KMT2D gene are the most common cause of KS and account for up to 75% of patients. Variants in KDM6A cause up to 5% of cases (X-linked dominant inheritance), while the etiology of about 20% of cases remains unknown. Current KS diagnostic criteria include hypotonia during infancy, developmental delay and/or intellectual disability, typical dysmorphism and confirmed pathogenic/likely pathogenic variant in KMT2D or KDM6A. Care for KS patients includes the control of physical and psychomotor development during childhood, rehabilitation and multi-specialist care. This paper reviews the current clinical knowledge, provides molecular and scientific links and sheds light on the treatment of Kabuki syndrome individuals.Kabuki syndrome (KS) is a rare developmental disorder principally comprised of developmental delay, hypotonia and a clearly defined dysmorphism: elongation of the structures surrounding the eyes, a shortened and depressed nose, thinning of the upper lip and thickening of the lower lip, large and prominent ears, hypertrichosis and scoliosis. Other characteristics include poor physical growth, cardiac, gastrointestinal and renal anomalies as well as variable behavioral issues, including autistic features. De novo or inherited pathogenic/likely pathogenic variants in the  Kabuki syndrome (KS) is characterized by distinct facial dysmorphism, growth retardation, psychomotor developmental delay and a wide spectrum of other manifestations affecting various body systems. Its genetic etiology has been proven approximately a decade ago . Since tKMT2D or KDM6A pathogenic/likely pathogenic variants (the genes responsible for KS). Alternatively, Whole-Exome Sequencing can be used [KS was first described in the early 1980s in Japan. It was named after the characteristic facial features which resemble the makeup of actors in the Kabuki theater in Tokyo. Initially, this syndrome was thought to be specific to the East Asian race; however, new data do not demonstrate clear prevalence of KS in any ethnic population. Total prevalence is estimated at 1 in 32 thousand [ be used . Even soKMT2D or KDM6A.A pathogenic or a likely pathogenic variant in aArched and broad eyebrows, with notching or sparsenessbShort columella and depressed nasal tipcLarge, prominent or cupped earsdPersistent fingertip padsTypical dysmorphic features including long palpebral features, eversion of the lower eyelid and two more of:International consensus diagnostic criteria for KS were established in 2019. These aimed to facilitate the process of diagnosing KS. It was concluded that a definite diagnosis of KS can be made in patients of any age with a history of infantile hypotonia, developmental delay and at least one of the major criteria:A list of supportive clinical features was agreed upon, including short stature, microcephaly, cleft palate, lip pits, hearing loss, congenital heart defects, feeding difficulties and immunological disorders. A probable diagnosis can be made in a patient with a history of infantile hypotonia, developmental delay and at least three of the supportive clinical features, and a possible diagnosis can be made in a patient with two of the supportive clinical features. These criteria may need to be expanded in the future; however, the authors argue that their use should already be implemented in order to make the KS diagnostic process universal .In the next chapters, we analyze the clinical characteristics of KS with regard to growth and endocrinology, neurology, cardiology, gastroenterology, laryngology, ophthalmology, oncology, nephrology and immunology. We also examine the effect of the KS molecular background on the clinical picture.KMT2D variants underlie Kabuki syndrome [KMT2D is also known as MLL2 (the latter name is considered outdated) [KDM6A gene on the X-chromosome. They claimed that KDM6A, which normally escapes inactivation, would be more likely expressed from an active X chromosome, so that the deletion of KDM6A would manifest itself in the affected individuals [KDM6A also cause KS. They examined 32 patients with KS diagnosed clinically who were negative for KMT2D variants. Two patients had nonsense KDM6A variants while one had a three base-pair deletion in KDM6A [KDM6A on the Y-chromosome. In the past, UTY was thought to have lost its demethylase activity and to be non-functional; however, knockout-mice models suggest that the gene is expressed in a cell. Female mice with homozygous KDM6A deletion do not survive, but hemizygous males do, as this is thought to be due to the UTY gene function. This suggests that UTY does play a role similar to KDM6A in metabolism and development [In 2010, Ng et al. performed the sequencing of exomes of 10 unrelated patients with Kabuki syndrome (KS) diagnosed clinically based on a specific scoring system. They demonstrated that syndrome . KMT2D iutdated) . Two yeaividuals . Miyake in KDM6A . UTY is elopment .KMT2D protein is a histone (H3) lysine methyltransferase protein, while KDM6A acts as a histone demethylase. Both proteins have complementary functions. KMT2D is responsible for cell-type specific gene expression during differentiation. It functions to trimethylate histone-3-lysine-4, opening the chromatin and activating homeobox and NESTIN genes during cell differentiation. KDM6A demethylates histone-3-lysine-27, closing the chromatin. Both genes affect the opening of chromatin and promote gene expression. Dosage sensitivity may be important in Kabuki syndrome. The switch between open and closed chromatin affects the access of transcriptional machinery to target genes, which may lead to the development of the disease [KMT2D’s catalytic activity. This in turn leads to an increase in methylated histone levels, which decreases homeobox gene expression, thus affecting global cell differentiation [KMT2D decreases mediator and polymerase levels, which leads to defects in cell-type specific gene expression and cell differentiation. KMT2D was identified as a mono and di-methyltransferase, which is essential for enhancer activation during cell differentiation [The  disease . Pathogentiation . Buildinntiation .KMT2D gene is located on chromosome 12. It is over 19 kilobase pairs in length and contains 54 exons. KMT2D codes for the enhancer histone-3-lysine-4 methyltransferase protein, one of six KMT proteins working as part of a chromatin modifier protein complex. Enhancers aid in gene expression regulation. They are often bound to transcription factors. KMT2D selectively binds to specific regions during various cell differentiation stages, activating gene expression depending on the differentiation stage [KMT2D loss-of-function variants inhibit neural crest development, suggesting that KS be considered a neurocristopathy. The expression of KMT2D in the neural crest during pre-migratory as well as migratory stages was found. KMT2D may be required for neural crest cell differentiation and consequent migration [KMT2D-caused transcription suppression led to the activity of hypoxia-response pathways. This proves that the loss of KMT2D function suppresses oxygen-responsive gene programs crucial for neural progenitor maintenance, ultimately leading to precocious neuronal differentiation [KMT2D and KDM6A in the development of tissue abnormalities, including craniofacial, heart and brain deformities. Variants in both KMT2D and KDM6A lead to improper cell differentiation, ultimately causing a characteristic dysmorphism and developmental delay [KDM6A variant and a phenotype including hypotonia, developmental delay, short stature, microcephaly, seizures, facial dysmorphism and cleft palate. KDM6A expression is significantly reduced in neural crest cells, leading to delayed neural development. The dosage sensitivity of KDM6A is associated with characteristic Kabuki syndrome dysmorphism.In humans, the igration . Cell-auntiation . A recenal delay . A fluorKMT2D and KDM6A proteins regulate the differentiation of mesenchymal cell lineages: myoblasts and osteoblasts. Mouse models have shown that lysine demethylase plays a crucial role in regulating mesenchymal lineage cell differentiation. Loss-of-function variants in lysine demethylase genes cause improper development of cells in this lineage and, presumably, several clinical neurological abnormalities that have been seen in KS patients. The anomalies present in KS children were also observed in mice with a loss-of-function KMT2D variant and brain development disorders: hypotonia, enhanced activity (corresponding to hyperactivity in KS children), a decreased auricular reflex (corresponding to hearing impairment) and motor coordination dysfunction [KMT and lysine demethylase proteins’ specific role in the regulation of myoblast differentiation was also recently examined using the C2C12 myoblast model system. KDM1A is the only one crucial for myogenic differentiation, while KDM6A (among others) is required for osteoblast differentiation. KDM1A, via histone-demethylation, represses the regulator of osteoblast differentiation, thus promoting myogenesis. KMT2D, however, is also required for osteoblast differentiation by the demethylation of a different receptor. These mechanisms of histone modification (lysine methylation) act as a signal, thus impacting the fate, rate and process of cell differentiation [function . KMT andntiation .KMT2D gene of the region encoding for methyltransferase (thus leading to the impairment of its function) resulted in impaired histone acetylation and methylation, eventually leading to a deficiency of the dentate gyrus granule cell layer and to reduced neurogenesis and hippocampal memory defects. A deficiency in postnatal neurogenesis directly results in the KMT2D defect as the base for intellectual disability in KS [The effect of the deletion in the ty in KS .KMT2D variant demonstrated sensory and psychomotor impairments, including hypotonia and impaired reflexes  and a reduction in the auricular reflex (suggesting hearing impairment) in mouse mutants. Increased general activity with decreased motor coordination, increased rearing and grooming and increased distance traveled and speed were observed. This study builds on Bjornsson et al.’s findings by presenting the KMT2D variant phenotype as more complex. This mouse model may shed light onto the wide range of psychomotor and behavioral impairments associated with KS in humans [n humans .KMT2D variant s likely cause reduced histone methylation due to impaired WRAD protein complex formation [KMT2D secondary structure through increased alpha-helical structure (coiled protein shape leading to dysfunction).Missense ormation . They maormation . NotableGrowth pattern in the pediatric population is dependent on several factors. These can be divided into intrinsic factors and external factors. Intrinsic factors (influenced by genetic factors) largely result from constitutive and familial growth or the presence of major congenital anomalies. External factors are shaped by prenatal care, labor mode, adaptation period, hormonal characteristics, feeding and nutrition during the first year of life and later into adolescence. On top of that, syndromic diagnosis by itself seems to carry important contributions to the growth pattern. Early observations of physical characteristics in Kabuki syndrome by Niikawa et al. suggested that birth weight and length were normal with ensuing postnatal growth retardation .KS patients commonly present with failure to thrive during infancy, feeding problems and reflux, with 65–74% of patients even requiring nasogastric tube insertion or gastrostomy. Conversely, during late childhood these children commonly present with obesity. Awareness and intervention by a dietician should be considered .Postnatal growth retardation was present in 50–70% of patients described in ensuing studies. Prenatal growth retardation was observed in a minority of analyzed cases and postnatal growth retardation was observed in the absolute majority, regardless of ethnic origins. . In a reKS children are generally born at term and with a normal birth weight. In one study, not one individual had a birth weight under the 3rd centile. Failure to thrive and feeding problems are observed in two thirds of patients. During the fifth year of life, progression