The role of MPP1/p55 and its palmitoylation in resting state raft organization in HEL cells.

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Lach et al., J. Biol. Chem., 2012, 287, 18974-18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.

Genome-scale RNAi profiling of cell division in human tissue culture cells.

Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.

Predictors of suicidality among Polish university students during COVID-19 pandemic.

PURPOSE OF REVIEW: The main purpose of the study was to assess university students' mental health and identify factors associated with the risk of suicidal thoughts, plans, and attempts during coronavirus disease 2019 (COVID-19) pandemic and distance learning. RECENT FINDINGS: The study was conducted in spring 2021 and comprised 10 760 Polish students. The survey employed modified versions of the C-SSRS, CIDI, WHO-5, GAD-7, the PTSD checklist for DSM-5 and CAGE-AID Questionnaire and included questions about panic attacks, COVID-19-related information and sociodemographic characteristics. The correlates of suicidality were examined using a series of logistic regression analyses. Almost 40% students experienced any suicidal thoughts and/or behaviours in the previous month: passive ideations only (15.8%), active ideations only (7.1%), plans without attempts (15%), and attempts (1.4%). Following variables were related to the increased risk for suicide attempts: severe anxiety [odds ratio (OR) = 11.39; 95% confidence interval (CI): 1.44-90.26], panic attacks (OR = 3.21; 95% CI: 1.75-5.91), and COVID-19 hospitalisation (OR = 11.04; 95% CI: 1.17-104.59). Major depression was associated with passive and active ideations, suicide plans, but not with attempts (OR = 1.37; 95% CI: 0.45-4.13). SUMMARY: University students present a high level of adverse mental health and increased risk of STBs during COVID-19 pandemic. A suicide prevention program tailored to this population is needed during and after the pandemic.

Light microscopy applications in systems biology: opportunities and challenges.

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology.

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

Unique Device Identification-Based Linkage of Hierarchically Accessible Data Domains in Prospective Surgical Hospital Data Ecosystems: User-Centered Design Approach.

BACKGROUND: The electronic health record (EHR) targets systematized collection of patient-specific, electronically stored health data. The EHR is an evolving concept driven by ongoing developments and open or unclear legal issues concerning medical technologies, cross-domain data integration, and unclear access roles. Consequently, an interdisciplinary discourse based on representative pilot scenarios is required to connect previously unconnected domains. OBJECTIVE: We address cross-domain data integration including access control using the specific example of a unique device identification (UDI)-expanded hip implant. In fact, the integration of technical focus data into the hospital information system (HIS) is considered based on surgically relevant information. Moreover, the acquisition of social focus data based on mobile health (mHealth) is addressed, covering data integration and networking with therapeutic intervention and acute diagnostics data. METHODS: In addition to the additive manufacturing of a hip implant with the integration of a UDI, we built a database that combines database technology and a wrapper layer known from extract, transform, load systems and brings it into a SQL database, WEB application programming interface (API) layer (back end), interface layer (rest API), and front end. It also provides semantic integration through connection mechanisms between data elements. RESULTS: A hip implant is approached by design, production, and verification while linking operation-relevant specifics like implant-bone fit by merging patient-specific image material (computed tomography, magnetic resonance imaging, or a biomodel) and the digital implant twin for well-founded selection pairing. This decision-facilitating linkage, which improves surgical planning, relates to patient-specific postoperative influencing factors during the healing phase. A unique product identification approach is presented, allowing a postoperative read-out with state-of-the-art hospital technology while enabling future access scenarios for patient and implant data. The latter was considered from the manufacturing perspective using the process manufacturing chain for a (patient-specific) implant to identify quality-relevant data for later access. In addition, sensor concepts were identified to use to monitor the patient-implant interaction during the healing phase using wearables, for example. A data aggregation and integration concept for heterogeneous data sources from the considered focus domains is also presented. Finally, a hierarchical data access concept is shown, protecting sensitive patient data from misuse using existing scenarios. CONCLUSIONS: Personalized medicine requires cross-domain linkage of data, which, in turn, require an appropriate data infrastructure and adequate hierarchical data access solutions in a shared and federated data space. The hip implant is used as an example for the usefulness of cross-domain data linkage since it bundles social, medical, and technical aspects of the implantation. It is necessary to open existing databases using interfaces for secure integration of data from end devices and to assure availability through suitable access models while guaranteeing long-term, independent data persistence. A suitable strategy requires the combination of technical solutions from the areas of identity and trust, federated data storage, cryptographic procedures, and software engineering as well as organizational changes.

