L. longipalpis, was produced using P. pastoris and crystallized in space group P212121.LJL143, a salivary protein from  Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P212121, with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59 000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly  Clinical manifestations of leishmaniasis vary from self-healing skin lesions to fever, anemia, skin destruction and death .Leishmaniasis is a neglected tropical disease that is endemic in over 88 countries and is caused by over 20 species and subspecies of parasitic protozoa of the genus Leishmania parasites are transmitted to humans by sandflies of the genus Phlebotomus and Lutzomyia in the Old and New Worlds, respectively . Approximately 250 ml of the starter culture was added to a 14 l fermentation vessel containing 5 l basal salt medium . The BSM was maintained at pH 5 by the addition of 14% ammonium hydroxide. Antifoam 204 was added to minimize foaming. About 16 h into fermentation a dissolved-oxygen (DO) spike from the depletion of glycerol was observed, which prompted the initiation of a fed-batch phase. During the fed-batch phase, 50% glycerol was added to the culture at 15 ml per litre per hour for 6 h. In the last 2 h of the fed-batch phase, the pH of the culture was ramped from 1 to 6.0 and the temperature was reduced by 2 K. The fed-batch phase was followed by an 8 h methanol-adaptation phase during which the agitation was increased to 700 rev min−1 and a methanol feed was ramped to 11 ml per litre per hour, which was maintained until the end of fermentation.DNA coding for the mature LJL143 peptide and a C-terminal hexahistidine tag was codon-optimized based on the yeast codon preference . The synthesized gene included EcoRI and XbaI restriction sites for subcloning into the pPICZα A vector Table 1. The plag for 30 min and filtered with a 0.22 µm membrane filter. The clarified supernatant was buffer-exchanged into the binding buffer  using a 3 kDa hollow-fiber cartridge . LJL143 was purified by immobilized metal-ion affinity chromatography (IMAC) using a 5 ml HisTrap FF column . Nonspecifically bound proteins were removed by sequential steps of washing with 5, 10, 25 and 55 mM imidazole prior to protein elution with 500 mM imidazole. LJL143 was purified to ∼99% as assessed by Coomassie-stained SDS–PAGE . The protein band was verified to be LJL143 by Western blotting using both anti-His-tag monoclonal antibody  and mouse antiserum against LJL143 .After 72 h of induction, the culture medium containing the secreted LJL143 protein was separated from the yeast cells by centrifugation at 12 000GE Fig. 1a. The pdy Fig. 1b and mo43 Fig. 1c.2.2.−1 in 50 mM sodium citrate buffer pH 5.0 using a 10K cutoff centrifugal concentrating device (Millipore). The protein concentration was measured by the absorbance at 280 nm prior to setting up crystallization experiments.Recombinant LJL143 was buffer-exchanged and concentrated to 23 mg mlInitial crystallization screens were carried out using commercial sparse-matrix screens from Hampton Research  at 298 K by vapour diffusion in sitting drops Table 2.2.3.M bis-tris pH 6.5, 28% PEG 2000 MME, 15% glycerol) and were flash-cooled directly in a stream of N2 gas at 113 K prior to collecting diffraction data. X-ray diffraction data were collected at the Baylor College of Medicine core facility using a Rigaku HTC detector  package  as precipitants. The best diffracting crystals were obtained after 48 h in the conditions detailed in Table 2a). Diffraction images reveal visible diffraction spots beyond 2.6 Å resolution . The crystal belonged to the orthorhombic space group P212121, with approximate unit-cell parameters a = 57.5, b = 70.2, c = 79.5 Å (Table 3http://www.ruppweb.org/Mattprob), a monomer is expected in the asymmetric unit , a Matthews coefficient of 2.39 Å3 Da−1 and an estimated solvent content of 48.5%.The typical yield of LJL143 was 50 mg per litre of fermentation supernatant. The resulting protein had an electrophoretic mobility of 42 kDa. The predicted theoretical molecular mass of LJL143 is 33 594.92 Da. LJL143 has two predicted N-glycosylation sites at on Fig. 2b. The cÅ Table 3. Based oBLAST analysis of LJL143 against the Protein Data Bank revealed no suitable homologues for molecular replacement. The closest structural homologue, 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase from Thermus thermophilus , shares only 21% sequence identity over a 26% query coverage. There are only ten S atoms out of a total of 4668 atoms, and our attempts at phase determination by single-wavelength anomalous dispersion using S atoms at the Cu Kα wavelength failed. Future studies will include other methods of experimental phase determination such as multi-wavelength anomalous dispersion phasing with selenomethionine-derivatized protein and heavy-atom derivatization for multiple isomorphous replacement (Ingram et al., 1956A


Besides the effects on the immune response, we have described how autophagy subversion by viruses may also concur to the enhancement of their replication and to viral tumorigenesis.We summarized the most recent findings on the role of autophagy in antiviral immune response. We described how viruses have developed strategies to subvert the autophagic process. A particular attention has been given to Epstein-Barr and Kaposi's sarcoma associated Herpesvirus, viruses studied for many years in our laboratory. These two viruses belong to  This occurs after they are degraded via proteasome and transported by transporters associated with antigen processing (TAPs). Differently, peptides that originate from extracellular antigens are delivered to the late endosomal/lysosomal compartment, where they are degraded by the lysosomal proteases and then presented in association with class II MHC. In the lysosomes, the invariant chain, which blocks class II MHC, is also degraded, rendering the MHC molecules available for peptide loading. Besides the differences in the sites of origin and processing, peptides presented via class I or II MHC activate different populations of T lymphocytes, which are CD8ectively . Howeverectively , are abl antigen . On the  antigen . Some of antigen  and the  antigen . LC3 is  antigen . After e antigen . In addi antigen . The bin antigen  and for  antigen . Autopha antigen . Intrigu antigen , it woul antigen . Retroviridae family, is able to induce the initial phases of autophagy by Env, an envelope fusogenic protein. On the other hand, it blocks the late autophagic phases to enhance viral production by expressing Nef, an accessory protein that interferes with the autophagosomal maturation [Among the strategies that allow viruses to escape from the immune control, the impairment of monocyte differentiation into functional DCs represents a common one –19. Baseturation , 25. Durturation –28. To dturation , 26, 29.turation –32 and cturation . Also Beturation –36. All turation .α-subfamily includes the neurotropic viruses Herpes Simplex viruses (HSV) 1 and 2 and Varicella-Zoster virus (VZV). Human cytomegalovirus (HCMV) and the human Herpesviruses 6 and 7 are members of the β-subfamily, while EBV and KSHV are members of the γ-subfamily [γ-Herpesviruses, given that our laboratory has for long time worked on the virus-host interaction of these viruses associated with several human cancers.Herpesviruses are large, double strand DNA viruses having a common particle structure . To dateubfamily . A commoubfamily . Althoug in vitro infection of monocytes induces apoptosis and results in an impairment of DC development [+ T cell response, playing a pivotal role in control of the EBV infection [+ T cell-mediated immune response against the EBV antigens, such as EBNA1, through the mechanism of cross-presentation [+ T cells [As for other Herpesviruses, EBV infection can be latent or lytic , 42. Durelopment , 46, altelopment . These anfection . Besidesentation . Autopha T cells . The inv T cells . Regardi T cells , it has  T cells . Since i T cells  and that T cells  and BGLF T cells , it woul+ T cells [+ T cells [EBV is associated with several different human cancers of B and epithelial cell origin, such as posttransplant lymphoproliferative disorder (PTLD), Hodgkin and non-Hodgkin lymphomas, nasopharyngeal carcinoma (NPC), and some forms of gastric carcinoma . Besides T cells , being p T cells  and CD8+ T cells , althoug T cells . As prev T cells . The mos T cells . Given tγ-Herpesvirus identified to date [ In vivo, KSHV mainly infects endothelial and B cells and establishes a lifelong latency in B lymphocytes of infected individuals, escaping from the host immune response [KSHV or Human Herpesvirus-8 (HHV-8) is the last human  to date . It show to date –64. Generesponse , 66. Sevresponse . KSHV isresponse , a multiresponse , and of response . It is iresponse , 68. Typresponse . Howeverresponse –74. In aresponse . Monocytresponse . The virresponse , 78. In vivo, DC functional impairment has been reported in patients with classical KS [As other Herpesviruses, KSHV has to cope with innate and adaptive immunity in order to establish a persistent infection in immunocompetent host. Latency is one of the strategies used by KSHV since viral gene expression is reduced. Conversely, during its lytic cycle, a high number of immunogenic proteins are produced , 79. Thesical KS  and a resical KS . Moreovesical KS . Furthersical KS . Interessical KS . In thissical KS , 96. Bessical KS . This issical KS . Of notesical KS , 100. Cosical KS  and/or Tsical KS , promotisical KS  and in tsical KS , 104. Sesical KS , 106 hassical KS , 108. v-sical KS . Besidessical KS , 110 andsical KS , 35. As sical KS . Always sical KS . K7 is isical KS , 113 whisical KS –116. As sical KS . Interessical KS . Altogetγ-Herpesvirus-associated cancers.A better understanding of how autophagy manipulation could influence antiviral immune response and control virus-induced tumorigenesis might help to discover strategies improving the outcome of the treatments of virus-associated malignancies. Moreover, since autophagy is activated and is involved in EBV and KSHV replication , 27, its


