SS genes were cloned and characterized from banana fruit. Among them, MaSSIII-1 showed the highest expression in banana pulp during fruit development at transcriptional levels. Further Western blot analysis suggested that the protein was gradually increased during banana fruit development, but drastically reduced during storage. This expression pattern was highly consistent with changes in starch granules, amylopectin content, and SS activity at the late phase of banana fruit development. Lastly, overexpression of MaSSIII-1 in tomato plants distinctly changed the morphology of starch granules and significantly increased the total starch accumulation, amylopectin content, and SS activity at mature-green stage in comparison to wild-type. The findings demonstrated that MaSSIII-1 is a key gene expressed in banana fruit and responsible for the active amylopectin biosynthesis, this is the first report in a fresh fruit species. Such a finding may enable the development of molecular markers for banana breeding and genetic improvement of nutritional value and functional properties of banana fruit.Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline during storage. The SS activity started to increase later than amylopectin and starch content. Secondly, four putative  Starch is the most widespread carbohydrate storage molecule in plants and plays a vital role in human nutrition, food industry and chemical manufacturing. The physicochemical properties of starch are strongly affected by its two key components, amylose and amylopectin . In highvia α-1,4-glycosidic linkages, and directly catalyzes the amylopectin biosynthesis  is not only one of the most highly consumed fruits in the world but also widely used as a staple food in the tropical and subtropical regions (MaGBSSI (DBE (SUCROSE PHOSPHATE SYNTHASE (SPS), SUCROSE SYNTHASE (SuSy), and INVERTASE  seedlings were obtained from the banana tissue culture center  and were grown at 28°C with 70% humidity, 200 μmol⋅m-2⋅s-1 light intensity, and long day condition (16 h light/8 h dark cycle). When banana seedlings produced five leaves, they were planted in the Institute of Tropical Bioscience and Biotechnology banana plantation  until harvest. Roots, stems, leaves, bracts, flowers, peels, and pulps at 60 days after emergence from the pseudostem (DAF) were collected separately using tweezers, frozen immediately in liquid nitrogen, and stored at -80°C until expression analysis in different tissues. For each biological replicate, two banana hands having a similar developmental stage from two plants were selected and six fingers from the hands were obtained. Banana pulps at 0, 10, 20, 30, 40, 50, and 60 DAF were collected, immediately frozen in liquid nitrogen, and stored at -80°C until starch/amylopectin quantification, mRNA and protein expression analysis.Banana (Banana hands (60 DAF) were separated into individual fingers representing the same developmental stage. For natural ripening treatment, banana samples were kept at 22°C and allowed to ripen in open air. In accordance with the previously published banana ripening stages , fruits Pulp samples were fixed in stubs using double-faced tape and coated with a 10 nm thick platinum layer using a Bal-tec MED 020 Coating system  before analysis with a FEI Quanta 600 FEG Scanning Electron Microscope . SEM observations were performed using the secondary electron mode operating at 15 kV.3)2, placed in a boiling water bath for 10 min, and centrifuged for 4 min at low speed . The supernatant was then transferred to a 20 mL volumetric flask. The total starch, amylose, and amylopectin contents of the extract were determined following Banana pulp was immersed in 0.5% (w/v) sodium bisulfite solution for 10 min to prevent browning, and then dried at 40°C for 20∼24 h. The dried pulp was milled to powder and suspended in 5 mL 80% (w/v) Ca, SSII (GSMUA_Achr6G23190_001), SSIII-1 (GSMUA_Achr11G18570_001), and SSIII-2 (GSMUA_Achr5G00700_001), were retrieved from the banana DH-Pang (AA group) genome sequence database1. Four primer pairs (Supplementary Table S1) were designed based on these sequences. The SS coding sequences were submitted to BLAST analysis to recover their corresponding genomic DNA sequences. Exon lengths were calculated by alignment of genomic DNA sequences with cDNA sequences, and introns were determined according to the “GC-AG” rule (RNA purification and cDNA synthesis were performed as previously described by AG” rule . The dedSupplementary Table S1). Moreover, PCR products were sequenced to confirm the specificity of primer pairs. Amplification efficiencies of primer pairs ranged from 0.9 to 1.1. The levels of MaSS and SISSIII-1 expression were quantified by qRT-PCR using an iQ5 real-time PCR detection system  with the SYBR ExScript RT-PCR Kit . ACTIN or GAPDH that were verified to be constitutive in expression and hence suitable to be used as internal controls were used as reference genes to normalize transcriptional levels of each MaSS gene and SISSIII-1 (Supplementary Table S1). Relative expression levels of four MaSS genes were analyzed in three technical replicates and calculated using the 2-ΔΔCT method  glycerol  and incubated at 30°C for 10 min. The supernatant (SS enzyme solution) was used to Western blot analysis.Pulp samples (0.5 g) were homogenized with a set of mortar and pestle on ice in an equal volume of solution, which consisted of 50 mM HEPES-NaOH (pH 7.4), 2 mM MgClglycerol . A 20 μLTM-N+ membranes . Membranes were probed with rabbit anti-MaSSIII-1 polyclonal antibody  and banana actin antibody (control) in 1:1,000 dilution in PBS-Tween 20 plus 3% BSA, respectively, followed by alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody  in 1:1,000 dilution. Positive signals on the membranes were detected with a 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) solution .For Western blot analysis, 30 μL of the SS enzyme solution from banana pulps at different developmental stages extracted by the above methods was loaded on each lane and separated on a 12% polyacrylamide gel. Proteins were then transferred onto HybondMaSSIII-1 coding region was inserted into the pCAMBIA-1302 vector under the transcriptional control of CaMV 35S promoter following a double digestion with Nco I and Spe I. The pCAMBIA-1302-MaSSIII-1 was transferred into A. tumefaciens strain LBA4404. Transgenic tomato (Solanum lycopersicum L.) plants were generated using the Agrobacterium-mediated transformation as previously described  agarose gel and transferred onto Hybond-N+ nylon membranes   . Probes t-tests, at significant levels of p < 0.05 (∗), and p < 0.01 (∗∗).Three biological replicates were performed for each sample, unless specified otherwise. Statistical analyses were performed using Microsoft Excel and SPSS . Analysis of variance was used to compare the statistical difference based on Student’s Figure 1A). As observed using SEM, starch granules in banana pulp were barely detectable at the initial sampling time, i.e., 0 DAF, but the oval-shaped starch granules become clearly visible and were significantly increased in size as the fruit developed from 10 (6.0∼8.2 μm) to 60 DAF (28.0∼35.7 μm). Morphological comparison of starch granules during this period of time indicated that the shape and number of starch granules remained consistent, but the size was significantly increased and maximized at 60 DAF .During banana fruit development the fruit size gradually increases and it becomes curled in a crescent shape as fruit develops .Starch granules in mature banana pulp were also observed using SEM at the 30 DPH during storage at 22°C, at 5 days’ intervals. Compared to the first time point (0 DPH), the quantity of starch granules per unit volume decreased rapidly over time. After 15 DPH of storage, starch granules were hardly detectable , implying that after maturity starch and amylopectin may have experienced a rapid degradation process. However, the SS enzyme activity increased later than starch and amylopectin accumulation at maturity and had an abrupt decline at the very beginning of storage .The total starch content and amylopectin content in pulp increased as fruit developed and peaked at 50 DAF  before decline at the 60 DAF time point and continued to decrease gradually during storage  genome sequence database2 revealed that MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 are located on chromosome 3, 6, 11, and 5, respectively . The sequence analysis also revealed markedly different primary structures as MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 contain 25, 6, 5, and 9 exons, respectively. The stop codon usage is also different as TAA was used by MaSSI and MaSSIII-1, but TGA was used by MaSSII and MaSSIII-2 .Full-length cDNAs encoding Supplementary Table S2), as previously identified in SS enzymes from amaranth (Supplementary Table S3), as previously identified in SSIII enzymes from wheat . MaSSIII-1 and MaSSIII-2 are aligned next to each other, forming a side branch in association with other SSIII sequences from monocot species. Similarly, MaSSI and MaSSII are clustered together with their respective orthologs from other species. This may also indicate that the divergence of the SSI, SSII and SSIII occurred prior to the speciation of these plant species, but the separation of SSIII-1 and SSIII-2 might be of a more recent history likely through a gene duplication and subsequent divergence.To elucidate phylogenetic relationship of banana MaSS genes were analyzed by qRT-PCR in seven different tissues, including roots, stems, leaves, bracts, flowers, peels, and pulp (60 DAF) . MaSSI was expressed at low levels in all tissues examined with the highest expression in pulps. MaSSII was also found to express low in all the vegetative tissues, including roots, stems, leaves, and bracts, and particularly low in flowers and fruit tissues. The expression patterns of MaSSIII-1 and MaSSIII-2 were very similar, with highest expression in pulps and leaves (starch content in leaves was approximately 22.67 ± 1.32%), but that MaSSIII-1 had the highest levels of expression.The spatial expression patterns of the four MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 during banana fruit development was also determined by qRT-PCR . Despite of being highly variable, the expression levels of MaSSI and MaSSII genes were found to be generally low throughout the entire banana pulp development. Both MaSSIII-1 and MaSSIII-2 showed a sharp increase of expression during the fruit development, the increase in expression occurred earlier in MaSSIII-1 than in MaSSIII-2, with the highest expression detected at the last sampling stage when banana had reached its maturity. However, the