Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6 Delta exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

Bridging large and small scales of water models using hybrid Molecular Dynamics/Fluctuating Hydrodynamics framework

This thesis presents a two-dimensional water model investigation and development of a multiscale method for the modelling of large systems, such as virus in water or peptide immersed in the solvent. We have implemented a two-dimensional ‘Mercedes Benz’ (MB) or BN2D water model using Molecular Dynamics. We have studied its dynamical and structural properties dependence on the model’s parameters. For the first time we derived formulas to calculate thermodynamic properties of the MB model in the microcanonical (NVE) ensemble. We also derived equations of motion in the isothermal–isobaric (NPT) ensemble. We have analysed the rotational degree of freedom of the model in both ensembles. We have developed and implemented a self-consistent multiscale method, which is able to communicate micro- and macro- scales. This multiscale method assumes, that matter consists of the two phases. One phase is related to micro- and the other to macroscale. We simulate the macro scale using Landau Lifshitz-Fluctuating Hydrodynamics, while we describe the microscale using Molecular Dynamics. We have demonstrated that the communication between the disparate scales is possible without introduction of fictitious interface or approximations which reduce the accuracy of the information exchange between the scales. We have investigated control parameters, which were introduced to control the contribution of each phases to the matter behaviour. We have shown, that microscales inherit dynamical properties of the macroscales and vice versa, depending on the concentration of each phase. We have shown, that Radial Distribution Function is not altered and velocity autocorrelation functions are gradually transformed, from Molecular Dynamics to Fluctuating Hydrodynamics description, when phase balance is changed. In this work we test our multiscale method for the liquid argon, BN2D and SPC/E water models. For the SPC/E water model we investigate microscale fluctuations which are computed using advanced mapping technique of the small scales to the large scales, which was developed by Voulgarakisand et. al.

Molecular markers and human history: A tale of two haplogroup systems

To chronicle demographic movement across African Asian corridors, a variety of molecular (sequence analysis, restriction mapping and denaturing high performance liquid chromatography etc.) and statistical (correspondence analysis, AMOVA, calculation of diversity indices and phylogenetic inference, etc.) techniques were employed to assess the phylogeographic patterns of mtDNA control region and Y chromosomal variation among 14 sub-Saharan, North African and Middle Eastern populations. The patterns of genetic diversity revealed evidence of multiple migrations across several African Asian passageways as well within the African continent itself. The two-part analysis uncovered several interesting results which include the following: (1) a north (Egypt and Middle East Asia) to south (sub-Saharan Africa) partitioning of both mtDNA and Y chromosomal haplogroup diversity, (2) a genetic diversity gradient in sub-Saharan Africa from east to west, (3) evidence in favor of the Levantine Corridor over the Horn of Africa as the major genetic conduit since the Last Glacial Maximum, (4) a substantially higher mtDNA versus Y chromosomal sub-Saharan component in the Middle East collections, (5) a higher representation of East versus West African mtDNA haplotypes in the Arabian Peninsula populations versus no such bias in the Levant groups and lastly, (6) genetic remnants of the Bantu demographic expansion in sub-Saharan Africa. ^

Validação de espécies de Centropomus (Centropomidae, Perciformes) do litoral do Rio Grande do Norte através de caracterização citogenética e molecular

The Centropomidae family consists of three genera, Centropomus, Lates and Psammoperca. Centropomus is the most diverse group, with six Centropomus species occur in the Western Atlantic Ocean C. poeyi Chávez, 1961, C. parallelus Poey, 1860, C. mexicanus Bocourt, 1868, C. pectinatus Poey, 1860 and C. ensiferus Poey, 1860. Some of these species are considered cryptic, because of its morphological traits showed low resolution for identification purposes. Despite showing great interest as a natural resource and fish culture, aspects of their diversity and karyotypic patterns are poorly understood. In this work morphological identification and comparison of mitochondrial 16S gene sequence were used to identify the species of the genus Centropomus occurring in Rio Grande do Norte, northeastern Brazil. Two sepecies were identified, C. undecimalis and C. mexicanus, which had the chromosomal aspects analyzed, through Classical cytogenetic method analyzes (conventional staining, C-banding, Ag-NORs), fluorochrome staining AT- and GC-specific, replication bands by incorporating of the base analog 5-Bromo-2’-deoxyuridine (5-BrdU), in situ chromosomal mapping of (TTAGGG)n sequences and in situ chromosome mapping 18S and 5S rRNA genes. Both species show 2n=48 acrocentric chromosomes, with ribosomal sites (Ag-NOR/18S rDNA/ Mitramycin+) in second chromosomal pair, in telomeric position on the long arm in C. mexicanus and interstitial in C. undecimalis. The nuclear organization pair (pair 2) shown a resolutive cytotaxonomic marker for these two species. The generated data reveal a lower species diversity than previously believed, suggesting that greater attention should be paid in taxonomic identification of the species, in view of optimize commercial actions exploitation, biological conservation and cultivation.