Hepatocellular carcinoma (HCC) has had a continuous increase worldwide in incidence over the last two decades. The development of HCC has a complicated process and correlates to numerous risk factors including hepatitis virus infection, alcoholism, food toxins, diabetes, obesity, smoking, congenital liver diseases, and errors of metabolism. Molecular links are also identified, which characterize tumorigenic intracellular pathways and suggest molecular targeted therapies.Each year, HCC is diagnosed in approximately 630,000 people worldwide and more than half of the new cases come from China. Correct understanding and interpretation of the details of carcinogenesis, establishment, and progression of this cancer is of paramount importance for its treatment with good outcome. There are many therapeutic modalities for treatment of HCC but evidence-based approach to the multidisciplinary management is substantial.This special issue contains the research papers focusing on the current general interest in HCC, including basic research, laboratory study, diagnosis, and treatment. Study on molecular aspects of HCC is a highlight of this special issue, involving microRNAs, genes, tumor markers, and so forth. We hope that these articles will stimulate the lasting exertion to unveil the mechanism of HCC development and progression and cast new insights into HCC establishment and management.Yong-Song GuanMohammad Ahmad al-ShatouriQing HeWei Mike Liu


Systemic administration of d-amphetamine  caused a transient increase in extracellular dopamine in the rACC, but an apparent increase in the cACC of the same animals was less clearly defined. Local infusion of d-amphetamine increased dopamine efflux in the rACC, only. Glutamate efflux in the rACC was increased by local infusion of dopamine (5–50 μM), which had negligible effect in the cACC, but only systemic administration of d-amphetamine increased glutamate efflux and only in the cACC. The asymmetry in the neurochemical responses within the rACC and cACC, to the same experimental challenges, could help explain why different subregions are recruited in the response to specific environmental and somatosensory stimuli and should be taken into account when studying the regulation of neurotransmission in the ACC.There is evidence for functional specificity of subregions along the rostrocaudal axis of the anterior cingulate cortex (ACC). The subregion-specific distribution of dopaminergic afferents and glutamatergic efferents along the ACC make these obvious candidates for coding such regional responses. We investigated this possibility using microdialysis in freely-moving rats to compare changes in extracellular dopamine and glutamate in the rostral (‘rACC': Cg1 and Cg3 (prelimbic area)) and caudal (‘cACC’: Cg1 and Cg2) ACC induced by systemic or local administration of This article is part of the Special Issue entitled ‘CNS Stimulants’. •Dopamine and glutamate efflux in two anterior cingulate subregions were compared.•d-amphetamine depended on subregion and route of drug administration.Responses to •These findings could help explain the disparate roles of the two subregions. The existence of subdivisions of the ACC along its rostro-caudal axis is inferred from detailed cytochemistry . FunctioIt has been suggested that there are reciprocal interactions between rostral and caudal zones of the ACC . This waThere is a strong rationale for predicting that dopaminergic transmission, at least, will vary along the ACC. The dopaminergic innervation of the ACC is strikingly heterogeneous. The density of dopaminergic terminals is characteristically highest within Cg3 (now known as the prelimbic cortex) and declines in a caudal direction . The Cg1e.g.,As regards glutamate, it is well established that this transmitter influences dopamine release in the prefrontal cortex and elsewhere in the brain  in a rostral and caudal region of the ACC following an acute d-amphetamine challenge. We chose this  stimulus because d-amphetamine increases the extracellular concentration of both dopamine and glutamate in the ACC and yet there has been no systematic investigation of the extent to which these responses generalise throughout its length.As a first step in characterising any differences in neurotransmission in subregions of the ACC that could underlie their functional reciprocity and specificity, we used The amount of dopamine and glutamate harvested by microdialysis probes (‘efflux’) was monitored in a rostral zone  area) and/or a caudal zone  of the ACC. These subregions are defined according to nomenclature in Findings from this study point to striking differences in the regulation of dopaminergic and glutamatergic neurotransmission in the rACC and cACC. These differences echo reports of functional reciprocity between these subregions  and coul2All procedures were carried out under the licensed authority of the UK Animals (Scientific Procedures) Act, 1986, and were approved by the local ethics committee at University College London.2.12/5% CO2, delivered at 2 L/min. Following loss of the righting reflex, rats were transferred to a stereotaxic frame and anaesthesia maintained via a face mask . The head was set in the flat-skull position  using blunt ear-bars. Core temperature was maintained at 37 °C using a homeothermic heating pad and rectal probe.Male outbred Sprague–Dawley rats (250–300 g) were obtained from the colony at University College London. They were housed in groups of four, with sawdust bedding, at 21 ± 1 °C and 55 ± 1% relative humidity, with a light–dark cycle of 12 h (lights on at 08.00 h) and free access to standard laboratory chow and water. Anaesthesia was induced by inhalation of 5% halothane in 95% O2, 1.3 mM), were implanted either uni- or contralaterally  with the tip at the following co-ordinates (mm from Bregma): (rACC) AP +2.5, ML ±0.6, DV −4.6; (cACC) AP +1.0 ML ±0.6, DV -3.6  efflux of dopamine was monitored in both subregions until it had stabilised, after which three basal samples were designated T−40-T0. At T0, rats were randomly assigned to groups given either d-amphetamine , or saline (1 mL/kg): these two treatments were administered in a counterbalanced sequence. This dose of d-amphetamine was chosen because it is known to increase dopamine efflux without inducing stereotypy . These concentrations were used because they increase dopamine efflux in other brain regions  at T0. This dose is the same as that in Experiment 1 and has been reported to increase glutamate efflux in the rostral zone of the medial prefrontal cortex (d-amphetamine: 1 μM (80 min), followed by 10 μM (80 min) and then 100 μM (160 min).l cortex . The secExperiment 3: Probes were implanted on contralateral sides of the brain for dual-probe microdialysis of the rACC and the cACC. The probes were used both to harvest extracellular glutamate and for local infusion of dopamine. The stock dopamine solution was kept on ice, in the dark, until loading into syringes for simultaneous infusion into both the rACC and cACC. The test concentrations of dopamine ) were similar to those used previously  was set at −280 mV and the analytical electrode set at +350 mV. For dopamine, the mobile phase comprised (mM): NaH2PO4 83, octanesulphonic acid (OSA) 0.23, EDTA 0.84, dissolved in 17% methanol, adjusted to pH 4.0 with orthophosphoric acid and pumped through the system at 1.15 ml/min. The conditioning electrode of the microdialysis cell (ESA 5014A) was again set at −280 mV but the analytical electrode was set at +180 mV. The yield of solutes is expressed as fmol/20 min (dopamine) or pmol/20 min (glutamate), with no adjustment for probe recovery. The limit of detection was 1 pmol (glutamate) and 2–4 fmol (dopamine). In two rats, the dopamine content of some baseline samples was below the limit of detection and was assigned a value of 0 fmol/20 min: both were from the group serving as saline-injected controls for the effects of systemic administration of d-amphetamine on dopamine efflux in the rACC (Experiment 1). Since there was no dopamine response in these animals, this will not affect the interpretation of the results. There were no animals with basal DA levels below the limit of detection in the local infusion experiments.Immediately after collection of the microdialysis samples, glutamate  and dop2.4(10) transformed data, provided this reduced statistical significance in the Mauchly test. When significance in this test remained, despite the transformation, the Greenhouse-Geisser ε correction was applied to the degrees of freedom. The Levene's test was applied to ensure equal variances across all levels of the between subjects' factors in the log(10)transformed data set. In all cases, a significant effect on the main factor(s), or interactions between them, was taken as the criterion for progressing to a post-hoc 2-way ANOVA. In experiments testing the effects of systemic drug administration, the data were divided into clusters (‘bins’) of consecutive samples. The bins comprised 3 samples  in order to assess the significance of any changes in transmitter efflux. In experiments involving local infusion of d-amphetamine or dopamine, the bins corresponded to samples collected during infusion of each test concentration . In these cases, only the last 3 samples in each bin were compared with the 3 basal samples and ‘bin’ was treated as another within-subjects’ factor in the ANOVA. ‘Subregion’ was treated as a ‘between-subjects’ factor in single-probe microdialysis and a ‘within-subjects’ factor in the dual-probe experiments. The criterion for significance was P ≤ 0.05.For each animal, mean basal efflux was calculated from the three consecutive samples taken immediately before the experimental challenge. These data were pooled to provide a measure of mean basal efflux for each experimental group. The significance of differences in transmitter efflux was assessed using multi-factorial split-plot ANOVA (SPSS PC) with repeated measures on the factor, ‘time’. ANOVA was carried out on the raw data unless the Mauchly test indicated statistically significant deviation from homogeneity of the variance/covariance matrix on the repeated measures factor (time). In those cases, ANOVA was carried out on log2.5d-Amphetamine sulphate and dopamine hydrochloride were obtained from Sigma–Aldrich (UK). When given by intraperitoneal injection, the drug was dissolved in 0.9% sterile saline and administered in a volume of 1 mL/kg. When infused locally into the terminal field, d-amphetamine was dissolved in the modified Ringer's perfusion solution. All reagents for the modified Ringer's solution and mobile phase were of chromatographic or AnalaR grade.3d-amphetamine but not during its local infusion. We saw no sign of behavioural stereotypy in either case.There was an increase in locomotor activity after systemic injection of 3.1d-amphetamine (3 mg/kg). Saline injection did not affect dopamine efflux in either region : F = 5.8; P = 0.04; ε = 0.45]. This