In plants, 4-coumarate:coenzyme A ligases (4CLs), comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species  were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS) genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR). We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes. Populus), Arabidopsis thaliana, maize (Zea mays), and rice (Oryza sativa) [Solanum tuberosum) and loblolly pine (Pinus taeda L.) are almost identical [Populus tremuloides), soybean (Glycine max), california poplar (Populus trichocarpa), and tobacco, are structurally divergent [The 4-coumarate:coenzyme A ligases (4CLs) play key roles in generating Coenzyme A (CoA) esters during the hydroxycinnamic acid production step of phenylpropanoid metabolism. The general phenylpropanoid pathway channels carbon flow into diverse branching pathways of secondary phenolic metabolism and generate various classes of phenolic secondary natural products, including flavonoids, lignin, suberins, and coumarins, which play important roles in plant development and environmental interactions . Accordi sativa) ,3,4,5. Mdentical ,7, whereivergent ,9,10,11.Malus domestica, Oryza sativa, Prunus persica, Pyrus bretschneideri, Prunus mume, and Arabidopsis, some genes encoding adenylate-forming enzymes are closely related to true 4CLs, but have no known specific biochemical function. These enzymes may function in the biosynthetic pathways of various metabolic and natural products in plants. For example, based on the phylogenetic relationship of 4CLs in Arabidopsis, four genes with distant similarity to true 4CL genes were identified [Arabidopsis 4CL-type adenylate-forming enzymes were classified as 4CL-like genes [The differential expression profiles of 4CL genes suggest that these genes, which function in the formation of different classes of benzene propane compounds, have undergone subfunctionalization ,12. As mentified . Subsequke genes . These 4ke genes ,16. Ehltke genes  identifike genes ,18,19.Arabidopsis 4CL-like gene (GenBank no. AEE29981) was found to encode a peroxisomal 12-oxo-phytodienoic acid, a CoA ligase related to jasmonic acid biosynthesis, which has since been designated OPCL1 [Pyrus bretschneideri Rehd.), yangmei (Prunus mume), peach (Prunus persica), and apple  in this report. Pears are among the most economically important fruit crops worldwide. Previous studies revealed that the stone cell content is an important factor impacting the taste of the fruit flesh in pears [Arabidopsis [Pb4CL1 gene related to lignin synthesis in the Chinese pear will help to improve the quality of pear fruit in the future.In contrast to true 4CLs, functions of 4CL-like genes are not related to flavonoid or lignin biosynthesis ,17, sugged OPCL1 ,24. Howein pears . Pear stin pears ,26. Accobidopsis , no studbidopsis , yangmeibidopsis , and peabidopsis  based onAEE, ACS, and 4CL, with species-specific identifiers used to distinguish among genes  [Pyrus bretschneideri Rehd. and the other species, including Arabidopsis, Prunus mume, Prunus persica, Malus x domestica, and Oryza sativa were downloaded from the Plant Genome Duplication Database [A map of the chromosome locations of 4CL-related genes in pear was produced using MapInspect software  based on their starting positions. A diagram of the exon/intron structures of these pear 4CL-related genes was generated using the online tool GSDS  based onitation) . ParametDatabase  and werecis-acting regulatory DNA elements in the promoter regions of the 4CL-related genes in the pear.To analyze the promoter regions of these genes, the upstream sequences of the 4CL-related genes were analyzed based on the positions of the genes provided in the pear GigaDB database . The PLAPb4CL/PbACS genes, pear fruits were sampled at 15, 39, 47, 55, 63, 79, 102, and 145 days after flowering (DAF). At least three fruits were harvested at each stage from 40-year-old pear trees grown in a horticultural field in Dangshan, Anhui, China. Total RNAs were extracted from the samples using Trizol reagent  according to the manufacturer’s instructions. DNase-treated RNA was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen). Gene-specific primers for quantitative reverse-transcription PCR (qRT-PCR) were designed to generate 80–200 bp products using Beacon Designer 7 software , and the tubulin gene [−ΔΔCT method [To investigate the expression patterns of T method . The reaThe experiments did not involve endangered or protected species. No specific permits were required for these locations/activities because the pears used in this study were obtained from a horticultural field in Dangshan, which were demonstration orchards at Auhui Agricultural University.Arabidopsis and Oryza sativa microarray data were downloaded from the ArrayExpress [Arabidopsis and Oryza sativa.Affymetrix yExpress , PLEXdb yExpress  and RiceyExpress . HierarcyExpress  to analyPrunus persica) [Malus x domestica) [Prunus mume) [Pyrus bretschneideri) [Oryza sativa and Arabidopsis (AtACN1 acts as an entry point to the glyoxylate cycle during seed germination [Using the sequence of the AMP-binding domain obtained from the Pfam database  as a quepersica) , apple (mestica) , yangmeius mume) , and peaneideri) . As a rebidopsis . The unrbidopsis . These amination . Arabidopsis ACN1 gene (AtCN1) [Arabidopsis genes for acyl-activating enzymes (AAEs) [Arabidopsis AAE gene (AtAAE) [Arabidopsis and Oryza sativa proteins represented in the bona fide 4CL clade have been described and annotated [PbACS genes are not present in cluster A. We propose that the gain or loss of some genes over the course of evolution may have helped the plant adapt to the environment. To further clarify the function of the 4CL genes in the pear, a phylogenetic tree was constructed for 4CL genes from the six species using the neighbor-joining method. Based on previous clustering approaches [Pb4CL1 and Pb4CL3 are primarily involved in lignin biosynthesis in the pear, whereas Pb4CL2 and Pb4CL3 are primarily involved in flavonoid biosynthesis.The large clade includes both  (AtCN1)  and manys (AAEs) . These As (AAEs) . In this (AtAAE) . The othnnotated ,3,4,48. proaches ,48, thesproaches ,48,50,51PbAEE5/PbAEE16.An unrooted phylogenetic tree containing all 4CL-related genes in the pear was constructed  using thPbAEE9 and PbACS2 having no introns, and PbAEE1 and PbAEE17 contains most introns (17) among the pear 4CL-related gene family. In addition, our analysis of exon/intron structure supports the phylogeny reconstruction. The 4CL-related genes within the same groups displayed similar exon/intron structures, particularly in terms of the number of introns, although there were some exceptions. For example, in group V, PbACS2 lacks introns, whereas PbACS7 and PbACS8 contain two introns. In addition, the intron length ranges from several bases to approximately 35,500 bases, which demonstrates the high sequence diversity among members of the 4CL-related gene family.The gene structures of the pear 4CL-related genes were drawn by GSDS  by compaArabidopsis and Oryza sativa genes, we can infer the functions of 4CL-related genes in the pear to some extent. To further investigate the origin and evolutionary process of pear 4CL-related genes in Rosaceae species, we performed a comparative analysis of the genomes of the pear, peach, and yangmei. Based on the plant genome duplication event database [Prunus persica, Pyrus bretschneideri, Prunus mume, Arabidopsis, and Oryza sativa  could not be conclusively mapped to these chromosomes because they were localized to scaffolds, which may have resulted from sequencing anomalies. The number of 4CL-related genes per chromosome varies widely. Chromosome 9 contains the largest number of pear 4CL-related genes (five), followed by chromosome 17 (four). Each of chromosomes 1, 2, 3, 6, 10 and 16 has only one 4CL-related gene. Relatively high densities of 4CL-related genes were found on the tops of chromosomal regions, such as chromosome 9. According to Holub’s definition of a gene cluster [In almost all Rosaceae plants, one or multiple genome duplication events have occurred over the course of evolution ,30,57. T cluster , we founPbAEE16/PbAEE5 on chromosome 9 has undergone tandem duplication, whereas nine gene pairs  have undergone segmental duplication (PbACS3/PbACS4) is located just outside the segmental duplication regions. However, their phylogenetic relationships and gene structures suggest that these genes have a similar evolutionary history, indicating that this gene pair has indeed undergone segmental duplication. We therefore assert that this gene pair has undergone gene fragment duplication  in the promoter sequences, the four sister pairs  were found to exhibit significant differences in their promoter regions, suggesting that the duplicated genes may not have some regulatory features in common, but may instead function in similar regulatory pathways. For example, each gene in these duplicated gene pairs contains at least one ABRE element in its promoter sequence.We also analyzed the promoter sequences in a 1500 bp-region upstream of the transcription start site (ATG) of the predicted ts, ABRE ,63, LTREts, ABRE , BOXP [6ts, ABRE , and BOXts, ABRE , were dets, ABRE . SurprisPbACS2 and PbACS6 in clade B, and Pb4CL3 in bona fide 4CLs were significantly increased at 15 DAF , were similar. However, the expression patterns of a few genes from the pear were different from those of Arabidopsis in the same cluster. For example, the expression of AtACS6 (in cluster C) was relatively high during each period, whereas the expression of PbACS2 was low beginning at 15 DAF , instead of in other tissues , no systematic analysis of these families has previously been reported in the Rosaceae. Using DNATOOLS software, we identified 149 4CL-related genes in four Rosaceae plants, the peach, yangmei, apple, and pear. We classified these genes and analyzed their evolutionary relationships (and those of rice and Arabidopsis), as well as their physical locations, promoter regions, collinearity, gene structures and expression patterns using qRT-PCR analysis. The results of this study provide a characterization of the 4CL-related gene family in pear and the phylogenetic relationships of 4CL-related genes among Prunus persica, Malus x domestica, Prunus mume, Oryza sativa, Pyrus bretschneideri, and Arabidopsis. To further explore the role of these genes in the pear, we also analyzed the expression profiles of several 4CL and ACS family genes in the pear, as well as those in Arabidopsis and Oryza sativa, in various tissues and during different developmental stages. The results suggest that only Pb4CL1 plays a key role in lignin biosynthesis and metabolic pathways in pear fruit. Our results clarify the biological function of 4CL/ACS genes in pear development and have a significant influence on our knowledge of woody plant 4CL-related genes.4CL enzymes participate in the regulation of lignin and flavonoid biosynthesis in plants. Proteins encoded by ACS genes may catalyze reactions involving different fatty acids or other acyl substrates. These genes are present in gene families in plants. Currently, although 4CL-related gene families were identified and characterized in several model plants (


