► Identifies key considerations in target selection and optimisation. ► Approaches to assign useful protein features and structure/function relationships. ► Comparison of latest crystallisation propensity predictors on nonredundant data. ► Discusses single point of reference target selection/optimisation resources. ► Guidance on using the SSPF Target Optimisation Utility (TarO).
Selection of protein targets for study is central to structural biology and may be influenced by numerous factors. A key aim is to maximise returns for effort invested by identifying proteins with the balance of biophysical properties that are conducive to success at all stages (e.g. solubility, crystallisation) in the route towards a high resolution structural model. Selected targets can be optimised through construct design (e.g. to minimise protein disorder), switching to a homologous protein, and selection of experimental methodology (e.g. choice of expression system) to prime for efficient progress through the structural proteomics pipeline.
Here we discuss computational techniques in target selection and optimisation, with more detailed focus on tools developed within the Scottish Structural Proteomics Facility (SSPF); namely XANNpred, ParCrys, OB-Score (target selection) and TarO (target optimisation). TarO runs a large number of algorithms, searching for homologues and annotating the pool of possible alternative targets. This pool of putative homologues is presented in a ranked, tabulated format and results are also visualised as an automatically generated and annotated multiple sequence alignment. The target selection algorithms each predict the propensity of a selected protein target to progress through the experimental stages leading to diffracting crystals. This single predictor approach has advantages for target selection, when compared with an approach using two or more predictors that each predict for success at a single experimental stage. The tools described here helped SSPF achieve a high (21%) success rate in progressing cloned targets to diffraction-quality crystals.
MSA, Multiple Sequence Alignment; PTM, Post Translational Modification; SSPF, Scottish Structural Proteomics Facility; MCC, Matthew’s correlation coefficient; AROC, Area Under the Receiver Operator Characteristic curve; Target selection; Crystallisation; Structural genomics; Structural biology; Bioinformatics; Construct design
Jpred (http://www.compbio.dundee.ac.uk/jpred) is a secondary structure prediction server powered by the Jnet algorithm. Jpred performs over 1000 predictions per week for users in more than 50 countries. The recently updated Jnet algorithm provides a three-state (α-helix, β-strand and coil) prediction of secondary structure at an accuracy of 81.5%. Given either a single protein sequence or a multiple sequence alignment, Jpred derives alignment profiles from which predictions of secondary structure and solvent accessibility are made. The predictions are presented as coloured HTML, plain text, PostScript, PDF and via the Jalview alignment editor to allow flexibility in viewing and applying the data. The new Jpred 3 server includes significant usability improvements that include clearer feedback of the progress or failure of submitted requests. Functional improvements include batch submission of sequences, summary results via email and updates to the search databases. A new software pipeline will enable Jnet/Jpred to continue to be updated in sync with major updates to SCOP and UniProt and so ensures that Jpred 3 will maintain high-accuracy predictions.
Summary: JABAWS is a web services framework that simplifies the deployment of web services for bioinformatics. JABAWS:MSA provides services for five multiple sequence alignment (MSA) methods (Probcons, T-coffee, Muscle, Mafft and ClustalW), and is the system employed by the Jalview multiple sequence analysis workbench since version 2.6. A fully functional, easy to set up server is provided as a Virtual Appliance (VA), which can be run on most operating systems that support a virtualization environment such as VMware or Oracle VirtualBox. JABAWS is also distributed as a Web Application aRchive (WAR) and can be configured to run on a single computer and/or a cluster managed by Grid Engine, LSF or other queuing systems that support DRMAA. JABAWS:MSA provides clients full access to each application's parameters, allows administrators to specify named parameter preset combinations and execution limits for each application through simple configuration files. The JABAWS command-line client allows integration of JABAWS services into conventional scripts.
Availability and Implementation: JABAWS is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws.
Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server.
Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org
Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.
Taro; Breast cancer; Antimetastatic activity; Tumor; Cancer therapy
Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to beta-lactam antibiotics. PBP2A crosslinks nascent peptidoglycan when the native TPs are inhibited by beta-lactams. Although mecA expression is essential for beta-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes MRSA strains to beta-lactams even though the beta-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and beta-lactams is noteworthy not simply because strategies to overcome methicillin-resistant S. aureus (MRSA) are desperately needed, but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The “synthetic lethality” of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic resistant bacterial infections.
Rising drug resistance is limiting treatment options
by methicillin-resistant Staphylococcus aureus (MRSA).
