 °C exhibited the best photocatalytic activity under visible-light irradiation with an optimal rate constant and apparent quantum efficiency that was 10 times higher than that of the single tetragonal phase BiVO4 and 5 times higher than that of the monoclinic phase BiVO4 1−x nanofibers through atmosphere sintering and investigated their photocatalytic performance and mechanism [x(GaN)1−x nanofibers and becomes colorless (k of 0.058 min−1 and apparent quantum efficiency (AQE) of 29.4%, which is about 40 times greater than that of the nanowires calcined at 600 °C (x(GaN)1−x nanofibers show very good stability in the cyclic test (x(GaN)1−x nanofibers, as shown in Surface defect engineering has been found to be the critical factor to improve photocatalytic activity, and can trap the photogenerated electrons or holes and modify the phonon transport ,130,131.echanism . The chaolorless b. By dynt 600 °C c. Moreovlic test d. The au2+-doped and N-decorated ZnO nanofibers (NFs) enriched with oxygen vacancy defects by electrospinning [O*), helping the holes release from the catalysts, and consequently delaying the recombination process  the fundamental principles and bottlenecks of the photocatalysis process; (2) UV and visible-light-response ceramic fiber photocatalysts prepared by electrospinning; (3) the strategies used to expand the light absorption range of ceramic fiber photocatalysts; and (4) various types of heterostructure used for efficient carrier separation. At present, electrospun ceramic nanofibers with adjustable band structure have great application potential in the field of photocatalysis, and the electrospinning technique offers a scalable, easy, and low-cost route to produce ceramic fiber photocatalysts. Due to the efforts of many research groups, electrospinning has been developed as a technique capable of producing a variety of ceramics and composite fibers with fine structure and controllable morphology.In the future, the photocatalytic efficiency and quantum yield of ceramic fiber photocatalysts should be enhanced for practical solar engineering. It is expected that novel strategies for improved light-harvesting and carrier separation will be realized in electrospun ceramic fibers. Moreover, the development of directly electrospun ceramic fabric or a mat with high solar energy conversion efficiency will become important, especially for the application to water splitting and hydrogen production. Regarding electrospinning technology, mass production of functional fibers with designed and controllable morphology will be one of the leading development directions. Then, combining the electrospinning techniques with other well-developed techniques for materials processing can increase the diversity of material and structure types, and open provide access to new properties and applications. As a result of the rapid innovation of science and technology, electrospinning will surely become an effective method for making 1D nanomaterials with a fine structure and ideal performance that is not limited to the field of photocatalysis.


Applying nanosized ultrasound contrast agents (nUCAs) in molecular imaging has received considerable attention. nUCAs have been instrumental in ultrasound molecular imaging to enhance sensitivity, identification, and quantification. nUCAs can achieve high performance in molecular imaging, which was influenced by synthetic formulations and size. This review presents an overview of nUCAs from different synthetic formulations with a discussion on imaging and detection technology. Then we also review the progress of nUCAs in preclinical application and highlight the recent challenges of nUCAs. Nanosized ultrasound contrast agents (nUCAs), as a complementary of microbubbles, are developed as contrast enhancers for ultrasound molecular imaging with the ability of penetrating through vasculature for extravascular imaging . High acTo date, the existing formulations of nUCAs are post formulations of microbubbles, such as centrifuge and filtration. However, post formulations may influence the stability of nUCAs . NotablyUltrasound imaging has been widely used in clinical fields because of relatively low price, no need of radiation, and noninvasiveness. The principle of early ultrasound imaging is linear imaging, but ultrasonic pulse through tissues and contrast agents would, respectively, produce nonlinear propagation and nonlinear vibration to produce nonlinear acoustic signal . In the late 1990s, harmonic signals generated by the nonlinear effects of tissues and contrast agents have been used in ultrasound imaging. According to the different sources of harmonic generation, harmonic imaging can be divided into tissue harmonic imaging and contrast harmonic imaging. A variety of experimental studies show that harmonic imaging has better spatial resolution, higher contrast, and clearer image sharpness.When the pressure of ultrasonic pulse has reached a certain threshold, the frequency of the emitted wave is double than that of ultrasound contrast agents, which can produce a second harmonic signal and ultraharmonic signal to achieve a better imaging. If the frequency fails to achieve threshold, subharmonics and ultraharmonics will still be produced, but the effect is not obvious. Notably, tissue does not