The goal of this review is to summarize the characterization of the wnt11f2 zebrafish mutant and to deliver some new insights concerning its role in skeletal development. In addition to the previously described defects in early development in this mutant as well as craniofacial dysmorphia, we show an increase in tissue mineral density in the heterozygous mutant that points to a possible role of wnt11f2 in high bone mass phenotypes.Wnt signaling is a key regulator of osteoblast differentiation and mineralization in humans and animals, mediated by the canonical Wnt/β-catenin and noncanonical signaling pathways. Both pathways are crucial in regulating osteoblastogenesis and bone formation. The zebrafish silberblick ( The extracellular Wnt signal stimulates several intracellular signal transduction cascades, including the canonical or Wnt/β-catenin-dependent pathway and the noncanonical or β-catenin-independent pathway, which can be divided into the Planar Cell Polarity (PCP) pathway and the Wnt/Ca2+ pathway.2Because of the high degree of conservation among species of the Wnt pathway and its extensive homology with its human homolog, zebrafish mutants represent key animal models to unravel the function of the Wnt signaling pathway in development and bone formation.wnt11f2 mutant, presents a mutation in a gene related to the Wnt noncanonical pathway. This fish is also known as silberblick (slb), and it was first characterized in 1996 in the frame of a large-scale screen for mutants affecting forebrain development by using N-ethyl-N-nitrosourea (ENU) as a potent mutagen to introduce random point mutations in the genome.3 Homozygous wnt11f2 mutant embryos were smaller at 24 hours post fertilization (hpf) than controls and the elongation of the body axis was delayed from the tailbud stage until the early somite stages (10–14 hpf), as indicated by a shorter and broadened notochord and an abnormally shaped prechordal plate.The wnt11f2 mutants,4 thus leading to incomplete separation of the optic stalk3 and fusion of the eyes to various degrees depending on the penetrance of the phenotype. For that reason, this mutant was called silberblick (slb), which in German means strabismus. At 5 dpf, the jaw was deformed. The mutants displayed a recessive phenotype with variability.Further characterization revealed that anterior migration of central nervous system cells was impaired in wnt11f2 mutant, then known as slb, was found to encode a homolog of the human WNT11 protein, and was thus initially named wnt11.5 Two mutants, carrying the variants tx226 (c.669G>A p.Trp 223) and tz216 (c.463G>T p.Gly 155) in this gene, were analyzed. In addition, the authors showed that microinjecting wnt11 mRNA into slb mutants was able to rescue their phenotype, confirming that the detected mutations in wnt11 are indeed responsible for the phenotype and suggesting that the tx226wnt11f2 allele represents a loss-of-function mutation rather than a dominant-negative form. Interestingly, microinjection of wnt11 mRNA into wild-type embryos often resulted in misshapen eyes, indicating that the amount of Wnt11 proteins needs to be well controlled for normal development.6 Since then, this mutant fish line has been used extensively to understand the function of Wnt signaling in early development of vertebrates.In the year 2000, the locus that affected the wnt11 homologs had been named incorrectly: the gene previously known as wnt11r (wnt11-related) is the true ortholog to the human WNT11, thus now called wnt11, while the gene previously known as wnt11 is present in birds and other teleosts but not existing in mammals is now called wnt11f2.7 Following the zebrafish nomenclature guidelines, a gene should be named after its mammalian ortholog, however, such confusion occurred before the full zebrafish genome had become available because phylogenetic analyses at that time revealed that the closest human family member to slb was WNT11.7In 2019, genomic analysis revealed that the two zebrafish wnt11f2 mutants; and only one article from 20218 was found using the “wnt11f2” search term. In contrast, the carefully manually curated international zebrafish network ZFIN (zfin.org) currently has 155 citations concerning the wnt11f2 zebrafish gene. This situation clearly reflects the confusion in comparative genetics and disease modeling due to incorrect nomenclature resulting from the presence of duplicate paralogs in zebrafish following the whole genome duplication in teleosts. Consequently, this review specifically aims to clarify the role of the wnt11f2 gene because the publications are not well aligned in just one platform.In PubMed, a simple search with the word “silberblick” resulted in 21 publications published between 1996 and 2013; searching for “wnt11 and zebrafish” identified 99 publications, although not all of them are related to zebrafish and the wnt11f2. In the first part of this review, we describe the morphological and genetic characterization of wnt11f2 zebrafish. In the second part, we add some new findings concerning bone formation using this fish model.Therefore, we focused on the characterization of 9 Several coordinated morphogenetic cell movements take place during gastrulation, including convergence and extension (C&E) movements mainly mediated by the noncanonical Wnt signaling pathway, also known as the PCP pathway. The PCP signaling pathway was first identified in Drosophila, and its cellular and molecular regulation is conserved from Drosophila to mammals.Vertebrate gastrulation is a complex morphogenetic process that organizes the embryo proper into the three germ layers: endoderm, mesoderm, and ectoderm.10 The wnt11f2 is maternally expressed11–13 and is one of the immediate early genes activated in mesoderm induction. The wnt11f2 is expressed in the dorsal region of the germ ring at sphere/dome stage (4–5 hpf). During gastrulation, wnt11f2 expression extends to the lateral and ventral germ ring while being downregulated in the shield and its axial derivatives. In addition to the germ ring, at the end of gastrulation, Wnt11f2 is expressed in restricted areas of the anterior paraxial mesoderm and anterior lateral neuroectoderm.5Wnt11f2 activity is required for cells to undergo correct C&E movements during gastrulation, as the tx226wnt11f2 mutants present an impairment of the Wnt/PCP signaling leading to a diminished body axis elongation.14During the gastrulation process, mesodermal and neuroectodermal cells move toward the dorsal midline and intercalate with one another, which leads to mediolateral narrowing (convergence) and anterior–posterior lengthening (extension) of the developing embryonic axis.wnt11f2 mutants between 10 and 12 hpf: C&E movements of both mesendodermal and neuroectodermal cells are reduced, which results in a shortened and broadened body axis that can be used to identify homozygous mutants at the end of gastrulation.4 The prechordal plate in the anterior axial mesendoderm appears to be abnormally shaped; the presumptive neural plate in tx226wnt11f2 embryos appears to be broader during gastrulation. The wnt11f2 is required in the nonaxial mesoderm to mediate cell intercalation along the anteroposterior axis that contributes to the extension of the body axis during late gastrulation.4 Shield and cell transplantation experiments performed by Heisenberg et al, where wild-type or mutant shields, or small groups of lineage-labeled nonaxial mesodermal cells were transplanted into tx226wnt11f2 mutant or wild-type embryos, respectively, to assess the movements, and the final location of the transplanted cells revealed that Wnt11f2 activity is required within lateral tissues of the gastrula, where it regulates mediolateral cell intercalations that underlie C&E movements.4A gastrulation phenotype is transiently visible in wnt11f2 embryos are predominantly affected in anterior regions of the gastrula suggests that other genes are involved in regulating C&E movements in more posterior regions. Therefore, in the absence of Wnt11f2, abnormal extension of axial tissue results in cyclopia and other midline defects in the head.5The observation that wnt5b partially overlaps functions with wnt11f2 to regulate C&E movements in lateral domains of the gastrula,15 confirming that both Wnt11f2 and Wnt5b are required during gastrulation for proper morphogenesis rather than cell fate specification. The wnt11f2/wnt5b double mutants show severe C&E cell-movement defects, and wnt5b RNA partially rescues the wnt11f2 mutant phenotype.15 At 3-somite stage, tz216wnt11f2 is required for C&E of both the mesoderm and ectoderm in the anterior and posterior region.8Also, tx226wnt11f2 mutants present slower and less direct migratory movements of these hypoblast cells.16 The authors observed that there is a misalignment in the tx226wnt11f2 hypoblast cells during the orientation process and the direction of movement, leading to less efficient movements of hypoblast cells toward the animal pole.16Wnt11f2 also controls the orientation and the velocity of the hypoblast cell migration in the germ ring at the onset of gastrulation; wnt11f2 mutants is the mitotic divisions in the dorsal epiblast cells, which exhibit less-pronounced animal–vegetal axis polarity relative to control cells in both the epiblast and perpendicular planes, meaning that there is a disruption in the cell division orientation in the mutants.17 In addition, it has been described that Wnt/PCP signaling plays an important role in the migration of the cranial neural crest18; together with other genes from the Wnt/PCP signaling such as Fzd7 and Disheveled, wnt11f2 controls the polarization of the protrusions formed in the neural crest, which allow the cells to migrate in a directed motion.20Another defect observed in the tx226wnt11f2 triggers the local accumulation of Fz7 at cell membranes along with the intracellular mediator Dsh and Wnt11f2 itself, modulating local cell contact persistence to coordinate cell movements during gastrulation.21 To summarize, gastrulation is the first large-scale morphogenetic process to occur during development. This process is in part regulated by Wnt/PCP pathway, and disruptions in the regulation of this pathway caused by a mutation in wnt11f2 in zebrafish cause defects in the mediolateral cell intercalation, the migration of hypoblast cells, the cell division orientation, and in cell adhesion.22The Wnt/PCP pathway also plays a role in cell adhesion. The 23This stage focuses on the primordia of the pharyngeal arches, present at early times but difficult to distinguish individually. In zebrafish, the pharyngula stage starts at around 24 hpf. The embryo is most evidently now a bilaterally organized creature, entering the pharyngula period with a well-developed notochord and a newly completed set of somites that extend to the end of a long postanal tail. The nervous system is hollow and expanded anteriorly.wnt11f2 in the wild-type zebrafish is at the developing somites and otic placodes at 24 hpf and also in the mesoendoderm and in midline structures during zebrafish heart morphogenesis.24 At 24 hpf, in about half of the wnt11f2 mutant embryos, the retinae are not properly separated anteriorly. The forebrain in wnt11f2 mutant embryos, although patterned normally, is broadened and shortened, possibly the result of defective C&E during gastrulation.25 The axonal scaffold appears normal, with the exception of a slight deformation at the anterior–ventral positions owing to the fusion of the eyes.4 It is possible that the major cause of the reduced resolution of bilateral eye fates in this mutant is related to the defect of forebrain morphogenesis.25 At 26 hpf, tz216wnt11f2 mutants have normal tails.26The expression of 4 Although wnt11f2 mutants show reduced medial–lateral cell intercalations in both anterior