Alternative Tag Research Articles

Resection of the adipose fin from rainbow trout acutely alters the cerebral transcriptome and respiratory frequency

As many as 100 million salmonid fish are tagged annually by removing the adipose fin, usually for recovery statistics and the fishing industry. In a multi-parametrical approach, we evaluated the response to resection of the adipose fin in rainbow trout Oncorhynchus mykiss over the course of 72 h. Both the concentrations of lactate and the cerebral transcriptomes differed in rainbow trout early after adipose fin clipping compared to the matching intact controls. The number of differentially expressed genes significantly increased, first in the midbrain and later in the forebrain, in fin-clipped compared to intact trout. Several of these genes were predicted to be involved in pain-induced and synaptogenesis-related pathways, suggesting that adipose fin resection may affect the architecture of neuronal networks. In an experimental setup applying swimming tunnels, we tested whether removal of the adipose fin had any consequences on the behavior of fin-clipped versus intact trout exposed to a regularly recurring water flow. Concomitant with increased and prevailing breath frequencies after repeated exposure to water flows, we observed a marked transcriptional response in the forebrain of fin-clipped trout compared with intact unchallenged controls at later time points after the resection. The resulting list of differentially expressed genes was assigned to a broad range of signaling pathways involved in trans-synaptic signaling, neuronal interaction dynamics, and neuroplasticity. Taken together, our findings suggest that resection of the potentially flow-sensory adipose fin alters pathways related to pain perception and likely enhances the physiological response to the current, probably indicating increased physical effort. We also deduce from the transcriptomic data that amputation of the adipose fin leads to reorganization of the high-level integration system in the brain. The short-term effects observed in the present study require further validation to estimate the prevailing stress for the organism. Nevertheless, we propose considering alternative tagging methods for salmonid fish that do not conflict with fish welfare, as the amputation of the adipose fin may have more far-reaching physiological effects than previously assumed.

An Examination of the Effects of Dorsal Fin-Mounted SPOT on Carcharodon carcharias, the Great White Shark

This opinion piece presents empirical evidence to examine possible negative consequences of the use of penetrative tagging as used on the great white shark (GWS). Tagging programs currently using this method attach SPOT (Spatial Positioning Only Tags) using corrodible bolts inserted through the dorsal fin while the shark is taken out of water. Such methods can cause harm to the tagged individual. Possible adverse effects include impacts on growth, tag biofouling, wounds, heightened stress, and hemorrhaging. This method may adversely impact dorsal fin structure and the shark’s hydrodynamics. As a result, data collected may not be reflective of natural behavior. Bolted SPOT are semi-permanently affixed to the shark but can have a battery life of approximately 3.5 years. Most of these tags (69%) ceased transmitting in less than 2 years. Alternative tagging technologies exist as more humane options.

Effects of handling and surgical implant on juvenile wild trout: Physiological and behavioral impact.

Fish involved in telemetry studies are usually handled, anaesthetized, and subjected to internal tag implantation, all of which have the potential to disrupt the fish's physiology, migratory patterns, food-seeking behavior, growth, and survival. As fish tagging is a widespread standard method in aquaculture, it is crucial to gain a better understanding of the short- and long-term effects on various aspects of fish welfare. The experimental outcomes of each phase of the surgical process and how it affects the fish's ability to recover during the post-tagging period, particularly in small salmonids, are poorly understood. Thus, it is essential to measure the extent of these adverse effects to precisely extrapolate differences in surgical intervention for tagged fish compared to their unmanipulated counterparts. In our study, we explored the post-tagging impacts on wild-captured brown trout (Salmo trutta) (1+) juveniles. We examined the time effect on fish recovery after each phase of the surgical procedure, which included anaesthetized fish, followed by surgery with/without internal tagging, within the first 2 and 25 h post-surgery. Common blood parameters (glucose levels and haematocrit) and endpoints related to swimming behavior, including fish movement, direction, and social distancing aspects, were studied. The findings of the study indicated no significant changes in glucose and haematocrit levels over the observational period. Fish subjected to anesthesia and tagging procedures exhibited a significant decrease in swimming activity, recovering to baseline levels 1 h after anesthesia and 24 h after surgical (tagging) procedures in recirculating aquaculture systems. Tagged fish showed less social proximity than non-tagged fish in the first 2 h post-surgery. A significant effect size was observed between nonsurgical (anesthesia) and surgical (non-tagged/tagged) groups, highlighting a more pronounced impact associated with surgical tag interventions. Our data indicate that the magnitude of behavioral response was significantly influenced by tag weight (~1.4% body/tag weight ratio in water) among manipulated fish groups. Consequently, our study reveals that wild-captured S. trutta juveniles, tagged with commercially available low-weight acoustic tags (V6, 69 kHz), experienced negative effects on swimming performance. In surgical studies, acknowledging potential influences is crucial for accurately inferring fish physiological and behavioral status. Emphasizing fish recovery potential in both short- and long-term periods is essential for quantifying tagging effects. Future research should prioritize exploring alternative tagging technologies and refining methodologies, with a particular focus on assessing telemetry's impact on socioeconomically relevant small salmonids.

