Mithril Mutae. A piece of Mithril, a legendary metal renowned for its strength, that was brought to life with alchemy. A bird found in the upper levels of outer Eos Tower of Ludibrium. It used to be able to fly, but a patch removed this ability. Drumming Bunny.
A bunny found in the upper-middle levels of outer Eos Tower of Ludibrium. It bangs its drum when it moves, and is similar to the Energizer Bunny. A yellow toy helicopter found in the lower-middle levels of outer Eos Tower of Ludibrium.
A purple toy plane that found in the lower-middle levels of outer Eos Tower of Ludibrium. A blue toy plane that resides near the lowest levels of outer Eos Tower of Ludibrium. It used to be able to fly, but a recent patch removed this ability. A bird found in the lower levels of the Eos Tower of Ludibrium. They resemble Chirppy, and have the same stats.
A young Homunculus, a person without a soul created through the powers of alchemy. Found deep in the Zenumist Laboratory of Magatia. It has the tendency to spawn in swarms. Reinforced Mithril Mutae. A piece of reinforced Mithril that was brought to life with alchemy. A toy octopus found in the lower-middle levels of inner Eos Tower of Ludibrium. Fairly fast and can jump, but otherwise not much of a threat. An evolved Homun, a failed Homunculus created by the powers of Alchemy.
A robot that looks like a remote that grew legs and walked off, created with alchemy. Neo Huroid. A strange robot with a roughly humanoid appearance, powered by the same stones that keep Orbis afloat and are used in alchemy experiments.
It can fire a small missile out of its mouth. A humanoid robot, connected to a giant chamber containing its mechanical heart. It has a poison mist attack. The strongest technological and monster found in the Alcadno Laboratory of Magatia. You are asked to retrieve its heart for a quest.
A phantom scientist who has seemingly lost his life while doing research, though he hasn't quite given up on his work yet. They can be found in a hidden map off of the Alcadno Laboratory, Area C A ghost NPC named Parwen is also found here. Specter Battle Hound. Specter Battle Hound Quest. Blue Speeyor. A dragon-like monster with leaves on its back, found in Heliseum's Twisted Forest. It can heal itself and nearby allies by 10, It can also cast Avoid Armor, which makes all non-critical attacks miss for a certain amount of time.
To attack you, it can charge energy and release it. Rurumo Silent Crusade. The corrupted version of Rurumo, which must be killed for the Silent Crusade. The potions have switched faces and it now has a dark aura. The closed-eye potion is pink instead of blue, the opened-eye potion is purple instead of red. It has the same abilities as the Rurumo in Mirror World. King Bloctopus. A more powerful Bloctopus.
It attacks by spitting out a small projectile with a long range, but moves slowly. Otherwise, it acts the same as any other Bloctopus. Found in the lower-middle levels of inner Eos Tower of Ludibrium. Block Golem. A huge Golem made out of the same bricks used to build Ludibrium. Plus you will get your tokens and no KSers and tons of people.
I would say Scarecrow to 60, sand rats to 70 and Iron Mutaes till I wouldnt go with Jesters until Jesters are an optimal spot up until level If you want to go past level 80 I'd suggest going to Mysterious Path 3, located in Singapore.
Then got an event chaos scroll and used it. It's the only decent item I have So I used one of those transformation potions from the boxes, and became Von Leon. I messed with peoples in the FM. No ads, no tracking, and no tolerance for hate and discrimination.
Ultimate Screen Talk. Screen Just a picture of really old maple. Ultimate 8. Therefore, we deduce that IONzymes may possess the lipoxidase-like activity in the process of inducing lipid peroxidation to destruct virus.
We found that IONzymes themselves rather than their supernatants destroyed viral particles Figure S In contrast, our recent study reported that Cys-nFeS Nanozymes release polysulfanes into supernatants for antibacterial activity[ 19 ].
The non-specific adsorption of IAVs on IONzymes may provide false-positive antiviral data because we analyzed virus titers using the samples after IONzymes being removed. To check it, IONzymes were mixed with DyLight labeled IAVs for 2 h and the supernatant and sediments were collected to perform immunofluorescence detection, respectively.
