Development of the means to efficiently and continuously renew missing and non-functional proteins in diseased cells remains a major goal in modern molecular medicine. While gene therapy has the potential to achieve this, substantial obstacles must be overcome before clinical application can be considered. A promising alternative approach is the direct delivery of non-permeant active biomolecules, such as oligonucleotides, peptides and proteins, to the affected cells with the purpose of ameliorating an advanced disease process. In addition to receptor-mediated endocytosis, cell-penetrating peptides are widely used as vectors for rapid translocation of conjugated molecules across cell membranes into intracellular compartments and the delivery of these therapeutic molecules is generally referred to as novel prospective protein therapy. As a broad coverage of the enormous amount of published data in this field is unrewarding, this review will provide a brief, focused overview of the technology and a summary of recent studies of the most commonly used protein transduction domains and their potential as therapeutic agents for the treatment of cellular damage and the prevention of regulated cell death. The hydrophobic nature of cellular membranes and the blood–brain barrier selectively restrict cells from taking up exogenous molecules larger than 500 Da. This is one reason why virus-mediated gene expression was considered for a long time as the most efficient and reliable approach for expressing bioactive proteins de novo, aiming to correct defects of proteins involved in a plethora of different genetic-metabolic disorders and diseases. Nevertheless, viral vectors are capable of integrating into the host chromatin and this may have serious consequences from long-term effects which limit the clinical and therapeutic applications of virus-associated material such as adenoviruses, herpes viruses or lentiviruses . An imprAlthough there seems to be little or no homology between the primary and secondary structures of the different PTDs, the efficacy of cellular transduction has been found to correlate strongly with the number of basic amino acids. Although there is no restriction on the size or type of the delivered cargo, the capacity for successful uptake of cargoes into cells increases when the cationic PTD is attached to lower molecular weight compounds , 14. CPPmodus operandi may be a novel chemotherapeutic approach that specifically targets the activity of CPP-conjugated anticancer drugs to tumor tissue.To elucidate the exact mechanisms of cellular entry, CPPs have been intensively studied for the last two decades. The most proposed feasibilities of cellular delivery of cargoes mediated by CPPs are illustrated in Fig. Beyond this, the nature of the link between the vector (CPP or PTD) and the cargo is also of importance. If these two elements are linked through a disulfide bridge, the cargo is rapidly released through the action of cytoplasmic glutathione. If the link is permanent, as in a fusion protein, then the final localization of the chimeric molecules will depend on the properties of both elements . NeverthAnother sophisticated approach used a transducible Tat-Cre recombinase reporter assay in viable cells where the functionality of the transduced cargo was indicated and confirmed through the genetic reconstitution of EGFP expression . Such prPharmacokinetic studies of the commonly used CPPs  physiological conditions in living mammals. Nevertheless, there are countless examples of successful applications of CPPs in treatment of human relevant injuries in the current literature, including decreased tumor growth and induction of cancer cell death (reviewed in ). Cargo s fusion protein significantly inhibits Fas-induced activation of procaspase-8 and downstream caspases, preventing cells from undergoing apoptosis. Systemic application of this protein prolongs survival and reduces multiorgan failure due to otherwise lethal Fas-receptor-mediated apoptosis in vivo [For a long time it was assumed that apoptosis was the only regulated form of cell death. Therefore, most successful applications of CPP-assisted delivery of proteins or peptides dealing with RCD have been reported in the context of apoptotic signaling pathways. Our own group discovered that incubation of lymphocytic Jurkat or BJAB cells with a recombinant Tat-FLIP in vivo . Given t in vivo . Protect in vivo . Both Ta in vivo  and our A related approach showed that the conserved N-terminal homology domain (BH4) of Bcl-xL alone, after fusion to the protein transduction domain of HIV Tat, closes voltage-dependent anion channels (VDAC) and thus efficiently inhibits mitochondrial cell death . The samEscherichia coli-induced sepsis improves survival. In such a study, Hotchkiss et al. proved an apoptotic signaling in this approach by application of a Tat-conjugated Bcl-xL fusion protein or Tat-BH4 peptide, respectively [Bcl-xL and the BH4 domain of Bcl-xL, respectively, are worthy of discussion in a further context of therapeutic CPP application. In a previous work, we were able to show that RIPK3-deficient mice, in contrast to C57BL/6 wild-type mice, were significantly protected from TNFα-induced and TNFα/zVAD-mediated hyperacute shock . Engagemectively . Thereinectively . Doubtleectively , but Cauectively . The disectively .Beside the transforming growth factor-β activated kinase-1 (TAK1 kinase), the adaptor MyD88 (myeloid differentiation primary response gene 88) is a suitable candidate which is able to mediate the decision to die by apoptosis or necroptosis , 76. MyDOxidative stress, or rather the formation of reactive oxygen species (ROS), plays a significant role in effecting cellular pathogenesis which contributes to regulated cell death in many neurological diseases such as stroke, brain trauma, Parkinson’s diseases and Alzheimer’s diseases . Like inIn an approach to ameliorate neurodegenerative diseases in patients, the group of Yadong Huang described in consecutive studies proteins consisting of human acidic fibroblast growth factor (haFGF) coupled to the Tat-domain of HIV –83. DeliAntigen-induced blockade of airway inflammation and hyperresponsiveness in mice were evaluated in an elegant study using a dominant negative Ras that was fused to the Tat protein transduction domain. At the cellular level, the CPP-carrying mutated Ras in this model inhibits the migration and infiltration of inflammatory cells, largely eosinophils and mononuclear cells as well as IL-4 and IL-5 production in the lung .per se cell-permeable and therefore need no further vehicles for transduction into target cells. That is why the immunosuppressive cyclosporin A would be an ideal component for treatment of inflammatory skin disorders mediated by dermal T lymphocytes. Unfortunately, this drug is only locally active and ineffective topically because of poor penetration into the skin. To overcome this hurdle, the aforementioned group of Rothbard conjugated a heptamer of arginine to cyclosporine A and demonstrated in a compelling fashion that, in contrast to the pure cyclosporin A, this modified drug was efficiently transported into the epidermis and dermis of mouse and human skin and as a result inhibited cutaneous inflammation [Additionally worth mentioning is an interesting study by Rothbard and colleagues which includes a category of molecules that normally do not require assistance for their uptake into cells. In general, small molecules which are used successfully for inhibition of different forms of RCD (e.g. necrostatin-1 or ferrostatin-1), or that operate as chemotherapeutic drugs (e.g. doxorubicin or cyclosporin A) are ammation . This apammation .Former studies performed in mice support that Tat-conjugated proteins can be efficiently transduced into neurons and protect the brain from mild or moderate ischemic injury . MammaliUnfortunately, stroke triggers a complex series of biochemical mechanisms and our knowledge of the molecular mechanisms of stroke pathophysiology is constantly advancing. In this context, some fundamental proteins like MAPKs  are involved in oxidative stress pathways and inflammation, while others like caspases, Bcl-2 family proteins, PARP-1, apoptosis-inducing factor (AIF), inhibitors of apoptosis proteins (IAPs), receptor interacting protein kinases (RIPKs) and heat-shock protein 70 (Hsp70) are involved in RCD pathways . The damA further clue to the applicability of CPPs in the course of RCD processes resulted from a study dealing with the transcription factor NF-κB, a central regulator of the immune response. It has been known for a long time that compounds which directly inhibit the binding of NF-κB to DNA may block inflammation and the associated tissue damage. Briefly, NF-κB essential modulator (NEMO) is a component of the IKK complex which participates in the activation of the NF-κB pathway. It was shown that the colon-targeted cell-permeable NF-κB inhibitory peptide TALDWSWLQTE is active against experimental colitis in vivo and indicates the therapeutic targeting of NF-κB for treatment of inflammatory bowel disease (IBD). Tissue permeability and colon availability of this NEMO binding domain peptide was examined spectro-photometrically using FITC-labeled constructs .d-isomer of p53 resulted in significant increases in lifespan and the generation of disease-free animals. This approach appears valuable because it was shown previously that p53 inhibits cystine uptake and thereby sensitizes cells to ferroptosis, an oxidative, iron-dependent, non-apoptotic form of RCD [Traditionally, therapeutics that restore genes inactivated during oncogenesis have been of superordinate interest in basic and translational science. It has long been speculated that direct reactivation of the endogenous tumor suppressor p53 in cancer cells will be therapeutically beneficial. So far, many different p53-derived peptides conjugated to various CPPs like penetratin or Tat have been demonstrated to restore the tumor suppressor function of p53 in cancer cells (reviewed in ). The grm of RCD . Ferroptm of RCD , 101. ItHowever, unequivocal controls are most important for applying the technology of CPPs. Correspondingly, the interpretation of necrotizing