increase was also evident when efflux after drug administration was compared with the basal samples . In the cACC of the same animals, the increase in dopamine efflux also reached criterion for statistical significance when compared with the saline group . However, unlike the rACC, the increase in dopamine efflux did not reach criterion for statistical significance when compared with the basal samples. The response to d-amphetamine in the cACC was also more erratic than in the rACC, but there was no difference in dopamine efflux in the two subregions within this time-frame   = 7.69; P = 0.02]  = 15,67; P < 0.001; ε = 0.623]. Specifically, infusion of the lower concentration of d-amphetamine (10 μM) increased dopamine efflux in the rACC but not the cACC  (d-amphetamine was substituted for the modified Ringer's solution (100 μM), there was a resurgence of dopamine efflux in the rACC, which reached maximum at T260 [63 ± 21 fmol/20 min]. Again, dopamine efflux in the rACC was greater than in the cACC . The apparent increase in the cACC at the higher concentration of d-amphetamine did not meet the criterion for statistical significance  , there was no statistical interaction between subregion and glutamate response  and the basal samples (T-40–T0): F = 6.21; P = 0.024; ε = 0.929]. The maximum increase occurred at T80 [+11.1 ± 7 pmol/20 min]. Apart from the samples collected from T160–T200 , efflux remained greater than that in the basal samples for the remainder of the experiment   = 4.19; P = 0.056; ε = 0.28]. However, efflux in the rACC during infusion with 50 μM dopamine was greater than in time-matched samples from the cACC  and the response to 5 μM dopamine in the rACC just missed the criterion for significance  . There is also evidence that dopamine transporters in the prelimbic cortex are concentrated in intervaricose regions of the nerve terminal (via polysynaptic loops), have a greater inhibitory influence on neuronal firing-rate than those in the ventral tegmental nucleus (A10)  . This rad-amphetamine could also rest on differences in factors such as: terminal heteroceptors that govern dopamine release in the anterior cingulate cortex  caused a clear increase in glutamate efflux in the cACC, this was not the case for the rACC.These empirical observations prompted us to investigate whether the net effect of all these processes has any bearing on glutamatergic transmission within these subregions. The effect of d-amphetamine differs in the two subregions. These findings are supported by published evidence to the extent that glutamate efflux in the rACC is not increased by systemic administration of d-amphetamine  that converge on somatodendritic regions of cortical pyramidal cells, which, together with the terminals of dopaminergic afferents from A9 and A10 nuclei, form neuronal ‘triads’ . Also, ain vitro, the pyramidal cell response to activation of dopamine receptors is time- and concentration-dependent; it can be mediated both directly and indirectly; and depends on pyramidal cell excitable state  but polysynaptic links are possible, also, such as indirect activation of glutamatergic thalamocortical neurones.However, the lack of any glutamate response to local infusion of 2+-dependent exocytosis of glutamate  are a prominent source of extracellular glutamate, which is extruded on a glial cystine/glutamate antiporter . This solutamate . Given tlutamate  and infllutamate . Indeed,striatum  and extrstriatum . We are d-amphetamine or dopamine challenge. Such regional disparities should be taken into account in electrophysiological and neurochemical studies of the ACC in vivo and in vitro. Moreover, these contrasting responses could contribute to the ‘functional reciprocity’ of subregions of the ACC  and RenaSci Ltd. The funders were not involved in defining the study objectives, the experimental design, interpretation of the results, or drafting of this paper.EA was in receipt of a Medical Research Council (UK) PhD studentship. This work was funded by the


K8.1 titres differed more between KS cases and controls, compared to LANA titres. These differences in titre between cases and controls suggest a role for lytic viral replication in the pathogenesis of HIV-related KS in this setting.HIV-associated Kaposi's sarcoma (KS) is a public health challenge in sub-Saharan Africa since both the causative agent, Kaposi's sarcoma associated-herpesvirus (KSHV), and the major risk factor, HIV, are prevalent. In a nested case-control study within a long-standing clinical cohort in rural Uganda, we used stored sera to examine the evolution of antibody titres against the KSHV antigens K8.1 and latency-associated nuclear antigen (LANA) among 30 HIV-infected subjects who subsequently developed HIV-related KS (cases) and among 108 matched HIV/KSHV coinfected controls who did not develop KS. Throughout the 6 years prior to diagnosis, antibody titres to K8.1 and LANA were significantly higher among cases than controls ( Kaposi's sarcoma (KS) is currently the most commonly reported cancer in Uganda and in other parts of sub-Saharan AfricaAmong individuals with HIV-associated KS in sub-Saharan Africa survival is generally poor.Infection with Kaposi sarcoma associated-herpesvirus (KSHV) and HIV, the major risk factor for Kaposi sarcoma, is common in sub-Saharan Africa. However, little is known about the evolution of KSHV antibody responses in HIV-infected individuals prior to the clinical onset of KS. This study shows that antibody titres against KSHV antigens K8.1 and latency-associated nuclear antigen (LANA) increase significantly in the six years leading up to HIV-associated KS. Titres of K8.1, a lytic antigen, rose more than LANA titres, indicating that the activation of genes in the lytic cycle of viral replication is important in KS development.To further our understanding of the patterns of antibody responses to KSHV over time, we conducted a case-control study nested within an actively followed population-based clinical cohort of HIV-infected adults living in rural Uganda.Our study was nested within an actively followed longitudinal clinical cohort of HIV-infected adults. These individuals were identified from within a larger general population cohort (GPC), established in 1989 in rural South West Uganda, to study the population dynamics of HIV disease in a typical rural Ugandan community.26HIV-infected adults identified within the GPC were invited to join the Rural Clinical Cohort (RCC)We identified, through the RCC study database, 30 individuals diagnosed with HIV-associated KS, between 2 October 1990 and 5 July 2010. All cases were confirmed by a review of clinic notes conducted independently by two clinicians. Five cases had histological verification of diagnosis, and the remainder had clinical documentation only. Controls were selected from among HIV-seropositive adults without a diagnosis of KS during the study period using the following procedure: each case was classified by sex, age band (≤ median age of 37 years or ≥38 years) and CD4 count band  at the time of their KS diagnosis. Potential controls were then examined to find the time points at which their characteristics  matched those of the case, and this time point was then taken as the pseudodiagnosis date for that control, which for simplicity will be referred to as the KS diagnosis date hereafter. Next, to ensure that all controls were KSHV seropositive at KS diagnosis, those fulfilling matching criteria were tested for KSHV antibodies using the methods described below, and 149 were found to be KSHV seropositive to either K8.1 or LANA at KS pseudodiagnosis. For each case, up to four individually matched controls were selected from the pool of 149 HIV and KSHV seropositive people, and they were included in the dataset as matched to the corresponding case. Results for HIV viral load were considered a suitable representation of an individual's viral load at KS diagnosis if measured within 100 days.All available serum samples taken up to and including the KS diagnosis sample were tested for antibodies against KSHV using a quantitative lytic K8.1 ELISA and latent LANA ELISA as previously described.t-test or χ2 test as appropriate. The change in titres (defined as the number of doubling dilutions) prior to KS diagnosis were analyzed using linear regression adjusted for matching factors age, sex and CD4 count. Correlation between repeated measurements on individual patients was adjusted for using generalized estimating equations (GEE) with an exchangeable correlation structure. To graphically present trends in titre, the GEE model was used to predict mean titres for cases and controls from a population of HIV-positive individuals in a 1:1 sex ratio, with a median age of 37 years and a CD4 count of between 200 and 500 cells per ml at KS diagnosis. Variability between individual patients was explored by constructing a multilevel mixed effects regression model incorporating random elements for both the titre level, the trend over time and covariance between the two. Random effects from the mixed models for each individual were estimated using the best linear unbiased predictor. Interactions between time prior to KS diagnosis and case-control status were examined in both models to assess whether patterns in titre over time differed between cases and controls. Tests for statistical significance were derived from likelihood ratio test statistics. All p values were two-sided, and significance was considered at the 5% level. Analyses were carried out using Stata 12SE .The characteristics of cases and of controls were initially compared using a Mann–Whitney test, Ethical approval for our study was obtained from the UVRI Science and Ethics Committee and from the Uganda National Council for Science and Technology.p = 0.006). Titres to K8.1 were higher among KS cases than controls at KS diagnosis  660–10,240] vs. 40 [IQR 0–80], p < 0.0001, p = 0.045) and titre  were observed for the LANA antigen at the KS diagnosis time point: 100% (30/30) of cases were seropositive compared to 88% (95/108) of the controls  and controls .Results were available for 30 cases and 108 matched controls. The median number of samples available from each individual was 3 (range 1–12). All cases at diagnosis and controls at pseudodiagnosis were seropositive to either K8.1 or to LANA but not necessarily to both. At the time of KS diagnosis 93% (28/30) of the cases were seropositive to K8.1 compared to 69% (74/108) of the controls .There was a significant interaction for K8.1 antibody titres between case-control status and time, such that the difference in K8.1 titres between cases and controls was largest at the time points closest to KS diagnosis Table . EstimatFig. 