AbstractQianotrechustenuicollis Uéno, 2000, was newly discovered in Cave Mahuang Dong of Shuanghe Dong cave system, the longest cave system of China in Suiyang County, northern Guizhou Province. To categorize this striking but still unknown species, a new genus and species are proposed: Shuangheaphaenopselegansgen. n., sp. n. Relationships of Shuangheaphaenops and other highly modified aphaenopsian genera from southern China Karsts are discussed.A remarkable aphaenopsian beetle, a sympatric species of  PageBreaksouthern Guizhou and northern Guangxi, such as Sinaphaenops Uéno & Wang, 1991, Dongodytes Deuve, 1993, Giraffaphaenops Deuve, 1992, Pilosaphaenops Deuve & Tian, 2008 and Uenotrechus Deuve & Tian, 1999  and Huanjiang (northernmost Guangxi) counties , but the hind part (elytra) is more likely similar to a Dongodytes (s. str.) species. It has three pairs of supraorbital setiferous setae on head and lacks lateromarginal setae on pronotum. It is also different from Sinaphaenops Uéno & Wang although its first and second male protarsomeres are dilated and spurred inward apically, a sexual modification appeared also in some Sinaphaenops species. Far more important, its elytral chaetotaxy is not similar to any other aphaenopsian genera mentioned above. Therefore, this interesting beetle represents a peculiar lineage within Chinese hypogean trechines. Here we describe this remarkable species, the first highly cave-adapted aphaenopsian species from northern Guizhou Province.In October, 2016, a cave biodiversity survey was carried out by our team in several counties of Zunyi and Tongren Districts, northern and northeastern Guizhou, in order to collect species of the genera As the longest cave system in China, Shuanghe Dong is connected by 42 caves or entrances, with total length coming up to 186.33 km . But theSCAU).The single blind beetle and the elytral debris for this study were collected by the naked eye using an aspirator inside the cave Mahuang Dong, and kept in 50% ethanol before study. Other cave beetles used for comparing were dry and mounted specimens of the insect collection of South China Agricultural University, Guangzhou, China  or from labrum to the elytral apex; the width of the body was taken as the maximum width of the elytra.Abbreviations of other measurements used in the text are as follows: length of head including mandibles, from apex of right mandible to neck constriction HLm length of head excluding mandibles, from front of labrum to occipital suture HLl maximum width of head HW length of prothorax, along the median line PrL length of pronotum, as long as PrL PnL maximum width of prothorax PrW maximum width of pronotum PnW width of pronotum at front PfW width of pronotum at base PbW length of elytra, from base of scutellum to elytral apex EL maximum width of combined elytra EWTaxon classificationAnimaliaColeopteraCarabidaeGenushttp://zoobank.org/0E7CBC8A-C92E-426F-A331-93156EE2B0A8Shuangheaphaenopselegans sp. n.Uenotrechus and Dongodytes species, presence of three pairs of supraorbital setae on head, two dorsal and preapical pores on elytra, the first and second protarsomeres in male distinctly modified.Large sized blind beetles, fore body evidently elongated and as long as elytra, shape intermediate between Uenotrechus Deuve & Tian, 1999, while hind part (elytra) to Dongodytes Deuve, 1993; large sized, with body and appendages thin and very elongate, fore body almost as long as hind part; three pairs of supraorbital setiferous pores present on head, with the posterior two pairs very close to each other; mandibles thin and elongated, feebly curved apically, longer than width of head, right mandible edentate though two vanished teeth can be faintly traced; labial suture PageBreakmoderately defined, separating of mentum and submentum, with the former bisetose and the latter 6-setose; mental tooth simple and thin, basal foveae quite narrow; antennae very long, the 10th and 11th antennomeres extending over apical margin of elytra. Prothorax dolioform, propleura distinctly tumid at basal half, evidently visible from above; pronotum barrel-shaped, distinctly elongated, longer than head excluding mandibles, narrower than head; without lateromarginal setae. Elytra similar to those of Dongodytes (s. str.) Deuve, 1993, narrowed anteriorly and dilated posteriorly, side margins narrowly bordered throughout, shoulders lacking; striae lacking though easily traceable; presence of two dorsal and preapical setiferous pores; the 1st pore in the humeral group of the marginal umbilicate series not transversely and backwardly shifted, the 5th and 6th pores in the middle group widely spaced. Protibia smooth, without longitudinal sulcus; the 1st and 2nd protarsomeres in male dilated and inwardly spurred at apices. Abdominal ventrites sparsely pubescent, each of ventrites IV-VII in male bisetose apically. Male genitalia moderately sclerotized, small, strongly curved ventrally in lateral view, with a quite large sagittal aileron; apical lobe very thin in dorsal view; parameres well developed, but much shorter than median lobe.Highly modified aphaenopsian trechines, fore part (head and thorax) of the body somewhat similar to Shuangheaphaenops can not be included in any lineage of the highly modified aphaenopsian genera known in southern China regarding to the peculiar morphological characteristics mentioned above, such as the peculiar facies and configuration of the body , vanished bidentate right mandible, and chaetotaxal patterns in which there are three pairs of supraorbital setiferous pores on head, lack of lateromarginal setae on pronotum, and unique pattern on elytral marginal umbilicate series, in particular, the humeral and middle groups.Shuangheaphaenops and Dongodytes (s. str.) which occurs only in northern Guangxi where is far distant from Cave Shuanghe Dong in northern Guizhou, the following characteristics are different: (1) head subparallel-sided, with three pairs of supraorbital setiferous pores, right mandibular teeth bidentate but almost vanished in Shuangheaphaenops ; (2) the 1st and 2nd protarsomeres of male distinctly modified in Shuangheaphaenops (indistinctly or not modified in Dongodytes); (3) pronotum much slender and lack of lateromarginal setae in Shuangheaphaenops ; and (4) the middle group (the 5th and 6th pores) of the marginal unbilicate series on elytra widely spaced each other in Shuangheaphaenops (versus close to each other in Dongodytes).Apart from the similarity in elytra and antennae between Uenotrechus Deuve & Tian, 1999, but Shuangheaphaenops has a slenderer head bearing three pairs of supraorbital setiferous pores, reduced bidentate teeth of right mandible, and much longer antennae , and pronotum without lateromarginal setae . In addition, head and elytra are glabrous in Shuangheaphaenops (versus PageBreakwhole body densely pubescent in Uenotrechus), the 1st pore of the marginal umbilicate series is located before the 2nd in Shuangheaphaenops , and both the 1st and 2nd protarsomeres in male are modified in Shuangheaphaenops (versus not modified in Uenotrechus).The fore body of this new genus is more or less similar to that of “Shuanghe + Aphaenops”. To indicate that the highly modified trechine genus occurs in Shuanghe Dong, the longest cave system in China.Guizhou (Suiyang) Fig. .Taxon classificationAnimaliaColeopteraCarabidaehttp://zoobank.org/392C6535-5261-47FF-A2F7-D7123A5D9DFA28°14'32"N, 107°17'24"E, 720 m, X-18-2016, leg. Wenbo Li, deposited in the insect collection of South China Agricultural University, Guangzhou, China (SCAU); additional material: an elytral debris, same cave and collecting date as above, leg. Mingruo Tang, in SCAU.Male, Cave Mahuang Dong, Shuanghe Dong cave system, Wenquan Zhen, Suiyang County, A large-sized, eyeless cave trechine beetle, highly modified in morphology, with very elongated and slender body which is about four times longer than wide, fore body about as long as elytra, antennae as long as body including mandibles, extending beyond elytral apex; body glabrous, except for basal half of pronotum which is covered with erected setae.HLm+PrL)/EL = 1.07. Habitus as in Fig. Length: 7.9 mm  or 7.2 mm (from labrum to elytra); width: 1.79 mm. Fore body (including mandibles) longer than elytra,  but pubescent on basal half. Underside of head and prothorax glabrous Fig. , abdominHLm/HW = 2.74, HLl/HW = 1.87); genae fairly developed, hardly dilated laterally, suddenly constricted posteriorly before occipital suture, making a well-marked but short neck constriction; subparallel-sided, widest at about middle from labrum to base of head; frons and vertex moderately convex, frontal furrows deep and well-marked, subparallel-sided, ended just behind the level of anterior supraorbital pores; clypeus transverse, 4-setose; labrum transverse, with frontal margin slightly protruding medially, 6-setose; anterior supraorbital setiferous pores located at about middle from frontal margin of labrum to base of head, while the posterior two pairs (which are very close to each other) at about 1/4 of head from base; palps long, slender and glabrous except for the 2nd labial palpomere PageBreakwhich is bisetose on inner margin; the 2nd labial palpomere 1.35 times longer than the 3rd; the 3rd maxillary palpomere 1.25 times longer than the 4th; suborbital pores at about middle from base to labial suture. Antennae thin and long, the 1st antennomere stouter than other, and the shortest, the 3rd the longest; the comparative length ratio of each antennomeres as: the 1st (8.5), 2nd (11.0), 3rd (18.0), 4th (17.0), 5th (17.0), 6th (16.5), 7th (13.5), 8th (11.0), 9th (11.0), 10th (10.0) and 11th (10.5).Head Fig.  elongatePrL/HLm = 0.80), but longer than head excluding mandibles (PrL/HLl = 1.17), widest at about 1/4 from base, twice as long as wide (PrL/PrW = 2.10), slightly wider than head (PrW/HW = 1.09), evidently wider than pronotum (PrW/PnW = 1.22), half as wide as elytra (PrW/EW = 0.49). Pronotum elongate, dolioform, two and half times longer than wide (PnL/PnW = 2.46), evidently narrower than head (PnW/HW = 0.89), base slightly wider than front (PbW/PfW = 1.09); lateral sides finely bordered throughout, base and front unbordered; nearly parallel-sided, fairly expanded at the widest part which is at about 3/7 from base, slightly sinuate before hind angles which are nearly rectangular, fore angle obtuse; median line well-marked, basal transversal impression very short; front slightly convex, base feebly concave. Scutellum fairly small.Prothorax shorter than head including mandibles (EL/PnL = 2.12), about twice as long as wide (EL/EW = 2.08); distinctly dilated posteriorly, widest at about apical 3/7 of elytra, lateral sides smooth, not ciliate, finely bordered throughout; distinctly convex; striae easily traceable though devoid, intervals moderately convex. Chaetotaxy: basal pore at subequal distant from scutellum to marginal gutter; two dorsal setiferous pores present on the 3rd stria at about 1/3 and 2/3 from base respectively; the preapical pore closer to suture than to apical margin; the marginal umbilicate pores not aggregated, the 8th pore near marginal gutter, the 4th and 5th pores distant from the gutter, others intermediary located; the humeral groups separately spaced, the 1st and PageBreakPageBreak4th pores distant from the 2nd and 3rd pores respectively which are close to each other, distance from the 1st pore to the 3rd slightly shorter than that from the 2nd to 4th; the middle group widely spaced, making distance of the 5th pore and 4th subequal to that of the 5th and 6th; apical group composed of three pores.Elytra Fig.  elongatest tarsomere shorter than, as long as, and longer than the 2nd–4th tarsomeres together in fore, middle, and hind legs, respectively.Legs thin and long, bearing short pubescence; fore- and middle femora sparsely setose; fore tibia smooth, without longitudinal furrow or sulcus; the 1PageBreakPageBreakMale genitalia Fig. : AedeaguPageBreakTo indicate the slender shape of this beautiful aphaenopsian beetle.China (Guizhou: Suiyang) Fig. . Known oQianotrechustenuicollis Ueno, 1998, an amphipod, a cave cricket (Figs Mahuang Dong Figs  is one oket Figs , a pseud


