Why do users continue to contribute to darknet Child Sexual Abuse Material forums? Examining social exchange, social capital, and social learning explanations using digital forensic artifacts.

BACKGROUND: The darknet hosts an increasing number of hidden services dedicated to the distribution of child sexual abuse material (CSAM). Given that by contributing CSAM to the forum members subject themselves to criminal prosecution, questions regarding the motivation for members contributing to darknet CSAM forums arise. OBJECTIVE: Building on insights gained from research into clearnet communities, here we examine the extent to which social incentives generated by the online CSAM community may explain members' posting behavior on darknet CSAM forums. PARTICIPANTS AND SETTING: We analyze digital forensic artifacts on the online behavior of members of a darknet CSAM forum that was shut down by law enforcement agencies in July 2015. METHODS: We apply group-based trajectory modelling (GBTM), social network analysis, and mixed-effect survival models. RESULTS: Applying GBTM three posting trajectories can be distinguished. Social network analyses finds the reply network to be more centralized than predicted by chance. Mixed-effect survival models show positive associations between the length of members' first post and the time since members' first registration on the forum and subsequent posting. Contrarily, the number of replies received appears to mitigate subsequent posting. CONCLUSIONS: Findings show posting activity on the forum to be concentrated in a minority of forum members who show posting trajectories that are both frequent and persistent. Results further suggest persistence in posting is motivated by social identity and, to a lesser extent, differential association processes.

Differential expression of eight transcripts and their roles in the cuticle of the blue crab, Callinectes sapidus.

Eight cuticle protein transcripts from Callinectes sapidus were sequenced and their expression determined across the molt cycle in both calcifying and arthrodial cuticle hypodermis using quantitative PCR, Northern blots, and in situ hybridization. Four transcripts, designated CsAMP, are found only in non-calcifying arthrodial membrane hypodermis. They all code for a Rebers-Riddiford-1 motif, known to bind chitin. CsAMP9.3 is most likely an exocuticle constituent since it is expressed only during pre-molt. The other three arthrodial transcripts are present both before and after ecdysis. One of these, CsAMP16.3, codes for a RGD cell-attachment motif that could be involved in anchoring chitin-protein fibers to pore canals, cellular extensions of the hypodermis in the cuticle. The other four transcripts, designated CsCP, were found only in calcifying hypodermis. CsCP14.1 contains an RR-1 motif, which is more commonly found in non-calcifying cuticle proteins. CsCP6.1 is expressed post-molt and contains a partial RR motif, suggesting that it could bind to chitin in the endocuticle. The other two transcripts from calcifying hypodermis do not code for RR proteins, but both contain three copies of a different insect cuticle motif. One of these, CsCP19.0, is expressed only post-molt while the other, CsCP15.0, is present both before and after ecdysis.

Expressed sequence tags from normalized cDNA libraries prepared from gill and hypodermal tissues of the blue crab, Callinectes sapidus.

Expressed sequence tags (ESTs) were produced from two normalized cDNA libraries from the blue crab, Callinectes sapidus. The gill library represented pooled RNA from respiratory and transporting gills after acclimation to either high or low salinity. The hypodermis library was from arthrodial and dorsal tissue from both pre- and post-molt crabs. Random clones were single-pass sequenced from the 5'-ends, resulting in 11,761 high quality ESTs averaging 652 bases. All the ESTs were assembled using Paracel Transcript Assembler software, producing 2176 potential transcripts-883 contigs and 1293 singlets. Of these, 1235 (56.7%) were sequenced only from the gill library, while 578 (26.6%) were exclusively hypodermal. There were 363 contigs containing ESTs from both tissues (16.7% of the putative transcripts). All contigs and singlets were compared to the public protein database using BLASTx, and descriptions of the three most similar proteins for each were recorded. Additional annotations included an Interpro analysis of protein domains and a listing of Gene Ontology (GO) categories inferred from similar proteins in GO-annotated databases. All sequences are available on a web page (http://firedev.bear.uncw.edu:8080/shaferlab/). The annotations can be searched, and BLAST alignment of user-inputted sequences against the putative transcripts is possible. In addition, the ESTs have been submitted to GenBank.

Identifying exoskeleton proteins in the blue crab from an expressed sequence tag (EST) library.

A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including discovery of genes for cuticular (exoskeletal) proteins, some of which may regulate mineralization. One of the expression libraries sequenced was from the hypodermis (the epithelium depositing the cuticle). RNAs used for cDNA synthesis were pooled from arthrodial and mid-dorsal hypodermis at both pre-ecdysis and post-ecdysis. This ensured representation from both calcifying and non-calcifying regions and from layers of cuticle deposited both before and after ecdysis. The EST database was mined for cuticular protein sequences in three ways. First, we searched for sequences coding for known cuticle-specific motifs like the Rebers-Riddiford chitin-binding sequence and a motif known only from proteins extracted from mineralized exoskeletons of other decapods. Second, we checked the associated annotations in the EST project for similarity to known cuticular proteins, often from insects. Third, BLAST was used to search the EST data for significant homology to published cuticular protein sequences from other crustaceans. In all, the database contains at least 73 contigs or singlets representing transcripts of cuticular proteins. Forty-five of these distribute among ten clusters of very similar transcripts, possibly representing alternative splicing or recent gene duplications. The rest share less similarity. We have obtained complete sequences for 25 of the transcripts, have produced phylogenetics trees comparing them with similar proteins from insects and other crustaceans, and have determined expression patterns across the molt in calcifying versus non-calcifying cuticle. The combination of homology analysis and gene expression analysis allows us to infer putative functions in cuticle synthesis and calcification.