A Molecular-scale Programmable Stochastic Process Based On Resonance Energy Transfer Networks: Modeling And Applications

While molecular and cellular processes are often modeled as stochastic processes, such as Brownian motion, chemical reaction networks and gene regulatory networks, there are few attempts to program a molecular-scale process to physically implement stochastic processes. DNA has been used as a substrate for programming molecular interactions, but its applications are restricted to deterministic functions and unfavorable properties such as slow processing, thermal annealing, aqueous solvents and difficult readout limit them to proof-of-concept purposes. To date, whether there exists a molecular process that can be programmed to implement stochastic processes for practical applications remains unknown.

In this dissertation, a fully specified Resonance Energy Transfer (RET) network between chromophores is accurately fabricated via DNA self-assembly, and the exciton dynamics in the RET network physically implement a stochastic process, specifically a continuous-time Markov chain (CTMC), which has a direct mapping to the physical geometry of the chromophore network. Excited by a light source, a RET network generates random samples in the temporal domain in the form of fluorescence photons which can be detected by a photon detector. The intrinsic sampling distribution of a RET network is derived as a phase-type distribution configured by its CTMC model. The conclusion is that the exciton dynamics in a RET network implement a general and important class of stochastic processes that can be directly and accurately programmed and used for practical applications of photonics and optoelectronics. Different approaches to using RET networks exist with vast potential applications. As an entropy source that can directly generate samples from virtually arbitrary distributions, RET networks can benefit applications that rely on generating random samples such as 1) fluorescent taggants and 2) stochastic computing.

By using RET networks between chromophores to implement fluorescent taggants with temporally coded signatures, the taggant design is not constrained by resolvable dyes and has a significantly larger coding capacity than spectrally or lifetime coded fluorescent taggants. Meanwhile, the taggant detection process becomes highly efficient, and the Maximum Likelihood Estimation (MLE) based taggant identification guarantees high accuracy even with only a few hundred detected photons.

Meanwhile, RET-based sampling units (RSU) can be constructed to accelerate probabilistic algorithms for wide applications in machine learning and data analytics. Because probabilistic algorithms often rely on iteratively sampling from parameterized distributions, they can be inefficient in practice on the deterministic hardware traditional computers use, especially for high-dimensional and complex problems. As an efficient universal sampling unit, the proposed RSU can be integrated into a processor / GPU as specialized functional units or organized as a discrete accelerator to bring substantial speedups and power savings.

Molecular analysis of evolutionary relationships of Phaseolus dumosus with the gene pool of common bean

Valuable genetic variation for bean breeding programs is held within the common bean secondary gene pool which consists of Phaseolus albescens, P. coccineus, P. costaricensis, and P. dumosus. However, the use of close relatives for bean improvement is limited due to the lack of knowledge about genetic variation and genetic plasticity of many of these species. Characterisation and analysis of the genetic diversity is necessary among beans' wild relatives; in addition, conflicting phylogenies and relationships need to be understood and a hypothesis of a hybrid origin of P. dumosus needs to be tested. This thesis research was orientated to generate information about the patterns of relationships among the common bean secondary gene pool, with particular focus on the species Phaseolus dumosus. This species displays a set of characteristics of agronomic interest, not only for the direct improvement of common bean but also as a source of valuable genes for adaptation to climate change. Here I undertake the first comprehensive study of the genetic diversity of P. dumosus as ascertained from both nuclear and chloroplast genome markers. A germplasm collection of the ancestral forms of P. dumosus together with wild, landrace and cultivar representatives of all other species of the common bean secondary gene pool, were used to analyse genetic diversity, phylogenetic relationships and structure of P. dumosus. Data on molecular variation was generated from sequences of cpDNA loci accD-psaI spacer, trnT-trnL spacer, trnL intron and rps14-psaB spacer and from the nrDNA the ITS region. A whole genome DArT array was developed and used for the genotyping of P. dumosus and its closes relatives. 4208 polymorphic markers were generated in the DArT array and from those, 742 markers presented a call rate >95% and zero discordance. DArT markers revealed a moderate genetic polymorphism among P. dumosus samples (13% of polymorphic loci), while P. coccineus presented the highest level of polymorphism (88% of polymorphic loci). At the cpDNA one ancestral haplotype was detected among all samples of all species in the secondary genepool. The ITS region of P. dumosus revealed high homogeneity and polymorphism bias to P. coccineus genome. Phylogenetic reconstructions made with Maximum likelihood and Bayesian methods confirmed previously reported discrepancies among the nuclear and chloroplast genomes of P. dumosus. The outline of relationships by hybridization networks displayed a considerable number of interactions within and between species. This research provides compelling evidence that P. dumosus arose from hybridisation between P. vulgaris and P. coccineus and confirms that P. costaricensis has likely been involved in the genesis or backcrossing events (or both) in the history of P. dumosus. The classification of the specie P. persistentus was analysed based on cpDNA and ITS sequences, the results found this species to be highly related to P. vulgaris but not too similar to P. leptostachyus as previously proposed. This research demonstrates that wild types of the secondary genepool carry a significant genetic variation which makes this a valuable genetic resource for common bean improvement. The DArT array generated in this research is a valuable resource for breeding programs since it has the potential to be used in several approaches including genotyping, discovery of novel traits, mapping and marker-trait associations. Efforts should be made to search for potential populations of P. persistentus and to increase the collection of new populations of P. dumosus, P. albescens and P. costaricensis that may provide valuable traits for introgression into common bean and other Phaseolus crops.