produce subharmonics and ultraharmonics. This further improves the contrast between tissue and contrast agents. However, the size of contrast agents has significant influence on the signal; a smaller size, such as NBs, may produce a low signal. In clinical practice, harmonic imaging contains fundamental wave and harmonic wave. The intensity of harmonic wave is much weaker than the fundamental wave, and the signal-to-noise ratio is low. Thus, it requires high sensitivity of equipment to detect and increase the difficulty of equipment development.in vivo , and then a high powered ultrasound pulse was applied to destroy the UCAs in the examined area. The difference in signals before and after destruction would be expressed as targeted UCA signals . Anotherin vivo .Nanobubbles (NBs) are nanoparticles (in the nanometer range) that are commonly composed of an encapsulating shell and gas core . Accordi6 or C3F8  . Gas vesGas vesicles have a protein shell with a hollow gas-filled core, with a dimension of ~200 nm and a thickness of ~2 nm. GVs are encoded by 8–14 genes, including the primary structural proteins GvpA and GvpC, and several secondary proteins that function as essential minor constituents or chaperones . GvpA isUltrasound molecular imaging relies on delivering UCAs to a specific site. There are two different classification methods of strategies involving the coupling of UCAs for targeting: 1) passive targeting or 2) active targeting .Passive targeting is related to the so-called enhanced permeability and retention (EPR) effect, which is a feature of many tumors and diverse inflammation sites. The pathological changes in blood vessels and normal blood vessels on the structure and morphology is different, the gap between the endothelial cells can be widened to 800 nm, and lymph is blocked at the same time. These two major reasons prompt nUCAs to seep in the enhancement of the lesion site and retention.via thiol-maleimide and coupled to a thiol-activated ligand. Albumin-shelled NBs also can bind to activated leukocytes via β2-integrins on leukocytes (Active targeting includes noncovalent conjugation and covalent conjugation. Noncovalent conjugation is similar to the modification and conjugation of ligands. Incorporation of phosphatidylserine in the shell of NBs results in activation and surface attachment of complement fragments because of a highly negative charged shell . In addiukocytes  and compukocytes . In a moukocytes . To reduukocytes .Ultrasound molecular imaging could potentially be used in early detection, monitoring treatment effects, and delivery of drugs. To date, there are various preclinical applications to explore the effect of targeted nUCAs .in vitro using PLGA-linked NBs to target residual tumors (New effective targets overexpressed on tumor tissue and neovasculature are regarded as a breakthrough for early accurate diagnosis and characteristic. Guo et al. constructed lipid nanobubbles combined with AS1411 to highly target nucleolin in triple-negative breast cancer, which can realize molecular imaging of tumor tissues and neovasculature to provide an early detection method . In addil tumors , as an al tumors . Using tl tumors .in vivo by quantitative analysis of the ultrasound contrast signal intensity. In order to monitor the survival of ovarian cells in the early transplantation, MU et al. developed AMH-targeted nanobubbles by integrating an AMH antibody onto the surface of NBs. Evidence showed that the ultrasound signal was relative with the expression of AMH in transplant time (The expression level of the target molecules, which reflects the diseased or abnormal status, can be evaluated ant time . In addiant time .in vitro silencing of targeted genes (Recently, NBs have been researched with regard to the efficient delivery of exogenous genes and drugs noninvasively . A recened genes .Microsized ultrasound contrast agents have been commercialized and used in diagnosis and adjuvant treatment with less and mild adverse reactions. In contrast, the research of nUCAs have been widely explored , while rThe feasibility of ultrasound molecular imaging studies has been demonstrated in numerous preclinical studies and used in different disease models. Therefore, ultrasound molecular imaging has been used in understanding the progression of disease mechanisms, which has also been used in preclinical testing of the efficacy of new drugs. This field is expected to expand with better standardization of target contrast agents and ultrasound imaging protocols. As more and more targeted nanosized ultrasound contrast agents are developed, the ability to characterize specific disease phenotypes will become critical. However, the widespread use of nUCAs in the clinic will depend on additional, extensive, and larger clinical trials that demonstrate safety. In addition, the integration of clinical information obtained from molecular imaging will need to be integrated into diagnostic and therapeutic pathways to improve diagnostic accuracy.Nanosized ultrasound contrast agents have been successfully used in early diagnosis and response to therapy.Nanosized ultrasound contrast agents have several challenges, such as the decrease in signal intensity, complex preparation process, the low concentration in the targeted area, and so on.


