Nasal nitric oxide (nNO) levels in allergic rhinitis (AR), healthy people or nonallergic rhinitis (NAR) have shown contradicting results in previous studies. By meta‐analysis, we reviewed studies that measured nNO in AR patients to assess nNO's ability to discriminate AR from healthy people or NAR.We systematically searched PubMed, Cochrane, Embase, Ovid, Web of Science, Wanfang Data, CNKI until December 15, 2020. Differences were expressed as standardized mean differences (SMD) with 95% confidence interval (CI), by random‐effects method.p = .001) or NAR . However, subgroup analysis based on measuring process and patient characteristics showed that no significant differences were detected in nNO between AR patients with nasal polyps or sinusitis or marked ostial obstruction and healthy controls.A total of 10 original studies with 561 AR patients, 327 healthy controls, 123 NAR patients were included in the narrative synthesis and 9 studies in the meta‐analysis. nNO in AR was significantly increased compared with healthy controls  in allergic rhinitis (AR) is significantly higher than healthy people and nonallergic rhinitis. However sinusitis, nasal polysis and marked ostial obstruction should be taken into consideration before nNO is applied to detect AR. AR will detriment patients' efficiency of work and study, decline their quality of life and impact on asthma control level in AR patients combined with asthma, causing heavy healthcare economic burden.Nitric oxide (NO) is a free radical gas, playing an important role in many biological mechanisms. In respiratory system, NO is continuously released from upper and lower airway and soaringly released following proinflammatory cytokines and stimuli inducement. Fractional exhaled nitric oxide (FeNO) has been used as a noninvasive tool to reflect eosinophilic inflammation in lower airway diseases. For example, high level of FeNO suggests possibility of asthma.22.1Our methods have been described detailly in the published protocol (PROSPERO registration: CRD42020160578). We systematically searched following databases until December 15, 2020: PubMed, Cochrane, Embase, Ovid, Web of Science, Wanfang Data, CNKI in accordance with the Preferred Reporting Items for Systematic Review and Meta‐Analyses for Diagnostic Test Accuracy. The search strategy used the following terms: “allergic rhinitis” AND “nasal nitric oxide” found within all fields. There was no constraint on the publication language or study design during searching.2.2Studies were included if they measured nNO in AR patients and healthy controls with information about nNO analyzer, sampling technique, sampling rate and AR diagnostic criteria. Studies were excluded if any of the following were presented (1) number of AR patients was less than 10; 2) the procedure of nNO measurement did not follow American Thoracic Society (ATS)/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005; the proc2.3After duplicate article exclusion, B.W. and Z.W., two of the authors, independently analyzed the found articles and carried out data extraction. Information like clinical characteristics of subjects, NO analyzer, NO sampling rate, sampling technique, AR diagnosis, nNO value and so forth was collected. If disagreement came up, a third investigator (Y.L.) was consulted, decision would be finally made by consensus.2.4The Quality Assessment of Diagnostic Accuracy Studies‐2 (QUADAS‐2) tool was used to evaluate the methodological quality of each study.2.5SD were included in the meta‐analysis. Otherwise, they would be excluded from meta‐analysis but still in qualitative synthesis. The reported nNO concentration (ppb) was converted into nl/min by formula ppb × sampling rate (L/min) to keep consistent between studies using different sampling rates.χ2 Cochran's Q test and I2 statics. In detail, I2 = 0% indicates no heterogeneity; 25%, low; 25%–50%, moderate; and more than 50%, high heterogeneity.SE of the SMD on the y‐axis), Egger test and the Begg and Mazumdar test. A p < .10 is considered statistically significant.Studies reporting nNO values with mean and 33.1In total, 1862 records were identified through a generalized search of all publications related to AR and nNO. After removing duplicates, 1092 records were screened by title and abstract. After screening titles and abstracts, 132 potentially eligible studies were selected for full review. Finally, 10 original research studies3.2The main demographic, study, index tests characteristics were presented in Tables Different brands of NO analyzers were used in the included studies: NIOX, ECOmedics CLD88sp, LR200, NIOX MINO, Sievers 280i, Sunvou. Among them, NIOX MINO and Sunvou are electrochemical analyzers while the rest are chemiluminescence analyzers. During measurement of nNO, participants were required to obtain velum closure while gas was sampled from one nostril. Several methods including holding breath, exhaling against resistance can be achieved to ensure velum closure. Sampling flow rate is required to range from 0.25 to 3 L/min according to ATS recommendation.3.3The QUADAS‐2 tool was used to evaluate the internal and external validity of each study.3.4p = .001). The heterogeneity of this outcome was significant (I2 = 92.7%) and it did not decrease after individually eliminating each study. It could be relevant with analyzer types, sampling rates, sampling techniques or population characteristics.Because only 1 study reported nNO values in media (range),Four studiesp = .021). There was a high degree of heterogeneity .Two studies3.5After quality assessment of all studies, there were no studies of low risk bias to perform sensitivity analysis.3.6Subgroup analysis was performed for studies measuring nNO with different measuring process and different patient characteristics. It was detailly shown in the Table Comparison 1: subgroup analysis by different NO analyzer typesIn the subgroup analysis, we analyzed the effects of NO analyzer types on nNO's ability to discriminate AR. There was no evidence for different effects of NO analyzer types between subgroups.Comparison 2: subgroup analysis by different NO sampling techniquesIn the subgroup analysis, we analyzed the effects of NO sampling techniques on nNO's ability to discriminate AR. There was no evidence for different effects of NO sampling techniques between subgroups.Comparison 3: subgroup analysis by different NO sampling flow ratesIn the subgroup analysis, we analyzed the effects of NO sampling flow rates on nNO's ability to discriminate AR. There was no evidence for different effects of NO sampling flow rates between subgroups.Comparison 4: subgroup analysis by AR patients with/without asthmaIn the subgroup analysis, we analyzed the concomitant of asthma on nNO's ability to discriminate AR. There was no evidence for different effects of asthma between subgroups.Comparison 5: subgroup analysis by AR patients with/without rhinitis symptomsIn the subgroup analysis, we analyzed the existence of rhinitis symptoms on nNO's ability to discriminate AR. There was no evidence for different effects of rhinitis symptoms between subgroups.Comparison 6: subgroup analysis by AR patients with/without nasal polypsIn the subgroup analysis, we analyzed the existence of nasal polyps on nNO's ability to discriminate AR. There was evidence for different effects of nasal polyps between subgroups. No significant differences of nNO were detected between AR patients with nasal polyps and healthy controls.Comparison 7: subgroup analysis by AR patients with/without sinusitisIn the subgroup analysis, we analyzed concomitant of sinusitis on nNO's ability to discriminate AR. To be more specific, sinusitis meant acute unilateral maxillary sinusitis or sinus inflammation in the studies enrolling AR patients with sinusitis.Comparison 8: subgroup analysis by AR patients excluding/not excluding smokingIn the subgroup analysis, we analyzed smoking on nNO's ability to discriminate AR. There was no evidence for different effects of smoking between subgroups.Comparison 9: subgroup analysis by AR patients with/without marked ostial obstructionIn the subgroup analysis, we analyzed the existence of marked ostial obstruction on nNO's ability to discriminate AR. The ostial obstruction was measured through a semiquantitative computed tomography scoring system or active anterior rhinomanometry.3.7p = .7251) and the Begg and Mazumdar test (p = .1179) indicated no publication bias.Publication bias was detected by visual examination to funnel plot Figure . While t4In this systematic review and meta‐analysis, we have shown that nNO in AR patients was significantly higher than healthy controls and NAR. The nNO measurement in accordance with ATS recommended standardized procedure fits for kids older than 4 years old and adults who are able to cooperate with sampling techniques to ensure velum closure. Subgroup analysis showed that when AR patients were concomitant with nasal polyps, sinusitis or marked ostial obstruction, it was hard for nNO to detect them.l‐arginine by NO synthase (NOS) in the respiratory system, which has three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).Gustafsson's group found endogenous NO was present in the exhaled air of humans and other mammals in 1991.There are two different ways of measuring the fractional concentration of nasal NO. If the measurement is obtained by nasal exhalation, it is called nasal FeNO. If the measurement is obtained by transnasal flow in series, it is called nNO.Using the same analyzer, same flow rate and same method, Nesic et al.Our study presented with some limitations. First, high degree heterogeneity significantly influences our results. Although it is hard to determine the exact source of heterogeneity, here are some possible sources: included studies were held in different countries and different inclusion and exclusion criteria were set, leading to diverse demographic and clinical characteristics; few studies gave detailed description on AR patients such as their AR symptoms; kids were involved in meta‐analysis, while nNO were age‐related in kids younger than 12 years old.All considered, our meta‐analysis found that nNO in AR patients are significantly higher than healthy controls and NAR. nNO serves as a potential indicator for discriminating AR. However, nasal polyps, sinusitis and marked ostial obstruction are supposed to be taken into consideration before nNO is applied to detect AR. In addition, referring to the role of FeNO played in asthma, it remains to be seen whether nNO could be used as an indicator of AR treatment responsiveness in future studies.The authors declare that there are no conflict of interests.All author drafted the manuscript. Conception was from Bingbing Wang, Zhenchao Wu, and Yi Liu. Bingbing Wang designed the study protocol, performed data extraction and checked the manuscript. Zhenchao Wu performed data extraction, data analysis and interpretation, revised the manuscript to make it more fluent. Feifei Wang, Zuojuan Yin and Lei Shi did the literature search and study selection. Administrative support from Yi Liu who was responsible for any disagreement arising from the whole work.I confirm that the manuscript has been submitted solely to this journal and is not published, in press, or currently submitted elsewhere.Supporting information.Click here for additional data file.


