Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.

The IkappaB kinase complex IKK is a central component of the signaling cascade that controls NF-kappaB-dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

The transcription factor nuclear factor B (NF-B

In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves.

Reef-building corals depend on intracellular dinoflagellate symbionts that provide nutrients. Besides sugars, the transfer of sterols is essential for corals and other sterol-auxotrophic cnidarians. Sterols are important cell components, and variants of the conserved Niemann-Pick Type C2 (NPC2) sterol transporter are vastly up-regulated in symbiotic cnidarians. Types and proportions of transferred sterols and the mechanism of their transfer, however, remain unknown. Using different pairings of symbiont strains with lines of Aiptasia anemones or Acropora corals, we observe both symbiont- and host-driven patterns of sterol transfer, revealing plasticity of sterol use and functional substitution. We propose that sterol transfer is mediated by the symbiosis-specific, non-canonical NPC2 proteins, which gradually accumulate in the symbiosome. Our data suggest that non-canonical NPCs are adapted to the symbiosome environment, including low pH, and play an important role in allowing corals to dominate nutrient-poor shallow tropical seas worldwide.