Over the past decades, clinical features and patients’ outcome of iatrogenic and HIV-related KS epidemiological subtypes have been widely described in large cohort series. Due to their lower incidence and the limited resources available in endemic KS countries, classical and endemic KS epidemiological studies remain scarce, thus increasing the challenge of such clinically heterogeneous chronic diseases’ management. In this large retrospective cohort study, six risk factors for treatment initiation were identified: time between first symptoms and diagnosis ≥1 year, endemic KS, total number of lesions ≥10, visceral or head/neck localization and edema. No response or treatment-free time difference was observed between the most frequently used therapeutic options: chemotherapy and interferon-alpha. Assessment for systemic treatment risk factors provides guidance for adequate follow-up and patients’ information on disease outcome. Absence of efficacy difference between systemic regimens allows treatment choice based on fitness.Background: Although several studies described the clinical course of epidemic and post-transplant Kaposi’s Sarcoma (KS), the lack of large cohorts of classic/endemic KS, precluded such characterization. Methods: We used multi-state modelling in a retrospective monocentric study to evaluate global disease evolution and identify risk factors for systemic treatment (ST) initiation. 160 classic/endemic KS patients consecutively diagnosed between 1990 and 2013 were included. Results: 41.2% of classic/endemic KS patients required ST. Cumulative incidence of ST after 2 years of follow-up was 28.4% . Multivariate analysis identified six risk factors for ST initiation: time between first symptoms and diagnosis ≥1 year, endemic KS, total number of lesions ≥10, visceral, head or neck localization and presence of edema. Type of ST, type of KS, age and time between diagnosis and ST were not associated with response. Mean treatment-free time during the first 5 years following ST was 44 months for interferon and 44.6 months for chemotherapy treated patients . Conclusions: Our study reveals ST risk factors in classic/endemic KS and highlights the clinical aggressiveness of the endemic KS subtype. No efficacy difference was observed between standard of care treatments, enabling treatment choice based on patient’s fitness. KS is an HHV-8 related lympho-angioproliferative disease with 4 clinical settings: iatrogenic (immunosuppressive therapy related), epidemic (HIV immune-deficiency related), endemic, and classic. Endemic KS develops in Sub-Saharian Africans whereas classic KS typically affects middle to elderly Mediterranean men with a male to female ratio ranging from 2:1 to 5:1 and an estimated incidence of 1.58 per 100,000 inhabitants per year in Sardinia  and 44.9 months  respectively .The mean treatment-free time during the first and the 5 first years post-treatment initiation was not significantly different between classic (5.6 and 45.3 months at 1 and 5 years respectively) and endemic KS (4.8 and 43.5 months at 1 and 5 years respectively)  a,b.During the first-year post-treatment initiation, the mean treatment-free time was higher in chemotherapy-treated patients (7.3 months) compared to interferon-treated patients (3.5 months) . However, this difference was no longer observed at 5 years post-treatment initiation , suggesting that the early difference observed is mainly related to a difference in treatment regimens length, rather than a real impact of either regimen on the disease course c,d.Our study provides an overview of clinical characteristics and therapeutic outcome in a large real-life endemic/classic KS cohort. Although classic/endemic KS are thought to have an indolent disease course, they can become symptomatic and require the use of systemic therapy . In our Defining an adequate follow-up frequency adapted to the clinical course of endemic/classic KS, remain challenging for physicians taking care of KS patients .There is no universally accepted staging classification for endemic/classic KS. Three staging systems have been proposed but deserve