Ophiophagus hannah) has a significant place in manycultures, and is a medically important venomous snake in the world. Envenomationby this snake is highly lethal, manifested mainly by neurotoxicity and localtissue damage. King Cobra may be part of a larger species complex, and is widelydistributed across Southeast Asia, southern China, northern and eastern regionsas well as the Western Ghats of India, indicating potential geographicalvariation in venom composition. There is, however, only one species-specificKing Cobra antivenom available worldwide that is produced in Thailand, usingvenom from the snake of Thai origin. Issues relating to the management of KingCobra envenomation , andchallenges faced in the research of venom (in particular proteomics), are rarelyaddressed. This article reviews the natural history and sociocultural importanceof King Cobra, cases of snakebite envenomation caused by this species, currentpractice of management , and major toxinologicalstudies of the venom with a focus on venom proteomics, toxicity andneutralization. Unfortunately, epidemiological data of King Cobra bite isscarce, and venom proteomes reported in various studies revealed markeddiscrepancies in details. Challenges, such as inconsistency in snake venomsampling, varying methodology of proteomic analysis, lack of mechanistic andantivenomic studies, and controversy surrounding antivenom use in treating KingCobra envenomation are herein discussed. Future directions are proposed,including the effort to establish a standard, comprehensive Pan-Asian proteomicdatabase of King Cobra venom, from which the venom variation can be determined.Research should be undertaken to characterize the toxin antigenicity, and todevelop an antivenom with improved efficacy and wider geographical utility. Theendeavors are aligned with the WHO´s roadmap that aims to reduce the diseaseburden of snakebite by 50% before 2030.King Cobra ( Snakebite envenomation is a medical emergency characterized by the toxicmanifestation of snake venom. It is a major public health problem, a commonenvironmental and occupational hazard affecting many impoverished and geopoliticallymarginalized populations in the tropics and subtropics . Close tOphiophagus hannah), a medically important elapid that has awide biogeographical distribution in Asia. This article reviews the current statusof King Cobra from the biological and medical perspectives, highlighting the impactof King Cobra bite on the community and the need for proper management. Recentadvances, controversies and challenges in the proteomics, toxicity andneutralization studies of King Cobra venom are discussed. In the past, snakebite envenomation received little attention globally despite itsdramatic socio-economic impact on the poor ,9,10. ItKing Cobra is the world’s largest venomous snake, with adults capable of growing upto 6 meters in length. It widely distributes throughout Southeast Asia on the MalayArchipelago (to the exceptions of territories located eastward from the Indonesianislands of Java and Sulawesi), in southern China (including the insular HainanProvince and Hong Kong SAR), and in parts of India   Figure . DependiHamadryas hannah(Cantor) in 1836. The genus Ophiophagus was proposed by Günther in1864, aptly attributing the name to its propensity to eat snakes. Ophiophagus hannah was formally accepted asthe valid name for King Cobra in 1945 [Naja genus; instead, it was more closely related tomambas (Dendroaspis spp.), based on more recent genetic analysis ofits phylogeny [Ophiophagus genus, although King Cobras from various regionsmay be part of a larger species complex. For instance, mitochondrial DNA analysis onKing Cobras in Thailand suggested a deep phylogenetic divergence between thenorthern and southern specimens [The taxonomy of King Cobra was first described as pecimens . Infactpecimens ,22.Howepecimens . Hence, Najaspecies (cobras) which are relatively more aggressive, and ambushing vipers/pitvipers which are resting motionless and often camouflaged. Additionally, despite itsfearless and aggressive reputation, field scientists often described the King Cobraas a placid snake, only manifesting aggressive behavior when disturbed or cornered,while preferring visual and acoustic threats display over attack. Typically, as asign of warning, it would raise its upper body above the ground, spreading its hoodwhile emitting a loud growling hiss [The King Cobra primarily feeds on other snakes, both venomous and non-venomous, butit probably also feeds on other reptiles and small mammals when food is scarce. A broading hiss ,25.Naja genus), and areconsidered as the descendant of Nagas, serpent/snake deities. In the 1940documentary film ‘Wheels across India’, Armand Denis recorded a religious ceremonyin Burma (Myanmar), where a priestess was kissing a King Cobra on its head severaltimes and making an offering to the snake as the God of Fertility  are thus exposed to a higher risk of envenomation [e.g., snake charming, snake dance) also led to documentedenvenomation cases [King Cobra is classified as a venomous snake of secondary medical importance (WHOCategory 2). By definition, venomous snakes listed in Category 2 are highly venomousspecies capable of causing morbidity, disability or death, for which exactepidemiological or clinical data may be lacking; and/or are less frequentlyimplicated  . The majnomation ,27. Clinnomation -31. The on cases -34.Naja spp.) in Asia. Envenomed patients commonly developintense local reactions characterized by pain and swelling resulting in tissuenecrosis, and descending paralysis that leads to respiratory failure - the principalmode of fatality in envenomation by King Cobra [The global epidemiological data of snakebite is scarce. For envenomation caused byKing Cobra, there were only nine clinical reports available in the literaturepublished between 1971 and 2020. Of these, three cases were reported from Myanmar,one each from Thailand and Malaysia, and four occurred outside Asia: two in the USA,one in the UK and one in the Netherlands. King Cobra envenomation in humanstypically manifests through local tissue damage and systemic neurotoxicity, with aconstellation of signs and symptoms similar to that caused by cobras,accompanied with signs of neuromuscular paralysis such as ptosis, limb weakness,dysphagia, dysarthria and flaccid paralysis. Paralysis of respiratory muscle resultsin asphyxia and generalized hypoxia, with death ensues from multiorgan failure[ failure,31,36,38i.e., Ophiophagus hannah MonovalentAntivenom (OhMAV) produced in Thailand was administered in eight out of the ninecases, indicated by the onset of neurological manifestation. The antivenom treatmentwas somewhat effective in reversing the systemic toxicity at high dosage rangingfrom 20 to 115 vials, indicating limited neutralization potency of the antivenom.In all cases reported, the diagnosis was made clinically through history taking oridentification of the biting snake. The treatment consisted of local effectmanagement through specialized wound care and control of secondary infection, andtreatment of systemic neurotoxicity with intubation and assisted ventilation ,29,31,36In general, when a bite occurs, the first thing the injured should do is to movebeyond the striking distance of the snake (to prevent repeated bite). The managementbegins with first aid as part of pre-hospital care, while the patient is transferredto the nearest medical facility that manages snakebite as soon as possible. The use of a tight  tourniquet, as widely practiced by many folks usinga belt, shoelace, clothing or even fence wire, severs the blood and lymphaticcirculation distally and thus potentially prevent or reduce the systemic spreadof venom from the bitten site. This practice, however, is highly controversialand now discouraged especially in bites that can cause tissue damage, astourniquets can concentrate the venom at the bitten site, promoting severenecrosis, gangrene, and resulting in the need to amputate, besides the dangersof “sudden release” . Pressurebandage immobilization (PBI), much advocated by Australian practitioners, is analternative that effectively reduces lymphatic drainage (the main route of venomspread) without compromising blood circulation. However, PBI too has a limitedrole in cytotoxic and hemotoxic bite, again, for the risk of worsened localtissue damage as seen in bites caused by most venomous Asiatic snakes, includingthe King Cobra. Thus, light bandaging along with immobilization of the bittenlimb has been a more common practice here while the victim is being transferredat the soonest for medical care. The identification of species is crucial for the selection of an appropriateantivenom, which is usually species-specific. The identification of snakespecies is, however, clinically challenging. Diagnosis based on the victim’sdescription is usually less reliable, while