We found that fluorescent-labeled viruses were largely existed in supernatants rather than in sediments Figure S18 , suggesting that the decrease of virus titer in supernatants was not due to that IONzymes adsorbed viral particles. Therefore, these analyses confirmed that the viral inactivation was from IONzymes themselves.
Importantly, IONzymes specifically act on viruses containing a lipid envelope, while showing little effect on non-enveloped viruses PCV In addition, it has previously been shown that polyunsaturated fatty acids PUFA -containing phospholipids found in membranes that are highly vulnerable to lipid peroxidation attack[ 38 ].
Hemagglutinin and NA are membrane proteins which are responsible for virus infection to host cells and M1 is a matrix protein to form a coat inside the viral envelope[ 40 - 42 ].
The lipid peroxidation may directly damage the transmembrane counterpart of hemagglutinin and NA or cause a structure change due to envelope disintegration. The western blot analysis verified that the three proteins were destructed. In contrast, we found that peroxidation cannot impact nucleoprotein NP , an internal influenza virus protein, encapsulating the virus genome to form a ribonucleoprotein particles RNP for the purposes of transcription and packaging[ 43 ].
Therefore, our results indicated that the peroxidation damage ignited by IONzymes in viral envelope only passes on the transmembrane proteins and adjacent matrix protein.
Previous a study reported that lipid peroxidation led to the disintegration of lipid membrane and the production of lipid-derived radicals, which ultimately damage the proteins[ 44 ], implying that IONzymes induce the lipid peroxidation of viral lipid envelope and may further locally produce an amount of radicals to destroy neighboring proteins. Importantly, IONzymes inactivate a broad spectrum of influenza A viruses. IAVs can be classified into various subtypes on the basis of antigenic differences in two major surface glycoproteins: hemagglutinin and NA protein, at least 18 hemagglutinin subtypes H1 to H18 and 11 NA subtypes N1 to N11 have been detected[ 45 ].
The common feature is that all subtypes of IAVs contain a typical lipid envelope. In addition, membrane-proximal cytoplasmic tails of HA and NA are highly conserved in sequence in all subtypes of influenza virus[ 46 , 47 ].
IONzymes treatment remarkably disintegrate envelope and destruct hemagglutinin and NA, we hypothesize that IONzymes are active against other viruses containing a lipid envelope. It has been well known that facemasks are very important to protect people from influenza infection in crowded spaces. For instance, live-poultry markets LPMs allows a high frequency of human-poultry contact and results in a five H7N9 influenza epidemic waves in China [ 33 ].
Therefore, such protective equipment is needed to intercept virus transmission from poultry to human. However, most facemasks only provide isolation protection for influenza virus but cannot inactivate the adsorbed virus. This may not only provide insufficient protection to the users, but also become a new contaminated source with high spreading risk because virus accumulated on facemasks still remains infectivity[ 48 ].
Our IONzyme-loaded facemask possesses an outstanding characteristic to fast inactivate multiple IAVs within the mask, implying potential broad applications of IONzymes in bioprotective PPEs against the emerging influenza threats, especially in high-risk places, such as hospitals and LPMs.
Although IONzymes demonstrated high antiviral activity in our study, the catalytic efficiency should to be further improved to allow its use in practical applications. Currently, high concentration of IONzymes and long incubation times are required in viral inactivation, indicating the activity may not be sufficiently high. Therefore, the activity of IONzymes need to be improved through adjusting nanoparticle size, component or surface modification, or designing a novel single-atom nanozyme[ 49 ] in the future study.
In addition, biosafety of IONzymes must be assessed prior use for practical applications. In summary, our findings reveal that IONzymes effectively induced lipid peroxidation in viral envelope and dysfunction of neighboring proteins responsible for host infection.
IONzymes may perform lipoxidase-like activity in the antiviral process. This viral inactivation via liperoxidation may contribute to understanding antiviral property of iron oxide nanoparticles and other inorganic nanomaterials.
