@article {110447, title = {DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration.}, journal = {Dev Biol}, year = {2022}, month = {2022 May 03}, abstract = {

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2022.04.011}, author = {Sarkar, Souradeep R and Dubey, Vinay Kumar and Jahagirdar, Anusha and Lakshmanan, Vairavan and Haroon, Mohamed Mohamed and Sowndarya, Sai and Sowdhamini, Ramanathan and Palakodeti, Dasaradhi} } @article {104418, title = {Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.}, journal = {J Med Genet}, volume = {59}, year = {2022}, month = {2021 Dec 16}, pages = {984-992}, abstract = {

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM.

METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 () gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect.

CONCLUSIONS: Our study demonstrates for the first time that the variants in the gene are associated with HCM, and early detection of the variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of gene variants.

}, issn = {1468-6244}, doi = {10.1136/jmedgenet-2021-107866}, author = {Jain, Pratul Kumar and Jayappa, Shashank and Sairam, Thiagarajan and Mittal, Anupam and Paul, Sayan and Rao, Vinay J and Chittora, Harshil and Kashyap, Deepak K and Palakodeti, Dasaradhi and Thangaraj, Kumarasamy and Shenthar, Jayaprakash and Koranchery, Rakesh and Rajendran, Ranjith and Alireza, Haghighi and Mohanan, Kurukkanparampil Sreedharan and Rathinavel, Andiappan and Dhandapany, Perundurai S} } @article {117968, title = {Smed-ETS-1 regulates cathepsin+ cell function and epidermal lineage landscape via basement membrane remodeling.}, journal = {J Cell Sci}, year = {2022}, month = {2022 Sep 29}, abstract = {

Extracellular matrix (ECM) is an important component of stem cell niche. Remodelling of ECM mediated by ECM regulators such as MMPs plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF), ETS-1 expressed in the cathepsin+ cell population in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outward from this region and progressively differentiate to maintain the terminal epidermis. Our data shows thickening of basement membrane in the absence of ets-1 results in defective migration of stem cells progeny. Furthermore, the absence of ets-1 led to a defective epidermal progenitor landscape, in spite of its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodelling in regulating tissue homeostasis and regeneration in planaria.

}, issn = {1477-9137}, doi = {10.1242/jcs.259900}, author = {Dubey, Vinay Kumar and Sarkar, Souradeep R and Lakshmanan, Vairavan and Dalmeida, Rimple and Gulyani, Akash and Palakodeti, Dasaradhi} } @article {104420, title = {Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.}, journal = {Stem Cell Reports}, volume = {16}, year = {2021}, month = {2021 Nov 09}, pages = {2736-2751}, abstract = {

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2021.09.013}, author = {Chandrasekaran, Abinaya and Dittlau, Katarina Stoklund and Corsi, Giulia I and Haukedal, Henriette and Doncheva, Nadezhda T and Ramakrishna, Sarayu and Ambardar, Sheetal and Salcedo, Claudia and Schmidt, Sissel I and Zhang, Yu and Cirera, Susanna and Pihl, Maria and Schmid, Benjamin and Nielsen, Troels Tolstrup and Nielsen, J{\o}rgen E and Kolko, Miriam and Kobol{\'a}k, Julianna and Dinny{\'e}s, Andr{\'a}s and Hyttel, Poul and Palakodeti, Dasaradhi and Gorodkin, Jan and Muddashetty, Ravi S and Meyer, Morten and Aldana, Blanca I and Freude, Kristine K} } @article {88157, title = {Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs).}, journal = {RNA}, year = {2021}, month = {2021 Jan 14}, abstract = {

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterise planarian tRFs, we first annotated 457 tRNAs in S.mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians - the shorter tRF-5s and itRFs, and the abundantly expressed 5{\textquoteright}-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5{\textquoteright}-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of Ago1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalogue of post-transcriptional regulatory systems in planarian, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

}, issn = {1469-9001}, doi = {10.1261/rna.077701.120}, author = {Lakshmanan, Vairavan and T N, Sujith and Bansal, Dhiru and Padubidri, Shivaprasad V and Palakodeti, Dasaradhi and Krishna, Srikar} } @article {100194, title = {Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.}, journal = {Life Sci Alliance}, volume = {4}, year = {2021}, month = {2021 Dec}, abstract = {

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

}, issn = {2575-1077}, doi = {10.26508/lsa.202101213}, author = {Chakravarthy, Anirudh and Nandakumar, Anirudh and George, Geen and Ranganathan, Shyamsundar and Umashankar, Suchitta and Shettigar, Nishan and Palakodeti, Dasaradhi and Gulyani, Akash and Ramesh, Arati} } @article {78005, title = {Modulation of β-catenin levels regulates cranial neural crest patterning and dispersal into first pharyngeal arch.}, journal = {Dev Dyn}, year = {2020}, month = {2020 May 19}, abstract = {

BACKGROUND: Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined.

RESULTS: Here, we report that a novel β-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane β-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of β-catenin levels, we show a requirement for down-modulating β-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in β-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch.

CONCLUSION: We suggest that β-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.