to obesity is prevalent in up to 57% of patients .KMT2D variant [KMT2D variant than in those without it . Patients with a mosaic KMT2D variant less frequently presented with short stature. Growth Hormone -deficiency is the most common endocrinological finding in KS patients. Endocrinological evaluation and even the possibility of rh-GH-therapy for KS-patients may be beneficial [Up to 70% of KS patients present with short stature. This has been suggested to be directly caused by the  variant . Genotypneficial .Indications for rh-GH-therapy in pediatric patients include confirmed GH-deficiency, Chronic Kidney Disease, Turner syndrome, Intrauterine growth restriction/Small for gestational age infants who fail to catch up to normal growth centiles, Prader-Willi syndrome, idiopathic short stature, SHOX gene haploinsufficiency and Noonan syndrome (USA) . GeneticKMT2D versus the KDM6A group. Additionally, throughout the treatment course, patients’ body proportions were not affected [The largest study to date by Schott et al. in 2017 analyzed the effect of growth hormone treatment on linear height and body proportions in KS patients. The result was a statistically significant linear height increase after one year’s worth of rh-GH-therapy. Children who received GH-treatment at a younger age tended to display greater height increases than those who started at an older age. No significant difference was described between the affected .KMT2D pathogenic variants showed growth patterns roughly two standard deviations below the norm, enabling growth monitoring using growth charts. It is crucial not to miss a separate non-syndromic etiology of disrupted growth . The limitation is that it is difficult to predict whether final KS patient growth rate will catch up to that of children not affected by KS. The normal distribution of Body mass index BMI in KS patient groups is much wider than in the general population, which suggests the increased presence of excess weight or even obesity in KS patients [Normative growth curves especially designed for children with KS based on the largest cohorts to date were recently created. They reaffirmed observations by Schott et al. in 2017 that patients . This obpatients ,27.There is support in the literature for evidence of a link between molecular genetics mechanisms and growth pattern in KS. First, a balanced interplay between gonadal and adrenal steroid hormones and GH is essential for the pubertal growth spurt. Patients with a deficiency in any of these hormones experience a deficient growth spurt.KMT2D is essential for a ligand-dependent estrogen-receptor-α activation [Molecular Genetics section.During puberty, estradiol promotes the epiphyseal fusion essential for bone growth and thus directly affects adult height in both sexes. Ligand-dependent estrogen-receptor-α acts as a mediator in estrogen-sensitive tissues. Molecularly, tivation . More deKMT2D variant causes a decrease in the GH/IGF-I axis activity, and therefore a diminished growth spurt and short stature.Decreased growth associated with decreased serum levels of Insulin-like growth factor-1 has been described in mouse models. The GH to IGF-I axis is highly active during puberty. The effect of estrogen on this axis has been theorized to happen through the direct action of testosterone/androgen receptors, or through the indirect action of estrogens on estrogen-receptor-α. This suggests a biological mechanism for a significantly diminished pubertal growth spurt in KS. Finally, testosterone stimulates GH-secretion in puberty and throughout adulthood. This effect depends on the level of aromatization of testosterone within estrogen . PerhapsKMT2D and KDM6A gene variant influences endochondral ossification at long bone growth plates by disrupting the estrogen receptors in developing skeletal tissues. Sex-based differences are not only dependent on hindered estrogen receptor activity as a result of gene variant. SOX9 has been linked to the pathogenesis of the KS phenotype by describing disrupted endochondral bone formation. In theory, KMT2D allows SOX9 to become uninhibited, thus activating chondrocyte differentiation (SOX9 being a well-described chondrocyte-differentiation mediator). Variants in KMT2D prevent this process from taking place. SOX9 has also been described as involved in inhibiting osteoblast differentiation. Abnormal SOX9 expression  prevents the differentiation of chondrocytes into osteoblasts, ultimately causing too many chondrocytes and too few osteoblasts in the skeletal system. This probably causes improper growth, specifically growth retardation in KS (shortening of long bones and ineffective bone formation) [KS mice phenotypes are comprised of skeletal growth retardation and decreased length and weight as a cause of shortened long bones . These frmation) .KDM6A variant have an increased risk of hyperinsulinism (and thus hypoglycemic episodes) than those with KMT2D pathogenic variants [KDM6A demethylation of the H3K27 protein may lead to the deregulation of β-cell development [KDM6A codes for a demethylase that acts on H3K27me3/me2 and induces a steady-state in proliferating cells [A wide variety of hormonal abnormalities can occur as a result of KS. Kabuki syndrome has been recently associated with hyperinsulinism and thus to transient neonatal or infantile hypoglycemia. Persistent hypoglycemia, however, is uncommon. KS patients with a variants . KDM6A delopment . KDM6A cng cells . Most acIt is hypothesized that KS is associated with excess gonadotropin secretion, which causes premature thelarche in 41% of patients . One patKMT2D variant s [Loss-of-function variant s associated with Kabuki syndrome (KS) lead to the dysregulation of the differentiation of neuronal and myogenic cells. Mechanistically, this may lead to several neurological abnormalities in KS patients. These include infantile hypotonia, developmental delay and intellectual disability, epilepsy, behavioral abnormalities including autistic-like features, and CNS malformations. Neuropsychological symptoms have more commonly been observed in patients with ariant s .Infantile hypotonia has been described as a cardinal feature in KS patients . SpecifiKMT2D gene deletions or variants causing a dysfunction of synthesis in the first half of the KMT2D protein [It has been suggested that developmental delay is more pronounced in patients with  protein . However protein . Varying protein . However protein ,42. Ther protein .Several studies ,44,45,46Autistic-like behaviors described in KS cases included: preference to be alone, selective interest in specific objects, stereotype behavior, attention instability and echolalia. Upon analysis using the CARS, ADOS and ADI-R scales (used in autism spectrum disorders), patients tended to fulfill the criteria of autism. They generally had low IQ. Some did present abnormal neurological findings; however, these did not seem to directly affect autism spectrum behaviors. It has been suggested that genetic variants that cause KS promote the development of autistic-like behaviors through epigenetic mechanisms, as in other diseases such as Rett syndrome, Fragile X syndrome, Prader-Willi and Angelman syndrome . Thus, tKMT2D variant —a rare phenomenon in KS patients—have been described. Systemic features in mosaic KS patients are not significantly different from those of non-mosaic patients. The difference was seen in psychiatric manifestations: anxiety disorder, multiple phobias, bizarre behavior (soliloquy), emotional dysregulation and autistic-like behavior [Three patients with a mosaic behavior . The intbehavior .A novel approach refers to IQ as a memory deficit in the context of KS. Histone deacetylase inhibitor protein HDACi-AR-42 affects hippocampal memory by improving adult neurogenesis . BuildinThe prevalence of epilepsy in patients with Kabuki syndrome has not been accurately established. Some studies suggest it is close to 5–16%, while other estimations are up to 36%. Seizures may result from infantile hypoglycemia or arise from abnormal electrical activity within the brain . A rangeKMT2D variant presented with characteristic KS dysmorphism as well as complex partial seizures. The seizures were partially responsive to antiepileptic drugs. Exon 39 of the KMT2D gene contains a region that encodes for a long polyglutamine chain. This chain is sensitive to variants which may cause CNS and other organ dysfunction [Four case reports describe patients with brain anomalies on imaging . This suggests that brain development and, more broadly, findings upon brain imaging are variable in the context of KS ,54,55,56function . KS has function ,60.KMT2D pathogenic variant presented with microcephaly and craniosynostosis [KMT2D variants and should be suspected in the context of KS even when facial dysmorphism is not present [Other rare single case reports of neurological abnormalities in the context of KS have been described. KS patients often present with nonspecific cerebral atrophy and microcephaly. CNS imaging is heterogenous in KS. A child with a confirmed nostosis , which mnostosis . A KS pa present . An adul present . Togethe present ,66.Most importantly, early intervention and rehabilitation have been described as beneficial for both patients and their caretakers. This is especially true if a collaborative treatment team of doctors, teachers, physical therapists, speech therapists and psychotherapists actively work in concert with the family. Specific psychotherapy techniques for KS patients should be considered on a case-by-case basis .Cardiological manifestations are cardinal features of Kabuki syndrome. The three most commonly observed cardiac defects in KS patients are atrial septal defect, (ASD), ventricular septal defect (VSD) and coarctation of aorta (CoA) . An analKMT2D variants present with CHD [KMT2D variants . Of septal defects, VSD and ASD-II are predominant. Bicuspid aortic valve has been reported in 1/8 of patients. Mitral stenosis, hypoplastic left heart syndrome, conotruncal defects, cardiomegaly, tetralogy of Fallot and other rare syndromes have been reported in under 5% of patients [Up to 80% of KS patients with with CHD . CoA is with CHD . CoA maywith CHD  other hewith CHD  or left with CHD . Taking with CHD . Togethepatients .KMT2D’s role in cardiogenesis has been demonstrated in mice. KMT2D deletions lead to decreased histone-3-lysine-4 methyltransferase expression at enhancers and promoters of cardiogenesis. This results in the downregulation of ion transport and cell cycle genes. Moreover, KMT2D-bound regions within cardiomyocytes have been defined [KMT2D knockdown mice hearts [KMT2D may be enough for normal heart function. A deletion of both copies leads to general disruption of myocardial development [KMT2D heterozygous frameshift deletion presented with VSD as well as a myriad of other cardinal KS features. Good general condition and prognosis was seen, and surgical correction of cardiological abnormalities was performed. This highlights the importance of a case-by-case surgical treatment approach to KS patients as well as genotype-phenotype-driven diagnostics [Xenopus frogs develop hypoplastic hearts with defective chamber development. Cardiomyocyte differentiation is severely affected by a loss of KMT2D function. KMT2D is required for the development of primary and secondary heart fields [KMT2D during endothelium and endocardium development has been suggested in zebrafish. Notch pathway inhibition leads to physiological cardiovascular development. Extrapolating on this finding, KMT2D may play a role in regulating vasculogenesis and angiogenesis (connective tissue development) [KMT2D’s function as a cardioregulatory gene in humans is not yet fully understood, nor is its role in the development of specific congenital cardiac diseases [KDM6A variants present with CHD, and those who do show mostly right-sided lesions [ defined . Variante hearts . Disordeelopment . On the gnostics . Knockdot fields . A link lopment) . Yet, KMdiseases ,76,77. F lesions . Mouse m lesions ,70.KMT2D variants have been suggested [Surgery is commonly indicated to treat congenital cardiac anomalies in KS patients. Usually, KS patients tolerate the surgical correction of CoA well, and