2b). Intercept estimates ranged from 3.0 to 8.4 dilutions among people with KS and were lower among controls (range 0.7–7.4 dilutions), with similar variance estimates within the two groups (c). While there was substantial overlap in distributions the shapes of the distributions were different. The slope estimates had no overlap between KS cases and controls: case K8.1 titres increased at rates between 0.44 and 0.67 dilutions per year whereas rates of change among controls ranged from a decrease of −0.19 dilutions per year to an increase of 0.14 dilutions per year (d). Thus, groups were easily distinguished based on slope. Variability in slope estimates among cases was similar to the variance among controls. There was a small negative covariance for K8.1 between the intercept (antibody titre at diagnosis) and slope (rate of change in antibody level) but this did not contribute significantly to the model fit.From the random effects model, predicted individual K8.1 antibody dynamics were clearly different for KS cases and controls b. Interco groups c. While per year d. Thus, a). Predicted individual LANA titre dynamics of cases and controls were comparable (b). Distributions of patient intercepts and slopes overlapped substantially; intercepts ranged from 1.00 to 10.81 dilutions among KS cases and from 0.11 to 9.92 dilutions among controls (c); rates of change in LANA titre (slope) ranged between −1.29 and 1.77 dilutions per year among individuals with KS and −0.60 and 1.26 dilutions per year among controls (d). There was a positive covariance for LANA between intercepts and slopes indicating that individuals with higher titres at diagnosis had higher rates of change of antibody level over time (b).The two LANA models were also comparable. From the GEE model, patients with KS had LANA titres approximately five times higher  than patients who did not have KS; however, there was no trend in the titres over time and there was no significant interaction between the effects of KS status and time Table a. Predicmparable b. Districontrols c; rates controls d. There ver time b.Given the infrequent use of ART Table  and consThis is the first longitudinal study examining patterns of antibody titres to both K8.1 and LANA in individuals with HIV-associated KS, living in a KSHV-endemic population in sub-Saharan Africa. The study has shown that individuals who develop HIV-associated KS have higher K8.1 and LANA antibody titres compared to those who do not, and that this is evident up to 6 years prior to KS diagnosis. In addition, K8.1 antibody titre increased with time toward KS diagnosis among cases, whereas individuals who did not develop KS had relatively stable titres over time.The results presented here are in accord with studies conducted among cohorts based in the West; antibody titres against KSHV antigens are higher among cases and increase prior to tumor diagnosis.6Like all herpesviruses, KSHV cycles between two distinct phases: latency, a quiescent state of persistent infection essential for the development of KS, and lytic reactivation, characterized by ordered increased gene transcription and in most cases, viral replication and host cell death.35The raised K8.1 titres among individuals who develop HIV-associated KS found in our study is in accord with the evidence that lytic gene expression may be important in KS tumorigenesis. It is likely that cellular immune responses are also critical to KS risk, as has been shown for transplant-associated KS.Our study had a number of limitations. The definition of case-control status in our study relied, in the majority of cases, on clinical diagnoses made at the time of a regular clinic visit. While large dermal KS lesions are relatively easy to diagnose, lack of histological confirmation may have resulted in some misclassification of cases and controls and small dermal lesions may have been missed. Further, no diagnostic tests were available to ensure that controls did not have visceral KS involvement or another KSHV-related pathology. Date of KS diagnosis was based on the clinical review date, which may have been affected by how regularly the HIV clinic was attended. Only simple linear titre dynamics could be investigated in our study because the majority of patients had only three samples available. The results of our study also rely on the validity of the measured antibody titre. The relatively small numbers of individuals on ART at KS diagnosis, in spite of low CD4 counts, reflects local policy  and limited access to ART in Uganda at the time when these cases were diagnosed.In our study, K8.1 titres provided a more useful means of differentiating patients developing KS from those who did not, compared to LANA titres. The differences in titre between cases and controls were sufficiently pronounced that the findings might have some clinical utility in terms of predicting which individuals may develop HIV-associated KS. K8.1 titres showed more marked differences than LANA titres and the slope of change in K8.1 titres completely distinguished cases from controls, suggesting that this may be a particularly useful parameter for such a purpose. In further research, development of a simple assay and investigation as to whether these parameters could be of use in other patient groups (such as transplant recipients) might be considered.For the related gammaherpesvirus, Epstein-Barr virus, age at infection is known to be an important determinant of outcome.In conclusion, during the 6 years prior to KS diagnosis, antibody titres against KSHV antigens K8.1 and LANA were significantly higher among cases compared to controls and K8.1 titres increased as diagnosis approached. These findings suggest strategies for identifying and understanding KS disease risk and support the hypothesis that lytic gene expression and replication are important in the pathogenesis of HIV-related KS in this setting.


The BRAF V600E mutation has recently been identified in a high percentage of papillary craniopharyngiomas , 2. The The patient initially presented aged 27 years in 1984 with galactorrhea, amenorrhea, hyperprolactinemia and a pituitary mass. Due to tumor progression while receiving bromocriptine she underwent trans-sphenoidal surgery (TSS) in 1992 with radiological remission but without a conclusive histological diagnosis. She was seen again in 2010 with bitemporal hemianopia and recurrent tumor, and underwent a second TSS followed by fractionated radiotherapy. Histology showed a papillary craniopharyngioma (Fig. The pretreatment MRI Fig. c showed The discovery of the activating BRAF mutation in melanoma and the development of molecular targeted therapy represented a major advance in the management of melanoma. Our case now extends the potential use of vemurafenib to a different indication. Craniopharyngioma is a benign non-metastatic tumor with a low proliferation rate that grows slowly but inexorably and invades surrounding tissues. Treatment mainstays include either trans-sphenoidal or trans-cranial surgery, with external beam radiotherapy. This tumor frequently arises during childhood and the use of radiotherapy has to be balanced with long term sequelae of therapy. Furthermore, some patients require repeated surgeries to achieve tumor control and patients with craniopharyngioma sustain severe morbidity both as a result of the tumor and the treatments. There are no recognised targeted or cytotoxic treatments available.In this case, proven to have a somatic BRAF V600E mutation, the tumor responded promptly and progressively to monotherapy with vemurafenib. It is noteworthy that regrowth was rapid with interruption of treatment which should be considered in future cases. The molecular targeted use of the BRAF inhibitor vemurafenib and the excellent tumor response support a pathogenic role for the BRAFV600E mutation in papillary craniopharyngioma. Craniopharyngiomas have not previously been considered as treatable with cytotoxic or targeted agents. BRAF inhibitors may be an effective treatment option as part of the multimodal therapeutic armamentarium for this condition. The successful use of vemurafenib in this patient is a proof-of-concept that justifies further clinical studies and represents a potential paradigm shift in the medical management of patients harbouring a craniopharyngioma.


The treatment of acute thromboembolic limb ischemia includes well-established surgical thrombectomy procedures and, in recent times, also percutaneous rotational thrombectomy using Straub Rotarex® system. This modality not only enables efficient treatment of such thrombotic occlusion but also in rare cases may imply the risk of perforation of the occluded artery. Herein, we report the case of a perforation of the superficial femoral artery (SFA) in an elderly female patient with thromboembolic limb ischemia. The perforation was successfully treated by implantation of self-expanding nitinol Supera stents and without the need for implantation of a stent graft. Peripheral artery disease (PAD) exhibits increasing morbidity and mortality during the last few decades  and is eEspecially patients with acute thromboembolic limb ischemia are in high risk of major amputation and death due to sepsis and multiorgan dysfunction. Such patients are usually older than 75 years and show further comorbidities, including atrial fibrillation and history of heart failure . This me∗120 mm balloon  was performed, which resulted in complete repair of the vascular injury of the SFA  in the SFA   and the  Figures . Due to  Figures . Local l Figures . However the SFA . Subsequ the SFA . After t the SFA  and the To the best of our knowledge this is the first case reporting the placement of self-expanding nitinol Supera stent for the management of a perforation in a peripheral artery.In the past decades, significant technical developments have occurred with endovascular therapy of acute thromboembolism, which offer several advantages over open surgical embolectomy techniques for the treatment of peripheral arterial thromboembolic occlusion . AlthougHerein we report the placement of a self-expanding nitinol Supera stent for the management of a perforation in a peripheral superficial femoral artery. Further novel options of complication management as described in our case may shift the treatment from surgical to even more endovascular treatment procedures in the future.


Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase , and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of “dopaminergic” neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory formation and arousal, in insects.The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine’s actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach,  Biogenic amines have been anatomically and biochemically identified in the central nervous system of vertebrates as well as invertebrates. In insects, these molecules regulate various physiological phenomena as neurotransmitters, neuromodulators, or neurohormones ,2. AmongDrosophila melanogaster, honey bee, Apis mellifera, and cricket, Gryllus bimaculatus [αs proteins, which in turn activate adenylyl cyclase to raise intracellular cAMP concentrations. In contrast, the D2-like receptors are coupled to Gαi proteins and inhibit adenylyl cyclase, or coupled to different intracellular second-messenger systems [2+ signaling pathways [Dopamine is synthesized from the amino acid tyrosine, the latter being converted into DOPA by tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine biosynthesis. DOPA is further converted into dopamine by DOPA decarboxylase (DDC) ,18. Receaculatus –21. All aculatus . The D1- systems . The INDpathways . DespiteComprehensive neuroanatomical studies of putatively dopaminergic neurons exist in flies ,12, the Periplaneta americana is amenable to behavioral experiments including various forms of learning paradigm [The cockroach, paradigm ,36,40–46paradigm –34,47. IPeriplaneta americana) were raised in crowded colonies at Hokkaido University  under a LD 12:12 h photoperiod at 27°C. Adult cockroaches of either sex were used for reduced silver impregnation while only adult males were used for immunocytochemistry. Male field crickets, Gryllus bimaculatus maintained under the same photoperiodic and temperature conditions at Hokkaido University were also used to test the specificity of an antiserum against tyrosine hydroxylase, as in American cockroaches  for 1 hour, and then brains were carefully dissected out. The dissected brains were fixed in a solution containing 4% PFA, 5% glacial acetic acid and 85% ethanol for 2 days, dehydrated, and then embedded in paraffin. Reduced silver staining was performed on 12-μm sections, as described elsewhere .Cockroaches with their wings removed were mounted on a plastic dish. A large window was made in the cuticle of the anterior side of the head, and the tracheae and fat bodies removed to expose the vertical lobes of the mushroom bodies. After removing hemolymph with a piece of Kimwipe, a glass microelectrode with crystals of Neurobiotin at the tip was stabbed into the vertical lobe under a dissecting microscope. The exposed brain region was then briefly washed with saline. The window was resealed with the excised cuticle and covered with a piece of Kimwipe soaked in saline, and then the cockroach left overnight at 4°C to allow the injected Neurobiotin to be taken up by neurites damaged by the microelectrode.2O2 and 0.3% nickel ammonium sulfate. After washing in PB, the sections were mounted on a gelatin-coated glass slide, dehydrated in a graded ethanol series, cleared in xylene, and mounted in Mount-Quick  beneath a cover slip.The Neurobiotin-injected tissues were fixed overnight at 4°C with 4% PFA and 1% glutaraldehyde (GA) in 0.067 M phosphate buffer  containing 1% saturated picric acid. After fixation, the brain was dissected and embedded in a gelatin/albumin mixture  and then post-fixed overnight at 4°C in 7–8% PFA in PB. The post-fixed tissues were sectioned in a frontal plane at a thickness of 40 μm with a vibrating blade microtome, then washed in PB, and incubated overnight at 4°C with streptavidin-horseradish peroxidase  in 0.01 M phosphate-buffered saline  containing 0.5% Triton X (PBST). The sections were developed with a solution of 0.032% 3,3’-diaminobenzidine tetrahydrochloride (DAB) in 0.1M Tris-HCl (pH 7.4) containing 0.0145% HP. americana brains. Preadsorption with 0.4 μM or more of the BSA-dopamine conjugate abolished immunoreactive signals.For anti-dopamine immunolabeling, a rabbit polyclonal antiserum against dopamine-bovine serum albumin (BSA)-GA conjugate (cat. No. AB122S) was purchased from Millipore  [For anti-tyrosine hydroxylase (TH) immunolabeling, a rabbit polyclonal antiserum raised against SDS-denatured rat TH (Cat. No. NB300-109) was purchased from Novus biologicals  .D. melanogaster synapsin  was used  [For immunolabeling neuropil structures, a mouse monoclonal antibody against was used . Synapsictively) .Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 4.5% stacking and 12% running gel. Cockroach or cricket brains were dissected out in PBS. After dissection, two brains were transferred into 50 μl of lysis buffer  containing 1.5 μl ß-mercaptoethanol , and immediately homogenized. The homogenate (containing two brains) was heated for 3 min at 100°C, transferred on ice and then centrifuged. The supernatant was subjected to SDS-PAGE and blotted on a Hybond-P PVDF membrane . As a molecular weight marker, Dr. Western  was used. After blocking the membrane with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20  for 30 min, it was incubated with the rabbit anti-tyrosine hydroxylase  in TBST for 2 h at 25°C. The membrane was rinsed in four changes of TBST, and incubated in donkey anti-rabbit IgG conjugated to HRP  in TBST for 90 min at 25°C. After being rinsed in four changes of TBST, the ECL Western Blotting Starter Kit  was used to visualize immunoreactivity.For dopamine immuno-labeling, brains were fixed with 2.5% GA and 1% sodium metabisulfite (SMB) in PB. For tyrosine hydroxylase immunolabeling, brains were fixed with 4% PFA in PB overnight at 4°C.The fixed tissues were embedded in a gelatin/albumin mixture, post-fixed, and sectioned as described above (0.5% SMB was added to the fixative for dopamine labeling). Non-specific binding sites were blocked with 5% normal goat serum (NGS) in 0.01 M PBST for 1 hour, and then incubated with respective primary antibodies at working dilutions in PBST containing 5% NGS listed in For dopamine and TH immunolabeling, we also applied an immunoperoxidase labeling method. Procedures prior to the secondary antibody incubation were as described above. After primary antibody incubation, the sections were incubated in goat anti-rabbit IgG conjugated to HRP  in PBST containing 5% NGS. After washing in PBST, sections were developed, mounted on glass slide, dehydrated, cleared and then mounted in Mount-Quick beneath a cover slip as described above.DAB-labeled and reduced silver stained preparations were imaged with a digital camera  mounted on a compound microscope . Several images at different focal planes were manually captured from single sections at intervals of 3–5 μm, and then superimposed into a single image with Zerene Stacker software .Sections labeled by immunofluorescence were imaged using a Zeiss LSM510 confocal microscope  equipped with Plan-Apochromat 20X/0.75 (Carl Zeiss). Alexa 488 was excited with an Ar laser at 488 nm and viewed through a 475-525-nm band-pass filter, and Cy3 was excited with a HeNe green laser at 543 nm through a 560-nm long pass filter. Confocal images were acquired at z-axis intervals of 1.5 to 2 μm and a resolution of 1,024 x 1,024 pixels. All confocal images are compressed from stacks of 10 to 15 optical sections. The size, contrast, and brightness of the images were adjusted using Photoshop CS4  and Corel Draw X4 .D. melanogaster brains [Immunoreactivity to an antiserum against dopamine was detected in all major neuropils of the cockroach brain except part of the central complex. In the brain, 15 different groups of cell clusters exhibit moderate to strong immunoreactivity . The numr brains . Both anP. americana consists of several divisions as in other insects, i.e. paired lobes , paired calyces , and a pedunculus,  immunoreactivity presented a fibrous appearance in the mushroom body. TH immunolabeling also provided moderate immunoreactive signals as in the dopamine immunolabeling. The locations of TH-positive cell clusters  are striTo confirm that the dopamine/TH-positive DCa1 neurons Figs  and 4J pP. americana. The terminology is based on that for the locust, Schistocerca gregaria [P. americana, the upper division covering the smaller lower division  of the  neurons . The numD. melanogaster TH [In addition to the anti-dopamine antiserum, we also employed an independent marker for dopaminergic neurons, a tyrosine hydroxylase (TH) antiserum. These two antisera immunolabeled almost the same groups of neurons with a notable discrepancy in the antennal lobe: local interneurons were immunolabeled by the dopamine antiserum  as reporaster TH . The conaster TH ,64. TheyP. americana contains ca. 200,000 intrinsic Kenyon cells per hemisphere [P. americana largely fall into three types (class I—III) [3 neurons) project their fibers to the vertical lobe [The mushroom body of the cockroach is substantially supplied with the dopamine-immunoreactive fibers, suggesting that dopamine engages in fundamental tasks of this neuropil area. The mushroom body of misphere . This ismisphere . The Kens I—III) ,67. Class I—III) . The Kens I—III) ,69. Seves I—III) ,33, and s I—III) ,36. Dends I—III) . The Kens I—III) ,53. Dopas I—III) –73. Likes I—III) , althougcal lobe . The PPLcal lobe ,77, cockcal lobe ,78. In tP. americana consists of the rod-like protocerebral bridge and a lip-like central body; its upper division covers the lower division, and both are connected to the ventro-laterally located lateral accessory lobe comprising dorsal and ventral shells (The central complex of the cockroach is innervated by numerous dopaminergic fibers, suggesting that dopamine plays important roles in this neuropil. The central complex is a conspicuous midline-spanning neuropil with higher-order visual inputs ,80. Thisl shells . The uppl shells , as impll shells . To visul shells , probablIn the fruit fly, a series of studies using mutants with structural defects in different compartments of the central complex and gene expression targeted by the Gal4/UAS system have revealed roles in visual spatial memory, and different areas of the central body are involved in different learning paradigms . So far,3 neurons in the honeybee [1 neurons in the honeybee [2 neurons in the honeybee [Comprehensive characterization of putatively dopaminergic neurons has been achieved in the honeybee , locust honeybee  and the honeybee ,75. Theihoneybee , DIP1 nehoneybee , PAM neuhoneybee ,12, and honeybee , and DIPhoneybee  may alsohoneybee , DP2 neuhoneybee , and PPMhoneybee ,12.On the other hand, there are considerable species-specific differences in the antennal lobe. In the cockroach and honeybee, either local interneurons or deutocerebral neurons ,24 innerDopamine’s actions in the cockroach nervous system are probably diverse, ranging from a role as a neurotransmitter and as a neuromodulator, as implied in this account as well as the previous ones ,11,87. ID. melanogaster. However, the dynamics of reinforcement is still poorly understood at the single cell level. The cockroach may be an ideal species to monitor the dynamic range of plasticity of single neurons because unlike D. melanogaster they are quite amenable to intracellular electrophysiological recording methods. The DCa1 neurons that are anatomically equivalent to the fruit fly’s PPL1 neurons are probably the most promising candidate to address physiologically the role of dopaminergic systems in the brain, in particular, the neural mechanism underlying aversive reinforcement.In many insects so far studied, dopamine provides an aversive value to sensory stimuli such as odor, color and visual patterns that are neutral before they are associated with aversion ,6,74,76.


Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.The yellow scorpion The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman’s degradation and tested on macrophages.The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity. Tityus serrulatus venom (Tsv) is composed of insoluble mucus, neurotoxic proteins that affect sodium or potassium channels, bioactive amines, hypotensins, proteinases, hyaluronidases, a bradykinin-potentiating peptide, a kallikrein inhibitor, allergenic proteins and other peptides whose biological functions are still not known [ot known . It is eot known .+-channel toxins (NaTx) and short-chain K+-channel toxins (KTx) [Neurotoxins are the most studied components of Tsv because of their interactions with ionic channels in excitable membranes and their role in the envenoming . Tsv neuns (KTx) .The family of potassium channels is comprised of the largest number of ion channels subtypes with high structural and functional diversities . These c+ blockers with two α-helices stabilized by two disulfide bonds [KTx are classified into four families: α, toxins constituted by 23-43 amino acids linked by 3-4 disulfide bonds; β, long peptides (~60 amino acid residues) stabilized by three disulfide bonds; γ, ether-a-go-go (ERG) channel blockers with 36-47 amino acid residues connected by 3 or 4 disulfide bonds; and κ, poor Kde bonds . Among tde bonds .+ and Na+ channels can affect many cells, such as macrophages, which participate in the inflammatory response of Ts envenoming [2 and LTB4 through TLR2 and TLR4 stimulation and peroxisome proliferator-activated receptor gamma (PPAR-γ) activation [Scorpion venoms and their isolated toxins are responsible for several immunological properties  observed after scorpion envenoming –11. Neurvenoming , 13. Intvenoming . Furthervenoming . Tsv alstivation .Until now, only the effects of few Ts toxins – namely of Ts1, Ts2, Ts5 and Ts6 – have been evaluated for macrophage activation –19.Tityus serrulatus venom. The major eluted peaks were analyzed by MALDI-TOF mass spectrometry and had their N-terminal sequence determined by Edman degradation. Additionally, the effect of a new β-KTx – Ts19 Frag-I, Ts4 and Ts3-KS were investigated for their cytotoxicity and cytokines and NO production on macrophages.Therefore, the present work purified the components present in the fractions VIIIA and VIIIB from et al. [Tsv was provided by the vivarium at the School of Medicine of Ribeirão Preto, University of São Paulo, Brazil, after extraction by the electrical stimulation method using 12 mV . Desiccaet al. . The fraThe amino acid residues of the N-terminal region from the eluted subfractions were sequenced by Edman degradation  on an auThe eluted subfractions were submitted to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry . The mass spectra were obtained in the linear positive mode after the equipment was calibrated with a standard peptide . Samples were resuspended in a solution of 80 % (V/V) acetonitrile (ACN) and 0.2 % (V/V) TFA and combined at the ratio 1:1 with 5 mg/mL 2,5-dihydroxybenzoic acid (DHB) matrix.The macrophage cell line J774.1 was obtained from the American Type Culture Collection . The cells were grown, total number of cells was counted, viability was determined and cells were plated, as previously described .et al. [The toxins (50 μg/mL) isolated from fractions VIIIA and VIIIB were incubated with the J774.1 macrophage line cells for 24 h. Then, the cell viability was evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich) , as descet al. . The asset al. .2−) in the supernatants was measured, at 540 nm, as an indicator of NO production according to the Griess method [The amount of nitrite  using specific antibodies and quantified at 405 nm, as previously described . The senp < 0.05 were considered statistically significant.Data are expressed as mean ± standard error of mean (SEM) and were analyzed using one-way ANOVA. Values of The fractions VIIIA and VIIIB, eluted from the cation exchange chromatography of Tsv, present low resolution in this chromatographic step (data not shown). For this reason, to isolate the toxins, these fractions were submitted to reversed-phase fast protein liquid chromatography (RP-FPLC) on a C18 column  , Ts19 Frag-I (peak 9.3) and Ts4 (peak 11) at 50 μg/mL was analyzed by MTT assay. We demonstrated that these toxins did not affect J774.1 cell viability when compared to non-stimulated cells   did not induce NO production when compared to non-stimulated cells (control). However, cells stimulated with peak 9.3  induced NO production by J774.1 cells (p < 0.05)  to stimulate macrophages was investigated through the production of cytokines. Ts3-KS was only tested for IL-6 production because of the low sample quantity. Ts4, Ts3-KS and peak 9.3 at 50 μg/mL induced IL-6 production  presents features of β-NaTx but with α-like activity [Ts2 , containing the sequence Gly-Lys-Lys in the C-terminal region, is processed by carboxypeptidases that remove the Lys residues. The remaining Gly-extended peptide is converted into a des-Gly peptide amine by an α-amidating enzyme to produce a serine-amide in its C-terminal end , herein + channels in synaptosome preparations [Ts8  was the first described member of β-KTx subfamily and was characterized as a selective blocker of voltage-gated non-inactivation Karations . Its matarations .Additionally, Ts4 , was the main toxin eluted from the fraction VIIIB, although it is also present in a high proportion in the fraction VIIIA. Ts4 causes allergic reaction, lachrymation, spasm of the hind legs in mice and dose dependent neurotransmitter release .2), an α-KTx from Androctonus australis hector scorpion venom, induces severe alterations in hepatic and pancreatic tissues by the activation of the inflammatory response with release of IL-6 and TNF-α [The α-KTx Ts6 induced NO and IL-6 production and inhibited the release of TNF-α . Kaliotond TNF-α . HoweverIn 2008, 27 amino acids residues of a new β-Ktx-like toxin from Tsv were identified by peptidomic analysis, whose precursor, known as Ts19, was determined through a transcriptomic study of the Ts venom gland , 34. PosT. stigmurus and TtrKIK from T. trivittatus and with Ts19, which presents a Leu in the C-terminal, we deduced that the amino acid residue to complete the entire sequence from Ts19 Frag-I is Leu. These 58 amino acid residues were submitted to ProtParam, a tool that predicted the pI 8.57. The composition of Ts19 Frag-I contains a high content of Lys residues, which explains the predicted basic isoeletric point. A similar result was observed experimentally with Ts15 [The corresponding molecular mass of the 57 amino acid residues of oxidized monoisotopic toxin (S-S) Ts19 Frag-I (peak 9.3) sequenced through Edman degradation was calculated as 6,458 Da. The average molecular mass of the same peak was determined as 6,575 Da through MALDI-TOF mass spectrometry, linear mode. The difference between these masses corresponds to the amino acid residue (Leu or Ile) of the C-terminal region. Since the Ts19 Frag-I shares high identity with the β-KTx-like toxins TstKMK from ith Ts15 . The theThe Ts19 Frag-I presents nine additional amino acid residues in the N-terminal region when compared with Ts19 Frag-II. Interestingly, the N-terminal region of Ts19 Frag-I starts with the amino acid residues KIK. Other toxins that have KIK in their N-terminal region showed cytolytic, antimicrobial and hemolytic activities , 8. The The peak 9.3 is constituted mainly (63.7 %) by Ts19 Frag-I  and by peptides of 6,985.2 Da and 7,441.5 Da, whose N-terminal sequences corresponded to Ts2 and Ts3-KS, respectively. The respective theoretical molecular masses of oxidized monoisotopic (S-S) Ts2 and Ts3-KS calculated by the Sequence Editor are 6,985 Da and 7,442 Da [The N-terminal of the peak 7.4 identified the toxin Ts3-KS. Its oxidized monoisotopic (S-S) molecular mass corresponds to 7,442 Da  while thThe toxins Ts3-KS (peak 7.4), peak 9.3 (Ts19 Frag-I) and Ts4 (peak 11) did not affect macrophage viability. In relation to cytokine modulation in macrophages, all tested toxins stimulated IL-6 production, although Ts3-KS proved to be the most potent stimulus. However, Ts3-KS and peak 9.3 did not change TNF-α production. Based on the peak 9.3 components , we eliminate the Ts2 participation in the peak stimulus since Ts2 is a potent inductor of TNF-α release even with low concentration (25 μg/mL) . MoreoveThe cytokines IL-6, IL-1, and TNF-α are elevated in most inflammatory states and have been recognized as targets of therapeutic intervention . On the In relation to IL-1β, the significant inhibition of this cytokine by Ts4 and peak 9.3 is highly interesting. In fact, Ts4 was considered non-toxic to mice due to its inability to induce the characteristics symptoms of toxicity produced by other scorpion toxins . However+ channels, they belong to different classes: α-KTx and β-KTx to Ts6 and Ts19 Frag-I, respectively [Finally, the NO release induced by peak 9.3 was highly groundbreaking. Ts6 toxin was the only known Ts toxin capable of stimulating this mediator release . Althougectively . Based oectively .Based on the literature, high NO levels in the serum or in peritoneal macrophage culture supernatants may be associated with such severe conditions as septic shock, hypertension and severe envenoming , 42. ThuThe toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide and Ts19 Frag-II, and a novel partially purified putative β-KTx, denominated Ts19 Frag-I, were isolated from the fractions VIIIA and VIIIB from Ts venom and analyzed through MALDI-TOF mass spectrometry and Edman degradation. The toxins Ts4, Ts3-KS and Ts19 Frag-I induce the release of IL-6 and do not show cytolytic activity. Additionally, Ts19 Frag-I induces the release of NO in macrophage cells. These results may contribute to elucidating not only the knowledge of macrophage immunomodulation after scorpion envenoming but also to the inflammatory actions of Ts toxins.