pancreatitis in regard to RCD is somewhat disputed. Some works describe that RIPK3 deficiency in mice partially protects against cerulein-induced pancreatitis (CIP), indicating the involvement of necroptotic signaling pathways in the course of this disease , 103. HoIn modern medicine, molecularly targeted imaging is gaining increasing significance. CPPs long ago became important tools for delivering and detection of fluorophores or luminescent nanocrystals such as quantum dots . FurtherSo far, a plethora of cargoes have been successfully delivered by CPPs or PTDs into a vast number of cell types and in over 25 clinical trials (Phase I and II) which have been performed predominantly using Tat-PTD, 8R and penetratin, evaluating this technique and its application as valuable and safe . The curN-methyl-d-aspartate receptor-postsynaptic density protein-95 interaction, a pro-death signaling pathway. This drug attenuates ischemic damage in the acute phase after stroke without affecting cerebral blood flow in the ischemic core or penumbra. A Phase II clinical trial proved the safety and efficacy of NA-1 in patients with iatrogenic stroke after endovascular aneurysm repair [At this time, the most promising candidate to become approved by the FDA may be NA-1 (Tat-NR2B9c), a peptide disrupting the m repair . Therefoper se are interesting for scientists but detrimental for a contemplated clinical evaluation. In such a manner, cationic CPPs, like the Tat-domain, sometimes evoke side-effects by remodeling the plasma membrane. Transduction of Tat-GFP fusion proteins causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine (PS) on the outer surface of the cell membrane. Remarkably, neither apoptosis nor necrosis is induced in these cells after exposure to Tat-GFP [Reports illustrating noticeable off-target effects of CPPs  Tat-GFP . This ob Tat-GFP . These f Tat-GFP .d-amino acids which are less susceptible to protease activity than the biologically used l-form could be considered an alternative for preparation of CPPs to increase serum stability. Such, it was revealed that d-isomeric Xentry was stable in serum for 4 h, much longer than the l-isomer which became inactive within 1 h [Despite extensive progress in recent years resulting in promising lines of preclinical evidence, more work is still needed and desirable for biomedical applications of therapeutically exploitable proteins. The low cell, tissue and organ selectivity of first generation CPPs in mammals in combination with the low protein transduction efficiency is a tremendous drawback of this technology and the therapeutic application of these carriers. In addition, the in vivo utility of CPPs may be compromised by the general capacity of polycationic peptides to activate mast cell secretion . Moreovethin 1 h .The challenge remains to find a compromise between avoiding the premature degradation of the cargo and obtaining a sufficiently effective drug release rate from the CPP-conjugate after internalization. Activatable cell-penetrating peptides (ACPPs) have an enhanced ability to penetrate their target cells within primary tumors or metastases compared to conventional CPPs due their cleavage by disease-associated matrix metalloproteinases and could eliminate background activity in healthy cells in such a manner . PotentiWithout any doubt, the current door-to-balloon-time appears to counteract the successful and effective prevention of RCD in myocardial infarction. In this regard, it is notable that organ transplantation is unavoidably associated with transient tissue hypoxia, which may cause ischemia–reperfusion injury (IRI), inflammation and rejection. Our own studies demonstrated that kidney ischemia–reperfusion is a form of acute kidney injury resulting in a cascade of cellular events prompting rapid cellular damage and suppression of kidney function. In this context, we have shown for the first time that necroptosis is a key element of IRI in the kidney and therefore will likely emerge as a promising target in solid organ transplantation . This is


Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated lymphoproliferative disorder. It most often occurs in patients with immunodeficiency and the clinical course ranges from indolent behavior to that of an aggressive malignancy. Pulmonary, central nervous system and dermatological manifestations are most common. To our knowledge this is the first reported case of LYG related to azathioprine therapy in Crohn disease.A twenty-six year old Caucasian woman with colonic Crohn disease on maintenance azathioprine therapy presented with right upper quadrant pain and fever. Diagnostic imaging revealed extensive liver, pulmonary and cerebral lesions. A diagnosis of LYG was made based on the pattern of organ involvement and the immunohistochemical features on liver and lung biopsy.Thiopurine therapy for inflammatory bowel disease is associated with an increased incidence of lymphoproliferative disorders. This report highlights the diagnostic challenges associated with LYG. As long-term thiopurine therapy remains central to the management of inflammatory bowel diseases it is essential that both patients and clinicians are aware of this potential adverse outcome. The infiltrate is angiocentric and typically invades the microvasculature. It is graded from I to III based on both the number of large B-cells and the proportion that stain positively for EBV-encoded small RNA (EBER) by in situ hybridization (ISH) [Here we present a case of LYG in a 26-year-old Caucasian female receiving azathioprine maintenance therapy for Crohn disease (CD) who presented with primary gastrointestinal complaints and fever. This report will highlight the diagnostic challenges associated with LYG and describe what we believe to be the first documented case of this rare disease in the setting of azathioprine treatment for CD.A 26-year-old Caucasian woman presented with three days of right upper quadrant pain, fever, and malaise. She had a six-year history of Crohn colitis initially treated with high dose mesalamine (4 g daily) then transitioned to azathioprine (2.5 mg/kg) after loss of response. She had been in clinical remission on azathioprine for the preceding 14 months. The patient had not been on any other immune modulating or biologic therapies.9/L, platelets = 80 x109/L), cholestatic liver enzyme elevation  and hepatic synthetic impairment . Azathioprine therapy was suspended. Investigations failed to identify a source of infection; however, magnetic resonance (MR) imaging revealed hepatomegaly with periportal, gastrohepatic, and retroperitoneal lymphadenopathy. A clinical diagnosis of azathioprine-associated pancytopenia with a concurrent non-A-E viral hepatitis was favored. She was administered G-CSF and transitioned to oral antibiotics. After clinical improvement, the patient was discharged from hospital with planned repeat MR imaging in one to two months.Six weeks later, she developed progressive abdominal pain, night sweats, and non-positional presyncopal symptoms. The patient did not report diarrhea or GI bleeding. Endoscopic evaluation of the upper GI tract and colon were normal with no evidence of inflammation or active Crohn disease. She had also developed her first outbreak of genital herpes  for which she was started on antiviral therapy. Repeat MR imaging now demonstrated extensive T2 hyperintense lesions distributed throughout her liver and both kidneys  during which a wedge biopsy of her lung was obtained. In the days prior to surgery she had developed right pleural effusion and lower lobe pneumonia. Cultures from her pleural fluid grew Streptococcus milleri and Prevotella species, therefore she received a six-week course of parenteral antibiotics.Now four months after her initial presentation, the VATS-acquired lung wedge biopsy demonstrated EBER positivity in an atypical CD20+ lymphocyte infiltrate with vascular invasion sufficient to confirm a diagnosis of grade II LYG , choledocholithiasis requiring cholecystectomy and admission to hospital with recurrent pneumonia. With therapy, all of her systemic lesions have steadily improved. This patient is alive and tolerating ongoing therapy at 12 months follow-up.Treatment was initiated with subcutaneous recombinant interferon-alpha three times weekly titrated up to 10 million units per mOur patient is the first reported case we are aware of with LYG in the context of CD and azathioprine use. Her presentation is atypical for LYG due to her young age, female gender, and the predominance of hepatic involvement preceding other pulmonary and CNS manifestations. In the larger context of LYG, only three percent of cases present with gastrointestinal symptoms; the majority primarily manifest pulmonary symptoms -4.In our case, the diagnosis of LYG was suspected based on pattern recognition on lung imaging and suggestive liver pathology. The negative EBV staining and lack of necrosis on the liver biopsy sample pointed away from LYG as the diagnosis. After open lung biopsy, however, the classic features of LYG were revealed, namely atypical large B-cells that were EBER-positive, vascular invasion and necrosis. These pathological findings along with the patient’s clinical presentation and pattern of organ involvement clinched the diagnosis of LYG. This case highlights the difficulty of establishing the diagnosis of LYG in many cases due to sampling error and histological heterogeneity in a given patient depending on the site biopsied .The current literature on LYG in association with CD or azathioprine is limited to two case reports. The first was a 17-year-old female patient with CD who developed LYG (stage III) with nasopharyngeal and pulmonary predominance following six years of 6-mercaptopurine therapy. She received systemic immunochemotherapy and was in clinical remission at four years follow-up [et al[Multiple population-based studies of inflammatory bowel diseases (IBD) have not shown an excess risk of lymphoproliferative disorders (LD) over baseline in part because it is difficult to rule out an effect from the disease itself in the population of patients who require more intensive therapy ,9. Howevet al-12. It set al.