and posterior mesendodermal domains, the extension of anterior regions seems most severely affected.5At 48 hpf, the eyes are slightly turned inward anteriorly, with no further obvious abnormalities detectable. Heisenberg and Nüsslein-Volhard propose that proper midline morphogenesis is essential for lateralization of the eye position.wnt5b but not wnt11f2tz216 embryos had significantly shorter body axes than control siblings and that wnt11f2tz216/wnt5b double-mutant embryos had the shortest body axes. Similarly, the gut tube was normal in wnt11f2tz216 embryos but slightly enlarged in wnt5b mutant embryos and significantly widened in wnt11f2tz216/wnt5b double-mutant embryos. These data suggest that at 48 hpf, wnt5b but not wnt11f2 is required for elongation of the body axis and formation of the gut tube, but wnt11f2 cooperates with wnt5b to regulate endoderm morphogenesis.8Assessment of the posterior body length at 48 hpf revealed that Zebrafish larvae show a clear and distinct swimming pattern in response to light and dark conditions following the development of a swim bladder at 4 dpf. Cartilage cells are distinctive in branchial arches in these later larval stages. The primordium of the operculum extends posteriorly to cover the first or even the second branchial arch. The first visible bone in zebrafish, the transversely oriented cleithrum, appears at 3 dpf.18 As neural crest cells migrate, they extend polarized protrusions allowing the cells to migrate in a directed motion. The Wnt/PCP elements Wnt11f2, Fzd7, and Disheveled control at least in part the polarization of these protrusions.27 Between 55 hpf and 3 dpf, wnt11f2 is expressed in the pharyngeal arches.28 In addition, wnt11f2 has a complex expression pattern, including expression in head neural crest that might eventually form the anterior basicranium. However, the expression domain is within early paraxial mesoderm, before mesoderm has migrated to reach where the head will form,5 with this observations are possible that genes responsible for severe craniofacial phenotypes might be remote to cartilage development itself.29 At 48 hpf the wnt11f2 is expressed in the lower jaw.30 By 4 dpf, most of the craniofacial cartilage elements of the zebrafish have formed.31 Indeed, the Wnt signaling pathways are known to regulate bone homeostasis.Craniofacial cartilage elements are derived from the cranial neural crest. As mentioned above, in zebrafish, Wnt/PCP signaling plays a role in the migration of the cranial neural crest cells.29 In wnt11f2 mutant zebrafish, the jaw is deformed.3 To understand what is causing this bone and cartilage defect, Sisson et al, in 2015,28 marked the outline of chondrocytes to assess the gross morphology of the cartilage elements. In tz216wnt11f2 fish at 4 dpf, the cartilage elements, especially the ceratohyal and Meckel's cartilage, were greatly deformed. The mutants showed disrupted placement of many of the cartilage elements derived from the premandibular, mandibular, and hyoid arches. However, the stacking of the chondrocytes seemed normal. In the wnt11f2 mutants, the prechordal plate is severely defective, which impairs the extension of axial tissues leading to defects in the head cartilage formation, chondrogenesis occurring near the end of embryogenesis.29Wnt11f2 function in zebrafish cartilage formation is to position the initial bilateral sites of chondrogenesis of the anterior basicranium and specify where migrating neural crest cells will settle down at early stages determining the fate of the cartilage morphology.wnt11f2 mutants deform a bilateral organization in the mutants into a one-dimensional array just along the midline. Wnt11f2 is important to determine the position of the initial normally bilateral sites of chondrogenesis of the anterior basicranium.29 Furthermore, molecular studies reveal that wnt11f2 function in Wnt noncanonical signaling pathway is crucial for embryonic midline development.24The tz216wnt11f2 eye phenotype can vary interindividually, together with the severity of the craniofacial defects. To determine whether the severity of the eye phenotype was related to the cartilage placement phenotype, Marlow et al compared embryos manifesting full cyclopia (class 5) to those with lesser degrees of synophthalmia (classes 2 and 3).32 The authors found a clear correlation between the progression of the eye-fusion phenotype and the disruption of the shape of the cartilage elements, especially the ceratohyal. However, the ability of the chondrocytes to stack was not disturbed. This observation suggests that the craniofacial defect seen in the tz216wnt11f2 mutant fish is due primarily to the eye field separation defect displacing the cartilage elements.Of note, the wnt11f2 signaling in zebrafish cartilage formation, we performed Alcian Blue staining, as described,33 to check the cartilage formation at 10 dpf in tx226wnt11f2 mutants , the wnt11f2 embryos showed normal expression of shh. The primary defect in wnt11f2 may be a reduction in medial–lateral intercalation of cells in the axial mesendoderm. Our results and those of Sisson et al21 confirm that wnt11f2 mutations affect craniofacial development indirectly through cyclopia resulting in incorrect head morphology. There is a link between the degree of severity of cyclopia and the degree of severity of craniofacial cartilage defects.The first description of the fgf8a gene expression  only dorsalized the embryos. Blocking canonical Wnt signaling by injecting a dominant-negative form of Tcf3 (ΔN-Tcf3) led to ventralization, without rescuing the wnt11f2 phenotype.5 In addition, the authors working with the wnt11f2 morpholino model showed that when they injected disheveled (dvl) protein, found a cardiac phenotype rescue and when they blocked dvl, they found a phenocopy of the wnt11f2 phenotype. These observations confirmed that dvl acts downstream of wnt11f2 in the Wnt noncanonical pathway.24After the discovery that a wnt11f2 truncated protein led to the ror2 was disrupted in zebrafish, a phenocopy of wnt11f2 was also found. Moreover, coexpression of low-dose ror2-TM mRNA together with injection of wnt11 morpholino, led to a more severe C&E and eye defects.38Similar observations were made when wnt11f2 is one of the representative noncanonical Wnts transducing PCP signals through fzd7 receptor.19The Wnt/PCP signaling pathway is required during the gastrulation process, controlling tissue polarity and cell movement by activating RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades. The A summary of the major actors during zebrafish gastrulation can be found in 40 This gradient, in turn, induces a gradient in the Vangl2 phosphorylation end establishment of tissue polarity.41 Similar effects were observed for Wnt5b, with PCP activation involved in Wnt5b-induced cell migration and chondrocyte differentiation.42In bone tissue, the PCP signaling pathway and tissue polarization have been associated with embryonal bone and joint formation, which involves cell migration, elongation, and gradient-dependent differentiation. For example, PCP is crucial during embryonal long bone cartilage elongation along the proximal–distal axis.wnt11f2/wnt5b double-mutant phenotype resembles the phenotype of embryos homozygous for kny, which encodes glypican 4 (gpc4). Coexpression of gpc4 RNA potentiated the activity of Wnt11f2 to rescue the wnt11f2 C&E phenotype.43 Furthermore, wnt11f2/gpc4 double mutants displayed a more severe phenotype than either mutant alone, including complete cyclopia featuring a single eye, which indicates that gpc4 acts as a positive regulator of Wnt11f2. The gpc4 is required for C&E of both the mesoderm and endoderm and interacts with wnt11f2.43 The gpc4 zebrafish mutants present broader and shorter body axis, the mutation is lethal after 5–7 dpf, but rescue experiments revealed that gpc4 has a role in regulating cartilage cell polarity, chondrocyte stacking, and endochondral ossification.44The gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia. Gpc4 can physically bind both Wnt5b and Wnt11f2 and regulates the formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells.8The transgenic expression of GFP-Wnt5b or Wnt11 initiate the noncanonical Wnt signaling pathway by binding to fzd2 and 7 receptors to regulate C&E movements in zebrafish.15Wnt11f2 and wnt11 show spatial and temporal patterns of expression compatible with a role during heart-tube assembly and their transcripts are expressed in neural ectoderm and mesoendoderm at 12 hpf. The expression pattern of wnt11 overlaps with that of wnt4a in the floorplate at 16 and 24 hpf and with that of wnt11f2 in the developing somites at 24 hpf. Of note, wnt11 transcripts are also expressed in the heart tube at 24 hpf.24 In studying hair cell orientation, Navajas Acedo et al45 reported a parallel role of PCP and Wnt pathway genes in regulating hair cell orientations in zebrafish neuromasts. Mutation in wnt11 disrupts hair cell orientation in neuromasts. However, heterozygous wnt11 and tz216wnt11f2 double mutants do not exhibit defects in hair cells, indicating that these two paralogs do not interact.wnt11f2 mutant zebrafish is an important animal model that can help in understanding bone formation and the role of Wnt signaling pathways in this process.The 24 The wnt11f2 mutant gastrulation phenotype is completely penetrant, whereas the eye phenotype is much more variable. This is also true for the deformities observed in the cranial cartilage, which were associated with the eye phenotype.Obviously, wnt11f2 is extremely important during gastrulation by enabling correct convergence–extension cell movements by activating the noncanonical Dvl/RhoA pathway.Future research should focus on the direct and indirect partners and signaling pathways that lead to the eye phenotype in homozygous mutants.wnt11f2 is involved in the canonical and noncanonical Wnt signaling pathway, which is associated with cyclopia, and this defect is also observed in case of disruption of Shh and Nodal pathways in zebrafish.46–48 Probably there is an interaction between these different signaling pathways in zebrafish, but further investigations are needed.The 49 and as many as 1 in 250 human fetus.50In humans, cyclopia is a rare form of holoprosencephaly, and different forms of holoprosencephaly affect ∼1 in 15,000 human live births51 Even if Wnt11f2 is absent in human, the noncanonical Dvl/RhoA pathway could be interesting to study to better understand holoprosencephaly. Moreover, wnt11f2 remains an important tool to study Wnt signaling in early development.Holoprosencephaly is commonly due to Shh pathway dysfunction, but the Nodal pathway is also involved.wnt11f2 mutants, although normal in their general development, skeletal morphogenesis, and reproductive capacity, present a significantly more highly mineralized bone skeleton.In this study, we show that the heterozygous 52 With this, we believe that the wnt11f2 zebrafish is a great model to better understand HBM etiology and to screen new bone anabolic drugs.Several rare genetic disorders with skeletal effects, like osteopetrosis and sclerosing bone dysplasia, are associated with generalized bone mineral density increase. Many of the HBM diseases are monogenic disorders related to the Wnt signaling pathway.WNT11 in humans exhibits the opposite phenotype.53 In a model of a WNT11 heterozygous mutant human osteoblast-like cell line generated by CRISPR-Cas9, we showed that the noncanonical pathway acts upstream of the canonical pathway, since in the absence of WNT11, the canonical pathway could not be rescued53 as was also observed in tx226wnt11f2 mutant zebrafish.5 This finding combined with our finding of HBM in wnt11f2 heterozygous zebrafish suggests that the noncanonical Wnt pathway is key to bone differentiation independent of bone patterning. Therefore, the phenotypic characterization of tx226wnt11f2 heterozygous adult would provide some insights into the potential genetic and cellular mechanisms of tx226wnt11f2 function in bone.Interestingly, we have recently shown that the heterozygous loss-of-function mutation of wnt11f2 mutant as a model to better understand HBM phenotype.In conclusion, we show that the noncanonical Wnt pathway acts as an inhibitory factor in bone mineralization in adults, and propose the heterozygous


