Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation.

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring.

Nicotine use remains highly prevalent with tobacco and e-cigarette products consumed worldwide. However, increasing evidence of transgenerational epigenetic inheritance suggests that nicotine use may alter behavior and neurobiology in subsequent generations. We tested the effects of chronic paternal nicotine exposure in C57BL6/J mice on fear conditioning in F1 and F2 offspring, as well as conditioned fear extinction and spontaneous recovery, nicotine self-administration, hippocampal cholinergic functioning, RNA expression, and DNA methylation in F1 offspring. Paternal nicotine exposure was associated with enhanced contextual and cued fear conditioning and spontaneous recovery of extinguished fear memories. Further, nicotine reinforcement was reduced in nicotine-sired mice, as assessed in a self-administration paradigm. These behavioral phenotypes were coupled with altered response to nicotine, upregulated hippocampal nicotinic acetylcholine receptor binding, reduced evoked hippocampal cholinergic currents, and altered methylation and expression of hippocampal genes related to neural development and plasticity. Gene expression analysis suggests multigenerational effects on broader gene networks potentially involved in neuroplasticity and mental disorders. The changes in fear conditioning similarly suggest phenotypes analogous to anxiety disorders similar to post-traumatic stress.

Metformin Affects the Transcriptomic Profile of Chicken Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecological malignancy in women. Metformin intake is associated with a reduced incidence of ovarian cancer and increased overall survival rate. We determined the effect of metformin on sphere formation, extracellular matrix invasion, and transcriptome profile of ovarian cancer cells (COVCAR) isolated from ascites of chickens that naturally developed ovarian cancer. We found that metformin treatment significantly decreased sphere formation and invasiveness of COVCAR cells. RNA-Seq data analysis revealed 0, 4, 365 differentially expressed genes in cells treated with 0.5, 1, 2 mM metformin, respectively compared to controls. Transcriptomic and ingenuity pathway analysis (IPA) revealed significant downregulation of MMP7, AICDA, GDPD2, APOC3, APOA1 and predicted inhibition of upstream regulators NFKB, STAT3, TP53 that are involved in epithelial-mesenchymal transition, DNA repair, and lipid metabolism. The analysis revealed significant upregulation of RASD2, IHH, CRABP-1 and predicted activation of upstream regulators VEGF and E2F1 that are associated with angiogenesis and cell cycle. Causal network analysis revealed novel pathways suggesting predicted inhibition of ovarian cancer through master regulator ASCL1 and dataset genes DCX, SEMA6B, HEY2, and KCNIP2. In summary, advanced pathway analysis in IPA revealed novel target genes, upstream regulators, and pathways affected by metformin treatment of COVCAR cells.

Caldichromatium japonicum gen. nov., sp. nov., a novel thermophilic phototrophic purple sulphur bacterium of the Chromatiaceae isolated from Nakabusa hot springs, Japan.

A novel thermophilic phototrophic purple sulphur bacterium was isolated from microbial mats (56 °C) at Nakabusa hot springs, Nagano prefecture, Japan. Cells were motile, rod-shaped, stain Gram-negative and stored sulphur globules intracellularly. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were the major pigments. Dense liquid cultures were red in colour. Strain No.7T was able to grow photoautotrophically using sulfide, thiosulfate, sulfite and hydrogen (in the presence of sulfide) as electron donors and bicarbonate as the sole carbon source. Optimum growth occurred under anaerobic conditions in the light at 50 °C (range, 40-56 °C) and pH 7.2 (range, pH 7-8). Major fatty acids were C16 : 0 (46.8 %), C16 : 1 ω7c (19.9 %), C18 : 1 ω7c (21.1 %), C14 : 0 (4.6 %) and C18 : 0 (2.4 %). The polar lipid profile showed phosphatidylglycerol and unidentified aminophospholipids to be the major lipids. The only quinone detected was ubiquinone-8. 16S rRNA gene sequence comparisons indicated that the novel bacterium is only distantly related to Thermochromatium tepidum with a nucleotide identity of 90.4 %. The phylogenetic analysis supported the high novelty of strain No.7T with a long-branching phylogenetic position within the Chromatiaceae next to Thermochromatium tepidum. The genome comprised a circular chromosome of 2.99 Mbp (2 989 870 bp), included no plasmids and had a DNA G+C content of 61.2 mol%. Polyphasic taxonomic analyses of the isolate suggested strain No.7T is a novel genus within the Chromatiaceae. The proposed genus name of the second truly thermophilic purple sulphur bacterium is Caldichromatium gen. nov. with the type species Caldichromatium japonicum sp. nov. (DSM 110881=JCM 39101).

The influence of adolescent nicotine exposure on ethanol intake and brain gene expression.

