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PubMed2006

Small interfering RNAs generated by recombinant dicer induce inflammatory gene expression independent from the TAK1-NFkappaB-MAPK signaling pathways.

Generation of mixtures of small interfering (si) RNAs by recombinant dicer avoids selection of efficient target sites within mRNAs but little is known about off-target effects of this approach. Using recombinant human dicer we generated siRNA mixtures (dsiRNA) directed against the protein kinase TAK1 and its subunit TAB1, important upstream molecules in the pathways activated by IL-1, TNF, and toll-like receptors (TLR). dsiRNA against TAK1 or TAB1 significantly suppressed their target proteins as well as TAK1-mediated activation of NFkappaB, p38 MAPK, and JNK, and of IL-8 transcription. However, microarray analysis of 136 endogenous inflammatory genes revealed that dsiRNA against TAB1 or TAK1 did not suppress IL-1 or TNF-induced genes but rather induced a broader range of 15 inflammatory genes as well as seven known interferon-response genes. The same genes were induced by dsiRNA directed against luciferase but not by a synthetic control siRNA molecule. Hence, our results show that complex mixtures of siRNA induce an inflammatory gene response that is independent from TAK1-mediated signal transduction. In the light of the increasing usage of enzymatically prepared libraries of siRNA these results provide important insight into potential off-target effects of this approach.

Gene Expression↗

PubMed2006

Identification of 2-arylbenzimidazoles as potent human histamine H4 receptor ligands.

A series of 2-arylbenzimidazoles was synthesized and found to bind with high affinity to the human histamine H(4) receptor. Structure-activity relationships were investigated through library preparation and evaluation as well as traditional medicinal chemistry approaches, leading to the discovery of compounds with single-digit nanomolar affinity for the H(4) receptor.

Aldehydes↗

PubMed2005

Direct interaction between metastasis-associated protein 1 and endophilin 3.

The yeast two-hybrid system was used to search for partners of mouse metastasis-associated protein 1 (Mta1). Screening of a cDNA library prepared from mouse embryo yielded positive clones coding for endophilin 3. The site of interaction was suggested to be the SH-3-binding domain of Mta1 and SH-3 domain of endophilin 3. This interaction was confirmed by GST pull-down assay in vitro and immunoprecipitation in vivo. The Mta1 and endophilin 3 transcripts were highly expressed in testis and brain. But, Mta1 localized mainly in nucleus and to a lesser extent in cytoplasm while endophilin 3 localized mainly in cytoplasm. If Mta1 functions in cytoplasm, it might be involved in the regulation of endocytosis mediated by endophilin 3.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida).

We report here for the first time the molecular characterization of a hyaluronidase from an aquatic source. SFHYA1 is the hyaluronidase found in the venom gland of stonefish, Synanceja horrida. Using a cDNA segment amplified with degenerate oligonucleotides based on the amino acid sequences of a conserved region in testicular-type hyaluronidases and a tryptic fragment of SFHYA1, clones encoding the precursor of this enzyme were isolated from a cDNA library prepared from stonefish venom glands. The deduced amino acid sequence of SFHYA1 shows that SFHYA1 is expressed as a precursor peptide with a 28-residue signal peptide for targeting it into endoplasmic reticulum. Mature SFHYA1 is a polypeptide composed of 449 residues containing three potential N-glycosylation sites, four putative hyaluronan-binding motifs [B(X)7B] and various residues implicated in substrate binding and catalysis. This cDNA was expressed in an active form in insect-cells but not in E. coli. Homology-based computational analyses suggested that SFHYA1 closely resembles the PH-20 family of hyaluronidases.

Amino Acid Sequence↗

PubMed2005

Initial characterization of kinocilin, a protein of the hair cell kinocilium.

