Drosophila melanogaster has served as an excellent model system for understanding the neuronal circuits and molecular mechanisms regulating complex behaviors. The Drosophila female reproductive circuits, in particular, are well studied and can be used as a tool to understand the role of novel genes in neuronal function in general and female reproduction in particular. In the present study, the role of Beadex, a transcription co-activator, in Drosophila female reproduction was assessed by generation of mutant and knock down studies. Null allele of Beadex was generated by transposase induced excision of P-element present within an intron of Beadex gene. The mutant showed highly compromised reproductive abilities as evaluated by reduced fecundity and fertility, abnormal oviposition and more importantly, the failure of sperm release from storage organs. However, no defect was found in the overall ovariole development. Tissue specific, targeted knock down of Beadex indicated that its function in neurons is important for efficient female reproduction, since its neuronal knock down led to compromised female reproductive abilities, similar to Beadex null females. Further, different neuronal class specific knock down studies revealed that Beadex function is required in motor neurons for normal fecundity and fertility of females. Thus, the present study attributes a novel and essential role for Beadex in female reproduction through neurons. Drosophila has served as an excellent model to unravel the neuronal regulation of multiple complex behaviors like memory and learning, aggression, courtship and female reproduction Beadex (Bx) was isolated as a spontaneous mutant with beaded wings  in Drosophila  Bx gene product (hypermorph) Bx itself codes for Drosophila LIM only (dLMO) domain containing protein Bx coding sequence leads to Bx loss of function and is evident as held-up wings phenotype. However, abrogation in the 3′UTR sequence, which harbors negative regulatory elements, leads to gain of function alleles of BxBx hypermorphs, increased Bx levels disrupt the functional complex of Apterous-Chip (Ap-Chip) due to the competitive binding of Bx to LIM-binding domain (LBD) of Chip Acheate and Scute through its positive interaction with Pannier (Pnr) in the sensory organ precursor (SOP) specification Lmo4, one of the vertebrate homologues of Bx, suppresses the formation of excitatory glutamatergic inter-neurons and promotes the development of GABAergic inter-neurons in mice Bx also affects Drosophila behavior in response to cocaine and ethanol anaplastic lymphoma kinase (Alk) in the brain of Drosophila Bx homologues in mice, Lmo4 and Lmo3, also affect the behavior of mice in response to cocaine and ethanol, through mechanisms similar to those in Drosophila The LIM domain consists of two tandemly organized zinc finger motifs with the conserved cysteine-histidine residues that co-ordinate zinc binding. LIM domain mediates its function through protein-protein interactions Bx role in multiple behaviors through neurons, the molecular mechanisms and the targets through which Bx effects its function are not clearly understood. In this study, we attribute a novel and essential function for Bx in Drosophila female reproduction. We show that multiple, post mating reproductive processes like ovulation, egg laying and sperm release are compromised in the Bx null females. We further provide evidence that Bx regulates these functions through the motor neurons that innervate the female reproductive tracts.Though several studies have reported 1118w females or female progeny of Canton-S males crossed with 1118w females were used as controls, unless specified. hpd-G14-1Bx was a kind gift from Pascal Heitzler #4368) Bx loss of function alleles show held-up wings phenotype) Bx locus on Y chromosome, DpW39Bx RNAi line was obtained from Vienna Drosophila RNAi Centre (VDRC Transformant ID#2917). All the Gal4 lines and GFP tagged reporter protein construct fly lines were procured either from Bloomington Drosophila stock centre, Indiana or National Center for Biological Sciences (NCBS) stock centre, Bangalore, India.All the flies were maintained at 23±2°C, under 12 hrs Light/Dark (L/D) cycles on cornmeal-sucrose-yeast agar media. Either 1118w and Bx hop-out flies was isolated using trizol (Sigma) following manufacturer's instructions. From the RNA, 2 µg was taken for making cDNA using first strand cDNA kit following manufacturer's protocol . Primers specific for Bx-RA and Bx-RB transcript, were designed as follows- Bx-RAFP 5′ – CTAATTGAGTCGAGTGTGCGTG -3′, Bx-RARP 5′ – AAGGAGGTTGGTTGTCGTCGTC -3′, Bx-RBFP 5′ – ATGGAGTACCTCTACAACGCTA – 3′ and Bx-RBFP 5′ – TTATTTCGGGACCCGTAC – 3′. House-keeping gene rp49 was used as control using following primers rp49FP 5′ – TTCTACCAGCTTCAAGATGAC – 3′ and rp49RP 5′ – GTGTATTCCGACCACGTTACA – 3′. In order to characterize the molecular lesion in hop-out Bx allele, genomic DNA was isolated from wild type (1118w) and isolated Bx mutant (7Bx). Primers were designed on either side of the p{GawB} hdp-G14-1Bx insertion site (assessed from Bx gene region are Bx-FP 5′ – GGCTCGTTGGTCTAGAGGTA – 3′ and Bx-RP 5′ – CATAATGGCATCTCCGCAAG – 3′. The location of the primers in the Bx gene is represented in Bx gene region was performed with ExTaq (TaKaRa). The resultant PCR products were analyzed through agarose gel electrophoresis and documented using Alpha DigiDoc RT2 . Further, the PCR product from the Bx mutant was cloned into TA vector  and sequenced . The resultant sequences were BLAST aligned against Drosophila genome (http://flybase.org/blast/) to determine the molecular lesion in the Bx mutant.RNA from whole body of 1–2 days old *, Elav-Gal4; +; +w  were crossed with *, UAS-SyteGFP; +; +w (BS#6924) males. For Dmef2-Gal4  In order to check the expression pattern of neuronal Gal4 lines in the female reproductive system, females of RNAiBx construct was brought together with UAS-Dcr2 (BS#24646) tsGal80  and grown at appropriate temperatures . For knock down across all the stages, progeny were grown from the early embryonic stage up to the test age (3–4 days old adult flies) at 29°C. For the pupal stage knock down, larvae were grown till wandering third instar stage at 18°C and were then shifted to 29°C till the adult flies eclosed. The eclosed flies were then kept at 18°C for the next 4–5 days. For the adult stage knock down of Bx, larvae and pupae were allowed to develop at 18°C and the freshly eclosed adult flies were kept at 29°C for next the 4–5 days.For efficient knock down studies, UAS-Virgin females (3–4 days old) of interested genotype and 2–3 days old Canton-S males (grown at 25°C) were isolated for crosses. Single pair matings were set up in the media vials. After the visual confirmation of mating and recording the duration of copulation, males were separated and the females were transferred into egg laying media vials  supplemented with yeast paste and kept in 25°C or 29°C incubator with 12 hrs L/D cycle. The females were removed 24 hrs later and the number of eggs laid was counted to assess the fecundity. Fecundity was plotted as the average number of eggs laid per female in 24 hrs. Further, same set of vials were incubated at 25°C and checked 24 hrs later to count the hatched eggs for the fertility assay. Fertility was plotted as percent hatched eggs. For assessing the ovulation ability of the females, 24 hrs post mating (hpm) with Canton-S males, reproductive tract was dissected and the percent of females showing the presence of egg in the uterus was recorded. Minimum of 15 females per genotype were used for all the experiments and the experiments were independently repeated at least three times. For oviposition assay, mated females were transferred on to petri plates with egg laying media and kept at 25°C or 29°C incubator for 6 hrs. The females were then removed and the percent of total eggs deposited on the media was recorded. Data for all the above assays were plotted as mean with standard deviation.Ovaries of the females mated with wild type males were dissected and the number of developing ovarioles was counted in each ovary. Following this, the same samples were fixed in 4% PF for 30 minutes and washed for 15 minutes (3×5 minutes) with PBTx. The washed samples were then incubated with Hoechst  for 30 minutes for nuclear staining, washed again in PBTx and mounted in Vectashield mounting media (Vector labs). The mounted samples were visualized and imaged using Zeiss fluorescent microscope (Zeiss Axio Observer Z1).dj-GFP males, which have GFP labeled sperm tails (BS#5417) To study the sperm storage and releasing ability, females of the interested genotype were mated with 2–3 days old Data was plotted, graphs were made and statistical test analysis was performed using Graph pad Prism 5 (Graphpad).Bx regulates the female reproduction or not, generation of loss of function or hypomorphic alleles of Bx was essential. New Bx alleles were generated by mobilizing P{GawB} element (hdp-G14Bx1-) inserted near 3′ end of the first intron , two constitutive exons (2 & 3) and about 70 bp of the 5′UTR  and normal mating duration (1118w) and other controls   . It is ihe media .7Bx and control females were analyzed to see if the reduced fecundity was due to abnormal ovary development. At gross level, it could be observed that the number of ovarioles was less in the 7Bx mutant females compared to those in the control females  and 7Bx mutant females. Thus, 7Bx mutant females have normal sperm storing ability. Over a period of 10–15 dpm, sperms stored in the storage organs are released for fertilizing eggs, thus depleting sperms from the storage organs dj-GFP sperms from the seminal receptacle of the control flies (1118w) were totally depleted by 10 dpm. However, the seminal receptacle of 7Bx mutant females retained dj-GFP sperms even after 10 dpm   , neithermuscles) . Elav-Gave tract . Knock d females . Even invulation , reducedvulation  and defevulation  with thehe ovary .Bx acts as a transcription co-activator in several developmental processes like wing disc morphogenesis and bristle development Bx function in the neurons was required during development for female reproduction, stage specific knock down