Mapping of C4 plant input from North West Africa into North East Atlantic sediments

Mapping the abundance of 13C in leaf-wax components in surface sediments recovered from the seafloor off northwest Africa (0-35°N) reveals a clear pattern of delta13C distribution, indicating systematic changes in the proportions of terrestrial C3 and C4 plant input. At 20°N latitude, we find that isotopically enriched products characteristic of C4 plants account for more than 50% of the terrigenous inputs. This signal extends westward beneath the path of the dust-laden Sahara Air Layer (SAL). High C4 contributions, apparently carried by January trade winds, also extend far into the Gulf of Guinea. Similar distributions are obtained if summed pollen counts for the Chenopodiaceae-Amaranthaceae and the Poaceae are used as an independent C4 proxy. We conclude that the specificity of the latitudinal distribution of vegetation in North West Africa and the pathways of the wind systems (trade winds and SAL) are responsible for the observed isotopic patterns observed in the surface sediments. Molecular-isotopic maps on the marine-sedimentary time horizons (e.g., during the last glacial maximum) are thus a robust tool for assessing the phytogeographic changes on the tropical and sub-tropical continents, which have important implications for the changes in climatic and atmospheric conditions.

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Trans-ethnic fine mapping highlights kidney-function genes linked to salt sensitivity

We analyzed genome-wide association studies (GWASs), including data from 71,638 individuals from four ancestries, for estimated glomerular filtration rate (eGFR), a measure of kidney function used to define chronic kidney disease (CKD). We identified 20 loci attaining genome-wide-significant evidence of association (p < 5 × 10(-8)) with kidney function and highlighted that allelic effects on eGFR at lead SNPs are homogeneous across ancestries. We leveraged differences in the pattern of linkage disequilibrium between diverse populations to fine-map the 20 loci through construction of "credible sets" of variants driving eGFR association signals. Credible variants at the 20 eGFR loci were enriched for DNase I hypersensitivity sites (DHSs) in human kidney cells. DHS credible variants were expression quantitative trait loci for NFATC1 and RGS14 (at the SLC34A1 locus) in multiple tissues. Loss-of-function mutations in ancestral orthologs of both genes in Drosophila melanogaster were associated with altered sensitivity to salt stress. Renal mRNA expression of Nfatc1 and Rgs14 in a salt-sensitive mouse model was also reduced after exposure to a high-salt diet or induced CKD. Our study (1) demonstrates the utility of trans-ethnic fine mapping through integration of GWASs involving diverse populations with genomic annotation from relevant tissues to define molecular mechanisms by which association signals exert their effect and (2) suggests that salt sensitivity might be an important marker for biological processes that affect kidney function and CKD in humans.

Transcriptome-Wide Mapping of Pea Seed Ageing Reveals a Pivotal Role for Genes Related to Oxidative Stress and Programmed Cell Death

Understanding of seed ageing, which leads to viability loss during storage, is vital for ex situ plant conservation and agriculture alike. Yet the potential for regulation at the transcriptional level has not been fully investigated. Here, we studied the relationship between seed viability, gene expression and glutathione redox status during artificial ageing of pea (Pisum sativum) seeds. Transcriptome-wide analysis using microarrays was complemented with qRT-PCR analysis of selected genes and a multilevel analysis of the antioxidant glutathione. Partial degradation of DNA and RNA occurred from the onset of artificial ageing at 60% RH and 50 degrees C, and transcriptome profiling showed that the expression of genes associated with programmed cell death, oxidative stress and protein ubiquitination were altered prior to any sign of viability loss. After 25 days of ageing viability started to decline in conjunction with progressively oxidising cellular conditions, as indicated by a shift of the glutathione redox state towards more positive values (>-190 mV). The unravelling of the molecular basis of seed ageing revealed that transcriptome reprogramming is a key component of the ageing process, which influences the progression of programmed cell death and decline in antioxidant capacity that ultimately lead to seed viability loss.

