Functional gene groups are concentrated within chromosomes,among chromosomes and in the nuclear space of the human genome

Genomes undergo changes in organization as a result of gene duplications, chromosomal rearrangements and local mutations, among other mechanisms. In contrast to prokaryotes, in which genes of a common function are often organized in operons and reside contiguously along the genome, most eukaryotes show much weaker clustering of genes by function, except for few concrete functional groups. We set out to check systematically if there is a relation between gene function and gene organization in the human genome. We test this question for three types of functional groups: pairs of interacting proteins, complexes and pathways. We find a significant concentration of functional groups both in terms of their distance within the same chromosome and in terms of their dispersal over several chromosomes. Moreover, using Hi-C contact map of the tendency of chromosomal segments to appear close in the 3D space of the nucleus, we show that members of the same functional group that reside on distinct chromosomes tend to co-localize in space. The result holds for all three types of functional groups that we tested. Hence, the human genome shows substantial concentration of functional groups within chromosomes and across chromosomes in space.     

Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Subject terms: Necroptosis, Cell death and immune response     

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background

Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown.

Methodology/Principal Findings

We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice.

Conclusions/Significance

TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.     

Translocation from nuclei to cytoplasm is necessary for anti A‐PCD activity and turnover of the Type II IAP BcBir1

Type II inhibitors of apoptosis (IAPs) belong to a subgroup of IAP‐related proteins. While IAPs are restricted to animals, Type II IAPs are found in other phyla, including fungi. BcBir1, a Type II IAP from Botrytis cinerea has anti apoptotic‐like programmed cell death (A‐PCD) activity, which is important for pathogenicity of this fungus. Here we report on the role of sub‐cellular localization of BcBir1 in protein turnover and anti A‐PCD activity. Expression of BcBir1 in Saccharomyces cerevisiae had no effect on sensitivity of the yeast cells to A‐PCD‐inducing conditions, whereas expression of a truncated N' part reduced sensitivity of the cells to these conditions. The full‐length BcBir1 protein was detected only in the yeast nucleus, whereas the N' part was observed both in the nucleus and cytoplasm. In B. cinerea, BcBir1 was mainly nuclear under optimal conditions, whereas under A‐PCD‐inducing conditions it shuttled to the cytoplasm and then it was completely degraded. Collectively, our results show that anti A‐PCD activity of BcBir1 occurs in the cytoplasm, the C′ end mediates regulation of steady state level of BcBir1 in the nucleus, and the N' end mediates anti A‐PCD activity as well as fast degradation of BcBir1 in the cytoplasm.     

The GPSM2/LGN GoLoco motifs are essential for hearing

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn ^ΔC mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn ^ΔC allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.     

Probing the active site of reconstituted human placental aromatase [Poster 02]

To gain some insight into the factors that determine active site-ligand binding and to delineate which sites of the androstene nucleus are critical for recognition by the aromatase enzyme, we compared the effect of various substituents located at the biologically important C-2, 6, 11, 16, 17, and 19 positions. Based on two independent measurements, namely optical difference spectra and inhibition of enzyme activity, we report: (a) oxygen functions at positions C-11 do not appear to induce difference spectra although they cause weak inhibition; (b) 2ß- and 16-hydroxyandrostenes induce type-I spectra and weak inhibition; (c) C-6 and C-19-substituted androgens are type-I spectrum inducers and strong inhibitors; (d) the presence of nonspecific binding sites on the aromatase enzyme cannot be ruled out.     

Lipid and insulin levels in obese children: changes with age and puberty

Objective: The goal was to describe the lipid profile and insulin changes seen in obese children and adolescents at different stages of puberty. Research Methods and Procedures: A cross‐sectional study was conducted by chart review of 181 obese (BMI > 95th) children and adolescents 5 to 17 years of age, who were referred to the Center for Atherosclerosis Prevention for cardiovascular risk reduction from January 2003 through December 2003. Results: Eighty (44.2%) subjects were <12 years of age, and 101 (55.8%) were ≥12 years. Severity of obesity as expressed by BMI standard deviation score did not differ between these age groups. A significant difference with lower serum levels of total cholesterol, non‐high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, and high‐density lipoprotein cholesterol was seen with older age and with advancing sexual maturity rating. Triglycerides, very‐low‐density lipoprotein cholesterol, and lipoprotein(a) levels remained elevated across age and pubertal stages. Insulin levels and insulin resistance as expressed by homeostasis model assessment were significantly higher with older age. Similar trends were observed both in obese boys and obese girls during puberty. Discussion: The most striking findings of this study are that in the 5‐ to 17‐year‐old obese population, the combination of elevated triglycerides and very‐low‐density lipoprotein cholesterol and low high‐density lipoprotein cholesterol levels place them at greater cardiovascular risk than their non‐obese peers, even when the changing patterns of lipids and lipoproteins seen during pubertal maturation are accounted for.