expression of MaSSIII-1 was about 10∼20-fold higher than that of MaSSIII-2 during the fruit development.The temporal expression of MaSS genes were also found to be differentially expressed . MaSSII showed consistently low levels of expression throughout the storage period, but the MaSSI expression was slightly increased during storage. The seemingly negative association between MaSSI expression and starch degradation needs to be further investigated. Although there can be a lack of congruency between transcript and activity, it is possible that there can be some starch biosynthesis occurring even during net degradation . The Western blot results was consistent with changes in starch granules , amylopectin content  and SS activity  during banana development and ripening, suggesting the expression of MaSSIII-1 protein might play an important role in regulating amylopectin metabolism in banana fruit during development and ripening.Western blot analysis with rabbit anti-MaSSIII-1 polyclonal antibody as a probe indicated that the size of MaSSIII-1 is approximately 90.0 kDa, which is consistent with the molecular weight (90.69 kDa) as predicted by the PeptideMass program. The expression of MaSSIII-1 protein was gradually increased during banana fruit development, but drastically reduced from 0 to 30 DPH of storage  were identified by Southern blot analysis . The fruit shape, starch granule morphology, gene expression, total starch content, amylopectin content, and SS activity were investigated in the wild-type (WT) and the MaSSIII-1 overexpressing transgenic plants.To further examine the function of MaSSIII-1 transgenic plants did not change , but the morphology of starch granules was significantly altered from immature green (IG) to orange-breaker (BR) in transgenic tomato plants, severe cracks in the surface of starch granules were observed . For fruit weight and volume, there are no significant differences between MaSSIII-1 transgenic plants and WT . Overexpression of MaSSIII-1 in tomato plants significantly increased the gene expression , SISSIII-1  expression in tomato fruit was lower than that of MaSSIII-1 transgenic plants . At IG and mature-green (MG) stages, total starch content, amylopectin content, and SS activity showed significant differences between transgenic plants and WT . Especially at the MG stage, the gene expression, total starch content, amylopectin content, and SS activity significantly increased 55∼120-fold, 13.47∼15.83 mg⋅g-1, 6.36∼9.17 mg⋅g-1, and 0.54∼0.80 U⋅min-1 in the transgenic fruits compared to WT, respectively.Compared to WT, the fruit shape of Despite of the extensive studies on cereal starch, little information is available regarding the dynamics of starch accumulation (net result of starch synthesis and degradation), SS activity, and amylopectin metabolism during fruit development and storage in fresh starchy fruits, such as banana. In tomato, starch is transiently accumulated during fruit development and degraded within the lifecycle of that organ, while starch synthesis and degradation are occurring simultaneously . HoweverGBSS genes have been cloned and identified in banana  in plants and play important roles in amylopectin biosynthesis . GBSS asn banana . In thisn banana . Moreoven banana . Sequenc SS gene . The MaS SS gene . Domain  SS gene . The ami SS gene .MaSSI and MaSSII were found to express in low levels in vegetative organs, flower and banana fruits, similar to their homologs in kidney bean . Such an observation is consistent with previous studies in rice, wheat, and Arabidopsis , suggesting these parameter increases may be attributed to MaSSIII because of increased transcript and polypeptide. Similar results were reported in wheat (MaSSIII-1 in tomato fruit showed severe cracks in the surface of starch granules and changed the starch granules morphology. This study suggests that MaSSIII-1 is a key gene in the amylopectin biosynthesis and therefore it could be used as a useful tool for marker assisted molecular breeding in banana.SSIII protein as a catalytic factor plays an important role in amylopectin metabolism . SSIII cin maize . Inhibitn potato . SSIII an potato . In fresin wheat  and ricein wheat , but thiSS genes encoding MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 were cloned and characterized. Expression pattern of only MaSSIII-1 was highly consistent with dynamic changes in starch granules, amylopectin content, and SS activity. MaSSIII-1 transgenic lines distinctly changed the morphology of starch granules. Overexpression of MaSSIII-1 in tomato plants significantly increased the amylopectin accumulation and SS activity in comparison to WT. This is the first report about the MaSSIII-1 gene involved in amylopectin metabolism in a fresh fruit species. These findings establish a solid foundation to further regulate the amylopectin metabolism in banana fruits or other fresh fruits using the MaSSIII-1 or its homologous genes.Starch synthase activity increased along with the amylopectin accumulation at later stages of banana fruit development, but declined during storage. Four ZJ and BX conceived and designed the experiments. HM, PS, QL, CJ, JL, and WH performed the experiments and carried out the analysis. HM, PS, and QL designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.ZJ and BX conceived and designed the experiments. HM, PS, QL, CJ, JL, and WH performed the experiments and carried out the analysis. HM, PS, and QL designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.


Lonchura maja).A novel variant of finch polyomavirus has been identified and sequenced from a diseased white-headed munia ( Gammapolyomavirus genus of the Polyomaviridae family, including the Aves polyomavirus 1, Anser anser polyomavirus 1, Pyrrhula pyrrhula polyomavirus 1, Corvus monedula polyomavirus 1, Serinus canaria polyomavirus 1, Cracticus torquatus polyomavirus 1, and Pygoscelis adeliae polyomavirus 1  in a flock of a Hungarian aviculturist. Histopathological examination revealed liver failure, nephritis, and myocarditis in the affected bird. From the pooled liver and kidney sample, polyomavirus DNA was detected by a broad-spectrum nested-PCR assay  and Gouldian finch , respectively (Pyrrhula pyrrhula polyomavirus 1 (The genome of the Hungarian polyomavirus strain, 14534/2011, was 5,284 bp in length and showed the highest overall nucleotide sequence homology of 91.1 and 91.2% with KX756154.The whole genome sequence of the strain 14534/2011 has been deposited in GenBank under the accession no.


Capsicum annuum). Herein, CaWRKY22, a member of a subgroup of IIe WRKY proteins from pepper, was functionally characterized in pepper immunity against Ralstonia Solanacearum. CaWRKY22 was found to target the nuclei, and its transcript level was significantly upregulated by Ralstonia Solanacearum inoculation (RSI) and exogenously applied salicylic acid (SA), Methyl jasmonate (MeJA), or ethephon (ETH). Loss-of-function CaWRKY22, caused by virus-induced gene silencing (VIGS), enhanced pepper’s susceptibility to RSI. In addition, the silencing of CaWRKY22 perturbed the hypersensitive response (HR)-like cell death elicited by RSI and downregulated defense-related genes including CaPO2, CaPR4, CaACC, CaBPR1, CaDEF1, CaHIR1, and CaWRKY40. CaWRKY22 was found to directly bind to the promoters of CaPR1, CaDEF1, and CaWRKY40 by chromatin immuno-precipitation (ChIP) analysis. Contrastingly, transient overexpression of CaWRKY22 in pepper leaves triggered significant HR-like cell death and upregulated the tested immunity associated maker genes. Moreover, the transient overexpression of CaWRKY22 upregulated the expression of CaWRKY6 and CaWRKY27 while it downregulated of the expression of CaWRKY58. Conversely, the transient overexpression of CaWRKY6, CaWRKY27, and CaWRKY40 upregulated the expression of CaWRKY22, while transient overexpression of CaWRKY58 downregulated the transcript levels of CaWRKY22. These data collectively recommend the role of CaWRKY22 as a positive regulator of pepper immunity against R. Solanacearum, which is regulated by signaling synergistically mediated by SA, jasmonic acid (JA), and ethylene (ET), integrating into WRKY networks with WRKY TFs including CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.The WRKY web, which is comprised of a subset of WRKY transcription factors (TFs), plays a crucial role in the regulation of plant immunity, however, the mode of organization and operation of this network remains obscure, especially in non-model plants such as pepper ( Arabidopsis and rice. However, the organization of transcriptional networks and their functional coordination to regulate plant responses to different stresses, especially in non-model plants, remains poorly understood.Being sessile, plants frequently encounter various biotic and abiotic stresses individually, and in some cases, collectively . To protArabidopsis and rice. A significant functional divergence among close structural homologs of WRKY proteins from different plant species was recently suggested  present in the promoters of their target genes . To testut W-box . These rCaWRKY40 was bound by CaWRKY22, we speculated that CaWRKY40 was transcriptionally regulated by CaWRKY22. To test this possibility, and to further assay the possible feedback regulation of CaWRKY40 by CaWRKY22, QRT-PCR was employed to check the possible modulation of CaWRKY22 by transient overexpression of CaWRKY40, or its silencing, as well as the possible effect of CaWRKY22 transient overexpression or silencing on the expression of CaWRKY40. The results showed that transcription of CaWRKY22 in CaWRKY40-transiently-expressing pepper leaves increased at 24 and 48 hpi compared to the control  triggered by flg22, chitin, or submergence modulating by the MAP kinase cascade   solution was used to destain the DAB-stained pepper leaves and kept in 95% absolute ethanol [Trypan blue and 3,3′-diaminobenzidine (DAB) staining was performed according to the previously published method of ,22,69. FCaWRKY22 silencing in pepper plants, following the method of our previous studies [Agrobacterium cells containing TRV1 and TRV2, TRV2-CaWRKY22, or TRV2-PDS (OD600 = 0.8) constructs were mixed in a 1:1 ratio, respectively. This mixture was infiltrated into cotyledons of 2-week-old pepper plants using a syringe without a needle. The Agrobacterium-inoculated pepper plants were grown in a growth chamber at 16 °C in the dark for 56 h with 45% relative humidity, and then transferred into a growth room at 25 ± 2 °C, 60–70 µmol photons m−2·s−1 and a relative humidity of 70%, under a 16-h light/8-h dark cycle.The Tobacco Rattle Virus (TRV)-based virus induced gene silencing (VIGS) system