cardiological prognosis is positive ,73,78. Suggested .KMT2D variants [Underlying connective tissue diseases may ultimately lead to cardiac defects in KS patients. Connective tissue developmental disorder in the context of KS has been proposed for decades, mostly while describing hip, knee and shoulder joint hypermobility in patients with variants . Digiliovariants . Perhapsvariants . Great vvariants  and doubvariants , may arivariants .Systemic vascular abnormalities in the context of KS have been recently reported. Gatto et al. describe a case of a KS patient who suffered a transient ischemic stroke due to occlusion of the right internal carotid artery. Despite the fact that cardiac and great vessel anomalies are common (as we demonstrate above), some rarer systemic vascular anomalies have been reported too, affecting among others small vessels of the brain . It is iBesides conotruncal, great vessel or connective tissue anomalies, an association of left-sided heart anomalies with KS has been theorized. The presence of normal X chromosomes can prevent the development of left-sided anomalies. Left-heart anomalies are of multifactorial origin . KS patiOne rare case of a child with KS, who died of untreated arrhythmia at the age of 11 months, was reported. The arrhythmia resulted from a cardiac conduction abnormality causing episodes of bradycardia unresponsive to pacing, which eventually led to asystole and death. On top of that, the child suffered from severe immunodeficiency. Autopsy revealed coronary sinus dilatation, tricuspid valve dysplasia, thickened chordae tendinae and right ventricular hypertrophy. Conduction abnormalities in KS children have not been extensively investigated .KMT2D variants, attention should be paid to the detection of left-sided obstructive lesions, while in patients with KDM6A variants, right-sided lesions should be the subject of attention [Most reports do not describe severe symptomatic heart disease in KS patients. In fact, some KS patients have remained cardiologically asymptomatic, or their symptoms have been masked by the underlying disease . Thus, ttention . Individttention . All patttention . If treaKabuki syndrome patients frequently present with poor feeding and nutrition, which leads to poor growth. They often have sensory issues that interfere with eating, rendering them more sensitive than other children. Textures, smells and temperatures of food may cause these children to present with aversions. Due to poorly coordinated suck and swallow reflexes, a nasogastric (NG) feeding tube or even a gastrostomy may be required ,88. ReflGastrointestinal (GI) anatomical dysmorphism has been described in single KS cases too, especially in females. It has even been suggested that KS in females is a risk factor for anorectal malformations . KS indiKMT2D has been found to be somatically mutated in hepatocellular carcinoma (HCC) and has also been confirmed as a hepatitis B virus integration site. Variants in KMT2D may result in abnormal enhancer regulation, which leads to changes in transcription, DNA breaks and tumor development. This also regulates hepatic metabolism and coactivates PPARy2, which leads to increased bile acid levels, which have been linked to hepatic tumor development (unspecific marker). Whether or not KS should be considered a risk factor for hepatocellular carcinoma remains unknown [Immunology section) [Hepatobiliary abnormalities, biliary atresia, hepatic fibrosis and sclerosing cholangitis have been documented in 2–21% of KS patients . Very li unknown . The asssection) . In one section) .Kabuki syndrome (KS) patients commonly present with external ear dysmorphism. In fact, this is one of the most characteristic findings of KS, present in upwards of 80% of patients . Outer eEar dysmorphism is often present together with clefting of the palate, lip or even the uvula. These dysmorphic traits increase the risk of otitis media in KS patients. Indeed, one of the key features of KS is recurrent acute otitis media (AOM) and the consequent necessity for recurrent antibiotic therapy. In vitro models show that recurrent AOM may be caused by disorders of eustachian tube development in vitro. This clinical picture is similar to that of other diseases characterized by craniofacial malformations . RecurreInner ear abnormalities (among them Mondini dysplasia) cause sensorineural hearing loss in KS patients. One KS patient manifested severe inner ear dysplasia, cochlear aplasia, semicircular canal dysplasia and unilateral vestibular enlargement. Another presented with bilaterally large ears, posteriorly rotated, underdeveloped antihelices and unilateral vestibular enlargement. Both children had profound sensorineural hearing loss, most likely due to recurrent AOM . Cases oImmunology section). Notably, all patients were positive for novel KMT2D variants, which have been demonstrated to affect B-cell development and the regulation of signaling necessary for lymphopoiesis. The potential effects of KMT2D on immunoglobulin levels are unknown. Regular intravenous immunoglobulin supplementation during young ages as a treatment for immune deficiency may be indicated [The chronic otitis media problem has been described in the context of KS immunological issues. The majority of patients had recurrent otitis media and hearing impairment, lower levels of memory cells, severe hepatitis-B antibody deficiency (despite vaccination) and immunoglobulin deficiency. Recurrent ear infections were attributed to congenital immunodeficiency , antibiotic and symptomatic treatment of chronic otitis media, and surgical correction of the underlying disease as a last resort . The bonKMT2D variants in neural crest cells on craniofacial dysmorphism development. Knockout mice demonstrated reduced frontonasal bone lengths and dysmorphism including cleft palate, hypoplastic mandible and cranial base ossification deficit. This was proven to be caused by altered osteochondral progenitor differentiation. Specifically, mutant neural crest cells led to defective secondary palatal shelf elevation with reduced extracellular matrix. Mutant cranial base chondrocytes do not properly differentiate and exhibit defective endochondral ossification [The craniofacial and dental features in the context of KS were outlined. The cardinal dysmorphism is a peculiar face with long or wide palpebral fissures, lower lateral eyelid eversion, arched eyebrows with the lateral third dispersed, prominent ears, depressed nasal tip, and skeletal and dermatoglyphic abnormalities. A typical combination of facial features is present in virtually every KS patient. Midface hypoplasia with or without mandibular hypoplasia is observed in some patients . Recent fication ,109. TabKMT2D and KDM6A genes in tooth development has been recently theorized. Currently, the maxillomandibular relationship, tooth size and dental arch forms are being studied. The expression of KMT2D and KDM6A in human tooth buds at 7–12 Hbd was recently demonstrated. Gene expression was observed in the dental epithelium of primary incisors, canines and molars at the bud and cap stages of tooth development. This suggests that loss-of-function KMT2D or KDM6A variants may underlie the observed orodental anomalies [KMT2D and KDM6A as epigenetic modulators of several biologic processes was recently discussed. Loss of their function leads to a variety of manifestations, in this context namely hypodontia, widely spaced teeth, absence of teeth, enamel hypoplasia, dental agenesis and caries [Oral anomalies are present in over 60% of KS cases. Documented orodental pathology cases include congenital tooth absence, malocclusion, high-arched palate, abnormal dentition, widely spaced teeth, hypodontia, conical incisors, screwdriver-shaped incisors, delayed tooth eruption and ectopic upper molars ,115,116.nomalies . The fund caries . Tooth bd caries . Incisord caries . KS cased caries ,117. Thed caries . Nursingd caries . The treatment of orodental symptoms includes orthodontic treatment to correct bite function in addition to esthetics. Extraction and implantation procedures are often used to correct disorders of tooth development and increase patient quality of life. Orthodontic treatment can also be aimed at correcting arch length discrepancy. Due to feeding difficulties and an increased risk of nursing bottle syndrome, dental and orthodontic care must be integrated with child speech therapy in order to maintain long-term effects of orthodontic treatment. This aims at quality of life improvement ,118,120.Cleft palate is a feature observed in 33–50% of KS cases. Pediatric follow-up is crucial in KS patients with clefting. Usually the first symptoms of cleft palate are speech development disorders and nasal leakage of milk during early infancy . They mention, however, that mental retardation often complicates the accurate correct diagnosis of cleft palate because the above symptoms may be attributed to it. Therefore, they suggest that cleft palates are more frequently present than reported. They highlight the importance of routine specialist examination for clefting . KS chilLower lip pits are another feature that has been reported in KS, albeit more rarely than other cardinal features. Two KS patients were initially diagnosed with Van der Woude syndrome . These patients presented with lower lip pits and cleft palate and were clinically diagnosed with Van der Woude syndrome in early infancy. Upon follow-up during the second to fourth years of life, it was consequently noticed that they exhibited clinical features that do not concur with Van der Woude syndrome: facial dysmorphism—widened palpebral fissures, eyelid eversion and prominent ear; chronic otitis media leading to hearing impairment; velopharyngeal insufficiency and delayed psychomotor development. These patients’ clinical picture resembled KS, which was later confirmed by genetic testing. These cases emphasize the need for the careful evaluation of patients with lip pits and raise awareness for differential diagnosis .Extraocular features have been defined as cardinal clinical diagnostic signs of Kabuki syndrome. They are present in nearly all KS patients  and inclCHD7—a gene coding for a helicase) [Coloboma is frequently reported in KS patients. It is sometimes associated with congenital heart disease and ear and renal defects, leading to the misdiagnosis of CHARGE syndrome before the presentation of typical facial dysmorphisms. It may affect the iris, choroid, retina or optic nerve. Coloboma derives from incomplete embryonic fissure closure in utero. Normally, the embryonic fissure develops from an invagination of the optic vesicle and leaves a gap that lets the hyaloid artery supply the inner eye, a process crucial for normal development. Iris or ciliary body colobomas result from incomplete anterior closure, choroid, retina or optic nerve colobomas result from incomplete posterior closure, whereas lens colobomas may be caused by incomplete closure of any segment. Eyelid colobomas are of a different embryologic origin and arise at a later stage of development. These are generally unrelated to disorders of globe development . CHARGE elicase) . Infrequelicase) .KMT2D variant [KMT2D variant [A wide array of rare ophthalmologic findings is present in almost all Kabuki syndrome individuals. Currently, it is not possible to directly associate these features with KS, yet a recommendation is widely supported that all KS patients should undergo ophthalmological assessment at the earliest age possible . Abnorma variant . Bilater variant ,133. Age variant . A KS pa variant .KMT2D variant was later confirmed in this patient. KMT2D proteins are active as part of a multi-subunit complex (see Molecular Genetics chapter) and have been known to interact with CHD7 and CHD8 proteins through the WAR complex. Additionally, KMT2D has been known to interact with the PAX-interacting protein 1 (PAXIP-1) as well as other transcription factors encoded by PAX2. These factors have a high rate of expression in the developing eye, which may also explain microphthalmia and eye developmental disorder in utero [Microphthalmia was described in several KS patients. In one neonate, extreme microphthalmia with an anomaly of globe