of Bx in the neurons was carried out. Bx knock down during the pupal development or in the adult stage separately did not cause any reproductive defects . This indicated that Bx function in the motor neurons is essential for female reproduction.As mentioned earlier, the reproductive defects in  muscles . Knock decundity  and fertecundity  comparedBx in Drosophila female reproduction. The Bx null females generated in this work showed normal ovariole development but with highly reduced fecundity and ovulation. Reduced fecundity correlates well with reduced ovulation. However, unlike earlier reported mutants which showed reduced fecundity and ovulation 7Bx mutant females. One possible reason for this could be the highly reduced abdominal cavity caused by the distended crop  during ovulation . In the females where Bx is knocked down neuronally, it is also possible that Bx functions in the ovarioles are unaltered, which might lead to accumulation of mature eggs in the ovarioles.The overall defects like reduced fecundity, sperm release from storage organs and abnormal oviposition  and 2 co females . This cl Bx null . This coBx plays an important role during the development of wing disc and SOP Bx function is essential during the development of the reproductive tract . During development, Bx might transcriptionally regulate the genes which affect the neuronal circuits innervating the female reproductive tract. However, Bx might work independent of Pnr, a known transcription factor that interacts with Bx, in regulating the female reproduction, since pnr knock down in the neurons does not affect fecundity or fertility (data not shown). Lmo4, a vertebrate homolog of Bx is known to affect the number of neurite outgrowths and their length in human SH-SY5Y neuroblastoma cells Lmo4, Bx also might regulate the projection of neurons on to target muscles during development. Though the present study does not show any evidence for such functions, it offers a look at the neuro-circuits regulating reproduction for dissecting the molecular mechanism of Bx function.Bx in these circuits did not cause any reproductive defects  Bx affects the female reproduction through these sensory neurons, Bx was knocked down in these sensory neurons using ppk-Gal4  Bx knock down in these sensory neurons did not cause any defect in fecundity or fertility  expression levels by Islet along with Lim3, another LIM-homeo domain protein 7Bx mutants and in the neuronal knock down of Bx, the functions of Islet and Lim3 are modulated (either increased or decreased activity) in the neurons due to their reduced interaction with Bx and this might result in altered neuronal function and the described phenotypes. This further emphasizes that Bx function is crucial during the female reproductive tract development. Drosophila Protein Interaction Map (DPiM) project has identified almost 100 proteins which interact with Bx in S2 cell lines 7Bx mutant flies and also in the neurons with Bx knock down, there by affecting the female reproduction.Reproductive defects in the  females . DrosophBx is essential for Drosophila female reproduction where it regulates ovulation, sperm release from the storage organs and oviposition. All the reproductive defects of Bx null females can be phenocopied by knocking down Bx in the neurons. More specifically, Bx plays an important role in female reproduction through the motor neurons. This work shows the importance of utilizing the female reproductive tract circuits in understanding the molecular pathways through which Bx might modulate neuronal functions.We conclude that Figure S1Copulation duration of 7Bx females is similar to that of wild type females. Both wild type and 7Bx mutant females showed normal duration of copulation when mated with wild type Canton-S males.(TIF)Click here for additional data file.Figure S2Innervations of Elav-Gal4 neurons onto the female reproductive tract muscles.Elav-Gal4 neurons showed innervations onto almost all the musculature of female reproductive tract like oviduct (OD), ovary sheath (S) and uterus (U). Blue- Phallodin-TRITC and Green-Syt-eGFP.(TIF)Click here for additional data file.Figure S3Bx knock down in the neurons show oviposition defect. Knock down of Bx in the neurons leads to defective oviposition where in close to 100% of the eggs was deposited on the surface of the media.(TIF)Click here for additional data file.Figure S4Mature eggs accumulation in 7Bx and the neuronal knock down of Bx females.7Bx females do not show accumulation of mature eggs in the ovaries unlike those of control ovaries (1118w). However, knock down of Bx in the neurons in females leads to accumulation of mature eggs in the ovaries unlike controls.(TIF)Click here for additional data file.Figure S5Innervation of VGlut-Gal4 and dTdc2-Gal4 classes of neurons onto the female reproductive tract muscles.VGlut Gal4 class of neurons showed projections only onto uterine musculature  but not to the oviduct or common oviduct . dTdc2-Gal4 class of neurons showed projections onto the oviduct  and uterine musculature . Blue-Phalloidin-TRITC and Green-Syt-eGFP.(TIF)Click here for additional data file.Figure S6Knock down of Bx in the glutamatergic neurons affects oviposition. Knock down of Bx in the glutamatergic neurons leads to oviposition defect similar to 7Bx mutant females, though to a smaller percentage. .(TIF)Click here for additional data file.Figure S7Crop distension in 7Bx mutant females reduces abdominal cavity space.7Bx mutant females showed increase in the abdominal size due to distension of crop caused due to reduced food movement along the gastric tract. This might lead to inhibition of mature egg accumulation in the Bx mutant ovaries. Red arrow indicates bulged abdomen and crop.(TIF)Click here for additional data file.Figure S8Bx does not regulate female reproduction through the sensory neurons in the female reproductive tract.Bx was knocked down in the sensory neurons in the female reproductive tract with ppk-Gal4. However, this does not reduce either fecundity (A) or fertility (B) of the females.(TIF)Click here for additional data file.


CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease.Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of  O- or N-linked glycosylation to form mature proteoglycans. Proteoglycans function at the cell membrane or as secreted components of the extracellular matrix  such as chondroitin and dermatan are a heterogeneous group of unbranched polysaccharides of variable length that consist of repeating disaccharides. With the exception of hyaluronic acid, GAGs are synthesized in the Golgi and then modified by enzymes including sulfotransferases. Chondroitin , heparin, dermatan, and keratin are all sulfated and subsequently appended to newly synthesized proteins via CHST11 and including an embedded MIR3922. Nullizygosity for the deletion was observed in the proband and not in three unaffected living siblings who subsequently underwent testing. When considered together with the known role for CHST11 in murine cartilage growth plate formation during development, our findings strongly suggest that chondroitin sulfation is required for normal development of the bony skeleton in humans. Our findings also support the hypothesis that CHST11 and/or MIR3922, a microRNA whose function has not previously been described, may act as tumor suppressors in the lymphocyte lineage.Although GAG-modifying genes such as sulfotransferases are required for normal GAG and proteoglycan synthesis, the role of these genes in human development and disease is less well understood. Here, we report a previously undescribed syndrome whose cardinal features include bilateral bony deformities of upper and lower extremities and susceptibility to malignant lymphoproliferative disease. The phenotype was present in two of seven siblings but in neither parent, suggestive of a genetic disorder with an autosomal recessive mode of inheritance. Whole-Genome Sequencing (WGS) of the proband revealed a biallelic 55 kb deletion at chromosome 12q23 spanning part of the locus for The proband is a 46-year-old woman who initially presented to the adult genetics clinic at Brigham and Women’s Hospital in 2005 for evaluation of a personal and family history of congenital hand/foot malformations and peripheral T-cell lymphoma. At 61 in. in height , she is shorter than all of her first-degree relatives except one similarly affected sister. She was born with bony abnormalities of the fingers and toes bilaterally, for which she underwent multiple surgical repairs in early childhood. On clinical exam, her fingers demonstrated brachydactyly with disproportionately short index fingers and either valgus or varus deformity at many of the interphalangeal (IP) joints Fig.. SimilarIn the left foot, plain films revealed fusion across the great toe IP joint; small middle phalanges of the second and third toes; and only two phalanges of the fourth toe with a well-defined lucency at the base of the proximal phalanx of the fourth toe, thought to be a separate ossicle Fig.. The fifThe proband’s medical history was further complicated at 44 years of age, when she developed several weeks of fever, flu-like symptoms, and pulmonary infiltrates unresponsive to antibiotics that culminated in respiratory failure and mechanical ventilation. Extensive testing for an infectious etiology was negative and she was diagnosed with acute respiratory distress syndrome (ARDS) of uncertain etiology. Her symptoms ultimately improved with high-dose steroids but recurred when steroids were discontinued. Surgical lung biopsy revealed an atypical lymphoid infiltrate consisting primarily of CD3+ T cells but with a minor component of CD20+ B cells. Immunoglobulin heavy chain polymerase chain reaction (PCR) did not identify any evidence of B-cell clonality. The histologic pattern of an angiocentric lymphoid infiltrate was suggestive of lymphomatoid granulomatosis, an Epstein–Barr virus (EBV)-associated lymphoproliferative disorder. However, additional evidence supporting this diagnosis including atypical lymphoid cells reactive for B-cell antigens or EBV-associated mRNAs was not identified. Bone marrow biopsy was unremarkable. The patient was