Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.     

The activity of the plant mitochondrial inner membrane anion channel (PIMAC) of maize populations divergently selected for cold tolerance level is differentially dependent on the growth temperature of seedlings

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.     

Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.     

Geographic differences in genetic susceptibility to IgA nephropathy: GWAS replication study and geospatial risk analysis

IgA nephropathy (IgAN), major cause of kidney failure worldwide, is common in Asians, moderately prevalent in Europeans, and rare in Africans. It is not known if these differences represent variation in genes, environment, or ascertainment. In a recent GWAS, we localized five IgAN susceptibility loci on Chr.6p21 (HLA-DQB1/DRB1, PSMB9/TAP1, and DPA1/DPB2 loci), Chr.1q32 (CFHR3/R1 locus), and Chr.22q12 (HORMAD2 locus). These IgAN loci are associated with risk of other immune-mediated disorders such as type I diabetes, multiple sclerosis, or inflammatory bowel disease. We tested association of these loci in eight new independent cohorts of Asian, European, and African-American ancestry (N = 4,789), followed by meta-analysis with risk-score modeling in 12 cohorts (N = 10,755) and geospatial analysis in 85 world populations. Four susceptibility loci robustly replicated and all five loci were genome-wide significant in the combined cohort (P = 5×10⁻³²–3×10⁻¹⁰), with heterogeneity detected only at the PSMB9/TAP1 locus (I² = 0.60). Conditional analyses identified two new independent risk alleles within the HLA-DQB1/DRB1 locus, defining multiple risk and protective haplotypes within this interval. We also detected a significant genetic interaction, whereby the odds ratio for the HORMAD2 protective allele was reversed in homozygotes for a CFHR3/R1 deletion (P = 2.5×10⁻⁴). A seven–SNP genetic risk score, which explained 4.7% of overall IgAN risk, increased sharply with Eastward and Northward distance from Africa (r = 0.30, P = 3×10⁻¹²⁸). This model paralleled the known East–West gradient in disease risk. Moreover, the prediction of a South–North axis was confirmed by registry data showing that the prevalence of IgAN–attributable kidney failure is increased in Northern Europe, similar to multiple sclerosis and type I diabetes. Variation at IgAN susceptibility loci correlates with differences in disease prevalence among world populations. These findings inform genetic, biological, and epidemiological investigations of IgAN and permit cross-comparison with other complex traits that share genetic risk loci and geographic patterns with IgAN.     

CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N‐ and C‐terminal domains suggests an evolutionary model involving a plasmid‐mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer–Villiger monooxygenase, allowed the recombinant bacteria to grow on long‐ and medium‐chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium‐chain alkanes overlapping AlkB and in the first step of sub‐terminal oxidation of long‐chain alkanes. It was also demonstrated that CYP116B5 is a self‐sufficient cytochrome P450 consisting of a heme domain (aa 1–392) involved in the oxidation step of n‐alkanes degradation, and its reductase domain (aa 444–758) comprising the NADPH‐, FMN‐ and [2Fe2S]‐binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).