captured specimen of the bitingsnake may not be always available - in fact, it is advisable not to capture thesnake to avoid secondary bite. Increasingly, people have the possibility ofgetting a photo or video of the offending snake in a bite by using the cellphone. This should be recommended, followed by the consultation with an expertwho is able to identify the snake.Naja spp.) and King Cobra, the envenomingeffects are rather similar, yet requiring totally different specific antivenomfor effective treatment. From the practical standpoint, King Cobra envenomationtypically occurs in captive environments and rarely in the wild, thereforespecies identification has not been much of a problem in King Cobraenvenomation, and the mono-specific King Cobra antivenom can be opted as thefirst-line treatment accordingly. In a worse scenario where the neurotoxicspecies cannot be identified, the patient will have to rely on the availabilityof a polyvalent antivenom that is able to cover the different neurotoxic speciesin the region . A brief guide on the syndromic identification of King Cobrabite and antivenom recommendation is illustrated in Alternatively, the syndrome-based approach is useful for diagnosis when thedescription of snake is unclear, or the offending snake photo or specimen isunavailable. This however can be equally challenging in areas where severalsympatric species that produce similar envenoming effects. In Asia, envenomationby the majority of cobra and krait species (land snakes) produces similarneuromuscular paralytic effect, as with the King Cobra. The kraits, nonetheless,do not cause a significant local envenoming effect, and thus it can berelatively easy to rule out krait envenoming based on clinical observation.Between cobras ,raised against the King Cobra venom of Thai origin. A polyvalent antivenom,Neuro Polyvalent Antivenom (NPAV) that can be used to treat the envenomationcaused by four medically important elapids in the region  is also available.These products are commonly used in the region, including Malaysia, as bothcountries shared similar medically important venomous herpetofauna [Naja) envenomation were used as analternative to treat King Cobra envenomation in China and Indonesia, but thetreatment outcome has been poor. Although hetero-specific antivenoms maycross-neutralize the venoms of closely related species which share compositionaland antigenic similarities [Naja cobras have variable venom antigenicity thatlimits immunorecognition of toxins and cross-neutralization by antivenomimmunoglobulins [The Thai Red Cross Society, Queen Saovabha Memorial Institute is the only antivenom producer in the world that manufactures the specificantivenom, etofauna . In Myanlarities , KingColobulins . Moreovelobulins .Snake venoms are integrated phenotypes used for predatory, digestive anddefensive purposes, and are subject to evolution with adaptation to changes inecological niches. Intraspecific variation of snake venom composition is awidely recognized phenomenon, and this has a huge impact on the effectiveness ofantivenom used. Compositional variation of snake venom within a species fromdifferent locales is often accompanied with variable expression and antigenicityof the key toxins, rendering the antivenom from a single production source lesspotent or even ineffective -45. AlthKing Cobra envenomation can result in extensive tissue necrosis, which requiresserial wound debridement, followed by reconstructive surgery using skin graftingand/or a flap, or, amputation in severe cases . To prevThe studies are usually conducted on reconstituted venom powder, obtained throughlyophilization (freeze-drying) or desiccation of crude venom milked from livesnakes. King Cobra venom can be readily collected by inducing the snake to bitethrough a film covering a clean container, or, by directly placing its fangs overthe edge of a collecting device. The venom of King Cobra is typically golden yellowin color, often viscous, and lyophilization yields powder with comparable colorationin different shades of yellow which is indicative of the presence of a flavinadenine dinucleotide (FAD)-containing flavoenzyme, L-amino acid oxidase (LAAO). 2(PLA2s) [The investigation of King Cobra venom properties dated back to the 1960s when Tu etal.  studied (PLA2s) , L-amino(PLA2s) , Kunitz-(PLA2s) , cobra v(PLA2s)  and ohan(PLA2s) . The glo50 ~0.05-0.20 µg/g, in mice), commonly found in the venoms ofmost Asian elapids  [50 = 4-80 µg/g) [Naja cobra venoms typically show cytolytic andcytotoxic activities [i.e., beta-cardiotoxin (β-CTX), was shown tobind to β-1 and β-2 adrenergic receptors of cardiomyocytes, and exhibit negativechronotropic activity [Molecular cloning and recent -omic studies demonstrated the diversity ofthree-finger toxins that include alpha-neurotoxins, muscarinic toxin-likeproteins (MTLPs), weak neurotoxins (WNTXs) and cardiotoxins/cytotoxins (CTXs) inKing Cobra venom ,56,57,59 snakes) -67. MTLP snakes)  while WN80 µg/g) ,70. CTXstivities . The CTXactivity ,72. CardQuantitative proteomics showed that 3FTxs are the most abundant toxins in KingCobra venom from Malaysia   and IndoThe SVMPs are multi-domain zinc-dependent enzymes of varying molecular weights.They are the principal toxins responsible for vasculature damage and mayinterfere with hemostasis, contributing to hemorrhage and coagulopathy inenvenomation caused by vipers and pit vipers ,74. CompO. hannah venom [2+ channels and blockade of smooth muscle contraction [Cysteine-rich secretory proteins (CRiSPs) were the third most abundant proteinfamily detected in King Cobra venom proteomes, constituting 6.5% to 8.7% oftotal venom proteins -58. Ophaah venom , was comtraction . Despite2s were represented by a rather smallnumber of subtypes and a low abundance  [2s in King Cobra venom consist of twodistinct subgroups: group IA, including acidic PLA2s which exhibitedmild proinflammatory activity but were non-lethal to mice at a high dose of 10µg/g [2s are commonly present in mostsnake venoms implicated in the pathophysiology of envenomation, the function ofPLA2 in King Cobra venom is likely ancillary without directlethal activity. In King Cobra venom proteome, PLAprotein) -58. The  10µg/g , and gro 10µg/g . While sKing Cobra venom is a rich source of snake venom LAAO - the intense yellowcoloration of the venom reflects the high content of FAD-containing LAAO in thevenom . LAAO waSeveral protein families of low abundances had been reported in proteomic studiesof King Cobra venom. Among these were Kunitz-type serine protease inhibitors(KSPIs), which accounted for 1.0-3.3% of total venom proteins -58. SnakOphiophagus venom factor (OVF) accounted for 2.8% of theMalaysian King Cobra venom proteome [Other minor protein families were reported rather variably in the King Cobravenom proteomes. Four subtypes of cobra venom factors (CVFs), includingproteome while noproteome ,58. CVF/proteome . Other pproteome ,60,62,63proteome ,59,61,63proteome ,63, phosproteome ,63, C-typroteome ,60,63, nproteome ,59,64, iproteome ,61-63, nproteome ,63, vascproteome ,57, nepr2) without a comprehensive profile ofall venom proteins. Possible interference from factors relating to the sex,developmental (ontogenic) stage and habitat of the snake cannot be excluded whenaddressing the variability of snake venom composition. Comparison of the findingsand interpretation for intra-species variation is, therefore, challenging. The proteomics of King Cobra venom sourced from different geographical locales hasbeen studied to varying depths. Specimens studied thus far were from Malaysia(peninsula) ,60,62,63The second and perhaps more influential factor is the methodology of analysis. Thedifferent approaches employed in each study resulted in varying depths of proteomicprofiling, which could be confounding when comparison is made, resulting inmisinterpretation of geographical venom variation. Proteomics in snake venomresearch aims to comprehensively profile the diversity (how many?) and proteinabundance (how much?) of all toxins present in a venom. The identification of highprotein diversity is usually achieved using protein decomplexation strategies thatdeploy chromatographic or electrophoretic separation methods of venom proteins,prior to mass spectrometry analysis -86. The i.e., Thailand, Malaysia, Indonesia and two Chinese localities(Hainan and Guangxi) . Their results demonstrated marked variations in the enzymatic activities andtaxon-specific lethality between the Chinese and the Southeast Asian King Cobravenoms. The venom samples from Southeast Asia exhibited higher metalloproteinase, acetylcholinesterase and alkaline phosphataseactivities than the Chinese samples. Taxon-specific lethality test was conducted inmice and lizards (Eutropis multifasciata) via intra-peritonealroute. It revealed that the Chinese samples were more lethal to mice with ani.p. LD50 of 0.5−0.6 µg/g, comparing