restarted on antibiotics and steroids and improved clinically. Her lung biopsy was reviewed at the National Cancer Institute and the overall impression was that of a reactive process, possibly an organizing pneumonia, rather than a primary lymphoproliferative disorder such as lymphomatoid granulomatosis, although the latter could not be definitively ruled out. The patient’s condition ultimately went without diagnosis.She subsequently underwent a second lung biopsy, again revealing nodular lymphoid infiltrates of primarily CD3+ T cells with scattered CD20+ B cells and CD68+ histiocytes. PCR for rearranged immunoglobulin heavy chain and T-cell receptor (TCR) genes did not demonstrate any evidence of B- or T-cell clonality, respectively. Flow cytometry of peripheral blood lymphocytes revealed no abnormalities. Overall, definitive diagnostic features of a T- or B-cell lymphoma were not observed. Based on clinical suspicion of malignancy, the patient was treated with rituximab followed by four cycles of chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The patient’s clinical symptoms improved in concert with partial regression of her pulmonary infiltrates. New wedge biopsies of the right middle and upper lobes revealed a nodular, angiocentric and bronchocentric atypical T-cell lymphoid infiltrate extending into intra-alveolar spaces. The infiltrate was composed of medium size and large atypical lymphoid cells admixed with small lymphocytes and histiocytes. Immunohistochemical studies revealed that the majority of large atypical lymphoid cells stained positively for the T-cell markers CD3, CD4, and CD8 (minority). CD5 expression was absent but for a small number of cells. The infiltrating T cells were also positive for CD43 and TIA-1 in a granular cytoplasmic pattern. EBV LMP-1 was negative, as were B-cell markers CD20 and CD79a and the natural killer cell marker CD56. Additional negative markers included CD30, CD15, CD34, MPO, and CD1a. Ki-67 stained approximately 20–30% of cells. EBV mRNA by in situ hybridization was also negative. On this occasion, TCR gamma gene rearrangement studies revealed evidence of clonality. The patient’s progressive clinical course, the cytologically atypical T-cell infiltrate, the immunophenotypic aberrancy (absence of CD5), the lack of EBV mRNA, and molecular evidence of clonality with an identical pattern observed at two different anatomical sites  were most consistent with a diagnosis of peripheral T-cell lymphoma. She received additional chemotherapy prior to undergoing a matched unrelated allogeneic bone marrow transplant from a male donor. Her clinical course has been complicated only by grade 1 graft versus host disease and several non-life-threatening infections. She continues to have no evidence of disease on periodic restaging positron emission tomography–computed tomography (PET-CT) scans.The proband’s family history is most notable for an older sister with similar congenital malformation of the hands/feet who succumbed to complications of an unnamed lymphoproliferative disorder in her early 20s. At a height of sixty inches, the deceased sister was also shorter than all other first-degree relatives. A review of her medical records from the 1980s revealed that she presented at 17 years of age with abdominal pain, nausea, and vomiting, subsequently found to be caused by a small bowel obstruction complicated by perforation and pneumoperitoneum. She also described a several week history of skin lesions of uncertain origin, fever, shortness of breath, and tachypnea. Radiographic imaging of the lungs revealed diffuse bilateral nodular infiltrates. Management of the small bowel perforation ultimately included partial ileal resection, right-sided colectomy, and jejunostomy. Per report, pathologic evaluation of the resected small bowel revealed dense lymphocytic infiltrates. Skin biopsy similarly revealed a lymphocytic infiltrate. Biopsy of the lung was notable for an angiocentric lymphocytic infiltrate resembling lymphomatoid granulomatosis, the same EBV-related condition initially suspected to underlie the proband’s pulmonary lymphoid infiltrates. A bone marrow biopsy was reportedly normal on two occasions. No additional molecular diagnostic testing was available and/or performed at the time. She was treated with cyclophosphamide, azathioprine, and prednisone with resolution of both pulmonary infiltrates and cutaneous manifestations. However, her course was subsequently complicated by new-onset seizures. Computed tomography (CT) imaging suggested involvement of the central nervous system (CNS) with the lymphoproliferative disorder. A brain biopsy was performed and the pathology was consistent with malignant lymphoma of the CNS, not otherwise specified. She ultimately died from complications of her disease at age 22.The proband and her deceased sister have five unaffected living siblings Fig.. No otheA search of online databases including OMIM and PubMed did not identify any named syndrome associated with both skeletal dysplasia and any type of lymphoproliferative disorder. Therefore, no specific candidate gene rose to a level of suspicion to warrant clinical testing. The results of chromosome breakage analysis with diepoxybutane (DEB) were normal and therefore did not support a diagnosis of Fanconi anemia (FA). Array-based comparative genomic hybridization using a custom-designed platform with 180,880 probes throughout the human genome did not reveal any clinically significant genomic gains or losses. Therefore, we performed WGS on genetic material obtained from the proband to identify rare homozygous variants that could potentially explain the phenotype in this family.Consent to perform WGS under Clinical Laboratory Improvement Amendments (CLIA) conditions was obtained under protocols approved by the Institutional Review Board (IRB) of Partners HealthCare, Inc. Explicit permission to publish the findings contained herein was obtained from the proband.μg/mL). The dishes were incubated at 37°C with 5% CO2 and examined daily for cell growth. When the cells became 90–100% confluent in culture, genomic DNA was isolated from fibroblasts using the Gentra Puregene Cell Kit  and subsequently used for the WGS sequencing.Due to the patient’s prior allogeneic bone marrow transplant, which resulted in reconstitution of the entire hematopoietic system by donor cells, we isolated and sequenced genomic DNA from cultured skin fibroblasts rather than from circulating (donor) hematopoietic cells  data sets were considered for analysis . Briefly, genomic DNA was randomly fragmented and then sequenced using 50 nt paired-end reads on an Illumina HiSeq 2000 Sequencer at an average depth of 30× coverage , exon 2 of CHST11, along with reference genes RPP30 and AP3B1  using genomic DNA from the proband’s buccal epithelia and cultured fibroblast cells. A multiplex PCR reaction was performed for 16 STR loci using the commercially available AmpFlSTR® IdentifilerTM PCR Amplification kit , following the manufacturer’s instructions. Resulting STR amplicons were separated and visualized using an ABI Prism®  Genetic Analyzer. STR alleles were scored using GeneMarker software v1.5 .To confirm that deletion of MBD3L5 and PRND. A homozygous missense variant in EPHA10 and a homozygous variant in OLA1 3′UTR were also identified. While the variants in these four genes appeared to be technically valid, there was insufficient evidence based on known expression and/or biological function to link these variants to the observed phenotype.Given the recessive mode of inheritance observed for the phenotype in this family, we searched the proband’s WGS data for genes harboring rare biallelic single-nucleotide and compound heterozygous variants Table. We idenCHST11 as well as the embedded microRNA MIR3922. By computational prediction, MIR3922 may target ZNF585A (Zinc Finger Protein 585A [19q13.12]), RNF214 (Ring Finger Protein 214 [11q23.3]), RAB10 (RAS-Related Protein RAB-10 [2p23.3]) and/or ERBB2  , chr19:55,253,424-55,295,014 (41.5 kb), and chr19:54,801,000-54,807,500 (6.5 kb). The first deletion included exon 2 of the gene The population frequency of these homozygous deletions was determined using genotyping data generated by Genome STRiP in the 1KG cohort Fig.. The twoCHST11 and MIR3922, saliva was obtained from the proband and genomic DNA was isolated from buccal epithelial cells. Droplet digital PCR (ddPCR) was performed to analyze the copy number of CHST11 and MIR3922 alleles. The average CHST11 copy number was confirmed to be 0 in the fibroblast cells and 1.23 in buccal epithelial cells (σ = 0.01). This unexpected result suggested the possibility of genomic heterogeneity secondary to a mixed population of host and donor-derived cells/genomes or due to chimerism from variable fusion of CHST11-null host DNA with CHST11-diploid donor genomic DNA  sister but were ultimately unsuccessful. Instead, we analyzed the copy number of CHST11 and MIR3922 in the proband’s unaffected siblings. A ddPCR analysis performed for the CHST11 and MIR3922 loci did not reveal homozygous deletion of CHST11 and MIR3922 in any of the three unaffected siblings who were tested  databases  were reported for the available sequences from hematopoietic or lymphoid tumor samples. However, somatic copy number variants in CHST11 were identified in several solid tumor samples. In the COSMIC database (accessed December 2013), 511 samples representing 8 of 27 solid tumor types were found to harbor copy number loss in the CHST11 locus. CHST11 copy number loss was most common in ovarian cancer (31.2% of samples). In addition, 28 and 33 nonsynonymous SNVs in CHST11 are reported in the Broad TumorPortal and COSMIC, respectively. Notable variants include missense substitutions in diffuse large B-cell lymphoma (p.P99Q) and CLL (p.R42L), a frameshift (p.F31 fs) in clear cell renal carcinoma, and nonsense variants in esophageal cancer (p.Q329*), lung adenocarcinoma (p.E325*), and liver cancer (p.E239*). The functional significance of these variants is presently unknown. No variant data are reported for MIR3922. Of note, a somatic translocation involving CHST11 ) was previously identified in a patient with B-cell chronic lymphocytic