Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Stable solution to <Emphasis Type="Italic">l</Emphasis><Subscript>2,1</Subscript>-based robust inductive matrix completion and its application in linking long noncoding RNAs to human diseases

Backgrounds

A large number of long intergenic non-coding RNAs (lincRNAs) are linked to a broad spectrum of human diseases. The disease association with many other lincRNAs still remain as puzzle. Validation of such links between the two entities through biological experiments are expensive. However, a plethora lincRNA-data are available now, thanks to the High Throughput Sequencing (HTS) platforms, Genome Wide Association Studies (GWAS), etc, which opens the opportunity for cutting-edge machine learning and data mining approaches to extract meaningful relationships among lincRNAs and diseases. However, there are only a few in silico lincRNA-disease association inference tools available to date, and none of them utilizes side information of both the entities simultaneously in a single framework.

Methods

The recently developed Inductive Matrix Completion (IMC) technique provides a recommendation platform among two entities considering respective side information about them. However, the formulation of IMC is incapable of handling noise and outliers that may be present in the datasets, while data sparsity consideration is another issue with the standard IMC method. Thus, a robust version of IMC is needed that can solve the two issues. As a remedy, in this paper, we propose Stable Robust Inductive Matrix Completion (SRIMC) that utilizes the l _2,1 norm based regularization to optimize the objective function with a unique 2-step stable solution approach.

Results

We applied SRIMC to the available association data between human lincRNAs and OMIM disease phenotypes as well as a diverse set of side information about the lincRNAs and the diseases. The method performs better than the state-of-the-art methods in terms of p r e c i s i o n @ k and r e c a l l @ k at the top-k disease prioritization to the subject lincRNAs. We also demonstrate that SRIMC is equally effective for querying about novel lincRNAs, as well as predicting rank of a newly known disease for a set of well-characterized lincRNAs.

Conclusions

With the experimental results and computational evaluation, we show that SRIMC is robust in handling datasets with noise and outliers as well as dealing with novel lincRNAs and disease phenotypes.

     

Turing learning: a metric-free approach to inferring behavior and its application to swarms

We propose Turing Learning, a novel system identification method for inferring the behavior of natural or artificial systems. Turing Learning simultaneously optimizes two populations of computer programs, one representing models of the behavior of the system under investigation, and the other representing classifiers. By observing the behavior of the system as well as the behaviors produced by the models, two sets of data samples are obtained. The classifiers are rewarded for discriminating between these two sets, that is, for correctly categorizing data samples as either genuine or counterfeit. Conversely, the models are rewarded for ‘tricking’ the classifiers into categorizing their data samples as genuine. Unlike other methods for system identification, Turing Learning does not require predefined metrics to quantify the difference between the system and its models. We present two case studies with swarms of simulated robots and prove that the underlying behaviors cannot be inferred by a metric-based system identification method. By contrast, Turing Learning infers the behaviors with high accuracy. It also produces a useful by-product—the classifiers—that can be used to detect abnormal behavior in the swarm. Moreover, we show that Turing Learning also successfully infers the behavior of physical robot swarms. The results show that collective behaviors can be directly inferred from motion trajectories of individuals in the swarm, which may have significant implications for the study of animal collectives. Furthermore, Turing Learning could prove useful whenever a behavior is not easily characterizable using metrics, making it suitable for a wide range of applications.     

The structure of the claspettes in the male genitalia of the subgenus <Emphasis Type="Italic">Ochlerotatus</Emphasis> Lynch Arribalzaga of the genus <Emphasis Type="Italic">Aedes</Emphasis> Meigen (Diptera,Culicidae) and their diagnostic significance

The structure of the claspettes (modified projections in the male genitalia) in species of the subgenus Ochlerotatus Lynch Arribalzaga of the genus Aedes Meigen is described in detail. A thorough investigation of the three-dimensional structure of the claspettes is necessary for identification of the Ochlerotatus species from Russia. The study of 30 species of Ochlerotatus demonstrated that frequently the use of the claspette structure as a diagnostic character was incorrect when the claspettes were described as flat structures. Examination of the three-dimensional structure of the claspettes has revealed characters differentiating Aedes diantaeus, A. intrudens and A. pullatus; A. communis, A. punctor, and A. hexodontus; A. annulipes, A. excrucians, and A. euedes, as well as some other species.     

Plant pressure sensitive adhesives: similar chemical properties in distantly related plant lineages

Main conclusion

A mixture of resins based on aliphatic esters and carboxylic acids occurs in distantly related genera Peperomia and Roridula , serving different functions as adhesion in seed dispersal and prey capture. According to mechanical characteristics, adhesive secretions on both leaves of the carnivorous flypaper Roridula gorgonias and epizoochorous fruits of Peperomia polystachya were expected to be similar. The chemical analysis of these adhesives turned out to be challenging because of the limited available mass for analysis. Size exclusion chromatography and Fourier transform infrared spectroscopy were suitable methods for the identification of a mixture of compounds, most appropriately containing natural resins based on aliphatic esters and carboxylic acids. The IR spectra of the Peperomia and Roridula adhesive resemble each other; they correspond to that of a synthetic ethylene–vinyl acetate copolymer, but slightly differ from that of natural tree resins. Thus, the pressure sensitive adhesive properties of the plant adhesives are chemically proved. Such adhesives seem to appear independently in distantly related plant lineages, habitats, life forms, as well as plant organs, and serve different functions such as prey capture in Roridula and fruit dispersal in Peperomia. However, more detailed chemical analyses still remain challenging because of the small available volume of plant adhesive.