was employed for  studies ,22,71,72Agrobacterium cells containing the 35S:CaWRKY22-flag vector were cultured to OD600 = 1.0 in LB medium containing the corresponding antibiotics overnight. These cells were then centrifuged at 7800 rpm, 28 °C for 10 min. The pellet was suspended into the induction medium  and adjusted to OD600 = 0.8. The suspension was infiltrated into pepper leaves using a needleless syringe. Later, the infiltrated plants were regularly observed for HR cell death, and harvested for trypan blue or DAB staining. RNA extraction used for detection of the expression of immunity associated genes.Total RNA was extracted from pepper leaves samples and wild type seedlings, using TRIzol reagent , were reverse transcribed by using the PrimeScript RT-PCR kit . To determine the relative transcription level of targeted genes, qRT-PCR and the corresponding data processing were performed, according to the method used in our previous studies ,21,22,722O thrice, and immediately incubated in 20 mL of double distilled water. Discs were kept in a shaker with gentle shaking (60 rpm) for 1 h at room temperature. Ion conductivity was recorded using a conductivity meter .Ion leakage was measured as previously described with slight modifications . The sixAgrobacterium cells carrying 35S:CaWRKY22-HA or 35S:CaWRKY40-HA were infiltrated into the pepper leaves at the eight leaf stage. The infiltrated leaves were harvested at the required time point and ~2 g leaves of pepper were fixed with 1.0% formaldehyde for 5 min. The chromatin was sheared by sonication to an average length of 300–500 bp and immuno-precipitated with antibody against hemagglutinin . The immuno-precipitated DNA was analyzed for enrichment of CaWRKY22 at the promoter region of targeted genes by common ChIP-PCR. The primers used for ChIP-PCR analysis are listed in The ChIP assay was carried out according to a previously described protocol . The AgrRalstonia solanccearum by directly targeting CaWRKY40 and incorporating into a WRKY network including CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.The data in the present study indicates that CaWRKY22 targets to nuclei, is up-regulated by RSI and confer enhanced immunity against


Purpose: The lack of biomarkers that can distinguish aggressive from indolent prostate cancer has caused substantial overtreatment of clinically insignificant disease. Here, by genome-wide DNA methylome profiling, we sought to identify new biomarkers to improve the accuracy of prostate cancer diagnosis and prognosis.COL4A6, CYBA, TCAF1 (FAM115A), HLF, LINC01341 (LOC149134), LRRC4, PROM1, and RHCG, were selected from Illumina Infinium HumanMethylation450 BeadChip analysis of 21 tumor (T) and 21 non-malignant (NM) prostate specimens. Diagnostic potential was further investigated by methylation-specific qPCR analysis of 80 NM vs. 228 T tissue samples. Prognostic potential was assessed by Kaplan-Meier, uni- and multivariate Cox regression analysis in 203 Danish radical prostatectomy (RP) patients (cohort 1), and validated in an independent cohort of 286 RP patients from Switzerland and the U.S. (cohort 2).Experimental design: Eight novel candidate markers, RHCG-TCAF1 was predictive of biochemical recurrence (BCR) in cohort 1, independent of the established clinicopathological parameters Gleason score, pathological tumor stage, and pre-operative PSA ): 2.09 (1.26 - 3.46); P = 0.004), and this was successfully validated in cohort 2 (HR (95% CI): 1.81 (1.05 - 3.12); P = 0.032).Results: Hypermethylation of the 8 candidates was highly cancer-specific (area under the curves: 0.79-1.00). Furthermore, high methylation of the 2-gene panel RHCG-TCAF1 panel adds significant independent prognostic value to established prognostic parameters for prostate cancer and thus may help to guide treatment decisions in the future. Further investigation in large independent cohorts is necessary before translation into clinical utility.Conclusion: Methylation of the  Prostate cancer is the most commonly diagnosed non-cutaneous malignancy in men in the Western world . The diaMethylation of CpG dinucleotides in promoter regions is an essential mechanism of long-term gene silencing. In human malignancies, aberrant hypermethylation of promoter-associated CpG islands (CGIs) is a well-established mechanism for tumor suppressor gene (TSG) silencing . While rCOL4A6 , CYBA , TCAF1 (TRPM8 channel-associated factor 1 (previously FAM115A)), HLF (hepatic leukemia factor), LINC01341 (long intergenic non-protein coding RNA 1341 (previously LOC149134)), LRRC4 (leucine rich repeat containing 4), PROM1 (prominin 1), and RHCG . Hypermethylation of all candidates was highly cancer-specific in surgical specimens as well as in diagnostic needle biopsy samples : 0.79-1.00). Finally, a 2-gene panel comprising RHCG and TCAF1 methylation was developed in the training cohort (203 Danish prostate cancer patients) and successfully validated in the validation cohort (286 Swiss/American prostate cancer patients) where it added significant independent prognostic value to routine clinicopathological parameters. This is the first study to show a prognostic biomarker potential for RHCG and TCAF1 methylation in prostate cancer.Here, we performed genome-wide DNA methylation profiling of 21 tumor (T) and 21 non-malignant (NM) prostate tissue specimens using the Illumina Infinium HumanMethylation450 BeadChip (450K array), and selected 8 novel methylation marker candidates associated to the promoter regions of 8 genes for further investigation: vs. NM samples, we identified 37,763 differentially methylated CpG sites , the majority of which were hypermethylated , and 9 true normal (N) prostate tissue samples , in addiTable 1N = 324). Next, filtering for gene association (according to Illumina annotations) generated a final list of 259 top candidate DMCs , associated to 163 genes , LRRC4, PROM1, RHCG, and TCAF1 (FAM115A) , generating a shortlist of the most differentially methylated CpGs  Figure . None ofTable 2Table 3vs. malignant cell lines, as was observed for NM vs. malignant tissue specimens in the 450K analysis. Moreover, we performed in-silico validation using 450K data from The Cancer Genome Atlas (TCGA) [N = 85; NM bladder, N = 10; kidney cancer, N = 244; NM kidney, N = 136). We found that hypermethylation of COL4A6, CYBA, LINC01341, and RHCG was highly prostate cancer-specific  in the 5 prostate cell lines. BS showed low methylation levels in NM cell lines  and high levels in malignant cell lines  for all 8 candidates, and fully corroborated the 450K results for these cell lines . Notablys (TCGA) , 7 for 2specific , suggestLINC01341 lacked annotation in both datasets, 6 of the remaining 7 candidate genes were downregulated in T vs. NM samples, consistent with epigenetic silencing through aberrant promoter hypermethylation , suggesting that hypermethylation of this region may stimulate transcription from the upstream TSS  and the large TCGA  RNA-seq datasets. While the non-coding hylation . TCAF1 wSS TSS1,  in at leCOL4A6, CYBA, HLF, LINC01341, LRRC4, PROM1, RHCG, and TCAF1 are frequent targets of hypermethylation in prostate cancer, and that aberrant promoter hypermethylation contributes to downregulation of 6 of these genes in this malignancy.Together, these data indicate that HLF) to 0.91 (LINC01341). At 96.7% fixed specificity (HLF) to 82.8% (TCAF1). To further examine the diagnostic potential, we performed qMSP analysis on 25 malignant and 50 NM (25 AN + 25 N) diagnostic needle biopsy (DNB) specimens. Again, highly significant cancer-specific hypermethylation was observed for all 8 candidates, with AUCs ranging from 0.97 (HLF) to 1.00   to 96% .To investigate the diagnostic potential of our 8 candidates, methylation levels were examined in 203 T and 30 NM prostate tissue samples cohort 1, Table , Table 1cificity , sensiti, TCAF1) . At 96% , TCAF1) , sensitiThese results from qMSP analysis of both surgical and DNB specimens confirmed that all 8 genes are highly frequent targets of prostate cancer-specific aberrant hypermethylation, and that our qMSP assays can be used on DNBs.To investigate their possible prognostic potential in prostate cancer, methylation levels of each candidate was initially compared to routine clinicopathological factors in radical prostatectomy (RP) cohort 1, as well as in an independent validation cohort including 286 RP patients  after RP. Initially, candidate gene methylation (as a continuous variable) was investigated in univariate Cox regression analysis in RP cohort 1. Here, RHCG and TCAF1 were significant predictors of BCR, independent of routine clinicopathological parameters (RHCG hazard ratio (HR) ): 1.60 (1.07-2.38), P = 0.021; TCAF1 HR (95% CI): 2.82 (1.54-5.17), P = 0.001, Table RHCG and TCAF1 were subsequently tested in the validation cohort, where both candidates were significant adverse predictors of BCR, independent of clinicopathological variables (RHCG HR (95% CI): 1.55 (1.23 - 1.94), adj. P = 0.001; TCAF1 HR (95% CI): 1.48 (1.18 - 1.85), adj. P = 0.001, Table RHCG and TCAF1 hypermethylation, in addition to routine clinicopathological parameters, were independent predictors of BCR in multivariate analysis in both RP cohorts.Next, in cohort 1, we performed multivariate Cox regression analysis including the 4 candidates significant in univariate analysis, in addition to GS, pT stage, surgical margin (SM) status, and pre-operative PSA. Here, both .60 1.07-.38, P = .60 1.07-.38, P = RHCG and TCAF1 methylation to a model of clinicopathological factors  improved the C-index from 0.769 to 0.782, whereas in cohort 2, adding RHCG and TCAF1 methylation to the clinicopathological model  increased the C-index from 0.703 to 0.718, indicating that RHCG and TCAF1 methylation improved the predictive accuracy in both cohorts and thus carries prognostic potential beyond that of routinely used clinicopathological factors.We applied Harrell's C-index to estimate the predictive accuracy of the multivariate model. In cohort 1, adding RHCG and TCAF1, respectively, were divided into high- or low- methylation by receiver operating characteristics (ROC) analysis (BCR status at 36 months follow-up) and analyzed for their ability to predict BCR. In cohort 1, high methylation of each candidate significantly predicted BCR in both Kaplan-Meier , where patients with high methylation of both genes were classified into the high-methylation group, and the remaining patients into the low-methylation group. In cohort 1, high methylation of dRHCG-TCAF1 significantly predicted BCR in univariate , where patients in cohort 1 were stratified into 3 groups based on whether they had high methylation of both candidates (high-methylation), one candidate (intermediate-methylation), or neither candidate (low-methylation). By Kaplan-Meier analysis, patients in the high-methylation group had significantly higher risk of BCR than patients in the 2 remaining groups, where patients with low methylation also had the lowest risk of BCR , further supporting their diagnostic potential. Moreover, as proof of principle, we demonstrated that our qMSP assays can be used on DNBs despite scarce sample amounts. While a molecular diagnostic test that can detect histologically verified prostate cancer in DNBs might have limited clinical utility, detection of methylation-based cancer field effects in morphologically normal prostate biopsies may be used to guide the need for repeat biopsy in men with exclusively cancer-negative DNBs but persistently elevated PSA, which remains a major clinical challenge , 11. AltRHCG, TCAF1, and the 2-gene panel RHCG-TCAF1 as novel independent adverse predictors of BCR after RP. Prior to this, only a few candidate methylation markers have demonstrated prognostic potential in more than one prostate cancer cohort by multivariate analysis adjusting for established clinicopathological parameters [PITX2 [GABRE~miR-452~miR-224 [C1orf114 (CCDC181) [C1orf114/HAPLN3 and AOX1/C1orf114/HAPLN3 [RHCG and TCAF1 as well as of the previously identified prognostic methylation marker candidates. These candidates should be investigated individually, as well as in combinations, to identify their true prognostic potential.Here, using two large prostate cancer patient cohorts, we identified and independently validated rameters . These as [PITX2 , 13, GAB~miR-224 , C1orf11CCDC181) , and the4/HAPLN3 . Furthere.g. by allowing active surveillance of low-risk patients and immediate treatment of high-risk patients. While the incremental gain in prognostic information obtained by adding parameters to existing models is often modest [RHCG-TCAF1 provided a C-index of 0.777 in cohort 1 and 0.717 in cohort 2. C-index analysis in reduced models, i.e. leaving out one variable at a time, demonstrated modest C-index contributions for any single variable (range 0.008-0.028 in cohort 1 and 0.011-0.032 in cohort 2), corresponding to a maximum contribution of 3.6% and 5.2% in cohort 1 and 2, respectively (data not shown). Moreover, the observed increments in C-index for our novel 2-gene methylation marker panel are comparable to those reported for other prognostic DNA methylation marker candidates [i.e. Decipher [Current prognostic prostate cancer classification systems rely on histopathological criteria, which cannot accurately predict whether a tumor will progress to clinically relevant disease or remain indolent. Novel biomarkers that enable distinction between aggressive and indolent cancer at the time of diagnosis could improve patient management significantly, n modest , the incn modest . The fulndidates , 13, 14,Decipher  and ProlDecipher .RHCG-TCAF1 has the potential to improve the accuracy of prostate cancer prognosis at the time of diagnosis, where only pre-operative clinicopathological parameters are available . Notably, more than 50% of prostate tumors are upstaged and/or upgraded after RP [Furthermore, as cancer-specific hypermethylation was detectable in DNBs, our results suggest that after RP , furtherRHCG in prostate tumors, which is consistent with previous reports of RHCG downregulation in kidney and oesophagal carcinomas [RHCG encodes an epithelial ammonia transporter that is widely expressed in the kidney, liver and intestinal tract, as well as in male reproductive organs, where it contributes to multiple components of fertility [RHCG in the normal and cancerous prostate, however, is unknown and further studies are warranted.The potential functional roles of our 8 candidate methylation markers in prostate physiology and malignancy are largely unknown. Here, we found that expression of 6 of these genes was significantly downregulated in prostate cancer, consistent with epigenetic silencing and suggesting a possible function in prostate carcinogenesis and/or tumor progression. More specifically, we observed promoter hypermethylation and downregulation of rcinomas , 22. RHCertility . The rolTCAF1 was recently identified as a positive regulator of TRPM8, an ionotropic testosterone receptor highly expressed in various organs, including the prostate [TCAF1 expression was unaltered or modestly upregulated in localized prostate tumors compared to NM prostate tissue samples, consistent with a previous study [TCAF1 in prostate cancer, is involved in alternative TSS-regulation in normal and malignant tissue [TCAF1 methylation and potential isoform-specific expression patterns in NM and malignant prostate cells to elucidate its possible role in prostate cancer tumorigenesis and progression.prostate , 25, butprostate . Accordius study . Severalt tissue . Moreovet tissue . Future PROM1 (CD133) expression, which is consistent with reports of PROM1 downregulation in several malignancies, including prostate cancer [PROM1 encodes a transmembrane glycoprotein widely used as a stem cell marker, but its potential role in prostate cancer is unknown. We also observed cancer-specific promoter hypermethylation and downregulation of COL4A6, which encodes a subunit of the epithelial basement membrane protein collagen IV. Our observations are consistent with reports of COL4A6 downregulation in a range of cancers, including prostate cancer [CYBA, HLF, and LRRC4. CYBA encodes a subunit of an NADPH oxidase, whereas HLF encodes a transcriptional activator of the proline and acidic-rich (PAR) protein family. LRRC4 is a proposed TSG involved in nervous system development and differentiation [LINC01341 [LINC01341 in the investigated datasets. Thus, further studies are needed to elucidate the possible role of LINC01341 in normal and malignant prostate biology.We observed cancer-specific promoter hypermethylation and downregulation of e cancer , 30. PROe cancer . Cancer-ntiation , and wasPROM1 CD1 expressie.g. radiation treatment or androgen deprivation), there are currently no established adjuvant lines of treatment for patients after RP. Thus, future studies should evaluate the prognostic potential of these novel candidate methylation markers in DNBs, and thus investigate whether they can predict prostate cancer aggressiveness at the time of diagnosis and thereby help guide treatment decisions.There are some limitations to the present study. The prognostic analyses for our candidates were based on RP specimens, and while it could be useful to identify high-risk patients in need of adjuvant therapy post-RP , thus indicating the robustness of our methylation marker candidates.Moreover, the RP cohorts used for analysis of prognostic biomarker potential were of moderately different compositions, with patients in cohort 1 suffering more BCRs and having tumors with higher pT stage and higher median pre-operative PSA compared to patients in cohort 2 ) from patients undergoing transrectal ultrasound-guided biopsy due to suspicion of prostate cancer were obtained from Dept. of Pathology, Aarhus University Hospital, DK, as described elsewhere . We obtained normal (N) biopsy samples from 25 patients with exclusively cancer-negative biopsies, tumor biopsies from 25 patients diagnosed with prostate cancer (T), and histologically normal (AN) biopsies from 25 patients with prostate cancer detected in other biopsies.Radical prostatectomy cohorts: Cohort 1 (training cohort) consisted of 566 curatively intended RP patients with histologically verified clinically localized prostate cancer (FFPE) from the Dept. of Urology, Aarhus University Hospital, DK (collected 1997-2009). All specimens were assessed by a trained pathologist. Punch biopsies were obtained from FFPE block-areas with > 80% cancer cells, as described previously [eviously , 36. Of Cohort 2 consisted of 448 and 117 curatively intended RP patients with histologically verified, clinically localized prostate cancer (FFPE) from Moffitt Cancer Center, U.S. (collected 1987-2006) and University Hospital Zurich, Switzerland (collected 1993-2001). Of the 565 patients, 19 were either lost to follow-up, had < 3 months follow-up, or suffered BCR within 3 months post-RP, 82 were excluded because of pre-/postoperative endocrine treatment or missing data for endocrine treatment, 6 were excluded due to positive lymph node status, 13 were excluded due to ethnicity (non-European descent), 118 lacked clinical data, and 41 lacked tissue in the FFPE block or had insufficient DNA concentration/quality. Final analyses comprised 286 samples Table .FFPE AN tissue samples from RP specimens and benign prostate hyperplasia (BPH) specimens  were sampled as described above for cohort 1 Table .For DNA purification from DNBs, 3-µm tissue sections were deparaffinized and DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to manufacturer's protocol. DNA from RP specimens (punch biopsies) was extracted in Denmark and Switzerland using gDNA Eliminator columns from the RNeasy plus micro kit (Qiagen) and the blood and cell culture DNA kit (Qiagen), respectively, as previously described . DNA fro2O), as well as serially diluted methylated DNA samples for standard curve analysis, were included on each plate. All reactions were run in triplicates. MYOD1 and aluC4 served as controls for DNA quality/quantity [MYOD1 was not amplified. Samples were considered negative for methylation if ≥ 2 methylation-specific reactions did not amplify. Bisulfite-converted DNA from DNBs was pre-amplified prior to qMSP . Associations between DNA methylation and clinicopathological variables were assessed by Wilcoxon rank-sum test and Spearman correlations. BCR (defined as PSA ≥ 0.2 ng/ml) was the clinical endpoint in survival analyses. Patients without BCR were censored at their last normal PSA measurement. In all cases, Hochberg . The proHochberg . For mulRHCG: ≥ 0.3608787; TCAF1: ≥ 0.519267). For the dichotomized 2-gene panel (dRHCG-TCAF1), patients were included in the high-methylation group if both genes were highly methylated. For the trichotomized 2-gene panel (tRHCG-TCAF1), patients were included in the high-methylation group if both genes were highly methylated, the intermediate group if one gene was highly methylated, and the low-methylation group if neither gene was highly methylated.Prognostic 2-gene model: For each gene, patients in cohort 1 (training cohort) were dichotomized into high/low-methylation groups by ROC analysis of BCR status at 36 months follow-up. Cohort 2 patients were dichotomized by the exact numerical cutoff values defined in cohort 1 (See Supplementary Materials and Methods.Data is available upon request.