Genetic variation of temperature-regulated curd induction in cauliflower: elucidation of floral transition by genome-wide association mapping and gene expression analysis

Cauliflower (Brassica oleracea var. botrytis) is a vernalization-responsive crop. High ambient temperatures delay harvest time. The elucidation of the genetic regulation of floral transition is highly interesting for a precise harvest scheduling and to ensure stable market supply. This study aims at genetic dissection of temperature-dependent curd induction in cauliflower by genome-wide association studies and gene expression analysis. To assess temperature dependent curd induction, two greenhouse trials under distinct temperature regimes were conducted on a diversity panel consisting of 111 cauliflower commercial parent lines, genotyped with 14,385 SNPs. Broad phenotypic variation and high heritability (0.93) were observed for temperature-related curd induction within the cauliflower population. GWA mapping identified a total of 18 QTL localized on chromosomes O1, O2, O3, O4, O6, O8, and O9 for curding time under two distinct temperature regimes. Among those, several QTL are localized within regions of promising candidate flowering genes. Inferring population structure and genetic relatedness among the diversity set assigned three main genetic clusters. Linkage disequilibrium (LD) patterns estimated global LD extent of r(2) = 0.06 and a maximum physical distance of 400 kb for genetic linkage. Transcriptional profiling of flowering genes FLOWERING LOCUS C (BoFLC) and VERNALIZATION 2 (BoVRN2) was performed, showing increased expression levels of BoVRN2 in genotypes with faster curding. However, functional relevance of BoVRN2 and BoFLC2 could not consistently be supported, which probably suggests to act facultative and/or might evidence for BoVRN2/BoFLC-independent mechanisms in temperature regulated floral transition in cauliflower. Genetic insights in temperature-regulated curd induction can underpin genetically informed phenology models and benefit molecular breeding strategies toward the development of thermo-tolerant cultivars.

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangifera indica) transcriptome for mapping and estimation of genetic diversity

The development of molecular markers for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here our identification of thousands of unambiguous molecular markers that can be easily assayed across genotypes of the species. With origin centered in Southeast Asia, mangos are grown throughout the tropics and subtropics as a nutritious fruit that exhibits remarkable intraspecific phenotypic diversity. With the goal of building a high density genetic map, we have undertaken discovery of sequence variation in expressed genes across a broad range of mango cultivars. A transcriptome sequence reference was built de novo from extensive sequencing and assembly of RNA from cultivar 'Tommy Atkins'. Single nucleotide polymorphisms (SNPs) in protein coding transcripts were determined from alignment of RNA reads from 24 mango cultivars of diverse origins: 'Amin Abrahimpur' (India), 'Aroemanis' (Indonesia), 'Burma' (Burma), 'CAC' (Hawaii), 'Duncan' (Florida), 'Edward' (Florida), 'Everbearing' (Florida), 'Gary' (Florida), 'Hodson' (Florida), 'Itamaraca' (Brazil), 'Jakarata' (Florida), 'Long' (Jamaica), 'M. Casturi Purple' (Borneo), 'Malindi' (Kenya), 'Mulgoba' (India), 'Neelum' (India), 'Peach' (unknown), 'Prieto' (Cuba), 'Sandersha' (India), 'Tete Nene' (Puerto Rico), 'Thai Everbearing' (Thailand), 'Toledo' (Cuba), 'Tommy Atkins' (Florida) and 'Turpentine' (West Indies). SNPs in a selected subset of protein coding transcripts are currently being converted into Fluidigm assays for genotyping of mapping populations and germplasm collections. Using an alternate approach, SNPs (144) discovered by sequencing of candidate genes in 'Kensington Pride' have already been converted and used for genotyping.

AFLP-based genetic mapping of the " bud-flowering" trait in heather (Calluna vulgaris)

Background: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait " flower type" .Results: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the " two-way pseudo-testcross" and the " integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris.Conclusions: All maps confirmed the independent inheritance of the most important horticultural traits " flower type" , " flower colour" , and " leaf colour". An AFLP marker for the most important breeding target " flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers. © 2013 Behrend et al.; licensee BioMed Central Ltd.