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Background Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis. Methods Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively. Results A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts. Conclusions The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis. [ABSTRACT FROM AUTHOR]

Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in realtime as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox. [ABSTRACT FROM AUTHOR]

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells. [ABSTRACT FROM AUTHOR]

Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from a male individual, it was inferred that this is a genome that switched from maternal to paternal transmission. Here we provide clear evidence that this genome is the maternal genome of M. trossulus. We have found that in this genome the tRNAGln in the coding region is apparently defective and that an intact copy of this RNA occurs in the CR, that one of the two conserved domains is missing essential motifs, and that one of the two first variable domains has a high rate of divergence. These features may explain the large size and mosaic structure of the CR of the maternal genome of M. trossulus. We have also obtained CR sequences of the maternal and paternal genomes of M. californianus, a more distantly related species. We compare the control regions from all three species, focusing on the divergence among genomes of different species origin and among genomes of different transmission routes. [ABSTRACT FROM AUTHOR]

Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site’s time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (OH) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (OL) suggested that the most probable site lies within the ND3 gene. By adopting this OL position we calculated times of exposure for 0FD (nondegenerate), 2FD (twofold degenerate), and 4FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0FD sites with exposure time, which is explicable as the result of biased usage of 4FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes. [ABSTRACT FROM AUTHOR]

Both the maternal (F-type) and paternal (M-type) mitochondrial genomes of the Mytilus species complex M. edulis/galloprovincialis contain a noncoding sequence between the l-rRNA and the tRNATyr genes, here called the large unassigned region (LUR). The LUR, which is shorter in M genomes, is capable of forming secondary structures and contains motifs of significant sequence similarity with elements known to have specific functions in the sea urchin and the mammalian control region. Such features are not present in other noncoding regions of the F or M Mytilus mtDNA. The LUR can be divided on the basis of indels and nucleotide variation in three domains, which is reminiscent of the tripartite structure of the mammalian control region. These features suggest that the LUR is the main control region of the Mytilus mitochondrial genome. The middle domain has diverged by only 1.5% between F and M genomes, while the average divergence over the whole molecule is ∼20%. In contrast, the first domain is among the most divergent parts of the genome. This suggests that different parts of the LUR are under different selection constraints that are also different from those acting on the coding parts of the molecule. [ABSTRACT FROM AUTHOR]

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI. [ABSTRACT FROM AUTHOR]

Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25-1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRTPCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is characterized by an accumulation of monoclonal plasma cells within the bone marrow (BM). Macrophages are an abundant component of myeloma BM microenvironment and support survival of the malignant cells and foster myeloma development and progression by suppression of the immune response. In our previous study, we found that MM patients overexpress pro-inflammatory cytokine interleukin-32 (IL-32). The present study aimed to investigate the role of IL-32 in myeloma progression and mechanisms of IL-32 on macrophages functions. We discovered that the expression of IL-32 was associated with the disease stage in myeloma patients. MM-derived exosomes containing IL-32γ promoted the expression of programmed death-ligand 1(PD-L1) by macrophages, thus promoting immune evasion. Mechanistically, myeloma-secreted IL-32γ acted via proteinase 3 (PR3) to enhance 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) dependent glycolysis and subsequent PD-L1 expression. Moreover, the PFKFB3-Janus kinase 1 (JAK1) axis might contribute to the expression of PD-L1 by macrophages. To sum up, we concluded that IL-32 was a critical mediator in myeloma progression, and targeting IL-32 signaling might be used as a potential strategy for treating myeloma. [ABSTRACT FROM AUTHOR]

Explores the genetics of mother-dependent sex ratio in blue mussels. Implications for doubly uniparental inheritance of mitochondrial DNA; Model for mother-dependent sex determination; Fitting the model to sex ratios produced by wild-caught animals.

The rapid development of high spatial resolution (HSR) remote sensing imagery techniques not only provide a considerable amount of datasets for scene classification tasks but also request an appropriate scene classification choice when facing with finite labeled samples. AlexNet, as a relatively simple convolutional neural network (CNN) architecture, has obtained great success in scene classification tasks and has been proven to be an excellent foundational hierarchical and automatic scene classification technique. However, current HSR remote sensing imagery scene classification datasets always have the characteristics of small quantities and simple categories, where the limited annotated labeling samples easily cause non-convergence. For HSR remote sensing imagery, multi-scale information of the same scenes can represent the scene semantics to a certain extent but lacks an efficient fusion expression manner. Meanwhile, the current pre-trained AlexNet architecture lacks a kind of appropriate supervision for enhancing the performance of this model, which easily causes overfitting. In this paper, an improved pre-trained AlexNet architecture named pre-trained AlexNet-SPP-SS has been proposed, which incorporates the scale pooling--spatial pyramid pooling (SPP) and side supervision (SS) to improve the above two situations. Extensive experimental results conducted on the UC Merced dataset and the Google Image dataset of SIRI-WHU have demonstrated that the proposed pre-trained AlexNet-SPP-SS model is superior to the original AlexNet architecture as well as the traditional scene classification methods. [ABSTRACT FROM AUTHOR]