In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism.Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism.  Manifestations can include hyperpigmentation, failure to thrive, and a poor response to illness, with hypoglycemia and hypotension. Reduced life expectancy is described, and the condition can be fatal if undetected. It is genetically heterogeneous, with some gene defects causing syndromic disease. Mechanisms of disease include steroid biosynthetic defects, adrenocorticotropic hormone (ACTH) resistance, adrenal dysgenesis, cholesterol synthesis disorders, and metabolic disorders incorporating peroxisomal and mitochondrial defects   . In brief, the number of variants was reduced by the following strategy: (i) identifying variants that were common to both individuals; (ii) excluding variants that were heterozygous; (iii) removing variants, annotated in SNP databases , with a minor allele frequency of greater than 0.01; and (iv) evaluating nonsynonymous coding variants, splice variants, and indels only. Finally, candidate variants in 2 genes (Chr10:69957117C>T in MYPN and Chr10:72628151G>A in SGPL1) were investigated for segregation with disease in the kindred by Sanger sequencing. Both variants segregated with disease in the family. MYPN encodes myopalladin, which is expressed in striated muscle and functions as a structural, signaling, and gene expression regulatory molecule in response to muscle stress. Variants in MYPN have previously been implicated in dilated cardiomyopathy, not a clinical finding in our patients, and although rare , there are 2 homozygotes noted in ExAC for this variant. Thus, we demoted this variant in MYPN and focused on the variant in SGPL1. This variant, Chr10:72628151G>A c.665G>A; p.R222Q, has a minor allele frequency of 1.656e-5; this represents 2/120744 alleles reported in the ExAC browser. Both are heterozygotes; one is from the South Asian population and the other non-Finnish European. No homozygotes are annotated in any database Affected individuals were mutation negative for the known genetic causes of PAI . WES wasSGPL1 encodes sphingosine-1-phosphate lyase (SGPL1), an important ER enzyme in sphingolipid catabolism . In kindred 3, a female (patient 5) presented with PAI at 6 months, developing nephrotic syndrome on follow-up at age 5.5 years. Patient 5 and her parents underwent WES and analysis through the GOSgene pipeline (SGPL1. Kindred 4 comprised 2 affected siblings (patients 6 and 7), manifesting PAI and nephrotic syndrome (< 1 year for both), who had a canonical splice site change . Patient 8, presenting with nephropathy (<1 year) and later developing PAI, had a frameshift mutation . We believe that the mutations in kindreds 3, 4, and 5 were novel and have not been annotated in any database. Concurrently, exome data from a cohort of 200 pediatric patients presenting with FSGS alone was examined for SGPL1 mutations, but none were identified. The frameshift and splice site mutations, p.S3Kfs*11 and p.S65Rfs*6, respectively, are predicted to result in markedly truncated mRNA message and are likely to be destroyed by nonsense-mediated mRNA decay, suggesting that the phenotype results from loss of enzyme activity and may represent a new defect of sphingolipid metabolism. The mutations p.R222Q and p.F545del are within highly conserved residues and are predicted to cause disruption to vital, highly conserved eukaryotic protein domains . Thyroid peroxidase antibodies were negative in all cases.Extraadrenal and -renal effects are described within our cohort. Generalized ichthyosis was present in most, though not all, patients . Skin biNeurodegenerative disease is seen in several of the sphingolipidoses associated with accumulating sphingolipid metabolites. Here, progressive motor and cognitive decline is described for 3 patients , with ataxia and sensorineural hearing loss also reported (patients 5 and 6) . Both PeFasting lipid profiling revealed raised total cholesterol and triglycerides in some of the patients , though + and low naive CD4+ and naive CD8+ cells, but normal proliferative capacity  and zona fasciculata (ZF), and between ZF and X-zone , the first enzyme in the steroidogenic cascade, while the classical pattern of subcapsular clusters of aldosterone synthase (CYP11B2) staining present in +/+Sgpl1 mice was replaced by a more continuous pattern in adrenals from –/–Sgpl1 mice  and others as signaling molecules . S1P has autocrine, paracrine, and distant effects, regulating cell migration, differentiation, survival, and other complex physiological processes by activating a subgroup of G protein–coupled receptors referred to as S1Pell fate .Lipids, including sphingolipids, have been shown to control steroid hormone biosynthesis in adrenal glands. Sphingolipid intermediates ceramide and sphingosine have been demonstrated to reduce steroidogenesis in vitro –28. Sphihttp://www.proteinatlas.org/) show SGPL1 expression in the tubules and glomeruli in normal human adult kidney; within the glomerulus, staining is apparently predominantly in podocytes , pulmonary , cardiac , urinary tract , thymic (atrophy), and hepatic abnormalities (disruption of liver homeostasis with defective hepatic lipid metabolism) (abolism) , 30, 39.abolism) , 39. Simabolism) . Inhibitabolism) . Partialabolism) . LymphodIn conclusion, we have identified a potentially progressive, disorder incorporating PAI and nephrotic syndrome, among other features. SGPL1 deficiency in humans has multisystemic effects, although the precise impact on sphingolipid metabolism and consequent pathogenic mechanism is yet to be elucidated and is likely to be tissue specific. Our findings highlight the importance of the sphingolipid metabolic pathway in adrenal function. A genetic diagnosis for patients with this form of PAI will be important for correct treatment, genetic counseling, and screening for comorbidities.Since strategies to modify SGPL1 activity are being explored in preclinical and clinical trials of several conditions, including multiple sclerosis and rheumatoid arthritis , 41, furhttps://www.dnanexus.com/) and Ingenuity variant analysis. SNPs with threshold coverage of at least 10 reads on the respective nucleotides were assessed.WES using the Illumina HiSeq 2000 Sequencer was conducted on 2 affected individuals (patients 2 and 3) (samples processed by Otogenetics Corp.). WES samples were prepared as an Illumina sequencing library, and in the second step, the sequencing libraries were enriched using the Agilent V4 Enrichment Kit. The captured libraries were sequenced and downstream analysis conducted via DNAnexus  were included.For confidence, call quality was set to be at least 20, read depth at least 10 in any sample; only data outside 0.1% of most exonically variable 100-base windows in healthy public genomes and outside 0.1% of most exonically variable genes in healthy public genomes  guidelines classification , being pVariants were included if they were homozygous in both cases and excluded if they were homozygous, compound heterozygous, hemizygous, heterozygous, haploinsufficient, or het-ambiguous. Variants were excluded if they were present in hetero- or homozygosity in 2 or more control subjects analyzed on the same platform.Taq DNA polymerase (Sigma-Aldrich). Cycling conditions were as follows: 95˚C for 5 minutes (1 cycle); 95˚C for 30 seconds, 55˚C for 30 seconds, and 72˚C for 30 seconds (30 cycles); and 72˚C for 5 minutes. PCR products were visualized on 1% agarose gel and sequenced using the ABI Prism Big Dye Sequencing Kit and an ABI 3700 automated DNA sequencer (Applied Biosystems) in accordance with the manufacturer’s instructions.Each exon of genes of interest, including intronic boundaries, was amplified by PCR using specific primers .The expression of SGPL1 or GAPDH  was set up in duplicate (per sample) on a Stratagene Mx3000P thermocycler using KAPA SYBR Fast qPCR Master Mix with 200 nM forward and reverse primers targeted to A commercially available SGPL1-human (Myc-DDK–tagged) SGPL1 expression vector  was used. Site-directed mutagenesis was carried out to prepare the R222Q and F545del constructs  were seeded in a 90-mm dish. Two days after transfection, cells were collected by trypsinisation, centrifuged, and washed with PBS. The pelleted cells, stored at –20°C till use, were adjusted to approximately 250 μl, with 0.25 M sucrose, 5 mM MOPS, pH 7.2, 1 mM EDTA, and 0.1% (v/v) ethanol, and sonicated  on ice. Lyase was measured as described before . Normal adult adrenals (n = 3) were placed in tubes with RIPA buffer supplemented with complete protease inhibitors and lysates obtained using a Precellys24 Tissue Homogenizer via bead beating technology. Lysates were then cleared and prepared as above. Samples were heated at 95–100˚C for 5 minutes and size-separated on 4%–12% SDS-PAGE gels . Proteins were then transferred to nitrocellulose membrane  using a semi-dry transfer blot (Bio-Rad) at 15 V for 1 hour. Blots were blocked with 5% nonfat dry milk in PBS–Tween 0.1% (blocking buffer) and immunolabeled overnight with a polyclonal anti-SGPL1 antibody . The following day, the membrane was washed 3 times in PBS–Tween 0.1% and then incubated with a goat anti-rabbit IRDye 800  at a 1:5,000 dilution, washed again, and visualized using the LI-COR Odyssey System.HEK-293 cells were transfected with the SGPL1 vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 hours, cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitors (Roche) and lysates cleared by centrifugation at 13,000 For visualization of SGPL1 in transfected mouse fibroblasts, lysates were size separated as above and membranes incubated with anti-FLAG antibodies , followed by alkaline phosphatase–conjugated secondary antibody . NBT/BCIP (Roche) was used for chromogenic detection.–/+Sgpl1 animals, obtained as described before  and embedded in paraffin. HFAs were obtained from the MRC–Wellcome Human Development Biology Resources (HDBR) – Institute of Genetics Medicine, Newcastle, United Kingdom). Sections were obtained using a microtome  at 6-μm thickness, deparaffinized, treated with 3% hydrogen peroxide in PBS for 30 minutes, and then, depending on the primary antibody (see below), treated or not with 10 mM sodium citrate, pH 6.0, for 20 minutes at 95oC in a water bath. Sections were then blocked with 5% normal goat serum (Sigma-Aldrich) in PBS 0.1% Triton X-100 (T-PBS) and incubated overnight with anti-CYP11B2 , anti-CP11A1 , and anti-SGPL1 . Sections were washed in T-PBS and incubated with a biotinylated goat anti-primary IgG secondary antibody  diluted 1:500 in T-PBS for 2 hours. Sections were washed, treated with avidin-biotin complex  according to the manufacturer’s instructions, rinsed again, and incubated with the ImmPACT DAB peroxidase substrate kit (Vector Laboratories). The reaction was stopped with H2O, and slides were counterstained with hematoxylin, dehydrated, and coverslipped using Vecta Mount (Vector Laboratories).Human adult adrenals, mouse adrenals, and kidneys from H&E staining was performed using standard procedures. For fibrotic changes in the kidney, Masson’s trichrome staining kit (Sigma-Aldrich) was employed. Images were acquired using a Leica DM5500B microscope equipped with a DCF295 camera (Leica) and DCViewer software (Leica), and then processed with Adobe Photoshop CS6 and Adobe Illustrator CS6.t test was used to generate P values. P values less than or equal to 0.05 were considered significant. Data are presented as mean ± SD in all figure parts in which error bars are shown.In studies in which statistical analyses were performed, a 2-tailed Student’s This study was approved by the local hospital ethics committees, and all parents  gave written informed consent. Approval was obtained from the Outer North East London Research Ethics Committee, reference number 09/H0701/12, the Marmara University Ethics Committee, reference number B.30.2.MAR.0.01.02/AEK/108, and the Vall d’Hebron Hospital Ethics Committee . All experiments involving mice were approved by the Ethical Committee for Animal Experimentation (ECD) of KU Leuven .SGPL1 deficiency has been described as the cause of Charcot-Marie-Tooth neuropathy in 2 siblings (Since the submission of this paper, siblings .LAM and RP designed the study. NR, TG, DB, IB, EB, HC, MFC, NC, F-C, UD, RK, BH, ES, EY, RP, and HLS recruited and clinically characterized patients. RP, LAM, FB, JCA, TG, and AB analyzed WES data. RP, AM, and LAM conducted Sanger sequencing and analysis of data. AM, EM, and RP generated mutant constructs, and PPVV conducted lyase activity experiments. PPVV generated and validated mouse lines. IH, LG, JH, and MS conducted mouse/human adrenal and kidney histology. RP and LAM prepared the draft manuscript. All authors contributed to the discussion of results and edited and approved the final manuscript.