further validation and are not commonly used in the real-life. They take into account KS lesions localization and skin lesions number, extension and evolution ,11,12. OInterestingly, our study shows that time from diagnosis to systemic treatment initiation does not influence response to treatment. This result allows physicians to adapt treatment initiation to patient’s quality of life requirements, as KS is a chronic disease with no curative therapeutic options available to date, the main objective of systemic therapy remaining an improved quality of life . PhysicaKS systemic therapy mainly relies on chemotherapeutic agents such as liposomal doxorubicin ,14,15,16Endemic KS develops in younger patients and tends to be difficult to treat and have poor clinical outcome with high lymphedema rates ,23. To oFinally, as KS therapy is now entering an exciting immunotherapy avenue, our data offers a baseline for clinical characteristics and outcome of endemic/classic KS in the pre-immunotherapy era. Indeed, KS tumors strongly express the T cell inhibitory molecules PD1 and PDL1  and seveLimitations of our study include its monocentric setting within a specialized outpatient’s clinic. Proportion of patients requiring a systemic treatment may therefore be overestimated, and KS management heterogeneity across different centers needs to be taken into account in our study results interpretation. The wide period of patient’s inclusion ranging from 1990 to 2013 also represents a limitation as this might be responsible for some degree of heterogeneity in terms of first-line treatment modalities.In this study, we report the clinical characteristics and outcome of a large monocentric cohort of endemic and classic KS. The endemic subtype, total number of lesions, visceral or head/neck localization, presence of edema at diagnosis and a higher time between first symptoms and diagnosis were independent risk factors for systemic treatment initiation. No response difference was observed between the standard of care treatments, chemotherapy and interferon, thus promoting the guidance of therapeutic choice following patient’s comorbidities.


Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193). Age-related macular degeneration (AMD) is the most common cause of blindness amongst the elderly in the industrialised world, affecting approximately 36 to 40 million people globally . While tThe complement system is a critical component of the immune system and can be activated via the classical, lectin, or alternative pathway. The first two pathways are activated by binding of their recognition molecules to IgG or IgM immune complexes, C-reactive protein, pentraxin 3, RNA, DNA, extracellular matrix proteins, altered self-structures such as beta-amyloid, apoptotic or necrotic cells, microorganism derived ligands, like LPS or carbohydrate structures on microorganisms or altered self-cells . The altHere, the generation of AAV viral vector constructs carrying the cDNA of human CFI is described and successful in vitro and in vivo expression of active protein in human cell lines and murine retina is shown. These encouraging pre-clinical studies have led to a first-in-human AAV gene therapy trial for late-stage AMD (NCT03846193).The cDNA of the most common human CFI sequence variant was downloaded from Genbank (Accession Number NM_000204.4) and ordered as gBlocks® Gene Fragments from Integrated DNA Technologies; this sequence is referred to as CFI. Four different codon optimised versions of the human CFI sequence were generated using publicly available codon optimising tools ; these codon-optimised (co) sequences are subsequently referred to as CFI.co-1, CFI.co-2, CFI.co-3 and CFI.co-4. All 5 constructs were further modified by insertion of a Kozak consensus sequence at the 5′-end. To generate pAAV.CFI, CFI cDNA sequences were cloned into a synthesised AAV genome backbone, consisting of 5′ and 3’ AAV2 inverted terminal repeats (ITR), a modified CAG promoter  and a bovine growth hormone polyadenylation site. Additionally, a modified Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was added