PCV13 was licensed by FDA based on safety and immunogenicity data compared with PCV7, and systematic reviews have shown that PCV13 is effective against acute otitis media, pneumonia, and invasive pneumococcal disease (IPD) in children  to 4 (very low certainty).**Streptococcus pneumoniae was detected in the middle ear fluid of 24% , proportion of participants meeting the serotype-specific IgG value of ≥0.35 μg/mL (response rate)  evaluated the immunogenicity of PCV15 compared with PCV13 in healthy infants and children  or PCV13  at ages 2, 4, 6, and 12–15 months (Two economic models (CDC model and Merck model) that assessed cost-effectiveness compared the use of PCV15 and PCV13 according to the currently recommended PCV13 4-dose series for children aged <2 years  for all children aged 2–59 months. In addition, risk-based PCV use is recommended for children aged 60–71 months with risk conditions, and persons aged 6–18 years with an immunocompromising condition, cerebrospinal fluid leak, or cochlear implant. For all recommendations, PCV13 and PCV15 can be used interchangeably. Interruption of the vaccination schedule does not require reinstitution of the entire series or the addition of extra doses.Infants aged 2–6 months. Four doses of PCV (either PCV13 or PCV15) are recommended. The primary infant series consists of 3 doses of PCV. Infants receiving their first dose at age ≤6 months should receive 3 doses of PCV at intervals of approximately 8 weeks . The fourth (booster) dose is recommended at age 12–15 months and ≥8 weeks after the third dose  who are medically stable enough to be vaccinated  are recommended. The first 2 doses should be administered with an interval of ≥4 weeks between doses. The third dose should be administered at age 12–15 months, ≥8 weeks after the second PCV dose.Children aged 12–23 months. When PCV is initiated at 12–23 months of age, 2 doses (either PCV13 or PCV15) are recommended, with an interval of ≥8 weeks between doses.Children aged 24–71 months. Unvaccinated healthy children aged 24–59 months should receive a single dose of PCV (either PCV13 or PCV15). Unvaccinated children aged 24–71 months with any risk condition should receive 2 doses of PCV (either PCV13 or PCV15) with an interval of ≥8 weeks between doses. Routine use of PCV is not recommended for healthy children aged ≥5 years who have not yet received a dose of PCV.Children and adolescents aged 6–18 years with an immunocompromising condition, cochlear implant, or cerebrospinal fluid leak. If a dose of PCV13 or PCV15 has not been administered previously, a single dose of PCV13 or PCV15 is recommended, regardless of whether the child has previously received PPSV23, even if PCV7 was received.Infants and children aged <24 months. Infants and children aged <24 months who have received ≥1 dose of PCV (either PCV13 or PCV15) should complete the vaccination series with either PCV13 or PCV15 . These children should receive a single dose of PPSV23 at age ≥2 years and ≥8 weeks after the most recent PCV dose . ChildreRevaccination with PPSV23 among children with immunocompromising conditions. Children aged ≥2 years who have an immunocompromising condition should receive a second dose of PPSV23 ≥5 years after the first PPSV23 dose.Recipients of hematopoietic stem cell transplants. Recipients of hematopoietic stem cell transplants are recommended to receive 3 sequential PCV doses followed by a dose of PPSV23 beginning 3–6 months after the transplant, as described in the General Best Practice Guidelines for Immunization  and the 23-valent pneumococcal polysaccharide vaccine (PPSV23) are recommended for U.S. children, and the recommendations vary by age group and risk group.On June 22, 2022, the Advisory Committee on Immunization Practices recommended use of PCV15 as an option for pneumococcal conjugate vaccination of persons aged <19 years, according to currently recommended PCV13 dosing and schedules. Risk-based recommendations on use of PPSV23 have not changed.Use of PCV15 as an alternative to PCV13 is expected to further reduce pneumococcal disease incidence in children and adolescents.


Drosophila melanogasterCyp6t3 gene. Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the “Black Box” step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, we find that Crispr generated null mutations inCyp6t3are viable and have normal developmental timing. Therefore, we conclude that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene’s expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of “off target” effects. Here we describe the genetic null mutant phenotype of the Drosophila melanogasterhave been identified and their activities ordered within a biosynthetic pathway. . Collectively, these genes are referred to as the Halloween group  and includephantom (phm), disembodied (dib), shadow (sad), shroud (sro),and spook (spo)which were first identified in the embryonic lethal screens of Nüsslein-Volhard, Wieschaus, and Jürgens as mutants that failed to produce differentiated cuticle . A closer examination of the embryo proper revealed that null mutants in these genes all exhibit a common phenotype characterized by arrest at embryonic stages 15-17 with failures of head involution, dorsal closure, and cuticle production . Subsequent genetic and biochemical analysis demonstrated that each of these gene products acts at a specific step in the biosynthetic pathway and their loss results in failure to synthesize ecdysone, and hence they all produce an identical loss-of-function phenotype.In all Arthropods, the steroid hormone ecdysone (E) acts to control molting and metamorphosis . During the past two decades, many of the enzymatic steps that convert dietary cholesterol to ecdysone in the insect; Ou et al. 2011; Christesen et al. 2017).Despite this progress, there remains uncertainty in the enzymes and reactions involved in an early step of the pathway termed the “Black Box”. The name arises because the chemical intermediates between the upstream 7-dehydrocholesterol (7dC) and the downstream 5beta-ketodiol have eluded isolation . Nevertheless, the activity of several gene products including Shroud (Sro), Spo/Spok, Cyp6t3 and Cyp6u1 have been suggested to act within the Black Box based on rescue experiments that involve feeding mutant larvae intermediate compounds that are thought to be upstream and downstream of the Black Box enzymes. In these experiments, feeding the downstream intermediate 5beta-ketodiol rescues loss-of-function larvae and enables them to progress through additional molts while feeding the upstream intermediate 7dC does not . , the source of larval ecdysone. The embryonic tissue source of E is still unknown. Strong RNAi knockdown in the PG of any of the Halloween gene products leads to first instar arrest. However, feeding the larvae various intermediates often enables them to progress into the second and third instar stage, provided the intermediate is downstream of the enzymatic block. These types of experiments suggested that the Sro and Spo/Spokenzymes are components of the Black Box.Cyp6t3(FBgn0033697) andCyp6u1(FBgn0033121) genes as encoding possible factors of the E biosynthetic pathway came about differently from classical genetic studies.Cyp6t3was identified as a gene that was specifically up-regulated in the PG in response to loss of the transcription factor DHR4. .DHR4mutants pupariate early likely because of precocious ecdysone production, and simultaneous knockdown ofCyp6t3in aDHR4mutant background suppressed the early pupation phenotype. In addition, RNAi knockdown ofCyp6t3in the PGs of wild type animals resulted in slow growth and large pupae, both of which were suppressed by feeding knockdown larvae ecdysone or 5beta-ketodiol but not 7-dC suggesting that, like Spo/Spok and Sro, this enzyme works at the Black Box step. Because of the low transcript abundance ofCyp6t3, it was also hypothesized thatCyp6t3might be a “bottleneck” in ecdysone biosynthesis, or even rate limiting.Cyp6u1 was identified in a genome wide transcriptome analysis of the PG as a P450 that showed high level expression specifically in the PG, and RNAi knockdown in the PG led to a developmental delay/arrest phenotype which was rescued by feeding ecdysone or 5beta-ketodiol but not 7-dC, again positioning this enzyme at the Black Box step.The identification ofCyp6t3 as an additional means to assess its function duringDrosophiladevelopment. Guide RNAs from both ends of theCyp6t3 cytochrome P450 domain were simultaneously injected into a Cas9 stock (Bestgene Inc.) for the purpose of generating mutations at each individual site as well as potential deletions between the sites. After screening the progeny of the G0 flies, we obtained 3 mutationsCyp6t3[Cr1], a 1 bp deletion at Guide 1, Cyp6t3[Cr2], a 2 bp deletion at Guide 2,andCyp6t3[Cr3], a 1 kbp deletion between Guides 1 and 2 . Translation of these mutated genes yields proteins with different premature stops or a large deletion within the Cytochrome P450 domain of Cyp6t3 . All three mutations are homozygous viable. For this reason alone, we deemedCyp6t3a non-essential gene for ecdysone biosynthesis.Since it has been reported that tissue-specific knockdown of genes can have more severe phenotypes than genetic null mutations , we made Crispr-mediated null mutations inCyp6t3 is not essential for animal survival, we reasoned that it could affect the rate of ecdysone biosynthesis. Previous work has shown that either raising or lowering the basal E biosynthetic rate leads to advanced or delayed timing of pupariation, respectively . To address this issue, we assessed the time to pupariation of variousCyp6t3 mutant combinations . In sum, we find no delay in developmental timing compared to the wild type control suggesting that the basal rate of ecdysone synthesis in theCyp6t3mutants is on par with wild type.Although loss ofCyp6t3 is not an essential component of the E biosynthetic pathway under lab growth conditions should not be surprising since, unlike the other Halloween genes that are highly conserved across all arthropods examined to date,Cyp6t3has been lost in some clades . In addition, as previously noted, the transcript abundance ofCypt6t3is very low by qRT-PCR  and did not make the cutoff in 2 different ring gland-specific transcriptional profiles, the only putative E biosynthetic P450 gene failing to do so . Despite these observations, the regulation ofCyp6t3transcription by DHR4 and its ability to suppress the precociousDHR4mutant phenotype when knocked down in the PG remains intriguing. It would be interesting to determine whether aCyp6t3,DHR[1] double mutant shows the same suppression of precocious pupariation as found when RNAi is used to knockdownCyp6t3in the PG of aDHR[1]mutant. It is also possible that Cyp6t3 is important under specialized growth conditions that modulate DHR4 activity. Another possibility is that Cyp6t3 function is redundant inD. melanogaster, and perhaps other species, with another P450 enzyme. Precedence for the existence of partial redundance in E biosynthetic enzymes in insects is exemplified by the Spo/Spok relationship . Both are thought to function within the Black Box but at different developmental stages, Spo acting embryonically and in female follicle cells while Spok acts in