leukemia (B-CLL)  was detected in saliva from our female proband. This suggests the contribution of donor cell-derived genomic DNA to the proband’s gastrointestinal tract epithelium, as has been previously described .Our analysis of existing genomic databases suggests that the Our review of the literature suggests that the combined phenotype of skeletal dysplasia and malignant lymphoproliferative disease is rare. A single case report describes an infant with short-limbed skeletal dysplasia and severe combined immunodeficiency who developed EBV-associated B-cell lymphoma at 10 months of age  and skeletal anomalies such as radial aplasia and thumb aplasia or hypoplasia, in addition to disturbances of several additional systems . The common molecular defect shared by all FA complementation groups is in DNA repair, resulting in hypersensitivity to DNA cross-linking agents.Notably, the phenotype observed in our proband resembles other rare Mendelian disorders that combine skeletal anomalies with specific hematopoietic derangements. The recessive disorder thrombocytopenia-absent radius (TAR) syndrome is characterized by low platelets and anomalies of the radius and may also present with eosinophilia, anemia, various other skeletal abnormalities, and disturbances of other systems . TAR has been associated with genetic variants in the gene CHST11 gene encodes a chondroitin 4-sulfotransferase localized in the Golgi apparatus that catalyzes the transfer of sulfate to position 4 of GalNAc in chondroitin. CHST11 is one of several known carbohydrate sulfotransferases, sharing four regions of homology including 5′ and 3′ phosphosulfate binding sites and two C-terminal alpha helical domains. The gene CHST3, for example, encodes a chondroitin 6-sulfotransferase that catalyzes the transfer of sulfate to position 6 of GalNAc in chondroitin.Chondroitin sulfate is the most common proteoglycan in cartilage and is present on the surface of cells in many tissue types as well as in the extracellular matrix. Chondroitin is an unbranched GAG that consists of alternating sugars of glucuronic acid and N-acetylgalactosamine . Sulfate moieties added at position 4 or 6 of GalNAc residues alter the physiochemical properties of chondroitin and chondroitin glycoconjugates. The CHST11 with a clinical phenotype underscores the importance of carbohydrate sulfotransferase function in human biology. Biallelic loss-of-function mutations in genes encoding other carbohydrate sulfotransferases have been previously described in rare syndromes with autosomal recessive inheritance. Homozygous or compound heterozygous mutations in CHST3, encoding a chondroitin 6-sulfotransferase, are associated with recessive spondyloepiphyseal dysplasia and congenital joint dislocations (OMIM 143095)   may be required for full expression of the combined phenotype, or it is possible that the genetic etiology of the lymphoproliferative disorder in each sister is distinct. Future studies could aim to directly test whether CHST11 expression is functionally required for the normal differentiation and/or proliferation of T lymphocytes, using cell lines or other in vitro models of lymphocyte differentiation. Moreover, it would be useful to assess the expression and function of MIR3922 in lymphocytes, both alone and in combination with reduced expression of CHST11. By analyzing mRNA and protein levels, it would also be of interest to assess whether CHST11 expression is reduced in sporadic hematologic malignancies, particularly because somatic dysregulation at the genetic level was not commonly observed in the Broad TumorPortal or the COSMIC database. Finally, additional evidence linking CHST11/MIR3922 deletion to our proband’s phenotype may be derived from ascertainment and genetic testing of additional families sharing a similar phenotype.In summary, we have identified


Enabling unique architectures and functionalities of microsystems for numerous applications in electronics, photonics and other areas often requires microassembly of separately prepared heterogeneous materials instead of monolithic microfabrication. However, microassembly of dissimilar materials while ensuring high structural integrity has been challenging in the context of deterministic transferring and joining of materials at the microscale where surface adhesion is far more dominant than body weight. Here we present an approach to assembling microsystems with microscale building blocks of four disparate classes of device-grade materials including semiconductors, metals, dielectrics, and polymers. This approach uniquely utilizes reversible adhesion-based transfer printing for material transferring and thermal processing for material joining at the microscale. The interfacial joining characteristics between materials assembled by this approach are systematically investigated upon different joining mechanisms using blister tests. The device level capabilities of this approach are further demonstrated through assembling and testing of a microtoroid resonator and a radio frequency (RF) microelectromechanical systems (MEMS) switch that involve optical and electrical functionalities with mechanical motion. This work opens up a unique route towards 3D heterogeneous material integration to fabricate microsystems. While monolithic microfabrication has been quite successful in the manufacturing of microsystems such as integrated circuits (IC) and microelectromechanical systems (MEMS)13672 (dielectric), Au , and epoxy-based SU8 (polymer) at the microscale. We refer to this approach as ‘micro-Lego’ due to the similarities to the commercial product, Lego, in the aspects of stacking and joining of different types of building blocks while at different scales. Four different materials are processed into inks, assembled into spatially organized 3D architectures via reversible adhesion-based transfer printing followed by thermal processing-based material joining. Depending on assembled material pairs, diverse joining mechanisms including fusion9132, Au and SU8. Furthermore, electrical connection between constituents on different steps of the RF MEMS switch is fulfilled to highlight 3D interconnection by micro-Lego. These 3D heterogeneous architectures are extremely challenging or inaccessible to reproduce via conventional monolithic microfabrication or other microassembly techniques such as those based on robotic pick-and-placeHere, we extend micro-masonry to an approach to assembling microsystems with four disparate classes of device-grade materials including Si (semiconductor), SiO2, sputter deposited Au and lithographically patterned SU8, and they are processed into ink arrays such that they are easily retrieved from donor substrates during transfer printing (92 (green), Au (yellow), and SU8 (brown) into the structures. It is worthwhile to note that the temperature of individual joining process should be considered when the sequence of assembling materials is set since materials such as SU8 or Au may not withstand the high temperature for Si-Si or Si-SiO2 fusion bonding. While 3D assembled structures demonstrated here do not exhibit device functionalities, these 3D structures realized from 2D inks at the microscale present the unparalleled heterogeneous material assembly capabilities of micro-Lego that can further be exploited in countless applications.printing . An ink rinting 9. This tr2, Si-Au and Si-SU8 interfaces created through micro-Lego. The procedure to make a Si specimen for a blister test and its dimensions are depicted in Gc is a material property termed critical energy release rate or toughness, which indicates the material’s resistance to fracture along any given path. Provided that a Si ink delaminates along the joining interface, the corresponding Gc indicates the toughness of the joining. The energy release rate G, on the other hand, is a loading parameter indicating the driving force for fracture. For the specimen geometry here, G is simply a function of the applied pressure pc and the central deflection of the Si ink dcdc that is a function of measured pc, specimen dimensions and material properties. At the moment when the Si ink delamination occurs, G reaches the toughness Gc. Three specimens are tested per each joining material pair and the resultant Gc are plotted in 2 and Si-SU8 pairs to compare the optimal and as conducted cases  microresonator of toroidal and wedge-disk geometries are typically fabricated using a combination of chemical etching and laser-reflow2 isotropic etch. To obtain microtoroids, the undercut is followed by physical reflow under high-power CO2 laser illumination (10.6 μm wavelength). The key challenge that persists with these methods is that ultra-high-Q silica resonators cannot be co-integrated with other planar photonic and electronic devices on a silicon substrate, since the resulting silicon substrate is nonplanar and frequently pitted. Micro-Lego, on the other hand, is capable of fabricating this geometry without any need for undercut as it merely joins pre-fabricated Si and SiO2 ring-shaped inks individually. Additionally, more complex multi-layered WGR geometries are now permissible  is assembled and tested. Thanks to their extremely high optical Q-factors, WGRs find extensive use in nonlinear optics171819missible  which arMicro-Lego is further utilized to assemble a series contact type RF MEMS switch. Common RF MEMS switches require 3D suspended architectures for electromechanical performances, which is complicated to produce using microfabrication due to limited material choice for a sacrificial layer as well as stiction during wet processes24Micro-Lego relies on thermal processing for material joining similar to conventional wafer bonding techniques9102 structure may fail to retain its original structure after thermal processing because of different thermal expansion coefficient between Si and SiO2. In this case, relatively low temperature (600 °C) in conjunction with oxygen plasma surface activation may be exercised in micro-Lego. To reproduce such a process for a blister test specimen, a SiO2 coated receiving substrate is activated using oxygen plasma followed by transfer printing and thermal processing. The test results yield 0.3 