Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.

Background:In order to the growing frequency of extendedspectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae, treatment of such infections requires rapid and reliable detection of this enzyme.This study aims to develop the rapid-simple-ESBL detection in these organisms by comparing with the double-disc synergy test/combined disc test.Methods: One hundred and forty-six isolates ofE. coliand 150 isolates ofK. pneumoniaeisolated from the clinical specimens were included in this study. The rapid-simple-ESBL (RS-ESBL) test was performed in phenol red glucose broth containing ceftazidime(CAZ) or cefotaxime(CTX) (2 g/ml) with or without clavulanic acid in microtiter plate. And the incubation period of this test was 4 hours. All of them were also tested for ESBL producer by double-disc synergy test/combined disc test.Results: Among 96 ESBL-producing E. coli isolates, 94(97.9%) and 95(99.0%) were identified as ESBL-positive by CAZ RS-ESBL and CTX RS-ESBL, respectively when compared with double-disc synergy test/combined disc test. Of 100 ESBL-producingK. pneumoniae, 97(97.0%) were ESBL-positive by both CAZ RS-ESBL and CTX RS-ESBL tests. All of the 100 non-ESBL producers were negative by CTX RS-ESBL test. Conclusion:The RS-ESBL test was proved to be a reliable, simple and rapid method for detecting ESBL in E. coli and K. pneumoniae.

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Background: Lower respiratory tract infections are among the leading causes of death in children but diagnosis and defining aetiology are challenging. Up to quarter of children treated for pulmonary tuberculosis (PTB), have microbiologically confirmed TB; most are treated based on clinical and radiological features. We wished to identify the presence of other potential respiratory pathogens in nasopharyngeal samples from children presenting for care with symptoms suggestive of tuberculosis.Methods & Materials: Nasopharyngeal swabs were collected from a cohort of children presenting with suspected PTB to Red Cross War Memorial Children's Hospital, Cape Town, South Africa, from July 2011 through to May 2012. Total nucleic acid was extracted and screened for the presence of 33 common respiratory pathogens using a multiplex real-time PCR assay, which includes probes for 21 viral, 11 bacterial and one fungal pathogen. Mycobacterial liquid culture was performed on sputum obtained from each participant. Children were categorised as definite TB (culture confirmed), not TB (improvement without TB treatment on follow-up) and possible TB (all others)Results: Nasopharyngeal swabs were obtained from 214 children, median age 36 months (interquartile range, [IQR] 5 -17 months). Overall, 34 (16%) of the children had definite TB, 86 (40%) had possible TB and 94 (44%) were classified as not TB. Moraxella catarrhalis (64%), Streptococcus pneumoniae (42%), Haemophilus influenzae (29%) and Staphylococcus aureus (22%) were the most common bacteria detected. Other bacteria detected include Mycoplasma pneumoniae (9%), Bordetella pertussis (7%)and ChlamydophiIa pneumoniae (4%). The most common viruses included metapneumovirus (19%), rhinovirus (15%), influenza C (9%), adenovirus (7%), cytomegalovirus (7%) and coronavirus OC43 (5.6%), the last of which was associated with definite TB (p = 0.024). M. catarrhalis and S. pneumoniae appeared concurrently in 49% of cases where at least one was detected. There was no clear difference in the distribution of respiratory pathogens between children with and without TB when assessed using linear discriminant analysis. Conclusion:There was no clear relationship between TB categorization and coinfection/colonization with other pathogens detected. Further work is needed to explore possible pathogen interactions and determine the prevalence, in a control group of children, of nasopharyngeal colonisation with the pathogens identified.