to samples fromIndonesia (1.1 µg/g), Malaysia (2.9 µg/g) and Thailand (3.5 µg/g), whereas theSoutheast Asian samples were more lethal to lizards  [50 not reported) than the two othersamples. Considering the very small sample number of this latter study, a conclusionfrom the geographic perspective could not be drawn as the variation observed islikely inter-individual difference.Earlier, Tan et al.  reportedot done) . Anotherot done) , and fou50 ~0.50µg/g) in comparison to the Malaysian (LD50 = 0.90 µg/g) and Thai(LD50 = 1.04 µg/g) specimens. In vivo neutralizationstudy revealed poor cross-neutralization of King Cobra venom lethalities bynon-specific antivenoms, i.e., the Chinese Najaatra Monovalent Antivenom (NaMAV) and Indonesian Serum Antibisa Ular(SABU), which are commonly used in the respective countries to treat King Cobraenvenomation [50), defined as the volume dose of antivenom needed toprotect half of the tested mice from venom-induced lethality. OhMAV was found moreefficacious in neutralizing the venom of Thai origin  than it was against the Malaysian (ED50 =129.09 µL), Indonesian (ED50 = 139.58 µL) and Chinese (ED50 =170.16 µL) venom samples. The observation strongly supports the species-specificityof OhMAV toward its immunogen of Thai origin, and underscores the impact of KingCobra venom variation on the effectiveness of antivenom treatment  were shown to varyconsiderably . The Chinomation . The spereatment .The existing data tend to support that the biochemistry and toxicity of King Cobravenom vary geographically, with a marked divergence between the Chinese and mostSoutheast Asian populations. From the ecological perspective, the variation intaxon-specific toxicity   suggestsN.kaouthia LNTX . The experimentalhetero-specific antibody bound to a short and a long neurotoxin in King Cobra venom,while weakly cross-neutralized the lethality of the venom. Another study by Liu etal. [O.hannah venom by the Taiwanese neurotoxic bivalent antivenom , which is raised against Bungarus multicinctus andNaja atra venoms. Similarly, the finding showed that TNBAVcross-immunoreacted with most O. hannah HPLC-eluted venom fractionsbut failed to cross-neutralize the venom toxicity in mice [i.e., OhMAV, to achieve effectiveneutralization in King Cobra envenomation [Antivenomics is applied to examine the immunorecognition of an antivenom to proteincomponents in a venom. Through the identification of toxins that are poorly boundimmunologically, the weakness of an antivenom can be identified for improvement. Theinformation on King Cobra antivenomics is however limited. Using a2DE-immunoblotting technique, Danpaiboon et al.  earlier u etal.  reported in mice . The phenomation . The difOphiophagus hannah) has a wide geographical distributionin Asia, and is a medically important venomous snake capable of causing fatalenvenomation. Epidemiological data of King Cobra envenomation, as with that ofsnakebite in the region, is relatively scarce. Worldwide, the only species-specificantivenom indicated for treating King Cobra envenomation  is produced in Thailand by theQueen Saovabha Memorial Institute, Bangkok, raised against the venom of Thai KingCobra. Clinical reports showed that high doses of the mono-specific King Cobraantivenom, often beyond 20 vials, were needed in the treatment of King Cobraenvenomation. Preclinical assessment of OhMAV neutralization activity revealedvariable efficacy against the venoms of King Cobra from different locales,indicating vast geographical variation in the composition and antigenicity of thevenom. Geographical variation of the venom, however, has not been comprehensivelyinvestigated despite a number of proteomic studies reported on King Cobra venom.Discrepancies in the venom proteomes reported across the different studies wereobserved, presumably due to differences in the sampling and method of analysis,which resulted in varying depths of venom profiling and incompatible quantitation ofprotein abundances. Comparison of King Cobra venom proteomes reported from differentstudies for the interpretation of geographical venom variability thus remainschallenging. Furthermore, mechanistic and antivenomic studies are also lacking toelucidate the clinical pathophysiology of envenomation and the variableeffectiveness of OhMAV in neutralizing King Cobra venom. King Cobra (v.s. captivity). A standard method of proteomic analysis shouldbe adopted, preferably one that incorporates the protein decomplexation strategy andhigh-resolution mass spectrometry coupled with deep database mining for betterdetection of protein diversity. The quantitation of protein should also bestandardized, and in this context, the term “percentage” should be used cautiouslyto denote relative protein abundance, so that the composition of a protein isdefined by its amount with respect to the total venom proteins. Furthermore, throughhigh-performance liquid chromatography of the venom (protein decomplexation), it ispossible to isolate the various venom proteins eluted for further functionalinvestigation. This will help identify the key toxins and elucidate their toxicity,providing deeper insights into the pathophysiology and treatment of envenomationaccording to the variability of venom. Furthermore, the antivenomic approach basedon either enzyme-link immunosorbent assay, immunoblotting or affinitychromatography, should follow to examine the degree of immunorecognition orimmunocapturing capacity of OhMAV toward the different toxin components in thevenom, for specimens originated from different geographical populations.Importantly, the immunologically-derived antivenomic findings must be interpretedagainst the in vivo efficacy of OhMAV, in which the venom andisolated toxin principles are subject to neutralization as outlined in functionalantivenomics [Future works should be tailored toward a comprehensive proteomic study of King Cobravenom from various locales, taking consideration into the potential variationarising from ontogenic factor and the environment of habitat (wildvenomics ,89. Thesvenomics . Althougvenomics , the appvenomics ,93. This


Innovative technology has been an important part of firefighting, as it advances firefighters’ safety and effectiveness. Prior research has examined the implementation of training systems using augmented reality (AR) in other domains, such as welding, aviation, army, and mathematics, offering significant pedagogical affordances. Nevertheless, firefighting training systems using AR are still an under-researched area. The increasing penetration of AR for training is the driving force behind this study, and the scope is to analyze the main aspects affecting the acceptance of AR by firefighters. The current research uses a technology acceptance model, extended by the external constructs of perceived interactivity and personalization, to consider both the system and individual level. The proposed model was evaluated by a sample of 200 users, and the results show that both the external variables of perceived interactivity and perceived personalization are prerequisite factors in extending the TAM model. The findings reveal that the usability is the strongest predictor of firefighters’ behavioral intentions to use the AR system, followed by the ease of use with smaller, yet meaningful, direct and indirect effects on firefighters’ intentions. The identified acceptance factors help AR developers enhance the firefighters’ experience in training operations. Augmented reality (AR) is defined as a system that has the following properties: (a) combines real and virtual objects, (b) is interactive in real-time, and (c) is registered in three dimensions . Feiner AR has been explored in all levels of education ,6, proviThe first potential of integrating AR in firefighting training is the cost effectiveness. Indeed, current firefighting training in the real environment requires important costs in infrastructures, such as a large training area, propane-fed firefighting props, fire extinguishers, ventilation, and ladders. The training facility has to deal with complex and varied scenarios, and their replication needs vehicles, dumpsters, pipes, containers, and even apartment floorplans. AR can handle all these infrastructures, providing considerable savings in consumables; thus, the preparation time for each training scenario is significantly reduced.Secondly, the use of AR leverages the trainees’ experiences to a wide variety of training scenarios. The firefighting area varies widely depending on the category and, as such, there is aerial support of wild firefighting , urban fApart from the cost effectiveness and the variety of scenarios offered, the AR adoption has no environmental charge, no uncontrolled fires, toxic gas emission, or heat burst. AR does not entirely supplement traditional training exercises; however, it offers the possibility to train in high-risk scenarios, without