Z' factor including siRNA design quality parameter in RNAi screening experiments.

RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z' factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z' factor, introduced by Zhang et al., ( 1) is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describe a new extension of the Z' factor, which integrates bioinformatics RNAi non-target compounds for screening quality assessment. Currently presented Z' factor is based on positive and negative control, which may not be sufficient for RNAi experiments including oligonucleotides (oligo) with lack of knock-down. This paper proposes an algorithm which extends existing algorithm by using additional controls generetaed from on-target analysis.

A Holistic Approach for the Identification of Success Factors in Secondary Cleft Osteoplasty.

Cleft lip and palate belong to the most frequent craniofacial anomalies. Secondary osteoplasty is usually performed between 7 and 11 years with the closure of the osseus defect by autologous bone. Due to widespread occurrence of the defect in conjunction with its social significance due to possible esthetic impairments, the outcome of treatment is of substantial interest. The success of the treatment is determined by the precise rebuilding of the dental arch using autologous bone from the iliac crest. A detailed analysis of retrospective data disclosed a lack of essential and structured information to identify success factors for fast regeneration and specify the treatment. Moreover, according to the current status, no comparable process monitoring is possible during osteoplasty due to the lack of sensory systems. Therefore, a holistic approach was developed to determine the parameters for a successful treatment via the incorporation of patient data, the treatment sequences and sensor data gained by an attachable sensor module into a developed Dental Tech Space (DTS). This approach enables heterogeneous data sets to be linked inside of DTS, archiving and analysis, and is also for future considerations of respective patient-specific treatment plans.

Enhanced CellClassifier: a multi-class classification tool for microscopy images.

BACKGROUND: Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories. RESULTS: We have developed a tool, Enhanced CellClassifier, which circumvents this obstacle. Enhanced CellClassifier starts from images analyzed by CellProfiler, and allows multi-class classification using a Support Vector Machine algorithm. Training of objects can be done by clicking directly "on the microscopy image" in several intuitive training modes. Many routine tasks like out-of focus exclusion and well summary are also supported. Classification results can be integrated with other object measurements including inter-object relationships. This makes a detailed interpretation of the image possible, allowing the differentiation of many complex phenotypes. For the generation of the output, image, well and plate data are dynamically extracted and summarized. The output can be generated as graphs, Excel-files, images with projections of the final analysis and exported as variables. CONCLUSION: Here we describe Enhanced CellClassifier which allows multiple class classification, elucidating complex phenotypes. Our tool is designed for the biologist who wants both, simple and flexible analysis of images without requiring programming skills. This should facilitate the implementation of automated high-content screening.

Kernelized Z' factor in multiparametric screening technology.

RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z' factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z' factor, introduced by Zhang et al. (1), is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describes a new extension of the Z' factor, which integrates multiple readouts for screening quality assessment. Currently presented multivariate Z' factor is based on linear projection, which may not be suitable for data with nonlinear structure. This paper proposes an algorithm which extends existing algorithm to deal with nonlinear data by using the kernel function. Using kernel methods for projections, multiple readouts are condensed to a single parameter, based on which the screening run quality is monitored.

The use of modern telemedicine technologies in an innovative optimal cardiac rehabilitation program for patients after myocardial revascularization: Concept and design of RESTORE, a randomized clinical trial.