Writing Effective Alt-text for Online Instructional Materials

Instructional materials are increasingly created and made available within an online space. Engaging materials utilize various types of images to break up walls of text and enhance learning. Describing these images with effective alternative text (alt-text) is subjective and contextually-based, requiring a treatment and knowledge beyond a simple checklist. This lesson is aimed to support educators in becoming more accessibility-literate, and to build in robust alt-text at the moment of creation. This lesson presents five different types of images. Each image type serves a distinct purpose within the context of online instructional materials. During the lesson, learners will work through each image type, with opportunities to practice examining HTML alternative tags and writing appropriate alt-text for each type of image.

Sublethal Effects of Acoustic Dummy Tag Implantation and External Tags in Silver Carp

AbstractTelemetry data collected via internally implanted transmitters are commonly used to monitor and manage invasive Silver Carp Hypophthalmichthys molitrix. However, the extent to which posttagging effects may influence fish behavior is generally overlooked in telemetry studies. Moreover, internal telemetry tags are often combined with an external tag that can also affect fish growth and survival. This study used two pond experiments to (1) evaluate the temporal effects of internal telemetry tags, external jaw tags, and the combination of the two tags on survival, relative weight, growth, and inflammation; and (2) compare the effects of two external tag types (jaw and loop tags) on Silver Carp. Response variables were evaluated at three time points: 1, 2, and 5 months posttagging. Growth and relative weight were lower in fish that received jaw tags than in the control group or in fish that received internal tags only; however, growth was different at 1 month posttagging but not at 2 months posttagging, indicating potential compensatory growth. Shedding rate of internal tags was 14%; after 5 months, inflammation was absent and tags were encapsulated in tissue. Survival was similar between loop‐ and jaw‐tagged fish (73%), although growth was lower in jaw‐tagged fish. Results indicate that survival effects and sublethal effects of telemetry tagging may be temporary, and researchers should examine detection data from within 1–2 months of tagging for possible postsurgery effects. Jaw tags reduced growth, and loop tags had reduced retention; therefore, further evaluation of alternative external tags is needed to optimize telemetry studies focused on Silver Carp. Additionally, researchers need to carefully evaluate which external tag to use to facilitate the returns of more expensive acoustic telemetry tags and whether external tags are necessary in a particular case.

Thermochemical and conformational studies of Ni(II) and Zn(II) ternary complexes of alternative metal binding peptides with nitrilotriacetic acid

Energy-resolved threshold collision-induced dissociation (TCID) of negatively-charged [amb5-H+M(II)+NTA-2H]− ternary complexes were studied for how they competitively dissociated into the products [amb5-3H+M(II)]− + NTA or [NTA-3H+M(II)]− + amb5, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contained one of the alternative metal binding-5 (amb5) heptapeptides with primary structures acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where the amino acids Aa1,2,6,7 included the potential metal binding sites of Asp, His, and/or Cys. The study included heptahistidine which represents the His affinity tag commonly used in immobilized metal affinity chromatography (IMAC) separation of recombinant proteins. Molecular modeling using PM6 located low energy, geometry-optimized conformers of the ternary complexes and their products which had collision cross sections that agreed with those measured by ion mobility. Using the molecular parameters, including vibrational and rotational frequencies, of the reactant and products from the molecular modeling, the energy-dependent intensities of the two product channels were modeled using a competitive TCID method to determine their threshold energies which equated to the two product channels 0 K dissociation enthalpies (ΔH0). Statistical mechanics thermal corrections provided the 298 K enthalpies (ΔH298) and Gibbs free energies (ΔG298) for the association reactions [amb5-3H+M(II)]− + NTA ↔ [amb5-H+M(II)+NTA-2H]− and [NTA-3H+M(II)]− + amb5 ↔ [amb5-H+M(II)+NTA-2H]−. These results were compared to determine which amb5 were effective as alternative tags for IMAC purification of recombinant proteins and is a routine that can be generally applied to compare the thermochemistry of metal ion ternary complexes.

Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Quantifying people's experience during flood events with implications for hazard risk communication.