ST14 gene, encoding the serine protease matriptase, have been associated with ichthyosis-hypotrichosis syndrome (IHS), a Mendelian disorder with skin and hair manifestations which include, in addition to ichthyosis and hypotrichosis, hypohidrosis and follicular atrophoderma. However, the understanding of the specific consequences of mutations in ST14 on the development of this syndrome is incomplete.Mutations in the ST14 was identified in a patient with IHS. This mutation affects an essential site within a ligand-binding domain of matriptase. Comparison with previous reports of IHS allowed further delineation of the phenotype of IHS in correlation with mutations present in these patients. Histological and ultrastructural analysis of skin and hair identified novel features in this disorder.Using a targeted next-generation sequencing array of 38 ichthyosis-associated genes on a large cohort of 180 ichthyosis patients from a primarily consanguineous background, a previously unreported homozygous p.Asp482Asn mutation in This study correlates genotypic and phenotypic features of the rare disorder, IHS, expands the spectrum of pathology associated with the disorder, and provides clinical evidence of the importance of the Asp482 amino acid, previously shown to have an essential role in matriptase activation. The inherited ichthyoses in the spectrum of Mendelian disorders of cornification affect the generalized integument with hyperkeratosis and scaling . The inh, ice pick-like depressions in the place of follicular units [The phenotype of IHS exists in a spectrum. The manifestations of ichthyosis are typically mild to moderate, but have been reported as severe in one case . Universar units –4. This ar units . Additioar units , pinguecar units , and pitST14, encoding the serine protease matriptase, have been reported in IHS since 2007, and up to date, six distinct mutations in this gene have been associated with IHS [ST14 at a functionally significant site of the protein, with a comprehensive clinical analysis, thus providing improved characterization of the genotypic and phenotypic features of IHS.Biallelic mutations in with IHS . Here, wThis study was approved by the institutional review board of the Pasteur Institute of Iran, and all subjects and the parents of minors gave written informed consent to participate in research and to publish their images. In this study, 180 extended families affected by nonsyndromic and syndromic forms of ichthyosis, diagnosed in various medical centers in Iran, were evaluated.ABCA12, ABHD5, AGPS, ALDH3A2, ALOX12B, ALOXE3, AP1S1, ARSE, CERS3, CLDN1, CYP4F22, EBP, ELOVL4, GJB2, GJB3, GJB4, GJB6, KRT1, KRT10, KRT2, KRT9, LIPN, LOR, NIPAL4, PEX7, PHYH, PNPLA1, PNPLA2, POMP, SLC27A4, SNAP29, SPINK5, ST14, STS, TGM1, TGM5, VPS33B, and ZMPSTE24), divided into 351 targets covered by 558 amplicon probes, which were designed to cover 99% of targeted bases. A total of 93.2% of the reads were aligned to the human genome, with the mean coverage of the target region being 493×. In addition, only 0.4% of bases of the target region were not covered by any sequence read, indicating that 99.6% of all target region bases were sequenced at least once.DNA was extracted from peripheral blood samples by salting-out method. Target enrichment was performed using the TruSeq Custom Amplicon kit . DesignStudio (Illumina) was used for library design. All coding exons, at least 20 bp of the intron at each intron-exon boundary, and up to 50 bp of 5′- and 3′-untranslated regions were targeted. The designed library contained 38 genes (broadinstitute.org), and predictive softwares were used to analyze the consequences of missense mutations including MutationTaster (mutationtaster.org), Sift v.1.03 (sift.jcvi.org), PROVEAN (provean.jcui.org), and PolyPhen-2 (genetics.bwh.harvard.edu).Reads were analyzed and aligned as previously described . VariantPCR was performed using Taq polymerase  according to the manufacturer’s instructions, with custom-designed primers. The PCR products were bidirectionally sequenced using an automated sequencer . Mutation positions are reported in reference to NM_021978.Hair samples obtained from the scalp of the proband were mounted on scanning electron microscopy sample holders, sputter coated with gold/palladium alloy, and imaged using a FEI FEG Quanta 250  operated at 5 kV. Multiple hairs were examined, and consistent findings were noted.The index case, an Iranian male of Persian decent, was born to consanguineous parents Fig. . No collHematoxylin and eosin staining of a 4 mm punch biopsy from flank skin revealed irregular acanthosis, mild basal layer hyperpigmentation, diminished granular layer, and mild basket weave hyperkeratosis extending focally to hair follicles associated with keratotic follicular plugging Fig. . ScanninST14 by using the NGS array, and the results were confirmed with bi-directional Sanger sequencing  array targeting 38 ichthyosis-associated genes . Among these patients tested, the proband in this case was found to have a homozygous missense mutation, c.1444G > A (p.Asp482Asn), in ing Fig. . UnfortuST14, expanding the genotypic spectrum of this syndrome. In likeness to the clinical presentation of previous IHS cases, our patient had ichthyosis and typical hair findings  an N-terminal transmembrane domain, (b) a sea urchin sperm protein-enteropeptidase-agrin (SEA) domain containing a cleavage site required for proteolytic activation of matriptase, (c) two adjacent C1r/C1s-urchin embryonic growth factor bone morphogenic protein (CUB) domains required for efficient N-terminal proteolytic processing, (d) four low density lipoprotein-receptor class A (LDLR-A) domains in tandem, and (e) the catalytic domain which encodes the serine protease portion of matriptase [ST14 in association with IHS. One abrogates the start codon, two are missense mutations, one is a frameshift deletion, and two are canonical splice site mutations  [The matriptase protein encoded by the triptase  Fig. 2)ST14 gene binding . Remarka binding . The spe 1G > A)  (Table 2ST14. This is the seventh mutation associated with IHS, with a unique combination of clinical and ultrastructural findings. This report provides corroborative evidence that the LDLR-A domains of ST14 are essential for matriptase function, particularly at its highly conserved aspartic acid residues.We report a case of IHS with a homozygous, previously unreported missense mutation in


SOX17 , CDO1 , ZFP42  and TAC1 . Conclusions: The present meta-analysis demonstrates that methylated SOX17, CDO1, ZFP42, TAC1, FAM19A4, FHIT, MGMT, p16, and RASSF1A are potential superior biomarkers for the screening and auxiliary detection of lung cancer.Background: DNA methylation in sputum has been an attractive candidate biomarker for the non-invasive screening and detection of lung cancer. Materials and Methods: Databases including PubMed, Ovid, Cochrane library, Web of Science databases, Chinese Biological Medicine (CBM), Chinese National Knowledge Infrastructure (CNKI), Wanfang, Vip Databases and Google Scholar were searched to collect the diagnostic trials on aberrant DNA methylation in the screening and detection of lung cancer published until 1 December 2016. Indirect comparison meta-analysis was used to evaluate the diagnostic value of the included candidate genes. Results: The systematic literature search yielded a total of 33 studies including a total of 4801 subjects (2238 patients with lung cancer and 2563 controls) and covering 32 genes. We identified that methylated genes in sputum samples for the early screening and auxiliary detection of lung cancer yielded an overall sensitivity of 0.46 (0.41–0.50) and specificity of 0.83 (0.80–0.86). Combined indirect comparisons identified the superior gene of  Lung cancer is the leading cause of malignant tumor death, with the morbidity and mortality of lung cancer gradually increasing over the past decades . Only 13p16 [RASSF1A [APC [MGMT [DAPK [RARβ [With the emergence and development of molecular epidemiology, which opens the “black box” of the disease process, molecular biomarkers have much potential to improve the understanding of the occurrence, development, and prognosis of disease for the early detection of these lesions at the pre-invasive stage, even predicting the progress of the disease ,5. Nowadp16 , RASSF1A[RASSF1A , APC [10PC [MGMT , DAPK [1MT [DAPK  and RARβPK [RARβ . Many stPK [RARβ . The devPK [RARβ ,16 have However, the DNA methylation of specific genes in sputum samples do not yet provide a convincing and superior gene panel with high sensitivity and specificity for clinical utility in lung cancer. In addition, there was still no comparative evaluation on the diagnostic accuracy of methylation in sputum samples for the screening and detection of lung cancer. Recently, network meta-analysis has been developed to assess the comparative effectiveness of several interventions and to synthesize evidence across a network of studies ,18. ThisWe followed the reporting guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement when conducting this review. The PRISMA statement has guidelines that include a four-phase flow diagram to systematically guide the inclusion and exclusion of research papers . In addiWe conducted a comprehensive literature search in PubMed, Ovid, Cochrane library, Web of Science databases, Chinese Biological Medicine (CBM), Chinese National Knowledge Infrastructure (CNKI), Wanfang, Vip Databases, and Google Scholar. The main search terms included: lung cancer or lung carcinoma or non-small cell lung cancer or “NSCLC”, “sputum or flema”, “diagnostic”, “sensitivity and specificity” and “methylation or hypermethylation or hypomethylation or demethylation”. All articles published until December 2016 were considered. In addition, the reference lists of all identified studies were manually searched to identify any additional studies.The articles, which could not be excluded based on the title and abstract, were retrieved for full-text review. Studies were included in this meta-analysis if they met the following criteria: (1) the diagnostic potential of sputum DNA methylation for lung cancer; (2) study design being case-control; (3) the patients being diagnosed with lung cancer by pathology; (4) provided data on the numbers of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN); (5) the methods of detecting methylation based on methylation-specific polymerase chain reaction (MSP) or quantitative methylation-specific PCR (qMSP).Studies were excluded from the meta-analysis for the following reasons: (1) abstracts, letters, reviews, expert opinions, case reports, or nonclinical studies; (2) studies had duplicate or overlapping data; (3) study was based on tissue, blood, or animals.Data extraction was conducted in duplicate by two investigators (Di Liu and Hongli Peng) based on title, abstract, author, year of publication, country of origin, sample size, assay methods, and diagnostic performance (sensitivity (SEN), specificity (SPE), TP, FP, FN, TN), target gene(s), and the score of the quality assessment of studies of diagnostic accuracy (QUADAS)  and the STARD and QUADAS guidelines were utilized to assess the methodological quality of each study. There are 25 items in the STARD initiative checklist, and a score of 1 was given when the item was yielded . FourteeI2 statistic, which measures the extent of inconsistency between studies, was also assessed [We used standard methods recommended for the direct meta-analysis which estimated the diagnostic test evaluation of DNA methylation compared with the gold standard . The numassessed . Spearmaassessed . Analysep-value of less than 0.05 was considered significant.For indirect comparisons, the comparative diagnostic accuracy of all biomarkers was estimated according to common comparator (the gold standard). We did not assume consistency  of two biomarkers without direct analyses. We took the step-wise approach, which was suitable for the simple star-network meta-analysis to obtain an indirect analysis. The Deeks’ test and Egger’s test were utilized to estimate the funnel plot asymmetry and the publication bias . The indP16, RASSF1A, APC, MGMT, PAX5, CGB, GATA, DAPK, RARβ, MAGE, HOXA, 3OST2, PRDM14, FAM19A4, PHACTR3, PCDH20, CXCL, Dal-1, Dab2, Dcr2, SULF2, Kifla, Jph3, SOX17, CDO1, ZFP42, TAC1, CDH1, H-cadherin, FHIT, RASSF2, TCF2l).The flowchart of included studies was presented in ZFP42: 0.87 (0.78–0.93), TAC1: 0.86 (0.77–0.92), SOX17: 0.84 (0.75–0.91), FAM19A4: 0.80 (0.74–0.85), and HOXA: 0.79 (0.63–0.89); whereas the higher of PLR were SOX17: 6.76 (2.34–19.54), FHIT: 5.93 (2.29–15.36), RASSF1A: 5.61 (3.73–8.43), MGMT: 4.78 (1.47–15.55), and p16: 4.71 (2.53–8.78).As shown in The graph of the SROC (summary receiver operating characteristic) cure is shown in SOX17, TAC1, ZFP42, CDO1 differed significantly from the other 28 methylated genes and were higher than 1, indicating that