Transcription factors of the nuclear factor κB (NF-κB) family are central coordinating regulators of the host defence responses to stress, injury and infection. Aberrant NF-κB activation also contributes to the pathogenesis of some of the most common current threats to global human health, including chronic inflammatory diseases, autoimmune disorders, diabetes, vascular diseases and the majority of cancers. Accordingly, the NF-κB pathway is widely considered an attractive therapeutic target in a broad range of malignant and non-malignant diseases. Yet, despite the aggressive efforts by the pharmaceutical industry to develop a specific NF-κB inhibitor, none has been clinically approved, due to the dose-limiting toxicities associated with the global suppression of NF-κB. In this review, we summarise the main strategies historically adopted to therapeutically target the NF-κB pathway with an emphasis on oncology, and some of the emerging strategies and newer agents being developed to pharmacologically inhibit this pathway. Just over 30 years ago, Ranjan Sen and David Baltimore discovered a protein complex that bound to a conserved immunoglobulin regulatory DNA sequence in activated B lymphocytes and that they referred to as “Nuclear Factor binding to the κ-Light-chain-enhancer B site” or, in short, nuclear factor κB (NF-κB) . DecadesRAS and PTEN mutations. These mechanisms are often responsible for inducing stable NF-κB activation in various types of solid malignancy, including colorectal, pancreatic, ovarian and breast carcinoma . R. R22]. RGenotoxic stress induced by ionising radiation or chemotherapeutic drugs results in canonical NF-κB activation, which enables cells to survive while the DNA damage is being repaired. This pathway of NF-κB activation involves the trafficking of signalling intermediates between the nucleus and the cytoplasm . TheIn addition to mediating this essential nuclear function, DNA damage-activated ATM is exported from the nucleus to the cytoplasm, where it binds to TRAF6 via its TRAF-interaction motif. This binding then activates the TRAF6 E3 ubiquitin ligase activity, leading to TRAF6-mediated self-polyubiquitination, in a reaction catalysed in conjunction with the E2 ubiquitin-conjugating enzyme, Ubc13 ,41,67. Tcyclin D1, BCL-2, LBcl-X, survivin and XIAP, have been implicated, each in diverse oncological contexts, as mediators of NF-κB-dependent tumour-cell survival leading to radiotherapy and/or chemotherapy resistance . B. B65]. Bymphomas  113]..CARD11 mThe gene encoding the adaptor protein, MYD88, which mediates NF-κB and MAPK activation downstream of all TLRs, with the exception of TLR3 , is recuNIK amplifications and c-IAP1/2 and TRAF3 deletions .,24.21,24The first proteasome inhibitors developed were molecules of the class of peptide aldehydes and were subsequently extensively used for preclinical research . The besThe human genome encodes two E1 ubiquitin-activating enzymes, UBA1 and UBA5, an estimated fifty E2 ubiquitin-conjugating (UBC) enzymes, and more than six hundred E3 ligases, conferring a large degree of substrate specificity to the ubiquitin cascade ,25,166. The F-box protein, β-TrCP, is involved in the signal-dependent recognition and degradative K48-linked ubiquitination of canonical IκBs and p100, as well as in the recognition and ubiquitination of a large group of other target substrates, including β-catenin, Snail, Emi1, Wee1, Cdc25A and Claspin ,179. FroWhile proteasome inhibitors, such as bortezomib, can provide significant clinical benefit in multiple myeloma and a few other indications, these agents inhibit NF-κB and many other essential cellular pathways that rely on the proteasome function ,162,166,Importantly, from a mechanistic standpoint, it is also unclear that the clinical response to proteasome inhibitors in patients with multiple myeloma and other B-cell malignancies results from the inhibition of NF-κB signalling, as it is becoming increasingly clear that the cellular accumulation of undigested proteins in these immunoglobulin-producing tumours can activate the unfolded protein response (UPR), thereby accelerating tumour-cell death . TherefoAmycolatopsis, has been shown to exhibit a potent and specific inhibitory activity on NF-κB nuclear import by means of its ability to directly bind to NF-κB dimers [Once liberated from IκBs, NF-κB dimers migrate into the nucleus where they regulate gene expression. This activation step offers a significant opportunity for therapeutic intervention, since, at least from a theoretical standpoint, a number of nuclear activities of NF-κB could be targeted with drug agents, including the post-translational modification of NF-κB proteins and their ability to dimerise, translocate into the nucleus, bind to DNA and interact with chromatin components, coactivators and corepressors and other transcription factors ,105. WhiB dimers ,184. StuB dimers ,186,187.Furthermore, sesquiterpene lactone (SL) compounds have been shown to inhibit the DNA-binding activity of RelA-containing NF-κB dimers by interacting with cysteine residue, C38, within RelA’s DNA-binding loop 1 (L1) . By bindSince NF-κB induces transcriptional programmes that affect all hallmarks of cancer ,191, an GADD45B), as a novel transcriptional target of NF-κB and effector of the NF-κB-dependent inhibitory activity on JNK signalling and apoptosis in response to TNF-α and other cues [GADD45B [RelA or any IKK subunit [−/−Gadd45b mice are viable, fertile and die of old age [Our group recently sought to obtain proof-of-concept for this principle in multiple myeloma, the paradigm of NF-κB-driven malignant diseases. Since a key pathogenetic function of NF-κB in multiple myeloma is to upregulate genes that block apoptosis and, despite its ubiquitous nature, NF-κB signalling induces highly tissue- and context-specific transcriptional programs ,79,192, her cues ,194. GADher cues ,196,197.her cues ,194. A nher cues ,198,199.her cues ,202,203.[GADD45B , and, un subunit , Gadd45b old age ,206, we We therefore developed the D-tripeptide inhibitor of the GADD45β/MKK7 complex, DTP3, which effectively disrupts this complex at nanomolar concentrations, in vitro, by binding to MKK7, and as a result, selectively kills multiple myeloma cells by inducing MKK7/JNK-dependent apoptosis, without toxicity to normal tissues ,201. We Further investigations will be required to determine the potential clinical benefit resulting from this approach in multiple myeloma patients, as well as its potential side-effects, propensity to develop drug resistance, and therapeutic efficacy in combination with other agents ,208. NevGADD45B, have been shown to be transcriptionally regulated by NF-κB, including those encoding various BCL-2-family members such as B-cell lymphoma-extra large (LBcl-X), myeloid cell leukaemia sequence 1 (MCL1), A1  and, at least in certain tissue contexts, B-cell lymphoma 2 (BCL-2) itself [L, MCL1, A1/BFL-1 and BCL-2 itself; multidomain proapoptotic effectors such as BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer (BAK); and BH3-only proteins, which convey apoptosis-initiating signals, such as BCL-2-interacting mediator of cell death (BIM), BH3 interacting-domain death agonist (BID), Bcl-2-associated death promoter (BAD), and p53 upregulated modulator of apoptosis (PUMA) [Several antiapoptotic genes, in addition to ) itself ,209. The) itself ,212,213.) itself . Accordi) itself . Congrue) itself , confirm) itself ,212,213.s (PUMA) ,212,213.s (PUMA) . Howevers (PUMA) ,215. A ns (PUMA) ,213,215.s (PUMA) ,216. ABTGiven the central role of aberrant NF-κB activation in the pathogenesis of the large majority of human diseases, the therapeutic targeting of the NF-κB pathway has been aggressively pursued by the pharmaceutical industry and academic laboratories for over two and a half decades. However, this goal has proven thus far an insurmountable challenge, due to the severe dose-limiting toxicities associated with the global suppression of NF-κB, resulting in the dismaying absence of a specific NF-κB or IKK inhibitor in the current anticancer armamentarium. Nevertheless, the past three decades, since NF-κB was first discovered in 1986, have witnessed tremendous advances in the understanding of the intricate signalling networks governing the NF-κB pathway and its multiple functions, and these efforts are now bearing some of the long-awaited fruits in the field of translational medicine, by pointing toward potential safer alternatives to therapeutic NF-κB inhibition. Undoubtedly, the targeting of upstream signalling mechanisms and protein-protein interactions governing the contextual and tissue-specific activation of NF-κB signalling and, at the opposite end of the spectrum, the non-redundant effectors of the diverse tissue-specific transcriptional programmes that NF-κB activates in order to exert its biological functions are amongst the most attractive strategies being developed to achieve contextual, cancer-cell selective therapeutic inhibition of the NF-κB pathway, and thereby circumvent the preclusive limitations of conventional NF-κB inhibitors. Perhaps, the most valuable lesson to be learnt from these initial therapeutic attempts is that the complexity of the intricate signalling networks governing the NF-κB regulation and function appears to hold the key to untangle the NF-κB therapeutic riddle and translate it into concrete clinical benefits. Indeed, while significant ground remains to be covered and the limited clinical successes obtained so far in select experimental clinical contexts have yet to transform into healthcare benefit for the broader patient population, the basic and translational knowledge unravelled by these studies on the biological complexity in the NF-κB pathway is providing tangible new opportunities for cancer-selective therapeutic intervention, which are certain to attract growing interest in the future and be further capitalised upon.