the PG during larval stages. One intriguing candidate for a redundant activity could beCyp6u1. This gene product is also a member of the P450 clade 6 subgroup but is highly conserved across Arthropoda . Examining the phenotype ofCyp6u1 null mutants andCyp6t3;Cyp6u1double mutants might provide insight on the redundancy issue.The fact thatIn summary, our studies do not resolve the enigma of the Black Box reaction mechanism in the E biosynthetic pathway. However, our elimination of Cyp6t3 involvement under standard growth conditions helps reduce the complexity of the necessary components. This should aid in simplifying the design of future studies aimed at understanding the details of the biochemical reactions catalyzed by the Blackbox enzymes and whether their regulation serves as a rate limiting step in E biosynthesis as previously suggested .Crispr mutant generationCyp6t3with no off-target sites. The targets with the PAM are: 5’ GGATCCCG¯AACCATAAGCGC CGG 3’ and 5’ GGATCAAA ¯ GACGCAGGGCTC CGG 3’. Sense and antisense oligonucleotides (Oligo Table) for the two guides RNAs were cloned into pU6-BbsI-chiRNA . Both guide RNAs were injected into vas-Cas9 embryos . Single G0 flies were screened by PCR using primerscyp6t3 01Fandcyp6t3 02R(Oligo Table) for the presence of deletions, making them candidates for further analysis. Ten to fifteen individual F1 progeny of G0 flies that displayed multiple deletion bands by PCR (possible Cr mutant/+) were then crossed to aCyO* balancer (BDSC 3198), homozygosed, the DNA region amplified by PCR and then sequenced for the presence of a mutation and its precise location. BothCyp6t3[Cr1] andCyp6t3[Cr2]mutations were obtained from a single G0 fly.The CRISPR Optimal Target Finder  was employed to identify two target sites withinClustal omega analysisused SnapGene Version 6.0.7 software.Developmental timingstinstar larvae started to emerge. 30-35 larvae were transferred to vial food  sprinkled with dry yeast and allowed to grow at 25° with a 12-hour light/dark cycle. Pupariation was scored every 2 hours with the data from 4 vials combined due to the speed of pupariation and lack of data points necessary for non-linear regression analysis in Prism .Crosses were set up in cages. Four-hour collections of embryos on yeast-apple juice plates  were aged about 16 hours, until 1Oligo TableFly lines


Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections. Plasmodium falciparum rapid diagnostic tests (RDT) that are a cornerstone of malaria control efforts in the high-burden, low-resource contexts in which malaria mortality is most acute  are affecting the accuracy of the RDTs. Infections with pfhrp2 deletions are missed by HPR2-based RDTs much of the time; infections with double deletions (missing both pfhrp2 and pfhrp3 genes) are invisible to RDTs and create false-negative results. Because these deletions represent an existential threat to recent gains made in malaria control, the World Health Organization (WHO) has emphasized the critical need for surveillance (Histidine-rich protein 2 (HRP2) is the primary target of the pfhrp2 and pfhrp3 deletions from the country come from a single report confirming their presence in 3 travelers to Australia . We performed malaria confirmation and speciation of 594 dried blood spot samples by multiplex PCR  underwent genotyping and molecular analysis for deletions in exon 2 of pfhrp2 and pfhrp3   from persons d pfhrp3 . Demograd pfhrp3 . We defipfhrp2 and 20.0% for pfhrp3  of sites. Pfhrp2 deletion rates in South Sudan were as high as or higher than the country’s immediate neighbors, where reported deletion rates from specific sites have varied from 26% in Ethiopia to 19% in Kenya, 6% in the Democratic Republic of the Congo, 3% in Uganda, and <1% in Sudan  among 518 genotyped PCR-positive samples was 15.6% for r pfhrp3 . Double r pfhrp3 . In 7/9 pfhrp2 and pfhrp3 deletions and double deletions , whereas age, MOI, and clinical severity were all predictors for pfhrp3 deletions. Only MOI was a significant predictor of double deletions  could be another way that age, disease severity, and deletion risk interact, because milder disease has previously been associated with pfhrp2 deletion surveillance, the transmission period should be considered (Sample collection in this study occurred at the end of the high-intensity malaria transmission season, when potentially high parasitic diversity but low prevalence could favor spread of gene-deleted organisms, making deletions easier to detect (pfhrp2 and pfhrp3 deletion surveillance. Consequently, the precise prevalence of pfhrp2 and pfhrp3 gene deletions causing false-negative results on RDTs in South Sudan was not generated to assess whether it is within the 5% threshold established by WHO (p-LDH RDT–positive samples, preventing us from evaluating the effects of deletions on malaria diagnoses.This study was limited because it was a secondary analysis of a study of molecular markers of antimalarial drug resistance and did not follow WHO protocols for pfhrp2 and pfhrp3 deletions is critical to designing effective public health strategies for malaria control. This study describes these deletions in a clinical cohort in a country with little previous endemic evidence of pfhrp2 and pfhrp3 deletion. Monoclonal infections were a principal predictor of deletions. We identified high levels of single and double deletions of pfhrp2 and pfhrp3, which if more widely present in this or other regions of South Sudan, could s eriously jeopardize HRP2-based RDT effectiveness moving forward. Future studies should be designed according to WHO protocol to produce precise estimates to measure the risk those deletions pose in South Sudan. Local variation in prevalence suggests the potential for deletion hotspots within the country and should be considered when designing malaria control strategies. Characterizing Plasmodium Falciparum pfhrp2 and pfhrp3 gene deletions in malaria-hyperendemic region, South SudanAdditional information about


The sizable transient changes in Ca2+ concentrations are necessary for the activation of signaling pathways, which rely on Ca2+ as a second messenger, including those involved with force generation, fiber type distribution and hypertrophy. However, prolonged elevations in intracellular Ca2+ can result in the unwanted activation of Ca2+ signaling pathways that cause muscle damage, dysfunction, and disease. Muscle employs several calcium handling and calcium transport proteins that function to rapidly return Ca2+ concentrations back to resting levels following contraction. This review will detail our current understanding of calcium handling during the decay phase of intracellular calcium transients in healthy skeletal and cardiac muscle. We will also discuss how impairments in Ca2+ transport can occur and how mishandling of Ca2+ can lead to the pathogenesis and/or progression of skeletal muscle myopathies and cardiomyopathies.In healthy muscle, the rapid release of calcium ions (Ca Innately, Ca2+ has flexibility in its bonding angles and lengths, allowing for an array of potential ligation patterns [2+-binding molecules [2+ signaling becomes apparent as it can be regulated by the location and expression of the Ca2+-binding proteins and the kinetics of the Ca2+-binding site.Calcium  can fluctuate from resting concentrations of 100 nM to values above 1000 nM during tetanus [2+]cyt is elevated above resting concentrations, Ca2+ interacts with two categories of Ca2+ binding molecules: buffers and sensors [2+ sensors elicit a downstream signal when binding occurs. The temporal range of Ca2+ signaling can be as brief as seconds but may also exist on the timescale of days [2+ signaling is effectively diminished when [Ca2+]cyt reverts back to resting concentrations. One strategy for increasing the rate of [Ca2+]cyt decay is through Ca2+ buffering. Buffers act to sequester Ca2+ without being directly incorporated into molecular signaling. The presence of Ca2+ buffers allows for the regulation of Ca2+ diffusion, which can indirectly affect molecular signaling pathways cyt is mediated primarily through the movement of Ca2+ across phospholipid membranes [2+]cyt is through Ca2+ sequestration in the sarcoplasmic reticulum (SR), a membrane bound organelle which surrounds the contractile myofilaments cyt but also in the regulation of the energy expenditure of the cell [2+ is also extruded across the plasma membrane to effectively reduce [Ca2+]cyt during the cardiac cycle cyt can result in the unwanted activation of proteolytic and apoptotic pathways, leading to muscle damage, dysfunction, and even disease. In this review, we discuss the role of Ca2+ transport in the maintenance of healthy muscle as well as the role it can have in the genesis and exacerbation of pathological states.Although Caembranes . Within ilaments . Ca2+ isdrolyzed ,10. SERCthe cell . Within ac cycle . The rol2+ release cyt [2+]cyt results in the immediate activation of SERCA, which acts to pump Ca2+ back into the SR. However, with the continuance of high frequency neural signaling the rate of Ca2+ outflow is greater than the ability for SERCA to re-sequester Ca2+. Consequently, [Ca2+]cyt rises within the myofiber and binds to troponin C resulting in the movement of tropomyosin and thus uncovering the myosin binding site on the thin filaments cyt begins.The RyR is a CaCa2+]cyt . Within ilaments ,17. Withilaments ,19. Musc2+]cyt to a peak is quickly followed by a decay, all within milliseconds [2+]cyt has a negative exponential relationship in which the initial rate of decay is rapid but as [Ca2+]cyt approaches pre-stimulation resting concentrations the rate of calcium uptake is attenuated. Within skeletal muscle tissue, the characteristics of a Ca2+ transient varies across different types of muscle fibers. Baylor and Hollingworth (2003) compared the ICTs of slow fibers from soleus tissue and fast fibers from EDL muscle [2+]cyt during Ca2+ release in the slow and fast fibre types; however, the peak [Ca2+]cyt amplitude was two times greater and the ICT half duration was ∼1.6 times shorter in fast EDL fibers compared with the slow soleus fibers cyt, PLN dissociates from SERCA and does not affect Vmax cyt is elevated, CAMKII will detect these elevations and respond by phosphorylating PLN, thus relieving its inhibitory effect on SERCA and allowing for faster calcium clearance cyt. MLN, on the other hand, is believed to be another inhibitory regulator of SERCA cyt rises due to SR Ca2+ release, the membrane potential effect becomes negligible, and the direction of the exchanger now changes to the forward mode with Ca2+ being extruded from the cytosol [+] and [Ca2+] can impact the direction of the exchanger, under physiological conditions, the direction is largely driven by the [Ca2+]cyt  to potentially help to overcome Na+-dependent inactivation by increasing the electrostatic potential [+ levels and works independent of the [Na+]cyt levels and (2) secondary or slow blockade that is dependent on [Na+]cyt levels, which enhance the affinity of NCX for H+ cyt during repeated muscle contractions when [Ca2+]cyt is very high cyt [2+ at an optimal ratio of 2 mol Ca2+:1 mol PV [2+ at resting [Ca2+]cyt (<100 nM) [2+ is dependent on the rate of dissociation of Mg2+ from the binding sites [2+]cyt, which is why it is often considered a slow Ca2+ buffer cyt ,111,112.Ca2+]cyt . In contCa2+]cyt . FurtherCa2+]cyt . PV is a1 mol PV ,116. The<100 nM) . The ratng sites . Consequ+ buffer . The binl muscle ,120. Howl muscle . Taken t2+ buffering, there is evidence showing that Ca2+ uptake through the mitochondrial Ca2+ uniporter is important for the maintenance of myocyte homeostasis [2+ uptake in cardiomyocytes does not alter [Ca2+]cyt transients or cardiac contractility, there is evidence that mitochondria