Herein we provide new evidence that wall teichoic acid (WTA) biogenesis
is a remarkable antibacterial target with the capacity to destabilize
the cooperative action of penicillin-binding proteins (PBPs) that
underlie β-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted
in the restoration of sensitivity of MRSA to a unique profile of β-lactam
antibiotics with a known selectivity for penicillin binding protein
2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously
approved drugs with a cryptic capacity to potentiate its activity
against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly
potentiated cefuroxime, and this synergy was abolished in strains
lacking tarO. The combination was also effective
in a Galleria mellonella model of infection. Using
both genetic and biochemical strategies, we determined the molecular
target of ticlopidine as the N-acetylglucosamine-1-phosphate
transferase encoded in gene tarO and provide evidence
that WTA biogenesis represents an Achilles heel supporting the cooperative
function of PBP2 and PBP4 in creating highly cross-linked muropeptides
in the peptidoglycan of S. aureus. This approach
represents a new paradigm to tackle MRSA infection.
An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.
Staphylococcus aureus or MRSA (Methicillin Resistant S. aureus), is an acquired pathogen and the primary cause of nosocomial infections worldwide. In S. aureus, teichoic acid is an essential component of the cell wall, and its biosynthesis is not yet well characterized. Studies in Bacillus subtilis have discovered two different pathways of teichoic acid biosynthesis, in two strains W23 and 168 respectively, namely teichoic acid ribitol (tar) and teichoic acid glycerol (tag). The genes involved in these two pathways are also characterized, tarA, tarB, tarD, tarI, tarJ, tarK, tarL for the tar pathway, and tagA, tagB, tagD, tagE, tagF for the tag pathway. With the genome sequences of several MRSA strains: Mu50, MW2, N315, MRSA252, COL as well as methicillin susceptible strain MSSA476 available, a comparative genomic analysis was performed to characterize teichoic acid biosynthesis in these S. aureus strains.
We identified all S. aureus tar and tag gene orthologs in the selected S. aureus strains which would contribute to teichoic acids sythesis.Based on our identification of genes orthologous to tarI, tarJ, tarL, which are specific to tar pathway in B. subtilis W23, we also concluded that tar is the major teichoic acid biogenesis pathway in S. aureus. Further analyses indicated that the S. aureus tar genes, different from the divergon organization in B. subtilis, are organized into several clusters in cis. Most interesting, compared with genes in B. subtilis tar pathway, the S. aureus tar specific genes (tarI,J,L) are duplicated in all six S. aureus genomes.
In the S. aureus strains we analyzed, tar (teichoic acid ribitol) is the main teichoic acid biogenesis pathway. The tar genes are organized into several genomic groups in cis and the genes specific to tar (relative to tag): tarI, tarJ, tarL are duplicated. The genomic organization of the S. aureus tar pathway suggests their regulations are different when compared to B. subtilis tar or tag pathway, which are grouped in two operons in a divergon structure.
All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat.
RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region.
The MyHits web server (http://myhits.isb-sib.ch) is a new integrated service dedicated to the annotation of protein sequences and to the analysis of their domains and signatures. Guest users can use the system anonymously, with full access to (i) standard bioinformatics programs (e.g. PSI-BLAST, ClustalW, T-Coffee, Jalview); (ii) a large number of protein sequence databases, including standard (Swiss-Prot, TrEMBL) and locally developed databases (splice variants); (iii) databases of protein motifs (Prosite, Interpro); (iv) a precomputed list of matches (‘hits’) between the sequence and motif databases. All databases are updated on a weekly basis and the hit list is kept up to date incrementally. The MyHits server also includes a new collection of tools to generate graphical representations of pairwise and multiple sequence alignments including their annotated features. Free registration enables users to upload their own sequences and motifs to private databases. These are then made available through the same web interface and the same set of analytical tools. Registered users can manage their own sequences and annotations using only web tools and freeze their data in their private database for publication purposes.
In the Taro District (population: 4434), the great tsunami of 11 March 2011 destroyed the central region including the clinic, the sole medical facility (one physician, 13 nurses and other staff) in the district, and many citizens were forced to live in evacuation centres.
The Taro District experienced massive damage during the tsunamis of 1896 and 1933. Since then countermeasures to tsunamis have been implemented. The great tsunami on 11 March 2011 caused catastrophic damage to the low-lying areas where approximately 2500 people lived; 1609 buildings were completely destroyed, and approximately 200 people died or were missing across the district.