downstream of the CFI cDNA sequence. WPRE was included to enhance short-term and long-term AAV-mediated transgene expression –23 and hAAV vector was prepared by co-transfection of the HEK-293 (ATCC) cell line with pAAV.CFI or pAAV.CFI.co (pAAV.CFI.co-1 to pAAV.CFI-co.4) and pDG (Plasmid Factory), the latter providing adenoviral helper sequences and packaging sequences (Rep/Cap of AAV serotype 2). This serotype was chosen to enable strong transduction levels of the RPE and a lower level of transduction in the neurosensory retina. HEK-293 cells were grown in DMEM (Sigma) supplemented with 10% heat-inactivated foetal bovine serum (Gibco), 1% 200 mM L-Glutamine (Sigma Aldrich) and 1% Penicillin-Streptomycin (Sigma Aldrich). Cells were harvested after 72 h and lysed to purify the viral vector particles. These were purified using iodixanol gradient centrifugation. Vector was concentrated on Amicon Ultra-15 centrifugal filter units (Merck Millipore) and stored at −70 °C. Vector purity was assessed by SDS PAGE and the titre was determined by quantitative PCR.All constructs were tested in vitro prior to in vivo expression studies in mice to confirm AAV, CFI transduction, expression and function. Cells were seeded in DMEM supplemented with 10% FBS at 1E + 06 (HEK-293) or 6E + 05 (ARPE-19) cells per well in a 6-well plate. Undifferentiated ARPE-19 cells were used for all experiments and an AAV carrying a GFP expression cassette was used as a negative control. For transfections, cells were incubated with 2.5 µg plasmid DNA and Lipofectamine LTX (Thermo Fisher Scientific) according to manufacturer’s recommendations. For transductions, cells were transduced at a MOI of 2.5E + 03 with each AAV construct for 16 h, after which cells were washed with PBS and incubated with fresh medium. Cells were incubated for a total of 72 h following plasmid or AAV delivery. Cells and supernatant were collected separately and stored at −70 °C. Cell pellets were divided for simultaneous analysis of protein and RNA expression.Female 8–10 week old C57BL/6 J mice (Charles River Laboratories) were used for all experiments. All animals were treated humanely in accordance with the UK Home Office Regulations under project license 30/3363. Mice were maintained on a 12:12-hour light/dark cycle. Mice were anaesthetised with a mixture of xylazine (10 mg/kg)/ketamine (80 mg/kg) in sterile saline; pupils were dilated with phenylephrine hydrochloride (2.5%) and tropicamide (1%). Proxymetacaine hydrochloride (0.5%) eye drops were used for additional local anaesthesia. An anterior chamber tap was performed prior to the subretinal injection using sterile 33 G needles (TSK Laboratory) and carbomer gel ; and a small circular glass coverslip was used to achieve good visualisation of the fundus. The injection was performed through the posterior retina using a 10 μL NanoFil syringe and a 35 G bevelled NanoFil needle (World Precision Instruments Ltd). Anaesthesia was reversed with atipamezole (2 mg/kg) in sterile saline. Mice were injected with AAV.CFI or AAV.CFI.co-1 viral vector at two different doses (1E + 07 genome copies (gc)/eye [low dose] and 1E + 08 gc/eye [high dose]) in a 2 µL total volume. Prior to injection, vectors were diluted in 0.001% PF68 (Gibco) in PBS (Gibco). Sham injections were performed using diluent only. Per viral vector and dose group, 6 mice were injected in both eyes. After harvesting, eyes were dissected as follows: first, a posterior eye cup dissection was performed in 50 µL of PBS followed by retinal detachment from the RPE/choroid/sclera complex (referred to as “RPE” onwards). After retina and RPE tissue were isolated, both plus the remaining eyeball tissue were flushed with the dissection fluid enriched PBS. These isolated ‘wash’ sample and eye tissues were immediately transferred to dry ice and stored at −70 °C to prevent protein degradation. Tissues from three mice were pooled for protein expression analysis as described above.RNA was extracted from cell pellets (in vitro) or homogenised eye tissues (in vivo) using an RNeasy mini kit (Qiagen) as per manufacturer’s instructions. For