can modulate [Ca2+]cyt in fast-twitch skeletal muscle fibers under certain conditions cyt. The first theory posits the lack of dystrophin results in transient tearing of the sarcolemma membrane during contractions. The membrane damage could allow for an influx of extracellular Ca2+ into the myofiber [2+ influx into the myofiber [2+]cyt can increase proteolytic activity and the production of ROS [2+ dysregulation [2+]cyt alone are enough to promote a shift toward a DMD like phenotype [2+]cyt by overexpressing SERCA in dystrophic tissue, which ameliorated the pathology cyt and decreased muscular degeneration and fibrosis cyt is decreased in these cells [2+]cyt cyt ,186.2+ release with HF could be due to defective coupling between the RyR and DHPR, reduced SR Ca2+ content could also contribute. This was assessed in one study using a canine model of HF by assessing the CICR gain, which is the amount of Ca2+ released from the SR for a given level of current density from the DHPR channel and ε, which is the effectiveness of coupling between DHPR activation and SR Ca2+ release cyt and inducing relaxation of the myocardium during diastole. Several studies have shown that SERCA Ca2+ uptake is reduced in failing hearts from both human and animal models cyt, it can actually increase the probability of arrhythmogenic events. In failing hearts, with increasing frequency of stimulation, the intracellular Na+ ([Na+]cyt) levels rise [+/H+ exchanger on the sarcolemma [+/K+ ATPase activity [2+]cyt and SERCA uptake of Ca2+ into the SR would also increase leading to SR Ca2+ overload cyt during E-C coupling must be rapidly lowered to re-establish resting concentrations and induce muscle relaxation, which is primarily accomplished by SERCA-mediated Ca2+ transport into the SR. During short contractions of fast-twitch rodent and amphibian myofibers, the cytosolic Ca2+ buffer PV also contributes to muscle relaxation by rapidly lowering [Ca2+]cyt. In the heart, Ca2+ extrusion from cardiomyocytes through the NCX contributes significantly to ventricular relaxation, and the PMCA and MCU also contribute to a very minor extent. Impairments in Ca2+ transport and dysfunctional Ca2+ homeostasis has consistently been shown to be involved with the pathogenesis and/or the progression of multiple myopathies within skeletal muscle and in HF. A better understanding of the function and regulation of muscle Ca2+ transport proteins can allow for greater insights into skeletal and cardiac muscle physiology and disease. Furthermore, this body of work can lead to novel approaches in the treatment of diseases involving Ca2+ dysregulation.Maintenance of Ca


Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18–22 weeks.NCT02521974 and NCT03554798) conducted in Panama, infants aged 18–22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model.In two similarly designed clinical trials (10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007  indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients.Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5ʹ untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 logThe data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses.Bill & Melinda Gates Foundation. Vaccination with Sabin oral poliovirus vaccine (OPV) results in robust intestinal and humoral immunity, which are key to the control of poliomyelitis, but the inherent genetic instability of the OPV strains is well documented.With the certification of the global eradication of wild type 2 poliovirus the routine use of the type 2 component of Sabin OPV was ceased globally in 2016. Although this switch has been successful in most regions, waning immunity to type 2 viruses in areas of low vaccine coverage has contributed to increasing numbers of outbreaks of type 2 cVDPV, with consequent increases in cases of paralysis.9Evidence before this studyThe Lancet four clinical studies that demonstrated the safety, tolerability, and immunogenicity of nOPV2 in adults, young children, and infants compared with monovalent Sabin OPV2 (mOPV2). The key driver of a change from mOPV2 to use of the novel vaccine in outbreak settings is its improved genetic and phenotypic stability, which has been described in two published reports in NPJ Vaccines from studies using shed stool samples obtained from adults and children administered mOPV2 or nOPV2. We did not conduct a literature search as the few published reports regarding novel OPV2 have been generated from the novel OPV2 consortium.The widespread transmission of type 2 vaccine-derived poliovirus constitutes a Public Health Emergency of International Concern. A novel type 2 oral polio vaccine (nOPV2) received a WHO emergency use listing in November, 2020, to respond to this crisis based on promising clinical data. We previously reported in two publications in Added value of this studyThis report represents the first rigorous comparison of genetic and phenotypic stability, conducted with samples from infants, a primary target population of nOPV2 use in outbreak settings, completing the assessment of nOPV2 in the previously reported clinical studies. We show that the vaccine viruses shed by infants administered nOPV2 have significantly reduced reversion to virulence compared with polioviruses shed by infants administered mOPV2. We also show the improved stability of the primary attenuation site, domain V, of nOPV2 compared with mOPV2.Implications of all the available evidenceThe data from this key study population provide a crucial demonstration of the better genetic and phenotypic stability of shed nOPV2 strains compared with shed mOPV2 in infants. These data suggest that nOPV2 should be associated with less paralytic disease and potentially a lower risk of seeding new outbreaks, the objective of the development of these new vaccines. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses.Novel OPV2 (nOPV2) was designed to provide similar protection as mOPV2 but with reduced risk of loss of attenuation.14WHO/UNICEF Estimates of National Immunization Coverage data  national coverage of a third dose of OPV was estimated to increase from 72% in 2015 to 86% in 2016 while the control phase 4 study was conducted and was estimated to be 88% in 2018 and 2019 while the phase 2 study was being conducted. In this Article, we used established methodsOur study supplements previous reports from similarly designed phase 4 and phase 2 studiesNCT02521974) conducted in 2015–16 before the global switch to bivalent OPV (bOPV) containing only types 1 and 3, in which mOPV2 was administered to 110 healthy infants age 18–22 weeks who received three doses of bOPV and one dose of inactivated polio vaccine at least 4 weeks before administration of mOPV2. The second is a phase 2 trial  in 2018–19, in which 150 healthy infants age 18–22 weeks received at least one oral dose of nOPV2 at least 4 weeks after receiving three doses of bOPV and one dose of inactivated polio vaccine.This study is an analysis of shed virus in samples obtained from two trials. The first is a phase 4 trial  virus.Polio Sabin Mono Two (mOPV2), a type 2 Sabin strain, was manufactured by GlaxoSmithKline Biologicals, Rixensart, Belgium. A dose consisted of 2 drops (0·1 mL) containing no less than 106·0 CCID50 in 1 mL.nOPV2, referred to as nOPV2 candidate 1 (S2/cre5/S15domV/Rec1/HiFi3) in earlier publications,10 CCID50 type 2 virus per g of stool was termed the exploratory endpoint specimen  as previously described.men EES; .15 Stool10 or 5 log10 CCID50 amplified virus from EES or control virus. Virus from EES were amplified in a single round of infection of Hep2C cells under conditions previously shown to be non-selective. These doses were selected for the assay to be sensitive to clinically relevant Sabin-2 reversion. The controls were 20 mice inoculated with Sabin-2 (SO + 2/II) at 6 log10 CCID50 doses and ten mice inoculated with SO + 2/II 5 log10 CCID50 doses. A Sabin-2 molecular construct with reversion at two genome sites (481a→g and 2908a→g) was inoculated into 20 mice at 4 log10 CCID50 dose level as a positive control. Back-titrations of the diluted samples confirmed that inoculum titres were within 0·5 log10 CCID50 of the nominal dose. Inoculated mice were monitored for paresis or paralysis over a 14-day period and assigned a clinical score per established protocol.17The virus titrations for EES and controls, and the mTgmNVT were described previously.50) was determined. For these tests, groups of ten Tg-PVR21 mice were inoculated with five different dose levels of each sample.For nOPV2 samples that induced 40% or more paralysis, the paralytic dose for 50% of mice  and its corresponding confidence interval. We used inverse probability weighting to account for stratified subsampling of M5 EES according to post-vaccination week, as described in the 50 values, averaged across mouse sex, were obtained by inverting the regression models. As an exploratory analysis, a similar model was fitted in R (version 4.0.0) to mouse paralysis data for each vaccine group and dose level to assess the relationship between frequency of mutation at key sites and mouse paralysis. See the A binomial logistic generalised linear regression mixed model with mouse sex as a factor and subject (EES)-specific random effects was fitted with SAS (version 9.4) to mouse paralysis data for each vaccine group separately, yielding estimates of paralysis rate. For comparison of paralysis rate of shed nOPV2 versus shed mOPV2, two additional similar models were fitted that also included a term to estimate the odds ratio of paralysis for the virus shed from nOPV2 recipients relative to mOPV2 recipients. This adjusted odds ratio (aOR) is adjusted for mouse sex imbalance owing to mice excluded from evaluation due to reasons unrelated to the inoculum. The primary inferential comparison between groups was predefined to be based on the model-estimated aOR (nOPV2/mOPV2 ratio) at 4·5 logThe funder of the study had no role in data collection, data analysis, or data interpretation. ASB was an employee of the study funder and was involved in the clinical trial design and writing of the report.Although the global cessation of OPV2 usage in routine immunisation required that the M2 and M5 studies were conducted non-contemporaneously, given the similar vaccination histories of the participants in both cohorts, and overall similar rates of viral shedding among nOPV2 and mOPV2 recipients early after vaccination,NGS was performed on shed virus (two replicates of 10% stool suspension per EES) from 36 EES from the M2 study ranging from 1 day to 28 days after mOPV2 administration. 34 EES were successfully sequenced, with two excluded due to insufficient DNA concentration following NGS library preparation. Data from the key genome sites are shown in NGS was performed on 52 EES (three of 55 EES had insufficient DNA for sequencing) ranging from 2 to 28 days following nOPV2 administration. SNPs in the key modified and known attenuating regions are summarised in Variation was also noted at position 459 in domain IV, corresponding to nucleotide 398 in Sabin-2. 