SOX17, TAC1, ZFP42, and CDO1 have a higher diagnostic accuracy. More information is shown in the SOX17 , CDO1 , ZFP42 , TAC1  (y: 0.75) .p < 0.001), indicating the heterogeneity of threshold effect. We also investigated the non-threshold effects; the results indicated the existence of significant heterogeneity in the overall sensitivity , specificity , PLR , NLR , and DOR . Therefore, a bivariate binomial mixed model was applied to summarize the pooled estimates in this study. To determine the sources of heterogeneity, we performed meta-regression to test the effect of ethnicity (Asian/others), sample size (n = 0–100/101–200/201–), and the quality of study (low/medium/high). Multivariable regression showed that ethnicity  and the sample size  had statistically significant differences, while the quality of study  showed no significant difference. Then, we conducted subgroup analysis based on ethnicity and the sample size, as shown in In the meta-analysis, computation of the Spearman’s rank correlation coefficient between the logit of sensitivity and that of 1-specificity of sputum DNA testing was 0.465 (p = 0.048) and the sample size  were of statistically significant bias for p16, while not significant bias for other candidate genes (p > 0.050).In addition, we performed a meta-regression to test the effect of ethnicity, sample size, and the quality of study with different genes. The results showed that ethnicity , CDO1 , ZFP42 , and TAC1 . A single DNA methylation biomarker cannot be expected to detect all cases of lung cancer. Some studies demonstrated that combined multiple methylated genes could improve the diagnostic value of cancers [FAM19A4 and PLR value of methylated RASSF1A, FHIT, MGMT, and p16 are relatively high, suggesting that they are comprehensive parameters for the screening test [RASSF1A and p16 genes are reported to be promising driving molecules in many cancers under the concept of precision medicine [FAM19A4, FHIT, and MGMT were reported to play important roles in the occurrence and deterioration of lung cancer [SOX17, CDO1, ZFP42, TAC1, FAM19A4, FHIT, MGMT, p16, and RASSF1A are useful in the screening and auxiliary detection of lung cancer.In the direct meta-analysis, we identified that methylated genes in sputum samples for the early screening and auxiliary detection of lung cancer yielded an overall sensitivity of 0.46 at the same specificity of 0.83. Furthermore, the PLR (positive likelihood ratio) was 2.72, NLR (negative likelihood ratio) was 0.64, and DOR (diagnostic odds ratio) value was 4.28, and the AUC (area under the curve) was 0.69, indicating a mid-level accuracy. Therefore, we should pick the superior genes for clinical utility as diagnostic biomarkers for lung cancer. Combined indirect comparisons identified the superior genes as  cancers ,60. We iing test . In addimedicine ,66,67. Ig cancer ,69,70,71To our knowledge, this study is the first systemic review and indirect meta-analysis to assess the comparative diagnostic effectiveness of the methylation profile of multiple candidate gens in sputum samples for the early screening and detection of lung cancer. According to the method of network meta-analysis ,27, we up = 0.001) and the sample size (p = 0.036) might be a source of heterogeneity for this meta-analysis. The results of subgroup analyses showed that large-sample studies had higher sensitivity than the small- and moderate-sample studies, while studies in Asia had lower sensitivity than other regions. However, we observed a large degree of heterogeneity among studies investigating methylation profile in sputum samples used for lung cancer. Threshold effect is one of the primary causes of heterogeneity among diagnostic accuracy studies . In the In addition, publication bias could exist in the meta-analysis. This meta-analysis was only based on published studies, therefore inducing the possibility of publication bias. The Deeks’ test and Egger’s test not only detect publication bias, but also indicate the heterogeneity due to the effect of ethnicity, sample size, the quality of study, etc. ,73. TherThe present network meta-analysis included 33 articles and 32 candidate genes, with the majority of genes only included in one article. These make it difficult to directly compare the diagnostic efficacy among multiple genes; thus, only indirect comparisons were evaluated in this study. However, the absence of direct comparisons may lead to bias. Pairwise meta-analysis and network meta-analysis were carried out sequentially for direct and indirect comparisons of migraine headache days among three interventions compared with those treated by three placebos, and the results showed that there was no significant inconsistency between the direct and indirect evidence for the majority of comparisons . AnotherBased on the focus of diagnostic accuracy studies, we identified other common limitations and insufficiency. Firstly, all the publications included in this analysis were reported on case-control studies, indicating that the selection bias could possibly lead to over-estimations of diagnostic accuracy compared with the cross-sectional study and cohort study ,77. In aSOX17, CDO1, ZFP42, TAC1, FAM19A4, FHIT, MGMT, p16, and RASSF1A are useful biomarkers in the screening and auxiliary detection of lung cancer. Our findings provide new avenues for assessing the comparative diagnostic effectiveness of several methylations in lung cancer based on the method of network meta-analysis. Further high-quality and large-scale studies are needed to confirm our analysis.In conclusion, despite these limitations, our meta-analysis advocates that methylated


Objective: Inflammation, oxidative stress, and decreased glucagon-like peptide-1 (GLP-1) are risk factors for cognitive impairment. Dipeptidyl peptidase-4 (DPP4) was identified as a novel adipokine capable of enhancing these risk factors. Hence, we investigated the relationship between plasma DPP4 activity and impaired cognitive function in elderly Chinese population with normal glucose tolerance (NGT).Methods: We performed a cross-sectional study using data from 1229 elderly participants (60 years or older) in Guilin. Plasma DPP4 activity, oxidative stress parameters, fasting active GLP-1, and inflammatory markers were measured in all participants. Impaired cognitive function was diagnosed according to the National Institute on Aging-Alzheimer’s Association workgroups criteria.Results: Participants in the upper quartile of plasma DPP4 activity had higher C-reactive protein (CRP), interleukin-6 (IL-6), 8-iso-PGF2a, nitrotyrosine, and lower GLP-1 and Montreal Cognitive Assessment (MoCA) scores compared with those in the lowest quartile (P < 0.001). The odds ratios (ORs) for increased CRP, IL-6, 8-iso-PGF2a, nitrotyrosine, and decreased active GLP-1 were higher with increasing DPP4 quartiles after adjustment for confounders . In the highest DPP4 quartile, impaired cognitive function risk was higher  than in the lowest quartile after adjustment for potential confounders. The risk for impaired cognitive function increased more with higher levels of DPP4 activity, nitrotyrosine and 8-iso-PGF2a (P < 0.05), but not with higher IL-6, CRP or lower GLP-1.Conclusion: Plasma DPP4 activity is significantly and independently associated with impaired cognitive function, mainly executive, in elderly Chinese population with NGT. The underlying mechanisms for this association may be partly attributed to the effect of DPP4 on oxidative stress. Plasma DPP4 activity might serve as a risk biomarker or therapeutic target for the prevention and treatment of impaired cognitive function. Mild cognitive impairment is accepted as an intermediate stage between normal cognitive functioning and dementia . Once miAccumulated evidence reveals that inflammation and oxidative stress may play critical roles in the pathogenesis of cognitive decline . MoreoveDPP4 activity inhibitors have now been widely used as antidiabetic drugs at clinical practice, however, no research has ever investigated the relationship between impaired cognitive function and plasma DPP4 activity in elderly Chinese population with normal glucose tolerance (NGT), or considered the feasibility of identifying plasma DPP4 activity as a biomarker or therapeutic target for the prevention and treatment of impaired cognitive function. Consequently, in this study, we investigated the relationship between impaired cognitive function and plasma DPP4 activities in a cross-sectional study of 1229 elderly Chinese population with NGT, since we aimed to further explore the underlying mechanisms and clinical implications for such relationship from a clinical perspective, the associations between plasma DPP4 activities and pathogenetic factors for impaired cognitive function mentioned above were evaluated as well.Because hyperglycemia may have a mutual effect with DPP4 and cognitive function , introduThe purpose, nature, and potential risks of the experiments were fully explained to the subjects, and all subjects gave written informed consent at the beginning of the study. The subjects had the full capacity to consent because they maintained general cognitive function and daily activities. We included only patients who had been fully able to understand and cooperate with study procedures.All participants completed a standard questionnaire containing questions about demographic characteristics, life style risk factors, education level, annual income, cognitive function, the histories of present and past illness and medical therapy. Anthropometric parameters such as blood pressure, body weight, height, and body mass index were measured and calculated as previously described . Venous Circulating levels of interleukin-6 (IL-6), C-reactive protein (CRP), 8-iso-PGF2a, nitrotyrosine, fasting active GLP-1, and DPP4 activity were measured as previously described ,b, 2016.Impaired cognitive function was diagnosed according to the National Institute on Aging-Alzheimer’s Association workgroups criteria . The cri2 or t-test. Pearson’s correlation analyses and partial correlation analyses were used to examine the correlations between DPP4 activity and other variables. The multivariate logistic regression analyses were used to calculate odds ratios (ORs) for impaired cognitive function, elevated proinflammatory markers, oxidative stress parameters, and decreased fasting active GLP-1. Owing to a lack of normal range of CRP, IL-6, 8-iso-PGF2a, nitrotyrosine, and fasting active GLP-1 in elderly Chinese population, the highest quartiles of CRP, IL-6, 8-iso-PGF2a, and nitrotyrosine were defined as increased, whereas the lowest quartiles of GLP-1 were defined as decreased.Statistical analyses were performed using SPSS version 16 statistical package . All continuous variables were expressed as means ± SD or medians (interquartile ranges), and all categorical variables were reported as frequency and percentage. Comparisons of means and proportions were performed with an analysis of covariance , χTable 1 showed the demographic characteristics and laboratory data of participants according to DPP4 activity quartiles. Patients with higher DPP4 activities tended to be relatively old (P = 0.006) with high levels of BMI, TG, IL-6, CRP, nitrotyrosine, and 8-iso-PGF2a , and lower fasting active GLP-1 and MoCA score (P < 0.001); Table 2 showed that participants with impaired cognitive function had higher nitrotyrosine, 8-iso-PGF2a, DPP4 activity, and lower MoCA score compared to those without impaired cognitive function. IL-6, CRP, and fasting active GLP-1 did not differ between two groups. After excluding participants with cardiovascular disease, IL-6 and CRP levels were still not found elevated in impaired cognitive function patients compared to healthy controls .r = 0.074, P = 0.009), BMI , IL-6 , CRP , nitrotyrosine , 8-iso-PGF2a  and negatively with fasting active GLP-1  and MoCA score . After adjustments for age, gender, BMI, and education level, these associations still remained statistically significant .Correlation analysis showed that plasma DPP4 activities were positively and significantly related to age  2.39–5.31] for