[Consistent with the findings in our patient, a comprehensive review of all LYG case series by Katzenstein et al. illustraet al.. Iatrogeet al..Here we present the first case in the literature of lymphomatoid granulomatosis in the setting of azathioprine therapy for Crohn disease. LYG is one of several EBV-driven conditions that should be considered when a lymphoproliferative disorder develops in an immunocompromised patient.The atypical features in our case underscore the importance of maintaining a high degree of clinical suspicion and diagnostic vigilance when evaluating non-specific abdominal symptoms frequently encountered in CD patients. This case highlights that even limited exposure to such therapy may induce LYG. Future research is needed into the pathogenesis and early detection of this disease to allow both clinicians and patients to make informed decisions and to raise awareness of this uncommon disorder.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.ALP: Alkaline phosphatase; CD: Crohn disease; CNS: Central nervous system; CT: Computed tomography; EBV: Epstein-Barr virus; EBER: Epstein-Barr virus encoded small RNA; GGT: Gamma glutamyl transferase; IBD: Inflammatory bowel disease; ISH: In situ hybridization; LD: Lymphoproliferative disorder; LYG: Lymphomatoid granulomatosis; MR: Magnetic resonance.The authors declare that they have no competing interests.WC and CG wrote the manuscript and performed the literature review. JP and PJB conceived of the report, participated in expert review and editing of the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/14/127/prepub


The present research used resting-state functional magnetic resonance imaging (fMRI) to examine whether the ability to generate creative ideas corresponds to differences in the intrinsic organization of functional networks in the brain. We examined the functional connectivity between regions commonly implicated in neuroimaging studies of divergent thinking, including the inferior prefrontal cortex and the core hubs of the default network. Participants were prescreened on a battery of divergent thinking tests and assigned to high- and low-creative groups based on task performance. Seed-based functional connectivity analysis revealed greater connectivity between the left inferior frontal gyrus (IFG) and the entire default mode network in the high-creative group. The right IFG also showed greater functional connectivity with bilateral inferior parietal cortex and the left dorsolateral prefrontal cortex in the high-creative group. The results suggest that the ability to generate creative ideas is characterized by increased functional connectivity between the inferior prefrontal cortex and the default network, pointing to a greater cooperation between brain regions associated with cognitive control and low-level imaginative processes. •Participants completed divergent thinking tasks and resting-state fMRI•Higher creative ability was related to greater rs-fc in the IFG and default network•Seed-to-voxel analysis further revealed greater rs-fc in the IFG and DLPFC•Controlled and spontaneous processes may cooperate more in the creative brain Recent literature reviews have been largely inconclusive on the brain basis of creative thought , raisingBut because these regions also correspond to large-scale networks with seemingly opposing functional roles , the ext1.1Divergent thinking has a long tradition in the creativity literature. The predictive power of divergent thinking tasks has fueled empirical interest in the cognitive basis of creative thought. An increasing body of research suggests that divergent thinking involves the top-down control of attention and cognition. Much of this evidence comes from latent variable studies showing effects of higher-order cognitive abilities, such as fluid intelligence , workingBehavioral evidence for the role of executive processes in divergent thinking has received support from electroencephalogram (EEG) and functional magnetic resonance imaging (fMRI) research. Several studies report task-related activation in brain regions associated with cognitive control, such as the inferior frontal gyrus (IFG) and inferior parietal cortex . Fink etThe inferior prefrontal cortex is commonly implicated in neuroimaging studies of divergent thinking . A recenBut the notion that divergent thinking is solely a controlled cognitive process is not fully consistent with the literature, as many of the same studies that report activation in brain regions associated with controlled cognitive processes also report activation in regions associated with spontaneous processes. Specifically, regions within the default mode network (DMN) have been reported in several recent studies of divergent thinking . The DMNFurther evidence for the role of the DMN in divergent thinking comes from two recent studies reporting associations between divergent thinking and functional connectivity in default mode regions . Both st1.2Although recent research supports the notion that divergent thinking recruits controlled cognitive processes in the inferior prefrontal cortex, the literature also implicates regions of the DMN, indicating that creative thought may also involve uncontrolled processes. The extent to which these seemingly disparate brain regions cooperate to support creative thought thus remains unclear. Because the brain is a highly complex system composed of functionally interconnected neural networks , the intThe present research sought to address the question of whether discrete brain regions linked with divergent thinking show increased functional connectivity in people of higher creative ability. To this end, we analyzed resting-state functional connectivity in participants of both high- and low-divergent thinking ability. Participants completed a battery of divergent thinking tasks in the lab and subsequently underwent resting-state functional imaging. In light of past research, we were particularly interested in exploring functional connectivity between the inferior prefrontal cortex and the core hubs of the DMN. This approach allowed us to determine whether regions within default mode and cognitive control networks exhibit stronger functional connectivity in highly creative individuals.22.1All participants were right-handed, native-German speakers with corrected-to-normal vision, and no history of CNS-affecting drugs or neurological disease. Participants provided written consent and were paid for their time. The study was approved by the local ethics committee at the University of Graz.2.2n=91). Participants were prescreened for this study based on their performance on a battery of six computerized divergent thinking tasks. We used a between-groups approach to characterize the intrinsic functional architecture of individuals of high-creative ability. Participants at the top 33% of the sample were assigned to the high-creative group, and participants at the bottom 33% of the sample were assigned to the low-creative group; the middle 33% was not included in the analysis. As described below, the two groups were carefully selected and matched to control for several variables associated with creative ability.The sample described here was taken from a larger pool of subjects that participated in previous MRI research at the University of Graz  to 4 (very creative) scale. We applied the Top 2 scoring procedure  and low-creative . A series of between-groups t-tests revealed that these groups did not differ significantly in terms of intelligence (p=.203), openness to experience (p=.101), age (p=.287), or gender (p=1.00). The high- and low-creative groups were, however, substantially different in terms of divergent thinking ability (p<.001). The two groups were thus equated on several variables associated with creativity, permitting an analysis of group differences in functional connectivity related to divergent thinking ability.We carefully matched the groups by iteratively removing participants until they were matched on intelligence, personality, age, and gender see . This pr2.3⁎-weighted functional images were acquired using a single shot gradient-echo EPI pulse sequence  and corrected online for head motion. The first two volumes were discarded to allow for T1 equilibration effects. Head motion was restricted using firm padding that surrounded the head. Data were acquired for five minutes while participants rested with their eyes closed. Following functional imaging, a high resolution T1 scan was acquired for anatomic normalization. Imaging data were then slice-time corrected and realigned using the Statistical Parametric Mapping (SPM) 8 package . Functional volumes were coregistered and resliced to a voxel size of 2 mm³, normalized to the MNI template brain , and smoothed with an 8 mm3 isotropic Gaussian kernel.Participants were scanned using a 3T Siemens Skyra system  with a 32-channel head coil. BOLD-sensitive T22.4http://www.nitrc.org/projects/conn; Functional connectivity analysis was implemented in MATLAB using the CONN toolbox  corrected (We then conducted a region of interest (ROI) analysis and a seed-to-voxel analysis. The ROI-to-ROI analysis allowed us to test hypotheses regarding the functional connectivity between the inferior prefrontal cortex and the DMN. We specified six 10 mm spherical clusters with peak- coordinates based on a reliability analysis of resting-state data cf. . Two ROIorrected .t-tests and Fisher׳s Z-transformed correlations were used to compute differences in functional connectivity between the high- and low-creative groups. Seed-to-voxel results are reported when significant at a voxelwise threshold of level of p<.001 uncorrected and a cluster-level threshold of p<.05 FDR corrected. All coordinates reported below refer to peak activations in anatomical MNI space.Next, we conducted a seed-to-voxel analysis. This allowed us to explore whether bilateral IFG was differentially connected to other brain regions outside of the DMN in the highly-creative group. Using the same seed ROIs in bilateral IFG defined above, temporal correlations were computed between these seeds and all other voxels in the brain. 