RESULTS: We attempted to identify genes that may be involved in biofilm formation in seven C. jejuni strains through construction of mutants using the EZ-Tn5 Transposome system. Only 14 mutants with reduced biofilm formation were obtained, all from one strain of C. jejuni. Three different genes of interest, namely CmeB (synthesis of multidrug efflux system transporter proteins), NusG (transcription termination and anti-termination protein) and a putative transmembrane protein (involved in membrane protein function) were identified. The efficiency of the EZ::TN5 transposon mutagenesis approach was strain dependent and was unable to generate any mutants from most of the strains used.
CONCLUSIONS: A diverse range of genes may be involved in biofilm formation by C. jejuni. The application of the EZ::TN5 system for construction of mutants in different Campylobacter strains is limited.
Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.
DESIGN: Intracanal samples were collected from primarily infected (n = 10) and post-treatment infected (n = 10) root canals of human teeth using sterile paper points. Bacterial DNA was amplified from seven hypervariable regions (V2-V4 and V6-V9) of the 16S rRNA gene, then sequenced using next-generation sequencing technology. The data was analyzed using appropriate bioinformatic tools.
RESULTS: Analyses of all the samples revealed eight major bacterial phyla, 112 genera and 260 species. Firmicutes was the most representative phylum in both groups and was significantly more abundant in the post-treatment (54.4%) than in primary (32.2%) infections (p>0.05). A total of 260 operational taxonomic units (OTUs) were identified, of which 126 (48.5%) were shared between the groups, while 83 (31.9%) and 51 (19.6%) disparate species were isolated from primary and post-treatment infections, respectively. A significant difference in beta, but not alpha diversity was noted using several different indices (p< 0.05). Differential abundance analysis indicated that, Prevotella maculosa, Streptococcus constellatus, Novosphigobium sediminicola and Anaerococcus octavius were more abundant in primary infections while Enterrococcus faecalis, Bifidobacterium dentium, Olsenella profusa and Actinomyces dentalis were more abundant in post-treatment infections (p <0.05).
CONCLUSION: Significant differences in the microbiome composition and diversity in primary and post-treatment endodontic infections were noted in our UAE cohort. Such compositional differences of microbiota at various stages of infection could be due to both intrinsic and extrinsic factors impacting the root canal ecosystem during disease progression, as well as during their therapeutic management. Identification of the key microbiota in primarily and secondarily infected root canals can guide in the management of these infections.
MATERIALS AND METHODS: This was a retrospective study of YOCRC (<50 years) over 8 years (January 2013 to December 2021). Immunohistochemistry staining of FOXP3, BRAFV600E, and MMR protein expression was performed using monoclonal antibodies. The staining intensity and percentage of positive cells were used to evaluate the staining using immunoreactive scoring. All data were analysed using descriptive and correlation statistics. A p-value of ≤ 0.05 was taken as statistically significant.
RESULTS: A total of 65 YOCRC patients were diagnosed, out of which 53.8% had proficient MMR (pMMR) with a mean age of 41, while 46.2% had deficient MMR (dMMR) with a mean age of 35.5. The pMMR with the BRAFV600E+ group expressed higher FOXP3+Tregs (54.2%) than the dMMR with the BRAFV600E+ group (22.9%). Patients with lower FOXP3+Tregs were observed more in dMMR with BRAFV600E- (47%) than in pMMR with BRAFV600E- (5.9%). There was a statistically significant association between the density of expressed FOXP3+Tregs with MMR and BRAFV600E status (p=0.002).
CONCLUSION: While most of the YOCRC had pMMR, others exhibited dMMR with loss of one or more MMR proteins. The presence of BRAFV600E demonstrated the YOCRC's sporadic nature. A high FOXP3+Treg expression was significantly associated with MMR and BRAFV600E status. Future research must be expanded to cover other hospitals to increase the sample size and include MLH1 hypermethylation testing.