elevated IL-6, 4.31 (2.86–6.49) for elevated CRP, 5.19 (3.45–7.82) for elevated nitrotyrosine, 6.39 (4.06–10.08) for elevated 8-iso-PGF2a, and 3.22 (2.17–4.77) for decreased active GLP-1 after adjustments for possible confounders.As shown in Among the 1229 elderly Chinese participants included in this study, 203 patients (16.5%) had impaired cognitive function. The impaired cognitive function prevalence according to plasma DPP4 activity quartiles were 9.1, 16.5, 15.0, and 25.4%, respectively.P = 0.002], however, this relationship was not attenuated after further adjustments for IL-6 or fasting active GLP-1 (Table 5). This independent relationship between DPP4 activity and impaired cognitive function still existed in a subgroup of participants without hyperuricemia and hypertriglyceridemia  for impaired cognitive function after adjustments for confounders. Interestingly, further adjustment for 8-iso-PGF2a reduced the magnitude of the OR for impaired cognitive function [2.26 (1.36–3.76), Figure 2C) and 8-iso-PGF2a , however, this increasing trend of impaired cognitive function risk were not observed in higher levels of IL-6 , CRP , and lower levels of fasting active GLP-1 . Even in the lowest quartiles of nitrotyrosine and 8-iso-PGF2a, the risks for impaired cognitive function were 3.35- to 3.86-fold higher in the highest DPP4 quartile than in the lowest quartile .The ORs for impaired cognitive function became more pronounced among subjects with both increased plasma DPP4 activities and higher circulating levels of nitrotyrosine  elevated plasma DPP4 activities were positively related to impaired cognitive function and negatively related to MoCA score in elderly Chinese population with NGT; (2) this relationship was paralleled by an increase in circulating oxidative stress parameters; (3) increased levels of proinflammatory markers and decreased levels of fasting active GLP-1 in peripheral circulation were not associated with an increased risk of impaired cognitive function.Previous findings have shown higher peripheral levels of inflammatory markers such as IL-6, CRP, TNF-α in patients with dementia compared to controls . Since iAside from inflammation, previous studies have provided a wealth of information in regard to the pathogenetic role of oxidative stress in the development of cognitive decline, even at the stage of impaired cognitive function, To examine the robustness of this relationship between DPP4 activity and impaired cognitive function and to obtain information on potential mechanisms that could mediate the observed relationship, we performed a set of logistic regression models comparing risk of impaired cognitive function between participants in the lowest DPP4 activity group and in highest DPP4 activity group. Interestingly, further adjustment for 8-iso-PGF2a yielded only a reduction of the impaired cognitive function risk across the DPP4 activity quartiles. Even within the lowest nitrotyrosine and 8-iso-PGF2a quartiles, the risks for impaired cognitive function were still 3.35- to 3.86-fold higher in the highest DPP4 quartile than in the lowest quartile. Thus, the positive relationship between increased plasma DPP4 activities and high impaired cognitive function risk in this non-diabetic Chinese population might not be merely the results of enhanced oxidative stress. Because of the pleiotropic effects of DPP4, the interactions between plasma DPP4 activities and other pathogenetic factors might promote the development of impaired cognitive function as well.in vivo and in vitro studies. It has been proved to reduce apoptosis and inflammation, protect neurons from oxidative stress, protect synaptic plasticity and memory formation from the detrimental effects of Aβ, and enhance neurogenesis in the brains (The neuroprotective actions of GLP-1, an incretin hormone secreted from L cells located in the distal ileum and colon, have been demonstrated in e brains . In thise brains , peripheBecause our study is cross-sectional, we cannot draw a causal conclusion that increased DPP4 activities promote the development of impaired cognitive function through its effect on oxidative stress. Although no direct evidence completed to date links impaired cognitive function back to the regulation of DPP4 activity, the parallel increase in oxidative stress and DPP4 activity could be interpreted in an opposite way.Our study has several limitations. One limitation is the cross-sectional nature of present study. We could not prove a causal association between plasma DPP4 activities and the development of impaired cognitive function. Second, postprandial GLP-1 levels and its relationship with impaired cognitive function were not evaluated in this study. Third, Specific cognitive domains, such as episodic memory, cannot be evaluated by MoCA. Finally, because of the sample size and epidemiological nature of this study, neuropsychological assessment and radiological survey cannot be conducted to evaluate the specific reasons leading to impaired cognitive function, such as Alzheimer’s disease, vascular dementia, or Parkinson’s disease.In summary, our study demonstrated a positive and significant relationship between plasma DPP4 activities and impaired cognitive function in elderly non-diabetic population, the underlying mechanisms for this relationship may be partly attributed to the enhancement of oxidative stress promoted by DPP4. DPP4 inhibitors have now been wildly used as antidiabetic drugs at clinical practice, moreover, protective effects of DPP4 inhibitors on cognitive impairment have also been reported in a retrospective longitudinal study in patients with hyperglycemia . HoweverTZ, BC, XH, and QL contributed to study concept and design. TZ, LQ, BC, XH, XZ, YL, HL, SQ, GL, and QL contributed to acquisition, analysis, or interpretation of data. TZ, BC, and XH performed drafting the work. TZ, LQ, BC, XZ, YL, HL, SQ, GL, and QL critically revised the manuscript for important intellectual content. LQ performed statistical analysis. TZ obtained funding. TZ supervised the study. All authors approved the final manuscript to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CNGB1 is associated with retinitis pigmentosa. We have identified and investigated a 34-year-old Chinese man with markedly have night vision blindness and loss of midperipheral visual field. The proband also lose his far peripheral visual field and also central vision. Proband’s retinal pigment deposits visible on fundus examination and primary loss of rod photoreceptor cells followed by secondary loss of cone photoreceptors. Target exome capture based next generation sequencing and Sanger sequencing identified novel nonsense mutation, c.1917G>A and a reported mutation, c.2361C>A, in the CNGB1 gene. Both the nonsense mutations are predicted to lead to the formation of a premature stop codon which finally results into formation of truncated CNGB1 protein product which finally predicted to be disease causing. According to the variant classification guidelines of ACMG, these two variants are categorized as “likely pathogenic” variants. Our findings expand the mutational spectra of CNGB1 and are valuable in the mutation-based pre- and post-natal screening and genetic diagnosis for retinitis pigmentosa.Retinitis pigmentosa (RP) is a severe hereditary eye disease characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal diseases. Germline mutations of  Retinitis pigmentosa (RP) is an inherited eye disease with dystrophy of retina dystrophy that finally results into loss of vision. It is genetically and phenotypically extremely heterogenous. According to inheritance pattern, disease onset and clinical symptoms, retinitis pigmentosa is showing extreme heterogeneity. There are almost one and half a million patients with RP, around the world . PatientBased on classical genetics, recessively inherited disease majorly caused by homozygous mutation of a candidate gene in consanguineous families , 9. For CNGB1 gene segregating with retinitis pigmentosa phenotype in the proband, with autosomal recessive (compound heterozygosity) inheritance.Here, in order to identify the molecular basis of RP in the proband of this Chinese family, we screened a panel of 60 genes associated with retinitis pigmentosa by targeted next-generation sequencing and confirmatory direct sequencing. In this study, we found novel heterozygous nonsense mutations of A 28 years old Chinese man of a non-consanguineous Chinese family with RP was studied. The pedigree contained only one affected individual   [NCBI Reference sequence NM_001297] of the human CNGB1 gene in the proband (II-1). Both of these heterozygous nonsense mutations of CNGB1 gene are predicted to form truncated CNGB1 protein in contrast with the wildtype CNGB1 protein consisting of 1251 amino acids. c.2361C>A, p.Tyr787* was previously reported by Xu et al., in 2014 [In our study, we found two heterozygous CNGB1 genes is associated with autosomal recessive retinitis pigmentosa (arRP) very rare. Clinical manifestations of our patient are same with all RP patients with mutations CNGB1 gene reported previously.Germline mutations in CNGB1 encode β-subunits of cyclic nucleotide-gated (CNG) channels plays a significant role for signal transduction pathways in both visual and olfactory system. These are ligand-gated channels regulated or controlled directly by second messenger like Cyclic guanosine monophosphate or cyclic adenosine monophosphate. CNG channels control the photo-transduction pathways in photoreceptor cells which is finally leads to photoreceptor cell hyperpolarization . HoweverCNGB1 gene causes formation of non-functional CNGB1 protein which finally affects the normal function of the CNG channel [Moreover, wild type α-subunit and β-subunit are required for normal function of CNG channel. Truncating mutation in  channel , 22.CNGB1 gene. Our study is significant for genetic screening and clinical diagnosis of retinitis pigmentosa.In conclusion, here, we report a Chinese patient who presented with retinitis pigmentosa, with novel mutations in the Proband and his parents of this Chinese family have given written informed consent as they are participating in this study. The Ethical Committee of the Department of Ophthalmology, Zhongnan Hospital of Wuhan University, Wuhan, China, reviewed and approved our study protocol in compliance with the Helsinki declaration. Diagnosis of the patients for retinitis pigmentosa has done by ophthalmologist.A proband of Chinese descend with retinitis pigmentosa, diagnosed in the Department of Ophthalmology, Zhongnan Hospital of Wuhan University, Wuhan, China, were enrolled in our study.ABCA4, AIPL1, ARL6, BEST1, C2orf71, CA4, CDHR1, CERKL, CLRN1, CNGA1, CNGB1, CRB1, CRX, CYP4V2, DHDDS, EYS, FAM161A, FLVCR1, ,FSCN2, GUCA1B, IDH3B, IMPDH1, IMPG2, KLHL7, LRAT, MAK, MERTK, NR2E3, NRL, OFD1, PDE6A, PDE6B, PDE6G, PRCD, PROM1, PRPF3, PRPF6, PRPF8, PRPF31, PRPH2, RBP3, RDH12, RGR, RHO, RLBP1, ROM1, RP1, RP2, ,RP9, RPE65, RPGR, SAG, SEMA4A, SNRNP200, SPATA7, TOPORS, TTC8, TULP1, USH2A, ZNF513) associated with retinitis pigmentosa and yielded an average of 6366534 reads per sample, with approximately 96.92% mapping to the targeted regions. The average sequencing depth of the target area is 217.08% with 99.73% coverage. The procedure for preparation of libraries was consistent with standard operating protocols. In each pooling batch, 10 to 33 samples were sequenced simultaneously on Illumina HiSeq 2500 Analyzers  for 90 cycles . Image analysis, error estimation, and base calling were performed using Illumina Pipeline software (version 1.3.4) to generate raw data. The raw reads were screened to generate – clean reads‖ followed by established filtering criteria. Clean reads with a length of 90 bp were aligned to the reference human genome from the NCBI database (Build 37) using the Burrows Wheeler Aligner (BWA) Multi-Vision software package with output files in - bam‖ format. The bamdata were used for reads coverage in the target region and sequencing depth computation, SNP and INDEL calling, and CNV detection. First, a novel three-step computational frame work for CNV was applied. Then, SNPs and INDELs were called using SOAPsnp software and Sam tools pileup software, respectively. A SNP or INDEL was be filtered if it could not follow the criterion: supported by at least 10 reads and >20% of the total reads. The frequency filter was set at 0.05. If a SNP frequency was more than 0.05 in any of the four databases , it would be regarded as a polymorphism, but not a causative mutation.DNA samples obtained from the proband (II-1) were sequenced using target exome-based next-generation sequencing. Roche NimbleGen’s  custom Sequence Capture Human Array was used to designed to capture 221340 kb of targeted sequence, covering 181 exons and flanking sequence (including the 100 bp of introns) of 60 genes (http://www.hgmd.cf.ac.uk/ac/index.php) and the Leiden Open Variation Database (http://www.lovd.nl/3.0/home), and then labeled as reported or novel . Sequence data comparisons and analysis were performed by DNASTAR SeqMan .CNGB1 was used.These two heterozygous novel nonsense mutations identified through targeted next generation sequencing were verified through Sanger sequencing using the primers: F1 5’-TTTACCAGTGAGGGACGGGC-3’, R1 5’-GTTTGTCTGGCTCCGGTAAGTA-3’. The reference sequence NM_001297 of


