Biomolecular imaging of host-pathogen interaction by Raman micro-spectroscopy

The ability of pathogens and their surrogate host cells to exchange molecules is at the core of all host-pathogen interaction processes. Small chemical molecule signals provide a rich vocabulary for cellular communication, and pathogens have evolved the capacity to modulate, mimic or hijack these signals in a defensive, exponentially increasing the scope and complexity of this chemical language between host cells and pathogens. The objective of this work is to use Raman microscopy (RMS) to analyze molecular changes during infection of human cells by intra- (Toxoplasma gondii) and extra-cellular (Acanthamoeba castellanii) protozoan parasites. The work is divided into two parts. Firstly, we studied host-pathogen biomolecular interaction by using RMS. Secondly, we employed stable isotopes substitution technique to induce spectral changes that are specific to the labelled molecules. In this approach mammalian cells are selectively grown in culture medium in which only certain molecules are substituted with stable-isotope counterparts, and the exchange of these molecules are monitored between individual mammalian cells and parasites in real-time. Our results show that Raman spectroscopy can be a great tool to understand the molecular processes that mediate the interaction between the pathogens and host cell by providing quantitative spatially- and temporally-resolved information regarding molecular trafficking and exchanges. A better understanding of these complex interactions will advance our understanding of microbial pathogenesis and potentially identify new therapeutic targets

Lightweight TDMA Protocol for wireless vehicular communications

Wireless vehicular communications (WVC) has been identified as a key technology for intelligent transportation systems (ITS) for a few years ago. IEEE 802.11p (WAVE: wireless access in vehicular environments) is the proposed standard for physical and MAC layer of WVC devices. The standard is extended from 802.11 protocols. The main objectives of the standard are to change the frame format and increase delay spread tolerance introduced by vehicle mobility, in which the channel bandwidth is scaled from 20 MHz in 802.11a to 10 MHz in 802.11p. However, to ensure interoperability between vehicles communicate in rapidly changing environment where a packet transmission should be completed in short time-frame is a problematic issue. This poster proposes WiFi-based TDMA technique with flexible time slots and guard bands to tackle this problem. The new TDMA sublayer is compatible with the 802.11p standard to ensure the feasibility of adoption by any vendor. The simulation results present the performance analysis and validate the efficiency of the proposed method

WiMAX-WiFi Synergy for Next Generation Heterogynous Network

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Faktor pendorong yang mempengaruhi keputusan melanjutkan pengajian ke institusi pengajian tinggi di kalangan staf kerajaan dan swasta

Pasaran kerja kini lebih bersifat dinamik akibat perubahan pesat dalam penggunaan teknologi dan kepelbagaian pengetahuan yang semakin disyaratkan. Pelbagai isu mula timbul apabila wujudnya persaingan dalam pemilihan kerjaya dan kriteria yang disyaratkan oleh majikan. Oleh itu, pendidikan dilihat sebagai aspek terpenting dalam menentukan hala tuju kerjaya dan sebagai penyelesaian bagi senario kehidupan masa kini. Oleh itu, kajian tinjauan berbentuk kuantitatif menggunakan instrumen soal selidik dilakukan kepada 150 kakitangan di tiga (3) organisasi di Kuala Lumpur untuk mengenalpasti faktor yang mendorong mereka terhadap keputusan melanjutkan pelajaran di Institusi Pengajian Tinggi (IPT) tempatan. Dapatan kajian menunjukkan faktor kendiri untuk melanjutkan pelajaran atas inisiatif sendiri merupakan faktor paling tinggi dalam mendorong keputusan kakitangan tersebut. Hasil kajian ini disarankan agar pihak majikan memberikan peluang serta insentif yang boleh menggalakkan pekerjanya untuk melanjutkan pelajaran bagi mempertingkatkan martabat ilmu dan profesion masing-masing

Iatrogenic vesicovaginal fistula repair-experience at Prince Hussein Urology and Organ Transplant Center