33.1t=3.00, p=.008), left IFG-PCC , left IFG-right IPL , and left IFG-mPFC . This suggests that the left IFG is more strongly connected to the DMN in participants with high divergent thinking ability.We began with the ROI-to-ROI analysis to examine the functional connectivity between bilateral IFG and default mode regions. This analysis revealed significantly stronger connectivity in the high-creative group in the left IFG and the entire default mode network: left IFG-left IPL  and right IFG-right IPL . The highly-creative group did not, however, show greater connectivity between the rightThe high-creative group also showed significantly stronger connectivity between right IFG and bilateral IPL: right IFG-left IPL (p<.05 FDR corrected), nor did this pattern emerge at a less conservative significance level (p<.05 uncorrected). Thus, compared to the low-creative group, the high-creative group showed different patterns of connectivity between the right IFG and regions of the DMN.IFG and the mPFC or the PCC at a conservative level of significance . The high-creative group also showed stronger connectivity between the left IFG and a cluster of voxels in right inferior parietal cortex . The highly-creative group also showed stronger connectivity between the left IFG and a cluster of voxels in left inferior parietal cortex . Taken together, the results of the seed-to-voxel largely confirm the results of the ROI-to-ROI results reported above.We thus performed a whole-brain, between-group seed-to-voxel analysis to examine potential group differences in functional connectivity between the left IFG and all other voxels in the brain see . In linek=154; 46, −68, 50). Novel to the current analysis, the high-creative group showed stronger connectivity between the right IFG and a cluster of voxels in left dorsolateral prefrontal cortex .Our final analysis contrasted the functional connectivity between the high- and low-creative groups in the right IFG and the rest of the brain see . In line4The present study explored divergent thinking-related functional connectivity between inferior prefrontal cortex and the DMN. We used resting-state functional imaging to analyze intrinsic connectivity differences in groups of high- and low-divergent thinking ability. Resultsrevealed increased functional connectivity between seed regions in inferior prefrontal cortex  and the DMN  associated with greater divergentthinking ability. This research extends past work by demonstrating that discrete brain regions commonly linked with divergent thinking in the creativity literature are actually more strongly functionally connected in highly creative individuals. The results further suggest that divergent thinking ability involves a greater cooperation between brain regions associated with controlled and spontaneous cognitive processes.A ROI-to-ROI analysis, contrasting groups of low- and high-divergent thinking ability, found greater functional connectivity between bilateral IFG and the DMN. This analysis revealed greater functional connectivity between the left IFG and all four default mode regions . Furthermore, the right IFG showed greater connectivity to bilateral IPL in the highly creative group, but not the mPFC and PCC. Taken together, these findings suggest that the ability to come up with creative ideas is associated with functional coupling between bilateral IFG and regions of the DMN.A seed-to-voxel analysis extended the ROI-to-ROI analysis by exploring the extent to which bilateral IFG showed greater functional connectivity with other brain regions in the highly creative group. These results were largely in line with the prior analysis: the left and right IFG showed greater functional connectivity with clusters of voxels in several regions of the DMN. The left IFG seed was significantly more connected to a large cluster of voxels in cingulate cortex; a second cluster was found in the right inferior parietal cortex, which overlapped with the target ROI in inferior parietal cortex. This analysis further revealed greater connectivity between the left IFG seed and a cluster of voxels in left inferior parietal cortex, overlapping with the left angular gyrus.The seed-to-voxel analysis also found that the right IFG seed was more strongly connected to right inferior parietal cortex. The right IFG seed was also more strongly connected to a cluster of voxels in left DLPFC—a region associated with controlled attention and working memory capacity —suggesti4.1The present study raises the intriguing possibility that creative thinking involves both controlled and spontaneous cognitive processes. But how might such seemingly opposing processes cooperate? Other theories have sought to explain the role of attention in creativity. One compelling theory suggests that a “failure to deactivate” regions of the default network during tasks requiring focused external attention may characterize high creative ability . TakeuchWe propose a similar, but more general account of the present findings based on a controlled attention view of creative thought . DefaultControlled attention appears to be particularly relevant to divergent thinking because salient, unoriginal ideas can impede the creative thought process. During an alternate uses task, for example, concepts that are strongly semantically associated with the prompt cue  are often the first responses produced  in the absence of an externally-presented task. If so, this may also explain the resting-state group differences found in the present study; that is, if highly creative people are more likely to engage in imaginative processes, they may also show stronger activity within regions of the default network at rest. One way to test this would be to administer experience-sampling probes in the scanner cf.  and ask


The use of patients’ own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs.MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists.We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs.We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner. In trofiling –5. DespiALK gene rearrangement significantly respond to the ALK TKI inhibitors [Today, the focus in personalized cancer medicine has moved towards the detection of novel predictive targets and the investigation of specific inhibitors of these targets , 6. Targhibitors –10. Howehibitors –13. The Whereas genomic profiles of carcinomas have mainly been acquired using solid tumor specimens, malignant pleural effusions (MPEs) have rarely been considered for these analyses. MPEs in the serosal cavities frequently occur in patients with metastatic lung, breast and ovarian cancer. The appearance of MPEs reflects the advanced or metastatic disease state beyond the organ of origin leading to significant therapeutic implications and worse prognosis . The MPEIn this study, we aimed at establishing a robust system for the efficient testing of targeted therapeutics in MPEs from patients with solid tumors. For this purpose we used the xCELLigence culture system, which enables impedance based live-cell monitoring of proliferation, cytostasis or cytotoxicity . Here, w7 cells and incubated for 30 minutes at 4°C. After washing, resuspended cells were passed through the MACS separator (Miltenyi Biotec). Release was performed by removing the column from the separator and flushing 5ml of buffer.All effusions processed in this study were acquired at the University Hospital Basel for routine diagnostics and were subjected to diagnostic analyses in the department of cytopathology of the Institute for Pathology. Effusions for culturing were processed as described . BrieflyResuspended effusion cells (from both Ficoll as well as MACS approach) were cultured in BD Primaria (BD Biosciences) flasks at 37°C and 5% CO2 with FBS (PAN-Biotech) or sterile filtered primary culture medium  and withThis study was approved by the local ethical committee in Basel, Ethikkommission Nordwest- und Zentralschweiz (EKNZ), with the approval number EKBB 284/11.Before culture and during each splitting, cytospins were performed on a Cytospin 4 Cytocentrifuge (Thermo Scientific Limited) at 800rpm for 2 minutes. Cytospins were fixed in Delaunay solution and standard immunocytochemistry procedures were applied using the automated BOND-MAX system . Antibodies used: TTF-1 , Ber-EP4  and Calretinin . All cytospins were evaluated by board certified pathologists .Cell growth experiments were performed by usage of the real-time cell analysis system xCELLigence (ACEA) as previously described . BrieflyALK gene, H522 cells are wild-type for ALK, ROS1 and CMET [ALK-rearranged cell line H3122 and the control cell line H522 with different concentrations of crizotinib. As expected, treatment with crizotinib led to a significant growth reduction in H3122 cells  and after each passage. We estimated tumor cell content by cytology and by immunocytochemistry (ICC) using markers previously confirmed to be positive when measured as part of routine clinical diagnostic procedure. In general, EpCAM    and Calr BerEP4)  were use BerEP4)  in effus BerEP4) . This in BerEP4) . We therSimilar to the cell line experiments described above , we treaEGFR gene  but no mutations in KRAS, BRAF and HER2, as well as no ALK gene rearrangement as determined by FISH. Based on these molecular characteristics, we assumed this lung cancer to be responsive to an EGFR-TKI. In addition, and as a control, we selected a pleural effusion from a 62 year old patient with metastatic TTF1 positive adenocarcinoma of the lung. Genomic analysis revealed neither an ALK gene rearrangement nor EGFR, BRAF or HER2 gene mutations, but an activating point mutation of the KRAS gene  and therefore this tumor could be considered a non-responder case to EGFR-TKI therapy. We thus processed both pleural effusions as described above and treated the cultured cells with the EGFR TKI erlotinib. The pleural effusion with the EGFR mutation showed a dose-dependent growth reduction upon treatment with erlotinib Click here for additional data file.S2 FigTreatment of a malignant mesothelioma with different concentrations of cisplatin (A), pemetrexed (B) and a combination thereof (C).(TIF)Click here for additional data file.S3 FigTreatment of normal mesothelial cells with pemetrexed (A) and cisplatin (B).(TIF)Click here for additional data file.


This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application.6 and 50x106 cells for intradermal injection into the recipient’s neck. Recipient serum was collected and tested for the presence of anti-donor antibodies prior to MSC injection and every 7 days after MSC injection for the duration of the 8-week study using the standard two-stage lymphocyte microcytotoxicity dye-exclusion test. In addition to anti-ELA-A2 antibodies, recipient serum was examined for the presence of cross-reactive antibodies including anti-ELA-A3 and anti-RBC antibodies.Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsatellite typing and used as allogeneic MHC-mismatched MSC recipients. MHC homozygote horses of known ELA-A2 haplotype were used as MSC and peripheral blood leukocyte (PBL) donors. One MHC homozygote horse of the ELA-A2 haplotype was the recipient of ELA-A2 donor MSCs as an MHC-matched control. Donor MSCs, which were previously isolated and immunophenotyped, were thawed and culture expanded to achieve between 30x10All MHC-mismatched recipient horses produced anti-ELA-A2 antibodies following injection of ELA-A2 MSCs and developed a wheal at the injection site that persisted for the duration of the experiment. Anti-ELA-A2 antibody responses were varied both in terms of strength and timing. Four recipient horses had high-titered anti-ELA-A2 antibody responses resulting in greater than 80% donor PBL death in the microcytotoxicity assays and one of these horses also developed antibodies that cross-reacted when tested on lymphocyte targets from a horse with an unrelated MHC type.Allogeneic MSCs are capable of eliciting antibody responses in vivo that can be strong and also cross-reactive with MHC types other than that of the donor. Such responses could limit the effectiveness of repeated allogeneic MSC use in a single horse, and could also result in untoward inflammatory responses in recipients.The online version of this article (doi:10.1186/s13287-015-0053-x) contains supplementary material, which is available to authorized users. The genetic background and age of the patient can also affect the quantity and quality of MSCs able to be cultured, making autologous MSC use impossible for some patients regardless of timing issues -4. Bankein vitro -13.in vitro using modified one-way mixed leukocyte reactions, it was found that MHC class II-positive MSCs caused significantly increased responder T-cell proliferation equivalent to that of the positive control of MHC mismatched peripheral blood leukocytes (PBLs) [in vitro mixed leukocyte reaction system is used to examine T-cell responses only, no conclusion could be made from this study as to the effect of MHC class I or class II status on the ability of MSCs to elicit a recipient immune response in vivo, particularly in terms of antibody production.The horse is a valuable species for assessing the safety and efficacy of MSC treatment. Autologous bone marrow-derived MSCs are used routinely in regenerative therapies for equine patients to treat musculoskeletal disorders including tendonitis, osteoarthritis, cartilage damage, and meniscal injuries -20. In as (PBLs) . As the in vitro, allogeneic MSCs do elicit cellular and humoral immune responses when implanted in immunocompetent or moderately immunosuppressed animals [in vivo using lymphocyte microcytotoxicity assays to measure recipient antibody responses. Our hypothesis was that MHC-mismatched MSCs would elicit recipient antibody responses and that these responses would be independent of MSC MHC class II expression.Previous studies in swine, rhesus macaques, and mice have shown that, in contrast to their behavior  animals -29. No sA schematic of the study design and methods is shown in Figure in vitro studies [Thoroughbred horses of known MHC haplotype belonging to the Equine Genetics Center at the Baker Institute for Animal Health of Cornell University  were used as MSC and leukocyte donors in these studies. All donor horses were MHC homozygotes of equine leukocyte antigen (ELA) haplotype ELA-A2 as determined previously by ELA serotyping, direct MHC gene sequencing, and microsatellite typing -33. Bone studies . Specifi studies . For con studies .Table 1Six healthy adult horses were used as allogeneic MHC-mismatched MSC recipients in these studies and identified by letters (Horses A to F) Table . These MAs a MHC-matched control, one Thoroughbred MHC homozygote horse of the ELA-A2 haplotype (Horse 2) belonging to the Equine Genetics Center at the Baker Institute for Animal Health of Cornell University was the recipient of MSCs from a different ELA-A2 donor. This horse was also screened and found to be negative for the presence of preexisting anti-ELA-A2 antibodies prior to use in this study.in vitro culture as described previously until 30 × 106 to 50 × 106 cells were attained . Serum was allowed to separate for 8 hours at 4°C and the tubes were then centrifuged at 800 × 6 cells/ml.Blood was collected via jugular venipuncture with extension sets  and 16 G needles into 250 ml evacuated containers  each containing 3,750 units of heparin . Plasma was allowed to separate for 20 minutes at room temperature and PBLs were then isolated from the plasma via carbonyl iron (Sigma-Aldrich) granulocyte depletion and Ficoll-Paque Plus  gradient centrifugation . This PBThe standard two-stage microcytotoxicity dye-exclusion test was used to detect recipient antibody responses as described previously for transplantation studies in the horse ,37. BrieTo determine the secondary response to MHC-mismatched allogeneic MSCs after possible immune priming, any MHC-mismatched recipient horse with a negative or weak primary antibody response was to receive a repeat, or second, injection of donor ELA-A2 MSCs on the right side of the neck at 5 weeks post initial injection.To test for antibody specificity against the ELA haplotype, the serially diluted antisera  were tested against fresh PBLs from a donor homozygote horse of the ELA-A3 haplotype (Horse 5 ) using tAll six MHC-mismatched recipient horses developed a wheal at the injection site that persisted for the duration of the experiment. For both MHC-mismatched recipient horses and the control MHC-matched recipient horse, vital parameters such as respiratory rate, heart rate, and body temperature remained within normal limits for the duration of the study.Lymphocyte viability after depletion of granulocytes via carbonyl iron and isolation using Ficoll-Paque Plus gradient centrifugation was >95%. Internal control sera known to be positive for antibodies against the donor PBLs resulted in 80 to 100% donor PBL death and known negative sera caused no detectable donor PBL death in all assays performed . All MHThe remaining two MHC-mismatched recipient horses (Horses A and D) had weak responses resulting in donor PBL death less than the predefined 80% cutoff used in the microcytotoxicity assay for reporting titers. Nevertheless, the antisera from both of these horses resulted in repeatable 10 to 35% donor PBL death at dilutions up to 1:32 on day 21 and up to 1:2,048 on day 28 post injection. The control MHC-matched recipient horse sera never showed any donor cell killing at any dilution at any time point (Table 6 cells injected into recipient Horse E) still produced a strong antibody response as compared with those produced by the two weak responders (Horses A and D), who received 46 × 106 and 50 × 106 cells, respectively.No correlation was found between MHC class II status of the donor MSCs and recipient antibody response in this study. Of the four recipient horses with strong antibody responses, two received MHC class II-negative MSCs and two received MHC class II-positive MSCs Table . SimilarHorses A and D with weak anti-ELA-A2 antibody responses following initial ELA-A2 MSC injection received a second injection of ELA-A2 MSCs at week 5 day 36) of the study .The results of this study demonstrate that allogeneic equine bone marrow-derived MSCs can elicit strong antibody responses after intradermal injection into MHC-mismatched recipients. Although the strength of the responses varied between recipients, all recipients developed antibodies against lymphocytes of the donor ELA-A2 haplotype. Four of the recipient horses generated strong responses in which antibodies were present by 7 to 14 days and persisted for the duration of the study. Overall, the responses seen in these horses were very similar to those reported previously in horses following ectopic trophoblast allotransplantation . Skin alin vivo [It is important to note that although FBS was used in the MSC culture media in this study, FBS cannot be the target of the anti-donor antibody responses observed. The microcytotoxicity assays used are extremely specific for antibodies in the recipient antisera that are targeting and killing donor PBLs. As the donor PBLs do not express bovine proteins and are never in contact with FBS during their isolation or culture within the assay, anti-FBS antibodies cannot be responsible for their death. While the use of FBS in MSC culture media up until the time of injection is certainly a potential concern for clinical application and should be avoided in a clinical setting or in future studies examining donor MSC viability in vivo , FBS wasThere are several conceivable reasons for the variation in the strength of the antibody responses observed in this study. The first is potential differences in recipient health and immune status. Although all recipient horses had normal physical examinations and were in fair to good body condition, it is interesting to note that the two weak responders (Horses A and D) were the only two recipient horses recently added to the research