A subtracted library prepared from vestibular sensory areas [Nat. Genet. 26 (2000) 51] was used to identify a 960bp murine transcript preferentially expressed in the inner ear and testis. The cDNA predicts a basic 124aa protein that does not share any significant sequence homology with known proteins. Immunofluorescence and immunoelectron microscopy revealed that the protein is located mainly in the kinocilium of sensory cells in the inner ear. The protein was thus named kinocilin. In the mouse, kinocilin is first detected in the kinocilia of vestibular and auditory hair cells at embryonic days 14.5, and 18.5, respectively. In the mature vestibular hair cells, kinocilin is still present in the kinocilium. As the auditory hair cells begin to lose the kinocilium during postnatal development, kinocilin becomes distributed in an annular pattern at the apex of these cells, where it co-localizes with the tubulin belt [Hear. Res. 42 (1989) 1]. In mature auditory hair cells, kinocilin is also present at the level of the cuticular plate, at the base of each stereocilium. In addition, as the kinocilium regresses from developing auditory hair cells, kinocilin begins to be expressed by the pillar cells and Deiters cells, that both contain prominent transcellular and apical bundles of microtubules. By contrast, kinocilin was not detected in the supporting cells in the vestibular end organs. The protein is also present in the manchette of the spermatids, a transient structure enriched in interconnected microtubules. We propose that kinocilin has a role in stabilizing dense microtubular networks or in vesicular trafficking.

Aging↗

PubMed2004

Vitellogenin of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae): gene organization and differential use by members of the genus.

The vitellogenin (Vg) gene of the parasitoid wasp, Encarsia formosa (Hymenoptera: Aphelinidae), has been cloned and sequenced. The gene codes for a protein consisting of 1814 amino acids in seven exons. The position of the six introns in the E. formosa gene align with those inferred for the Vg gene of the honeybee, Apis mellifera. The position of two introns in the hymenopteran sequences are shared with every full-length insect Vg gene characterized to date. The deduced amino acid sequence of the E. formosa Vg gene most closely resembles that of the ichneumonid parasitoid, Pimpla nipponica (38% identity). The gene product, less the putative signal peptide, contains large quantities of serine (11.3% of total residues) but lacks the extensive polyserine tracts found in the Vgs of insects outside the apocritan Hymenoptera. The gene also codes for the highest level of lysine (9.5%), and lowest levels of phenylalanine (2.6%) and tyrosine (2.3%), observed in any insect Vg characterized to date. The mature gene product retains 12 cysteine residues in positions conserved in other insect Vgs. Ovary homogenates suggest that processed Vg is stored in the egg as an uncleaved molecule of approximately 200 kDa. Vg expression was examined in three additional Encarsia species. The protein was found in female E. sophia and E. luteola, but not in male E. luteola or female E. pergandiella. Despite extensive screening of a phage library prepared from E. pergandiella genomic DNA, a Vg gene was not detected in this species.

Amino Acid Sequence↗

PubMed2005

Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae).

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.

Amino Acid Sequence↗

PubMed2005

The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: sequence, properties, immunocytochemical localization and function.

CDNAs coding for five procathepsin L-like proteinases (pCALs) were cloned and sequenced from a cDNA library prepared from Tenebrio molitor larval midguts: pCAL1a (with the isoforms pCAL1b and pCAL1c), pCAL2, and pCAL3. All the pCALs have the active residues Cys 25, His 169, Asn 175, and Gln 19 (papain numbering), the ERFNIN motif of papain-like enzymes and their sequences are homologous to cathepsin L enzymes. pCAL1a was expressed in bacterial systems. It is auto-catalytically activated at low pH, has kinetic properties and N-terminal sequence identical to hemocyte cathepsin L-like proteinase (CAL) and was used to raise antibodies. Semi-quantitative RT-PCR data showed that mRNAs for pCAL2 and pCAL3 were transcribed in midgut and in lesser amounts in hemolymph, whereas that for pCAL1a was transcribed in these tissues and also in fat body, Malpighian tubules, and carcass. Imunochemical detection recognized pCAL1a translation in all tissue homogenates, except anterior midgut. At this region, the presence of pCAL2 is suggested on the grounds of electrophoretical migration and high recovery of CAL2 activity from anterior midgut cells and from isolated midgut contents. Immunocytochemical localization data revealed that pCAL1a occurs in lysosome-like vesicles in all tissues, except anterior midgut, where a labelling considered to correspond to pCAL2 is found in large acidic granules being released by apocrine secretion. Putative pCAL2 was also detected in midgut contents, probably in the form of CAL2, the major luminal CAL, which was purified to homogeneity. A cladogram of insect CALs result in a monophyletic branch with lysosomal T. molitor enzymes and enzymes from five insect orders and in a polyphyletic array of coleopteran sequences, including digestive CALs from T. molitor. The data suggest that only Coleoptera have digestive CALs that may originate by gene duplication and independent evolution relative to the gene encoding the lysosomal enzyme.