quantification of CFI RNA, iTaq Universal SYBR Green Supermix (Bio-Rad) was used as per manufacturer’s instructions; primer sequences are shown in Table Cell pellets (in vitro) or homogenised eye tissues (in vivo) were resuspended in radioimmunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor cocktail (Sigma). Tissues of 3 eyes were pooled to ensure detection of CFI protein. Protein concentrations of cell and tissue lysates were determined using a BCA protein assay kit (Thermo Scientific Pierce) as per the manufacturer’s instructions. Western blotting was performed by separation of proteins by means of SDS PAGE, followed by semi-dry blotting and immunostaining of CFI, GFP or β-actin bands. Equal volumes of cell culture supernatants were loaded and for lysates, normalised amounts of protein, determined by BCA protein assay, were loaded for each group. CFI was detected with a mouse monoclonal antibody to human CFI  or a goat anti-human CFI . GFP was detected with rabbit polyclonal antibody to TurboGFP  and β-actin was detected with mouse anti-β-actin . Protein bands were visualised using Clarity Western ECL Substrate (Bio-Rad) and analysed with an Odyssey® Fc Imaging System. Immunoblots of transfected cells were analysed using Image J software. In brief, bands were selected as region of interest, plotted as histograms and the area under the curve was calculated for each lane.Eyes were removed and a hole was made just behind the ora serrata. The eyeball was placed in 4% (w/v) paraformaldehyde in 0.12 M phosphate buffer, pH 7.2 for 1 hour at room temperature. Next, the anterior segment and the lens were removed, and the eyes were prepared for sections or whole mount. Eye cups used for sections were cryopreserved with increasing concentrations of sucrose rxin 0.12 M phosphate buffer, pH 7.2 , embedded in OCT (Tissue-tek) and frozen in a mould at −70 °C. Sections were cut at a thickness of 10 µm on a cryostat and mounted onto glass slides . The slides were air dried for 2 h at room temperature and stored at −70 °C. For eye cups used for whole mounts, retina and RPE/choroid were separated by gently peeling the retina off the RPE and stored separately in PBS at +4 °C.Sections were blocked and permeabilised by incubation at room temperature for 60 min in PBSGT solution (0.2% (w/v) gelatine, 0.25% (v/v) Triton X-100 in PBS). Whole mounts were blocked and permeabilized by incubation at room temperature for 2 h in 0.2% (w/v) gelatine, 1.5% (v/v) Triton X-100 in PBS. Subsequently, sections and whole mounts were incubated with primary antibodies diluted in PBSGT solution overnight at room temperature. CFI was detected with rabbit anti-CFI  and phalloidin  was stained with Alexa Fluor 488 phalloidin . After washing in PBST solution (0.1% (v/v) Triton X-100 in PBS), sections and whole mounts were incubated with goat anti-rabbit secondary antibody coupled to Alexa Fluor594  and stained with DAPI in PBSGT solution for 1.5 h at room temperature. The slides were washed with PBST solution and subsequently cover-slipped with mounting medium .A C3b cleavage assay was performed to confirm functional activity of CFI expressed after subretinal injection in mice. In this assay, the C3 alpha chain is degraded by CFI into iC3b which can be detected by immunoblotting as a reduction of the alpha chain at 116 kDa and appearance of the two iC3b breakdown bands at 68 and 43 kDa. 20 µL of dissection ‘Wash’ sample collected during dissection of eyes was incubated with 1 µg purified C3b (Complement Technology Inc.) and 0.5 µg purified CFH (Complement Technology Inc.) for 1 hour at 37 °C. The same volume of ‘wash’ sample was standardised amongst all in vivo study conditions to enable accurate comparison of FI functional activity. C3b cleavage products were detected by western blotting using a goat anti-human C3 antibody  and Peroxidase conjugate AffiniPure Donkey Anti-goat IgG .Statistical analysis was performed using GraphPad Prism 8.4.0. If data were normally distributed with equal variance then parametric tests were used (ANOVA) with Tukey’s or Sidak’s multiple comparisons tests . If data were not