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Detecting and monitoring illegal harvesting pressure on wild populations is challenging due to the cryptic nature of poaching activities. Although change in population age structure has been suggested as an indicator of harvesting pressure, few studies have tested its validity when based on short-term field surveys. Using data from rapid demographic assessment surveys carried out in 2009 at six sites in Tanzania, we examined whether African elephant populations experiencing contrasting levels of poaching pressure showed significant differences in their age structure, operational sex ratio (i.e. adult males to adult females), dependent individual to adult female ratio at the group level, and proportion of tuskless individuals. We also compared similar metrics between the population sampled in Ruaha National Park in 2009 and again in 2015 following a suspected increase in poaching. Elephant populations experiencing medium and high levels of poaching in 2009 were characterised by fewer calves and old individuals, a reduced number of adult males relative to adult females, and a lower ratio of calves to adult females within groups. We also found a higher proportion of tuskless individuals in poached populations (>6%). Changes in age structure in the Ruaha population between 2009 and 15 were similar to those observed across sites in 2009. Our findings are consistent with previous work documenting how the loss of older individuals – targeted for their larger tusks – decreases recruitment and survival of elephant calves. Illegal killing for ivory is a huge threat to the survival of African elephants. In this context, the present study contributes towards validating the use of age structure as an indicator of poaching pressure in elephant populations, but also in other wildlife populations where illegal offtake is targeted at specific age classes.

Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy., Cytokine-induced signaling acts as an ON/OFF switch dependent on the presence of ligands. Here the authors construct synthetic cytokine receptors responsive to synthetic ligands able to activate canonical signaling pathways.

Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating that the level of endogenous sgp130 in the bloodstream is not sufficient to block IL-6 trans-signaling via sIL-6R. Importantly, addition of the single-domain antibody VHH6, which specifically stabilizes IL-6·sIL-6R complexes but did not bind to IL-6 or sIL-6R alone, drove free IL-6 into IL-6·sIL-6R complexes and boosted trans-signaling but not classic signaling, demonstrating that endogenous sIL-6R has at least the potential to form complexes with IL-6. Our findings indicate that even though high concentrations of sIL-6R and sgp130 are present in human serum, the relative ratio of free IL-6 to IL-6·sIL-6R allows for simultaneous classic and trans-signaling.

Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E134/S135) and IL-11 (G116/S117) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitut...

Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic “cargo internalization receptors” (CIRs) for tracking of individual cell populations by 1H/19F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For 19F MR visibility, the GFP carrier was equipped with “contrast cargo”, in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1–3). Exposure of CIR+ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence micros...

Interleukin-6 (IL-6) is a proinflammatory cytokine of the IL-6 family, members of which signal through a complex of a cytokine-specific receptor and the signal-transducing subunit gp130. The interaction of IL-6 with the membrane-bound IL-6 receptor (IL-6R) and gp130 stimulates "classic signaling," whereas the binding of IL-6 and a soluble version of the IL-6R to gp130 stimulates "trans-signaling." Alternatively, "cluster signaling" occurs when membrane-bound IL-6:IL-6R complexes on transmitter cells activate gp130 receptors on neighboring receiver cells. The soluble form of gp130 (sgp130) is a selective trans-signaling inhibitor, but it does not affect classic signaling. We demonstrated that the interaction of soluble gp130 with natural and synthetic membrane-bound IL-6:IL-6R complexes inhibited IL-6 cluster signaling. Similarly, IL-11 cluster signaling through the IL-11R to gp130 was also inhibited by soluble gp130. However, autocrine classic and trans-signaling was not inhibited by extracellular inhibitors such as sgp130 or gp130 antibodies. Together, our results suggest that autocrine IL-6 signaling may occur intracellularly.

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.

Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E

Interleukin (IL)-6-deficient, but not IL-6 receptor (IL-6R)-deficient mice present with a delayed skin wound healing phenotype. Since IL-6 solely signals via the IL-6R and glycoprotein 130 (gp130), Il-6r-deficient mice are expected to exhibit a similar phenotype as Il-6-deficient mice. However, p28 (IL-30) and ciliary neurotrophic factor (CNTF) have been identified as additional low-affinity ligands of the IL-6R/gp130/LIFR complex. IL-6 plays an inflammatory and regenerative role in inflammatory bowel disease (IBD). In the present study, we compared Il-6r-deficient mice with mice treated with neutralizing IL-6 monoclonal antibody (mAb) in a model of dextran sodium sulfate (DSS)-induced colitis. Our results, in agreement with those of previous reports, demonstrated that IL-6 mAbs slightly attenuated DSS-induced colitis during the regeneration phase. Il-6r-deficient mice and mice with tissue-specific deletion of the Il-6r in the myeloid cell lineage (LysMCre) with acute and chronic DSS-induced colitis were, however, indistinguishable from wild-type mice. Our data suggest that IL-6 and IL-6R have an additional role in colitis, apart from the IL-6/IL-6R classic and trans-signaling.

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) is coordinated by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The soluble IL-6R is mainly generated by ADAM10- and ADAM17-mediated ectodomain shedding. Little is known about the role of the 52-amino acid-residue-long IL-6R stalk region in shedding and signal transduction. Therefore, we generated and analyzed IL-6R stalk region deletion variants for cleavability and biological activity. Deletion of 10 amino acids of the stalk region surrounding the ADAM17 cleavage site substantially blocked IL-6R proteolysis by ADAM17 but only slightly affected proteolysis by ADAM10. Interestingly, additional deletion of the remaining five juxtamembrane-located amino acids also abrogated ADAM10-mediated IL-6R shedding. Larger deletions within the stalk region, that do not necessarily include the ADAM17 cleavage site, also reduced ADAM10 and ADAM17-mediated IL-6R shedding, questioning the importance of cleavage site recognition. Furthermore, we show that a 22-amino acid-long stalk region is minimally required for IL-6 classic signaling. The gp130 cytokine binding sites are separated from the plasma membrane by ~96 Å. 22 amino acid residues, however, span maximally 83.6 Å (3.8 Å/amino acid), indicating that the three juxtamembrane fibronectin domains of gp130 are not necessarily elongated but somehow flexed to allow IL-6 classic signaling. Our findings underline a dual role of the IL-6R stalk region in IL-6 signaling. In IL-6 trans-signaling, it regulates proper proteolysis by ADAM10 and ADAM17. In IL-6 classic-signaling, it acts as a spacer to ensure IL-6·IL-6R·gp130 signal complex formation.

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.

The local-loop bypass issue - where businesses with heavy local communication requirements establish their own exchange networks, bypassing the local telephone company - is well known and has major implications for local telephone operating companies (LOCs). Although cost is usually the primary reason in any bypass decision, other factors including specific service requirements play a significant role. This article describes the special service needs of electricity supply industries (ESIs) and some of the reasons for their desire to reduce their reliance on LOCs. The authors suggest that because of increasing competitive pressures, the information and communication needs of the ESIs will grow substantially, further reinforcing the desire for independent metropolitan area communication networks. A prototype of such a network, NetComm, is described.

