Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Solution structure of termite-derived antimicrobial peptide,spinigerin, as determined in SDS micelle by NMR spectroscopy

Spinigerin is a linear antibacterial peptide derived from a termite insect. It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria, but has no hemolytic activities against human erythrocytes. In this study, we present a three-dimensional solution structure of spinigerin in SDS micelles. According to CD data spinigerin has an alpha-helical conformation in the presence of TFE, DPC micelles, and SDS micelles. The three-dimensional structure of spinigerin as determined by NMR spectroscopy contains a stable alpha-helix from Lys4 to Thr23. Spinigerin (4-21), an 18-residue fragment from Lys4 to Leu21, contains a similar content of alpha-helical structure compared to native spinigerin and was found to retain antibacterial activity, too. Therefore, this alpha-helical structure and the strong electrostatic attraction between four Lys and three Arg residues in spinigerin and the negatively charged polar head groups of the phospholipids on the membrane surface play important roles in disrupting membrane and subsequent cell death.     

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

Controlled activity of mouse astrocytes on electrospun PCL nanofiber containing polysaccharides from brown seaweed

The central nervous system (CNS), once injured, rarely recovers original function mainly due to its limited regeneration ability. Astrocytes are cells that play critical roles in neural regeneration. Several biomaterials have been studied to replace and regenerate lost tissues within injured CNS. Seaweeds have extracellular polymeric substances (EPS) with bioactive properties such as antiviral and antioxidant properties. In this study, astrocyte activity was assessed, after being cultured on an electrospun polycaprolactone (PCL) nanofibrous mat containing a brown seaweed EPS. Laminarin and fucoidan, two main components of EPS extract from the brown seaweed, were concluded to increase or decrease astrocyte activity with respect to their concentration. When the concentration was under 10 ??g/ml, the astrocytes tended to increase their viability. In contrast, over 10 ??g/ml EPS in media suppressed the viability of astrocytes. In addition, when contained in PCL nanofiber, the EPS extract was also proven to influence astrocyte activity in the same way as the case when astrocytes were exposed to EPS in solution. This implies that the brown seaweed EPS?CPCL nanofiber mat can be used for temporal control of astrocyte activity by EPS concentration. Through this research, we propose that the electrospun EPS?CPCL nanofiber could be used as a nanomedicine or scaffold to treat CNS injuries.     

LC–MS/MS-based metabolome analysis detected changes in the metabolic profiles of small and large intestinal adenomatous polyps in <Emphasis Type="Italic">Apc</Emphasis><Superscript><Emphasis Type="Italic">Min</Emphasis>/+</Superscript> mice

Introduction

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is inactivated in the initiation of colorectal neoplasia. Apc ^Min/+ mice, which possess a heterozygous APC mutation, develop numerous adenomatous polyps, which are similar to those observed in familial adenomatous polyposis (FAP) in humans. However, unlike FAP patients, Apc ^Min/+ mice predominantly develop adenomatous polyps in the small intestine. The metabolic changes associated with the development of polyps in the small and large intestine remain to be investigated.

Objectives

The objective of this study was to elucidate the metabolic changes associated with intestinal polyp formation.

Methods

We compared the metabolite levels of pairs of polyp and non-polyp tissues obtained from the small intestines (n = 12) or large intestines (n = 7) of Apc ^Min/+ mice. To do this, we analyzed the tissue samples using two methods, liquid chromatography-tandem mass spectrometry (1) with a pentafluorophenylpropyl column for cation analysis, and (2) with a C18 reversed phase column coupled to an ion-pair reagent for anion analysis.

Results

Pathway mapping of the metabolites whose levels were significantly altered revealed that the polyp tissue of the small intestine contained significantly higher levels of intermediates involved in glycolysis, the pentose phosphate pathway, nucleotide metabolism, or glutathione biosynthesis than in the equivalent non-polyp tissue. In addition, significantly higher levels of methionine cycle intermediates were detected in the polyp tissues of both the large and small intestines. Organ-dependent (small vs. large intestine) differences were also detected in the levels of most amino acids and urea cycle intermediates.

Conclusion

Our results indicate that various metabolic changes are associated with polyp development, and understanding these alterations could make it possible to evaluate the treatment response of colorectal cancer earlier.

     

An 8 kb Nucleotide Sequence at the 3' Flanking Region of the sspC Gene (184{degrees}) on the Bacillus subtilis 168 Chromosome Containing an Intein and an Intron

As part of the Bacillus subtilis genome sequencing project,we determined the complete nucleotide sequence of an 8000-bpfragment downstream of the sspC gene (184°) of the B. subtilis168 chromosome. The sequence analysis shows that the sspC geneis located inside of the SPß region, which differsfrom the current genetic map of B. subtilis 168. This regioncontains 12 putative ORFs (yojQ through yojZ and sspC). A homologysearch for the deduced products of the ORFs shows signi.cantsimilarities to enzymes involved in deoxyribonucleotide metabolism:ribonucleotide reductase (Nrd) E, NrdF, thioredoxinand dUTPase.Interestingly, this DNA fragment includes two split genes, yojPcontaining conserved motifs of an intein and yojQ and yojS withan 808-bp intervening sequence for a putative intron structure.In addition, the yojR gene includes a putative new DNA replicationterminator.     

Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-beta stimulation of human lung fibroblast contraction of collagenous matrix and induction of alpha-SMA and the role of alpha-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels +/- -TGF-beta1. TGF-beta1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-beta1 upregulated alpha-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. Alpha-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-beta1, suggesting alpha-SMA is required for gel contraction. Thus, Smad3 mediates TGF-beta1-induced contraction and alpha-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-beta1.