herd, while the four strong responders were existing members of the university teaching or research herd. It is possible that the immune systems of the two weak responders were not as strong as the other recipients due to factors such as improper nutrition prior to their arrival at the university and/or stress from having gone through the auction and associated transport as well as being placed with new herd members. Such factors could increase endogenous glucocorticoid levels and thereby cause immune suppression -43. As sThere is also the possibility that some of the strong responders may have had a previous primary response against the ELA-A2 haplotype. While this seems unlikely given that the majority of these horses had long-term known histories and health records with the university, it is possible that the recipient mare could have been pregnant in the past or that the geldings could have been exposed to ELA-A2 antigens through another mechanism. In particular, recipient Horse C had a response that is more typical of a secondary, or primed, immune response than of a primary immune response. This horse developed detectable antibodies by 7 days post injection and had very high titers that remained high for the duration of the study. While all recipient horses were screened for the presence of the circulating anti-ELA A2 antibodies prior to MSC injection, there was no practical way to screen them for past exposure prior to the MSC injections performed in this study.Although the authors are of the opinion that the recipients themselves are the likely cause of the observed variability in antibody responses, one must also consider the fact that different MSCs were used. While this was done intentionally to test for possible differences in response to MSC MHC class II expression, this difference could have affected the results in several ways because MSCs are not only heterogeneous in their basal MHC class II expression, but also dynamic in their MHC expression as has been demonstrated previously with interferon gamma stimulation . It is pLastly, the observed results for the two weak responders that received repeat MSC injections might be misleading due to the fact that both horses had circulating anti-ELA-A2 antibodies at least very close to the time of the repeat injection. It is possible that the donor MSCs injected in week 5 may have been targeted by circulating antibodies and destroyed before they could induce a secondary antibody response. This would be a potential explanation for why Horse D did not develop a further antibody response.The doses of MSCs used in this study were clinically relevant and proportionally similar on a body weight scale (approximately 0.1 million MSCs/kg body weight) to those originally used in allogeneic MSC studies in other species -27. ReceIn this study only bone marrow-derived MSCs were examined and they were delivered via intradermal injection. In addition, semi-allogeneic recipients were not included. The effect that more clinically relevant injection site locations may have on recipient antibody response remains to be determined, as does the effect of the MHC haplotypes of the clinical MSC donor and recipient. Clinical patients are not expected to be MHC homozygotes or completely matched in terms of their MHC haplotype. Given the frequency distribution of MHC haplotypes in breeds such as the Thoroughbred or Standardbred, it is possible that some recipients would be semi-allogeneic to randomly selected donor MSCs -33. Futuin vitro to an extent that the MSCs are no longer immunogenic in vivo.Finally, perhaps the most important finding of this study in terms of clinical application is the potential for the development of strong, long-lasting, and possibly cross-reactive antibody responses after MSC transplantation. Cross-reactivity with diverse MHC class I antigens happens over time as the immune response progresses and the recipient develops broader and stronger antibodies against other MHC epitopes that may be shared against other MHC haplotypes . While ain vivo, which can be strong and also cross-reactive with MHC haplotypes other than the donor type. These results highlight the potential risk of clinical allogeneic MSC application.This study is the first to investigate the antibody response to allogeneic MSC transplantation in the horse. Using known MHC-mismatched donors and recipients, we have shown that allogeneic MSCs are indeed capable of eliciting antibody responses


Hypsibius dujardini, and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade  Advances in high-throughput sequencing technologies are revolutionizing the field of genomics by allowing researchers to generate large amount of data in a short period of time . These tFor instance, difficulties associated with the cultivation of bacterial and archaeal organisms  have perOn the other hand, researchers who study eukaryotic genomes generally focus on the recovery of a single organism, which, in most cases, simplifies the identification of the target genome in assembly results. However, sequences of bacterial origin can contaminate eukaryotic genome assemblies due to their occurrence in samples , DNA extHypsibius dujardini by exploiting some of the best practices of high-throughput sequencing available today , constructed paired-end Illumina libraries according to the TruSeq RNA-seq protocol, and sequenced their cDNA libraries with a read length of 100 bp.We obtained the RNA-seq data using the NCBI accession id JNA272543. Brieflyhttp://github.com/meren/illumina-utils) for quality filtering of short Illumina reads using ‘iu-filter-quality-minoche’ script with default parameters, which implements the quality filtering described by We used illumina-utils  (availabOur workflow with anvi’o to identify and remove contamination from a given collection of scaffolds consists of four main steps. The first step is the processing of the FASTA file of scaffolds to create an anvi’o contigs database (CDB). The resulting database holds basic information about each scaffold in the assembly . The second step is the profiling of each BAM file with respect to the CDB we generated in the previous step. Each anvi’o profile describes essential statistics for each scaffold in a given BAM file, including their average coverage, and the portion of each scaffold covered by at least one read. The third step is the merging of all anvi’o profiles. The merging step combines all statistics from individual profiles, and uses them to compute hierarchical clusterings of scaffolds. The default organization of scaffolds is determined by the average coverage information from individual profiles, and the sequence composition information from the CDB. This organization makes it possible to identify scaffolds that distribute similarly across different library preparations. The final step is the visualization of the merged data on the anvi’o interactive interface. The anvi’o interactive interface provides a holistic perspective of the combined data, which allows the identification of draft genome bins, and removal of contaminants.http://github.com/meren/anvio) to process scaffolds and mapping results, visualize the distribution of scaffolds, and identify draft genomes following the workflow outlined in the previous section, and detailed in http://theseed.org/) and imported these results into the CDB using the program ‘anvi-populate-genes-table’ to store the information about the locations of open reading frames (ORFs) in scaffolds, and their taxonomical and functional inference. We profiled individual BAM files using the program ‘anvi-profile’ with a minimum contig length of 1 kbp, and the program ‘anvi-merge’ combined resulting profiles with default parameters. For the analysis of https://inkscape.org/).We used anvi’o v1.2.2  anvi’o files to regenerate PRJNA309530), and (3) the FASTA files for bacterial genomes we identified in the raw assemblies from Boothby et al. and Koutsovoulos et al.The URL The use of multiple library preparations and sequencing strategies is likely to result in more optimal assembly results . Hence, Through the anvi’o interactive interface we selected 14,961 scaffolds from the Boothby et al. assembly that recruited large number of short-reads in a consistent manner . This 18Our mapping results indicate the presence of non-target sequences in the assembly that recruit reads only from long-read libraries. One interpretation could be that most of the contamination in Boothby et al.’s assembly originated from Moleculo libraries, post DNA-extraction . HoweverChitinophaga and Thermosinus in Boothby et al.’s assembly , 24,841 scaffolds that were longer than 1 kbp from the raw assembly were clustered based on sequence composition and coverage values in data from the two Illumina sequencing libraries (the inner dendrogram). Scaffolds longer than 40 kbp were split into sections of 20 kbp for visualization purposes. The second layer shows the GC-content for each scaffold. The next two view layers represent the log-normalized mean coverage values for scaffolds in the two sequencing datasets. Finally, our scaffold selections  are displayed in the outer layer. In the right panel (curation step II), the 15,839 scaffolds from the tardigrade selection from step I were clustered based on sequence composition only for more precise curation. Additional scaffold selections  are displayed in the outer layer.Click here for additional data file.10.7717/peerj.1839/supp-3Table S1Click here for additional data file.10.7717/peerj.1839/supp-4Table S2Click here for additional data file.10.7717/peerj.1839/supp-5Table S3H. dujardini from As they are identified in the raw assembly by Click here for additional data file.