Amino Acid Sequence↗

PubMed2022

Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids.

Cell-free DNA (cfDNA) has become the predominant analyte of liquid biopsy; however, recent studies suggest the presence of subnucleosomal-sized DNA fragments in circulation that are likely single-stranded. Here, we report a method called direct capture and sequencing (DCS) tailored to recover such fragments from biofluids by directly capturing them using short degenerate probes followed by single strand-based library preparation and next-generation sequencing. DCS revealed a new DNA population in biofluids, named ultrashort single-stranded DNA (ussDNA). Evaluation of the size distribution and abundance of ussDNA manifested generality of its presence in humans, animal species, and plants. In humans, red blood cells were found to contain abundant ussDNA; plasma-derived ussDNA exhibited modal size at 50 nt. This work reports the presence of an understudied DNA population in circulation, and yet more work is awaiting to study its generation mechanism, tissue of origin, disease implications, etc.

Biological sciences↗

PubMed2026

Effect of repeated mass drug administration on the transmission of yaws: a retrospective genomic epidemiology study.

BACKGROUND: Yaws, a neglected tropical disease caused by Treponema pallidum subspecies pertenue (T p pertenue), has evaded eradication, in part due to a high proportion of asymptomatic cases. Repeated mass drug administration (MDA), whereby an entire population is repeatedly treated irrespective of disease, could provide a solution. Here, we aimed to investigate the effect of MDA on the genomic epidemiology of T p pertenue. METHODS: We conducted a retrospective genomic epidemiology study on samples collected during a cluster-randomised trial of mass administration of azithromycin for yaws eradication in the Namatanai District of Papua New Guinea. Participants were in 38 wards (administrative units encompassing several villages) in three local-level government areas (LLGs). The experimental group received an initial round of MDA followed by two further rounds 6 months and 12 months after the first round. The control group received one round of MDA followed by two rounds of treatment targeting clinical cases and contacts only, on the same schedule as the MDA in the experimental group. A follow-up survey on both groups was done 18 months after the first MDA round. Swab samples were collected at each round from ulcerative and nodular skin lesions, and blood was collected by finger-prick for serological testing at 18 months. Metadata on ulcer size (cm) and duration (days) were recorded at each round, and treponemal and non-treponemal antibodies were recorded at 18 months. Samples from swabs positive for T p pertenue underwent library preparation and whole-genome sequencing. We examined the phylogenetic relationships between genomes, linking them with geospatial and patient metadata to understand the impact of MDA on T p pertenue diversity and transmission. FINDINGS: Swabs collected from 297 individuals with active yaws from April 30, 2018, to Nov 2, 2019, yielded 222 good-quality Tp pertenue genomes. We identified 20 sublineages of T p pertenue in the control group and 21 in the experimental group at the beginning of the study. At the end of the study, there were 13 sublineages in the control group and three in the experimental group, of which two persisted in both groups. Three sublineages not detected at baseline were observed in the control group after commencing MDA. The two sublineages that persisted in both groups had non-synonymous mutations in penicillin-binding proteins. One of these sublineages evolved macrolide resistance in three individuals and was associated with lowered treponemal antibody (p=0·0036) and longer ulcer duration (p=0·015). Despite the study taking place within a small island, sublineages were geographically clustered, with pairs of samples from the same ward (odds ratio 7·1, 95% CI 5·7-8·8; p<0·0001) or neighbouring wards (4·3, 3·3-5·4; p<0·0001) more likely to share the same sublineages compared with pairs from different LLGs. Additionally, older individuals were more likely to share sublineages than were younger individuals (1·5, 1·2-1·9; p<0·0001). INTERPRETATION: Repeated MDA was successful in reducing and maintaining the genetic diversity of T p pertenue at a low level but was associated with the development of macrolide resistance. Yaws re-emergence after MDA was attributed to multiple sublineages, of which the majority were detected in the population before MDA. Participants within the same ward were more likely to share sublineages than those that were more widely geographically separated, suggesting that re-emergence was driven by local transmission. These findings could inform future yaws elimination strategies. FUNDING: European Research Council, EU, Provincial Deputation of Barcelona, Barberà Solidària Foundation, Wellcome, and Fundació "la Caixa".

