Designed Auto-assembly of Nanostreptabodies for Rapid Tissue-specific Targeting in Vivo

Molecular medicine can benefit greatly from antibodies that deliver therapeutic and imaging agents to select organs and diseased tissues. Yet the development of complex and defined composite nanostructures remains a challenge that requires both designed stoichiometric assembly and superior in vivo testing ability. Here, we generate nanostructures called nanostreptabodies by controlled sequential assembly of biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads such as other antibodies to create bispecific antibodies as well as organic and non-organic moieties. When injected intravenously, these novel and stable nanostructures exhibit exquisite targeting with tissue-specific imaging and delivery, including rapid transendothelial transport that enhances tissue penetration. This “tinkertoy construction” strategy provides a very flexible and efficient way to link targeting vectors with reporter and/or effector agents, thereby providing virtually endless combinations potentially useful for multipurpose molecular and functional imaging in vivo as well as therapies.     

Key Edaphic Properties Largely Explain Temporal and Geographic Variation in Soil Microbial Communities across Four Biomes

Soil microbial communities play a critical role in nutrient transformation and storage in all ecosystems. Quantifying the seasonal and long-term temporal extent of genetic and functional variation of soil microorganisms in response to biotic and abiotic changes within and across ecosystems will inform our understanding of the effect of climate change on these processes. We examined spatial and seasonal variation in microbial communities based on 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) composition across four biomes: a tropical broadleaf forest (Hawaii), taiga (Alaska), semiarid grassland-shrubland (Utah), and a subtropical coniferous forest (Florida). In this study, we used a team-based instructional approach leveraging the iPlant Collaborative to examine publicly available National Ecological Observatory Network (NEON) 16S gene and PLFA measurements that quantify microbial diversity, composition, and growth. Both profiling techniques revealed that microbial communities grouped strongly by ecosystem and were predominately influenced by three edaphic factors: pH, soil water content, and cation exchange capacity. Temporal variability of microbial communities differed by profiling technique; 16S-based community measurements showed significant temporal variability only in the subtropical coniferous forest communities, specifically through changes within subgroups of Acidobacteria. Conversely, PLFA-based community measurements showed seasonal shifts in taiga and tropical broadleaf forest systems. These differences may be due to the premise that 16S-based measurements are predominantly influenced by large shifts in the abiotic soil environment, while PLFA-based analyses reflect the metabolically active fraction of the microbial community, which is more sensitive to local disturbances and biotic interactions. To address the technical issue of the response of soil microbial communities to sample storage temperature, we compared 16S-based community structure in soils stored at -80°C and -20°C and found no significant differences in community composition based on storage temperature. Free, open access datasets and data sharing platforms are powerful tools for integrating research and teaching in undergraduate and graduate student classrooms. They are a valuable resource for fostering interdisciplinary collaborations, testing ecological theory, model development and validation, and generating novel hypotheses. Training in data analysis and interpretation of large datasets in university classrooms through project-based learning improves the learning experience for students and enables their use of these significant resources throughout their careers.     

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome

Background

Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes?

Results

A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser.

Conclusion

We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.     

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.     

Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.     

ALLELOPATHY AS AN EMERGENT,EXPLOITABLE PUBLIC GOOD IN THE BLOOM‐FORMING MICROALGA PRYMNESIUM PARVUM

Many microbes cooperatively secrete extracellular products that favorably modify their environment. Consistent with social evolution theory, structured habitats play a role in maintaining these traits in microbial model systems, by localizing the benefits and separating strains that invest in these products from ‘cheater’ strains that benefit without paying the cost. It is thus surprising that many unicellular, well‐mixed microalgal populations invest in extracellular toxins that confer ecological benefits upon the entire population, for example, by eliminating nutrient competitors (allelopathy). Here we test the hypotheses that microalgal exotoxins are (1) exploitable public goods that benefit all cells, regardless of investment, or (2) nonexploitable private goods involved in cell‐level functions. We test these hypotheses with high‐toxicity (TOX+) and low‐toxicity (TOX?) strains of the damaging, mixotrophic microalga Prymnesium parvum and two common competitors: green algae and diatoms. TOX+ actually benefits from dense populations of competing green algae, which can also be prey for P. parvum, yielding a relative fitness advantage over coexisting TOX?. However, with nonprey competitors (diatoms), TOX? increases in frequency over TOX+, despite benefiting from the exclusion of diatoms by TOX+. An evolutionary unstable, ecologically devastating public good may emerge from traits selected at lower levels expressed in novel environments.     

Cutting edge: sustained expansion of CD8+ T cells requires CD154 expression by Th cells in acute graft versus host disease

Brief treatment with alphaCD154 Ab has been shown to prevent acute graft versus host disease (aGvHD). We extend these data to show that in the absence of CD154 function, donor T cells are unable to expand or generate high level anti-host CTL activity. Using transgenic (Tg) alloreactive CD8+ T cells adoptively transferred into allogeneic recipients, we show that short-term expansion of the CD8+ Tg T cells occurred in the absence of Th cells, and this short-term expansion could be facilitated with an agonistic alphaCD40. While CD40 agonism could enhance short-term expansion, sustained expansion of CD8+ Tg T cells required bona fide CD154-expressing CD4+ alloreactive Th cells. While CD154 was necessary for CD8+ Tg T cell sustained expansion, IL-2 was also implicated as essential. These observations suggest alphaCD154 therapy in GvHD is effective because the treatment causes an abortive CD8 alloresponse leading to the exhaustion or deletion of alloreactive CD8+ clones preventing the development of disease.     

On the localization,the fine structure,and the chemical composition of crystalline inclusions in theThesium humifusum haustoria

Summary TheThesium humifusum haustoria onMedicago marina roots, fixed in October–November, frequently contain in the cytoplasm of their cells inclusions consisting of sticks 0,3 m in thickness and 8 to 10 m in lenght, alone or associated in stacks of 3 or 6 units. These sticks consist of fibres, 10 nm in thickness, oriented in the same direction and separated from the others by a gap of 8 nm; these fibres seem to be composed of helically wound filaments and a less electron-dense matrix.The chemical composition of these inclusions was studied by enzymatic digestion in ultra thin sections; pronase digested the cytoplasmic paracrystals. This demonstrates that they are composed primarily of protein.The physiological significance of the inclusions is discussed: presumably the haustorium functions as a storage organe.     

Ultrastructure of the protocerebral neurosecretory cells of larval Galleria mellonella, in situ and after culture of the brain in vitro

Three major groups of neurosecretory cells are described in the larval brain of Galleria mellonella at two different times during the last larval instar and in larval brains after 72 hr of culture in vitro. The medial group in vivo consists of four distinct neurosecretory cell types, based on characteristic size and morphology, while the posterior and lateral groups each contain a single distinct type of neurosecretory cell. Morphological differences between the same neurosecretory cells at the different times during the last instar are most apparent in the lateral L-1 cells and in the medial M-2 cells, where pleiomorphism is particularly evident in the size, density and accumulations of neurosecretory granules. The only neurosecretory cells in which apparent synthesis of neurosecretory granules is still observed after culture of the brain in vitro are the medial M-2 cells. The other neurosecretory cell types show no accumulation of neurosecretory granules nor new synthesis of neurosecretory material, but are similar to neurosecretory cells in the brain in vivo in all other respects. The morphology of the neurosecretory cells in the larval brain in vivo and in vitro is discussed in relation to their appearance at the light microscopic level and to a known neurohormonal function of the brain which is maintained during 72 hr in vitro.