Nicotine and alcohol are often co-abused. Adolescence is a vulnerable period for the initiation of both nicotine and alcohol use, which can lead to subsequent neurodevelopmental and behavioral alterations. It is possible that during this vulnerable period, use of one drug leads to neurobiological alterations that affect subsequent consumption of the other drug. The aim of the present study was to determine the effect of nicotine exposure during adolescence on ethanol intake, and the effect of these substances on brain gene expression. Forty-three adolescent female C57BL/6J mice were assigned to four groups. In the first phase of the experiment, adolescent mice (PND 36-41 days) were exposed to three bottles filled with water or nicotine (200 µg/ml) for 22 h a day and a single bottle of water 2 h a day for six days. In the second phase (PND 42-45 days), the 4-day Drinking-in-the-Dark paradigm consisting of access to 20% v/v ethanol or water for 2h or 4h (the last day) was overlaid during the time when the mice did not have nicotine available. Ethanol consumption (g/kg) and blood ethanol concentrations (BEC, mg %) were measured on the final day and whole brains including the cerebellum, were dissected for RNA sequencing. Differentially expressed genes (DEG) were detected with CuffDiff and gene networks were built using WGCNA. Prior nicotine exposure increased ethanol consumption and resulting BEC. Significant DEG and biological pathways found in the group exposed to both nicotine and ethanol included genes important in stress-related neuropeptide signaling, hypothalamic-pituitary-adrenal (HPA) axis activity, glutamate release, GABA signaling, and dopamine release. These results replicate our earlier findings that nicotine exposure during adolescence increases ethanol consumption and extends this work by examining gene expression differences which could mediate these behavioral effects.

Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes.

Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. The pathophysiological roles and clinical value of EVs are under intense investigation, yet most studies are limited by technical challenges in the isolation of nanoscale EVs (nEVs). Here, we report a lipid nanoprobe that enables spontaneous labelling and magnetic enrichment of nEVs in 15 minutes, with isolation efficiency and cargo composition similar to what can be achieved by the much slower and bulkier method of ultracentrifugation. We also show that the lipid nanoprobes, which allow for downstream analyses of nucleic acids and proteins, enabled the identification of EGFR and KRAS mutations following nEV isolation from blood plasma from non-small-cell lung-cancer patients. The efficiency and versatility of the lipid nanoprobe opens up opportunities in point-of-care cancer diagnostics.

Divergent Ah Receptor Ligand Selectivity during Hominin Evolution.

We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150-1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins.

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.

Origin and evolution of the cystic fibrosis transmembrane regulator protein R domain.

The Cystic Fibrosis Transmembrane Conductance Regulator protein (CFTR) is a member of the ABC transporter superfamily. CFTR is distinguished from all other members of this superfamily by its status as an ion channel as well as the presence of its unique regulatory (R) domain. We investigated the origin and subsequent evolution of the R domain along the CFTR evolutionary lineage. The R domain protein coding sequence originated via the loss of a splice donor site at the 3' end of exon 14, leading to the subsequent read-through and capture of formerly intronic sequence as novel coding sequence. Inclusion of the remaining part of the R domain coding sequence in the CFTR transcript involved a lineage-specific gain of exonic sequence with no homology to protein coding sequences outside of CFTR and loss of two exons conserved among ABC family members. These events occurred at the base of the Gnathostome evolutionary lineage ~550-650 million years ago. The apparent origination of the R domain de novo from previously non-coding sequence is consistent with its lack of sequence similarity to other domains as well as its intrinsically disordered structure, which has important implications for its function. In particular, this lack of structure may provide for a dynamic and inducible regulatory activity based on transient physical interactions with more structured domains of the protein. Since its acquisition along the CFTR evolutionary lineage, the R domain has evolved more rapidly than any other CFTR domain; however, there is no evidence for positive (adaptive) selection in the evolution of the domain. The R domain does show a distinct pattern of relative evolutionary rates compared to other CFTR domains, which sheds additional light on the connection between its function and evolution. The regulatory function of the R domain is dependent upon a fairly small number of sites that are subject to phosphorylation, and these sites were fixed very early in R domain evolution and have remained largely invariant since that time. In contrast, the rest of the R domain has been free to drift in sequence space leading to a more star-like phylogeny than seen for the other CFTR domains. The case of the R domain suggests that domain acquisition via the de novo creation of coding sequence, and the novel functional utility that such an event would seemingly entail, can be one route by which neo-functionalization is favored to occur.

Transposable element distribution, abundance and role in genome size variation in the genus Oryza.

BACKGROUND: The genus Oryza is composed of 10 distinct genome types, 6 diploid and 4 polyploid, and includes the world's most important food crop - rice (Oryza sativa [AA]). Genome size variation in the Oryza is more than 3-fold and ranges from 357 Mbp in Oryza glaberrima [AA] to 1283 Mbp in the polyploid Oryza ridleyi [HHJJ]. Because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from 12 representative Oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. Particular attention was paid to the role played by the most important classes of transposable elements (Long Terminal Repeats Retrotransposons, Long interspersed Nuclear Elements, helitrons, DNA transposable elements) in shaping these genomes and in their contributing to genome size variation. RESULTS: We identified the elements primarily responsible for the most strikingly genome size variation in Oryza. We demonstrated how Long Terminal Repeat retrotransposons belonging to the same families have proliferated to very different extents in various species. We also showed that the pool of Long Terminal Repeat Retrotransposons is substantially conserved and ubiquitous throughout the Oryza and so its origin is ancient and its existence predates the speciation events that originated the genus. Finally we described the peculiar behavior of repeats in the species Oryza coarctata [HHKK] whose placement in the Oryza genus is controversial. CONCLUSION: Long Terminal Repeat retrotransposons are the major component of the Oryza genomes analyzed and, along with polyploidization, are the most important contributors to the genome size variation across the Oryza genus. Two families of Ty3-gypsy elements (RIRE2 and Atlantys) account for a significant portion of the genome size variations present in the Oryza genus.