Despite proven efficacy of cardiac rehabilitation (CR) in reducing the all-cause mortality in patients after myocardial revascularization, the penetration of CR, due to patient-related factors and referral rates remains limited. To improve the outcomes, home-based tele-rehabilitation (TR) has been proposed recently. In theory TR enhances the effects of standard CR procedures due to implementation of an intelligent monitoring system designed to ensure optimal training through on-demand transmission of vital signs, aimed at motivating the patients through daily schedule reminders, setting daily goals and creating a platform for mutual feedback. Several meta-analyses assessing various studies comparing these two methods (CR and TR) have proven that they are at least equally effective, with some of the research showing superiority of TR. Although there was a small sample size, lack of long-term follow-up, reporting effects of TR itself, no integration with tools designed for coaching, motivating and promoting a healthy lifestyle constitutes an important limitation. The latter carries a hopeful prognosis for improvement when utilizing a broad-spectrum approach, especially with use of dedicated technological solutions exploiting the fact of a large and yet rapidly increasing penetration of smartphones, mobile PCs and tablets in the population. The above-mentioned findings worked as the basis and rationale for commencing the RESTORE project aimed at developing and delivering state-of-the-art, comprehensive TR for patients after myocardial revascularization and evaluating its molecular aspect in view of how it influences the atherosclerosis progression attenuation. This paper presents the current state and rationale behind the project based on up-to-date TR efficacy data.

In vivo analysis of covering materials composed of biodegradable polymers enriched with flax fibers.

BACKGROUND: The objective of this study was to investigate the in vivo effect of bioactive composites with poly(lactic acid) (PLA) or polycaprolactone (PCL) as the matrix, reinforced with bioplastic flax fibers, on the surrounding muscle tissue. METHODS: Materials of pure PLA and PCL and their composites with flax fibers from genetically modified plants producing poly-3-hydroxybutyrate (PLA-transgen, PCL-transgen) and unmodified plants (PLA-wt, PCL-wt) were placed subcutaneous on the M. latissimus dorsi for four weeks. RESULTS: The analysis of histological samples revealed that every tested material was differently encapsulated and the capsule thickness is much more pronounced when using the PCL composites in comparison with the PLA composites. The encapsulation by connective tissue was significantly reduced around PCL-transgen and significantly increased in the cases of PLA-transgen and PLA-wt. In the collected muscle samples, the measured protein expression of CD45, lymphocyte common antigen, was significantly increased after the use of all tested materials, with the exception of pure PCL. In contrast, the protein expression of caveolin-1 remained unchanged after treatment with the most examined materials. Only after insertion of PLA-wt, a significant increase of caveolin-1 protein expression was detected, due to the improved neovascularization. CONCLUSION: These data support the presumption that the new bioactive composites are biocompatible and they could be applicable in the medical field to support the regenerative processes.

New nano-hydroxyapatite in bone defect regeneration: A histological study in rats.

Many types of bone substitute materials are available on the market. Researchers are refining new bone substitutes to make them comparable to autologous grafting materials in treatment of bone defects. The purpose of the study was to evaluate the osseoconductive potential and bone defect regeneration in rat calvaria bone defects treated with new synthetic nano-hydroxyapatite. The study was performed on 30 rats divided into 5 equal groups. New preproduction of experimental nano-hydroxyapatite material by NanoSynHap (Poznan, Poland) was tested and compared with commercially available materials. Five mm critical size defects were created and filled with the following bone grafting materials: 1) Geistlich Bio-Oss®; 2) nano-hydroxyapatite+ß-TCP; 3) nano-hydroxyapatite; 4) nano-hydroxyapatite+collagen membrane. The last group served as controls without any augmentation. Bone samples from calvaria were harvested for histological and micro-ct evaluation after 8 weeks. New bone formation was observed in all groups. Histomorphometric analysis revealed an amount of regenerated bone between 34.2 and 44.4% in treated bone defects, whereas only 13.0% regenerated bone was found in controls. Interestingly, in group 3, no significant particles of the nano-HA material were found. In contrast, residual bone substitute material could be detected in all other test groups. Micro-CT study confirmed the results of the histological examinations. The new nano-hydroxyapatite provides comparable results to other grafts in the field of bone regeneration.