Semantic drift is a well-known concept in distributional semantics, which is used to demonstrate gradual, long-term changes in meanings and sentiments of words and is largely detectable by studying the composition of large corpora. In our previous work, which used ontological relationships between words and phrases, we established that certain kinds of semantic micro-changes can be found in social media emerging around natural hazard events, such as floods. Our previous results confirmed that semantic drift in social media can be used to for early detection of floods and to increase the volume of ‘useful’ geo-referenced data for event monitoring. In this work we use deep learning in order to determine whether images associated with ‘semantically drifted’ social media tags reflect changes in crowd navigation strategies during floods. Our results show that alternative tags can be used to differentiate naïve and experienced crowds witnessing flooding of various degrees of severity.

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications

BackgroundInclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties.ResultsThree different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content.ConclusionsThis study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

Exploring Accessibility in DOAJ: A Case Study

This study explores the accessibility of HTML articles indexed in the Directory of Open Access Journals (DOAJ). The researchers tested a random sample of journals that possessed the DOAJ seal of approval, added content in 2019, were published in English and had 50 or more indexed articles. From this sample, HTML full-text articles were randomly selected for accessibility testing, using the W3C Easy Checks. 131 journals on 22 platforms were reviewed. All content reviewed provided unique web page titles. The majority of articles reviewed allowed for increases in text size and had meaningful heading structure. The application of alternative text tags for images and form labels was inconsistent, as was the ability to use keyboard controls for page navigation. Further, most content did not clearly indicate cursor location. The most egregious accessibility issues came from design choices, which generated a variety of contrast errors. More in-depth studies of DOAJ and other OA indexes and content providers are recommended, as is requiring adherence to WCAG 2.0 accessibility criteria as a condition of article submission.

Tailoring the properties of (catalytically)-active inclusion bodies

BackgroundImmobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically.ResultsWe here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein.ConclusionWe here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Bifunctional cleavable probes for in situ multiplexed glycan detection and imaging using mass spectrometry.

In situ analysis of glycans is of great significance since they mediate a range of biological activities. Aberrant changes of glycosylation are closely related to cancer onset and progression. In this work, bifunctional laser cleavable mass probes (LCMPs) were developed for in situ glycan detection from both cells and tissues using laser desorption ionization mass spectrometry (LDI-MS). Specific recognition of glycans was achieved by lectins, and inherent signal amplification was achieved by the conversion of the detection of glycans to that of mass tags which overcame the low ionization efficiency and complicated mass spectra of glycans. Multiplexed glycan profiling was easy to implement due to the simple and generic synthetic route to LCMPs and serial alternative mass tags, which offers high sensitivity, low interference and in situ detection of glycans. Moreover, as an excellent inherent matrix, LCMPs facilitated direct glycan detection from the cell surface and tissue imaging using LDI-MS. Intrinsic and fine glycan distribution in human cancer and paracancerous tissues was strictly demonstrated by MS imaging to explore the correlation between glycosylation and various cancers. This approach presented a versatile LDI-MS based platform for fast and in situ multiplexed glycan engineering, thus providing a new perspective in glycobiology and clinical diagnosis.

SYNTHESIS AND CHARACTERIZATION OF FATTY ACID METHYL ESTER MIXTURES DERIVED FROM ACORN KERNEL OIL

Abstract Biodiesel fuels are produced via transesterification of a triacylglycerol (TAG, e.g. vegetable oil, waste cooking oil, or animal fats) with a short-chain alkyl alcohol in the presence of a suitable catalyst. Alternative TAG sources, ones not derived from plants used as human food sources, have been of particular recent interest. In this work, the oil extracted from the endosperm of acorns, acorn kernel oil (AKO), was used as an alternative TAG source for the synthesis of biodiesel fuels. Acorns were collected from various species of oak trees (Quercus spp.) in the city of Nacogdoches, Texas. AKO was extracted from the acorn endosperm. The AKO was then subjected to acid-catalyzed and base-catalyzed transesterification with methanol and ethanol to produce acorn kernel oil methyl esters (AKOME) and acorn kernel oil ethyl esters (AKOEE) respectively. Concentrated H2SO4 was used as the acid catalyst and K2CO3 was used as the base catalyst. The effect of using a room temperature ionic liquid on percentage conversion for base-catalyzed transesterification was also investigated. Product mixtures were characterized using 1H-NMR spectroscopy. The NMR data were used to confirm the presence of transesterified products as well as to quantify the percentage conversion for the reaction. Percent conversion results ranged from 96 to 98% for AKOME products and 96 to 97% for AKOEE products.