Web-based self-help interventions that aim to reduce problematic substance use are able to reach “hidden” consumer groups in the general population who often fear stigmatization and thus avoid institutional addiction treatment. In Western European countries, including Switzerland, cocaine is the most widely used psychoactive substance after alcohol, tobacco, and cannabis. Although approximately one in six users develop serious problems of dependency, only a minority seeks help from psychiatrists or in outpatient counseling centers or psychiatric hospitals. Offering web-based therapy treatment may potentially reach users who hesitate to approach institutional treatment services and help them reduce their cocaine use before they get into more serious trouble.The study will use a three-arm randomized controlled trial (RCT) design to test the efficacy of a web-based self-help intervention with or without guided chat counseling compared with that of a waiting list control condition in reducing or stopping cocaine use. The primary outcome measure will be the weekly quantity of cocaine used. Secondary outcome measures will include the number of cocaine use days in the past 30 days, the severity of cocaine dependence, the use of alcohol, tobacco, and/or other illicit drugs, changes in mental health symptoms, and treatment retention. The self-help intervention will consist of eight modules that are designed to reduce cocaine use and depression symptoms. These modules are based on the principles of Motivational Enhancement Therapy and Cognitive Behavioral Therapy, such as Behavioral Self-Management. The three individual chat therapy sessions will be based on the same therapy approaches and will be tailored to participants’ self-help data and aim to assist the reinstatement of social rewards and the improvement of social support and relationships.This study will be the first RCT to test the effectiveness of a web-based self-help intervention in combination with or without chat counseling in reducing cocaine use. The expected findings will contribute substantial knowledge that may help design effective guided and unguided web-based treatment for cocaine users. Moreover, the study will elucidate to what extent a therapeutic alliance with cocaine users can be established in a guided Internet-delivered setting. Additionally, the present study will investigate changes in social support with specific guided therapy interventions that aim to ameliorate social support and social perceptions and compare these changes with those in an unguided self-help interventionISRCTN12205466. Registered 24 February 2015.Current Controlled Trials  Although the prevalence rates of problematic cocaine use and cocaine dependence are unclear, the lifetime prevalence of cocaine use has almost doubled in the Swiss general population (aged 15–24 and 25–34 years) over the past ten years [It is assumed that quasi-controlled, occasional use of cocaine is much more prevalent than addicted use and that only a few users are currently in treatment . NeverthCognitive Behavioral Therapy (CBT) has been successful in treating cocaine dependence, while intense research on psychopharmacological approaches over the past three decades has not identified any compelling evidence of effective medicinal treatments [Contingency Management or the Community Reinforcement Approach, which reward cocaine abstinence with money or use positive reinforcement, have primarily been reported in the United States and seem hardly applicable to European countries due to health care policy or health insurance-related differences [Outpatient treatment that is based on eatments . Nevertheatments . Furtherferences , 10.Web-based interventions that aim to reduce problematic cocaine use might fill an important gap by providing support for problematic cocaine users or those with the initial dependence symptoms who have not responded well to the present institutional treatment offerings. The Internet is a useful tool to reach hidden populations, such as illicit drug users . FurtherBehavioral Self-Management (BSM), relapse prevention [Motivational Enhancement Therapy (MET) [However, web-based interventions have primarily focused on reducing alcohol and cannabis use –17. Web-evention , 22 and py (MET) . Web-baspy (MET) . Similarpy (MET) , 25. Thepy (MET) –28. The py (MET) , 30. It py (MET) .In a previous study, we demonstrated the feasibility and potential effectiveness of the initial web-based self-help intervention, Snow Control (without chat), which was designed to reduce problematic cocaine use , 32. OveThe present study aims to investigate and compare the effectiveness of the revised web-based self-help intervention, Snow Control 2.0, with tailored chat counseling that is based on CBT, MET, BSM, and social problem solving in reducing problematic cocaine use. More specifically, a three-arm randomized controlled trial (RCT) will be conducted to test the effects of a self-help intervention with and without chat counseling compared with those of a waiting list control group.The first-arm intervention consists of three individual chat-counseling sessions that are based on MET and CBT and the web-based self-help intervention from study arm 2. These chat sessions are tailored to the data that are derived from participants’ self-help interventions. Chat counseling will explore the currently available social support for cocaine users, help improve their valuable relationships, and respond to their individual requests. The web-based self-help intervention from study arm 2 is based on classical CBT for the treatment of cocaine dependence , BSM 2121, MET , CBT foro Registration processo Personal companion o Examination of the pros and cons of changing cocaine consumption patterns to address motivationo Goal setting and introduction to e-mail reminderso Introduction to the cocaine consumption diary and its fully automated progress charts and statisticso Introduction to the “My Snow Control” folder: individuals can review their acquired module documents o Introduction to the emergency button for immediate responses to frequently asked questions and access to emergency contactsPart 1: Introductiono Module 1: Strategies for goal achievemento Module 2: Identifying your risk situationso Module 3: Caring about your needs: reduction of stress and depression symptoms and sleep hygieneo Module 4: Managing cocaine cravingso Module 5: Managing relapseso Module 6: Six-step program to tackle your problemso Module 7: Say “no” to bolster refusal skillso Module 8: Preserving achievementsPart 2: ModulesThe web-based self-help intervention (study arms one and two) is organized in two parts. Part 1 can be accessed after successful registration and completion of the baseline assessment. Part 1 has to be worked through systematically. Part 2 is presented in a clearly arranged menu  will support behavioral changes through MET. Participants in the chat condition can discuss the modules of the web-based self-help intervention and review the development of their consumption diary; they are also motivated to foster and review socially rewarding contacts. Social reward sensitivity is assumed to be limited in the self-help only study arm 2, which does not include sensitizing individual counseling.o Personalized feedback according to the baseline assessment and potential cocaine-related physical and mental health riskso Review of successful strategies for change that are related to topics other than cocaineo Imagining daily routines, structuring the day and managing boring situations after cocaine cessation/reduction with new rewards; exploring the aim to live with cocaine abstinence or with low cocaine use and the dissonances with its current use; and mobilizing social support and the option of inviting a relevant third party to join chat session 2o Review of and agreement on the overall cocaine use objectiveo Review strategies to deal with cocaine cravings and perspectives for chat session 2Chat session 1: Starting point and objective agreemento Exchange of experiences regarding the agreed-upon objective since the beginning of the interventiono Review of the patterns that relate to the consumption diary goals and achievementso Review and consolidation of web-based self-help moduleso Review of social rewards prior to cocaine useo Experience and improvement of existing social support and relationshipso Revision of intervention objectives and perspectives for chat session 3Chat session 2: Exchange of experiences, social support, and relationshipso Exchange of experiences regarding the agreed-upon objective since the beginning of the interventiono Review of the patterns that relate to the consumption diary goals and achievementso Consolidation of web-based self-help modules and identification of the best five strategies for long-term successo Experience of intention to change and improvement of social support and relationshipso Definition of long-term objectives and consolidation of strategies for long-term successChat session 3: Review and consolidationThe chat sessions will be structured as follows:Health care professionals will be assigned as online counselors, who will contact registered users during their 6-week programs to arrange three chat counseling sessions. To conveniently manage their interactions with clients, counselors will have access to a specific user management area to add arranged chat dates, define current statuses, and add personal comments about their clients. With this tool, counselors