Recent emergence and growing use of terahertz (THz) radiation for medical imaging and public security screening raise questions on reasonable levels of exposure and health consequences of this form of electromagnetic radiation. In particular, picosecond-duration THz pulses have shown promise for novel diagnostic imaging techniques. However, the effects of THz pulses on human cells and tissues thus far remain largely unknown. We report on the investigation of the biological effects of pulsed THz radiation on artificial human skin tissues. We observe that exposure to intense THz pulses for ten minutes leads to a significant induction of H2AX phosphorylation, indicating that THz pulse irradiation may cause DNA damage in exposed skin tissue. At the same time, we find a THz-pulse-induced increase in the levels of several proteins responsible for cell-cycle regulation and tumor suppression, suggesting that DNA damage repair mechanisms are quickly activated. Furthermore, we find that the cellular response to pulsed THz radiation is significantly different from that induced by exposure to UVA (400 nm).

Plants are able to maintain the memory of stress exposure throughout their ontogenesis and faithfully propagate it into the next generation. Recent evidence argues for the epigenetic nature of this phenomenon. Small RNAs (smRNAs) are one of the vital epigenetic factors because they can both affect gene expression at the place of their generation and maintain non-cell-autonomous gene regulation. Here, we have made an attempt to decipher the contribution of smRNAs to the heat-shock-induced transgenerational inheritance in Brassica rapa plants using sequencing technology. To do this, we have generated comprehensive profiles of a transcriptome and a small RNAome (smRNAome) from somatic and reproductive tissues of stressed plants and their untreated progeny. We have demonstrated that the highest tissue-specific alterations in the transcriptome and smRNAome profile are detected in tissues that were not directly exposed to stress, namely, in the endosperm and pollen. Importantly, we have revealed that the progeny of stressed plants exhibit the highest fluctuations at the smRNAome level but not at the transcriptome level. Additionally, we have uncovered the existence of heat-inducible and transgenerationally transmitted tRNA-derived small RNA fragments in plants. Finally, we suggest that miR168 and braAGO1 are involved in the stress-induced transgenerational inheritance in plants.

Bacterial cooperative associations and dynamics in the biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle, and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that maybe unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.