actually encountering any physical risk of the trainees’ health. Despite the reported side effects experienced by the head-mounted display (HMD) , most usFor all the aforementioned advantages, using AR to train firefighters is a novel training approach that will improve the training of qualified firefighters. Some preliminary studies have already been conducted in the field of firefighting training ,17,18,19https://etraining.naftes.eu/, accessed on 5 April 2021) AR application. NAFTES is freely available through the Google Play store. The suggested model includes the basic TAM variables, namely, usefulness, ease of use, and intention to use, extended by two more constructs, namely, perceived interactivity and personalization, which are addressed to evaluate the human–AR system interaction and the contextual affordances to support the training of the ship crew and officers. NAFTES was developed for two different variants, suitable for mobile  and wearable devices (HoloLens), aiming to help trainees achieve their learning goals. The trainees can either select the mobile variant to use their personal devices and interact in the augmented ship space, or combine the AR glasses and see-through lenses. Furthermore, the system holds personalization in its incorporated mechanisms, since it provides tailored information to the users, to help towards fulfilling their learning needs. During the training phases, the system visualizes objects, information, and videos that are available to the users, according to their location, activity and actions, and delivers the corresponding assessment units to them. Regarding the level of expertise, the system offers further help to inexperienced users and supports them during their interaction with it. In the case of a diagnosed difficulty in using the application, the system proposes the use of the AR Walker application to familiarize the participants with the equipment. Moreover, it provides two different simulation scenarios for novice and advanced users, in order to match their different levels of proficiency. As such, the system provides a high degree of adaptability to trainees who have the ability to select the learning path, based on their interest, existing knowledge or the demands of their role.We assessed the acceptance of AR technology for firefighting training, by proposing an acceptance model specific for a “Naval Fire Fighting Training & Education System” (NAFTES) (NAFTES assumes the following different facets: (a) introductory training (IT); (b) theoretical (classroom) training (THT); and (c) hands-on training (HOT). IT is based on an advanced learning management system (LMS), which allows the trainees to be introduced in the fire training fundamentals. THT is supplemented by multimedia material , which is to be integrated in the LMS, and retrieved and presented during classroom hours. Finally, HOT is an advanced portable system, which will be carried by trainees during their presence in the simulated engineering room. The system is in the form of human–AR applications, which will overlay warnings or explanatory text to the trainees’ viewport. Such applications will be executed in optical head-mounted displays (Google glasses), which will retrieve material from the LMS. While creating 3D environments is not new, developers have traditionally had to use 2D interfaces to build 3D experiences. Using AR, the full 3D space is used, which is unprecedented, and defines many of the imminent changes in human–computer interaction (HCI). Another important feature of such devices is their indoor localization capability. The NAFTES platform is complemented by a post-training assessment module, which is appended in the IT–THT–HOT workflow.The AR firefighting training (AR-FFT) application provides two fire simulation scenarios, namely, an electric fire and a cooking oil fire. The participants have already been trained in theory, and are familiar with the protocol they have to follow in each of the aforementioned cases. The AR application helps them to evaluate their knowledge in a simulation environment. The users navigate in fire-prone areas in a ship by using QR codes. The trainees have to follow printed marks, placed in bright spots near the corresponding equipment they describe, which reveal useful instructions. The personnel are familiarized with the firefighting equipment and processes. The application also provides information material and questions on the corresponding theory, aimed at self-evaluation.For example, the instructor chooses to simulate the electric fire scenario, and places five signs  in specific positions on the ship deck. The trainees start the training simulation, searching for the center of the fire , a commoAfterwards, further instructions to follow are displayed, and the trainees have to find the next correct check point and follow the new instructions that appear. According to the protocol, on incident identification, the personnel must find the nearest telephone to report the occasion to the damage control center and provide useful information .The next steps of the safety protocol are displayed in Finally, the scenario suggests that in the case of no meaningful results regarding the previous actions, all the personnel should exit the dangerous area and seal the compartment .When completing all the check points correctly, the trainees have successfully accomplished the particular protocol.Davis  proposedThe related literature review is structured into the analysis of six different studies on AR acceptance in educational settings, and the possible extensions of their models. Yusoff et al.  evaluateWojciechowski and Cellary  presenteWang et al.  conducteMao et al.  evaluateIbili et al.  developePapakostas et al.  examinedAlthough there has been a lot of research on the adoption of AR for training, few studies have concentrated on the context of firefighting training ,28,29, wOne of the main criteria of effectively managing a naval fire emergency is good preparedness. AR is a promising technology that assists training and improves the abilities that are needed to deal with risky and unexpected circumstances. So far, little research has been made to investigate the acceptance of such a technology by firefighters.Contrarily, there is still much to be done to mitigate the shortcomings of AR technology in this area. Without sufficient user studies and through the use of traditional training methods, there is little real-world evidence on the actual effectiveness of AR training. The case of technology acceptance among firefighters needs to be addressed.Is AR acceptable by firefighters as part of their training to develop their skills? (RQ1);How do the technical aspects of using AR affect trainees’ experiences of using it? (RQ2);What effect does the AR application’s content have on the trainees’ user experience? (RQ3).Consistent with the previously stated goals, the main objective of this study is to present a modified TAM model, evaluating the user experience of AR in firefighting training. The existing literature explains the acceptance of AR technology in fields such as aviation, engineering, and military, however it does not consider the specific usage constraints in the domain of firefighting. From this, it can be deduced that there is much room for future investigation in this area. To fill the gap in the literature, we propose a detailed, domain-specific acceptance model, in which both interactivity and cognitive affordances were investigated, and thus their influence on user acceptance was measured. The related research questions that form the hypotheses of the proposed model are as follows:We evaluated the AR firefighting application by expanding a TAM model, according to the research statement. Furthermore, the use of augmented reality in naval training, especially in the field of firefighting, has been shown to have a high potential for making training sessions more cost-effective, safer, and scenario-independent.Davis  proposedIn this study, the final version of the model TAM, consisting of the variables of perceived usefulness, perceived ease of use, and behavioral intention, is proposed and found to be useful in predicting the use of AR for firefighting, and the following hypotheses are proposed:Hypothesis 1 (H1).PEOU of the AR firefighting application positively affects its PU.Hypothesis 2 (H2).PEOU of the AR firefighting application positively affects its BI.Hypothesis 3 (H3).PU of the AR firefighting application positively affects its BI.The belief of a system user may be influenced by external factors, referred to as external variables . In addiLee and Lee  providedIn the context of the current research, PI results in firefighters’ positive beliefs about the usefulness and ease of use of the AR application, which in turn influences their behavioral intentions in the naval firefighting training. We advanced the TAM model