can follow their clients’ progress in reducing their cocaine use through clearly arranged charts, monitor their clients’ module progress, and look up previous chat histories. Specific lists will help counselors track their clients . All written dialogues during chat counseling sessions will be recorded in a database for further analysis.Participants who are randomized to the waiting list will have the opportunity to participate in the web-based self-help intervention 6 months after registration. Follow-up measures for these participants will be assessed online or through telephone interviews for those participants who cannot be reached online at the 6-month follow-up.Snow Control 2.0 is a website that is based on Drupal 7, a content management system with a responsive design for computer screens, tablets and smartphones.www.snowcontrol.ch by providing a unique username and e-mail address. Registered users will be asked to take care to not disclose any personal information about their real identities. For this purpose, they will be advised to register an anonymous e-mail account with a third-party e-mail provider. To prevent the creation of accounts with invalid e-mail addresses, the registration process will not be complete until a verification e-mail link has been clicked and a personal password been set.Any Internet user can register an online account via In a three-arm RCT, the current study aims to compare the effectiveness of three interventions—guided chat-counseling and web-based self-help (arm 1), web-based self-help alone (arm 2) and a waiting list control condition (arm 3)—in reducing cocaine use in problematic cocaine users. This study will be conducted following the CONSORT-EHEALTH  who are an extension of the CONSORT statement , 38. TheThe study population will be recruited through the revised Snow Control 2.0 website, several websites from local outpatient treatment centers, and websites with nightlife prevention offers. In addition, advertisements will be placed in Internet-forums and (commuter) newspapers. Moreover, we plan to send mailings, including flyers, to local addiction counseling centers, general practitioners, psychotherapists and psychologists.In reducing problematic cocaine use, tailored chat counseling in combination with the web-based self-help intervention (study arm 1) is more effective than the waiting list control condition (study arm 3).In reducing problematic cocaine use, the web-based self-help intervention (study arm 2) is more effective than the waiting list control condition (study arm 3).In reducing problematic cocaine use, chat counseling in combination with the web-based self-help intervention (study arm 1) is more effective than the web-based self-help intervention alone (study arm 2).We hypothesize that web-based self-help interventions, which involve interactions with real counselors, will be more effective in reducing cocaine use among problematic cocaine users compared with self-help interventions only and the waiting list condition. We will test the following detailed study hypotheses with respect to the main outcome, i.e., a reduction in the weekly quantity of cocaine used between the baseline assessment and the 6-month follow-up:Participants in study arm 1 will demonstrate increased social support at the 6-month follow-up compared with those in study arms 2 and 3.After 6 weeks of intervention, participants in study arm 1 will be more satisfied overall with the received intervention than those in study arm 2.Participants in study arm 1 will show higher intervention retention rates than those in study arm 2.Participants in study arm 1 will show greater self-help intervention adherence than those in study arm 2.Increased working alliance ratings will predict better outcomes for participants in study arm 1.Analogous hypotheses apply to the secondary outcomes, including the severity of cocaine dependence, alcohol and other drug use, and mental health symptoms, of study arms 1 and 2 and the waiting list control group. Moreover, we have the following specific expectations:The primary outcome measure is the weekly quantity of cocaine used, a very promising measure for this type of intervention in problematic cocaine users . Moreoven = 144 in each study group would reveal 80 % power  to detect this difference based on calculations performed with G-Power software. Thus, we aim to recruit a total of 432 study participants.Based on the results of our previous study , we expeThe Snow Control 2.0 webpage will explain the study rationale to the participants. The participants will then be informed of the following: (1) study inclusion and exclusion criteria  will also be invited to the first chat session at least three times over a two-week period if they do not respond to the first invitation. Each invitation will contain at least three dates for chat sessions, as suggested by their counselor. If they are unable to attend the chat session on any of these dates, participants can suggest dates to their counselor. This procedure will be repeated until a first chat date has been set. If participants do not show up on the agreed chat date, they will be sent a new chat date until the first chat appointment is realized. If participants do not reply after three invitations to schedule chat appointment, they will no longer receive chat requests from their counselor, unless they actively make an effort to contact their chat counselor within 6 weeks of the intervention. The second and third chat appointments will be scheduled at the end of the preceding chat session. If participants do not show up for the subsequent sessions, they will be sent new suggestions for chat dates (twice for each session). Finally, yet importantly, the participant and counselor may terminate the counseling if they agree that it is no longer needed at the end of each chat session.Data will be analyzed according to the intention-to-treat principle (ITT). For the ITT analyses, we will apply the multiple imputations procedure (MICE) of STATA, which imputes missing data using all available data on a person. Baseline measurements will be compared using t-tests and chi-square tests. Differences between primary and secondary outcome variables between the baseline and follow-up assessments will be tested using the generalized estimating equation (GEE) models. The GEE model is a repeated-measures regression model that considers the correlations between the repeated measures from each person . We willWe will conduct additional exploratory regression analyses to determine whether baseline variables predict the frequency and quantity of cocaine use, the severity of cocaine dependence (SDS), reduced psychiatric symptoms (MHI-5), social support (SSQ-6) at follow-up and treatment retention. For these analyses, we will use linear, multinomial, or binary regression models, depending on the scale of the outcome measure. Similar regression analyses will be conducted for working alliance  in relation to primary and secondary outcomes for the participants who receive the additional chat counseling in study arm 1 and for client satisfaction (CSQ-4) sum score in study arms 1 and 2. The treatment retention and client satisfaction of study arms 1 and 2 will be compared using chi-square tests at week 6 (intervention end).This RCT will be executed in compliance with the Helsinki Declaration and has been approved by the ethics committee of the Canton of Zurich on February 2, 2015 (KEK-ZH-No. 2014–0611).To the best of our knowledge, this study will be the first RCT to test the effectiveness of a web-based self-help intervention with and without chat counseling in reducing problematic cocaine use. This study will have important implications for the ongoing discussion about whether interactive web-based interventions with personal contacts is more effective in reducing substance use in problematic users than individual self-help interventions. Compared with the previous Snow Control web-based self-help intervention , 32, we To confirm the effectiveness of the self-help intervention and chat counseling, these parts will be translated and implemented in Safe Zone, a Swiss addiction portal . Thus, tThe weekly quantity of cocaine used is the main outcome in the present study. This outcome was very promising in the first Snow Control study , as consApart from active withdrawal of informed consent, we do not maintain any specific criteria for dropouts. We learned from the consumption diaries of earlier studies , 18 thatThe greatest challenge for the Snow Control 2.0 trial is the expected strong attrition rate during intervention and low numbers for the follow-up assessment. We will address these issues as follows: (1) We will impute missing data. (2) The baseline assessment requires approximately 20 min, which will attract more motivated participants and prevent the participation of less motivated individuals. (3) By allowing the participants to choose their personal companions, the intervention was more tailored to individual participants. The provision of optional contents allows more in-depth information and exploration when required. Moreover, personalized motivation-enhancing e-mail feedback supports individual progress in reducing problematic cocaine use. (4) Participants who complete the 6-month follow-up assessment will be compensated with 40 Euro. Moreover, participants who complete the 6-month follow-up assessment will be entered in a lottery to win 400 Euro (online voucher or online charitable donation).