Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development

A transgenic mouse line named iUBC-KikGR was generated, which expresses the photoconvertible fluorescent protein Kikume Green-Red (KikGR) under the control of the human Ubiquitin C promoter. KikGR is natively a green fluorophore, which can be converted into a red fluorophore upon exposure to UV light. KikGR is expressed broadly throughout transgenic embryos from the two-cell stage onward and in the adult. Specificity of photoconversion can range from the entire embryo to a region of an organ, to a few individual cells, depending on the needs of the experimenter. Cell movements, tissue reorganization, and migration can then be observed in real time by culturing the tissue of interest as an explant on the microscope stage. The iUBC-KikGR transgenic line represents a singular genetic reagent, which can be used for fate mapping, lineage tracing, and live visualization of cell behaviors and tissue movements in multiple organs at multiple time points.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

The equilibrium population size of a partially migratory population and its response to environmental change

A partially migratory population consists of non‐migrant and migrant individuals that share a common site during one period of the annual cycle. In this paper, we derive the expected equilibrium population sizes of migrants and non‐migrants and show how the abundance of one type is dependent on the other because their dynamics are coupled through density‐dependent effects. We present an approach for developing hypotheses about how changes in the environment will influence partially migratory populations and for formulating testable predictions about the effects of future changes on the proportions of migrants and non‐migrants. We apply this approach to a hypothesis put forward by Berthold that improved environmental conditions at the shared site will generally increase the number of non‐migrants and decrease the number of migrants, and to a study by Nilsson et al. which observed an increase in the number of migrants in a partially migratory blue tit Cyanistes caeruleus population in Sweden, but an overall maintenance of the proportion of migrants.     

A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro–stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.     

The relationship between the pleiotropic phenotypic effects of a mutation fixed by selection

A pleiotropic model of mutation is presented that allows for correlations between the effects of a new mutation and for the distribution of mutational effects to vary from being leptokurtic to normally distributed. Using this model I quantify how selection transforms the correlation between the effects of a new (random) mutation into the correlation between the effects of a mutation that is fixed by selection and contributes to an adaptation. Results suggest that under most conditions the correlation between the effects of a fixed mutation is less than the correlation between the effects of a new mutation. I also generalize previous results that quantified the expected size of a fixed mutation's effect on a character given an observed effect of that mutation on another character. In agreement with previous results, work here suggests that as the observed effect becomes large and beneficial the expected effect on another character approaches the expected effect of a new (random) mutation given the observed effect. Lastly, these theoretical results are related to recent empirical work that found beneficial mutations had a positive correlation in their pleiotropic effects.     

The morphology and relationships of the walking mud spiders of the genus Cryptothele (Araneae: Zodariidae)

We revise the relationships of the spider genus Cryptothele after reexamination of the morphology of the spinnerets, leg tarsal claws and maxillae with scanning electron microscopy. Cryptothele species have a particular conformation of the spinning field of the anterior lateral spigots that is typical of zodariids and close relatives: the field of major ampullate gland spigots, together with their strain sensilla, are invaginated within the field of piriform gland spigots. The implantation of the teeth on the inner side of the leg tarsal claws is also consistent with its placement among zodariids. We added Cryptothele to a morphological dataset of zodariid genera, together with the outgroups Homalonychus (Homalonychidae) and Penestomus (Penestomidae). The phylogenetic analysis concludes that the genus Cryptothele is a member of the subfamily Cydrelinae, which by priority is here considered a junior synonym of Cryptothelinae. Cryptothele specimens cover most of their body with soil particles which become consolidated as mud, and the debris is probably held in place by curved setae covered by long barbs. The spinnerets, which can be retracted and hidden, as well as the booklungs, are surrounded by a crown of thick setae that are densely covered by short barbs, protecting those areas against soil particles. Cryptothele are probably specialized to prey on termites, and their phylogenetic placement indicates that this diet specificity evolved two times independently in zodariids.     

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants.     

AnthWest,occurrence records for wool carder bees of the genus Anthidium (Hymenoptera,Megachilidae, Anthidiini) in the Western Hemisphere

This paper describes AnthWest, a large dataset that represents one of the outcomes of a comprehensive, broadly comparative study on the diversity, biology, biogeography, and evolution of Anthidium Fabricius in the Western Hemisphere. In this dataset a total of 22,648 adult occurrence records comprising 9657 unique events are documented for 92 species of Anthidium, including the invasive range of two introduced species from Eurasia, A. oblongatum (Illiger) and A. manicatum (Linnaeus). The geospatial coverage of the dataset extends from northern Canada and Alaska to southern Argentina, and from below sea level in Death Valley, California, USA, to 4700 m a.s.l. in Tucumán, Argentina. The majority of records in the dataset correspond to information recorded from individual specimens examined by the authors during this project and deposited in 60 biodiversity collections located in Africa, Europe, North and South America. A fraction (4.8%) of the occurrence records were taken from the literature, largely California records from a taxonomic treatment with some additional records for the two introduced species. The temporal scale of the dataset represents collection events recorded between 1886 and 2012. The dataset was developed employing SQL server 2008 r2. For each specimen, the following information is generally provided: scientific name including identification qualifier when species status is uncertain (e.g. “Questionable Determination” for 0.4% of the specimens), sex, temporal and geospatial details, coordinates, data collector, host plants, associated organisms, name of identifier, historic identification, historic identifier, taxonomic value (i.e., type specimen, voucher, etc.), and repository. For a small portion of the database records, bees associated with threatened or endangered plants (~ 0.08% of total records) as well as specimens collected as part of unpublished biological inventories (~17%), georeferencing is presented only to nearest degree and the information on floral host, locality, elevation, month, and day has been withheld. This database can potentially be used in species distribution and niche modeling studies, as well as in assessments of pollinator status and pollination services. For native pollinators, this large dataset of occurrence records is the first to be simultaneously developed during a species-level systematic study.