J/m2, which is lower than for Si-SiO2 pair joined at the temperature of 1000 °C, but still on par with other known wafer-scale joining strength2 surface results in slightly higher joining strengthWhile thermal processing conditions for material joining in micro-Lego are adopted from conventional wafer bonding techniques, optimal wafer-scale thermal processing conditions are not necessarily exactly replicated at the microscale when implementing micro-Lego processes. For example, an assembled Si-SiOThis work was intended to develop a novel transfer printing and thermal process based microassembly technique to enable highly organized delicate microscale 3D architectures with high structural integrity that are challenging otherwise. The joining strength data obtained through blister tests  as well 2, Au, and SU-8 is presented here. The method employs reversible adhesion-based transfer printing and thermal processing-based material joining. The interfacial joining characteristics between dissimilar materials are quantitatively studied through blister tests to validate the structural integrity of assembled structures and devices. Utilizing this approach, a few microsystems, including a microtoroid resonator and a RF MEMS switch, are fabricated and their device performances are examined. This work finds a unique way for 3D heterogeneous microsystems, with relevance not only to MEMS but also to electronics, photonics, metamaterials, and other fields.In summary, microassembly of four different classes of materials including Si, SiO2 and SU8 inks are prepared on individual donor substrates as described in Si, Au, SiOReceiving substrates with rims covered or fomred by four different materials are made as depicted in 2 (1 μm thick) disc inks are brought and joined together on a silicon receiving substrate. The edge of the SiO2 disk ink is then reflowed with CO2 laser (10.6 μm wavelength) illumination into the requisite microtoroid geometry. The SiO2 microtoroid resonator is tested by measuring optical transmission, using a tunable 1520–1570 nm fiber-coupled laser and photodetector as depicted in Si (20 μm thick) ring and SiOCPW line is prepared by sputter deposition and selective etching of 5 nm thick Cr and 100 nm thick Au on a silicon on insulator (SOI) substrate followed with etching of the below Si device layer using reactive ion etch (RIE). Si spacer inks are assembled on the CPW substrate. Au inks are then assembled on the Si spacer inks for electrical interconnection between patterned Au lines and top surfaces of Si spacer inks. The Si/Au composite beam ink is transferred and joined over the assembled Au inks at each end. The Si/Au composite beam ink fabrication procedure is further described at Materials and Method section in How to cite this article: Keum, H. et al. Microassembly of Heterogeneous Materials using Transfer Printing and Thermal Processing. Sci. Rep.6, 29925; doi: 10.1038/srep29925 (2016).


T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. Although there are many contributing arms of an effective immune response, T cells are one of the most significant players. T cell receptors display an impressive breadth of specificity for a wide variety of antigens owed largely to the process of T cell development in the thymus where VDJ rearrangement can generate a diverse repertoire . EngagemThe study of the affinity parameters that govern a productive recognition event is of great interest as these fundamental mechanisms inform our understanding of immune responses ranging from autoimmunity to viral infection. Though responses to antigens that have differential binding affinities to TCR have been studied extensively, understanding the impact of disrupting binding to MHC has been highlighted to a lesser extent. Like their APL counterparts, recognition of MHC variant peptides (MVP) can alter T cell phenotype, but in this case the TCR affinity for antigenic pMHC is likely maintained with the parameter of pMHC stability driving the sub-optimal triggering. For instance, we have previously shown an MHC variant peptide of myelin oligodendrocyte (MOG) can anergize polyclonal encephalitogenic T cells by triggering negative signaling mediated by Src homology tyrosine phosphatase (SHP-1). This sub /mrml. Geometric mean affinities are reported ± SEM. This assay is also described in detail in [Human RBCs  were biotinylated using Biotin-X-NHS (EMD4 Biosciences) and subsequently labeled with streptavidin (Thermo Scientific) and biotinylated peptide:MHC-I monomers. P14 T cells were purified from spleens using the EasySep CD8+ T cell Enrichment Kit (Stemcell). Single cells were aspirated onto opposing glass micropipettes in a cell chamber mounted on the stage of an inverted light microscope. An electronically controlled piezoelectric actuator controlled repeated T cell contact with a stationary pMHC-coated RBC for two seconds. Binding events were visualized as an elongation of the RBC membrane upon retraction of the T cell. Cells were brought into contact 50 times in order to calculate an adhesion frequency (Petail in  and 61]a). Quantb. Cells were plated at 2x105 cells per well in a flat bottom 96 well plate and loaded with peptides of interest. Cells were placed at 37°C to induce downregulation of unoccupied MHC molecules and harvested at indicated time points to assay for expression of H-2Db by flow cytometry. Half life was calculated by using data from 1/2 = (t*ln(2))/ln where t1/2 = half life, t = time elapsed, N0 = initial H-2Db expression after loading, Nf = final H-2Db expression at final time point assayed.RMA/S cells were incubated at room temperature overnight to allow for surface expression of empty H-2DStatistical analyses were performed using GraphPad Prism 6 software (Software for Science). Significance was calculated using two-way ANOVA, unpaired t test, and Wilcoxon matched pairs signed rank tests.


Functional annotations of large plant genome projects mostly provide information on gene function and gene families based on the presence of protein domains and gene homology, but not necessarily in association with gene expression or metabolic and regulatory networks. These additional annotations are necessary to understand the physiology, development and adaptation of a plant and its interaction with the environment.RiceCyc is a metabolic pathway networks database for rice. It is a snapshot of the substrates, metabolites, enzymes, reactions and pathways of primary and intermediary metabolism in rice. RiceCyc version 3.3 features 316 pathways and 6,643 peptide-coding genes mapped to 2,103 enzyme-catalyzed and 87 protein-mediated transport reactions. The initial functional annotations of rice genes with InterPro, Gene Ontology, MetaCyc, and Enzyme Commission (EC) numbers were enriched with annotations provided by KEGG and Gramene databases. The pathway inferences and the network diagrams were first predicted based on MetaCyc reference networks and plant pathways from the Plant Metabolic Network, using the Pathologic module of Pathway Tools. This was enriched by manually adding metabolic pathways and gene functions specifically reported for rice. The RiceCyc database is hierarchically browsable from pathway diagrams to the associated genes, metabolites and chemical structures. Through the integrated tool OMICs Viewer, users can upload transcriptomic, proteomic and metabolomic data to visualize expression patterns in a virtual cell. RiceCyc, along with additional species-specific pathway databases hosted in the Gramene project, facilitates comparative pathway analysis.http://www.gramene.org/pathway/.Here we describe the RiceCyc network development and discuss its contribution to rice genome annotations. As a case study to demonstrate the use of RiceCyc network as a discovery environment we carried out an integrated bioinformatic analysis of rice metabolic genes that are differentially regulated under diurnal photoperiod and biotic stress treatments. The analysis of publicly available rice transcriptome datasets led to the hypothesis that the complete tryptophan biosynthesis and its dependent metabolic pathways including serotonin biosynthesis are induced by taxonomically diverse pathogens while also being under diurnal regulation. The RiceCyc database is available online for free access at The online version of this article (doi:10.1186/1939-8433-6-15) contains supplementary material, which is available to authorized users. Oryza sativa) continue to be the flagship plant models and share a significant fraction of reported literature on gene functions and phenotypes associated with plant development and metabolism. Therefore, newly sequenced plant genomes and transcriptomes depend heavily on rice and Arabidopsis genome annotations for projecting genome, gene product and pathway annotations. In the majority of cases gene product annotations involve InterPro . Through this database we present a snapshot of the substrates, metabolites, enzymes, reactions and pathways of primary and intermediary metabolism in rice. In this report we will provide database insights based on RiceCyc version 3.3. Beside discussing the development of RiceCyc we will demonstrate the capabilities of RiceCyc as a platform for integrating and analyzing multiple omics data sets in terms of knowledge discovery. To emphasize the latter, we present here the first detailed report on full-scale annotation and integrated analysis of the serotonin, auxin IAA (indole-3-acetic acid) and tryptophan biosynthesis pathways in terms of stress and diurnal regulation, using the RiceCyc platform and publicly available rice transcriptomic data sets.In order to respond to and survive environmental challenges, plants, as sessile organisms, have developed a multitude of anatomical, morphological, growth habit and developmental adaptations that are based on underlying genetic variation. Gene duplication and alternative splicing of gene transcripts, commonly seen in plant species, provide plasticity and “sub-functionalization” that can contribute to plants’ ability to endure external stresses with reproductive success Freeling . While cThe RiceCyc pathway database currently features 316 metabolic pathways, 2,103 enzymatic reactions, 87 transport reactions and 1,543 compounds and metabolites. 