development, hyperplastic primary vitreous and hypoplastic optic chiasm were all present . KMT2D vin utero . Ocular A summary of other rare ophthalmologic findings that were reported in under 10 KS patients in the literature is presented in KMT2D is expressed in most cells and tissues. Its pathogenic variant results in the interruption of histone methylation related to gene expression, thus affecting normal growth and development as well as many other processes described in other chapters. Its role in development, metabolism, cell differentiation and tumor suppression has been recently described [KMT2D has been defined as one of the most commonly mutated genes in a number of cancers such as gastric cancer, lymphoma and medulloblastoma [KMT2D promotes lung tumorigenesis and upregulates tumorigenic processes such as glycolysis in a recent mouse model [KMT2D loss-of-function variant has also been demonstrated; however, the rarity of such variants makes it difficult to assess their pathogenicity exactly [KMT2D loss-of-function variant causes the proliferation of neoplastic cells [KMT2D deficiency attenuates cancer cell migration, promoting tumor growth [KMT2D is required for effective H3K4 methylation [escribed . Defectiblastoma . The lunse model . The onc exactly . Using ric cells . This paic cells . Additior growth . The stuhylation .KMT2D and KDM6A belong to the same beta-globin and estrogen-receptor regulator multiprotein complex  and interact with each other [KDM6A has been shown to interact with CBP transcription-activator protein in a breast cancer cell-line MCF-7 in a Drosophila model. Clinically, this variant has been found in many tumor types, including multiple myeloma, esophageal squamous cell carcinoma, renal cell carcinoma, glioblastoma, and breast, urinary tract, pancreatic and colorectal cancers. KDM6A has been demonstrated as a tumor suppressor that leads to slow cell growth [KDM6A in the context of urinary tract, pancreatic and lung tumors and acute myeloid leukemia [KDM6A has a pro-oncogenic role. The loss of KDM6A in human breast cancer cells causes a decrease in estrogen-induced cell proliferation in vitro. This is, however, based on one recent study and conclusions as for in vitro pro-oncogenic trends of KDM6A are yet to be determined [ch other ,150. Recl growth ,152,153.leukemia . Converstermined .KMT2D variants, i.e., restricted to cancer tissue, is well known. However, the significance of germline  pathogenic variants in this gene in KS patients is yet to be determined. KS patients may present with a wide range of tumors. The new research on growth hormone therapy (see Growth chapter) suggests that it may worsen tumor burden [The above described role of somatic r burden . Cancer In a 3-year-old KS patient in whom Wilms tumor was diagnosed, no negative impact on lifespan was demonstrated and the tumor was successfully managed according to procedures used in non-KS patients . NeuroblKMT2D variant. She underwent donor liver transplantation. On biopsy, besides characteristics of hepatocellular carcinoma, the explanted liver exhibited hepatic adenomas with a low Ki-67 proliferation index [KMT2D has been shown to be mutated in hepatocellular carcinoma cases, and has even been suggested to be associated with earlier recurrence of disease, greater microvascular invasion and a more aggressive phenotype [Hepatoblastoma was described in one 6-year-old clinically diagnosed KS patient. Prognosis was assessed to be favorable and this patient was treated with chemotherapy (which led to mild hematotoxicity). Later, a right lobe hepatectomy was performed, followed by adjuvant chemotherapy, successfully leading to complete remission . Hepatocon index . The asson index ,159. KMThenotype ,160. It henotype ,161. DesCTNNB1. KS may predispose patients to pilomatricoma formation because CTNNB1 plays a role in the WNT pathway, which is regulated by KMT2D [Pilomatricoma—a benign hair cell tumor characterized by lobules of epithelial cells—has been associated with several genetic syndromes  ,163,164.by KMT2D ,163. Surby KMT2D .KMT2D variant. She presented with a recurring neck mass despite excision and received a favorable prognosis. The tumor was successfully excised [Giant cell fibroblastoma, a soft-tissue malignancy that is especially rare in childhood, was reported in a 12-year-old female patient with a  excised .Spinal ependymoma was diagnosed in a 23-year-old KS patient. Ependymoma is a tumor originating from the ependymal lining of the ventricular system. Most cases are located intracranially; however, spinal presentation is less common. The patient presented with hypoesthesia of the feet and lumbar pain. Laminotomy tumor resection was performed and no neurological complications were observed. Ki-67 index was low upon biopsy. This case sheds light on atypical symptom presentation in KS patients—even though ependymoma does not typically present in the spine, spinal abnormalities are typical in KS patients .KMT2D germline variant. He presented with clubfeet, hirsutism, hypotonia, and feeding problems during infanthood; chronic otitis media, obesity and failure to thrive during late childhood; and confirmed Immunoglobulin IgA and IgG deficiencyType 1 diabetes mellitus1 and pancreatic failure in young adulthood. He required pancreatic transplant thrice, after which he developed Ebstein-Barr virus-positive Hodgkin Lymphoma and underwent chemotherapy, achieving complete remission. KMT2D is one of the most frequently mutated genes in follicular lymphomas as well as in diffuse large B-cell lymphomas. By the loss of H3K4 methylation at enhancer DNA regions, truncating variants of the KMT2D protein lead to the loss of enzymatic activity and haploinsufficiency, causing malignant transformation [KMT2D variant causing KS as well as a translocation t  causing Burkitt lymphoma in an EBV-positive KS patient. KS patients may thus be at increased risk for hematological cancers. Decreased histone regulation may cause DNA damage due to KMT2D variant, and that may increase the risk for other permanent DNA damage to occur, in this case, t  translocation [Several KS patients with hematological malignancies were reported. Hodgkin Lymphoma was reported in a patient with a severe multiorgan phenotype and ormation ,169. A cormation . Severalormation ,173,174.KMT2D and KDM6A encode, among others, proteins acting in the COMPASS complex, which serves as a chromatin opener. Loss of function of KMT2D and KDM6A epigenetically leads to defective B-lymphocyte terminal differentiation and thus hypogammaglobulinemia and reduced memory B-cell numbers [KMT2D variant had reduced serum IgA and elevated IgM. The bone marrow, spleen and intestine of KMT2D-mutated mice contained fewer IgA-secreting cells. There were more B-lineage cells in the Peyer patches; however, there were fewer Peyer patches overall. This was also found in vivo. KMT2D epigenetically controls the gene ITGB7, which encodes for an adhesion protein that mediates intestinal homing (the process by which white blood cells target both inflamed and non-inflamed regions of the gut to provide an immune response). KMT2D insufficiency has deleterious effects on B-cell differentiation, explaining this mechanism of immunodeficiency in KS patients [KS patients present with numerous immunopathological manifestations.  numbers . B-cell patients .KMT2D takes part in chromosomal translocations that create chimeric proteins. Translocation partners exist in a super elongation complex, which is required for Hox gene expression in white blood cells as well as in leukemic cells  [chapter) . The COMKMT2D or KDM6A). An increased risk of immune thrombocytopenic purpura development with missense variants than with truncating variants has been suggested [Otolaryngology chapter), hypogammaglobulinemia, increased risk of autoimmune disease, increased risk of immune thrombocytopenic purpura, vitiligo, autoimmune hemolytic anemia, thyroiditis and other rare autoimmune cases [Considering the genotype, the prevalence in KS patients of immune deficiency or autoimmune manifestations does not differ by gene type . The routine administration of IVIG has not been proven to be effective in every KS patient. IVIG should only be considered in case of low total serum IgA [MTOR inhibitors, as a more targeted treatment for immunodeficiencies associated with KS, are a subject of recent research, and singular case studies present the remission of cytopenia (see Table 6) [KS patients are more likely to develop Common Variable Immunodeficiency, which is known to increase the risk of autoimmune disease development, especially immune thrombocytopenic purpura and autoimmune hemolytic anemia . The KS erum IgA ,183. MTOTable 6) .KMT2D variant increases the risk of renal manifestations, but not of urinary tract anomalies [KMT2D epigenetic role in the regulation of renal development. Dysfunctional KMT2D may ultimately lead to renal dysplasia. However, the specific role of KMT2D in renal development has not been established [Up to 40% of KS patients present with urinary system abnormalities. Of these, about half present with renal malformations and half with urinary tract anomalies. The concomitant presentation of renal and urinary tract abnormalities is rare. Renal malformations and diseases include, in order of prevalence, horseshoe kidney, renal hypodysplasia, renal ectopy and renal duplication and renal insufficiency (single reported cases). Urinary tract malformations include, in order of prevalence, hydronephrosis and ureteral duplication. No phenotype correlation between KS and vesicoureteral reflux or ureteropelvic junction obstruction seems to be prevalent . Considenomalies ,189,190.Several KS patients with severe renal insufficiency have been reported. Some of these patients also presented with renal dysplasia. One KS patient presented with severe renal insufficiency during the first month of life due to dysplastic kidneys. She had a positive family history of Polycystic Kidney Disease. Poor prognosis was concluded . AnotherHNRNPK, which codes for a ribonucleoprotein. The variant causes a multiorgan development disorder among other effects, ultimately leading to chronic kidney disease and kidney failure. Renal anomalies leading to chronic kidney disease are not unheard of in KS patients too. Renal ultrasound, kidney function evaluation and, when appropriate, early dialysis are recommended in both Au Kline Syndrome and KS patients too [Kabuki syndrome has often been compared to Au Kline syndrome while considering kidney diseases. Au Kline Syndrome is caused by a variant in ents too .Generally, the treatment of renal anomalies in KS patients is no different than in patients without KS . SeveralRare urinary system manifestations reported in the literature are presented in It is critical that general practitioners are aware of KS. Dysmorphic features in KS patients may appear similar; however, the entire clinical picture may vary widely between patients. This is especially true considering issues pertaining to specific organ systems, as we have demonstrated in our review. Early genetic diagnostics to detect the underlying syndrome are crucial as they confirm KS as well as classify the KS subtype. The early recognition of abnormal medical issues in KS patients is important to prevent complications and to improve prognosis, especially considering that the treatment of certain conditions in KS patients is no different in patients without KS. A greater understanding of the clinical presentation of KS will allow doctors to provide quality primary care to patients and to their families.Further research should focus on the specific genetic pathomechanism of the various clinical states we describe in order to potentially develop novel treatments. Mouse models and computer models are being studied. Existing treatments  should be another subject of further research. Large cohorts of KS patients should be recruited for a longitudinal study as current findings remain limited. Finally, an in-depth analysis of KS patients’ quality of life should be performed in order to shed light on the most helpful quality of life improvement techniques as well as, we hope, to prepare for the coming age of genetic therapy.


