inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010-2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

A scaling law in CRISPR repertoire sizes arises from the avoidance of autoimmunity.

Some prokaryotes possess CRISPR-Cas systems that use DNA segments called spacers, which are acquired from invading phages, to guide immune defense. Here, we propose that cross-reactive CRISPR targeting can, however, lead to "heterologous autoimmunity," whereby foreign spacers guide self-targeting in a spacer-length-dependent fashion. Balancing antiviral defense against autoimmunity predicts a scaling relation between spacer length and CRISPR repertoire size. We find evidence for this scaling through a comparative analysis of sequenced prokaryotic genomes and show that this association also holds at the level of CRISPR types. By contrast, the scaling is absent in strains with nonfunctional CRISPR loci. Finally, we demonstrate that stochastic spacer loss can explain variations around the scaling relation, even between strains of the same species. Our results suggest that heterologous autoimmunity is a selective factor shaping the evolution of CRISPR-Cas systems, analogous to the trade-offs between immune specificity, breadth, and autoimmunity that constrain the diversity of adaptive immune systems in vertebrates.

How nonuniform contact profiles of T cell receptors modulate thymic selection outcomes.

T cell receptors (TCRs) bind foreign or self-peptides attached to major histocompatibility complex (MHC) molecules, and the strength of this interaction determines T cell activation. Optimizing the ability of T cells to recognize a diversity of foreign peptides yet be tolerant of self-peptides is crucial for the adaptive immune system to properly function. This is achieved by selection of T cells in the thymus, where immature T cells expressing unique, stochastically generated TCRs interact with a large number of self-peptide-MHC; if a TCR does not bind strongly enough to any self-peptide-MHC, or too strongly with at least one self-peptide-MHC, the T cell dies. Past theoretical work cast thymic selection as an extreme value problem and characterized the statistical enrichment or depletion of amino acids in the postselection TCR repertoire, showing how T cells are selected to be able to specifically recognize peptides derived from diverse pathogens yet have limited self-reactivity. Here, we investigate how the diversity of the postselection TCR repertoire is modified when TCRs make nonuniform contacts with peptide-MHC. Specifically, we were motivated by recent experiments showing that amino acids at certain positions of a TCR sequence have large effects on thymic selection outcomes, and crystal structure data that reveal a nonuniform contact profile between a TCR and its peptide-MHC ligand. Using a representative TCR contact profile as an illustration, we show via simulations that the statistical enrichment or depletion of amino acids now varies by position according to the contact profile, and, importantly, it depends on the implementation of nonuniform contacts during thymic selection. We explain these nontrivial results analytically. Our study has implications for understanding the selection forces that shape the functionality of the postselection TCR repertoire.

Detection of congenital uterine malformation by using transvaginal three-dimensional ultrasound.

This study assessed the clinical application of transvaginal three-dimensional ultrasound (3D TVUS) in the diagnosis of congenital uterine malformation. A retrospective study was performed on 62 patients with congenital uterine malformation confirmed hysteroscopically and/or laparoscopically. The patients were subjected to transvaginal two-dimensional ultrasound (2D TVUS) and 3D TVUS. The accuracy rate was compared between the two methods. The accuracy rate of 3D TVUS was (98.38%, 61/62), higher than that of 2D TVUS (80.65%, 50/62). 3D TVUS coronal plane imaging could demonstrate the internal shape of the endometrial cavity and the external contour of the uterine fundus. It allowed accurate measurement on the coronary plane, and could three-dimensionally show the image of cervical tube, thereby providing information for the diagnosis of some complex uterine malformation. 3D TVUS imaging can obtain comprehensive information of the uterus malformation, and it is superior to 2D TVUS for the diagnosis of congenital uterine malformations, especially complex uterine anomaly.

CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000.

Small non-coding RNAs (ncRNAs) are important components of many regulatory pathways in bacteria and play key roles in regulating factors important for virulence. Carbon catabolite repression control is modulated by small RNAs (crcZ or crcZ and crcY) in Pseudomonas aeruginosa and Pseudomonas putida. In this study, we demonstrate that expression of crcZ and crcX (formerly designated psr1 and psr2, respectively) is dependent upon RpoN together with the two-component system CbrAB, and is influenced by the carbon source present in the medium in the model plant pathogen Pseudomonas syringae pv tomato DC3000. The distribution of the members of the Crc ncRNA family was also determined by screening available genomic sequences of the Pseudomonads. Interestingly, variable numbers of the Crc family members exist in Pseudomonas genomes. The ncRNAs are comprised of three main subfamilies, named CrcZ, CrcX and CrcY. Most importantly the CrcX subfamily appears to be unique to all P. syringae strains sequenced to date.

[Effect of IL-15 on the proliferation, differentiation and anti-apoptosis of CD34+ cells in patients with MDS].

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.