CD4 T-cells are important for long-term control and clearance of several fungal infections in humans, particularly those caused by Cryptococcus species. Understanding the mechanisms underlying protective T-cell immunity against fungal infection is critical for developing mechanistic insights into the pathogenesis of the disease. Here, we describe a protocol that enables analysis of fungal-specific CD4 T-cell responses in vivo, using adoptive transfer of fungal-specific T-cell receptor (TCR) transgenic CD4 T-cells. While the protocol here uses a TCR transgenic model reactive to peptide deriving from Cryptococcus neoformans, this method could be adapted to other fungal infection experimental settings.

The molecular basis of Z-DNA recognition and stabilization is mostly discovered via X-ray crystallography. The sequences composed with alteration of purine and pyrimidine are known to adopt Z-DNA conformation. Due to the energy penalty for forming Z-DNA, the small molecular stabilizer or Z-DNA-specific binding protein is required for DNA to adopt Z conformation prior to crystallizing Z-DNA. Here we described the methods ranging from preparation of DNA and Z-alpha protein to crystallization of Z-DNA in detail.

One of the few available systems for gene expression in (hyper)thermophilic Archaea is the virus-based shuttle vector pMJ05 for Sulfolobus solfataricus. Although it is still not fully developed and there are some difficulties arising from the large size of the vector (>20 kb), it has successfully been used for the production of foreign and own proteins in S. solfataricus. Most often, the development of genetic tools for Archaea is held back by the lack of an efficient transformation system. In the case of pMJ05, this difficulty has been alleviated by using the Sulfolobus virus SSV1 as the vector backbone. The ability of the pMJ05 plasmid to spread in the culture as a virus, the availability of an effective selection marker (pyr) and of tunable promoters (araS and tf55α) make this system one of the first choices for heterologous gene expression in (hyper)thermophilic Archaea.