Water, one of the most priceless sources of life, is becoming dangerously threatened and contaminated due to population growth, industrial development, and climatic variations. The drainage of industrial, farming, and municipal sewage into drinking water sources pollutes the water. The textile processing industry is one of the major consumers of water. Herein, the idea of water-free dyeing of electrospun poly  PICT nanofibers is proposed. For this, two different deep eutectic solvents (DE solvents) were introduced as an alternative to water for the dyeing of PICT nanofibers in order to develop a water-free dyeing medium. For this, C.I. disperse red 167 was used as a model dye to improve the aesthetic properties of PICT nanofibers. PICT nanofibers were dyed by conventional batch dyeing and ultrasonic dyeing methods to investigate the effect of the dyeing technique on color buildup characteristics. Dyeing conditions such as dyeing time, temperature and, dye-concentration were optimized. Morphological and chemical characterization observations revealed a smooth morphology of dyed and undyed PICT nanofibers. The ultrasonically dyed nanofibers showed higher color strength and increased tensile strength compared to conventionally dyed nanofibers. Further, the consumption of electrical and thermal energy was also calculated for both processes. The results confirmed that the ultrasonic dyeing method can save 58% on electrical energy and 25% on thermal energy as compared to conventional dyeing. Recently, the characteristics and synthesis of polyesters based on isosorbide are gaining more attention in terms of exploring potential applications. Poly  PICT is one of the bio-based polymers and is widely used in different applications because of its ease of production, eco-friendly character, green source of raw material , unique molecular structure, good transition temperature, and mechanical properties ,6,7,8.For advanced apparel applications, the enhancement of the aesthetic properties of nanofibers is equally important as their functional characteristics ,5. Some Water, one of the most priceless sources of life, is becoming dangerously threatened and contaminated due to population growth, industrial development, and climatic variations. The drainage of industrial, farming, and municipal sewage into drinking water sources pollutes the water. The textile processing industry is one of the major consumers of water . In the Recently textile industries have become very conscious about limiting the consumption of electrical energy during processing, especially in those processes which increase the production cost . Many teHerein, dye-ability disperse dye [Disperse dye, Foron Rubine S-GFL 150  (Red-167:1) was assessed keeping in mind the better color strength and chemical compatibility of disperse dyes with a polyester-based substrate. PICT nanofibers were dyed conventionally and ultrasonically in order to compare the dyeing efficiency, electrical, and thermal energy consumption. The color strength and colorfastness of ultrasonically dyed samples and conventionally dyed samples were measured in order to measure the effect of both processes on color buildup. Samples were examined with SEM and FTIR for the physicochemical properties. Furthermore, tests of the shrinkage and tensile strength of samples were also carried out. Moreover, the electrical and thermal consumption for both processes was also calculated in order to assess the energy consumption for both processes.n ~ 26,000, Choline chloride (>98%) with a molecular weight of 139.62 g/mol, Furfuryl alcohol (98%) with a molecular weight of 98.10, Chloroform having a molecular weight of 119.38, and Trifluoroacetic acid (TFA) having a molecular weight of 114.02 were supplied by Sigma-Aldrich, USA. Ethylene Glycol with a molecular weight of 62.068 was purchased from Wako, Japan. Disperse dye, Foron Rubine S-GFL 150  (Red-167:1) were obtained from Clariant (Pakistan). PICT with an average molecular-weight of M PICT nanofibers were prepared using an electrospinning technique. The PICT polymer was dissolved in chloroform: TFA (3:1) and stirred for 2 h. In order to obtain bead-free nanofibers with stable electrospinning, PICT nanofibers were prepared using different polymer concentrations from 6% to 14% and optimized at 10% concentration. The applied voltage to the polymer solution was 10.6 kV using a high voltage power supplier  for electrospinning, where the distance between tip and collector was kept at 16 cm. The nanofiber samples were pealed off after air drying for 24 h for further characterizations.Deep eutectic solvents have emerged as an alternate to the conventional organic solvents and ionic liquid to make organic transformations more efficient. Choline chloride, ethylene glycol, and furfuryl alcohol were the raw chemicals for the preparation of the deep eutectic solvents. Two types of deep eutectic solvents were prepared by alternating the mixing of choline chloride, ethylene glycol, and furfuryl alcohol. The first solvent was prepared by mixing choline chloride and furfuryl alcohol at 1:2, and the second solvent was prepared by mixing choline chloride and ethylene glycol at 1:2, and these solvents were named as CC:FF and CC:EG, respectively . After mBoth conventional and ultrasonic methods were used for the batchwise dyeing of PICT nanofibers with disperse dye and two types of deep eutectic solvents used as a dyeing medium. Ultrasonic equipment  with a fixed frequency of 38 kHz and output power of 320 W was used to carry out the ultrasonic dyeing of PICT nanofibers, and the intensity of the sonication bath was set at 0.98 W/cm2 (intensity = Power/bath area: 320–328). For dyeing, a 10 mg mass of PICT nanofibers having an average thickness of 120 µm was used. The ratio of dye liquor to the mass of nanofibers was maintained at 20:1. All samples were dyed by conventional and ultrasonic methods by optimizing at different dyeing parameters. The dyeing temperature was checked at , dyeing time at , and the dye concentration  were used to check color buildup of disperse dyes for dyeing PICT nanofibers. The results were optimized for both the conventional and ultrasonic dyeing of PICT nanofibers and compared with respect to dyeing efficiency and color strength.The color strength of conventionally and ultrasonically dyed PICT nanofibers was measured using a data colors spectrophotometer and assessed by K/S values, calculated by Equation (1):The changes in the chemical structure of dyed and undyed PICT nanofibers were assessed by the Fourier transform infrared spectroscopy on IR prestige—21 by Shimadzu Japan. All the dyed and undyed samples were analyzed by using ATR-FTIR mode. To analyze the physical structure, a scanning electron microscope model (JEOL JSM 6010LA), at a voltage of 30 kV with the highest magnification of 500,000× was used. The samples were sputtered by Pd-Pt before the morphology analysis.The color fastness test was carried out for checking the dyed PICT nanofibers. Color fastness to washing was performed in a gyro-wash , under the standard method of ISO 105, C10: 2006. The color fastness to light was also investigated according to ISO 105—B02 by Appollo The tensile strength of prepared PICT nanofibers was investigated using the Universal Tensile Testing Machine . The PICT nanofibers having a 120 µm average thickness were assessed, the tensile strength and tensile stress of all samples were compared, and Young’s modulus values were also calculated. 2 with a constant average thickness of 120 µm. The shrinkage of PICT nanofibers after dyeing was calculated by using Equations (4) and (5) for lengthwise shrinkage, and widthwise shrinkage, respectively.A shrinkage test was performed by taking the PICT nanofiber sample size of 5 × 5 cmLs is lengthwise shrinkage, L is the original length of the samples, L∆ is the change in the original length after dyeing. In Equation (3), Ws is the widthwise shrinkage. W is the original width of samples. w∆ is the change in the original width after dyeing. In Equation (4), Rs is the residual shrinkage after dyeing [In Equation (2), r dyeing .The energy consumption of conventionally and ultrasonically dyed PICT nanofibers was calculated by the electrical energy Equation (5) as given below:Similarly, the thermal energy consumption of conventionally and ultrasonically dyed PICT nanofibers was calculated by the thermal energy Equation (6) as given below:PICT nanofibers were dyed with C.I. disperse Red-167:1 by ultrasonic and conventional methods at different temperatures. The dyeing temperature of PICT nanofibers was studied in the range of 30 °C to 80 °C by keeping the time constant at 30 min. The time optimization for the dyeing of PICT nanofibers is just as important as temperature. At an optimized temperature, dyeing was carried out for different time intervals from 10 min to 50 min. Red disperse dye was used to dye PICT nanofibers in the CC:EG medium and CC:FF medium. The color build-up properties of PICT nanofibers as per dye concentrations are given in Ultrasonically and conventionally dyed PICT nanofibers have been compared with respect to color strength as shown in −1 and 1241 cm−1 confirms the presence of C=O and C-O in dyed and undyed PICT nanofibers. The spectra further showed the stretching of C-O at 1108 cm−1. The bending peak at 721 cm−1 confirms the presence of C=C in PICT nanofibers before and after dyeing. The functional groups of Red-167:1, CC:EG, and CC:FF could not be observed because a small number of respective chemicals were utilized to carry out the dyeing process and were rinsed well after dyeing [An FTIR spectroscopy was carried out to examine the change in the chemical structure of the dyed and undyed PICT nanofibers shown in r dyeing . UltimatTo analyze the PICT nanofiber’s properties, it is necessary to perform the XRD on dyed and undyed samples. The color fastness test was performed on the dyed PICT nanofibers which are presented in PICT nanofibers after exposure to sonication showed substantial shrinkage so it was necessary to analyze the shrinkage in PICT nanofibers after dyeing. In For the dyeing of PICT nanofibers, electrical energy consumption was compared between the conventional dyeing method and ultrasonic dyeing method as given in The thermal energy was also compared between the ultrasonic dyeing and conventional dyeing method as shown in The PICT nanofibers were successfully dyed in DE solvents with disperse dye using ultrasonic and conventional dyeing methods. Physiochemical outcomes confirmed the presence of dye molecules on the fiber surface without altering the chemical structure of the electrospun PICT nanofibers. Colorimetric studies and colorfastness tests suggest better color buildup properties of the ultrasonic-assisted dyeing of PICT nanofibers with DE solvent than that of the conventional dyeing method. It was found that shrinkage was the ultimate reason for increased color strength in ultrasonically dyed PICT nanofibers. Further, Ultrasonic dyeing in DE solvents proved to be a better choice for environmentally friendly and cleaner production, this cleaner approach can save electrical energy (58.6 and 58.7%) and thermal energy (25 and 25.4%) with CC:FF and CC:EG mediums, respectively. The crystallinity and tensile strength were increased in PICT nanofibers after dyeing.