by employing the interactivity of the crew and the officers, as a prerequisite variable, and therefore, the following hypotheses are composed:Hypothesis 4 (H4).PI of the AR firefighting application positively affects its PU.Hypothesis 5 (H5).PI of the AR firefighting application positively affects its PEOU.Furthermore, personalization has been found to be a major component of many information technology-based systems . TraininIn the context of the current research, the perceived value for personalization (PP) might play an important role with regard to the firefighters’ positive attitude towards the naval firefighting training AR application. We advanced the TAM model by employing the personalization as the second prerequisite variable of the proposed TAM model, and the hypotheses included in the model are as follows:Hypothesis 6 (H6).PP of the AR firefighting application positively affects its PU;Hypothesis 7 (H7).PP of the AR firefighting application positively affects its PEOU.The flow of the research model is shown in This study tested the proposed TAM model at the training of ship crews in firefighting operations. The participants were 200 volunteer firefighters, first responders, advisers working in the crisis management sector, and teachers from vocational education providers, all of whom had the opportunity to test, firsthand, the AR application of NAFTES. The AR application has been developed for both mobile devices and HoloLens glasses. The participants have been demonstrated both technologies; however, for the sake of the experiment, they used the mobile AR training system running in their own mobile devices. The reason for this decision is the cost of each equipment , as HoloLens is not yet considered an economically viable approach. Their previous experience regarding the use of AR technology was just for entertainment purposes through gaming. The volunteers were aged between 18 and 60 years old. The measurements of gender and age were derived from a randomly selected sample and do not have an impact on our research findings. The demographics analysis is shown in The study was carried out for a period of six months, in which the participants had the chance to interact with, and validate the capabilities of, AR. The users’ feedback was evaluated in order to reveal the AR’s strengths in real applications. Data were collected via a questionnaire, which was delivered to the participants. The participants were asked to evaluate all five constructs, namely, PI, PP, PU, PEOU, and BI, by rating their training experience using a 7-point Likert scale, ranging from (1) strongly disagree to (7) strongly agree. The variables of the proposed model are assessed using indicators , adoptedThe structural equation modeling (SEM) approach is used to analyze the collected data. The partial least squares (PLS) algorithm was used to build the model for the regression analysis of the variables. By estimating the individual item loadings on their respective variables, and then estimating the causal relationships among the variables, PLS allows for an optimal assessment of the measurement and structural models at the same time .PLS is a strong candidate for data analysis because of the exploratory nature of our sample, where the primary goal is to understand the variance of the dependent variables induced by the independent variables, rather than the goodness of fit between the model and data .In this analysis, PLS is used to test seven hypotheses and assess the relationships between the variables. SmartPLS v.3.3.3, a software tool for PLS-SEM developed by Ringle et al. , was useIn the current study, we used a reflective measurement model, which assumes that the measures represent the indication of an underlying construct . We initThe descriptive statistics of the constructs’ indicators of the proposed model present the mean values (M), the standard deviation (SD), the excess kurtosis and skewness . The indAfter the descriptive statistics analysis, we assessed the measurement model’s reliability, by measuring the indicator reliability and the internal consistency . The indMoreover, the discriminant validity assessment has the goal to ensure that a reflective construct has the strongest relationships with its own indicators. In order to examine the discriminant validity of the constructs, we firstly calculated the square root of the AVE in each variable, comparing its value to the other variables’ correlation values . The modRegarding the results shown in Lastly, the discriminant validity was measured in two parts. Based on the results shown in After the assessment of the measurement model, we used the PLS algorithm to evaluate the path coefficients, and to test the hypotheses along with their significance levels. We ran bootstrapping estimation with the 2000 resampling method, and obtained the t-statistics corresponding to each path of our model.We initially evaluated the coefficient of determination (R2). The R2 values can describe the level of predictive accuracy, as high R2 values are indicative of the quality of the model. The coefficient value of the paths of the inner model are assessed in order to measure the level of significance and show how strong the effect of a variable is on another variable. The path coefficient values range from −1 to +1, and show significance when its value is greater than 0.200 . Figure p < 0.01), H2 , H3 , H4 , H5 , H6 , and H7 .We used a two-tailored t-test, with a significance level of 1%. The critical t-statistics value is 2.580 for a significance level of 1% . A summary of the results from the structural model’s hypotheses testing is shown in 2) [2) [2 is applicable only to reflective endogenous constructs [2 values  ,59, and 2) [2) . Q2 is anstructs , as BI, 2 values  based onve model .2 effect size , PP to both PU (f2 = 1.027) and PEOU (f2 = 0.687), and PI to PU (f2 = 0.416). PI has a medium effect on PEOU (f2 = 0.270), while PEOU has a small effect on BI (f2 = 0.040) and PU (f2 = 0.102). All of the significant paths of the proposed model show at least a weak effect size greater than 0.020, while the majority of the four out of a total of seven paths show large effect sizes, much greater than 0.350.Regarding the fThis study contributes to the acceptance of AR technology by firefighters, by combining the variables PU, PEOU, and BI from the core TAM model , and twoRegarding the results of the outer model, the indicators’ reliability and the internal consistency are confirmed. Indeed, all the factor loadings ranged from 0.833 to 0.992, which are greater than the 0.700 threshold limit. As far as the internal consistency of the variables is concerned, the CA values ranged from 0.902 to 0.981, while the CR ranged from 0.940 to 0.986. Sagnier et al.  chose toFurthermore, the model’s discriminant validity is assessed by two means, first by the AVE square root of each variable, and secondly by the HTMT ratio of correlations. Pal and Patra  also use2) value of 0.910, meaning that PU and PEOU explain 91.0% of the variance of BI. Moreover, PI and PP explain 84.0% of PEOU, while PI, PP, and PEOU explain 94.6% of the variance of PU.Regarding the results of the inner model, the findings showed that the BI target endogenous variable has a coefficient of determination , while PEOU has a weaker effect on BI (0.142). Both hypothesized path relationships between PU and BI, and PEOU and BI, are statistically significant as their t-statistics values, 17.790 and 2.876, respectively, are greater than the cut-off value of 2.580. Furthermore, PEOU is highly affected firstly by PP (0.593) and secondly by PI (0.363). As far as PU is concerned, it is affected firstly by PP (0.542), secondly by PI (0.294), but also from PEOU (0.182).From the evaluation results presented in this section, it can be observed that the behavioral intention to use the AR application is highly affected by usefulness, while ease of use has a much weaker effect. In our study, we found a positive, yet comparably smaller, effect of the perceived ease of use on user acceptance of AR, either directly on the behavioral intention to use, or indirectly through the perceived usefulness. It seems reasonable considering the domain of firefighting, which involves critical situations with people’s lives at stake. Firefighters demand an AR application for their training, firstly absolutely useful for real-life scenarios, and secondly to be easy to use. They seem willing to tolerate a certain amount of difficulty to handle a technology application, as far as the usability is proper. These findings are in line with previous studies ,64, whicThe perceived usefulness is also very well predicted by the external variables of interactivity and personalization, which were included in our proposed model in order to evaluate the system and the individual level, respectively. As far as the perceived interactivity is concerned, we found