459u→c increased in frequency in later EES and is fixed in a day 28 EES. By contrast to mOPV2 EES, mutations which result in reversion of VP1-143 were observed consistently, with several nOPV2 EES at 50% variant frequency or higher (fixed in at day 28 EES). Changes involved in the reversion of VP1-143 are observed at nucleotides 2969 and 2970 and changes at both positions were sometimes detected within the same sample. The 2969 and 2970 variants are reported as SNPs but could represent dinucleotide polymorphisms. Generally, the data suggest these two locations of mutations might undergo more rapid selection for nOPV2 than mOPV2.No reverting polymorphisms (defined as strengthening of a dinucleotide pairing in the stem regions of domain V) were observed in the novel domain V (nucleotides 468–535) of shed nOPV2 . Low-levTo confirm whether the culture-amplified virus was sufficiently representative for neurovirulence assessment, the frequency of mutation in stool samples was compared with the frequency of mutation in the amplified sample for mOPV2 and nOPV2 . PolymorTo provide a sufficiently robust evaluation of potential neurovirulence of shed virus in mice, preclinical and previous clinical data determined that 36 EES per vaccine (mOPV2 and nOPV2) would provide sufficient power for comparison. No subsampling was therefore necessary for mOPV2 EES, and 36 of the 51 nOPV2 EES  were subsampled with simple random sampling within strata defined by post-vaccination week, focusing on those EES most distal to vaccination .10 CCID50 dose and 76·7% at the 5 log10 CCID50 dose  indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients.For nOPV2, rates of paralysis increased with later EES days  and a doShed mOPV2 virus reverted rapidly and showed high paralysis rates in the mouse model. NGS data support the high levels of reversion observed for the shed mOPV2 virus . Reversi10 dose and 17 of 36 showed 10% or more paralysis at the 5 log10 dose. A single day 28 EES (M5-2-351) showed 40% paralysis at the 4 log10 CCID50 dose and 60% paralysis at the 5 log10 CCID50 dose. This day 28 EES had fixed mutations at nucleotide 123 in cre5, VP1-143 and at nucleotide 459 with additional fixed changes at nucleotide 556 within domain V and in the 3D polymerase .For nOPV2 , six of 50 (50) contain higher levels of 459u→c in domain IV (398u of Sabin-2) and VP1-143 substitutions. Virus from the day 28 EES with fixed mutations at key sites shows the lowest (more virulent) PD50 estimate of 5·3 log CCID50. The PD50 of the day 28 EES aligns with data from a series of virus strains constructed to incorporate key polymorphisms in the nOPV2 background and evaluate their impact on neurovirulence (50 6·3 log), but notably higher than the observed PD50 of 3·5 log for a partially-reverted day 7 shed Sabin-2 virus.10 CCID50 dose, suggesting that PD50 would generally be less than 4 log for these Sabin-2 viruses.nOPV2 EES with higher neurovirulence were evaluated in multi-dose neurovirulence tests to determine the PD50 . In geneirulence  and is s10 CCID50 dose level  consistent with the presence of cre5, domain IV and VP1-143 mutations in the shed virus. These mutations appear to accumulate rapidly in nOPV2 . Sequencing data can be accessed on the Sequence Read Archive database (accession number PRJNA786548). Algorithms for polymorphism selection are described in the methods. R codes for tables and figures specific to this publication are available from the corresponding author upon reasonable request.We declare no competing interests. The National Institute for Biological Standards and Control, University of California, San Francisco, claim intellectual property rights associated with the nOPV2-c1. ET works for the manufacturer of nOPV2. JS and BK are employees of Viroclinics, which is paid by PATH for the conduct of next-generation sequencing and mouse neurovirulence tests. ASB was an employee of the study funder and was involved in study design and writing of the report but had no role in data collection or analysis.


BRAFV600E mutation represent a unique subset of central nervous system tumors. Targeted therapies including BRAF and MEK inhibitors are now being explored as possible new treatment options.High-grade gliomas (HGG) with BRAFV600E mutations treated with BRAF inhibitors.We report an 18-year-old female with a grade 3 pleomorphic xanthoastrocytoma treated upfront with dabrafenib and trametinib. We also conducted a systematic literature review of patients with HGG and BRAFV600E mutations treated with BRAF inhibitors were retrieved through our systematic review of the literature. Only 1 young patient with an anaplastic ganglioglioma was treated upfront with a BRAF inhibitor with a curative intent. Best response reported with radiation therapy and systemic therapy was a stable disease (SD) for 18 patients (56.3%) and progressive disease (PD) for 9 patients (28.1%). Responses to treatment regimens that included BRAF inhibitors were reported in 31 patients and included 4 complete responses (12.9%), 23 partial responses (74.2%), 2 SDs (6.5%), and 2 PDs (6.5%). Despite local recurrences resected surgically, the patient has been on dabrafenib and trametinib for more than 54 months. Thirty-two patients with HGG and Our patient had durable disease control with dabrafenib and trametinib. Given favorable responses reported in patients with HGG treated with BRAF inhibitors, we believe that upfront targeted therapy is a possible treatment approach that should be studied in the context of a clinical trial. BRAFV600E mutation.BRAF inhibitors appear to be an effective approach for the treatment of HGG with BRAFV600E mutation.BRAF inhibitors should be studied as a possible option for upfront treatment in HGG with BRAFV600E mutation.We reported the evolution of a young patient treated upfront with a combination of BRAF/MEK inhibitors. This approach without prior chemotherapy or radiation has rarely been reported. We conducted a systematic review of the literature and report that responses are more frequent in patients treated with BRAF inhibitors when compared to standard regimens. Results from our review and our clinical experience suggest that clinical trials should investigate the possibility to use BRAF and MEK inhibitors in upfront therapy for HGG with BRAFV600E mutation is the second most frequent mutation in pediatric low-grade glioma (LGG) but the alteration has also been reported in a subset of primary and secondary high-grade glioma (HGG).1 The overall survival of HGG BRAFV600E mutation is more favorable than other subgroups including HGG H3.3 G34 R/V but despite standardized treatment strategies including surgery, radiation therapy, and chemotherapy the prognosis is poor.3 New treatment approaches are currently being investigated including BRAF inhibitors (BRAFi) with or without MEKi for LGG and HGG with BRAFV600E mutation. However, there are currently limited data available on the outcome of patients treated with BRAFi. Following our experience with 1 patient with HGG treated upfront with BRAFi and MEKi, we aimed to better understand the expected outcome in similar patients treated with targeted therapy by conducting a systematic review of the literature.We describe a case report of a young patient with a HGG treated with BRAFi and MEKi.BRAFV600E mutations and treated with BRAFi . Relevant articles were then reviewed in detail and included. Given the nature of the study, no ethical board approval was required. The family and patient gave their consent for this case report.A systematic review of the literature in the PubMed and Embase databases was conducted for original articles on HGG including glioblastoma (GBM), anaplastic astrocytoma, anaplastic ganglioglioma, and grade 3 pleomorphic xanthoastrocytoma (PXA) with th BRAFi .4 ArticlOur patient was diagnosed with epilepsy at 14 years of age when she presented with focal seizures characterized by “déjà vu phenomenon” followed by an alteration of consciousness. Electroencephalogram showed intermittent dysfunction in the right temporal region. Magnetic resonance imaging (MRI) at diagnosis of epilepsy was normal  and B. S2) including a right-side amygdalohippocampectomy  and trametinib (2 mg PO daily by mouth once daily) was initiated.Treatment was initially well tolerated except for mild dry skin. No cardiac dysfunction was detected. After 16 months of treatment, a grade 1 retinal pigment epithelial detachment with minimal visual acuity impairment was observed. Trametinib was dose reduced by 50% to 1 mg daily.BRAFV600E mutation was confirmed by immunohistochemistry and PCR. Given the slow progression and presence of areas with low-grade features in the recurrence, methylation profiling was requested on both the initial tumor and the recurrence. Both profiles were highly consistent with grade 3 PXA (score: 0.98). RNAseq did not identify a resistance mechanism or significant alteration such as telomerase reverse transcriptase promoter mutations. Copy number variation methylation was very similar between recurrence and diagnosis. Reexamination of primary tumor pathology revealed areas compatible with PXA including scattered eosinophilic granular bodies and xanthomatous change .A total of 23 articles were included. Of those, 21 articles were case reports or small case series.th BRAFi –3. Media8 Fourteen (43.8%) had leptomeningeal dissemination at 1 point during their follow-up including 2 patients at diagnosis.Progressive disease (PD) was reported in 29 patients (90.6%) and death was reported in 16 patients (50 %) with a median overall survival of 24 months after diagnosis (range 2–112 months). All patients died of their disease except for 1 patient who suffered from an extensive intracerebral hemorrhage related to vemurafenib according to the authors.20 Best response achieved on systemic therapy before BRAFi  was a stable disease (SD) for 18 patients (56.3%) and PD for 9 patients (28.1%).In terms of treatment, 26  had focal radiation therapy following resection and 10 patients (31.3%) received a second round of focal radiation therapy. Five patients did not receive radiation therapy: 3 patients due to poor neurological status and 2 patients due to young age (1.5 and 4 years old). Most patients also received prior systemic treatment before BRAFi ; 12 patients (37.5%) had received more than 2 lines of systemic therapy (range 1–4). Only one young patient with an anaplastic ganglioglioma was treated upfront with a BRAFi with a curative intent.Median time between diagnosis and the start of treatment with BRAFi was 11 months (range 2–98 months). The treatment regimens varied: 15 (46.9%) patients were treated with combination of dabrafenib plus trametinib; 7 (23.3%) patients received dabrafenib monotherapy; 7 (23.3%) patients were treated with vemurafenib monotherapy; 2 (6.7%) patients received vemurafenib followed by dabrafenib; 1 (3.3%) patient received combination of vemurafenib plus cobimetinib. Median treatment time with BRAFi was 9 months (range 1.5–32 months) and 7 (23.3%) patients were still receiving targeted therapy at the time of reporting. Response was reported in 31 patients and included 4 complete responses , 23 partial responses , 2 SDs (6.5%), and 2 PDs (6.5%).BRAFV600E mutations treated with BRAFi in monotherapy or the combination of BRAFi and MEKi were also reviewed.27 Kaley et al. reported 24 adults with CNS tumors and BRAFV600E mutations treated with vemurafenib including 15 patients with HGG including 6 patients with GBM, 5 with anaplastic astrocytoma, 3 anaplastic gangliogliomas, and 1 HGG not otherwise specified. All patients with high-grade tumors had received prior treatment including radiation therapy (15/15) and most had received chemotherapy (12/15). Using the response evaluation criteria in solid tumors 1.1 criteria, 2 patients had a PR . They reported a median overall survival for GBM and anaplastic astrocytoma of 11.9 months.26 In a recent study by Wen et al., adult patients with BRAFV600E mutation were treated with combination of dabrafenib and trametinib.27 The study included 45 HGG (31 GBM). All patients received prior therapy including radiation therapy followed by chemotherapy or concurrent chemoradiotherapy. They observed an overall response rate (ORR) of 33% with 3 CR and 12 PR. SD was seen in 22% and PD in 42% of patients. Median duration of treatment was 14.9 months for HGG. Median overall survival was 13.7 months for GBM and 45.2 months for other HGG.27Two clinical trials involving patients with HGG and