6,643 protein coding genes i.e. about 14% of the total ~47,894 protein coding and tRNA genes that are either known and/or predicted from rice   allows users to perform comparisons between any two or more species by selecting their choices and look for common or species-specific pathways, reactions, metabolites etc. Each pathway and reaction details page also provides a cross-species comparison option to compare details of the specific pathway or reaction between any two or more species  illustrates the biochemical pathway map within the context of the cell metabolome  for validating and mapping microarray probe IDs, gene names or gene symbols to a locus ID in user provided data files. If the gene IDs are missing in the data file but if there are mappings from the microarray platform specific probe ID to a specific gene, the tool regenerates a valid expression data file for expression data analysis within the OMICs Viewer. The ability to visualize the expression patterns in virtual cell-like simulations and projections on the pathways and reactions helps the user to pinpoint and interpret interesting expression patterns as compared to examining a tabulated text file or annotation and function enrichment data derived from tools such as Blast2GO and BinGO.The tool Cellular Overview   which connects from the ‘Modifications’ hyperlink to the curation webpage (http://gramene.org/pathway/ricecyc_deleted_pathways.html).Computationally predicted databases suffer both from false negatives and false positives. The primary reference pathway database, MetaCyc that we have used for creating RiceCyc only houses experimentally demonstrated pathways, enzymes, reactions and compounds listed in its reference library which are derived from several organisms including plants. We used taxonomic filtering options to filter out non-plant pathways. This was successful only if the reference library was flagged with the appropriate taxonomic designation. To further reduce the false positives escaping the taxonomic pruning we evaluated the projections against the curated list of plant pathways and reactions provided in the frame format by PlantCyc-PMN , BioPAX level-2 and 3, and SBML (Stromback and Lambrix ftp://ftp.gramene.org/pub/gramene/pathways/ricecyc/) and the installation of PathwayTools . The local database setup on desktops running windows, linux, mac or unix environments offer additional operability, such as, faster speed, the opportunity to update the database with proprietary data, and the ability to perform programmatic queries including comparing species wide cellular overviews of pathway networks from multiple species.The complete RiceCyc database is available online as well as for download from CCA1 is reported to control genes related to plant defense and growth hormone metabolism in Arabidopsis  genes. It was reported  and β (ASB) subunits  show synchronized cycling, with upregulation around 12 h and 36 h (dusk) . Similarly, the enzyme tryptophan synthase  fungal pathogen Magnaporthe grisea - causing rice blast disease and 3) by the angiosperm parasitic weed Striga hermonthica. The above pathogens induced the genes ASB1 and ASA2, PAT (LOC_Os03g03450), PAI, IGPS, TSA (LOC_Os07g08430) and TSB1. Together these genes cover all six reaction steps in the tryptophan biosynthesis pathway. Interestingly, all the above genes/enzymes also exhibit synchronized diurnal cycling as described previously  shows preferred induction after Striga and Xanthomonas treatments whereas ASB2, ASA1, and TSB Type-2 show preferred induction after Xanthomonas treatment.The tryptophan biosynthetic pathway is known for its role in providing precursors for plant defense-related secondary metabolic compounds and the pathway genes are known to be induced by pathogens and elicitors  is reported not to be induced by pathogens or elicitors . XA21 is a pattern recognition receptor kinase that mediates resistance to the Gram-negative bacterium Xanthomonas oryzae pv. oryzae  having the indole-3-glycerol phosphate lyase activity and tryptophan synthase β-subunit (TSB) are known to be induced by biotic and abiotic stresses in Arabidopsis  by X. oryzae in the current study suggests a specific role for the ‘type 2’ TSB, at least in rice.Tryptophan synthase α-subunit , a neurotransmitter is well known to play several key physiological and behavior-associated roles in animals. Serotonin, its precursor molecules and interactors such as their receptors and transport proteins, are also known to show diurnal modulation in animals. Interestingly the serotonin molecule is also known to be present in a wide range of plant species (Roshchina De novo synthesis of IAA in plants is complex and can occur via tryptophan (Trp)-dependent or Trp-independent pathways. To date, a complete de novo auxin biosynthetic pathway has not been well-defined for plants (Zhao nts Zhao . The gennts Zhao . TherefoArabidopsis tryptophan amino transferase 1 (TAA1) gene mutants defective in the conversion of L-tryptophan to IPA  in contrast to the currently described YUC route of IAA synthesis , LOC_Os01g47530 (OsMPK8), LOC_Os03g57220 (GIP1), LOC_Os05g07860 (SAB10) and LOC_Os11g36180 (XA21) were also components of the “100 protein interactors” that were reported to be the key regulators within the rice biotic and abiotic stress response network that have been identified through Y2H screening  through activation of defense response genes in the nucleus  interacts with anthranilate synthase-β subunits ASB1 and ASB2 that are components of the key regulatory reaction of the tryptophan biosynthetic pathway. In addition, SAB10 (LOC_Os05g07860), a component of the ethylene mediated abiotic stress response subnetwork in rice  and both α subunits of this key regulatory enzyme complex. Many of these interactions based on co-induction and expression were reported in rice and Arabidopsis and are consistent with our analysis . These amino acids are reported to provide a protective role during induced oxidative stress and play a critical role in the initiation of hypersensitive response and immunity in Arabidopsis  gene, which encodes a cytochrome P450 monooxygenase that bears tryptamine 5-hydroxylase activity. It catalyzes the conversion of tryptamine to serotonin. This is a plant-specific reaction that is not found in humans  annotations to the rice gene products based on gene-gene homology evaluated by Ensembl Compara datasets. The final step was curation and quality check, where a human expert would review and accept, modify or reject the computational predictions. The rice genes used for the current build are based on MSU6 rice genome annotations. However, in the future we will be updating them with RAP (Rice Annotation Project) identifiers and upcoming revised IRGSP  annotations for Oryza sativa ssp. japonica cultivar Nipponbare.The rice protein sequences were downloaded from Gramene’s Rice Ensembl genome. The proteins were submitted to an annotation workflow that included HMM-based annotations from InterPro, SMART, PRINT, Prosite and Pfam. The second round of annotations involved cellular localization prediction based on Predotar for plastid and mitochondria . Functional enrichment analysis using GO category over-representation was carried out using the network visualization program Cytoscape . It is primarily a genome-scale network based on predicted protein-protein interactions and co-expression.The analysis of diurnal gene expression was conducted as described in Filichkin, Breton et al. . The datAfter building the RiceCyc pathway database, it was hosted on a webserver for online use, following installation guidelines provided by the PTools software Additional file 2: Table S2: Gene expression values of diurnally cycling rice genes mapped to RiceCyc pathways. The headers of data columns indicate the diurnal sampling time point starting with the beginning of the light period (0 h). (XLSX 379 KB)Additional file 3: Figure S1: Expression pattern and over-represented metabolic processes of diurnally cycling genes. (A) Hierarchical clustering of gene expression pattern of diurnally cycling genes in rice, mapping to RiceCyc metabolic pathways. Mean centered expression levels are represented with green denoting downregulation, and red indicating upregulation. (B) Over-represented gene ontology categories of gene set represented in (A). (PPTX 505 KB)Additional file 4: Figure S2: Gene expression analysis of the genes associated with enzymes catalyzing IAA biosynthesis. (A) A zoom-in view of IAA biosynthetic pathway. (B) Heat map of expression patterns of all cycling genes in the pathway with the gene ID and reaction of the pathway. Colored circles are used to identify individual reactions. (C) Biotic and abiotic stress induction of tryptophan biosynthetic genes based on data compiled in Genevestigator for rice. Expression data available for perturbations were filtered for significance level  and fold change . (PPTX 221 KB)Additional file 5: Figure S4: Tissue type expression pattern of genes in tryptophan biosynthesis (A), serotonin biosynthesis (B), and IAA biosynthesis (C) from data compiled in Genevestigator. (PPTX 178 KB)Additional file 6: Figure S3: Rice tryptophan biosynthetic enzyme interactome based on RiceNet. Blue circles on the periphery of the interactome represent tryptophan biosynthetic enzymes. Green circles represent interacting proteins identified from RiceNet. Three of the five anthranilate phosphoribosyltransferase paralogs  did not share interactions with other tryptophan biosynthetic enzymes and clustered separately . (PPTX 2 MB)http://www.functionalnet.org/ricenet/) to find proteins that interact with tryptophan biosynthetic pathway enzymes. Nodes represent genes/proteins while edges represent interactions. Column-A is node/interactor-1, column-B is relationship (network edge) and Column-C is node/interactor-2. (XLSX 33 KB)Additional file 7: Table S3: Rice tryptophan biosynthetic enzyme interactors based on the RiceNet. We probed the rice probabilistic functional network, RiceNet (Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Below are the links to the authors’ original submitted files for images.


Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both  RNA plays a central role in many cellular processes, acting as a major player in gene expression control46A modification of the mRNA, the RNA looping, and the direct entry driven by Internal Ribosome Entry Site (IRES) elements10111415In eukaryotic mRNAs, a variety of mechanisms have been proposed to allow the ribosomal machinery to recognize the translation start codon. These are the scanning of the 5′UTR following cap-dependent recruitment of the translation machinery, the cap-independent mechanisms guided by the mThe RNA sequences and secondary structures of picornaviruses show higher heterogeneity than in hepatitis C virus (HCV). Accordingly, the picornavirus IRES elements are grouped into five different types ; each type harbors conserved sequence motifs and a common RNA structure core maintained by evolutionarily conserved substitutions18Picornavirus IRES elements are organized in modular domains. In type I and type II IRES, each domain consists of one or more stem-loops that provide the binding site for RBPs and various eIFs, with the exception of eIF4E20212224Experimental evidence supports the involvement of RNA structural motifs for IRES activity262724303133RNA viruses in general, and FMDV in particular, are characterized by a high genetic variability3822in silico design can be successful in constructing complex functional RNA elements.In spite of the mutational analysis carried out in the picornavirus IRES Py tract, it remains elusive whether having the Py tract in a unique structural conformation is an absolute requirement for IRES activity. To answer this question we have designed candidate RNA sequences adopting different conformations of domain 5, but harboring a pyrimidine tract of the same length as that of wild type IRES. Hence, the pyrimidine tract could be either unpaired or base-paired in stem-loops with different stability. For this, we have made use of the FMDV IRES, a type II IRES element whose secondary structure is well characterized25To design RNA structural motifs with unique folding patterns, potentially affecting initiation of protein synthesis, we focused our attention on domain 5 of the FMDV IRES, located immediately upstream of the functional translation initiation codon . ModificThe experimentally determined RNA structure of domain 5 consists of three elements , of whic22in silico design and posterior filtering process produced sequences with high probability of having the Py base-paired. Among these sequences (which were arbitrarily named), sequence I-20 was selected because it had the highest probability for the Py tract to be base-paired. Moreover, four additional sequences  with moderate and low probability among the filtered sequences were selected in order to later establish correlations between measures and IRES activity. Measures shown to have high correlation with IRES activity can then be used as optimization criteria in subsequent design rounds.The target structure used as input to RNAiFoldAs devised, the selected candidates (RNA family I) had a pyrimidine tract of the same length as the wild type RNA , and U/Cin vitro synthesized RNA. As shown in 35S-labeled luciferase (LUC) polypeptide to chloramphenicol acetyl transferase (CAT) polypeptide in rabbit reticulocyte lysates (RRL) was reduced in all selected candidates relative to the wild type RNA. Note that the activity of RNAs I-2, I-3, I-4, and I-7 was very similar. However, the activity of RNA I-20 was reduced to a higher extent than all other candidates  activity expressed from bicistronic RNAs in transfected BHK-21 cells . The samild type . Again, p values and absolute SHAPE differences in To gain information about the RNA structure of the selected candidates, we chose a representative member of the family, RNA I-3, to analyze its local RNA flexibility in solution using SHAPE methodology . SHAPE rUsing RNAstructure software to predict the secondary structure including SHAPE reactivity data for wild type RNA and candidate I-3 , we showTo further prove the effect of structural accessibility of the pyrimidine tract on IRES activity, we plotted the parameters used to select final candidates of family I against the efficiency of protein synthesis measured in cell-free systems . This daSince the reduced activity of RNA I-20 could be related to the higher stability of the hairpin sequestering the Py tract, we attempted to generate a second round of candidates, selected on the basis of adopting a stable hairpin that sequesters the Py tract within a long stem-loop. In order to generate sequences for this family, we again used RNAiFold with the target structure shown in in vitro assay indicated a strong decrease of luciferase synthesis , confirming that the selected candidate harbored a modified RNA structure. As observed before for candidate I-3, the RNA structure model of candidate II-B obtained by imposing SHAPE reactivity on RNAstructure  separated by 84 nts21Concerning the differences among type I and type II picornavirus IRES elements, experimental evidence points to a differential role of the spacer structure on protein synthesis. For instance, in the case of FMDV RNA, the presence of a highly stable hairpin (six repetitions of the Xma linker) within the spacer  but not the presence of unstructured sequences up to 99 nts corresponding to the polylinker sequence of pGem3, hinders IRES activityThe lack of conserved pathways to recognize start codons among different IRES elements2332515354To determine potential similarities in Py accessibility of different IRES elements, we have analyzed the Rfam families of viral IRES for structural analogy. We used INFERNAL 1.1It is worth noting that expression vectors used to express more than one protein in eukaryotic cells usually contain type II picornavirus IRES elements, due to their high efficiency and their proven resistance to inhibitory conditions for cap-dependent translation. Given that spacers of variable sequences and length, often inherited from previous constructs, are found in expression vectors harboring IRES elements, our rational design provides useful data for the definition of RNA structural features of this spacer region affecting internal initiation of translation. In summary, we show here that a critical step for FMDV IRES function is the presence of an unpaired Py tract, along with the immediately upstream conserved stem-loop. All the selected candidates that form a stable hairpin sequestering the Py tract within the stem of a downstream hairpin show a significant decrease in protein synthesis despite conservation of the Py residues. To our knowledge, this is a novel finding since previous studies investigated the effect of modified Py sequences but did not investigate structural accessibility of the Py tract. Thus, future studies aimed to develop improved expression vectors should take into account the accessibility of this conserved structural motif.H. sapiens. A high-throughput version of the hybrid computational/experimental method described in this paper could generate sufficient data to train a regression support vector machine to predict translation efficiency of IRES elements. Such a tool could possibly provide insights concerning the ratio of protein products from genes that contain an IRES element.Several machine learning algorithms have been developed57RNAiFold computes all sequences that fold into a given characteristic secondary structureFamily I was designed to fold into the target structure shown in Family II was designed to fold into the target structure shown in We computationally estimated the accessibility of the polypyrimidine tract at 30 °C for each candidate using three different methods  and 2: F2 10 mM, 15 min at 37 °C and then inserted into the HindIII and XhoI restriction sites of pBIC, previously linearized with the same enzymes. Colonies that carried the correct insert were selected for further studies. Prior to expression analysis, the nucleotide sequence of the entire length of each region under study was determined (Macrogen).Insertion of the candidate sequences into the bicistronic plasmid pBICIn vitro transcription was performed for 1 h at 37 °C using T7 RNA polymerase, as describedin vitro were translated in 70% rabbit reticulocyte lysate (RRL) (Promega) supplemented with 35S-methionine (10 μCi)The relative IRES activity obtained in assays conducted with monocistronic RNAs (100 ng of the IRES-firefly luciferase RNA and 25 ng of the renilla luciferase RNA) was monitored in a luminometer using the dual system (Promega).5 cells were prepared 20 h after transfection in 50 μl of 50 mM Tris-HCl, pH 7.8, 120 mM NaCl, 0.5% NP40. Luciferase and chloramphenicol acetyl transferase (CAT) activities were measured as describedPlasmids carrying the candidate sequences upstream of the luciferase reporter gene were assayed in BHK-21 cells. Transfection of 90% confluent monolayers was carried out using cationic liposomes 1 h after infection with the Vaccinia virus VT7F-3 expressing T7 RNA polymerase. This assay excludes the presence of cryptic promoters since the transfected plasmid is transcribed in the cell cytoplasm by the T7 RNA polymerase. Extracts from 10in vitro. RNAs (2 pmol) were treated with N-methylisatoic anhydride (NMIA)in vitro synthesized RNA was renatured by heating at 95 °C for 2 min, snap cooling on ice for 2 min, and subsequently incubated in a final volume of 18 μl of folding mix  for 20 min at 37 °C. Then, RNA were incubated with dimethyl sulfoxide (DMSO) (untreated RNA) or 6.5 mM NMIA for 45 min at 37 °C, precipitated and resuspended in 10 μl of 0.5X TEt-test.Monocistronic constructs were linearized with SphI prior to synthesis of RNA transcripts http://www.clustal.org). The RNA sequences were obtained from GenBank; only domain 5 unique sequences were included in the alignment. The phylogenetic tree was inferred by the neighbour-joining method from the multiple-sequence alignment using MEGA4 program (www.megasoftware.net). Robustness of evolutionary relationships was assessed by 1000 bootstrap replicates. Bootstrap values are shown as percentages, only nodes with percentage values higher than 50% are indicated.The alignment of forty-six sequences of FMDV IRES field isolates was performed using CLUSTALX software with default parameters (http://weblogo.berkeley.edu) was generated from the multiple-sequence alignment of the IRES sequences. The overall height of each stack indicates the sequence conservation at that position (measured in bits), and the height of symbols within the stack reflects the relative frequency of the corresponding nucleic acid at that position.The sequence logo (http://varna.lri.fr/).Secondary RNA structure prediction accuracy can be improved incorporating SHAPE reactivity values as constraints in RNAstructure softwareHow to cite this article: Fernandez-Chamorro, J. et al. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements. Sci. Rep.6, 24243; doi: 10.1038/srep24243 (2016).