Dopaminergic fibers originating from area A11 of the hypothalamus project to different levels of the spinal cord and represent the major source of dopamine. In addition, tyrosine hydroxylase, the rate-limiting enzyme for the synthesis of catecholamines, is expressed in 8-10% of dorsal root ganglia (DRG) neurons, suggesting that dopamine may be released in the dorsal root ganglia. Dopamine has been shown to modulate calcium current in DRG neurons, but the effects of dopamine on sodium current and on the firing properties of small DRG neurons are poorly understood.The effects of dopamine and dopamine receptor agonists were tested on the tetrodotoxin-resistant (TTX-R) sodium current recorded from acutely dissociated small (diameter ≤ 25 μm) DRG neurons. Dopamine (20 μM) and SKF 81297 (10 μM) caused inhibition of TTX-R sodium current in small DRG neurons by 23% and 37%, respectively. In contrast, quinpirole (20 μM) had no effects on the TTX-R sodium current. Inhibition by SKF 81297 of the TTX-R sodium current was not affected when the protein kinase A (PKA) activity was blocked with the PKA inhibitory peptide (6–22), but was greatly reduced when the protein kinase C (PKC) activity was blocked with the PKC inhibitory peptide (19–36), suggesting that activation of D1/D5 dopamine receptors is linked to PKC activity. Expression of D1and D5 dopamine receptors in small DRG neurons, but not D2 dopamine receptors, was confirmed by Western blotting and immunofluorescence analysis. In current clamp experiments, the number of action potentials elicited in small DRG neurons by current injection was reduced by ~ 30% by SKF 81297.We conclude that activation of D1/D5 dopamine receptors inhibits TTX-R sodium current in unmyelinated nociceptive neurons and dampens their intrinsic excitability by reducing the number of action potentials in response to stimulus. Increasing or decreasing levels of dopamine in the dorsal root ganglia may serve to adjust the sensitivity of nociceptors to noxious stimuli. V1.1, NaV1.2, NaV1.6, and NaV1.7) and TTX-R (NaV1.8 and NaV1.9) voltage-dependent sodium channels , the inhibitory effect of 10 μM SKF 81297 on the TTX-R sodium current was substantially affected. In collected results Figure C,D, the We next tested whether D1/D5 dopamine receptors inhibition of TTX-R sodium channels is mediated by changes in the voltage-dependence of activation or inactivation of the channels. The steady-state activation curve had a midpoint of −15.1 ± 4.2 mV and a slope of 5.0 ± 1.8 mV (n = 17) in control  sensitive to capsaicin were included in the study, and among those about 88% were also IB4(+). All these features suggest that the dopamine-sensitive neurons correspond to small nociceptive neurons -70.Western blot and immunofluorescence analysis confirmed the expression of D1 and D5 dopamine receptors in dorsal root ganglia, but no evidence was found for the expression of D2 dopamine receptors. These results were consistent with those obtained with electrophysiology in which the D2 agonist quinpirole had little effect. Using polymerase chain reaction (PCR) and DNA sequencing, transcripts of D1-D5 dopamine receptors, including D2 dopamine receptors, have been reported in dorsal root ganglia ,31. A poIn most DRG neurons, the inhibitory effects of dopamine or SKF 81297 on TTX-R sodium current required many minutes to develop fully. In most of the neurons tested, the inhibitory effect of the drug did not reach a steady-state even after 15 min. This is slower than the inhibitory effect of dopamine on TTX-S sodium current in hippocampal or prefrontal cortex neurons ,59, but V1.8 channels in DRG neurons with increases in current amplitude and changes in the kinetic properties ), where V is the step membrane potential in millivolts, V1/2 is the half-maximal voltage in millivolts, and k is the slope factor in millivolts. Sodium channels availability was determined by using 500 msec prepulses from −120 to + 30 mV followed by a test pulse to −10 mV. The test pulse current is normalized to its maximal value. Solid lines are best fits to the Boltzmann function: 1/(1 + exp[(V-V1/2)/k]), where V is the prepulse membrane potential, V1/2 is the half-maximal voltage, and k is the slope factor in millivolts. In current clamp experiments, reported voltages were corrected for the – 8 mV junction potential between the potassium methanesulfonate-based internal solution and the Tyrode’s solution present when zeroing pipette current. The junction potential was measured using a flowing 3 M KCl bridge [Currents and voltages were controlled and sampled using a Digidata 1440 A interface and pClamp 10 software . Current or voltage signals were filtered at 10 kHz  and digitized at 50 kHz. Analysis was performed using pClamp 10 and Igor Pro , using DataAccess  to import pClamp files in Igor. Small DRG neurons (diameter 18 to 25 μm) were initially selected by measuring the diameter from images captured to computer by a CCD camera Oly-150  using a video acquisition card . A more accurate measurement of cell diameter was obtained from measurements of whole-cell capacitance assuming a membrane capacitance of 1 μF/cml bridge . Action l bridge . UsuallyDRG: Dorsal root ganglia; TTX: Tetrodotoxin; TTX-R: Tetrodotoxin-resistant; TTX-S: Tetrodotoxin-sensitive; TH: Tyrosine hydroxylase; EEDQ: Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.The authors declare that they have no competing interests.WG performed research and analyzed data. ES analyzed data. MR performed research and analyzed data. MP designed research, performed research, analyzed data, and wrote the paper. All authors read and approved the final manuscript.


A convenient and inherently more secure communication channel for encoding messages via specifically designed molecular keys is introduced by combining advanced encryption standard cryptography with molecular steganography. The necessary molecular keys require large structural diversity, thus suggesting the application of multicomponent reactions. Herein, the Ugi four-component reaction of perfluorinated acids is utilized to establish an exemplary database consisting of 130 commercially available components. Considering all permutations, this combinatorial approach can unambiguously provide 500,000 molecular keys in only one synthetic procedure per key. The molecular keys are transferred nondigitally and concealed by either adsorption onto paper, coffee, tea or sugar as well as by dissolution in a perfume or in blood. Re-isolation and purification from these disguises is simplified by the perfluorinated sidechains of the molecular keys. High resolution tandem mass spectrometry can unequivocally determine the molecular structure and thus the identity of the key for a subsequent decryption of an encoded message. Designing molecular keys and combining advanced encryption standard cryptography with molecular steganography is a secure way for encoding messages. Here, the authors use the Ugi four-component reaction of perfluorinated acids to create a library of 500,000 molecular keys for encryption and decryption. Although ultimate security will never be reached, a constant progress and improvement of cryptography and steganography, both evolving with the needs and possibilities of new technologies on the one hand and the steady increase of the capabilities of adversaries on the other hand, is required. A core task of cryptography is encryption, i.e., to convert messages into unintelligible ciphertexts that can only be decrypted by a receiver who possesses a dedicated decryption key. Steganography, in contrast, hides the fact that a message (encrypted or not) is being transmitted. For a higher degree of security, decryption keys can be concealed by steganography, i.e., via chemicals4. Today, more sophisticated methods are discussed for data encryption, such as fluorescent materials11 or multi-analyte fluorescent molecular sensors14. Furthermore, DNA was exploited for secure communication19. Molecular logic gates25, molecular computing systems28, and systems based on authorizing password entries38 also contributed to this field. Other approaches utilized NMR chemical shifts39, microorganism colonies40, antibodies41, 3D photonic crystals42, and molecular tags  serving as barcodes for the identification of chemical libraries via gas chromatography43.The idea to use chemicals for secret communication dates back to ancient times when secret inks were first applied44, Serpent, or Blowfish can protect data reliably, but require a secret key for their operation. This secret key must be known by both the encryptor and the decryptor, and should be chosen uniformly at random. Typically, secret keys are short , but it is not a priori clear how encryptor and decryptor receive common secret keys. Popular methods to distribute secret keys involve the use of asymmetric cryptography . The disadvantage of using asymmetric cryptography (besides being considerably less efficient than symmetric cryptography) is that state-of-the-art asymmetric encryption schemes require specific algebraic structures, and thus may potentially be more prone to a structured cryptanalysis.Considering cryptography, state-of-the-art symmetric encryption schemes, like the Advanced Encryption Standard However, hiding of the secret key via steganography  can introduce an additional level of security. Even if the number of known chemical compounds is tremendously large and steadily growing, their application as molecular keys is limited considering certain requirements: First, a systematic synthesis methodology should be applied providing molecules with a high level of structural complexity. Moreover, simple, robust, and reproducible procedures should be used. Finally, the products should be thermally and chemically as inert as possible and be designed for simple isolation, purification, and analysis.46. In the field of MCRs, the Ugi reaction was appealing, because four components are combined to a single product in a straightforward one-pot reaction47. In the Ugi reaction . Variation of the precursor components creates molecular diversity with minimal synthetic effort50.Multicomponent reactions (MCR) represent highly suitable synthetic tools to provide molecular keys, fulfilling these criteria. In a MCR, three or more precursor components are combined to one reaction product, containing moieties of all precursors1), via a secret communication channel based on molecular keys, which can be easily hidden in various media, transferred nondigitally1, isolated via F-tags and unambiguously read out via ESI-MS/MS . In our system, the well-established AES algorithm is combined with an effective hiding and transportation of the encryption key. The latter consists of systematically selected and tailor-made molecules that can be transported in a concealed fashion via a non-digital channel.Herein we report a means to advance chemical communication systems , enabling a highly simplified purification via fluorous solid phase extraction (F-SPE)In Fig. Our molecular keys provide a robust steganographic channel that can be used to transport, i.e., cryptographic keys. The transmitted keys can hardly be recognized, because (i) an adversary does not know that a key is hidden in a molecule; (ii) only the recipient knows where the molecular key is located/stored ; (iii) information on how to extract and analyze the molecule, convert the chemical structure into digital information and apply the decryption is necessary; and (iv) since the keys are transferred physically, an attacker cannot rely on computing power alone.55. The user can thus choose the appropriate system for the required