The Taro National Health Insurance Clinic, the sole medical facility in the Taro District, was required to play a central role in a variety of activities to care for residents in severely affected areas. First of all, evacuees needed to move to neighbouring hospitals or safer evacuation centres because lifeline services were cut off to the first evacuation centre. Then, the clinic staff worked in a temporary clinic; they visited the evacuation centres to assess the public health and medical situation, cared for wounded residents, managed infection control and encouraged a normal lifestyle where possible. Additional medical, pharmaceutical and logistical support was received from outside the district.
There was no noticeably severe damage to health, although there was manifestation of and deterioration in lifestyle-related diseases (e.g. diabetes, hypertension, obesity). Health care activities gradually returned to their pre-disaster levels. At the end of July 2011, the evacuation centres closed, and all evacuees moved to temporary accommodations.
Isolated rural health practitioners were required to be involved in a wide variety of activities related to the disaster in addition to their routine work: e.g. preventive health (public health and safety activities), routine medical care, acute medical care, psychological care, post-mortems and recovery of medical facilities. Although the whole health care system returned to near-normal six months after the disaster, it is important to plan how to develop more resilient medical systems to respond to disasters, especially in rural areas. This article describes my experience and lessons learnt in responding to this disaster.
Kāneʻohe Bay, which is located on the on the NE coast of Oʻahu, Hawaiʻi, represents one of the most intensively studied estuarine coral reef ecosystems in the world. Despite a long history of anthropogenic disturbance, from early settlement to post European contact, the coral reef ecosystem of Kāneʻohe Bay appears to be in better condition in comparison to other reefs around the world. The island of Moku o Loʻe (Coconut Island) in the southern region of the bay became home to the Hawaiʻi Institute of Marine Biology in 1947, where researchers have since documented the various aspects of the unique physical, chemical, and biological features of this coral reef ecosystem. The first human contact by voyaging Polynesians occurred at least 700 years ago. By A.D. 1250 Polynesians voyagers had settled inhabitable islands in the region which led to development of an intensive agricultural, fish pond and ocean resource system that supported a large human population. Anthropogenic disturbance initially involved clearing of land for agriculture, intentional or accidental introduction of alien species, modification of streams to supply water for taro culture, and construction of massive shoreline fish pond enclosures and extensive terraces in the valleys that were used for taro culture. The arrival by the first Europeans in 1778 led to further introductions of plants and animals that radically changed the landscape. Subsequent development of a plantation agricultural system led to increased human immigration, population growth and an end to traditional land and water management practices. The reefs were devastated by extensive dredge and fill operations as well as rapid growth of human population, which led to extensive urbanization of the watershed. By the 1960’s the bay was severely impacted by increased sewage discharge along with increased sedimentation due to improper grading practices and stream channelization, resulting in extensive loss of coral cover. The reefs of Kāneʻohe Bay developed under estuarine conditions and thus have been subjected to multiple natural stresses. These include storm floods, a more extreme temperature range than more oceanic reefs, high rates of sedimentation, and exposure at extreme low tides. Deposition and degradation of organic materials carried into the bay from the watershed results in low pH conditions such that according to some ocean acidification projections the rich coral reefs in the bay should not exist. Increased global temperature due to anthropogenic fossil fuel emmisions is now impacting these reefs with the first “bleaching event” in 1996 and a second more severe event in 2014. The reefs of Kāneʻohe Bay have developed and persist under rather severe natural and anthropogenic perturbations. To date, these reefs have proved to be very resilient once the stressor has been removed. A major question remains to be answered concerning the limits of Kāneʻohe Bay reef resilience in the face of global climate change.
Reef resilience; Hawaii; Climate change; Coral reefs; Kāneʻohe Bay; Corals; Natural history; Eutrophication
The RNA response element TAR plays a critical role in HIV replication by
providing a binding site for the recruitment of the viral transactivator protein
Tat. Using a structure-guided approach, we have developed a series of
conformationally-constrained cyclic peptides that act as structural mimics of
the Tat RNA binding region and block Tat-TAR interactions at nanomolar
concentrations in vitro. Here we show that these compounds
block Tat-dependent transcription in cell-free systems and in cell-based
reporter assays. The compounds are also cell permeable, have low toxicity, and
inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and
CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and
CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic
L50 exhibited an IC50 ∼250 nM. Surprisingly, inhibition of
LTR-driven HIV-1 transcription could not account for the full antiviral
activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic
inhibition curve with the first phase occurring after HIV-1 entry into the host
cell and during the initiation of HIV-1 reverse transcription. The second phase
coincides with inhibition of HIV-1 transcription. Reconstituted reverse
transcription assays confirm that HIV-1 (−) strand strong stop DNA
synthesis is blocked by L50-TAR RNA interactions in-vitro.