significance in the role of the communication, the user control, and the time. By implication, it suggests that firefighters (a) appreciate two-way communication through the AR application, (b) enjoy the flexibility of controlling a mobile device for their training, thus operating virtual objects, and (c) need fast-delivered messages and information, as their reactions have to be immediate and effective.As far as the perceived personalization is concerned, we also found significance for the application’s usability. Indeed, firefighting is a process where numerous operations occur simultaneously. As such, firefighters can participate in fire service training through various training formats, encouraging personal and professional growth. The AR firefighting application offers guidance and support, in order to assist personally every trainee to navigate through the competitive process of attaining a career as a firefighter. The participants experience one-on-one personal coaching through virtual classroom training sessions.The perceived interactivity positively affects not only the system’s usability, but also the perceived ease of use. Indeed, in the fire service, the hands-on portion of the training is based on the interactivity of the training platform. The must-have features, as well as the right support to assist with the AR functionality, give credits to the trainees’ ease to use the AR application, increasing the retention and the actual system usage.The perceived personalization equally affected usefulness and ease of use. A training system tailored to the firefighters’ needs provides the personnel self-paced training, with greater consistency. The better the AR application fits to the firefighters needs, the easier and more convenient the training is. On the contrary, traditional in-person training sessions are typically one-size-fits-all. The trainees attend the same training session, regardless of their roles, i.e., ship crew or officers.In summary, our findings provide three meaningful implications for the user acceptance of AR firefighting training. The first one is that the external variable of perceived interactivity has a positive impact on the perceived usefulness and perceived ease of use. The second one is that the second external variable of perceived personalization has also a positive, but stronger than interactivity, impact on the perceived usefulness and perceived ease of use. The third one is that usability is the strongest predictor of behavioral intention; however, the direct and indirect effect of ease of use is still meaningful. The perception of interaction experienced by firefighters is about controlling the content, the communication, and the speed, and the personalization on the feedback of their actions. These factors influence the intrinsic motivation of the firefighters, their perception of the ease of use of the AR application, and the perceived usefulness. The contribution in the field of AR, based on the study of the proposed TAM model, is that the use of AR technology may be prevalent in firefighting training, and may become an integral part of the educational procedure. This assumption is justified by the fact that firefighting is a high-risk job and firefighters are exposed to danger, so they prioritize their safety. The evaluation results show that AR can potentially become a useful technology upgrade for an immersive training, so that firefighters would be prepared for high-risk incidents.In our proposed model, usability was found to be the strongest predictor of the intention to use AR in firefighting training. The variable of usability is crucial and, as such, the results can be expanded in other domains such as aviation and military. Indeed, the aforementioned domains base their training on highly usable applications, combining both interactive and adaptive techniques.The presented technology acceptance model contributes to achieving an in-depth understanding as to why firefighters accept or reject an AR firefighting training application . The training advantages of cost-effectiveness, variety of real-life scenarios, and safety, offered by the AR platform, made us consider a novel, well-established model to measure the user experience and usability of the training application. The results of the conducted evaluation additionally provide insights into the significance and the comparative importance of the external variables, thus providing better support for their ongoing development.Regarding the limitations and the future steps of this research, the study involved trainees from Greece, who possibly exhibited some common features in terms of user experience. Therefore, it is possible that our findings may possess limited generalization, so future research should involve more countries, aiming at enhancing the quality of the training of ship crews in firefighting operations.


In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders. Skeletal muscle accounts for more than 40% of the human body weight and stores approximately 50–75% of human protein in various forms as the largest protein pool . Skeletavia transcriptional regulation of miRNAs and long non-coding RNAs (lncRNAs)   and c-MylncRNAs) .lncRNAs are a class of non-coding RNAs longer than 200 nucleotides in length and with almost no coding ability . lncRNAsWe previously discovered dozens of uncharacterized lncRNAs from mouse C2C12 myoblast differentiation versus myoblast proliferation using gene chip analysis. A co-expression network revealed that one lncRNA, Gm13398, exhibited relatively high expression and was co-expressed with Myog during myoblast differentiation . In the 2. For the myogenic differentiation experiment, C2C12 myoblasts were seeded in six-well plates, and when they reached 80–90% confluence, they were placed in DMEM containing 2% horse serum .Mouse C2C12 myoblasts and 293T cells  were cultured in Dulbecco’s modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum  at 37°C in 5% COTM Real-Time PCR System  using SYBR-Green Mix , with the expression levels normalized over an 18s period. Relative RNA expression was calculated using the 2–ΔΔCt method. The primer sequences are listed in Total RNA was extracted from cultured cells or tissues using TRIzol reagent  according to the manufacturer’s protocol. Complementary DNAs were synthesized from 500 ng of total RNA using a Prime Script RT reagent kit . The quantitative polymerase chain reaction (qPCR) assays were performed in the StepOnePlusin situ hybridization (FISH), cells were fixed, permeabilized, and hybridized with lnc-GD2H probe in buffer overnight at 42°C. Then, nuclei were stained with DAPI. Images were captured using a laser scanning confocal microscope  at 37°C.Oligonucleotides  targeting lnc-GD2H were modified with fluorescein isothiocyanate. Briefly, for lnc-GD2H fluorescence Total protein was extracted using RIPA lysis buffer containing phenylmethylsulfonyl fluoride, then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes . The membranes were incubated with primary antibodies against Tubulin , CDK2 , CDK4 , CDK6 , c-Myc , myosin heavy chain (MyHC) , Myog , Mef2a , Mef2c , and NACA . The membranes were then incubated with horseradish peroxide-conjugated goat anti-mouse  or anti-rabbit  secondary antibody. Band intensities were determined using a chemiluminescent imaging system .C2C12 myoblasts were infected with negative control lentiviruses (lv-OE-NC) or those containing lnc-GD2H (lv-OE-lnc-GD2H) or c-Myc (lv-OE-c-Myc) at a multiplicity of infection of 100 following the manufacturer’s instructions. The lv-OE-NC, lv-OE-lnc-GD2H, and lv-OE-c-Myc lentiviruses were synthesized by Hanbio (China). Then, stably transfected cells were selected and maintained using puromycin .TM RNAi MAX , following the manufacturer’s instructions.siRNAs against lnc-GD2H, c-Myc, and NACA were synthesized by Gene Pharma (China). For siRNA transfection, cells were transfected with the indicated siRNAs using LipofectamineCell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay . Stably transfected lv-OE-NC, lv-OE-lnc-GD2H, and lv-OE-c-Myc overexpression myoblasts were cultured in 96-well plates. siRNA-transfected NC (siNC), lnc-GD2H, and c-Myc myoblasts were also cultured in 96-well plates. After 24, 48, or 72 h of siRNA and overexpression, CCK-8 reagent was added to each well for 1 h at 37°C. Then, the absorbance was measured at 450 nm using an automatic microplate reader .For 5-ethynyl-2′-deoxyuridine (EdU) staining, stably transfected lv-OE-NC, lv-OE-lnc-GD2H, and lv-OE-c-Myc overexpression myoblasts were cultured in 96-well plates. siRNA-transfected NC, lnc-GD2H, and c-Myc myoblasts were also cultured in 96-well plates. After 72 h of siRNA and overexpression, EdU reagent  was added at a final concentration of 10 μM and the plates incubated for 2 h at 37°C. Then, cells were harvested for EdU staining according to the manufacturer’s instructions.First, lnc-GD2H-overexpressing and control stable cell lines were cultured in 96-well plates and allowed to differentiate for 72 h. The harvested cells were fixed, blocked with goat serum , incubated with primary anti-MyHC antibody , and then incubated with Alexa Fluor 594-conjugated AffiniPure goat anti-mouse IgG secondary antibody . DAPI  was used to stain the cell nuclei. Images were captured using a Leica DMIL LED fluorescence microscope . Fusion indices were calculated as the percentage of nuclei with two or more nuclei in the fused myotubes in MyHC-positive cells out of the total nuclei .