Ventricular arrhythmias arise from disruptions in the normal orderly sequence of electrical activation and recovery of the heart. They can be categorized into disorders affecting predominantly cellular depolarization or repolarization, or those involving action potential (AP) conduction. This article briefly discusses the factors causing conduction abnormalities in the form of unidirectional conduction block and reduced conduction velocity (CV). It then examines the roles that sodium channels and gap junctions play in AP conduction. Finally, it synthesizes experimental results to illustrate molecular mechanisms of how abnormalities in these proteins contribute to such conduction abnormalities and hence ventricular arrhythmogenesis, in acquired pathologies such as acute ischaemia and heart failure, as well as inherited arrhythmic syndromes. On the one hand, ventricular arrhythmias can lead to sudden cardiac death (SCD), which accounts for around 400,000 deaths in the United States Ventricular arrhythmias can be categorized into disorders affecting cellular depolarization or repolarization 2INa, which determines the AP upstroke (phase 0) max brings the membrane to threshold much more quickly. Secondly, increased myocardial excitability given by 1/ means that smaller inward currents are required to reach threshold. The resting membrane potential also influences INa through regulation of the inactivation kinetics of sodium channels i) depends on the resistance of both the myoplasm i would increase CV. Membrane capacitive load is determined by the surface area and volume ratio m) prolongs the time needed to bring membrane to threshold, thereby reducing CV. These processes determining CV can be described mathematically by the cable theory CV of the propagating APs depends on both active and passive properties of the cell membrane . Active Ito inhibition enhances conduction between cell pairs, suggesting that repolarization currents are able to influence CV Additional factors can modulate the CV of the AP wave. Firstly, gap junctions, high-resistance ion channels located between successive myocytes Unidirectional conduction block can occur in homogeneous cardiac tissues in the presence of functional asymmetries 3V1.5 α-subunit is encoded by the SCN5A gene INa, therefore determines myocardial excitability and CV of the APs. Depolarization also initiates fast inactivation 2 + and the Ca2 +-binding protein calmodulin are present at the carboxyl terminus 2 +/calmodulin-dependent Kinase II phosphorylates the sodium channel, causing a negative shift in the voltage-dependence of steady-state inactivation without altering the voltage-dependence of steady-state activation or the peak INaVoltage-gated sodium channels are responsible for the initiation and propagation of APs in excitable cells. Each channel consists of a pore-forming α-subunit, a modulatory β-subunit and other regulatory proteins. Of these, the NaINa consists mainly of a tetrodotoxin-insensitive component, attributable to the cardiac isoform NaV1.5 V1.1, NaV1.3, and NaV1.6 3.1SCN5A have been described  and sodium–calcium exchanger (INCX), and the outward currents mediated by the voltage-gated delayed rectifier potassium channels (IK) IKr and IKs, respectively) make up IK. Persistent sodium current activation leads to delayed cellular repolarization and the development of early after-depolarization phenomena by the two mechanisms. Firstly, depolarizing shifts in the membrane potential can reactivate the L-type calcium channels ICa,L that further depolarizes the membrane. This sets up a positive feedback loop, triggering an AP. Secondly, at membrane potentials negative to the threshold of ICa,L activation (but before full repolarization), spontaneous calcium release from the sarcoplasmic reticulum can activate INCXINa , similar to that of LQTS type 3 Two main types of autosomal dominant inherited arrhythmias resulting from mutations in SCN5A gene are observed in Brugada syndrome INa is balanced by the transient outward potassium current, Ito in the early plateau phase of the AP. Because higher Ito density is observed in the epicardium compared to the endocardium, it has been postulated that repolarization occurs more readily in the epicardium, resulting in a loss of dome morphology Secondly, loss-of-function mutations in the SCN5A have been associated with the development of Sick Sinus Syndrome (SSS) SCN5A+/− mice modelling Lenègre disease showed progressive conduction defects associated with myocardial fibrosis SCN5A mutations SCN5A gene In contrast, autosomal recessive mutations of SCN5A, acquired alterations in channel properties have been observed. Experimental models of heart failure have demonstrated positive shifts in the resting membrane potential, which causes partial inactivation of sodium channels INa, T), peak INa leading to a decrease in dV/dtmax. Increased late component of INa  has been observed in heart failure and also in post-infarction modelling, leading to action potential prolongation 2 + transient, sarcoplasmic reticular Ca2 + content and Ca2 + removal INa, LINa is likely to be a contributing factor for sudden death in patients with failing ventricles.In addition to the above congenital abnormalities in ATP, is regulated by ATP levels inside the cells ATP channels are activated upon ATP depletion and ADP accumulation ATP channels might not have opened at least during early ischaemia +]o is increased and pH is reduced. A rise in [K+]o would initially cause membrane hyperpolarization, but as steady state is reached, there is a positive shift in the resting membrane potential as described by the Goldman field relationship +]o+]o increases the proportion of inactivated sodium channels, which would reduce dV/dtmax and therefore the CV of the propagating AP INa. Thus, the effects of high [K+]o on CV are biphasic, first increasing then decreasing the speed of conduction max during combined hyperkalaemia, acidosis and hypoxia in guinea pig ventricular muscle 2 + have been described. Beta adrenergic stimulation increases cAMP and activation of protein kinase A INa. However, there may also be a PKA-independent mechanism that reduces INa, likely through a G-protein coupled signaling pathway. In contrast, calcium binds to the conserved C2 domain of protein kinase C INaA particular type of potassium channel, K4i and determines CV 2 +iiGap junctions were first described as hexagonal arrays of protein molecules in the plasma membrane of Mauthner cell synapses of goldfish INa at the end of the cells Gap junctions represent the electrical coupling pathway between cardiomyocytes, whereas anchoring complexes termed fascia adherens junctions and desmosomes mediate mechanical coupling between them. Together these structures form the highly organized intercalated disk 4.1+/− mice showed 45 to 50% decrease in Cx43 expression. In these systems, CV was either unaltered Inherited mutations in GJA1, the gene that encodes for Cx43, have been associated with an autosomal dominant condition called oculodentodigital dysplasia 2 +i has been associated with reduced conductance as well as uncoupling of gap junctions. PKC-mediated phosphorylation, a calcium-dependent process, of the serine residue at the 368 position, has been linked to reduced conductance Acquired abnormalities in gap junctions occur in several pathological conditions, such as ischaemia and infarction 5Understanding the mechanisms of conduction abnormalities allows effective pharmacological therapy to be developed for the treatment of ventricular arrhythmias. Several studies have focused on the arrhythmic effects of gap junction downregulation For heart failure, fibrosis is a significant pro-arrhythmic factor. In hypertensive rats, fibrosis can be prevented by the ACE inhibitor enalapril 6This article reviewed the physiological mechanisms by which alterations in structure and function of gap junctions and sodium channels lead to conduction abnormalities and in turn, to ventricular arrhythmogenesis. It is clear that AP conduction is not a simple phenomenon solely determined by biophysical parameters of ion channel conductances, and resistive and capacitive properties of myocytes. It is also influenced by myocardial cell size, ion channel localization, cellular architecture and source–sink relationships. For example, in heart failure, the extensive electrophysiological and structural remodelling are key factors in arrhythmogenesis. For instance, up- and downregulation of ion channels alter transmembrane currents, and fibrosis affects axial resistance and membrane capacitance. Conduction is a fascinating aspect of cardiac electrophysiology and there is much to be learnt. A systems approach is needed for the development of effective therapeutic agents that can prevent or abolish arrhythmias. Without a full appreciation of the pharmacological effects on the above factors, these agents may be at best, ineffective but at worst, will themselves induce malignant ventricular arrhythmias.