Enamel shear bond strength of different primers combined with an orthodontic adhesive paste.

AIM: The aim of this study was a comparison of shear bond strength (SBS) on tooth enamel of different primers combined with the adhesive paste Transbond XT. MATERIALS AND METHODS: Forty bovine teeth were used in order to create 40 test blocks. The blocks were divided into four groups of 10 blocks each: group A - sample primer (SP); group B - Opal Seal (OS); group C - Transbond Plus SEP (TSEP); group D - Transbond XT Primer (TXT). After surface preparation and application of the primer, respectively, two stainless steel brackets were fixed on each tooth by using Transbond XT. Accordingly, 80 brackets were debonded (n=20). Shear bond strength and adhesive remnant index (ARI) scores were evaluated. Statistical analyses were performed by using the Student's t-test and Mann-Whitney U test. RESULTS: All tested groups revealed high shear bond strength in a similar size range. There were no significant differences between the groups concerning shear bond strength. The ARI scores of group C showed significantly lower ARI scores (0 and 1) than that of group D. Apart from that there was no statistical difference. CONCLUSION: In combination with the adhesive paste Transbond XT, all tested primers were suitable for fixing orthodontic brackets. The primers could be changed according to the clinical situation.

Study protocol for a randomized controlled pilot-trial on the semiocclusive treatment of fingertip amputation injuries using a novel finger cap.

INTRODUCTION: Fingertip amputation injuries are common in all ages. Conservatively treated fingertips can regenerate skin and soft tissues to form a functionally and cosmetically excellent new fingertip. Little is known about this ability that, in humans, is confined to the fingertips. Even less is known about the role of the bacteria that regularly colonize these wounds without negative impact on regeneration and healing.As an alternative to surgery, self-adhesive film dressings are commonly used to establish a wet chamber around the injury. These dressings leak malodorous wound fluid eventually until the wound is dry. Having that into consideration, we have therefore developed a silicone finger cap that forms a mechanically protected, wet chamber around the injury for optimal regeneration conditions. It contains a puncturable reservoir for excess wound fluid, which can be thus routinely analyzed for diagnostic and research purposes.This study protocol explains the first randomized controlled trial (RCT) on the semiocclusive treatment of fingertip amputations in both children and adults comparing traditional film dressings with the novel silicone finger cap. Being the first RCT using 2 medical devices not yet certified for this indication, it will gather valuable information for the understanding of fingertip regeneration and the design of future definitive studies. METHODS AND ANALYSIS: By employing an innovative pseudo-cross-over-design with a dichotomous primary endpoint based on patients preference, this pilot study will gain statistically significant data with a very limited sample size. Our RCT will investigate acceptance, safety, effectiveness, and efficacy of this novel medical device while gathering information on the clinical course and outcome of conservatively treated fingertip injuries. A total of 22 patients older than 2 years will be randomly assigned to start the conservative treatment with either the traditional film-dressing or the novel finger cap. The treatment will be changed to the other alternative for another 2 weeks before the patient or the guardian is confronted with the decision of which method they would prefer for the rest of the treatment (if required). ETHICS AND DISSEMINATION: Ethical approval (EK 148042015) of the study protocol has been obtained from Institutional Review Board at the TU Dresden. The trial is registered at the European Database on Medical Devices (EUDAMED-No.: CIV-15-03-013246) and at ClinicalTrials.gov (NCT03089060).

SiRNA sequence model: redesign algorithm based on available genome-wide libraries.