Passive integrated transponder (PIT) tagging black tiger shrimp, Penaeus monodon: Applications for breeding programs

Passive integrated transponder (PIT) tagging provides a practical method of long term and high-throughput tagging and tracking of shrimp in a commercial context. Identification of individual broodstock can allow informed mating decisions necessary to identify individuals in a breeding program. The effects of PIT tags on mortality were assessed following tag insertion into one of three separate treatment locations in the abdominal muscle of juvenile Penaeus monodon, while animals in a control treatment were tagged with an alternative tag type currently used commercially: an external eye stalk tag. 336 animals were tagged in total and their survival tracked over 28days in three replicate indoor tanks consistent with a commercial maturation facility. Low mortality was observed across all treatments over 28days (<14% mortality in the control, and <8% in PIT tagged treatments), with no significant difference in mortality rates among the treatments and control treatment (χ2=5.26, P=0.15). Therefore, it can be recommended that PIT tagging at any of the locations described could be adopted and used in a commercial shrimp breeding scenario.

Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylationfrom various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, insome cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.

Feasibility of Using Passive Integrated Transponder Technology for Studying the Ecology of Juvenile Striped Mullet (Mugil cephalus) in Streams

Passive integrated transponder (PIT) tags are an important technology used to study ecology and behavior of animals including several stream fishes. In this study we investigated feasibility of using PIT technology to study ecology of juvenile Mugil cephalus (striped mullet) in Hawaiian streams. We quantified retention rates of PIT tags relative to an alternative tagging approach, visible implant elastomer (VIE) tags in the postorbital adipose tissue, for juvenile M. cephalus through a laboratory study. Next we conducted a field study of movement patterns of PIT-tagged juvenile mullet in a lowland Hawaiian stream (Waiāhole Stream) from early to mid-August 2014. For the laboratory portion of the study, 30 individuals (5–7 cm fork length) were randomly distributed among three treatments: PIT, VIE, and control (no tag) and held for 48 days. Tag retention was 100% for PIT-tagged and 0% for VIE-tagged fish. For the field study, 19 individuals (14.6–18.7 cm fork length) collected from Waiāhole Stream were implanted with PIT tags, and the movement of tagged individuals between the stream and its stream-mouth estuary/Kāne‘ohe Bay was inferred using a stationary PIT antenna array for 7 days. During that period, the antenna array registered 189 detections (97% during daylight hours, one missed detection) from five individuals, translating into 27 visits in total. Trip lengths to the estuary ranged from 45 min to 22 hr, with longer forays (≥10 hr) occurring primarily at night and shorter forays (<10 hr) occurring throughout daylight hours. Our results suggest that PIT tags are a feasible and effective marking technology for studying ecology of individual M. cephalus in streams, which paves the way for future research using this approach to study other aspects of their ecology.

Who needs particles? A challenge to the classification of particles as a part of speech in Russian

In 1985, Zwicky argued that ‘particle’ is a pretheoretical notion that should be eliminated from linguistic analysis. We propose a reclassification of Russian particles that implements Zwicky’s directive. Russian particles lack a coherent conceptual basis as a category and many are ambiguous with respect to part of speech. Our corpus analysis of Russian particles addresses theoretical questions about the cognitive status of parts of speech and practical concerns about how particles should be represented in computational models. We focus on nine high-frequency words commonly classed as particles: esce, tak, ved’, slovno, daže, že, li, da, net. We show that the current tagging of particles in the manually disambiguated Morphological Standard of the Russian National Corpus is not entirely consistent, and that this can create challenges for training a part-of-speech tagger. We offer an alternative tagging scheme that eliminates the category of ‘particle’ altogether. We show that our enriched scheme makes it possible for a part-of-speech tagger to achieve more useful results. Our analysis of particles provides a detailed account of various sub-uses that correspond to different parts of speech, their relationships, and relative distribution. In this sense, our study also contributes to the study of words that exhibit part-of-speech ambiguities.

Evolving effective behaviours to interact with tag-based populations

Tags and other characteristics, externally perceptible features that are consistent among groups of animals or humans, can be used by others to determine appropriate response strategies in societies. This usage of tags can be extended to artificial environments, where agents can significantly reduce cognitive effort spent on appropriate strategy choice and behaviour selection by reusing strategies for interacting with new partners based on their tags. Strategy selection mechanisms developed based on this idea have successfully evolved stable cooperation in games such as the Prisoner's Dilemma game but relies upon payoff sharing and matching methods that limit the applicability of the tag framework. Our goal is to develop a general classification and behaviour selection approach based on the tag framework. We propose and evaluate alternative tag matching and adaptation schemes for a new, incoming individual to select appropriate behaviour against any population member of an existing, stable society. Our proposed approach allows agents to evolve both the optimal tag for the environment as well as appropriate strategies for existing agent groups. We show that these mechanisms will allow for robust selection of optimal strategies by agents entering a stable society and analyse the various environments where this approach is effective.

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

BackgroundAlternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression.ResultsThe human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system.ConclusionsIn this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.