Adolescent↗

PubMed2004

Spatial and temporal expression patterns of Xenopus Nkx-2.3 gene in skin epidermis during metamorphosis.

Using PCR cloning, the mRNA of XNkx-2.3 gene, a Xenopus tinman homologue, was identified in a cDNA library prepared from thyroid hormone (T(3))-treated tadpole skin. Quantitative RT-PCR and RNase Protection Assay confirmed the expression of XNkx-2.3 in adult frog skin and its amount was similar to the amount found in heart. In situ hybridization indicated that XNkx-2.3 was expressed in the frog epidermis. Further analysis of XNkx-2.3 expression patterns demonstrates that it shares great similarities with a 63 kDa keratin, a well-characterized marker for skin maturation, in the following aspects. First, XNkx-2.3 was expressed in tadpole skin during metamorphosis (stages 55-59), but not in pre-metamorphic (stage 54) skin. Secondly, XNkx-2.3 expression in skin responded to T(3) stimulation because it could be precociously induced by T(3) at pre-metamorphic stage, both in tadpoles and in cultures of skin explants. Finally, the T(3)-induced appearance of XNkx-2.3 in head skin occurred earlier and at higher level than that in tail skin. These data suggest that XNkx-2.3 may be an important factor for skin maturation and may also serve as a good marker to indicate the maturation of Xenopus epidermis.

Animals↗

PubMed2006

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei.

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.

Amino Acid Sequence↗

PubMed2004

Expression analysis of the Arabidopsis peroxidase multigenic family.

Class III peroxidases form a numerous multigenic family in higher plants, whose expression is particularly sensitive to internal or external events. Arabidopsis thaliana genome harbours 73 genes encoding peroxidases. Since they exhibit homologies ranging from 28% to 93% at the nucleotide level, the risk of cross-hybridisation may be important when measuring the level of transcripts by blotting techniques, using whole cDNA sequences. We developed a procedure to assess the expression of all peroxidase genes on one membrane, with a high specificity. The method was based on the determination for each gene of a short specific sequence (amplicon) exhibiting at the most 70% homology with any other sequences of the Arabidopsis genome. Amplicons specific for each of the 73 peroxidase genes and two pseudogenes were blotted on a nylon membrane that was hybridised with radiolabelled cDNA libraries prepared from mRNAs of Arabidopsis roots, stems, leaves and flowers. Many genes were expressed at a low level, often in all organs, while sixteen genes were rather strongly expressed, in two to four organs. Some genes with no ESTs reported in databases were found to be expressed and this was confirmed by RT-PCR. Isoelectric focusing analysis revealed that the isoperoxidase pattern was similar in leaves, stems and flowers, but was quite different in roots. To our knowledge, only one similar study has been performed on the cytochrome P450 family, using microarrays, but this is the first work describing the expression profile of a whole large multigenic family using specific macroarrays.

Arabidopsis↗

PubMed2026

Portable metagenomics for preventive surveillance and outbreak control in livestock and poultry: Pathogen detection, resistome profiling, and antimicrobial stewardship.

Conventional diagnostics for livestock and poultry outbreaks commonly rely on culture or targeted PCR panels, which may be too slow or too narrow to guide early control decisions. Portable metagenomics, particularly real-time nanopore sequencing, offers a route to broad pathogen detection, antimicrobial-resistance gene profiling, and outbreak investigation within an integrated workflow. This implementation-focused review evaluates how near-point-of-care metagenomics may support preventive veterinary medicine through earlier detection, surveillance, cohorting, biosecurity decisions, and antimicrobial stewardship. We synthesize sample-to-answer workflows for enteric and respiratory disease in food-producing animals, including sampling, nucleic-acid extraction, host depletion or target enrichment, library preparation, sequencing, bioinformatics, quality control, and interpretation. Applications in calf diarrhea, bovine respiratory disease, poultry outbreaks, mastitis, and resistome monitoring are considered alongside the central limitation that detection alone does not establish causation. Pathogen and resistance-gene signals must therefore be interpreted with clinical signs, lesions, epidemiology, controls, and confirmatory testing. We also propose a minimum reporting checklist, intended as a practical framework rather than a validated consensus standard. Portable metagenomics is not a replacement for conventional diagnostics, but appropriately validated workflows can reduce uncertainty during time-sensitive outbreaks and support more judicious antimicrobial use.