Morphine decreases DDX49 expression in HCC cells. Our results suggest that DDX49 contributes to HCC, and that morphine may exert anti-cancer effects by down-regulating it.Pain in hepatocellular carcinoma (HCC) is a frequent cause of low quality of life, and morphine is routinely used as a first-line opiate analgesic in HCC. Morphine may exert not only analgesic effects but also anti-cancer effects via unknown mechanisms. Here we show that morphine can inhibit HCC cell proliferation. We further show that DEAD-box helicase 49 (DDX49) is up-regulated in HCC tumors, and that knocking down the DDX49 gene decreases tumor formation  The prevalence of liver cancer is increasing worldwide. Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths . AdvanceIn addition to exerting analgesic effects, morphine appears to be able to suppress tumor proliferation in gastric, lung, prostate and breast cancers , 6. SincHere we provide evidence that morphine does inhibit HCC tumor proliferation and metastasis, and that it exerts these effects in part by down-regulating DEAD-box helicase 49 (DDX49) , which wEighty surgical specimens of HCC tissues and paired adjacent non-cancer tissues were obtained from patients with a mean age of 62 yr  at the Tumor Hospital of Guangxi Medical University between January 2016 and June 2017. Tissue samples were frozen in liquid nitrogen and stored at -80° C. For the present study, HCC and normal liver tissues were confirmed by a pathologist.Before surgery, patients had provided written informed consent for their clinical samples and medical data to be analyzed and published in an anonymized format for research purposes. The Ethics Committee approved the analysis of the surgical samples.2. Morphine hydrochloride was obtained from Humanwell Healthcare Group .The liver cancer cell lines QGY-7703 and SMMC-7721 were obtained from the Cell Bank at the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences . The cells were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum , penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37° C in a humidified chamber under 5% CO3) were seeded into 96-well culture plates, allowed to attach, then exposed to morphine for 72 h. Cell survival was determined as described [We used a commercial MTT assay  to assess the proliferation of QGY-7703 cells and SMMC-7721 according to the manufacturer’s instructions. Cells (5×10escribed .5) were cultured in 6-well sterile plates in complete RPMI-1640 medium. When a confluent monolayer had formed, a sterile 200-μl pipette tip was used to make a scratch across the monolayer in the middle of the plate. At 24 h later, the cells that had moved into the wounded area were photographed under a microscope and analyzed using Image J. Each experiment was performed at least three times.Cells (5x104) were pretreated for 72 h with morphine at 0.01, 10, or 1000 μmol/L, then seeded into the upper chamber. Dulbecco’s modified Eagle medium supplemented with 10% FBS was added to the lower chamber as an attractant for the cancer cells. The culture plates were incubated at 37° C for 24 h. Cells that had migrated through the membrane into the lower chamber were fixed using 4% paraformaldehyde , stained with 1% crystal violet , and imaged under a microscope . Experiments were conducted three times.For migration assays, transwell inserts  were inserted into a 24-well plate. QGY-7703 and SMMC-7721 cells (2×104) were placed into the upper chamber of transwells in which the membrane had been pre-coated with 50 μl Matrigel . The plates were incubated for 36 h at 37° C, then processed in the same way as in migration assays.For invasion assays, QGY-7703 and SMMC-7721 cells  and 0.1% Triton X-100. Flow cytometric analysis was conducted using the EPICS XL-MCL FACS system . Data were analyzed using MultiCycle Software for Windows .For apoptosis assays, QGY-7703 and SMMC-7721 cells were cultured for 24 h, then processed using the Annexin V/FITC Apoptosis Detection Kit  according to the manufacturer’s instructions. Cells were analyzed using the EPICS XL-MCL system.Total RNA was isolated from QGY-7703 cells using TRIzol  according to the manufacturer's protocol. Probe synthesis, hybridizations, and analysis of microarray images were performed by Shanghai Genechem .Lentiviral plasmids encoding DDX49 (LV-DDX49-OE) or the antisense sequence to target 5'-AGGAGCAGATCAAGAAGAA-3' in the DDX49 gene (LV-DDX49-KD) were prepared by Shanghai Genechem. The antisense sequence knocked down DDX49 expression by at least 90% (data not shown) in QGY-7703 and SMMC-7721.Efficiency of transfection was measured under a fluorescent microscope based on expression of GFP encoded in the lentiviral plasmids. Transfected cells were seeded into 96-well plate at a density of 2000 cells/well (100 μl per well) and incubated for 24 h at 37° C. The Celigo system  was then used to assay proliferation in each well every day for 5 days.Total protein was extracted from cell lines using RIPA buffer, and protein concentration was estimated using a commercial BCA kit . An aliquot (35 μg per well) was fractionated by SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 30 min, then incubated overnight at 4° C with anti-DDX49 antibody , followed by a secondary antibody . Blots were immunostained with antibody against GAPDF  as a loading control. Immunostained blots were analyzed using an imaging system .t test, while differences among more than two groups were assessed using one-way analysis of variance (ANOVA). Differences associated with p<0.05 were considered statistically significant. All statistical analyses were performed using SPSS 13.0 .Data were reported as mean ± standard error of the mean (S.E.M). Differences between two groups were assessed for significance using Student’s This study was carried out in accordance with the Guidelines for Human Specimens for Research in the Guangxi Zhuang Autonomous Region, from the Medical Ethics Committee of Guangxi Zhuang Autonomous Region. The study protocol was approved by the Tumor Hospital of Guangxi Medical University.P < 0.05), which rose to 40% at the highest concentration of 1000 μmol/L (P < 0.05). Morphine also significantly suppressed migration and invasion of both HCC cell lines .QGY-7703 cells were transfected with DDX49-shRNA or negative-control shRNA (control) and injected into nude mice. Analysis of tumor size after 6 weeks showed that knocking down DDX49 led to significantly smaller tumors  stage A showed that DDX49 levels were significantly higher in cancerous tissues , 4E. NexMorphine (10μM) significantly down-regulated DDX49 in HCC cells (P < 0.05).To identify the functional role of DDX49 in HCC tumorigenesis, we infected QGY-7703 cells with either DDX49 overexpression vector (LV-DDX49-OE) or lentivirus mediated DDX49 silencing vector (LV- DDX49-KD) to increase or knock down the DDX49 expression, respectively. Then cells were treated with morphine for 48h. Overexpression of DDX49 in HCC cells reversed cell proliferation inhibition effects of morphine compare to negative control vector-transfected . HCC celTo further study the role of morphine in the regulation of cell apoptosis, cells were treated with LV-DDX49-OE or LV- DDX49-KD. Cells with DDX49 over-expressing lentivirus decreased cell apoptosis compare to the negative control virus, P < 0.01. While cells with DDX49 knocking down increased cell apoptosis . CompareKnocking down DDX49 in QGY-7703 cells significantly reduced their ability to form colonies, while overexpressing DDX49 remarkably increased their ability to form colonies .Our data showed that DDX49 has a direct regulation on cell proliferation. This prompted us to investigate whether DDX49 has a regulation role in cancer metastasis. We subsequently investigated the migration and invasion of QGY-7703 cells following the DDX49 deletion or overexpression and then treat with morphine for 48h. We found that DDX49 deletion decreased cells migration  and invain vitro and in xenograft-bearing animals [Improving pain control in cancer patients is a priority for patients and clinicians. Morphine is a first-line opioid analgesic for patients with various types of cancer, including HCC . Several animals –13. The Moreover, morphine has been shown to induce apoptosis and cell cycle arrest –17 in vaWe found that morphine antagonized the effects of DDX49 overexpression while reinforced the effects of DDX49 knockdown. These effects of morphine likely reflect the drug’s ability to down-regulate DDX49 expression. Our results suggest that morphine inhibits HCC progression in part by down-regulating DDX49.Our experiments suggest that one way in which morphine inhibits HCC is to reduce levels of activated (phosphorylated) MAPK, which presumably reduces downstream activation of target proteins that remain to be identified. MAPKs direct cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflammatory cytokines. Further studies should explore in detail what downstream processes are affected by morphine in HCC. They should also build on our finding that DDX49 may increase MAPK expression in HCC.Whatever the mechanism(s) through which morphine and DDX49 affect HCC, we found that DDX49 expression level differentiated HCC tissue from adjacent healthy liver tissue with good sensitivity and specificity, based on receiver operating characteristic curves. Further study should explore the diagnostic potential of this biomarker, alone and in combination with other biomarkers such as AFP.Our results should be interpreted with caution given several limitations in the study. The morphine concentrations that we used differ from physiological concentrations in humans. In fact, animal studies have given conflicting results about the effects of morphine on cancer: for example, one study showed that 40 μM morphine promoted glioblastoma proliferation , while mDespite these limitations, our study provides evidence that DDX49 can help drive HCC and that morphine may inhibit disease progression in part by down-regulating DDX49.All authors of the study agreed to submit the manuscript to this journal.Supplementary Figure 1Supplementary Table 1


Background: Dopamine, a key neurotransmitter in the autonomic nervous system participating in the homeostatic balance between sympathetic and parasympathetic divisions, is involved in food intake regulation. Objective: We investigated whether dopamine is altered by acute fasting or overfeeding diets with varying macronutrient content. Design: Ninety-nine healthy subjects underwent 24-h dietary interventions including eucaloric feeding, fasting, and five different overfeeding diets in a crossover design. Overfeeding diets  included one diet with 3%-protein (low-protein high-fat overfeeding—LPF: 46%-fat), three diets with 20%-protein, and a diet with 30%-protein (44%-fat). Urine was collected for 24 h and urinary dopamine concentration was quantified by high-performance liquid chromatography. Plasma pancreatic polypeptide (PP) concentration, an indirect marker of parasympathetic activity, was measured prior to and after each diet after an overnight fast. Results: During 24-h of fasting, dopamine decreased on average by ~14% compared to eucaloric conditions, whereas PP increased by two-fold (both p < 0.001). Lower dopamine during 24-h fasting correlated with increased PP . Similarly, on average urinary dopamine decreased during LPF by 14% (p < 0.001) and lower dopamine correlated with increased PP . No changes in dopamine and PP concentrations were observed during other overfeeding diets . Conclusions: Dopamine concentrations decrease during short-term fasting and overfeeding with a low-protein diet. As both dietary conditions have in common protein deficit, the correlation between dopamine and PP suggests a compensatory mechanism underlying the shift from sympathetic to parasympathetic drive during dietary protein deprivation. However, whether dietary interventions that affect daily energy balance such as fasting and overfeeding alter urinary dopamine concentrations has not been evaluated in humans.A chronic imbalance between energy intake and expenditure is responsible for the world-wide increased prevalence of obesity