Arabidopsis transfer DNA (T-DNA) mutants for 6 of the maize bZIPs, among which the mutants atbzip20 and atbzip47, representing ZmbZIP54 and ZmbZIP107, showed obviously inhibited growth of roots and above-ground parts, compared with wild type. Five highly Pb-tolerant and 5 highly Pb-sensitive in maize lines were subjected to DNA polymorphism and expression level analysis of ZmbZIP54 and ZmbZIP107. The results suggested that differences in bZIPs expression partially accounted for the differences in Pb-tolerance among the maize lines. Our results contribute to the understanding of the molecular regulation mechanisms of TFs in maize under Pb stress.Pb can damage the physiological function of human organs by entering the human body via food-chain enrichment. Revealing the mechanisms of maize tolerance to Pb is critical for preventing this. In this study, a Pb-tolerant maize inbred line, 178, was used to analyse transcription factors (TFs) expressed under Pb stress based on RNA sequencing data. A total of 464 genes expressed in control check (CK) or Pb treatment samples were annotated as TFs. Among them, 262 differentially expressed transcription factors (DETs) were identified that responded to Pb treatment. Furthermore, the DETs were classified into 4 classes according to their expression patterns, and 17, 12 and 2 DETs were significantly annotated to plant hormone signal transduction, basal transcription factors and base excision repair, respectively. Seventeen DETs were found to participate in the plant hormone signal transduction pathway, where basic leucine zippers (bZIPs) were the most significantly enriched TFs, with 12 members involved. We further obtained 5  As a widely grown food crop, maize is important in supporting the growing world population. Maize production is threatened by lead pollution of soil, which results from atmospheric Pb deposition, sewage irrigation, waste accumulation, and metal mine acid waste water. Pb accumulates in roots, stems and other organs, and seriously affects the growth and development of plants . More secis-elements in the promoter regions of several stress-related genes and thus up-regulate the expression of many downstream genes, resulting in the imparting of abiotic stress tolerance . Th. ThOryzaBased on KOG, GO and pathway analyses, we sought to identify the TF genes involved in responding to the heavy metal Pb. As a result, a total of 464 genes (1.99%) expressed in CK or Pb treatment samples were annotated as TFs from the RNA-seq. They were classed into 50 TF families according to their DNA-binding domains. The bZIP, GRAS, MADS-box, K-box, homeodomain-TF and CBF/NF-Y families were the largest, in which 99, 47, 40, 38, 31 and 28 members were expressed in at least one of the four libraries, respectively, followed by TCP, jumonji, SBP-box, Pathogenesis, TFIIB and WRKY. The proportion of these 12 families was 76.98% . The resp-value < 0.05, and log2 fold change < −1 or > 1). Counts of 230, 231, and 227 DETs were identified for stage I, stage II, and stage III, respectively  TFs in Pb treatment samples were identified as differentially expressed TFs (DETs) compared with CK . The expression changes of these genes at 0 h (CK) and Pb treatment stages were consistent with the results of RNA-seq B. The rehan 0.05 . This suTo explore significant differences in gene expression profiles between CK and stage I, stage II, and stage III, the co-modulated DETs were analysed using the K-means clustering algorithm. The 262 DETs were categorized into 4 distinct clusters . Class Ip < 0.05) pathways, including plant hormone signal transduction , basal transcription factors , and base excision repair   . HormoneAtbZIP20/AHBP-1b/TGA2, AtbZIP22/TGA3, AtbZIP26/OBF5/TGA5, AtbZIP45/TGA6, AtbZIP46/PAN, AtbZIP47/TGA1 and AtbZIP57/OBF4/TGA4) of bZIPs  have been proven to play crucial roles in abiotic and biotic stress in Arabidopsis [A total of 17 TFs were detected that participate in the plant hormone signal transduction pathway, where bZIPs were the most significantly enriched with 12 members involved . Among tbidopsis ,24,25. Arabidopsis mutant library for orthologues of the 12 maize bZIP genes among T-DNA insertion mutants [AtbZIP20, orthologue of ZmbZIP54), tga-2 , tga-3 , tga-4 , and tga-5  via the TAIR mutant seed stock [To reveal the potential relations between bZIP genes and Pb tolerance, we searched the  mutants . We succed stock . The T-DArabidopsis thaliana wild type (Columbia): Pb(NO3)2 50 mg/L; Pb(NO3)2 100 mg/L; Pb(NO3)2 150 mg/L; Pb(NO3)2 200 mg/L and Pb(NO3)2 250 mg/L. We found that seedling growth was seriously affected at a concentration of Pb(NO3)2 200 mg/L. When it reached 250 mg/L, Arabidopsis growth was significantly suppressed. Leaves turned yellow and soon died. Thus, a Pb concentration of Pb(NO3)2 200 mg/L was considered a suitable treatment.To identify the most appropriate concentration of Pb, five concentration levels were set for AtbZIP20 or AtbZIP47 reduced the Pb tolerance of Arabidopsis, but did not significantly influence its growth under normal conditions. As is well known, the bZIP family contains evenly spaced leucine residues, that allow dimerization, and a basic DNA-binding domain [tag2 showed the strengthened inhibition of root growth under phytoprostane or jasmonic acid relative to the wild type [tga2 under Pb stress in our research. As oxylipins, jasmonates, and phytoprostanes regulate stress responses and diverse physiological and developmental processes [AtbZIP47 could play a role in defense response to phytoprostane and jasmonic acid under Pb stress; whereas, mutant tag1 did not show obvious phenotype changes under phytoprostane nor jasmonic acid when compared with the wild type [tag1 under Pb stress. A possible explanation is that the response of AtbZIP20 to Pb stress is independent of jasmonates and phytoprostanes. The mutants and WT (Columbia) were then subjected to Pb treatment. The results indicated that plant growth was inhibited by Pb stress for both the wild type and the mutants; whereas only tga-1 and tga-2 showed significantly different responses compared with the wild type. Specifically, there were no differences in plant growth between WT (Columbia) and the tga-1 or tga-2 mutants under control conditions. However, growth inhibition of the mutants was more severe than that in the WT under Pb stress. As shown in g domain  that funild type , which irocesses ,30. Maksrocesses . Combiniild type , which ibZIP genes are annotated as DNA-binding transcription factors, and are expected to be present in the nucleus [ZmbZIP54 and ZmbZIP107 with the GFP gene and transformed them into Oryza protoplasts by the PEG method [Oryza protoplasts transformed with the fusion gene vectors ZmbZIP54-GFP or ZmbZIP107-GFP, but in both the nucleus and the cytoplasm of the Oryza protoplasts transformed with the control vector, which showed that ZmbZIP54 and ZmbZIP107 proteins were specifically located in the nucleus.Plant  nucleus . To veriG method , respectZmbZIP54 and ZmbZIP107 for the 10 lines. Sequence analysis indicated that there are no significant genotype polymorphisms among the various lines -induced oxidative stress [ZmbZIP54 and ZmbZIP107 in roots. Differences in Pb tolerance among maize lines have been previously reported ,34. To ous lines . We subs 08WSC51 A. In conve lines B. Remark bZIP TF . Over-exe stress . Based o


To discuss the clinical value of immunoglobulin gene rearrangements in the diagnosis of B-cell lymphoma.A total of 209 cases of B-cell lymphomas and 35 cases of reactive lymphoid hyperplasia were selected for DNA extraction and PCR amplification using the BIOMED-2 primer system. Gel electrophoresis of heteroduplexes was used to analyze immunoglobulin gene rearrangements.A total of 209 cases of B-cell lymphoma, including 69 extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue, 63 diffuse large B-cell lymphomas, 39 follicular lymphomas, 15 small lymphocytic lymphomas, 6 plasmacytomas, 6 mantle cell lymphomas, 7 nodal marginal zone B-cell lymphomas, and 4 lymphoplasmacytoid lymphomas, were examined. Immunoglobulin gene rearrangements were found in all 209 cases, with 93 IGHA, 122 IGHB, 98 IGHC, 167 IGK, 100 IGL, 167 IGHA/B/C, 204 IGH/IGK, 209 IGH/IGK/IGL, 129 IGH+IGK, 81 IGH+IGL, 83 IGK+IGL and 68 IGH+IGK+IGL gene rearrangements. Immunoglobulin gene rearrangements were not found in the 35 cases of reactive lymphoid hyperplasia. IGH and IGK gene rearrangements were mainly found in mantle cell lymphomas, small lymphocytic lymphomas, extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue and diffuse large B-cell lymphomas. The IGH gene rearrangement was mainly found in lymphoplasmacytoid lymphomas and follicular lymphomas. IGK and IGL gene rearrangements were mainly found in plasmocytoma, and the IGK gene rearrangement was mainly found in nodal marginal zone B-cell lymphomas.The BIOMED-2 standardized immunoglobulin gene rearrangement detection system is an important tool in B-cell lymphoma diagnosis. Analysis of IGH, IGK and IGL gene rearrangements is valuable in confirming the classification of B-cell NHL. Lymphoma is a malignant tumor derived from lymphoid tissue and lymph nodes, which can be classified into two types, Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) , 2. NHL According to the expression status of different immune phenotypes, morphological analysis and classification standards of lymphatic and hematopoietic system tumors formulated by the WHO in 2015 , the selIG gene rearrangements were found in all 209 B-cell lymphoma samples Table , which iIn the 209 cases of B-cell lymphoma analyzed, IGH and IGK gene rearrangements were mainly found in MCL Figure , CLL Fi and DLBCThe B cell receptor (BCR) on the B cell membrane surface identifies and combines with antigens and mediates the immune response . The BCRGene rearrangement is a normal physiological process that occurs during B lymphocyte maturation. In the embryonic state, the B lymphocyte immunoglobulin gene is composed of a variable region (V), diverse region (D), joining region (J) and constant region (C). These regions are nonconsecutive on the chromosome and are separated by insertion sequences of different lengths. After a certain stage of lymphocyte development, these regions realign to assemble into a structural gene, i.e., they undergo gene rearrangements, in a process that is catalyzed by a recombinase. The rearranged gene of each lymphocyte is unique, and therefore, B-cells can recognize millions of different antigens. If a single cell can evade physiological regulation due to the presence of specific factors, it will grow uncontrollably in a process called cloning hyperplasia, and malignant lymphoma will ensue. This cloning hyperplasia can lead to special gene rearrangements becoming dominant, which can lead to the generation of a clone detection index for malignant lymphoma –21. MaliIn 2003, 47 organizations from 7 countries in Europe developed the BIOMED-2 multiplex PCR system, which contains 107 primers divided into 18 multiplex PCR tubes . This neIn this study, the BIOMED-2 system was employed to analyze IG gene rearrangements in 244 samples of paraffin embedded lymphoid tissue. Of these, 209 B-cell lymphoma samples were positive for IG gene monoclonal rearrangement, but the 35 lymphadenosis samples did not show IG gene monoclonal rearrangement bands , suggesting that the detection rate was 100%, which is markedly