normally distributed, then non-parametric tests were performed  with Dunn’s multiple comparison test. Analyses performed for each data set are stated in the respective figure legends.Five AAV viral vectors were generated carrying either the cDNA sequence of the human CFI gene or codon optimised versions of the CFI sequence. A schematic diagram of the vectors is shown in Fig. Generated pAAV.CFI and pAAV.CFI.co-1 to pAAV.CFI.co-4 plasmid constructs were transfected into the ARPE-19 cell line to confirm expression of CFI. Cells were co-transfected with a Turbo-GFP plasmid to ensure that cells were equally transfected. CFI was expressed from each construct and protein was detected as a band at 63 kDa Fig. , upper. pAAV.CFI and pAAV.CFI.co-1 were selected and packaged into AAV2 capsids. HEK-293 and ARPE-19 cells were transduced with AAV.CFI and AAV.CFI.co-1 at an MOI of 1E + 04. CFI protein expression and secretion were confirmed by immunoblotting and again did not show differences in secreted levels of CFI Figure .Female 8 to 10-week-old C57BL/6 J mice received a subretinal injection via the transscleral route with two doses (1E + 07 [low dose] or 1E + 08 vg/eye [high dose]) of AAV.CFI or AAV.CFI.co-1 and CFI expression was analysed after 4 weeks. The expression of CFI in lysates of mouse retinal tissues was confirmed by RT-qPCR analysis. The tissue type, dose and injection material had a significant influence on the relative mRNA levels of CFI. A strong dose effect was observed in AAV.CFI in all tissues analysed Fig. ; howeverWestern blotting of lysed tissue fractions revealed CFI protein with bands detected at 50 kDa (heavy chain) and 30 kDa (light chain) Fig. . Both AAImmunohistological analysis of eye cups was performed to confirm RPE expression and to determine in vivo location of CFI. Phalloidin and DAPI co-staining were used to distinguish the different retinal layers. CFI protein was detected in eyes of mice injected with AAV.CFI and AAV.CFI.co-1 and localised mainly in two compartments: strong expression was detected in the RPE and weaker expression in both the outer and inner segments of photoreceptors Fig. . WholemoDue to the small volume in mice, it is not easy to directly analyse the vitreous humour for presence and processing of secreted CFI protein. During dissection of eyes, tissues were rinsed with PBS and used as “wash” sample for functional testing of CFI protein by a C3b cleavage assay. Presence and activity of CFI is measured by degradation of C3b and detected by immunoblotting. Figure This paper describes the development and characterisation of five vectors for a gene therapy to treat AMD. These results show for the first time, in vivo expression of active human CFI protein in mice. By subretinal injection, it is possible to target the majority of AAV.CFI constructs to the RPE which sits between Bruch’s membrane and photoreceptors. CFI is a complement regulator that only through post-translational modification is converted from a single polypeptide chain (pro-CFI) into the dimeric active enzyme. Furin cleaves pro-CFI into a heavy and a light chain  and onlyOf note, western blot and RT-qPCR analysis do not correlate with respect to which tissue has the highest CFI expression; however, a dose-effect is observed in both analyses with AAV.CFI resulting in higher CFI expression levels than AAV.CFI.co-1. The reason for the discrepancy between both methods could be low sample numbers and the fact that three tissue samples were pooled for immunoblot analysis.Codon optimisation as employed in this study is often used to increase levels of recombinant proteins. Most amino acids are encoded by multiple synonymous codons but the relative expression of individual tRNAs can vary dramatically across species and even cell or tissue types . Codon oCurrent drug development efforts and clinical studies for the treatment of AMD and/or geographic atrophy focus on inhibition of the complement system, anti-inflammatory drugs, neuroprotection, reduction of toxic by-products, improvement of mitochondrial function, and stem cell therapies . After aSupplementary Material