Living phosphatic stromatolites in a low‐phosphorus environment: Implications for the use of phosphorus as a proxy for phosphate levels in paleo‐systems

In the geological record, fossil phosphatic stromatolites date back to the Great Oxidation Event in the Paleoproterozoic, but living phosphatic stromatolites have not been described previously. Here, we report on cyanobacterial stromatolites in a supratidal freshwater environment at Cape Recife, South African southern coast, precipitating Ca carbonate alternating with episodes of Ca phosphate deposition. In their structure and composition, the living stromatolites from Cape Recife closely resemble their fossilized analogues, showing phosphatic zonation, microbial casts, tunnel structures and phosphatic crusts of biogenic origin. The microbial communities appear to be also similar to those proposed to have formed fossil phosphatic stromatolites. Phosphatic domains in the material from Cape Recife are spatially and texturally associated with carbonate precipitates, but form distinct entities separated by sharp boundaries. Electron Probe Micro‐Analysis shows that Ca/P ratios and the overall chemical compositions of phosphatic precipitates are in the range of octacalcium phosphate, amorphous tricalcium phosphate and apatite. The coincidence in time of the emergence of phosphatic stromatolites in the fossil record with a major episode of atmospheric oxidation led to the assumption that at times of increased oxygen release the underlying increased biological production may have been linked to elevated phosphorus availability. The stromatolites at Cape Recife, however, form in an environment where ambient phosphorus concentrations do not exceed 0.28 μM, one to two orders of magnitude below the previously predicted minimum threshold of >5 μM for biogenic phosphate precipitation in paleo‐systems. Accordingly, we contest the previously proposed suitability of phosphatic stromatolites as a proxy for high ambient phosphate concentrations in supratidal to shallow ocean settings in earth history.     

Mesoderm-inducing factors: a small class of molecules

Mesoderm-inducing factors (MIF's) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs-the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other. The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13,000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6.7 (WEHI) and 7.7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose. All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used. Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiations factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Genetic structure of cherry fruit fly (Rhagoletis cingulata) populations across managed,unmanaged, and natural habitats

The cherry fruit fly (CFF), Rhagoletis cingulata Loew (Diptera: Tephritidae: Trypetini), is endemic to eastern North America and Mexico, where its primary native host is black cherry [Prunus serotina Ehrh. (Rosaceae)]. Cherry fruit fly is also a major economic pest of the fruit of cultivated sweet (Prunus avium L.) and tart (Prunus cerasus L.) cherries. Adult CFF that attack wild black cherry and introduced, domesticated cherries in commercial and abandoned orchards are active at different times of the summer, potentially generating allochronic isolation that could genetically differentiate native from sweet and tart CFF populations. Here, we test for host‐related genetic differences among CFF populations in Michigan attacking cherries in managed, unmanaged, and native habitats by scoring flies for 10 microsatellite loci. Little evidence for genetic differentiation was found across the three habitats or between the northern and southern Michigan CFF populations surveyed in the study. Local gene flow between native black cherry, commercial, and abandoned orchards may therefore be sufficient to overcome seasonal differences in adult CFF activity and prevent differentiation for microsatellites not directly associated with (tightly linked to) genes affecting eclosion time. The results do not support the existence of host‐associated races in CFF and imply that flies attacking native, managed, and unmanaged cherries should be considered to represent a single population for pest management purposes.     

Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection

Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.     

Evaluation of organic insecticides for management of spotted‐wing drosophila (Drosophila suzukii) in berry crops

Spotted‐wing drosophila, Drosophila suzukii (Matsumura), is an invasive pest affecting fruit production in many regions of the world. Insecticides are the primary tactic for controlling D. suzukii in organic as well as conventional production systems. Organic growers have a greater challenge because fewer insecticides are approved for use in organic agriculture. The most effective organically approved product is spinosad, but alternatives are needed because of label restrictions limiting the number of applications per year, toxicity to beneficial arthropods and the risk of developing resistance. We evaluated several organically approved insecticides against D. suzukii in laboratory assays and field trials conducted on organic blueberry and raspberry farms. Spinosad was consistently the most effective insecticide, but a few other insecticides such as azadirachtin + pyrethrins, Chromobacterium subtsugae and sabadilla alkaloids showed moderate activity. None of the treatments had long residual activity. Mortality started to decline by 3 days after treatment, and by 5 days after application, the treatments were not different from the controls. These products may be useful in rotation programmes, necessary for reducing reliance on spinosad and mitigating resistance. Cultural and biological control approaches are needed in fruit production for D. suzukii management, but insecticides will likely continue to be the dominant management tactic while these other approaches are being optimized and adopted.     

Designer TGFβ superfamily ligands with diversified functionality

Transforming Growth Factor--beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.