and its related comorbidities over the past decades. Beyond environmental and genetic factors, several neurotransmitters including dopamine and neuropeptides such as pancreatic polypeptide, neuropeptide Y, and peptide YY are involved in the central regulation of food intake and in the reward effects of feeding , and thu2 receptor in the mesolimbic and striatum regions, suggesting that low-protein diet may be a determinant of central dopaminergic function [In animal models, chronic food restriction strongly influences both the activity of dopamine circuits of motor control, motivation, arousal, reinforcement, and reward, and multiple feeding behaviors controlled by dopamine ,7. In rafunction . In vivofunction .Following-up our recent human study showing an association between dopamine and ad libitum food intake, in the current clinical study we evaluated whether urinary dopamine concentrations were altered by 24 h of fasting and five different overfeeding diets with varying macronutrient content to elucidate the physiologic mechanisms induced by acute changes in energy intake and daily energy balance.clinicaltrials.gov identifier: NCT00523627) aimed to characterize the short-term metabolic responses to 24-h fasting and different overfeeding diets. From 2008 to 2015, ninety-nine healthy individuals aged 18–45 y completed the study and had available measurements of urinary dopamine concentrations during 24-h eucaloric conditions (baseline)  . ParticiUpon admission to the clinical research unit, a weight-maintaining diet  was provided daily to the participants and its caloric intake was calculated using unit-specific equations based on gender and weight. Body weight was recorded daily in the morning after an overnight fast and was maintained within 1% of the admission weight by adjusting the WMD by ±200 kcal/day if needed. On the second day of admission, dual-energy X-ray absorptiometry (DXA)  was performed to assess body composition with fat mass (FM) and fat free mass (FFM) calculated from percentage body fat (%fat). An internally validated regression equation was used to make DXA measurements comparable between different DXA machines.A 3-h OGTT was performed after three days on the WMD and only subjects with normal glucose regulation based on ADA criteria continued the study. Plasma insulin and glucose concentrations were obtained by an automated immunoenzymometric assay  and the glucose oxidase method , respectively.2 production and O2 consumption were recorded every minute, averaged, and extrapolated to 24 h. The 24-h RQ was calculated as the ratio of 24-h CO2 production to 24-h O2 consumption, and 24-h EE was calculated using the Lusk’s equation [Twenty-four-hour energy expenditure (EE) and respiratory quotient  were assessed in a large, open-circuit, whole-room indirect calorimeter (respiratory chamber) as previously described ,15. Brieequation .The experimental protocol for dietary interventions is shown on Subsequently, subjects underwent six dietary assessments (fasting and five different overfeeding diets) given for 24 h in the whole-room indirect calorimeter with three-day period on the WMD between each intervention when subjects resided on the ward. The order of diets was randomized for each participant to reduce the sequence effects in the repeated-measures analysis of dopamine concentrations.The energy intake of each overfeeding diets was equal to twice the baseline 24-h EE value (200% of energy requirements). The overfeeding diets included: three overfeeding diets with normal protein content (20%): standard , high-carbohydrate , and high-fat  overfeeding diet; one overfeeding diet with a high protein content : 44% fat, 26% carbohydrate); one overfeeding diet with low protein content : 51% carbohydrate, 46% fat). Only sessions when subjects consumed ≥95% of total energy intake provided with the overfeeding diet were included in the data analysis. During 24-h fasting, only water was provided, and subjects were instructed to keep hydrated. Because of logistical difficulties or significant return of interventional diets , not all the 99 subjects had valid dopamine measurements for all the dietary interventions. The HPF diet was added to the study protocol after the first 20 individuals completed their baseline admission, therefore the sample size of this diet was smaller compared to the other overfeeding diets.p = 0.01, https://neurology.testcatalog.org/show/CATU accessed on 15 July 2020), the normal range for urinary dopamine is 65–400 mcg/24 h. Twelve subjects showed measurements above the normal range but sensitivity analyses excluding those subjects yielded similar results (data not shown).Urine was collected inside the calorimeter during each 24-h session and then stored at −70°C for subsequent measurements. Urinary dopamine concentrations were measured by Mayo Clinic Laboratories  via high-performance liquid chromatography, a reliable measurement technique . The uriBlood samples collected after an overnight fast both prior to and after each dietary intervention were used for the measurements of plasma pancreatic polypeptide (PP), an indirect marker of parasympathetic activity , and insData are presented as mean with standard deviation (Gaussian variables) or as geometric mean with its 95% confidence interval  as indicated. The total area under the curve (AUC) of glucose and insulin concentrations during the OGTT was calculated using the trapezoidal rule. The Spearman’s correlation was used to assess the effect of storage time on dopamine measurements. Differences in dopamine concentration among ethnicities were assessed via ANOVA with post-hoc adjustments (Tukey method) while the unpaired Student t-test was used to evaluate differences according to gender. The Pearson correlation coefficient was used to quantify the associations between dopamine and anthropometric and demographic parameters. The intraclass correlation coefficient (ICC) was calculated for dopamine measurements during all dietary conditions via a linear mixed model analysis accounting for diet (fixed effect) and repeated measurements (using a compound symmetry covariance structure) to quantify within-subject consistency. Differences in dopamine concentration during 24-h fasting and overfeeding diets from eucaloric conditions were evaluated by Dunnett’s post-hoc test to correct for multiple comparisons. A sensitivity analysis was performed by including only participants with valid dopamine data for all dietary interventions, which led to similar results . To test10 transformed before analyses to meet the assumptions of parametric tests . The individual changes in PP and insulin concentration after each dietary condition were calculated as the difference between the post-diet value minus the pre-diet value and analyzed by Student’s t-test. Statistical analysis was performed using the SAS statistical software package .Values of PP and insulin were logp = 0.6).Clinical characteristics of study cohort are reported in p = 0.009) and was on average higher in Blacks compared to Whites . Urinary dopamine was positively correlated with BMI . In a multivariable regression model, both BMI  and ethnicity (p = 0.02), but not gender or age (both p > 0.08), were independent determinants of urinary dopamine concentrations .Urinary dopamine differed by ethnicity . No associations were observed between dopamine and fasting glucose, 2-h OGTT glucose, glucose AUC during OGTT, fasting insulin, and 2-h OGTT insulin .Urinary dopamine was also positively associated with insulin AUC during the OGTT , indicating within-subject consistency of dopamine measurements across diets. There was no period effect (p = 0.67) on dopamine concentrations, indicating no carryover effects across dietary interventions.The individual urinary dopamine concentrations during each dietary condition are shown in p < 0.001), whereas no overall changes were observed during any normal-protein overfeeding diet . Based on these results for dopamine, our subsequent analyses focused on 24-h fasting and 24-h low-protein overfeeding diet.The changes in urinary dopamine from eucaloric condition during each dietary intervention are reported in p < 0.001, p < 0.001, p < 0.001, p = 0.03). These inverse associations were observed also after adjustment for %fat . Similar results were observed in a sensitivity analysis including only men (data not shown).During 24-h fasting, urinary dopamine decreased on average by 14% or −39.4 mcg/24 h  or PP (p = 0.18) concentrations.Following 24-h fasting, plasma insulin concentrations decreased on average by ~50% . Urinary dopamine during 24-h low-protein overfeeding was negatively associated with plasma PP concentration, such that a lower urinary dopamine concentration during 24-h low-protein overfeeding correlated with increased PP concentration . Similar results were observed after adjustment for percentage body fat, age, gender, and ethnicity . There were no significant associations between 24-h urinary dopamine excretion rate and plasma PP concentration during energy balance and during normal/high-protein overfeeding diets .No associations were found between insulin concentration and urinary dopamine or PP concentrations  both during 24-h fasting and low-protein overfeeding, but not during overfeeding diets with normal or high protein content. Interestingly, we observed a two-fold increase in plasma pancreatic polypeptide (PP) concentrations following 24 h of fasting and this increase was inversely associated with the urinary dopamine concentration during the intervening 24-h fasting period; similarly, urinary dopamine excretion during low-protein overfeeding was also inversely related to the change in PP following this diet.During 24-h fasting, urinary dopamine decreased from eucaloric conditions by ~14%. Dopamine is implicated in the regulation of food intake and plays a role in feeding behavior . HoweverIn humans, no significant changes in fasting plasma dopamine concentrations were observed at the 14th and 29th day during long-term intermittent fasting conditions (Ramadan period) . ConversThe regulation of feeding might be affected by pancreatic signals, which convey information regarding food ingestion to brain regions . Pancrea1, D2, D4, and D5) [Direct stimulation of the gastrointestinal tract after nutrient ingestion stimulates the release of dopamine in brain circuits controlling food intake , includi and D5) . Further and D5) . As the  and D5) , we migh2 receptors in the striatum [2 receptors downregulation [After 24 h of low-protein high-fat overfeeding diet, urinary dopamine excretion rate decreased from eucaloric condition to the same extent observed during 24-h fasting (−14%). Very few studies have evaluated the dopamine response to overfeeding diets with different protein content. Following L-dopa administration, rats fed a high vs. low protein diet have an attenuated increase in striatal dopamine concentrations , suggeststriatum . In a migulation . In thisgulation .In humans, a small cross-over study including six men fed a single high-protein meal (60 g of protein) showed increased urinary dopamine excretion after 5 h compared to no-protein meal . In humaAs we observed a reduction in urinary dopamine concentrations during 24 h of low-protein high-fat overfeeding diet that contained approximately 1/3 protein compared to the eucaloric diet , we mighOur study has several limitations. First, the female subgroup was small; however, the main results were confirmed in sensitivity analyses including only men. We did not perform brain neuroimaging to assess the neural mechanisms involved in dopamine function , which wAcute, short-term (24 h) dietary interventions with low-protein content (fasting and low-protein overfeeding diet), but not normal/high-protein overfeeding diets, decreased urinary dopamine excretion rate which, in turn, was associated with increased plasma PP concentration. The interaction between dopamine and PP may underlie a compensatory mechanism to acute dietary protein deficit in the autonomic nervous system, reflecting a fasting-induced increased parasympathetic drive with a concomitant reduction in sympathetic activity.