application. Importantly, the molecular keys are designed for simplified purification utilizing fluorous tags, as well as for straightforward characterization (tandem-MS), requiring only trace amounts of the key at all stages.Data storage capacity is admittedly much higher for DNA than for synthetic molecules, which might change in the near future with the development of sequence defined macromoleculesAn exemplary way of key transmission is presented in Fig. The encryption of secret documents/files , the molecular key was purified via F-SPE, further demonstrating the major advantage of the herein used F-tagged molecular keys. The molecular key was obtained in excellent purity and sufficient quantity for subsequent analysis (see Methods section). Alternatively, the molecular key was concealed and transported in a commercial perfume, adsorbed onto instant coffee powder, green tea, or on sugar  are determined. In mathematic terms, a four-dimensional problem is solved considering four different parameters. The four parameters are obtained via four different molar masses from high resolution tandem mass spectrometry (the monoisotopic mass + three fragments). Tandem mass spectrometry (tandem-MS) can be applied universally, minimal sample preparation is necessary and the detection limits are very small, thus requiring only trace amounts of substance to synthesize, transport and obtain the information. In Fig. m/z of 595.1615 Da for [M + Na]+. After entering this mass into the analysis script  are displayed in a distribution plot  should be avoided due to undesired side reactions . Since the molecular keys are designed for tandem mass spectrometric analysis, the dataset for one component  should not contain isomers . Isotope labeled components can be beneficial for MS purposes, but were also excluded from our database. Diastereomeric mixtures of molecular keys synthesized from racemic starting components can be treated and utilized similar to stereomerically pure molecular keys  was dissolved in a minimal amount of methanol, subsequently the amine (1.70 eq.) was added and the resulting mixture was stirred for 60 min over sodium sulfate for the imine formation . The perfluoro acid (1.00 eq.) was dissolved in a minimal amount of methanol and added to the imine at room temperature. The resulting mixture was stirred for 2 min. Subsequently, the isocyanide (1.70 eq.) was added to the stirring mixture. The reaction was stirred for 1–3 d at room temperature. If a precipitate was formed during the reaction, additional methanol was added. If a long perfluorinated chain was present, tetrahydrofuran was utilized in order to homogenize the reaction mixture. The crude reaction mixture was dried under reduced pressure and purified via column chromatography . Typical yields: 20–85%.In a 10  mL round bottom flask, the aldehyde  was dissolved in a minimal amount of methanol. Subsequently, the amine (1.30 eq.) was added and the resulting mixture was stirred for 60 min over sodium sulfate. The perfluoro acid (1.00 eq.) was dissolved in a minimal amount of methanol and added to the imine at room temperature. The resulting mixture was stirred for 2 min. Subsequently, the isocyanide (1.20 eq.) was added to the stirring mixture. The reaction was stirred for 3 d at room temperature. The crude reaction mixture was dried under reduced pressure and purified via column chromatography . Typical yields: 2%–35%. The lower yields compared to GP1 are most probably due a larger excess of components and higher concentrations favoring product formation for GP1.For F-SPE separation, we suggest to utilize 1 g of perfluorinated silica gel per 50 mg of mixture subjected (5% by weight). However, in literature higher loadings of 5%–15% by weight are reported.: Flush the perfluorinated silica gel with dimethylformamide (0.2 mL per g silica gel), gently applying positive pressure.Step 1Step 2: Precondition the column with methanol/water (8:2) (3 mL per g silica gel).: Suspend the mixture to be purified in a minimal amount of dimethylformamide and load onto the preconditioned column.Step 3: Elute the organic fraction with 2 mL methanol/water (80:20) per g silica gel. We suggest fractionating into tubes of appropriate size.Step 4: Elute with 4 mL methanol per g silica gel to collect the fluorous fraction. Note: For long chain perfluorinated compounds, additionally elute with the same amount of tetrahydrofuran/acetone (1:1). Again, fractionating into tubes is desirable. TLC of the fractions indicates the complete elution of the desired compound.Step 5: In order to recycle the F-SPE silica gel, flush with acetone and tetrahydrofuran and air dry. We do not suggest reusing the same silica gel more than three times.Step 6c-hexane/ethyl acetate (3:1) was performed to yield the desired perfluorinated Ugi product.After separation, the fractions were tested for purity via TLC, combined and concentrated under reduced pressure. If unreacted perfluoro acid or residual dimethylformamide were present in the fluorous fraction, a short silica gel filter column, eluting with Note: For purifying the molecular keys extracted from the various disguises 5 g of F-SPE silica gel were utilized.The molecular keys can be hidden in many creative ways. Since the molecular keys are flouro-tagged compounds, they can easily be isolated from various types of surrounding media. The main requirement to the surrounding environment or the matrix material is the absence of any perfluorinated alkyl chains. Furthermore, the extraction of the molecular key should be possible with reasonable effort.The following extraction examples were performed with the molecular key A(005)-B(002)-C(004)-D(007).Adsorbed onto paper: 4.0 mg of the molecular key were dissolved in 0.5 mL methanol and subsequently transferred dropwise onto the top right corner of an envelope. Intermediately, the drops were quickly evaporated by gently blow-drying with a heat gun. For further protection and disguising, the stamp was placed just above the covered area , from green tea 3.6 mg (90%), and from sugar 3.4 mg (87%), of the molecular key were recovered.Adsorbed onto instant coffee powder, tea, or sugar: 4.0 mg of the molecular key were dissolved in 0.5 mL dichloromethane and added dropwise onto 1 g of the substrate powder, each drop was placed onto a different spot and allowed to evaporate separately. For the extraction, the powder was ground with a mortar and stirred three times in 5 mL dichloromethane for 3 min. The combined extracts were and filtered over celiteNote: For the extraction from sugar no further purification via F-SPE was necessary (fully water-soluble substrate).From blood: 5.0 mg of the molecular key were dissolved in 0.5 mL ethanol and concentrated under reduced pressure until a transparent film of the molecular key with a minimized amount of ethanol was obtained. 5 mL of pig blood were added into the vial and gently stirred for 5 min. The blood should be stored and transported under cooling. For the extraction, the blood was diluted with 10 mL water and extracted with 20 mL dichloromethane three times . The organic extracts were washed separately with 10 mL water twice. The organic phases were dried over sodium sulfate (emulsion broke upon drying), evaporated under reduced pressure, and purified via F-SPE. In this protocol, 4.5 mg (91%) of the molecular key were recovered.Note: The extraction from blood was intended to demonstrate a challenging extraction example for the herein presented F-tagged molecular keys from complex biological media. Extraction from biological media would be far more challenging for cryptographic keys encoded in DNA or other biomolecules. The authors also want to clearly state that it is unethical to transport molecular keys in living organisms.For MS analysis, even smaller amounts of substance are required so in principle a lower concentration of the molecular key could be employed. However, the herein obtained mass of several milligrams were sufficient to further conduct TLC, GC-MS and NMR experiments for purity determination. GC-MS evidenced a purity of 99%, see Supplementary Figure If the molecular key is adsorbed on a solid support, we propose to extract with dichloromethane several times (at least three) in order to fully recover the molecular key. Subsequently, the dichloromethane extract is washed with water and purified via F-SPE. If the molecular key is transported in solution, we propose concentration, and liquid/liquid extraction with dichloromethane and water. The combined organic layers are dried prior to F-SPE.m/z), which is used in the computer assisted readout  and successively move on to higher fragmentation energy levels . However, the chosen energy should be as low as possible in order to avoid the further fragmentation of fragments. Furthermore, the observation of heavier/larger fragment species is more likely for lower fragmentation energies. In Supplementary Figure The next step towards the analysis of a molecular key is to isolate the [M + Na]m/z) recorded at 35 eV (a) and at 50 eV (b) collision energy are displayed. The heavier fragments indicated by the diamond cluster and asterisk can only be observed in the 35 eV energy spectrum . The lighter fragments indicated by the black circle and left-pointing triangle can solely be observed in the 50 eV spectrum (b). The corresponding fragment assignment is depicted in the corresponding table of Supplementary Figure However, in some cases, two tandem-MS spectra at different fragmentation energies are necessary to observe enough relevant fragments. In Supplementary Figure In addition, tandem-MS fragment analysis can distinguish between isomers (molecules with same mass but different structures). In Supplementary Figure m/z  = O.The analysis of tandem-MS spectra at different energies turned out to be time consuming. At this point, a computer script . The resulting possibilities are listed by the script and the masses of probable fragments are directly displayed. After entering additional fragments, the resulting possibilities are further refined until the structure is determined. The fragment masses are entered with two decimals . If accidentally a wrong number is entered, the REDO command will return to the initial selection of possibilities after entering the [M + Na]+ mass. After entering enough fragments, the selection of possibilities is reduced to one distinct molecule. With the alphanumerical information of the key  was further confirmed by GC-MS -C1H NMR spectrum (a) split signals can be observed for the protons on position CH1 and NH6. The signal between 5 and 6 ppm with a relative integral of two does not couple to other protons, as indicated in the COSY experiment (b). The HSQC experiment  correlations) indicates two cross signals and hence two carbon bound species (CH1) in the respective area. The HMBC spectrum  was utilized to further confirm the molecular structure. The 19F stacked spectrum g of the molecular key (bottom of g) displays two new AB signals (originating from the CF214 group next to the newly formed amide bond), if compared to the precursor.All substances synthesized in the context of this work were characterized via 1D and 2D NMR. Representative spectra for a molecular key [A(005)-B(002)-C(004)-D(007)] are displayed in Supplementary Figure All relevant data is included as supplementary information and is also available from the corresponding author.Availability of computer codes: All relevant scripts are included as supplementary information. The analysis script is provided in Supplementary Software Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Software 1Supplementary Software 2