     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Role of ships’ hull fouling and tropicalization process on European carcinofauna: new records in Galician waters (NW Spain)

Ten unusual decapod crustacean species are reported for the coasts of Galicia (NW Spain), eight of them recorded for the first time in this area. Three species: Pilumnopeus africanus, Charybdis hellerii and Pachygrapsus gracilis, are non-indigenous species. The reports of Panopeus africanus and Inachus aguiarii represent the northernmost localities of these African species, whose previous North limits were in the Southwestern European coasts. Remaining five species: Xaiva biguttata, Bathynectes longipes, Parthenopoides massena, Monodaeus couchii and Homola barbata are scarcely known species found within their distribution range. Updated data about these species are given, both from the point of view of their distribution and identification (morphological and molecular methods), as well as the potential pathways for introduction of the non-indigenous ones. According to evidence presented in the present study, biofouling still remains an important vector of species transmission and surely has been undervalued with respect to ballast water.     

Soil spore banks of ectomycorrhizal fungi in endangered Japanese Douglas-fir forests

Interactions between trees and ectomycorrhizal fungi are critical to the growth and survival of both partners. However, ectomycorrhizal symbiosis has barely been explored in endangered trees, and no information is available regarding soil spore banks of ectomycorrhizal fungi from forests of threatened trees. Here, we evaluated soil spore banks of ectomycorrhizal fungi from endangered Japanese Douglas-fir (Pseudotsuga japonica) forests using bioassay approaches with congeneric P. menziesii and Pinus densiflora seedlings in combination with molecular identification techniques. Rhizopogon togasawariana was predominant in soil propagule banks and was found in all remaining P. japonica forests when assayed with P. menziesii, while no colonization of this fungus was observed on Pinus seedlings. Given the observed specificity of R. togasawariana for P. menziesii and its phylogenetic position within the Pseudotsuga-specific Rhizopogon lineage, its geographical distribution is likely restricted to the remaining Japanese Douglas-fir forests, indicating a high extinction risk for this fungus as well as its endangered host. Spore banks of R. togasawariana remained highly infective after preservation for 1 year or heat treatment at 70 °C, suggesting an ecological strategy of establishing ectomycorrhizal associations on regenerating Japanese Douglas-fir seedlings after disturbance, as observed in other Rhizopogon–Pinaceae combinations. Therefore, the regeneration of Japanese Douglas-fir seedlings may depend largely on the soil spore banks dominated by R. togasawariana, which has co-evolved with the Japanese Douglas-fir for over 30 million years. More attention must be paid to underground ectomycorrhizal fungi for the conservation of endangered tree species, especially in the era of human-induced mass extinction.     

Evaluation of molecular techniques for identification and enumeration of <Emphasis Type="Italic">Raoultella terrigena</Emphasis> ATCC 33257 in water purifier efficacy testing

Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID^® 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10⁶ CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.     

Potential for biocontrol of the exotic starfish<Emphasis Type="Italic">, Asterias amurensis</Emphasis>, using a native starfish

Asterias amurensis is a starfish native to the northern Pacific that was introduced into southern Australia in the 1980s. It is widely viewed as one of Australia’s most serious invasive marine pests, but there are few methods available to control new or established populations. The role of Coscinasterias muricata in controlling the distribution of Asterias in Port Phillip Bay, and its potential to eliminate new infestations of Asterias were investigated. Laboratory feeding trials, where alternative mussel prey were available, showed Coscinasterias consumed Asterias at the rate of ~45/year. Field surveys in Port Phillip Bay showed that most Coscinasterias occurred at depths shallower than 15 m, while most Asterias occurred at depths greater than 15 m, and the ratio of Asterias/Coscinasterias only exceeded 45 at depths greater than 15 m. Consequently, if laboratory and field feeding rates are similar, Coscinasterias would be expected to exert significant control over Asterias populations at depths shallower than 15 m, and augmenting Coscinasterias populations at sites of new Asterias infestations may help eliminate newly-established populations.     