The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

Annotation and specificity of existing genome-wide small interfering RNA libraries.

Large-scale RNA interference experiments, especially the ones based on short-interfering RNA (siRNA) technology have become increasingly popular over the past years. Obviously, the sequence (and structure) of the corresponding siRNA is a key factor in obtaining reliable results in these large-scale studies, and the companies use a variety of algorithms to design them. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Nevertheless, as the genome annotations are still continuously changing, siRNAs may become obsolete, so siRNA reagents should be periodically re-annotated according the latest version of sequence database. In this article, existing siRNA design algorithms, design parameters, and siRNAs are evaluated. A new approach for systematic analysis and re-annotation of siRNAs libraries produced in the last decade is introduced here.

1Click1View: interactive visualization methodology for RNAi cell-based microscopic screening.

Technological advancements are constantly increasing the size and complexity of data resulting from large-scale RNA interference screens. This fact has led biologists to ask complex questions, which the existing, fully automated analyses are often not adequate to answer. We present a concept of 1Click1View (1C1V) as a methodology for interactive analytic software tools. 1C1V can be applied for two-dimensional visualization of image-based screening data sets from High Content Screening (HCS). Through an easy-to-use interface, one-click, one-view concept, and workflow based architecture, visualization method facilitates the linking of image data with numeric data. Such method utilizes state-of-the-art interactive visualization tools optimized for fast visualization of large scale image data sets. We demonstrate our method on an HCS dataset consisting of multiple cell features from two screening assays.

An siRNA designing tool with a unique functional off-target filtering approach.

Investigations have revealed that silencing unwanted transcripts or off-targeting can induce false positive phenotype during RNA interference (RNAi)-based gene function study. But still the standard computational approaches towards small interfering RNA (siRNA) off-target minimization fall short in terms of addressing this false positive phenotype issue. Some of these off-targets may interfere with the biochemical pathway being investigated. It may also inadvertently target cell's metabolic pathways with unquantifiable consequences on the processes of user's interest. Here, we report the development of a siRNA selection tool that, for the first time, implements a functional off-target filtering that aims to minimize false positive phenotypes arising from inadvertent targets that are functionally similar or related to the direct target gene, along with a multi-parametric classifier (support vector machine) for optimized selection of potent siRNAs. The functional off-target filtering minimizes the number of off-target genes which are functionally related to the direct target gene, i.e. involved in a common biological process and may have similar phenotype. A text-mining algorithm is used to find related biological processes associated with the direct target and each off-target transcript by comparison of the biological processes associated with these genes. It also gives the user a choice to select one or more off-targets that may be potentially more harmful, from a predicted off-target gene list to be filtered out. Testing with huge set of biologically validated siRNAs from three different sources showed consistent good performance of our tool in terms of effective siRNA selection. It outperformed four potent siRNA selection algorithms of present day in terms of specificity in the selection of highly efficient siRNAs when compared on a common test set. A genome wide testing with potent siRNAs used in high-content screening confirmed validation of 2767 designed siRNAs in terms of phenotypic output. This tool presently supports siRNA designs for human genes and is freely available at http://gyanxet-beta.com .

SeedSeq: off-target transcriptome database.

Detection of potential cross-reaction between a short oligonucleotide sequence and a longer (unintended) sequence is crucial for many biological applications, such as high content screening (HCS), microarray nucleotide probes, or short interfering RNAs (siRNAs). However, owing to a tolerance for mismatches and gaps in base-pairing with target transcripts, siRNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs (off-targets) besides their intended target transcripts (on-targets). The reference sequence (RefSeq) collection aims to provide a comprehensive, integrated, nonredundant, well-annotated set of sequences, including mRNA transcripts. We performed a detailed off-target analysis of three most commonly used kinome siRNA libraries based on the latest RefSeq version. To simplify the access to off-target transcripts, we created a SeedSeq database, a new unique format to store off-target information.