BRAFV600E mutation.We report a case of a teenager with a grade 3 PXA who initially presented with focal seizures. Her first MRI was normal, and the lesion was identified 16 months later when seizures recurred with symptoms of intracranial hypertension. The patient was treated upfront with a combination of dabrafenib and trametinib. We conducted a systematic review of the literature and summarized current data on treatment with BRAFi of HGG with 28 The initial MRI as part of the epilepsy workup usually reveals the tumor but in rare cases, the lesion is not detected. In an adult series of patients with HGG, 4.7% had normal imaging at presentation (9/193).29 MRIs are usually repeated in patients with refractory epilepsy, especially if they were well controlled in the past or if they presented with new neurological symptoms to rule out an ongoing process not initially recognized.In children, only 1%–3% of patients with epilepsy have an underlying tumor.20Given the extent of resection, tumor location, and family preference to avoid radiation therapy, we decided to treat this patient upfront with dabrafenib and trametinib without radiation. This approach has rarely been reported. In our systematic review, only 4 patients received upfront BRAFi including 3 patients with poor neurological status precluding the use of radiation and chemotherapy. Only 1 young patient with an anaplastic ganglioglioma was treated upfront with a BRAFi with a curative intent. The patient has been stable for more than 23 months according to the authors.BRAFV600E mutation.Responses to BRAFi in patients with HGG vary in the literature. In our literature review, 27 patients (87.1%) presented a significant decrease in their tumors . This high response rate needs to account for a possible publication bias toward selection of case reports with favorable responses to BRAFi. However, other studies have revealed a high rate of response in HGG with BRAFV600E mutations treated with BRAFi.2 A total of 11 children with HGG were reported . Detailed treatment and disease course were not provided but all patients received prior radiation therapy and 9 received between 1 and 3 lines of systemic therapy. Four patients (36%) responded (1 CR and 3 PRs) and all patients except 1 progressed within 18 months. Median time to progression was 10 months.Nobre et al. reported a large cohort of patients with gliomas and BRAFV600E mutations. Sixteen (84.2%) received upfront radiation therapy followed by BRAFi+/−MEKi and 3 patients underwent biopsy with upfront treatment with BRAFi+/−MEKi. They reported an ORR of 64.3% when including CR and PR. Only 1 patient had PD as the best response. They showed a favorable 18-month progression free survival (PFS) of 83% compared to 42% in the BRAF-mutant historical cohort. They reported a 3-year overall survival (OS) of 82% compared to 44% in the BRAF-mutant historical cohort.30Recently, Rosenberg et al. reported an original series of 19 pediatric patients with HGG and BRAFV600E mutation. They reported an ORR of 56% in patients with pediatric HGG and 38.2% in patients with adult HGG treated with BRAFi+/−MEKi.31Andrews et al also conducted a large systematic review of glioma with BRAFV600E mutations (NCT02684058). After completing at least 1 line of treatment (radiation and/or chemotherapy) patients received a combination of dabrafenib and trametinib. The ORR was 56.1% for the entire cohort and 66.7% for PXA grade 3 (4/6 patients). The 12 months PFS was 44.1% and the OS was 32.8%.32Recently, Hargrave et al. presented the results of the recently completed Novartis trial for pediatric HGG with 33 Therefore, the use of BRAFi might be more effective with at least transient improvement in tumor control, neurological function, and quality of life. Given the small number of patients in our literature review, we did not observe significant differences in response rates between pediatric patients, adolescents/young adults, and adults. It will be interesting to see if upcoming studies report different outcomes based on age.In recurrent HGG, response rates with chemotherapy have rarely exceeded 5%, with an overall survival of 5–9 months and progression-free survival of less than 3 months.34 This should be investigated in the setting of a clinical trial. However, given the rarity of HGG with this alteration, it may take several years and the involvement of a large consortium to answer this question. Currently, the Children’s Oncology Group is conducting a study using dabrafenib and trametinib for HGG following local radiation therapy (NCT03919071).It is possible that prior treatments negatively impact the outcome of these patients. Radiation and chemotherapy could induce new mutations facilitating resistance to targeted therapy. Data are currently very limited to support this hypothesis but given the poor outcomes of patients with HGG, new treatment approaches (including upfront targeted therapy) and a modification of sequences of treatment should be considered. Treatment with BRAFi could be initiated at diagnosis and radiation therapy with chemotherapy could be reserved for progression. In some cases, targeted therapy could be continued during radiation and chemotherapy in select patients to avoid the rebound phenomenon.36 The combination of dabrafenib and trametinib was used in the Novartis trial for HGG (NCT02684058).32 Despite limited data, we used this approach to optimize tumor control.In 16 patients (50%) a combination of BRAFi and MEKi were used. The benefit of this combination for HGG have not been determined yet but studies in adults with melanoma have reported a reduction in both death and progression with similar toxicity.BRAFV600E mutation . This is higher than what is generally, reported in GBM where approximatively 25% of patients have metastases within the central nervous system at 1 point during their follow-up.37 It will be important to investigate whether HGG with BRAFV600E mutations are more likely to have leptomeningeal dissemination in future large studies.In our literature review, leptomeningeal dissemination was frequently reported in patients with HGG and 38 The term anaplastic is no longer used for diffuse astrocytoma and it was not possible to know if some of these tumors would be classified differently. Nevertheless, we included case reports of patients with features suggestive of higher-grade lesions that have been historically treated aggressively with resection, radiation, and chemotherapy.Another limitation of this systematic review of the literature is the accuracy and classification of diagnosis. Diagnoses were based on descriptions and not in accordance with the latest WHO classification of CNS tumors.We report a case of grade 3 PXA treated upfront with dabrafenib and trametinib. Despite local recurrences, our patient had a favorable and durable disease control. We reviewed the current literature, and this treatment approach has rarely been reported. Given the favorable response seen in patients with HGG treated with the combination of BRAFi and MEKi, we suggest that upfront targeted therapy treatment is feasible and should be studied in the context of a clinical trial.vdac174_suppl_Supplementary_Figure_S1Click here for additional data file.


SMN genes. These elements within the SMA genes may play key roles in understanding this early-onset neurodegenerative disease as well as how transposable elements can impact gene expression. Understanding the roles of transposable elements in SMA may provide key insights into other neurodegenerative diseases.Transposable elements are DNA sequences that can move throughout the genome. They play essential roles in gene regulation and function. Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality worldwide. Since transposable elements have been linked to other genetic diseases, we examined the genomes from SMA patients as well as healthy genomes for the presence of transposable elements. We identified distinct transposable elements that may impact gene expression by affecting promoter activity or transcriptional termination of the Survival Motor Neuron 1 (SMN1) gene but retention of its nearly perfect orthologue SMN2. Both genes are highly enriched in TEs. To establish a link between TEs and SMA, we conducted a comprehensive, in silico analysis of TE insertions within the SMN1/2 loci of SMA, carrier and healthy genomes. We found an Alu insertion in the promoter region and one L1 element in the 3′UTR that may play an important role in alternative promoter as well as in alternative transcriptional termination. Additionally, several intronic Alu repeats may influence alternative splicing via RNA circularization and causes the presence of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that could lead to SMN1 exons deletions and, ultimately, SMA. TE characterization of the SMA genomic region could provide for a better understanding of the implications of TEs on human disease and genomic evolution.Transposable elements (TEs) are interspersed repetitive and mobile DNA sequences within the genome. Better tools for evaluating TE-derived sequences have provided insights into the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the  Transposable elements (TEs) are interspersed repetitive DNA sequences with the ability to mobilize in the genome. This mobility is mediated by element-encoded proteins such as DNA transposase or reverse transcriptase and occurs within the genome of virtually all walks of life, including prokaryotes, unicellular and multicellular eukaryotes and even large DNA viruses ,2. TEs cRetrotransposons are divided in two major subclasses based on the presence or absence of a long terminal repeat (LTR) sequence. Long and short interspersed nuclear elements  comprise the two types of non-LTR retrotransposons. The only active, autonomous TE family in humans is the LINE-1 family, although most of these sequences are no longer mobile due to various forms of rearrangements, mutations and 5′-truncation ,8,9. SINInitially considered inert remnants of evolution and so called genomic parasites, TEs are now recognized as important players in genomic evolution, genome organization and gene regulation—due primarily to the advances in genome sequencing and better analysis tools Once co-opted by the host genomes, TEs provide important sources of new regulatory sequences that can act as alternative promoters, tissue specific enhancers, splice sites, polyadenylation signals, insulators, termination sites and transcriptional factor binding sites, thereby altering nearby gene expression in cis ,13,14,15While TEs play a beneficial role in genome evolution, their presence can also be detrimental to the host and cause several problems to normal gene expression and to genome organization, stability and integrity ,8,11,19.Survival Motor Neuron 1 (SMN1) but retention of the paralogous Survival Motor Neuron 2 (SMN2) gene [SMN1 and SMN2 are nearly identical except for 20 single nucleotide differences, with the C to T transition in exon 7 (c.840C > T) being the most functionally relevant difference [SMN1 is located in a highly unstable region of the large arm of chromosome 5 (5q13.2), a region of the genome that is enriched with repeated sequences, pseudogenes and transposable elements [SMN1 and SMN2 are both highly enriched in TEs—especially Alu and L1 repeats—spanning both genes [SMN1 introns have an impact on the regulation of the splicing patterns as two Alu elements can give rise to new alternative SMN exons as well as on circularization events of SMN RNA that result from inverted Alu repeats [SMN1 introns also makes this gene prone to deletion events caused by Alu/Alu recombination events [Attention to the contribution of TEs to neurodegenerative diseases has been rising in the last few years . Spinal N2) gene ,27,28. Sfference . SMN1 iselements ,31,32. Ielements ,31. SMN1th genes . The hig repeats ,34,35. Sn events ,37,38.SMN1 and SMN2 (SMN1/2) genes, have only used the reference gene sequence in their analysis [SMN1/2 loci of whole genome sequences from SMA patients, SMA carriers and healthy individuals. The results of this comprehensive bioinformatic analysis could provide important insights into the potential involvement of TEs in SMA onset as well as help understand the roles of TE dynamics in genome evolution, gene regulation and human disease.Prior studies on the organization of TEs within the analysis . As someSMN1/2 gene sequences from 20 SMA carriers, 22 non-carriers and 37 SMA patients obtained from different sources. Genome sequencing data from the “1000 Genomes Project” cohort [SMN1 and SMN2 copy numbers for the MNDRL cohort were confirmed by digital droplet PCR  that is available in Ensemble.Our sample database consisted of ” cohort  were sel” cohort  previous” cohort . The SMAplet PCR  [42,43,4SMN1 reference sequence by Geneious Mapper tool [SMN1 and a consensus sequence of the mapped reads which was used for predicting TE insertions.The raw next generation sequencing (NGS) data obtained were processed and then mapped against per tool  (GeneiouSMN1/2 sequences obtained and the SMN1 and SMN2 Consensus Coding Sequences (CCDS) [Homo sapiens” to ensure that the best cut-off was applied to each model thereby ensuring more accurate predictions of TE location and orientation. Overlapping Dfam matches (nearly perfect overlaps) are automatically removed by Dfam so as to remove model redundancy.All s (CCDS)  isoformss (CCDS) ,48. ThisSMN1/2 sequences and CCDS were aligned using Clustal Omega version 1.2.2 multiple sequence alignment (MSA) program [SMN1/2 transcriptional elements and motifs, including the promoter elements and other regulatory sequences were either described previously [All the TE annotated  program    .SMN1/2 promoter exhibits differences in TE insertion sites and subfamilies present between samples. We hypothesize that the untranslated regions may be subjected to less evolutionary pressure thereby allowing more diversity in TE insertions. With respect to the gene region downstream of exon 7, our first analysis of SMN1/2 gene reference sequence obtained from Ensembl showed several TE insertions belonging to various subfamilies (SMN1/2 is located within the terminator region of the gene and primarily serves as the 3′UTR region of the gene [SMN1 deletion), SMA carriers, non-carriers or healthy genomes, all samples show this L1 insertion inside exon 8  in Dfam showed a L1 element at the 3′ end of the CCDS, indicating that these complete SMN1 transcripts have an imbedded TE sequence derived from the L1MC5a insertion in exon 8 , does not contain this L1 insertion  contains the same L1MC5a insertion in a similar 3′ location to that seen in the longest SMN1 transcript (results not shown). Similar to SMN1, the remaining and more common SMN2 transcripts isoforms  do not exhibit any L1 insertion in their sequence (results not shown).Since SMN1/2 introns are highly enriched in Alu-derived repeats with many of them in an inverted orientation  [SMN expression.While these domesticated TEs may be integrated in the gene regulatory network, the significance of the remaining motifs inside the AluJb sequence remains to be investigated. The TSSs present inside the Alu sequence are tissue-specific and/or developmental stage-specific TSSs, with TSS2 being used as a fetal transcription start site and the use of TSS3 is still unknown . This AllncRNAs) . Future SMN1/2 3′UTR region serves as an alternative terminator for the genes transcription by giving rise to longer transcripts. We will experimentally confirm this hypothesis in future studies using rapid amplification of the 3′cDNA ends (3′RACE) in control and SMA cells to identify alternative 3′UTRs.The majority of human genes use alternative polyadenylation sites that are embedded in TEs, suggesting that these can influence the 3′ end processing of host gene transcripts ,72. L1 aSMN1/2, that functions as the 3′UTR of the gene, is an example of a relatively rare event. Furthermore, the L1 insertion in exon 8 does not belong to the active L1 elements  of the human genome that are composed only of the L1PA1 and L1PA2 subfamilies [Two factors may explain how this L1 element became fixed in this gene region. First, exon 8 has a lower percentage of G + C content 36.4%) when compared with the whole gene region (42.3%). The lower G + C content may have favored an L1 insertion as these elements have a bias towards lower G + C regions of the genome . Second,6.4% whenfamilies . TherefoSMN1 isoform transcripts could explain why these transcripts are less common than their shorter counterparts. Interestingly, weakly expressing genes were found to be rich in LINE insertions what can be explained by the ability of L1 elements to disrupt transcriptional elongation based on the presence of strong polyA signals in their sequences that possibly function as transcriptional terminators [SMN1/2 reported in this study, are only moderately selected against and may provide a gradual mechanism of evolution by which retrotransposons alter the expression profile and influence crucial gene networks in the human genome [3′UTR retrotransposon insertions reduce mRNA expression ,67,79. Tminators . We arguminators ,82. 3′UTn genome ,67,83.SMN2 as it shares the same TE insertional patterns in its sequence with SMN1. Accordingly, the longest SMN2 transcript isoform presents this L1 insertion in its 3′ region, in an identical sequence position as in SMN1 longest transcript. This hints to this retrotransposon having the same alternative terminator role in SMN2 transcription to that in SMN1 transcription. We hypothesize that this alternative terminator role can have implications in SMA severity by reducing SMN2 mRNA expression due to the presence of this L1MC5a element within SMN2. The confirmation of this hypothesis will require further analysis using a combination of experimental and in silico methodologies.Additionally, it is possible that this L1MC5a element has the same transcription terminator function in SMN1 reference sequence explains the high levels of circularization of SMN1/2 transcripts as Alu repeats located in introns 4 and 5 are especially active in this process [SMN1/2 introns may explain how these genes generate several circular RNAs, we believe that circRNA biogenesis occurs in healthy individuals and SMA patients to the same extent. It is possible, however, that dysregulation of these Alu repeats in SMA-affected genomes may lead to an increased formation of circRNAs coded by SMN1. These higher levels of circRNAs formation and the widespread alternative circularization of SMN1/2 pre-mRNA may have a still undiscovered role in SMA onset or may contribute to worse SMA phenotypes, owing to circRNAs ability to interfere with the coding capacity of human genes [SMN1/2 may function as a potential biomarker for the genes’ overall transcriptional/splicing stability since higher circRNAs levels indicate aberrant RNA splicing events that may be linked to SMA.Circular RNAs (circRNAs) are a widely expressed class of non-colinear RNAs generated in a diverse set of eukaryotic organisms . Due to  process ,53,58. P process . Additio process . In futu process . The prean genes ,58,84. ASMN genes, exon 6B [SMN1 occurred among Alu elements of the (S) subfamily supporting the idea that sequence identity between the two elements at a locus—alongside proximity—appear to be proportional to their chances of successful recombination [SMN1/2 canonical exons. In a wider spectrum, Alu and other TEs provide transcriptome diversity and ultimately result in the diversification of the human proteome.The two exonization events within the  exon 6B ,57 and e exon 6B , have bebination ,90,91. TSMN1 gene, we cannot draw definitive conclusions about the involvement of Alu elements in the deletion events in a disease context. Our results show that in the analyzed SMA patients and in the remaining samples, the critical Alu elements responsible for these deletions are also present in the same position and orientation in the SMN1/2 sequence as in the gene reference sequence and as described previously [per se is not the reason for the deletion events. Their presence in SMN1/2 introns is a source of sequence homology that can be responsible for genomic rearrangements and consequently disease in some genomes [de novo mutations in SMN1 and are not inherited from carrier parents [SMN1 are responsible for de novo deletions in germinative cells due to unequal recombination since TE silencing mechanisms are often relaxed in these developmental stages [SMN1/2 genomic region leading to disease-causing deletions of SMN1 exons and potentially whole gene deletions under specific circumstances. Alu elements invasion of SMN1/2 makes these genes very susceptible to Alu-mediated deletions, that have critical consequences to genome stability and host health.Some insertional polymorphisms that were observed in these Alu repeats between the analyzed samples may be the result of normal interpersonal sequence variability. Accordingly, polymorphic Alu elements account for 17% of structural variants in the human genome, clearly establishing a link between individual TE polymorphisms and human genetic variation . Since teviously ,37,38. A genomes . TE reco parents . It is ll stages ,19. Alu SMN genes revealed a pervasive invasion of its sequence by TEs that we believe may severely impact these genes’ regulation structure, expression and overall genomic stability. The several TEs present inside these genes, especially Alu and L1 elements that are highly enriched in the promoter and intronic regions of the gene, seem to play important roles in gene expression, novel exon creation, alternative splicing and deletion events known to lead to SMA. Additionally, a L1 element insertion in the 3′UTR region of the gene is also responsible for a domestication event that gave the gene an alternative terminator, therefore increasing the diversity of SMN1 transcripts and being a prime example of how a TE insertion inside a protein-coding gene can create a gradual mechanism of evolution by which retrotransposons alter the human transcriptome.Our analysis of the SMN1/2 completed in this work serves as a starting point for further investigations on the impacts of TEs in human disease and particularly, their role in SMA onset and severity. While the TEs identified in this in silico analysis were present in both SMN1 and SMN2, it is possible that they may affect the regulation of these genes differently. Future studies will further characterize the effects of these TEs in SMN1 and SMN2 on gene regulation under healthy conditions as well as in SMA.The in silico analysis of


B1 has a strong inhibitory effect on the melanoma cell line A357 at 100 µM (76.15% inhibition).A series of novel triazoloquinolinone and imidazoquinazolinone derivatives were designed and synthesised, and their biological activities against SHP2 protein and melanoma A357 cell line were evaluated in vitro. The results show that some target compounds have moderate to excellent inhibitory activity on SHP2 protein and melanoma A357 cell line. Structure-activity relationships (SARs) showed that both imidazoquinazolinone and triazoloquinazolinone derivatives have good SHP2 protein kinase and melanoma cell line A357 inhibitory activity. The results of molecular docking also showed that the cores of imidazoquinazolinone and triazoloquinazolinone have a certain affinity for SHP2 protein at the same time. Compared with SHP244, the target compounds have quite good liver microsomal stability and has more drug potential. The most promising compound