Development of haplotype-specific molecular markers for the low-molecular-weight glutenin subunits

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the major components of gluten, and their allelic variation has been widely associated with different wheat end-use quality parameters. These proteins are encoded by multigene families located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3); the genes at each locus are divided by large intergenic and highly recombinogenic regions. Among the methods used for the LMW-GS allele identification, polymerase chain reaction (PCR)-based molecular markers have the advantages of being simple, accurate, and independent from the plant stage of development. However, the available LMW-GS molecular markers are either incapable of capturing the complexity of the LMW-GS gene family or difficult to interpret. In the present study, we report the development of a set of PCR-based molecular markers specific for the LMW-GS haplotypes present at each Glu-3 locus. Based on the LMW-GS gene sequences available in GenBank, single nucleotide polymorphisms (SNPs) specific for each Glu-3 haplotype were identified and the relevant PCR primers were designed. In total, we developed three molecular markers for the Glu-A3 and Glu-B3 loci, respectively, and five molecular markers for the Glu-D3 locus. The markers were tested on 44 bread wheat varieties previously characterized for their LMW-GS genic profile and found to be equally or more efficient than previously developed LMW-GS PCR-based markers. This set of markers allows an easier and less ambiguous identification of specific LMW-GS haplotypes associated with gluten strength and can facilitate marker-assisted breeding for wheat quality.     

DNA-Based Identification of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> s.l.: a Multiplexed Amplification Refractory Mutation System (MARMS) and High Resolution Melting Curve Analysis (HRMA)

The wide distribution of Valeriana officinalis as a herbal remedy as well as the considerably higher concentration of putative mutagenic valepotriate metabolites in other drug-delivering valerian species like Valeriana procera Kunth and Valeriana jatamansi Jones ex Roxb. illustrate the necessity of secure authentication of roots of Valeriana officinalis s.l., especially as the morphologically similar roots of the acutely toxic Veratrum album can be mistaken for those of Valeriana officinalis. We developed two DNA-based systems, a multiplex amplification refractory mutation system (MARMS), and a high-resolution melting curve analysis (HRMA) assay, both based on a sequence mutation within the atpB-rbcL region. With both methods, identification of Valeriana officinalis s.l. was possible. With the HRMA, the characteristic melting curve of 33 samples of Valeriana officinalis s.l. and of two commercial samples of Valerianae radix was distinct from the melting curves of all other Valeriana species (60 accessions), and from the closely related genera Centranthus and Valerianella. Since adulteration of Valeriana with toxic Veratrum species was reported previously, Veratrum primers were included in a multiplex PCR-HRM analysis. This system allowed the detection of a Veratrum admixture down to the level of 0.01 %. Although the advantages, in terms of sensitivity, specificity and practicality of the HRM for analysis of degraded plant material were superior to the MARMS assay, both methods are suitable for routine analysis. The results demonstrated the general ability of HRMA to detect specific (toxic) adulterations in drugs in a semiquantitative way.     

Molecular understandings on ‘the never thirsty’ and apomictic <Emphasis Type="Italic">Cenchrus</Emphasis> grass

The genus Cenchrus comprises around 25 species of ‘bristle clade’ grasses. Cenchrus ciliaris (buffel grass) is a hardy, perennial range grass that survives in poor sandy soils and limiting soil moisture conditions and, due to the very same reasons, this grass is one of the most prevalent fodder grasses of the arid and semi-arid regions. Most of the germplasms of Cenchrus produce seeds asexually through the process of apomeiosis. Therefore, the lack of sufficient sexual lines has hindered the crop improvement efforts in Cenchrus being confined to simple selection methods. Many attempts have been initiated in buffel grass to investigate the various molecular aspects such as genomic signatures of different species and genotypes, molecular basis of abiotic stress tolerance and reproductive performance. Even though it is an important fodder crop, molecular investigations in Cenchrus lack focus and the molecular information available on this grass is scanty. Cenchrus is a very good gene source for abiotic stress tolerance and apomixis studies. Biotechnological interventions in Cenchrus can help in crop improvement in Cenchrus as well as other crops through transgenic technology or marker assisted selection. To date no consolidated review on biotechnological interventions in Cenchrus grass has been published. Therefore we provide a thorough and in depth review on molecular research in Cenchrus focusing on molecular signatures of evolution, tolerance to abiotic stress and apomictic reproductive mechanism.     

Identification of birch pollen species using FTIR spectroscopy

In this study, the morphology and chemical composition of pollen grains of six birch species (Betula utilis Doorenbos, B. dahurica, B. maximowicziana, B. pendula, B. pubescens and B. humilis) were examined to verify which of these features allow distinguishing them in a more unambiguous way. For this purpose, scanning electron microscopy and light microscopy, as well as Fourier transform infrared (FTIR) spectroscopy and curve-fitting analysis of amide I profile, were performed. The microscopy images show that the pollen grains of B. pubescens, B. pendula and B. humilis are similar in diameter and significantly smaller than those of others species, with the largest diameter observed for B. utilis Doorenbos. However, the results obtained from FTIR spectroscopy indicate that the chemical compositions of B. pubescens and B. pendula are similar, but B. humilis is outlaying. Summarizing, it is not possible to unambiguously state, which feature or which technique is the best for differentiating between the six chosen birch species. However, the study showed that both techniques have potential for identification of birch pollen species.     

New sterigmatocystin-producing species of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Versicolores</Emphasis> from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.