These findings are consistent with genetic evidence that TAR plays critical
roles both during reverse transcription and during HIV gene expression. Our
results suggest that antiviral drugs targeting TAR RNA might be highly effective
due to a dual inhibitory mechanism.
The HIV-1 transactivator protein (Tat), together with the elongation factor
P-TEFb binds to an HIV-1 RNA secondary structure in the 5′-UTRs of nascent
viral mRNAs (TAR) and promotes transcription elongation. This process has been
an attractive target for drug development but previous inhibitors that bind
either Tat or TAR have been plagued by poor inhibition of virus replication,
limited cell penetration, and off-target effects. In this article, we describe a
series of rationally designed cyclic peptides that block Tat-TAR interactions.
L50, the most potent of these compounds, inhibits a wide range of HIV-1 strains
from around the world. Remarkably, L50 inhibits two distinct steps in the HIV-1
lifecycle. As expected, L50 inhibits Tat-dependent HIV-1 transcription, but the
majority of its anti-HIV activity is due to a block in reverse transcription,
i.e. synthesis of the proviral DNA from the RNA genome. L50 inhibition of
reverse transcription reveals an important role for TAR RNA during reverse
transcription as well as providing one of first examples of a drug with a dual
mechanism of action.
Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome.
In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats.
A remarkable range of environmental conditions is present in the Hawaiian Islands due to their gradients of elevation, rainfall and island age. Despite being well known as a location for the study of evolutionary processes and island biogeography, little is known about the composition of the non-marine algal flora of the archipelago, its degree of endemism, or affinities with other floras. We conducted a biodiversity survey of the non-marine macroalgae of the six largest main Hawaiian Islands using molecular and microscopic assessment techniques. We aimed to evaluate whether endemism or cosmopolitanism better explain freshwater algal distribution patterns, and provide a baseline data set for monitoring future biodiversity changes in the Hawaiian Islands.
1,786 aquatic and terrestrial habitats and 1,407 distinct collections of non-marine macroalgae were collected from the islands of Kauai, Oahu, Molokai, Maui, Lanai and Hawaii from the years 2009–2014. Targeted habitats included streams, wet walls, high elevation bogs, taro fields, ditches and flumes, lakes/reservoirs, cave walls and terrestrial areas. Sites that lacked freshwater macroalgae were typically terrestrial or wet wall habitats that were sampled for diatoms and other microalgae. Approximately 50% of the identifications were of green algae, with lesser proportions of diatoms, red algae, cyanobacteria, xanthophytes and euglenoids. 898 DNA sequences were generated representing eight different markers, which enabled an assessment of the number of taxonomic entities for genera collected as part of the survey. Forty-four well-characterized taxa were assessed for global distribution patterns. This analysis revealed no clear biogeographic affinities of the flora, with 27.3% characterized as “cosmopolitan”, 11.4% “endemic”, and 61.3% as intermediate.
The Hawaiian freshwater algal biodiversity survey represents the first comprehensive effort to characterize the non-marine algae of a tropical region in the world using both morphological and molecular tools. Survey data were entered in the Hawaiian Freshwater Algal Database, which serves as a digital repository of photographs and micrographs, georeferenced localities and DNA sequence data. These analyses yielded an updated checklist of the non-marine macroalgae of the Hawaiian Islands, and revealed varied biogeographic affinities of the flora that are likely a product of both natural and anthropogenic dispersal.
Algal distribution; Biodiversity survey; Biogeography; Cyanobacteria; Dispersal; Freshwater algae; Hawaiian Islands; Molecular characterization; Taxonomy; UPA
The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.
Twenty-two cover crops were evaluated for their ability to reduce damage by root-knot nematode, Meloidogyne javanica, to taro, Colocastia esculenta, in a tropical cropping system. Cover crops were grown and incorporated into the soil before taro was planted. Barley, greenpanic, glycine, marigold, sesame, sunn hemp, and sorghum x sudangrass DeKalb ST6E were poor or nonhosts to the nematode as measured by low population changes of nematodes in soil between cover crop planting and taro planting. Alfalfa, buckwheat, cowpea, lablab, Lana vetch, mustard, oat, okra, rhodes grass, ryegrain, ryegrass, siratro, sweet corn, and wheat allowed nematode populations to increase dramatically. Taro yields were greatest in the marigold plots and lowest in the ryegrain plots. Taro corm weight decreased with increasing initial nematode population (Pi) (r = 0.22, P = 0.056). Siratro, ryegrass, and Blizzard wheat plots had higher taro yield than plots with similar Pi's but planted to other cover crops. These cover crops may have antagonism to other soil microorganisms or their decomposition products may be toxic or adversely affect the nematodes. Cover crops can be an effective and valuable nematode management tactic for use in minor tropical cropping systems such as taro.