higher than that of other detection systems  and consTo avoid false negatives, primers for the functional fragments of IGH, IGK and IGL gene were designed in the BIOMED-2 system. For B-cell lymphomas localized in the germinal center or precursor germinal center , 27, andWith the introduction of the IGK light chain, 204 IGH/IGK clonal rearrangements were detected, indicating a detection rate of 98%. When V-J and Kde fragments were included in IGK, this rearrangement occurred in all IG λ positive and one third of the IG κ positive B-cell lymphomas –33. MoreFurthermore, IGH and IGK gene rearrangements were mainly found in MCL, CLL, MALT and DLBCL. IGH gene rearrangement was mainly found in LPL and FL, IGK and IGL gene rearrangements primarily in plasmocytoma, and IGK gene rearrangement primarily in MZL. The differences in IGH, IGK and IGL gene rearrangement rates in DLBCL, MALT, FL, CLL, MCL, MZL, plasmocytoma and LPL indicated that analysis of the gene rearrangement rate could be clinically important in the classification of different B-cell NHLs.The BIOMED-2 system required that the load of oncocytes be between 1% and 5%. If the number of oncocytes were too low, the monoclonal bands would not be clear in a polyclonal background . The locIn conclusion, a higher detection rate and specificity can be achieved by using the BIOMED-2 system to diagnose malignant B-cell tumors. However, a detailed understanding of the results would require new insights into lymphoma development. Additionally, when analysis of gene rearrangements is used to diagnose lymphoma, it is necessary essential that these data be combined with PCR results, clinical data, tissue morphology and immunohistochemical results to enable a comprehensive assessment of the disease.A total of 209 excised B-cell lymphoma samples collected by the pathology department at Xiangya Hospital from June 2014 to December 2016 were used for the study. Of the 209 cases, 126 were male and 83 female. The age of the patients from whom the samples were obtained ranged from 6 to 83 years (mean age 51). Following classification, there were 69 MALT lymphomas, 63 DLBCL, 39 FL, 15 SLL, 6 plasmacytomas, 6 MCL, 7 MZL, and 4 LPL. The control group was comprised of 35 cases of reactive lymphoid hyperplasia.All specimens were fixed in 10% neutral formalin, entrapped through conventional paraffin embedding, and processed into 4 μm serial sections with conventional HE staining. The immunohistochemical S-P method was employed to mark immune phenotypes. LCA , CD3 , CD45RO , CD20 , CD10 , CD15 , CD30 , Bcl-2 , Bcl-6 , CyclinD-1 , PAX-5 , kappa light chain  and lambda light chain  were used.The IdentiClone™ B-Cell Clonality Assay kit was purchased from Invivoscribe Technologies. The 20 bp DNA ladder and 10× loading buffers were purchased from Takara. GelRed dye was purchased from BioTium, AmpliTaq Gold DNA polymerase from ABI, and DNA FFPE Tissue Kit from Xiamen Aide Biomedicine Limited.Ten paraffin sections of 8 μm thickness were transferred to 1.5 mL sterile centrifuge tubes. Genomic DNA was extracted using the extraction kit, and an ultraviolet spectrophotometer was used to determine the concentration and purity of the DNA.H family primers: GGCCTCAGTGAAGGTCTCCTGCAAG, GT CTGGTCCTACGCTGGTGAAACCC, CTTGGGGGGT CCCTGAGACTCTCCTG, CTTCGGAGACCCTGTCC CTCACCTG, CGGGGAGTCTCTGAAGATCTCCTGT, TCGCAGACCCTCTCACTCACCTGTG;The BIOMED-2 primer system –37 was uH family primers: CTGGGTGC GACAGGCCCCTGGACAA, TGGATCCGTCAGCCCC CAGGGAAGG, GGTCCGCCAGGTCCAGGGAA, TGG ATCCGCCAGCCCCCAGGGAAGG, GGGTGCGCCA GATGCCCGGGAAAGG, TGGATCAGGCAGTCCCC ATCGAGAG, TTGGGTGCGACAGGCCCCTGGACAA;IGH tube B VH family primers: TGGAGC TGAGCAGCCTGAGATCTGA, CAATGACCAACATG GACCCTGTGGA, TCTGCAAATGAACAGCCTGAG AGCC, GAGCTCTGTGACCGCCGCGGACACG, CAG CACCGCCTACCTGCAGTGGAGC, GTTCTCCCTGC AGCTGAACTCTGTG, CAGCACGGCATATCTGCAG ATCAG.IGH tube C VH primer: CCAGTG GCAGAGGAGTCCATTC.IGH tubes A, B AND C JIGK tubes A and B Vk family primers: TCAAG GTTCAGAGGCAGTGGATCTG, GGCCTCCATCTCC TGCAGGTCTAGTC, CCCAGGCTCCTCATCTATGAT GCATCC, CAACTGCAAGTCCAGCCAGAGTGCAT CC, CCTGCAAAGCCAGCCAAGACATTGAT, GACCGATTTCACCCTCACCCTCACAATTAATCC;λ primers: Vλ1/2 ATTCTCT GGCTCCAAGTCTGGC, Vλ3 GGATCCCTGAGCGAT TXTXTGG; Jλ primer: CCCTGGTTCGAGTGGCAG GATC. There were also 5 pairs of BIOMED-2 internal control primers. To analyze the integrity of the extracted DNA, the amplified fragments with these primers should be 100, 200, 300, 400 and 600 bps long. The housekeeping gene β-actin was used as the internal control. The products of PCR were analyzed by polyacrylamide gel electrophoresis.IGK tube A Jk primers: CCCTGGTTCCACCTC TAGTTTGCATTC, CCCTGTGCTGACCTCTAATTTG CATTC; IGK tube B Jk primer: CGTGGCACCGCGA GCTGTAGAC. IGL tube VCompared with the standard, samples that showed 1 or 2 unusual clear bands different from the background product in a polyacrylamide gel were considered as positive. Additionally, the following 4 judgment standards proposed by McCarthy were applied: (1) the width of the electrophoresis bands should not be more than 1 mm with straight sides; (2) the molecular weights of the electrophoresis products should be within the desired values; (3) if no smeared band and no primer dimer are found, the samples should not be regarded as negative but the result interpreted as caused by an abortion of PCR amplification; (4) all the other bands that exceed the molecular weight range are nonspecific bands –42.


CCL24 is one chemotactic factor extensively studied in airway inflammation and colorectal cancer but less studied in hepatocellular carcinoma (HCC) retrospectively. So HCC tissue microarray (TMA) was used to estimate relationship between CCL24 and prognosis, cell experiments were conducted to study its influence for HCC cell biological behavior. CCL24 was injected to nude mice to monitor tumor formation and pulmonary metastasis; qRT-PCR, western blot and Immunohistochemistry were used to explore potential mechanism. CCL24 plays roles in target cells via its downstream CCR3, or it is regulated by Type 2 helper T cells (Th2 cell) factors, so immune related experiments were conducted. Meanwhile, Rho GTPase family have close relation not only with T cell priming, but with neovascularization; CCL24 contributes to neovascularization in age-related macular degeneration via CCR3, so Rho GTPase family, Th2 cell factors, Human Umbilical Vein Endothelial Cells were used to uncover their trafficking. Ultimate validation was confirmed by small interfering RNA. Results showed CCL24 expression was higher in caner tissues than adjacent normal tissues, it could contribute to proliferation, migration, and invasion in HCCs, could accelerate pulmonary metastasis, promote HUVECs tube formation. Th2 cell factors were irrelevant with CCL24 in HCCs; and RhoB, VEGFA, and VEGFR2 correlated with CCL24 in both mRNA and protein level. Downstream RhoB-VEGFA signaling pathway was validated by siRhoB and siVEGFA inhibition. In a word, CCL24 contributes to HCC malignancy via RhoB-VEGFA-VEGFR2 angiogenesis pathway and indicates poor prognosis, which urges us to study further CCL24 effects on diagnosis and potential therapy for HCC. Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide with an ascending trend in recent years for its various pathogenesis , includi+ epithelial cells, CD31+ endothelial cells, and CD68+ macrophages [+ malignant T cells to the skin in skin-specific cutaneous T-cell lymphoma (CTCL) [CCL24, known as eotaxin-2, MPIF-2, or Ckβ-6, had been studied in allergies and eosinophilic esophagitis for many years since its discovery in 1997 . CCL24 wrophages , especiarophages . Meanwhirophages ; anotherrophages . As the rophages ; CCR3 pla (CTCL) . CertainIn vivo tumorigenesis in nude mice was also analyzed by CCL24 interference. Subsequent CCR3, Type 2 helper T cells (Th2 cell) factors, Rho GTPase family, VEGFA, etc. were used to explore their relevance with HCC, qRT-PCR, western blot and Immunohistochemistry were used to explore potential mechanism, ultimate speculation or conclusion was investigated by siRNA validation.In our study, we initially investigated the expression of CCL24 in fresh HCC tissues and HCC cell (HCCs) lines, the prognosis role of CCL24 was also analyzed. Biological behaviors such as proliferation, migration, and metastasis of HCCs lines were induced by CCL24 overexpression or CCL24 knockdown. First, we compared mRNA levels in 20 fresh HCC tissues with their paired adjacent normal tissues. Results revealed that CCL24 expression was remarkably higher in HCC tissue than adjacent normal tissues . Meanwhile, the OS and TTR of the CCL24Low group were both longer than those of the CCL24High group  of the patients had suffered recurrence and 35.6% (112/315) had died. The 1-, 3-, and 5-year overall Survival (OS) rates in the CCL24y Figure . The medP = 0.013, HR = 1.783; P = 0.012, HR = 1.666; Table In univariate analysis, γ-GT, tumor number, tumor size, microvascular invasion, TNM stage, BCLC stage and CCL24 level were associated with OS, whereas, γ-GT, liver cirrhosis, tumor number, tumor size, microvascular invasion, TNM stage, BCLC stage and CCL24 level were associated with TTR. In multivariate analysis, γ-GT, tumor size, TNM stage and CCL24 level were associated with OS, whereas, γ-GT, liver cirrhosis, tumor number, microvascular invasion, BCLC stage and CCL24 level were associated with TTR. Univariate and multivariate analyses demonstrated that CCL24 expression in tumor cells served as an independent risk factor for both OS and TTR  compared to less metastatic HCC cell lines  and a normal liver cell line (L02)  , we explInterestingly, the migration and invasion ability of HUVECs changed regularly after different HCC conditioned medium inference. Results showed the migration and invasion of the Huh7-CCL24 group were higher than those of the Huh7-Vector group , transcatheter arterial chemoembolization (TACE), percutaneous ethanol injection (PEI), or external radiotherapy for patients with HCC recurrence might influence the OS results.In conclusion, CCL24 contributed to the malignant biological behavior of HCC through the RhoB-VEGFA-VEGFR2 pathway and was inversely correlated with worse prognosis. This new discovery illustrated the complexity of archenteric cancers and the potential homogeneity of HCC in embryology. Meanwhile, this was the first study to investigate the role of CCL24 in HCC, which will shed light on the diagnosis and target therapy of HCC.The seven HCC cell lines, normal liver cell line L02, human umbilical vein endothelial cells, and nude mice used in this study are described in the Supporting Information.Two independent cohorts consisting of 315 HCC patients and 20 paired fresh cancer/adjacent normal tissue samples were included in this study. The specific information and follow-up process are described in the Supporting Information.2 test or Fisher's exact test was adopted to compare qualitative variables. t test or Pearson's correlation test was used to analyze quantitative variables. Kaplan–Meier analysis was used to analyze survival. Survival curves between the different groups were calculated using a log-rank test. Cox proportional hazards model was adopted to determine the univariate or multivariate hazards. A P value of <0.05 was considered statistically significant.All data were analyzed with SPSS 19.0 software . Pearson χFor other descriptions of the materials and methods used in this study, see the Supporting Information.