In addition, as a typical nanomaterial, IONzymes exhibit features such as multifunctionality, high biocompatibility, high stability and low cost, which will render them competent for serving as a scalable biocidal part of equipment protective against viral infection. Therefore, we propose that IONzymes represent a general antiviral agent against influenza viruses and other enveloped viruses, such as HIV, Ebola virus, Zika virus, West Nile virus, and Nipah virus, all of which represent considerable challenges to national healthcare systems.
All experiments involving live viruses and animals were performed in negative-pressure isolators with high efficiency particulate air HEPA filters in animal biosafety level 3 ABSL-3 facility in accordance with the institutional biosafety manual. One part of the SY strain was purified on a discontinuous sucrose density gradient according to our previously described method[ 51 ]. Fluorescent labeling of influenza virus was performed using our established method described previously[ 51 ].
Unincorporated dye was removed by using commercial fluorescent dye removal columns Thermo Fisher Scientific. Newcastle disease virus NDV, envelope virus [ 52 ] and Porcine circovirus 2 PCV-2, non-enveloped virus [ 53 ] were also isolated, identified, and stored by our laboratory. IONzymes were prepared according to our previously described method[ 13 ].
IONzymes were synthesized in one-step in a solvothermal system by combining FeCl 3 and sodium acetate NaAc in ethylene glycol. The size distribution of these IONzymes has been characterized previously[ 54 ]. Briefly, 0. Next, 3. The enzyme kinetics for peroxidase-like activities of IONzymes were also evaluated. After centrifugation, supernatant was collected. The magnet was placed under the pipes to pull down IONzymes and then supernatant was also collected to detect the levels of lipid peroxidation using a commercial MDA detection kit according to the manufacturer's instruction Nanjing Jiancheng Bioengineering Institute, Nanjing, China.
Samples supernatant were primary-fixed with 2. The images of influenza virus were captured using a Tecnai 12 transmission electron microscope Philips, Netherlands. Protein extracts were resolved on SDS-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, blocked with PBS containing 0. Protein bands were visualized using enhanced chemiluminescence Thermo Scientific, USA on radiographic film.
Two-fold serial dilutions of the samples from column 2 to column 11 were performed. To identify influenza virus positive wells, the HA assay was performed[ 57 ].
Supernatants were analyzed using a neuraminidase assay kit Beyotime Institute of Biotechnology, Nantong, China according to the manufacturer's instructions. Fluorescence was released with an emission wavelength of nm and an excitation wavelength of nm and monitored using a multifunctional microplate reader Tecan. NA activity is shown as the intensity of fluorescence above the background values for supernatant without virus.
After being washed, the cells were observed using a Leica fluorescence microscopy. Annexin V possesses high affinity for phosphatidylserine PS , which is translocated from the inner to the outer leaflet of the plasma membrane in the process of apoptosis. PI cannot permeate live cells and early apoptotic cells but stains necrotic cells, binding tightly to the nucleic acids in the cell. The cells were washed and examined under a fluorescence microscope.
Cells positive for PCV-2 viral antigens were counted in six fields of view. Animals were monitored daily for weight loss and mortality over a period of 14 days as described previously[ 59 ]. Three mice of each group were euthanized at 7 days p. Each tissue sample was homogenized in 1 mL of PBS containing antibiotics and centrifuged at 6, rpm for 10 min, and 0.
Virus titers were calculated as described[ 27 ]. Basing on the traditional facemasks with absorbent filter cotton, different concentrations of IONzymes 0. After drying by airflow for 30 min, IAVs was sprayed onto the eighth layer the outermost layer.
After incubating for 0. After washing and extrusion, viral suspensions were harvested, and the population of viable virus was then measured using HA and TCID 50 methods. Unpaired Student's two-sided t -test was employed to determine the differences between the two groups. Data were combined from at least three independent experiments unless otherwise stated. Fedson DS. Influenza, evolution, and the next pandemic. Evol Med Public Health. Hu J, Liu X.
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