}, issn = {1097-0177}, doi = {10.1002/dvdy.208}, author = {Javali, Alok and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Sambasivan, Ramkumar} } @article {60607, title = {Dynamic expression of tRNA-derived small RNAs define cellular states.}, journal = {EMBO Rep}, volume = {20}, year = {2019}, month = {2019 Jul}, pages = {e47789}, abstract = {

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5{\textquoteright}-tsRNAs. We report the identification of a set of 5{\textquoteright}-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5{\textquoteright}-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5{\textquoteright}-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5{\textquoteright}-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5{\textquoteright}-tsRNAs in defining distinct cell states.

}, issn = {1469-3178}, doi = {10.15252/embr.201947789}, author = {Krishna, Srikar and Yim, Daniel Gr and Lakshmanan, Vairavan and Tirumalai, Varsha and Koh, Judice Ly and Park, Jung Eun and Cheong, Jit Kong and Low, Joo Leng and Lim, Michelle Js and Sze, Siu Kwan and Shivaprasad, Padubidri and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi and DasGupta, Ramanuj} } @article {65299, title = {Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.}, journal = {FEBS Lett}, year = {2019}, month = {2019 Sep 17}, abstract = {

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a non-canonical enzyme, phenylalanine hydroxylase (PAH). Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescue the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

}, issn = {1873-3468}, doi = {10.1002/1873-3468.13607}, author = {Sarkar, Arunabha and Mukundan, Namita and Sowndarya, Sai and Dubey, Vinay Kumar and Babu, Rosana and Lakshmanan, Vairavan and Rangiah, Kannan and Panicker, Mitradas M and Palakodeti, Dasaradhi and Subramanian, Sabarinath Peruvemba and Ramaswamy, Subramanian} } @article {47057, title = {FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.}, journal = {iScience}, volume = {9}, year = {2018}, month = {2018 Nov 07}, pages = {399-411}, abstract = {

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2{\textquoteright}O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2{\textquoteright}O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2018.11.007}, author = {D{\textquoteright}Souza, Michelle Ninochka and Gowda, Naveen Kumar Chandappa and Tiwari, Vishal and Babu, Rosana Ottakandathil and Anand, Praveen and Dastidar, Sudhriti Ghosh and Singh, Randhir and Selvaraja, Bhuvaneish and Pal, Rakhi and Ramesh, Arati and Chattarji, Sumantra and Chandran, Siddharthan and Gulyani, Akash and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {22019, title = {Cytoplasmic poly (A) binding protein (PABPC2) critically regulates epidermal maintenance and turnover in planarian Schmidtea mediterranea.}, journal = {Development}, year = {2017}, month = {2017 Aug 14}, abstract = {

Identifying key cellular events that facilitate stem cell function and tissue organization is critical for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic Poly A binding protein, SMED-PABPC2. Knockdown (KD) of Smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggested epidermal lineage transcripts, including zfp-1, to be translationally regulated by SMED-PABPC2. Together, our results uncover a novel role of SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing, and subsequent processes for regeneration.

}, issn = {1477-9129}, doi = {10.1242/dev.152942}, author = {Bansal, Dhiru and Kulkarni, Jahnavi and Nadahalli, Kavana and Lakshmanan, Vairavan and Krishna, Srikar and Sasidharan, Vidyanand and Geo, Jini and Dilipkumar, Shilpa and Pasricha, Renu and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi} } @article {21471, title = {Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm.}, journal = {Sci Adv}, volume = {3}, year = {2017}, month = {2017 Jul}, pages = {e1603025}, abstract = {

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

}, issn = {2375-2548}, doi = {10.1126/sciadv.1603025}, author = {Shettigar, Nishan and Joshi, Asawari and Dalmeida, Rimple and Gopalkrishna, Rohini and Chakravarthy, Anirudh and Patnaik, Siddharth and Mathew, Manoj and Palakodeti, Dasaradhi and Gulyani, Akash} } @article {12201, title = {MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia.}, journal = {Cell Death Dis}, volume = {8}, year = {2017}, month = {2017 Jan 12}, pages = {e2547}, abstract = {

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

}, issn = {2041-4889}, doi = {10.1038/cddis.2016.471}, author = {Arya, Deepak and Sachithanandan, Sasikala P and Ross, Cecil and Palakodeti, Dasaradhi and Li, Shang and Krishna, Sudhir} } @article {11620, title = {Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea.}, journal = {G3 (Bethesda)}, volume = {6}, year = {2016}, month = {2016 Oct 13}, pages = {3035-3048}, abstract = {

In eukaryotes, 3{\textquoteright} untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3{\textquoteright}UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3{\textquoteright}UTRs for \~{}14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40\% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3{\textquoteright}UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration.

}, issn = {2160-1836}, doi = {10.1534/g3.116.031120}, author = {Lakshmanan, Vairavan and Bansal, Dhiru and Kulkarni, Jahnavi and Poduval, Deepak and Krishna, Srikar and Sasidharan, Vidyanand and Anand, Praveen and Seshasayee, Aswin and Palakodeti, Dasaradhi} }