Animals↗

PubMed2005

cDNA cloning of a novel P-I lebetase isoform Le-4.

In order to better understand the function of fibrinolytic enzyme lebetase isoforms and the synthesis of disintegrins we have isolated a cDNA encoding the most basic isoform (Le-4) from the cDNA library prepared from the poly(A)(+) RNA of the venomous gland of an individual Vipera lebetina snake. The truncated 5'-sequence of 1112 basepairs encodes the mature protein with 203 amino acid residues with calculated isoelectric point and size of 5.6 and 22,930 Da, respectively. Multiple comparison of the deduced amino acid sequence of the metalloprotease part of Le-4 is related to short reprolysins, identities were within the range of 60--87%. The two lebetase isoforms are synthesized in different way: Le-4 is synthesized with metalloprotease domain only; Le-3 is synthesized with metalloprotease and disintegrin-like domain and processed posttranslationally. The sequence of the disintegrin-like part of Le-3 is identical to A-chain of the heterodimeric disintegrin VLO5 from Vipera lebetina obtusa venom (Calvete et al., 2003).

Amino Acid Sequence↗

PubMed2006

Molecular cloning and characterization of chicken NK-lysin.

NK-lysin is an anti-microbial and anti-tumor protein expressed by NK cells and T lymphocytes. In a previous report, we identified a set of overlapping expressed sequence tags constituting a contiguous sequence (contig 171) homologous to mammalian NK-lysins. In the current report, a cDNA encoding NK-lysin was isolated from a library prepared from chicken intestinal intraepithelial lymphocytes (IELs). It consisted of an 850 bp DNA sequence with an open reading frame of 140 amino acids and a predicted molecular mass of 15.2 kDa. Comparison of its deduced amino acid sequence showed less than 20% identity to mammalian NK-lysins. The tissue distribution of NK-lysin mRNA revealed highest levels in intestinal IELs, intermediate levels in splenic and peripheral blood lymphocytes, and lowest levels in thymic and bursa lymphocytes. Following intestinal infection of chickens with Eimeria maxima, one of seven Eimeria species causing avian coccidiosis, NK-lysin transcript levels increased 3-4-fold in CD4+ and CD8+ intestinal IELs. However, cell depletion experiments suggested other T lymphocyte subpopulations also expressed NK-lysin. The kinetics of NK-lysin mRNA expression indicated that, whereas infection with E. acervulina induced maximum expression only at 7-8 days post-infection, E. maxima and E. tenella elicited biphasic responses at 3-4 and 7-8 days post-infection. Finally, recombinant chicken NK-lysin expressed in COS7 cells exhibited anti-tumor cell activity against LSCC-RP9, a retrovirus-transformed B-cell line. We conclude that chicken NK-lysin plays important roles during anti-microbial and anti-tumor defenses.

Amino Acid Sequence↗

PubMed2006

A 38-kDa protein from Babesia gibsoni and its antibody response in an experimentally infected dog.

A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.

Animals↗

PubMed2006

Babesia gibsoni rhoptry-associated protein 1 and its potential use as a diagnostic antigen.

A cDNA encoding the rhoptry-associated protein 1 (RAP-1) homologue was obtained by immunoscreening an expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1740bp. Computer analysis suggested that the sequence contains an open reading frame of 1425bp encoding an expected protein with a molecular weight of 52kDa. Based on the sequence similarity, this putative protein was designated as the B. gibsoni RAP-1 (BgRAP-1). The BgRAP-1 gene was expressed in the Escherichia coli BL21 strain, and the recombinant BgRAP-1 was used as the antigen in the enzyme-linked immunosorbent assay (ELISA). The results can differentiate between the B. gibsoni-infected dog sera and the Babesia canis infected dog sera or the normal dog sera. Furthermore, the antibody response against the recombinant protein was maintained during the chronic stage of infection, indicating that the recombinant BgRAP-1 protein might be a useful diagnostic antigen for the detection of antibodies to B. gibsoni infection in dogs.

Amino Acid Sequence↗