Background: Despite the advances in technology in urology practice, and the surgical approach in dealing with iatrogenic vesicovaginal fistula repair, the most important is to achieve continent rate with minimum morbidity.Methods: From January 2006 to December 2017, the medical records and operative notes of 52 female (mean age 37 year) who had undergone transabdominal transvesical operative repair of their vesicovaginal fistula (VVF) at this institution were reviewed retrospectively. CT urography and diagnostic cystourethroscopy were the modalities of diagnostic tools. Trans-abdominal, transvesical repair with omental flap interposition were performed within 4-6 months in all cases. Patients were evaluated at two to three weeks initially, then at three months interval and later annually.Results: In present study, the most common presentation of VVF was urine leakage through vagina. In two third of the patients the etiology was due to hysterectomy procedure, regarding the location of the fistula, 94.2% of the fistulas located high in the posterior wall of the urinary bladder (supratrigonal), with the mean size of 2.2cm (range 5-25mm). 49 patients had single fistula (94.2%). The mean operative time was 110 minutes (range 60-130 minutes) and the mean post-operative urethral catheterization was 21 days (range 17-24 days). Almost all patients were continent after a mean of five months.Conclusions: Iatrogenic VVF is one of the distressing complications of gynecological procedure; delayed transabdominal transvesical approach with omental flap interposition is associated with excellent and durable results with minor morbidity. Standardization of the technique is a key success in the outcome of the repair

Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging

Improved understanding of the mechanism of nutrient’s uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman micro-spectroscopy (RMS) and coherent anti-Stokes Raman spectroscopy (CARS) to visualise the time-dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE-19) cells were prelabelled with deuterated Phe (L-Phe(D8)) and the uptake of the host derived L-Phe(D8) by A. castellanii trophozoites was measured by RMS for up to 48 hours post infection (hpi). This approach revealed a time-dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L-Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16-18% L-Phe(D8) from L-Phe(D8)–supplemented culture medium after 3h, 24h and 48h hpi. CARS microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite

Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography

Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in-vitro. However, obtaining quantitative molecular information from the Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least square fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 256 mg/m, while proteins were distributed more uniformly and reaching concentrations as high as ~5012 mg/ml. The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman map of fixed cells (n=10), we found a linear relationship between the scores corresponding to the first component (PC1) and cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n=10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were obtained concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in-vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman Spectroscopy

Label-free imaging using Raman micro-spectroscopy (RMS) was used to characterize the spatio-temporal molecular changes of T. gondii tachyzoites and their host cell microenvironment. Raman spectral maps were recorded from isolated T. gondii tachyzoites and T. gondii-infected human retinal cells at 6 hr, 24 hr and 48 hr post-infection. Principal component analysis (PCA) of the Raman spectra of paraformaldehyde-fixed infected cells indicated a significant increase in the amount of lipids and proteins in the T. gondii tachyzoites as the infection progresses within host cells. These results were confirmed by experiments carried out on live T. gondii-infected cells and were correlated with an increase in the concentration of proteins and lipids required for the replication of this intracellular pathogen. These findings demonstrate the potential of RMS to characterize time- and spatially-dependent molecular interactions between intracellular pathogens and the host cells. Such information may be useful for discovery of pharmacological targets or screening compounds with potential neuro-protective activity for eminent effects of changes in brain infection control practices

Optical sectioning in multi-foci Raman hyperspectral imaging

In this study, we compared the depth-discrimination and speed performance of multi-foci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator (LC-SLM) was employed for the generation of multi-foci laser beams, and a digital micromirror device (DMD) was used as a software-configurable reflective pinhole array. The patterns of the laser-foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance-to-size ratios were tested and compared. After optimisation of the laser foci pattern, we demonstrated the feasibility of multi-foci Raman hyperspectral microscopy for recording depth-resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth-discrimination and short acquisition times (20 minutes for single plane confocal image) was achieved

Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging

Improved understanding of the mechanism of nutrient’s uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman micro-spectroscopy (RMS) and coherent anti-Stokes Raman spectroscopy (CARS) to visualise the time-dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE-19) cells were prelabelled with deuterated Phe (L-Phe(D8)) and the uptake of the host derived L-Phe(D8) by A. castellanii trophozoites was measured by RMS for up to 48 hours post infection (hpi). This approach revealed a time-dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L-Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16-18% L-Phe(D8) from L-Phe(D8)–supplemented culture medium after 3h, 24h and 48h hpi. CARS microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite