Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.     

Recruitment of protein phosphatase 2A to dorsal ruffles by platelet-derived growth factor in smooth muscle cells: Dephosphorylation of Hsp27

In this study, we investigated the mechanism underlying Hsp27 dephosphorylation in smooth muscle cells. We found that protein phosphatase 2A (PP2A) dephosphorylates Hsp27. In addition, Hsp27 dephosphorylation was regulated by membrane cholesterol content. We showed that PDGF induced a three-fold increase in the proportion of PP2A activity regulated by cholesterol in the Triton-insoluble fraction of cell lysates. Moreover, cholesterol depletion decreased the amount of PP2A recovered in Triton-insoluble fraction. Thus, PDGF might regulate a small pool of PP2A associated with lipid rafts. Isolation of detergent-resistant membrane fragments by Optiprep-gradient density indicated that this pool of PP2A was not associated with caveolae, but was recovered in a higher density fraction (DRM-H) with ganglioside GM1, α-actinin, Hsp27 and p34, a component of Arp2/3 complex. These proteins were also present in dorsal ruffles containing GM1 but not caveolin-1. Phosphorylated Hsp27 levels detected in dorsal ruffles were variable. Cholesterol depletion, which inhibits dorsal ruffle formation, decreased PP2A levels and increased the Hsp27-P to Hsp27 ratio in DRM-H. These findings suggest that Hsp27 is dephosphorylated by PP2A in dorsal ruffles, in non-caveolar lipid raft microdomains. However, similarly to p34, non-phosphorylated Hsp27 is associated to non-raft membrane domains at the leading edge of lamellipodia.     

Hydroxychloroquine decreases human MSC‐derived osteoblast differentiation and mineralization in vitro

We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC‐derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ‐treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC‐derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up‐regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ‐treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC‐derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC‐derived osteoblast. The discrepancy between high BMD and decreased MSC‐derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.     

Declines in moth populations stress the need for conserving dark nights

Given the global continuous rise, artificial light at night is often considered a driving force behind moth population declines. Although negative effects on individuals have been shown, there is no evidence for effects on population sizes to date. Therefore, we compared population trends of Dutch macromoth fauna over the period 1985–2015 between moth species that differ in phototaxis and adult circadian rhythm. We found that moth species that show positive phototaxis or are nocturnally active have stronger negative population trends than species that are not attracted to light or are diurnal species. Our results indicate that artificial light at night is an important factor in explaining declines in moth populations in regions with high artificial night sky brightness. Our study supports efforts to reduce the impacts of artificial light at night by promoting lamps that do not attract insects and reduce overall levels of illumination in rural areas to reverse declines of moth populations.     

Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2

Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD–2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors.     

Growth of harmful marine algae in multispecies cultures

Mixtures of harmful and harmless algae were grown in discontinuouslydiluted batch cultures under ammonium, nitrate and phosphatelimitation, and at different irradiances (20–500 µjnolquanta m^–2 s^–1). The species used were Chrysochromulinapolylepis, Emiliania huxleyi type B, Rhodomonas sp., the dinoflagellalesFibrocapsa japonica, Gymnodinium simplex, Gyrodinium aure-olum,Heterocapsa triquetra, Heterosigma carterae, Prorocentrum micansand Alexandrium tamarense, the diatoms Chaetoceros socialis,Cymatosira belgica, Ditylum brightwellii, Laudcria borealis,Odon-telta aunla, Pseudonitzschia pungens, Streptotheca tamesis,and the cyanobacterium Synechococcus. Their growth responsein the mixed algal cultures is discussed in relation to theirabundance in different natural habitats. In comparison withthe other non-diatoms, the mixotrophic C.polylepis grew fastunder all tested nutrient and light limitations.Emiliania huxleyigrew well under nitrogen (N) limitation (with nitrate as N source)and at irradiance levels from 15 up to 500 µmol quantam^–2 s^–1. No growth of calcifying cells could bedetected under N limitation when ammonium was used as N source.Rhodomonas grew reasonably well under ammonium-N limitationand grew fast at the highest irradi-ance. The dinoflagellateswere poor competitors compared to the Prymnesiophyceae. Theenvironmental fitness of the Prymnesiophyceae appears to beclosely related to the reproductive capacity of the vegetativestage, whereas the natural distribution of dinoflagellates seemsmore closely dependent on the generative reproduction-relatedspecific life cycle characteristics of the individual species.The marine diatoms include a mixture of both types of species.Some marine diatom species clearly have the capability to outcompetenon-diatoms under different types of nutrient and light limitationswhen silicate is in excess. Other diatoms seem to be poor competitors.     

Effect of oxygen on growth and respiration of potato tuber callus

Growth and oxygen uptake of potato callus is faster in an oxygen-enriched atmosphere (70% oxygen, v/v; oxygen-callus) than in air (20% oxygen, v/v; air-callus). Especially the non-mitochondrial, so-called residual respiration is increased in oxygen-callus. The capacities of the mitochondrial respiratory pathways (cytochrome pathway, V_cyt and alternative pathway, V_alt) are also higher in this callus. In both callus types only a small part of the alternative pathway capacity is used in uninhibited respiration. The lower oxygen uptake of air-callus at normal air oxygen pressures is partially due to diffusional impedance. Measurement of the respiratory parameters of air-callus in oxygen-saturated medium leads to higher values than measurement in air-saturated medium, although these values are still lower than those of oxygen-callus.ATP-production was calculated from the oxygen-uptake data and compared with the dry weight production of the callus to give values of 10.0 and 10.8 g dry weight produced.-mol ATP^-1, for air-callus and oxygen-callus respectively. As no harmful side-effects are observed, cultivation of callus under elevated oxygen pressures may be useful, when rapid callus-growth is necessary.Abbreviations AA antimycin A; A; - BHAM benzohydroxamate - DW dry weight - FW fresh weight - 8-OHQ 8-hydroxyquinolin - RC respiratory control - SHAM salicylhydroxamate     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.     

Apolipoprotein gene cluster on chromosome 19

Summary Recently, using an APOE cDNA probe, we discovered an Hpa I restriction fragment length polymorphism (RFLP) that appeared to be strongly associated with the expression of type III hyperlipoproteinemia (Klasen et al. 1987). In the present report it is shown that the same Hpa I RFLP can be revealed with both the APOC1 and APOC2 cDNA probes. This enabled us to localize the polymorphic Hpa I site between the APOE and APOC1 genes and to localize the APOC2 gene approximately 22 kb 3 of the APOC1 pseudogene on chromosome 19.