Staphylococcus aureus and Pseudomonas aeruginosa bacteria, respectively, with 5% VB loading. Moreover, a significant reduction in virus titer (37%) was observed with the 5% VB/PAN/ZnO nanofiber sheet. Hence, VB-loaded PAN/ZnO nanofibers have great potential to kill enveloped viruses such as influenzas and coronaviruses and could be the ideal candidate for the development of nanofiber-based PPE, such as facemasks and surgical gowns, which can play a key role in the protection of frontline health workers and the general public in the COVID-19 pandemic.Designing novel antiviral personal protective equipment (PPE) is crucial for preventing viral infections such as COVID-19 in humans. Here, we fabricate an electrospun nanofiber-based Viroblock (VB)-loaded polyacrylonitrile (PAN)/zinc oxide (ZnO) hybrid nanocomposite for PPE applications. Five different concentrations of Viroblock  were added to PAN/ZnO solution and loaded for electrospinning. The developed samples reflected antibacterial activity of 92.59% and 88.64% against  However, the limited affinity of the aforementioned functional nanomaterials towards textile-based PPE is a great challenge. Researchers are overcoming this challenge by using different binders or in situ growth of nanomaterials on the surface of the fabric. Seino et al. developed antiviral textile through the immobilization of silver nanoparticles on the surface of cotton fabric by using a radiochemical process [2 nanoparticles for antibacterial and antifungal protective clothing [+2 ions as compared to zeolite-coated textile [In recent times, the world has progressed in various domains, but viral infections persist to grow day by day, which not only affects human lives but also the global economy . Many vi process . Norazi clothing . Shaheenclothing . Severalclothing . Imai et textile . Sumit e textile . Neverth textile , which m textile . To fix  textile ,23. Cui  textile . Rashid  textile . Ji et a textile . In anot textile . Chowdhu textile . Multifu textile .Herein, considering the current COVID-19 pandemic situation and excessive demand for protective clothing, we are the first to present a robust methodology to develop efficient antiviral and antibacterial electrospun nanofibrous membranes by incorporating HeiQ Viroblock (VB) and ZnO nanoparticles in a polyacrylonitrile (PAN) electrospun solution. Zinc oxide is a multifunctional material with exceptional properties  protector) ,31, but All the chemicals were purchased from different sources and used in this study without further additional purification. Polyacrylonitrile  was used as the main precursor material for the fabrication of nanofibers due to its better fouling properties, high chemical stability, and high mechanical strength . PolyacrIn this study, a total of seven different solutions were prepared. Pure PAN solution (10 wt%) was prepared by dissolving 1 g of PAN powder in 9 g of N,N-dimethylformamide (DMF) solvent. The solution was allowed to stir magnetically on the magnetic stirrer for about 10 h at a stirring speed of 500 rpm to form a homogeneous solution. For the PAN/ZnO composite solution, 5 wt% ZnO nanoparticles were added and allowed to stir magnetically at 500 rpm for approximately 15 h. Moreover, different solutions of PAN/ZnO loaded with VB  were prepared by allowing the composite solution to stir magnetically at 500 rpm for approximately 23 h. In all the prepared VB-loaded ZnO/PAN composite solutions, the concentration of ZnO nanoparticles remained constant.For the fabrication of pure PAN electrospun nanofibers, PAN solution was poured in 10 mL of a disposable syringe, which was connected to the pipette tip. A copper wire acting as a positive electrode was fitted inside the syringe. The solution in the form of nanofibers was drawn through the tip of the syringe under the applied voltage of 17 kV. These extruded fibers were collected on the aluminum foil wrapped on the negative charged rotating cylinder (collector). The distance between the collector and tip of the syringe was kept at 15 cm while maintaining the flow rate of 1 mL/h. Similarly, this process was repeated for the fabrication of PAN/ZnO and VB-loaded PAN/ZnO electrospun nanofibers. The illustration scheme and possible reaction of fabrication of electrospun nanofibers are shown in The surface morphology of the prepared electrospun nanofibers was analyzed by using a scanning electron microscope (SEM) , by applying 12 kV voltage. ImageJ software (version 1.49) was used to calculate the average diameter of electrospun nanofibers by taking 200 random measurements from each prepared sample .To check the uniform distribution of nanoparticles and VB in electrospun nanofibers, TEM analysis was used. High-resolution TEM  was used to capture the TEM images at a working voltage of 200 kV ,38.−1 to 4000 cm−1. The spectra were recorded with 128 scans with a resolution of 4 cm−1.The chemical interaction between PAN, ZnO NPs, and Viroblock (VB) was analyzed by Fourier Transform Infrared (FTIR) . Spectra of all the prepared samples were recorded from 400 cmPseudomonas aeruginosa and Staphylococcus aureus contact with nanofibers, the bacterial plate method was applied for various time points.Quantitative antibacterial analysis of prepared electrospun nanofibers was conducted by following the AATCC-100 standard . Accordiv/v) silane solution (2 × 105 CFU/mL). Moreover, the bacterial inoculum was cooled down to slow down the bacterial growth. The nanofibers were inoculated with 200 μL of cold and diluted bacteria and afterward placed in 20 mL of cold silane solution and shaken for a minute to detach the bacteria. The treated mixture was serially diluted in a 1:10 ratio and spread on a media agar plate for incubation at 37 °C for 24 h. The rate of inhibition or antibacterial efficiency was calculated by using the following formula:The bacterial culture broth was incubated overnight. The triplicate method  was usedR = antibacterial efficiency (reduction percentage);A = no. of bacteria in the broth detached from the membrane after inoculation at 0 h;B = no. of bacteria recovered from the broth after 24 h of contact with a treated membrane.The antiviral activity was evaluated by the following procedure. Firstly, a sample suspension of disinfectant was mixed with an infectious dose (ELD50) of avian influenza (AI) virus with an equal volume 1:1 (1 mL disinfectant + 1 mL virus infectious dose) and vortexed for 5 s followed by incubation for 2 h at 2–8 °C in a shaking incubator. After that, 100 µL of each mixture was inoculated via the chorioallantoic sac route with 8 embryonated eggs of each group. The hole-in-eggs were then sealed with molten wax and incubated for 48–72 h and were daily monitored to examine the viability of embryos. After inoculation, eggs were chilled to 24 h to settle down the Red Blood Cells (RBCs), eggshells were removed from the top of the eggs, and 10 mL allantoic fluid (AF) was harvested and centrifuged at 5000 rpm for 5 min for the removal of debris from AF. Supernatant was collected to check the virus growth. To check the virus titers, 50 µL of saline in a U-shaped bottom 96-well microtiter plate was used; 50 µL of tested AF was added in the first column of the 96-well plate, and subsequent wells were added typically with two folds up to the eleventh column. The final well served as a negative control with no virus. After serial dilution, 0.7–1% of chick RBCs were added to each well and mixed gently. The plates were incubated for 30 min at room temperature. Following the incubation period, the assay was analyzed by agglutination reaction and button formation .To investigate the effect of ZnO NPs and VB on the morphology of pristine PAN, PAN/ZnO, and PAN/ZnO/VB nanofibers, TEM images were studied, as shown in −1 to 3500 cm−1 corresponding to the –OH functional group. This band increased largely as ZnO and VB were added to the electrospun nanofibers. The –OH band had more depth in the case of spectrum B than spectrum A, which might be due to the hydrophilic nature of ZnO, which allowed more –OH radical to be absorbed onto electrospun nanofibers. However, by increasing the concentration of Viroblock, this band further increased, and the broadness of the band decreased, which might have been due to the presence of highly reactive silver ions (Ag+) in the VB that could attract more hydroxyl radicals from the atmosphere, as shown in (b–g). In all spectra, the characteristic peaks appeared at 2918.7, 2852.43, and 1453.26 cm−1, which corresponded to –CH group vibration and stretching due to alkene. The sharp peaks appeared at 1731.5 and 2242.61 cm−1 indicating C=O stretching and –C≡N stretching due to acrylamide [−1 were related to C–H stretching, C=O stretching, –OH bending, and N–H bending, respectively. In all spectra (except a and b), a minute peak appeared at 2991.37 cm−1 corresponding to the –OH group, which might be due to the presence of silver ions (Ag+), as well as polar liposomes present in the Viroblock. These silver ions and liposomes tend to attract moisture from the atmosphere. In −1 was due to the vibrational bond of ZnO; however, this peak did not appear in the case of pristine PAN electrospun nanofibers [To understand the structure of prepared electrospun nanofibers, FTIR analysis was performed, as shown in rylamide . The chanofibers .Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, respectively, as metal oxide nanoparticles exhibited superior antibacterial properties, which suppressed the growth of both bacteria compared to the pure electrospun PAN nanofibers. Moreover, metal oxide nanoparticles provide reactive sites for the interaction of bacteria and nanofibers [+2 and Ag+ ions [Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Furthermore, +2 or Ag+ ions can easily penetrate the cell wall of Gram-positive bacteria rather than Gram-negative-bacteria [Antibacterial activity of pristine PAN nanofibers, PAN/ZnO nanofibers, and PAN/ZnO nanofibers loaded with various concentrations of VB was observed by qualitative analysis followed by the AATCC-100 standard method. The antibacterial activity of all prepared electrospun nanofibers was tested against Gram-positive bacteria  of Viroblock were chosen for the antiviral activity, while pristine PAN nanofibers were used as a control sample. The plaque assay method was used to check the antiviral activity of prepared electrospun nanofibers. After three hours of contact with the virus, a significant decrease in the virus titers was observed, as shown in Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). Further, higher antibacterial activity was recorded for Gram-positive bacteria (Staphylococcus aureus) than Gram-negative bacteria (Pseudomonas aeruginosa), corresponding to the thicker and more complex permeability of the barrier cell wall of the latter. It was also found that as the concentration of the VB increased, the antibacterial activity also increased significantly. The 5%VB-loaded PAN/ZnO electrospun nanofibers were used to check the antiviral activity against enveloped influenza virus. After three hours, a 37.5% reduction in virus titer was found in the case of 5%VB-loaded nanofibers as compared to pristine PAN nanofibers, which reveal the excellent antiviral activity of 5% VB-loaded nanofibers as compare to the pristine PAN nanofibers. From these results, it can be concluded that these VB-loaded PAN/ZnO electrospun nanofibers have great potential for the development of personal protective equipment such as facemasks and surgical gowns, which can play a key role in the protection of frontline health workers and the general public in the COVID-19 pandemic and onward.This study is focused on the fabrication of antiviral nanofiber sheets by using the electrospinning technique. PAN/ZnO nanofibers were loaded with five different concentrations of Viroblock . From the SEM analysis, it was confirmed that all the prepared electrospun nanofibers had smooth and bead-free surface morphologies. Uniform distribution of the ZnO and Viroblock in nanofibers was justified by using TEM analysis. FTIR analysis confirmed the physical interaction between PAN, ZnO, and Viroblock. Qualitative antibacterial analysis proved the strong antibacterial activity against both Gram-positive bacteria (