TM Complex is a multicomponent food product that meets the requirements of a food for special medical purpose. As a complete, high-energy diet it consists of properly balanced nutrients, vitamins and minerals. The aim of this study was to assess the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results showed that NutramilTM Complex reduced the viability and proliferation of breast and prostate cancer cells and that this process was associated with the induction of apoptosis via activation of caspase signalling. Data showed elevated levels of p53 tumour suppressor, up-regulation of p38 MAPK and SAPK / JNK proteins and downregulation of anti-apoptotic ERK1/2, AKT1 and HSP27. Treatment with NutramilTM Complex also affected the expression of the BCL2 family genes. Results also showed down-regulation of anti-apoptotic BCL-2 and up-regulation of pro-apoptotic members such as BAX, BAD, BID. In addition, we also observed regulation of many other genes, including Iκβα, Chk1 and Chk2, associated with apoptotic events. Taken together, our results suggest activation of the mitochondrial apoptotic pathway as most likely mechanism of anti-carcinogenic activity of NutramilTM Complex.Nutramil Breast cancer is one of the most common type of cancer affecting women around the world , and is Numerous studies showed that selected nutrients and non-nutrients can act preventively in cancer. They included, among others, diets rich in vegetables , fruits, oily sea fish and soybeans; thus, rich in vitamin E, C, carotenoids  selenium, omega-3 fatty acids, glucosinolates, polyphenols and etc. –6.TM Complex is a Food for Special Medical Purpose (FSMP), intended for people whose nutritional requirements cannot be met by normal food, which follows the European Union Directive 1999/21/EC regarding its composition and levels of vitamins and minerals. It can be used as a complete, all-day meal for the hospitalised patient, or used as a supplement. The choice depends on nutritional status, individual energy needs, ability to take other meals as well as general health or disease progression. NutramilTM Complex is also used to prevent malnutrition in associated with lack of appetite, anorexia, Alzheimer's disease, Parkinson's disease, cancer, irritable bowel syndrome, partial gastric or intestinal resection, etc. In addition, it is also gluten- and lactose-free.NutramilTM Complex has decreased the viability of breast and prostate cancer cells [TM anti-cancer properties as a multi-component product. However, limited data is available on some individual substances that constitutes the composition of NutramilTM Complex. In this manuscript, we present the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results suggest that observed reduction in viability of cancer cells can be associated with induction of apoptosis.In our pilot studies, Nutramiler cells , 8. To oTM Complex (NC) is given in the TM Complex without calcium caseinate (NC-CC) and calcium caseinate alone (CC) as one of the main components of the formulation. Analyses were blinded as all samples were numerically encoded. Their decoding took place at the end of the study.Composition of the NutramilTM) was purchased from the American Type Culture Collections. Cells were cultured in appropriate EMEM medium  according to the ATCC protocol with an addition of 10% FBS .The human breast adenocarcinoma cell line MCF-7 (estrogen receptor (ER) positive cell line, progesterone receptor positive and HER2 negative, ATCC® HTB22TM) and LNCaP cell lines  were purchased from the American Type Culture Collections. Cells were cultured in appropriate EMEM and RPMI 1640, respectively, medium  according to the ATCC protocol with an addition of 10% FBS .The human prostate carcinoma DU145 . Cells were cultured in appropriate RPMI 1640 medium  according to the protocol with an addition of 10% FBS .3 cells per well), 12-well plates (9x104 cells per well) or 6-well plates (2x105 cells per well). 24 h after seeding, growth medium was replaced with a medium containing: 1) NC, 2) NC-CC or 3) CC. The final applied concentrations of each treatment were 1, 2, 3, 4, 5 and 10% for 24, 48 and 72 h. Untreated cells in culture medium were used as a negative, untreated control (UC).Cells were seeded on 96-well plates  was used as a positive control at the final concentration of 1.5 μM.Cell viability was determined by Crystal Violet Assay  and Cytotoxicity Detection Kit LDH  according to the manufacturer's protocol.Each treatment included 3 biological and 4–5 technical replicates.Cell proliferation was determined with 5’-bromo-2’-deoxy-uridine (BrdU) Labeling and Detection Kit III (Roche), according to manufacturer's instruction.Each treatment included 3 biological and 3 technical replicates.AKT1, APAF1, BAD, BAX, BCL2, BID, CASP3, CASP8, CDKN2A, CYCS, FADD, FAS, FASLG, HRAS, IGF1, IGF1R, KRAS, MYC, NRAS, RRAS, TP53, YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ, YWHAZ. Results were normalized using at least two reference genes  and were calculated using the 2-ΔΔCT method [Reverse transcription was performed using 1 μg of total RNA isolated from the cells using the Maxima first Strand cDNA Synthesis kit for RT-qPCR (Thermo Scientific). A quantitative verification of genes was performed with TaqMan® Array Human C-MYC and Apoptosis (Thermo Fisher Scientific) according to the manufaturer’s protocol and StepOnePlus™ system. Analysed genes: T method .Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856, Cell Signaling Technology, MA, USA. Assay target proteins were P44/42 MAPK (ERK1/2) phosphorylation, AKT phosphorylation, BAD phosphorylation, HSP27 phosphorylation, SMAD2 phosphorylation, p53 phosphorylation, p38 MAPK phosphorylation, SAPK/JNK phosphorylation, PARP cleavage, Caspase-3 cleavage, Caspase-7 cleavage, total Ikβα, Chk1 Ser345 phosphorylation, Chk2 phosphorylation, Ikβ α phosphorylation, eIF2α phosphorylation, TAK1 phosphorylation, Survivin and α-Tubulin as a reference protein.http://imagej.nih.gov/ij/). Results are shown as a mean±SD normalized to the internal reference protein (α-Tubulin). Untreated negative control (UC) was set as 100% expression level.Images were acquired by briefly exposing the slide to standard chemiluminescent film. Densitometry analysis was performer using ImageJ . Protein extract was separated on a polyacrylamide gel and transferred to a nitrocellulose filter  by wet-electroblotting. Subsequently, the immobilized proteins were incubated with the appropriate primary antibody: cytochrome c (#11940), Smac/Diablo (#2954), HtrA2/Omi (#9745) and β-Tubulin (#2128) . Finally, the appropriate secondary antibody conjugated with horseradish peroxidase  was applied. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate . Western blot stripping buffer  was used to remove the antibodies from the membrane.Whole cell lysis was carried out using Cell Lysis Buffer  according to the manufacturer’s protocol, with the addition of Protease Inhibitor Cocktail . Total protein was quantified with Pierce BCAAll experiments were performed in at least three independent experiments and measured in triplicates. Shapiro-Wilk’s test was applied to assess normality of distribution. An independent samples t-test was applied to compare unpaired means between two groups and P≤0.05 was considered statistically significant. All analyses were performed using Statistica ver.12 .TM Complex showed a cytotoxic effect on all examined cells, both cancer and non-malignant in a dose–and time–dependent manner  and medium-chain triglycerides (MCTs) (20%). It provides very important mono-unsaturated oleic acid, essential fatty acids, as well as long-chain triglycerides (LCTs). MCTs provide fast and lasting energy and are easily absorbed by the body. In addition, they also have a positive effect on calcium absorption.NutramilTM Complex decreases the viability of breast and prostate cancer cells [In our previous pilot studies, we have shown that Nutramiler cells , 8. In cTP53 in all examined cell lines. We also measured promoted expression of CDKN2A in prostate cancer lines 2D3 may not be directly translated into the observed properties of the formulation; however, some studies suggest that cholecalciferol may be metabolized in some tumour cells, especially in breast cancer cells [in vitro on lymphoma cells using ascorbic acid (form of vitamin C present in NutramilTM Complex) showed a decrease in the viability of cancer cells without affecting normal cells [Nutramilman body . Some ofer cells . Studiesal cells . In turnal cells .6 and B12 can cause DNA damage and lead to cancer [TM Complex, with reduced survival of cancer cells. Another component of NutramilTM Complex is the sodium selenite, which anti-tumor properties have been demonstrated in animals [in vivo studies involved nude mice bearing human colorectal carcinoma SW480 cell line xenografts, which were injected peritoneally for 21 days with sodium selenite. Results showed significant tumour suppression. In addition, authors showed reduced expression of Bcl-xL proteins and increased expression of pro-apoptotic proteins, including Bax, Bad and Bim as well as Caspase-9 [TP53 and PTEN suppressors and down-regulation of BCl-2 expression. In promyelocytic leukemia NB4 cells, sodium selenite induced reactive oxygen species generation that was associated with early events that triggers endoplasmic reticulum stress mitochondrial apoptotic pathways [For B complex vitamins, deficiencies in Bo cancer , 39. Ribo cancer  and by eo cancer . The effo cancer , 43; on o cancer , 44. Vito cancer . It has o cancer . However animals  and in c animals –51. One aspase-9 . In studpathways .TM formulation with potential anti-neoplastic significance is magnesium. This element participates in many metabolic processes and redox reactions [+ and Cl- channels, that regulate the membrane potential and increase the probability of Ca2+ channel opening. Also, the activity of individual ions affects their concentration in the cytoplasm [Zinc whose anti-neoplastic properties have been described for breast cancer cells and prostate cancer  is also eactions . Magnesieactions , while heactions . Epidemieactions . It is aytoplasm . The corytoplasm . Finallyytoplasm .TM Complex exhibits anti-tumor properties. Moreover, our data suggest that it activates apoptotic events via mitochondrial-induced pathway , the in vitro studies are a valuable and multi-faceted source of information and may explain the adequacy of further in vivo research.In conclusion, our study, as one of very few, show the effect of complete food medical supplement on cancer cells. It is well known that cells integrate multiple signals from a variety of sources before following either pro- or anti-apoptotic pathway. Taking into account the significant reduction if cancer cell growth Figs  with sim pathway . This isS1 Table(DOCX)Click here for additional data file.