® Reporter Assay System  was normalized to Renilla luciferase activity. The data are presented as the mean ± standard deviation (SD) of three independent experiments.The c-Myc plasmid together with the wild-type lnc-GD2H promoter P1 , the truncated lnc-GD2H promoter P2 , three predicted simultaneously mutated c-Myc binding sites in the lnc-GD2H promoter , or the NACA and lnc-GD2H plasmid together with the Myog promoter were transfected into 293T cells using FItran transfection reagent  in 96-well plates. The potential binding sites of NACA in the Myog promoter region were mutated from GGAGAGAGTAG to CCTCTCTCATC at the site −331 to −341 (Myog promoter-Mut1) and from GCACAGAAGAG to CGTGTCTTCTC at −1,356 to −1,366 (Myog promoter-Mut2). Firefly activity observed with the Dual-LuciferaseLinearizing DNA was biotin-labeled and incubated with proteins extracted from C2C12 overnight at room temperature. Next, streptavidin beads  were added to each reaction mixture, which were then incubated for 2 h at 4°C. Finally, the beads were washed in buffer , and the bound proteins were used for western blotting.The ChIP assay was performed using the ChIP kit  according to the manufacturer’s instructions. Briefly, DNA that had undergone ChIP was eluted, reverse X-linked, and purified. Each ChIP reaction was performed using 1 μg of NACA antibody , and IgG was applied as the isotype control. Fold enrichment was quantified using qPCR.in vitro transcription. Biotinylated RNAs were mixed with proteins extracted from C2C12 cells, followed by targeting of the RNAs with streptavidin beads . The co-precipitated proteins were used for mass spectrometry.Briefly, vectors carrying lnc-GD2H and antisense lnc-GD2H were linearized with the corresponding restriction enzymes to prepare template DNAs for After SDS-PAGE following RNA pulldown, the differentially expressed and obviously present band in the Coomassie brilliant blue staining lane was excised and used for MS analysis. The UniProt database was used for identifying proteins. Proteins were identified and ranked based on percentage cover  and the number of unique peptides. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD025292.RNA immunoprecipitation assay was conducted using RIP Kit  according to the manufacturer’s instructions. Briefly, C2C12 cells were lysed with RIP lysis buffer, and incubated with the antibody against NACA  for RIP. Then Protein A/G beads were added to the lysates to pull down antibody-protein-RNA complex. The co-precipitated RNAs were analyzed using qPCR. Total RNAs (input control) and the negative control (IgG) were assayed at the same time to confirm that the examined signals were from RNAs binding to NACA.Stably transfected lv-OE-NC or lv-OE-lnc-GD2H C2C12 overexpression myoblasts and siNC or silnc-GD2H myoblasts were cultured in six-well plates and then serum-starved for 24 h. Next, artificial wounds were scratched on the monolayers across the well using 10 μL pipette tips, and the scratched areas were photographed every 6 h until 24 h after wounding. The wound size was determined by calculating the ratio between the surface area of the wound after scratch of 6 h, 12 h, 24 h and the area of the initial wound (0 h).via the inhalation of isoflurane, and all efforts were made to minimize suffering.Six-week male C57BL/6 mice were purchased from the Animal Laboratory of Sun Yat-sen University . The study was approved by the Ethics Committee of Guangdong Second Provincial General Hospital (no: 2017-YJS-033). For CTX injection, muscle injury was induced by injecting 100 μL of 10 μM CTX (dissolved in PBS) into the right tibialis anterior muscles of the mice on day 0. Then, 2.5 nmol silnc-GD2H/siNC oligonucleotides were prepared by pre-incubating with Lipofectamine 2000 for 15 min and injected on days −1, 1, and 4. The muscles of six mice were harvested on days 3 and 6, and RNA and proteins were then extracted for qPCR, western blotting, and hematoxylin and eosin (H&E) staining. Mice were sacrificed To assess tissue morphology, freshly isolated tibialis anterior muscles were fixed with 4% paraformaldehyde and stained with H&E. Next, the sections were analyzed under a microscope . Six microscope images per field were captured. Muscle fiber cross-sectional area (CSA) in the images was measured using ImageJ software .1 was used to predict the c-Myc binding sites in the lnc-GD2H promoter sequence. The universal protein knowledge base UniProt2 was used to retrieve the proteins identified based on MS. The online RNA-protein interaction prediction tool RPISeq3 was used to predict the possible interactions between lnc-GD2H and NACA; random forest (RF) classifier and support vector machine (SVM) classifier values greater than 0.5 were considered “positive.”The transcription factor prediction database JASPARt-test. A P-value < 0.05 was considered to indicate statistical significance.All experimental values are presented as the mean ± SD in graphs plotted using GraphPad Prism 5.0 . Comparisons between two groups were performed using the To explore the role of lnc-GD2H in myogenesis, we first observed the cell morphology under a microscope  and deteMouse C2C12 myoblasts were selected to investigate the potential role of lnc-GD2H in myogenesis. Stably transfected cells were selected to examine the overexpression effect of lnc-GD2H. The qPCR results showed lnc-GD2H expression was significantly activated in lnc-GD2H stably transfected lentivirus-infected myoblast cells (lv-OE-lnc-GD2H) compared with control myoblast cells (lv-OE-NC) during myoblast proliferation . DiffereConversely, knockdown of lnc-GD2H with two independent siRNAs  inhibiteRecently, c-Myc was reported to bind to and control dozens of lncRNAs to regulate skeletal muscle development . In the Luciferase reporter experiments showed that c-Myc enhanced the promoter activity of lnc-GD2H . TherefoBased on the lnc-GD2H expression profile during C2C12 myoblast differentiation, lnc-GD2H also potentially played a regulatory role in modulating myoblast differentiation. To investigate whether lnc-GD2H affects myoblast differentiation, immunofluorescence staining, qPCR and western blotting were used to analyze myoblast differentiation after lnc-GD2H overexpression or knockdown. The results showed that overexpression of lnc-GD2H led to a significant acceleration in C2C12 differentiation based on enhanced immunostaining and higher protein levels of MyHC , which iin vitro, incubated with proteins extracted from C2C12, captured by streptavidin beads, and then subjected to SDS-PAGE analysis. The same proteins from SDS-PAGE were then used for MS  analysis, and the specific proteins binding to lnc-GD2H were detected. Proteins that potentially interact with lnc-GD2H detected using MS are listed in Next, the molecular mechanisms underlying the promotional role of lnc-GD2H in myoblast differentiation were investigated. RNA FISH with differentiating C2C12 cells indicated that lnc-GD2H was distributed in the cytoplasm and nucleus . Many nuIn addition, we also analyzed the possible interaction between lnc-GD2H and NACA using the online database RPISeq; the results showed that lnc-GD2H would very likely bind to NACA based on RF classifier and SVM classifier values greater than 0.5 . To furtNext, we predicted there were two NACA DNA binding sites located in the promoter region of the Myog gene based on a previous report . The lucThere was no significant difference in the expression of NACA during myoblast proliferation and differentiation . Overexpin vivo assay of injury-induced regeneration. First, silnc-GD2H and siNC were injected three times, on the day before CTX injection (−1), and on days 1 and 4 post-CTX injection. The muscles were then harvested at the assigned times for analyses as illustrated .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier The animal study was reviewed and approved by Ethics Committee of Guangdong Second Provincial General Hospital.in vitro and analyzed the data. RC, YS, and SZ performed the experiments in vivo and analyzed the data. RC and SL wrote the manuscript. All authors read and approved the final manuscript.RC, CL, and TJ conceived and designed the experiments. RC, SL, YS, HS, and SZ performed the experiments 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.