Four differentially expressed cDNAs in Callinectes sapidus containing the Rebers-Riddiford consensus sequence.

Decapod crustaceans such as Callinectes sapidus, the blue crab, provide unique opportunities to study proteins involved in biomineralization. Subsequent to each molt, the previously deposited soft cuticle is calcified while the postecdysial layers are simultaneously deposited and mineralized. Though the majority of the exoskeleton hardens, morphologically similar cuticle at the joints, called arthrodial membrane, remains flexible. It seems reasonable that hypodermal cells producing these cuticle types should be synthesizing proteins that regulate mineralization. Data presented here are consistent with this hypothesis, showing that transcripts coding for proteins containing the chitin-binding Rebers-Riddiford (RR) consensus sequence (Gx(8)Gx(7)YxAxExGYx(7)Px(2)P) are differentially expressed. Two RR-containing transcripts, CsAMP8.1 and CsAMP6.0, are found only in arthrodial membrane and are expressed uniformly both before and after ecdysis. They have high sequence homology with RR-containing proteins from uncalcified portions of the cuticle of Cancer pagurus, Penaeus japonicus, and Homarus americanus. The other two transcripts, CsCP8.5 and CsCP8.2, are expressed solely in premolt and in hypodermis depositing calcifying cuticle rather than arthrodial membrane. They have high sequence homology with calcification-associated peptides containing the RR sequence obtained from the calcified cuticle of Procambarus clarkii. This suggests possible involvement in the postmolt mineralization of the pre-ecdysial cuticle.

Expression of a serine proteinase homolog prophenoloxidase-activating factor from the blue crab, Callinectes sapidus.

Tanning, or sclerotization, of crustacean cuticle provides initial reinforcement by cross linking cuticular proteins attached to the cuticle chitin-fiber matrix. This process is catalyzed in part by phenoloxidase, which is under the control of a serine protease activation cascade. The cDNA of a prophenoloxidase-activating factor (PPAF) was cloned and sequenced from the hypodermal tissue of the blue crab, Callinectes sapidus. It codes for a serine proteinase homolog containing a single clip domain. If it is involved in sclerotization, its transcription might be expected to be molt-cycle related. Expression patterns were determined by quantitative PCR and Northern blotting in hypodermis underlying both arthrodial and dorsal (calcifying) cuticles. Transcript levels in pre-molt RNA from both hypodermis types were high, suggesting that the PPAF produced may be incorporated into the pre-ecdysial cuticle layers and then activated at ecdysis to regulate tanning. After a decrease at ecdysis, a second increase in PPAF mRNA occurred at three to four hours post-molt in arthrodial membrane hypodermis but not dorsal hypodermis. This suggests that cuticle deposited after ecdysis may tan in the non-calcifying regions but may not tan where calcification occurs. The PPAF gene is also transcribed at low levels in the hemocytes of intermolt crabs, but not in the hepatopancreas.

Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization.

A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.

Pharmacodynamic interaction between propofol and remifentanil regarding hypnosis, tolerance of laryngoscopy, bispectral index, and electroencephalographic approximate entropy.

BACKGROUND: The purpose of this investigation was to describe the pharmacodynamic interaction between propofol and remifentanil for probability of no response to shaking and shouting, probability of no response to laryngoscopy, Bispectral Index (BIS), and electroencephalographic approximate entropy (AE). METHODS: Twenty healthy volunteers received either propofol or remifentanil alone and then concurrently with a fixed concentration of remifentanil or propofol, respectively, via a target-controlled infusion. Responses to shaking and shouting and to laryngoscopy were assessed multiple times after allowing for plasma effect site equilibration. The raw electroencephalogram and BIS were recorded throughout the study, and AE was calculated off-line. Response surfaces were fit to the clinical response data using logistic regression or hierarchical response models. Response surfaces were also estimated for BIS and AE. Surfaces were visualized using three-dimensional rotations. Model parameters were estimated with NONMEM. RESULTS: Remifentanil alone had no appreciable effect on response to shaking and shouting or response to laryngoscopy. Propofol could ablate both responses. Modest remifentanil concentrations dramatically reduced the concentrations of propofol required to ablate both responses. The hierarchical response surface described the data better than empirical logistic regression. BIS and AE are more sensitive to propofol than to remifentanil. CONCLUSIONS: Remifentanil alone is ineffective at ablating response to stimuli but demonstrates potent synergy with propofol. BIS and AE values corresponding to 95% probability of ablating response are influenced by the combination of propofol and remifentanil to achieve this endpoint, with higher propofol concentrations producing lower values for BIS and AE.