Objectives: This study aimed to: (i) evaluate the effects of Malaysian Trigona honey on bacterial structure and (ii) assess the anti-virulence potential of this honey by examining their impacts on the expression of selected genes (involved in stress survival and biofilm formation) in a test organism.
Materials and Methods: Trigona honey's impacts on the bacterial structure (cell morphology) and the expression profiles of select Pseudomonas Aeruginosa and Streptococcus Pyogenes genes were examined using scanning electron microscopy (SEM) and real-time PCR (RT-qPCR) analysis, respectively.
Results: SEM showed that the decreased cell density deformed, disrupted, and damaged cells for both bacteria. RT-qPCR showed that the expression of fleN, fleQ, and fleR genes of P.aeruginosa were decreased, 4.26-fold, 3.80-fold and 2.66- fold respectively. In addition, scpA, ftsY, and emm13 of S.pyogenes were decreased, 2.87-fold, 3.24-fold, and 4.65-fold respectively.
Conclusion: Our results indicate that Trigona honey may be an effective inhibitor and virulence modulator of P. aeruginosa and S. pyogenes via multiple molecular targets. This deduction needs to be investigated in vivo.
RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM.
CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.
MATERIALS AND METHODS: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification.
RESULTS: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR.
CONCLUSION: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.
METHODS: MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.
RESULTS: We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA's anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.
CONCLUSIONS: FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.
METHODS: Eighty (80) male, 6-week-old Sprague Dawley rats were grouped in to four groups, the first group was irradiated with (940 nm) diode laser, second group with LIPUS, and third group with combination of both LLLT and LIPUS. A forth group used was a control group in an incomplete block split-mouth design. The LLLT and LIPUS were used to treat the area around the moving tooth once a day on days 0-7, then the experiment was ended in each experimental endpoint (1, 3, 7, 14, and 21 days). For amount of tooth movement, models were imaged and analyzed. Histological examination was performed after staining with (hematoxylin and eosin) and (alizarin red and Alcian Blue) stain. One step reverse transcription-polymerase chain reaction RT-PCR was also performed to elucidate the gene expression of RANK, RANKL, OPG, and RUNX-2.
RESULTS: The amount of tooth movement, the histological bone remodeling, and the RT-PCR were significantly greater in the treatment groups than that in the control group. Among the treatment groups, the combination group was the highest and the LIPUS group was the lowest.
CONCLUSION: These findings suggest that LLLT and LIPUS can enhance the velocity of tooth movement and improve the quality of bone remodeling during orthodontic tooth movement.