Kidney renal papillary cell carcinoma (KIRP) accounts for 10%–15% of renal cell carcinoma (RCC), patients with KIRP tend to have a poor prognosis, and there was a lack of effective prognostic indicators for this type of cancer. Currently, owing to the availability of The Cancer Genome Atlas (TCGA), long non-coding RNAs (LncRNAs) have been discovered to indicate a prognostic value in some tumors. In that regard, we analyzed lncRNA-sequencing data of KIRP in TCGA, and among 780 differentially-expressed lncRNAs, we selected 37 lncRNAs which were able to assist the prognosis. In addition, by using the multivariate cox regression analysis, the prognosis index (PI) that consisted of 7 lncRNAs  could predict the progression and outcomes of KIRP with accuracy. More importantly, the PI was considered an independent indicator for prognostication of KIRP. Moreover, having categorized patients with KIRP into cohorts of high risk and low risk, according to the PI, we found that the key genes and pathways varied in these two groups. Overall, these LncRNAs, especially the PI, may be conceived as biomarkers and helpful for determining the different pathological stages for KIRP patients. However, their biological functions need to be further confirmed. Kidney cancer is a neoplasm with heterogeneity, of which epithelial renal cell carcinoma (RCC) represents the major proportion. Morphologically, RCC includes various histological subtypes—kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and malignancies of chromophobe, collecting duct, and subtypes with classification. Of these subtypes, KIRP ranks second in terms of morbidity rate, comprising 10%–15% of cases, following KIRC with incidence of 75%–80% [Approximately 30% of RCC patients have been found to exhibit distant metastasis when diagnosed, thus the prognosis of RCC remains poor. Clinically, patients with other RCC subtypes tend to have desirable outcomes; however, patients with KIRP were more likely to experience obviously worse clinical courses , 6, 7.Currently, several biomarkers for KIRC that have been detected include von Hippel-Lindau (VHL) –11, vascThe Cancer Genome Atlas (TCGA) has rendered the results of KIRP available for researchers –30, whicAnalysis of the DELs was conducted to compare the expressions of 13,198 lncRNAs between KIRP and normal kidney tissues. A total of 780 DELs Figures  were colIt was demonstrated by the univariate Cox proportional hazards regression that 37 of these 271 differentially-expressed lncRNAs displayed remarkable prognostic value. Then multivariate Cox proportional hazards regression analysis confirmed that seven differentially-expressed lncRNAs showed prognostic significance for KIRP, containing actin filament associated protein 1 antisense RNA1(AFAP1-AS1), GAS6 antisense RNA 1 (GAS6-AS1), RP11-1C8.7, RP11-21L19.1, RP11-503C24.1, RP11-536I6.2, and RP11-63A11.1  was performed to distinguish relevant biological processes and signaling pathways . We alsoIn the high-risk group, 3502 differentially expressed genes (DEGs) were obtained, including 2269 up-regulated and 1233 down-regulated genes . MeanwhiP = 0.0318)   than in that of normal controls  Figure . The GAS) Figure . AFAP1-A) Figure . These rIn the current study, we analyzed lncRNA-sequencing data of KIRP in TCGA, and used multivariate Cox regression analysis to obtain seven lncRNAs to form a pool, which included AFAP1-AS1, GAS6-AS1, RP11-1C8.7, RP11-21L19.1, RP11-503C24.1, RP11-536I6.2, and RP11-63A11.1. The PI was also calculated. Surprisingly, the PI was discovered to be an independent indicator for KIRP with high prognostic significance. Additionally, after categorizing the KIRP patients into high risk and low risk groups according to the PI, we observed alterations in key genes and pathways between these two cohorts. To our knowledge, this current study pioneered research into the prognostic value of lncRNAs in KIRP.RCC represents over 90% of renal carcinomas, among which the subtype KIRP ranked second in the incidence rate, following KIRC –5. Remaret al. [In recent years, researchers have had a great opportunity to identify a wide range of novel biomarkers for various malignancies thanks to TCGA, but the roles of lncRNAs in kidney cancers were rarely reported. In 2015, Malouf GG et al.  conducteWe singled out the differentially-expressed lncRNAs and then gathered 7 lncRNAs by univariate and multivariate Cox proportional hazards regression, each of which would bear clinical significance such as TNM stage, distant metastasis, and tumor stage, etc. to some extent. More interestingly, the PI composed of these seven lncRNAs displayed considerable predictive potential for disease progression, and could even qualify itself as an independent indicator for prognosis with its clinical significance. Furthermore, taking GEO dataset into validation, we collected 67 KIRP-related series. The clinical and prognostic value of lncRNAs AFAP1-AS1 and GAS6-AS1 could be partially verified by GSE2748 and GSE48352. However, the clinical efficacy of these lncRNAs and the PI required further confirmation with a larger sample size and joint effort.Having classified the patients into high risk and low PI cohorts according to their PI scores, we examined the signaling pathways of differentially-expressed genes in the two cohorts and found that the major pathways of the high risk group included KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION, KEGG_TGF_BETA_SIGNALING_PATHWAY, and KEGG_MAPK_SIGNALING_PATHWAY; these were utterly different from its low counterpart, of which the main pathways involved KEGG_OXIDATIVE_PHOSPHORYLATION and KEGG_REGULATION_OF_AUTOPHAGY. It was assumed that the pathway differences helped clarify the underlying molecular mechanisms which produced contrasting outcomes. Nonetheless, the relationships between these pathways and KIRP required experimental verification.For the present, the clinical implications and mechanisms of lncRNAs in other diseases appeal to our research team. By literature research, of the seven lncRNAs, we only retrieved reports on AFAP1-AS1 and GAS6-AS1 in other diseases, whereas reports were lacking on RP11-1C8.7, RP11-21L19.1, RP11-503C24.1, RP11-536I6.2, and RP11-63A11.1. It was thus necessary to conduct in-depth investigation.in vitro and in vivo experiments explored the function and molecular mechanism of AFAP1-AS1. The knockdown of AFAP1-AS1 significantly prohibited cell migration and invasion in nasopharyngeal carcinoma (NPC) and lung cancer cells [in vivo. AFAP1-AS1 also promoted the cell proliferation and invasion through up-regulating the RhoA/Rac2 signaling [A large number of er cells , 33. Silignaling , 35. Silignaling . The knoignaling , 38. In ignaling . AFAP1-Aignaling , 41. AFAignaling , 43.Clinical researche into lncRNA AFAP1-AS1 in multiple tumors was diversified. AFAP1-AS1 might act as therapeutic targets in NPC, and anti-PD-1 immune treatment was considered suitable for patients with co-expression of AFAP1-AS1 and PD-1 , 44. Meaet al. [et al. [Owing to the extensive research on AFAP1-AS1, Liu et al.  performe [et al.  investigP < 0.001). In addition, reduced expression of GAS6-AS1 was adversely linked with lymph node metastasis (P = 0.032) as well as advanced tumor node metastasis stage (P = 0.003). Via univariate and multivariate analysis, GAS6-AS1 was considered to act as an independent predictive factor for overall survival duration (P = 0.036). Moreover, GAS6-AS1 level showed a negative relationship with GAS6 mRNA level. Aberrant GAS6-AS1 expression could participate in the development of NSCLC via affecting of its host gene, rendering it possible in becoming a diagnosis target in NSCLC patients [Studies on GAS6-AS1 were also rather scarce, and only relevant studies on lung cancers were retrieved. It was reported that down-regulation of GAS6-AS1 expression was observed in tumor tissues in 50 cases of non-small cell lung cancer (NSCLC) compared with adjacent normal tissues , which contained 321 KIRP tissues and 32 tumor-free adjacent normal tissues up to May 10, 2017. The clinical parameters, including age, tumor size, status of lymph node metastasis, status of distant metastasis, clinical TNM stage, and overall survival, etc. were collected and analyzed. Due to the public availability of the data from TCGA, additional approval by the ethics committee was not required. The data was used and processed according to TCGA Human Subjects Protection and Data Access Policies.TCGA has been a major source of valuable data for 33 types of tumors, including for mRNA expression, protein expression, various mutations, amplifications, etc. In this study, we collected sequencing data of KIRP from the TCGA website (http://www.gencodegenes.org/) for further analysis, and a subset of expression profiles of 13,198 lncRNAs was eventually obtained. The differentially expressed lncRNAs were subsequently filtered by edgeR R package, with Padj < 0.05 and |log2FC| > 2 of expression level when comparing tumor and adjacent normal kidney tissue. The differentially expressed lncRNAs, which had been log2 transformed, were illustrated in a volcano plot and heatmap.The current KIPR TCGA dataset was comprised of gene counts for 60,244 mRNAs. In this study, we chose the lncRNAs with descriptions from GENCODE  was used to examine the differential expression of these lncRNAs between KIRP and non-cancerous kidney tissues. The relationships between DEL expression and clinical progress were evaluated via Student’s T-Test, Spearman correlation, and K-M Curve. The PI, which was used for OS prediction, was created on the basis of the linear combination of the expression level multiplied by the regression coefficient that was derived from the multivariate Cox regression model (β). The calculation formula is as follows: PI = βDEL1 × exprDEL1 + βDEL2 × exprDEL2+ ··· + βDELn × exprDELn. [The univariate Cox proportional hazards regression was applied to obtain the differentially-expressed lncRNAs that were strongly associated with OS, with the significance level set at 0.05. The multivariate Cox regression model was also used to calculate the prognosis value of these differentially-expressed lncRNAs. Next, the clinical role of these DELs with prognostic value was assessed. Student’s xprDELn. –55.Patients with KIRP were categorized into high risk and low risk cohorts according to the cut-off of the individual infection point of PI. The impact of PI on the OS of KIRP cases was measured by univariate and multivariate Cox proportional hazards regression analysis. Several clinical parameters, such as age, tumor stage, and cancer status, etc. were adjusted. Hazard ratio (HR) and 95% confidence intervals (CI) were evaluated. In order to gauge the accuracy of the prognostic model for time-dependent disease outcomes, we utilized the R package “survival ROC” to conduct ROC curve analysis within 5 years as the defining point . Kaplan-p-value less than 0.05 and a false