Interleukin (IL-)6 trans-signaling via the soluble IL-6 receptor (sIL-6R) activates all cells of the body, whereas Interleukin (IL-)6 classic signaling via the membrane-bound IL-6R is limited mainly to hepatocytes and lymphocytes. Accordingly, the phenotype of IL-6:sIL-6R expressing transgenic mice is much severe as compared to single IL-6 transgenic mice. Due to occurrence of free IL-6, classic signaling is also activated during simultaneous expression of IL-6 and sIL-6R complicating the background-free analysis of IL-6 trans-signaling in vivo. IL-6 is physically trapped in Hyper-IL-6, the fusion protein of IL-6 and the sIL-6R, and specifically induces only IL-6 trans-signaling. Interestingly, Hyper-IL-6 transgenic mice have not been described to date, likely due to toxicity issues which prevents establishment of Hyper-IL-6 transgenic mice. Split-intein mediated protein ligation enables the production of toxic proteins. Here, we efficiently adapted this technology to protein ligation of the designer cytokine Hyper-IL-6 (I-H-IL-6) within the cellular secretory compartment using gp41-1 and IMPDH split-inteins. The presence of non-ligated IL-6 did, however, still enable induction of IL-6 classic signaling. To prevent the production of non-ligated biologically active IL-6, biologically inactive IL-6 split-cytokines were developed using structure-guided modeling. Biologically inactive split-IL-6 variants were combined with split-intein mediated protein ligation, resulting in background-free generation of Hyper-IL-6 (Split2-H-IL-6) in vitro and in vivo. The split2-cytokine protein ligation was applied for a second Hyper-cytokine and therefore likely represents a general strategy to generate highly potent and background-free composite Hyper-cytokines in vitro and in vivo.

Cosmopolitans like Gillian Brock, Charles Beitz, and Thomas Pogge argue that the principles of justice selected and arranged in lexical priority in Rawls’ first original position would—and should for the same reasons as in the first—also be selected in the second original position. After all, the argument goes, what reasons other than morally arbitrary ones do we have for selecting a second set of principles? A different, though undoubtedly related, point of contention is the cosmopolitan charge (most famously, made by Pogge) that Rawls fails to consider the unfavorable conditions that owe themselves to global factors. Perhaps there was a time when interconnectedness and interdependency between states was not a factor; but in the current global order, this certainly is not the case. While this paper will address other related cosmopolitan concerns mentioned in Brock’s work, it is these two points that are perhaps the two biggest threats to the Rawlsian project and, as such, it is these two points that will be the primary focus of this paper.

The report discusses fabrication of high technology electronics equipment, particularly digitally based systems, by specifying, designing and fabricating equipments on a fully automated basis. A hypothetical universal machine is proposed, operating under computer netted control, combining developments in the automated design, numerical control, and integrated circuit packaging fields. The intended results include computer-engraved PC boards, computer modified equipment, automatic testing, engineering design change drawings, computer repair and maintenance, and computer-made, fully assembled printed circuits at remote locations.

The report discusses technical and economic feasibility of widespread computer-netting technology to aid DOD procurement effectiveness, and describes procurement automation system development, the software and hardware required, and provides a hardware configuration. Next steps are logically detailed in a procurement automation system. Cost-benefit analysis and a survey of procurement automation systems presently in use is provided. It is indicated that savings in excess of $1 billion per year might be achievable in the future.

Since we do not know of any completely workable solutions to the problems we have considered, we shall settle for a few general observations: 1) The public availability of socially useful computer communications services is and has been held back by legal battles that are now under way between the potential suppliers. 2) No simple resolution of these issues in the near future seems likely in view of the past conceptual separation of computers and communications doctrines. 3) The current policy is to determine whether the nation shall or shall not have certain computer communications services, by the adversary process. In this process, often only the voices of the loudest adversary suppliers are heard. 4) Although there can be no certainty that better alternatives cannot be devised, we believe that such a possibility assumes a higher probability if the key actors come from the technical community sectors more representative of the future consumers. 5) If we are to have the new services that are possible, we need an approach that makes better use of the technologists' dreams and goals rather than have future prospects excessively bound by lawyers paid to preserve the interests of their clients, irrespective of any secondary consequences. 6) We cannot be sanguine about this possibility, as technological statesmanship is too easily corrupted by the same forces that have placed us in this predicament. Furthermore, even if not corrupted, beneficial cooperation can too readily be regarded as simply collusion. 7) Although we do not have any clear answer, we do know that present approaches are not taking us where we want to go very rapidly and that alternative approaches should at least be considered.