S. P. Livingston

The range of DNA sequences used to study the interrelationships of the major arthropod groups (chelicerates, myriapods, hexapods and crustaceans) is limited. Here we investigate the value of two genes not previously employed in arthropod phylogenetics. Histone H3 data were collected for 31 species and small nuclear ribonucleic acid U2 data for 29 species. The sequences provided a total of 460 sites and 192 parsimony-informative characters. H3 analyses showed substantial codon usage bias, but had a low consistency index (0.26). Consistency indices were higher for the U2 data (0.49), suggesting that the class of snRNAs may provide several phylogenetically useful genes. The present data are not by themselves sufficient to clarify major arthropod group relationships. Partitioned data for H3 and U2 are incongruent according to Incongruence Length Difference tests. Although the most parsimonious trees, based on combined analyses of all taxa, differ substantially from morphology-based trees, anomalous groupings are weakly supported with only one exception. The trees uphold monophyly of Onychophora, Branchiopoda, and Malacostraca (rather than the rival Phyllopoda). Cladistic analyses constraining the monophyly of morphologically defined classes do not significantly distinguish between the main rival hypotheses of major clade relationships. Combined (‘spliced’) analysis of both genes improves topological congruence with morphological groupings relative to that of either partition. Character congruence between H3, U2, and morphology is increased by downweighting (but not excluding) transitions and third codons. Analyses of four-taxon statements using PHYLTEST found significant support for the basal position of the Crustacea among the euarthropods. This support may be due to the similarity of chelicerates, myriapods and hexapods in percentage GC content.

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12,700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial-specific bacteriophage PO4. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage-resistant mutants was identified in which the transposon had inserted near sequences that cross-hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN-dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild-type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72-75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA-D. PilS and PilR clearly belong to the family of two-component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.

, we here examined a cohort of 131 tumour sections from 78 cases across 6 cancer types by Visium spatial transcriptomics (ST). This was combined with 48 matched single-nucleus RNA sequencing samples and 22 matched co-detection by indexing (CODEX) samples. To describe tumour structures and habitats, we defined 'tumour microregions' as spatially distinct cancer cell clusters separated by stromal components. They varied in size and density among cancer types, with the largest microregions observed in metastatic samples. We further grouped microregions with shared genetic alterations into 'spatial subclones'. Thirty five tumour sections exhibited subclonal structures. Spatial subclones with distinct copy number variations and mutations displayed differential oncogenic activities. We identified increased metabolic activity at the centre and increased antigen presentation along the leading edges of microregions. We also observed variable T cell infiltrations within microregions and macrophages predominantly residing at tumour boundaries. We reconstructed 3D tumour structures by co-registering 48 serial ST sections from 16 samples, which provided insights into the spatial organization and heterogeneity of tumours. Additionally, using an unsupervised deep-learning algorithm and integrating ST and CODEX data, we identified both immune hot and cold neighbourhoods and enhanced immune exhaustion markers surrounding the 3D subclones. These findings contribute to the understanding of spatial tumour evolution through interactions with the local microenvironment in 2D and 3D space, providing valuable insights into tumour biology.