discovery rate (FDR) value <0.25 were deemed significantly enriched. If no significant gene set could be obtained, then the gene sets were listed ascendingly according to the orders of p-value and FDR. The results were generated by GSEA.GSEA, also known as functional enrichment analysis, could facilitate the identification of types of genes or proteins that are over-represented in a large set of genes or proteins which may be connected with disease phenotypes. GSEA distinguishes itself from other pathway analysis owing to its enrichment process–the gene expressions in each cohort are first calculated and then enriched according to their expressions. The calculation is as follows: calculate the enrichment score (ES) that embodies the amount to which the genes in the set are over-represented at either the top or bottom of the list. The Molecular Signatures Database (MsigDB) accommodates a large group of annotated gene sets that can be utilized with most GSEA Software. In the current study, a total of 60,483 genes were inputed for GSEA. Gene sets with a 2FC| > 2. The inconsistent DEGs between high-risk and low-risk groups were sent for further signaling pathways analysis to unveil different possible molecular mechanisms. DAVID database was used for the annotation and visualization of DEGs in different risk groups. GO terms and KEGG pathway of DAVID were considered as significant with Padj < 0.05.DEGs were calculated between high-risk and low-risk groups and normal tissues, respectively. DEGs were identified using the edgeR package with Padj<0.05 and |log* OR malignan* OR adenocarcinoma). The levels of lncRNA expression between different groups were analyzed by Student’s t-test. Survival analysis was performed using Kaplan–Meier method.The correlative microarrays from GEO DataSets were gathered to validate the clinical roles of the seven lncRNAs; the following search terms were adopted:  AND papillary AND (cancer OR carcinoma OR tumor OR neoplas


Here, we report the complete genome sequence of an H1N1 avian influenza virus (AIV), which was isolated from the feces of migratory birds in the Republic of Korea during the winters of 2014 and 2015. Full-genome sequencing and phylogenetic analysis revealed that all genome segments belonged to the Eurasian lineage. The multiple alignments of full coding nucleotide sequences were performed by MUSCLE algorithm. Phylogenetic analysis based on nucleotide sequences was constructed with the neighbor-joining method with 1,000 replicates in Molecular Evolutionary Genetics Analysis (MEGA), version 6.06 , and cDNA was synthesized using SuperScript III reverse transcriptase  with the Uni-12 primer (5′-AGCRAAAGCAGG-3′), as per the manufacturer’s protocol. PCR products were amplified with universal primer sets targeting the full genomes of influenza virus using the Phusion master mix kit  (The complete genomic lengths of 8 segments containing the polymerase basic 2 (PB2), PB1, polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), nonstructural (NS), and matrix (M) genes represented 2,280, 2,274, 2,151, 1,701, 1,497, 1,410, 982, and 838 nucleotides, respectively. In HA protein, the cleavage site possesses only a single basic amino acid (PSIQSR↓GLF), which is characteristic of low-pathogenicity avian influenza virus (AIV)  and amanOur study is meaningful for furthering the understanding of molecular evolution in the Eurasian lineage of H1N1 avian influenza virus and will facilitate future investigations of the epidemiology of this virus.KX066868 to KX066875.The genome sequence of the avian H1N1 influenza virus, A/wild bird/Korea/SK14/2014, was deposited in GenBank under the accession numbers


Cronkhite-Canada syndrome is a rare syndrome consisting of extensive gastrointestinal polyposis and ectodermal changes including cutaneous hyperpigmentation, alopecia, and onychodystrophy. We report the case of a 45-year-old Caucasian male patient who failed multiple treatments over 2 years including steroids, azathioprine, adalimumab, and cyclosporine. He had recurrent and prolonged hospitalizations because of diarrhea, abdominal pain, weight loss, and malnutrition. Sirolimus was initiated with a significant clinical and endoscopic benefit apparent within, respectively, 2 and 8 weeks. An ongoing remission was achieved and maintained for over 6 months after prednisone tapering. We review the current evidence on treatment of Cronkhite-Canada syndrome and suggest the incorporation of sirolimus in that algorithm. We report the case of a 45-year-old man with diarrhea, abdominal pain, weight loss, and malnutrition who was diagnosed with CCS and resisted to a panoply of treatments before finally responding to sirolimus.Cronkhite-Canada syndrome (CCS) is a rare noninherited condition characterized by diffuse gastrointestinal hamartomatous polyposis and ectodermal changes including cutaneous hyperpigmentation, alopecia, and onychodystrophy. Main complaints are diarrhea, abdominal pain, and weight loss. Mean age at onset is 59 years. Microscopic examination regularly shows inflammatory cell infiltration with mononuclear cells and eosinophils. Etiology remains uncertain, but immune dysregulation plays a large role. Prognosis in CCS remains poor with a 5-year mortality of 55%.12/A/E, folic acid, copper, manganese, IgA, IgM, C-reactive protein, Helicobacter pylori serum antibody, procalcitonin, and IgG4 were within normal limits. Autoimmune workup was negative. Endoscopy (H pylori were negative. IgG4-positive-cell count/high-power field was modestly increased (19 cells per field). Methylprednisolone 20 mg intravenous 3×/d was started on arrival and cyclosporine was continued. Facing poor clinical response, cyclosporine was switched to sirolimus (4-6 mg 1×/day aiming for blood levels 8-10 ng/mL). Prednisone 50 mg/day initially (following intravenous methylprednisolone) was progressively decreased. Frank improvement was noted at week 2 on sirolimus with resolution of abdominal pain and progressive restoration of the capacity to eat although diarrhea (400-800 g per day) reappeared with oral feeding. At week 4, prednisone was weaned off completely without any flare-up. At week 8, the patient had gained 5 kg, early upper endoscopy assessed partial healing, and the patient was finally discharged from the hospital with home parenteral nutrition (decreased from 7 to 5 nights per week), sirolimus 5 mg 1×/day, and free diet. Parenteral nutrition was stopped at week 12 as the patient could eat a normal oral diet. Dysgeusia and asthenia disappeared, and he had only 3 to 4 stools per day. Hemoglobin and serum albumin were normalized. Six months later, he had regained a normal life. He was back to work and returned to playing hockey. After 8 months of sirolimus, upper and lower endoscopies showed a decrease in the number and size of the polyps  was introduced without response after 2 months. In January 2017, the patient was hospitalized for severe malnutrition, weight loss, and incapacity to eat due to gastric outlet obstruction by a polypoid antral mass. When transferred at our center in February 2017, his treatment included parenteral nutrition, cyclosporine, prednisone, cotrimoxazole 3×/week, iron, calcium, and vitamins K and D 1×/wk. On admission, he had microcytic anemia (93 g/L), hypoalbuminemia (17 g/L), vitamin D deficiency (22 nmol/L), low ferritin (9.4 µg/L), low immunoglobulin G (IgG), low selenium, and elevated fecal calprotectin . Creatinine, liver function tests, vitamins Bndoscopy  showed ne polyps .3 They should be slowly tapered and discontinued when a sustained response is obtained. Azathioprine maintained remission without relapse in 5 patients of the Mayo Clinic for a median of 4.5 years.4 It is the most recognized steroid-sparing agent in CCS. As for anti–tumor necrosis factor drugs, high tissue tumor necrosis factor-α level was reported. Infliximab induced complete remission and maintenance in one case.5 Infiltration by IgG4-positive plasma cells was also present in half of examined CCS polyps6 to a lesser extent than in IgG4-related disease, but to our knowledge, no experience was reported with a B-cell depletion therapy such as rituximab. Other treatments reported in the literature include various combinations of histamine-receptor inhibitor, proton pump inhibitor, mesalamine, sulfasalazine, antibiotics, antiplasmin agent, and cromolyn sodium.3 Treatment of H pylori was favored when present as remissions were reported after eradication.7 Surgery  has been used to improve hypoproteinemia in some cases. Calcineurin inhibitors repress T-cell activation by blocking interleukin-2 production. Success was reported in 2 patients on cyclosporine and 1 patient on tacrolimus.810 mTOR inhibitors block interleukin-2 response, but to our knowledge, experience in CCS is lacking. Arguments in favor of using sirolimus in our patient with refractory CCS were the following: (1) sirolimus (rapamycin) 5 ng/mL11 and everolimus12 have been successfully used in adult refractory inflammatory bowel disease; (2) it also led to positive outcomes in 10/14 pediatric patients with refractory inflammatory bowel disease13; (3) and mostly, sirolimus reduced the quantity and size of polyps in Peutz-Jeghers syndrome mouse model, possibly via an antiangiogenic effect.14 In the present case report, sirolimus provided significant clinical and endoscopic benefit apparent within, respectively, 2 and 8 weeks, with an ongoing remission of 6+ months in a patient who failed multiple treatments over 2 years including steroids, azathioprine, adalimumab, and cyclosporine. We suggest that sirolimus could be a valuable therapeutic option for CCS patients.We report the case of a patient with CCS refractory to all suggested treatments who achieved impressive clinicoendoscopic remission and maintenance when treated with sirolimus. Current standard treatment of CCS is based on the 500+ cases reported in the literature. All patients should receive aggressive nutritional support and parenteral nutrition as needed. Corticosteroids induced and maintained remission in an important proportion of the reported cases with prednisone 40 mg/day being the recommended dose.


Scientific Reports 10.1038/s41598-017-14980-y, published online 31 October 2017Correction to: The original version of this Article contained a typographical error in the Abstract.“Under oxic conditions the CH4 production showed a dependence on the water content.”now reads:“Under anoxic conditions the CH4 production showed a dependence on the water content.”.In addition, in the original version of this Article, Affiliation 1 was incorrectly listed as ‘GFZ German Research Centre for Geosciences, Section 5.3 Geomicrobiology, Telegrafenberg, 14469, Potsdam, Germany’. The correct affiliation is listed below:GFZ German Research Centre for Geosciences, Section 5.3 Geomicrobiology, Telegrafenberg, 14473, Potsdam, Germany.This has now been corrected in the PDF and HTML versions of the Article.