barley; Colocagia esculenta; control; cover crop; Crotalafia juncea; glycine; greenpanic; Hordeum vulgare; management; marigold; cover crop; Crotalaria juncea; glycine; greenpanic; Hordeum vulgate; management; marigold; Meloidogyne javanica; nematode; Neonotonia wightii; Panicum maximum; root-knot nematode; sesame; Sesamum indicum; sorghum x sudangrass DeKalb ST6E; sunn hemp; sustainable agriculture; Tagetes erecta; taro; tropical cover crops
TropGENE-DB, is a crop information system created to store genetic, molecular and phenotypic data of the numerous yet poorly documented tropical crop species. The most common data stored in TropGENE-DB are information on genetic resources (agro-morphological data, parentages, allelic diversity), molecular markers, genetic maps, results of quantitative trait loci analyses, data from physical mapping, sequences, genes, as well as the corresponding references. TropGENE-DB is organized on a crop basis with currently three running modules (sugarcane, cocoa and banana), with plans to create additional modules for rice, cotton, oil palm, coconut, rubber tree, pineapple, taro, yam and sorghum. The TropGENE-DB information system is accessible for consultation via the internet at http://tropgenedb.cirad.fr. Specific web consultation interfaces have been designed to allow quick consultations as well as complex queries.
An annotated genomic sequence of the corn anthracnose fungus Colletotrichum graminicola has been published previously, but correct identification of gene models by means of automated gene annotation remains a challenge. RNA-Seq offers the potential for substantially improved gene annotations and for the identification of posttranscriptional RNA modifications, such as alternative splicing and RNA editing.
Based on the nucleotide sequence information of transcripts, we identified 819 novel transcriptionally active regions (nTARs) and revised 906 incorrectly predicted gene models, including revisions of exon-intron structure, gene orientation and sequencing errors. Among the nTARs, 146 share significant similarity with proteins that have been identified in other species suggesting that they are hitherto unidentified genes in C. graminicola. Moreover, 5′- and 3′-UTR sequences of 4378 genes have been retrieved and alternatively spliced variants of 69 genes have been identified. Comparative analysis of RNA-Seq data and the genome sequence did not provide evidence for RNA editing in C. graminicola.
We successfully employed deep sequencing RNA-Seq data in combination with an elaborate bioinformatics strategy in order to identify novel genes, incorrect gene models and mechanisms of transcript processing in the corn anthracnose fungus C. graminicola. Sequence data of the revised genome annotation including several hundreds of novel transcripts, improved gene models and candidate genes for alternative splicing have been made accessible in a comprehensive database. Our results significantly contribute to both routine laboratory experiments and large-scale genomics or transcriptomic studies in C. graminicola.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-842) contains supplementary material, which is available to authorized users.
Colletotrichum graminicola; Anthracnose of corn; RNA-Seq; Genome annotation
Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI′J′ and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI′J′) could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI′J′ is due to poor translation of the TarJ′ enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.
Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression.
Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α).
There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062).
TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-620) contains supplementary material, which is available to authorized users.
Tumor microenvironment; Angiogenesis; tRNA synthetase; Serous papillary ovarian cancer; Database analysis; Multivariate Cox analysis
The regulation of protein function through reversible phosphorylation by protein kinases and phosphatases is a general mechanism controlling virtually every cellular activity. Eukaryotic protein kinases can be classified into distinct, well-characterized groups based on amino acid sequence similarity and function. We recently reported a highly sensitive and accurate hidden Markov model-based method for the automatic detection and classification of protein kinases into these specific groups. The Kinomer v. 1.0 database presented here contains annotated classifications for the protein kinase complements of 43 eukaryotic genomes. These span the taxonomic range and include fungi (16 species), plants (6), diatoms (1), amoebas (2), protists (1) and animals (17). The kinomes are stored in a relational database and are accessible through a web interface on the basis of species, kinase group or a combination of both. In addition, the Kinomer v. 1.0 HMM library is made available for users to perform classification on arbitrary sequences. The Kinomer v. 1.0 database is a continually updated resource where direct comparison of kinase sequences across kinase groups and across species can give insights into kinase function and evolution. Kinomer v. 1.0 is available at http://www.compbio.dundee.ac.uk/kinomer/.